Method of treating patient's oral diseases (versions)

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, and aims at treating patient's oral diseases. Cultured tissue constructs of fibroblast dermal cells are implanted onto the oral tissue. The cultured tissue constructs comprise the cultured cells and endogenous-produced extracellular matrix components without the exogenous matrix or network sustaining or scaffold links.

EFFECT: group of inventions enables providing more simple and less painful treatment of the oral diseases requiring the implanted tissue constructs to be used.

33 cl, 36 ex, 21 dwg

 

The technical field to which the invention relates.

The invention relates to the field of tissue engineering, in particular to the use of cultured tissue construct for regeneration and repair of tissues of the mouth.

The level of technology

The area of tissue engineering combines bioengineering methods with the principles of the life Sciences for understanding the structural and functional relationships in normal and pathological mammalian tissues. The aim of tissue engineering is the design and end use of biological substitutes to restore, maintain or improve tissue functions. Thus, by means of tissue engineering can design and produce bioengineered tissue in the laboratory. Bioengineered tissue may include cells that are typically associated with native mammalian tissues or person with scaffolds synthetic or exogenous matrix.

New bioengineered tissue must be functional for the transplant into the body of the owner and must continuously be included in the body of the host body or gradually bioconservatism cells from the patient-recipient. Manufacturer equivalent tissue without supporting link or without scaffold throws scientific challenges in the field of creation of new bioengineered tissue.

The majority of the processing is stopping the soft tissue of the oral cavity is carried out with the use of autologous tissue sky. Although this procedure can work very well when performing certain operations, it has the disadvantage of requiring a "donor site", which can be very painful for patients. This additional donor site is a very poor source of tissue, therefore, only a small number of teeth can be handled at one time. As a result it may be necessary to conduct multiple operations, or the dentist has the ability to ensure only the "worst teeth", even though several teeth may remain uncured, although the transplant procedure can also be useful for them.

Disclosure of inventions

The invention relates to methods for treating conditions of the oral cavity of the patient by implantation of cultured tissue construct into the tissue of the oral cavity of the patient.

Cultured tissue constructs of the invention contain cultured cells and components of the endogenous-produced extracellular matrix components without requiring exogenous matrix or network support or the us items. Thus, the invention primarily is to produce human cells and components of the human matrix produced by these cells, for use in the body of man.

The implant cultured tissue constructs contains fibroblasts that produce components of the extracellular matrix or without the addition of extracellular matrix components, network support or without the addition of the us elements.

Cultured tissue constructs contain fibroblasts that produce components of the extracellular matrix in the system with deterministic composition and/or without the use of uncertain biological components or components non-human origin, such as bovine serum or extracts of organs.

In addition, cultured tissue constructs can be made by sequential seeding of cells of different types that produce cultured tissue construct that mimics the cellular composition and tissue structures of the native tissue. In particular, this cultured tissue construct contains at least a layer of epithelial cells deposited on the layer of cultured cells fibroblasts. In addition, the tissue construct is produced or samsobeats cultured cells without the need for the us element or adding exogenous components of the extracellular matrix.

The implant cultured tissue construct of the present invention also provides a gel mixture of collagen the CSOs solution and agent, causing the failure.

In addition, implants cultured tissue construct of the present invention contain a layer of collagen gel, which contains collagen together with the agent that causes contraction, located on the gel layer is acellular collagen.

In yet another embodiment, the present invention epithelial cells added to a layer containing collagen gel and the agent causing the failure.

Strength properties of tissue constructs do autonymy in circulation, they are easy to peel off from the apparatus for culturing, in which they are formed and directly implanted without the need for any support or carrier in clinical or experimental applications. Tissue constructs of the invention indicated for the treatment of patients with lesions in the tissue of the oral cavity, such as a cut in the gums, loss of the interdental papilla, alveolar failure, damaged implant oral cavity, defects in the separation zone of the roots of teeth, and for patients in need of tissue reconstruction after resection of tumors of the maxillofacial region.

Brief description of drawings

Figure 1 is a graph showing the increase in the concentration of collagen, which is determined by the analysis of hydroxyproline in comparison with the number of cells in the dermal is onstructi, obtained from the cells of the foreskin of a newborn human and described in Example 1.

Figure 2 is a graph of data illustrating the contraction of hydrated collagen grid cells fibroblasts.

Figure 3 is a graph of data illustrating the contraction of the cells fibroblasts hydrated collagen meshes with different collagen content.

Figure 4 is a graph of data illustrating the contraction of the cells fibroblasts hydrated collagen meshes containing various amounts of fibroblast cells.

Figure 5 is a graph of data showing the ability of fibroblast cells to cause contraction of hydrated collagen mesh when using cells of different levels of doubling the population.

6 is a graph of data illustrating the effect of 10.0 μg/ml of inhibitor of cytochalasin In the ability of fibroblast cells to cause contraction of hydrated collagen mesh.

7 is a graph of data illustrating the effect of 0.36 μg/ml of inhibitor colcemid on the ability of fibroblast cells to cause contraction of hydrated collagen mesh.

Fig is a graph of data illustrating the effect of cytosine arabinoside on the ability of fibroblast cells to cause the counter is of hydrated collagen mesh.

Fig.9 is a graph illustrating the effect of thrombin on the contraction of collegeevy.myblogvoice at a concentration of thrombin 4.0 units/ml

Figure 10 is a graph illustrating the contraction of the collagen mesh, depending on the concentration of platelets and thrombin at a concentration of 4.0 units/ml

Figures 11-14 is a graphic illustrating the contraction of collagen meshes depending on the function, type and concentration of the used collagen.

Fig is a graph illustrating the effect of inhibitors of cytochalasin In and colcemid on the contraction of hydrated collagen meshes platelets.

Fig represents the image in isometric, partial section view of one apparatus according to the present invention.

Fig is a full-scale image in the isometric system, shown in Fig.

Fig is a section along the line 3-3 of the device shown in Fig.

Pig is a three-dimensional image, partially rejected, other apparatus in accordance with the present invention.

Fig - expanded image (top) of the apparatus shown in Fig.

Fig is a detailed section along the line 6-6 of the apparatus shown in Fig.

The implementation of the invention

From Britanie relates to methods for treating disorders of the oral cavity of the patient by implantation of cultured tissue construct.

It should be noted that the terms "cultured tissue construct", "genetically engineered living tissue", "construct the cell matrix, layer the cell matrix, equivalent skin", "living tissue" and "living connective tissue" may be used interchangeably, as embodiments of cultured tissue constructs of the present invention.

Still design currently living tissue constructs collected cells are not fully and should be based on either the addition or introduction of exogenous components of the matrix, or the inclusion of synthetic parts for structures or supports, or the use of both of these parts.

Bioengineered tissue constructs described in this document, exhibit many of the natural properties of the tissue, from which we derive its cells. Produced thus tissue constructs can be used to ensure state of the oral cavity of the patient.

One preferred implementation is a construct of the cell matrix containing the first type of cells and endogenous-produced extracellular matrix, in which the first type of cells are able to synthesize and excrete extracellular matrix to generate the construct in the cell matrix.

Another preferred variant implementation, not only at the em of a two-layer construct, containing the first type of cells and endogenous-produced extracellular matrix and the layer of cells of the second type located on or within the construct of the cell matrix formed by the first cell type.

More preferred implementation is a construct of the cell matrix containing fibroblasts, for example, those that come from the dermis, for the formation of a cultured dermal construct.

Another preferred variant implementation is a construct of the cell matrix containing fibroblasts, for example, those that come from the dermis, for the formation of a cultured dermal construct with a layer of keratinocytes, cultured for education epidermal layer to form the resulting two-layer cultured skin construct. Cultured skin constructs of the invention are many physical, morphological and biochemical characteristics of natural leather.

In an even more preferred embodiment, the construct of the cell matrix is a tissue construct, which is similar to the dermal layer of the skin - human dermal construct, which is formed in the system is deterministic (certain) composition containing cells of human origin and do not use the it chemically undefined components during their cultivation.

In the most preferred embodiment, the tissue constructs of the invention are made in the system of a certain chemical composition, containing cells of human origin, but not containing chemically undefined components or biological components or cells of human origin.

In another embodiment of the present invention cultured tissue constructs contain a gel mixture containing a solution of collagen and the agent causing the failure.

In a preferred variant of the invention, the cultured tissue constructs contain a first layer containing a cell-free collagen gel and a second layer located on the first layer, the second layer contains a second collagen gel containing collagen and the agent causing the contraction. In an even more preferred embodiment of the invention the second layer of collagen with an agent that causes contraction, seeded with cells keratinocytes.

A: the Use of cultured tissue constructs:

Cultured tissue construct of the invention can be used to treat disorders of the oral cavity, such as a cut in the gums, loss of the interdental papilla, the failure of the alveolar ridge, the effects of the damaged implant oral or p the following resection of oral and maxillofacial tumors.

It is recognized that health requires around each tooth was functional stationary part of the gums. In the absence of this fabric is often atrophy of the edge of the gums, leading to loss of part of the cortical plate, creating a worse prognosis for the teeth. In addition, often find inflammation of the mucous membrane in front of the teeth in the absence of functional zones of a fixed part of the gums, in spite of good home care for the oral cavity. This inflammation can cause osteoporosis around the teeth.

Since the late 1960-ies of this type of problem regularly corrected by free autogenous graft. The mucous membrane is removed from the front side of the examined teeth, and keratinized tissue is harvested from the palate and placed in the form of a seam on the layer of the graft. First, the graft is supported plasmatic circulation, and then subjected to revascularization of the surrounding layer. The success of this type of transplant reaches 100%. However, in many patients there is a strong desire to identify donor material is used instead of the palatal tissue. As a result, the number of surgical sites needed for this procedure, of course, can be reduced by half. After a few years began to use the replacement donor materials.

In recent years has been the use of freeze-dried skin of a corpse, as recently as the donor material was used acellular dermal implant. Although very remote, but possible, risk of infection associated with these types of dermal materials (mainly the risk of Contracting AIDS and hepatitis) relatively hinders their use. In addition, the resulting aesthetic appearance of the implants that use this donor material, usually not quite perfect.

In: Cultured tissue constructs containing the structural layer, at least one type of cells that produce extracellular layer, with the endogenous-produced extracellular matrix

One preferred embodiment of the invention provides a structural layer, at least one type of cells that produce extracellular matrix components and endogenous-produced extracellular matrix, for brevity denoted by the term "matrix", matrix fully synthesized and going through the cultivation of cells. This layer in this document is called "construct cellular matrix or layer of cellular matrix as cells secrete and see by themselves inside and along their matrix. As disclosed in co-pending application for U.S. patent, under serial No. 09/523809, toregister the trated 03/03/2000, and incorporated in this document by reference in its General form, cultured tissue constructs do not require, and thus do not include components of exogenous matrix, namely, the components of the matrix that are not produced by cultured cells, but are introduced by other means. In the preferred embodiment, it is shown that the construct of the cellular matrix produced by dermal fibroblasts has a predominant concentration of collagen, similar to its concentration in natural skin. By electron microscopy it was shown that matrix by nature is fibrous, including collagen, which shows a four-stage structure bands with a length of 67 nm, and packing of fibrils and fibrillar ligaments, similar to natural collagen.

By electrophoresis SDS page-ordinator with the delayed decrease of the detected presence in these constructs collagen as type I and type III is the predominant types of collagen found in natural human skin. Using standard techniques of immunohistochem tissues dermal construct the cell matrix becomes positive with respect to decorin, dermatologit the proteoglycan, which is known to be associated with the collagen fibrils and, as expected, regulates the diameter of the fibrils in ivo. Decorin you can also visualize the construct through PAM. Produced by tissue also stained positively with respect to tenascin - glycoprotein of the extracellular matrix found in, for example, mesenchyme or tissues during recovery. It was shown that like tissue when restoring in vivo, in tissue produced in culture increases the ratio of collagen type I to collagen type III as education matrix.

Not trying to associate with theory, suggest that the cells fill up quickly in the open space between them together with a loose matrix, similar to granulation tissue, consisting mainly of collagen type III and fibronectin, and then reconstruct this loose matrix dense matrix composed mainly of collagen type I. it has Been shown that the generated cell matrix contains glycosaminoglycans (GAGS)such as hyaluronic acid (ha), fibronectin; proteoglycans, in addition to decorin, for example, biglycan and versican and profile sulfated glycosaminoglycans, such as digitalready acid, di-chondroitin-0-sulfate, di-chondroitin-4-sulfate, di-chondroitin-6-sulfate, di-chondroitin-4,6-sulfate, di-chondroitin-4-sulfate-UA-2S and di-chondroitin-6-sulfate-UA-2S. These structural and biochemical characteristics manifest themselves during the times the development of the construct in culture and become clearly evident, when the construct is approaching its final form. The presence of these components in fully formed kultivirovanii dermal construct cell of the matrix indicates that the construct has structural and biochemical characteristics approaching that of normal dermis.

While the above list is a list of biochemical and structural features of cultured construct the cell matrix generated from dermal fibroblasts, it should be understood that the cultured constructs the cell matrix formed from other types of fibroblasts will develop many of these and other features, phenotypic tissue from which they originated. In some cases, the fibroblasts can be induced for expression phenotypically components or by chemical exposure or exposure, physical exertion or by transgenic methods.

Another preferred embodiment of the invention is the layer of the cell matrix, in which there is a second layer of cells, have at it. The second layer of cells grown on the layer of the cell matrix for the formation of bioengineered two-layer tissue construct. In a more preferred embodiment, the cells of the second layer on the origin of Vlada epithelial. In the most preferred embodiment, the second layer contains the cultured human keratinocytes, which together with the first layer of the cellular matrix, construct the cell matrix formed from dermal fibroblasts and endogenous matrix for the formation of the dermal layer, form a living construct of the skin. When the epidermal layer is fully formed, it is a multilayered, stratified and well-differentiated layer of keratinocytes, which are manifested in the form of the basal layer, nadesalingam layer, granular layer and stratum corneum. Skin construct has a well-developed basal membrane, located at the dermal-epidermal junction, as shown by the method of transmission electron microscopy (TEM). Basal membrane looks very thick around polydesmida, markgrafenheide fibrils that are composed of collagen type VII, which is evident by the PAM method. Fixing fibrils may look coming out of the basement membrane, capturing fibrils of collagen in the dermal layer. These locking fibrils and other components of the basal membrane, are keratinocytes. It is also known that while the keratinocytes are able to select for themselves the components of the basal membrane, recognized the basal membrane will not be formed in the absence of fibroblasts. Immunohistochemically coloration of skin construct of the present invention also showed the presence of laminin - protein basal membrane.

In a preferred method of the invention for the formation of the construct of the cell matrix, the first type of cells, type of cells that produce extracellular matrix, plated on the substrate, cultivated and encouraged to synthesize and highlight ordered the extracellular matrix around them to form the construct of the cell matrix. Drugom preferred method of the invention, the surface of the construct of the cell matrix seeded with cells of the second type of cells that are cultivated for the formation of a two-layer tissue construct. In the preferred method, a full-layer skin construct, having features similar to natural human skin, is produced by culturing fibroblasts, such as dermal fibroblasts, under conditions sufficient to induce the synthesis matrix to form a cell matrix of the dermal cells and matrix of the dermal layer, which is seeded with epithelial cells such as keratinocytes and cultured under conditions sufficient for the formation of fully differentiated, stratified epidermal layer.

Thus, one way to get cloth is o constructs of the present invention includes:

(a) culturing at least one type of cells that produce extracellular matrix, in the absence of exogenous components of the extracellular matrix or structural supporting element; and,

(b) stimulation of cells obtained at stage (a) to the synthesis, isolation and organize extracellular matrix components for the formation of a tissue construct, consisting of cells and matrix synthesized by the cells; at this stage (a) and (b) can be carried out simultaneously or sequentially.

For the formation of a two-layer tissue construct containing the construct of the cell matrix and the second cell layer thereon, the method further includes a step of: (C) culturing the cells of the second type formed on the surface of the tissue construct to generate the two-layer tissue construct.

The type of cells that produce extracellular matrix, for use in the invention can be any type of cells capable of producing and highlight components of the extracellular matrix and regulate components of the cell matrix to construct the cell matrix. You can cultivate several types of cells that produce extracellular matrix, to form a construct in the cell matrix. Cells of different types or sources of tissue can Kul is to wirawati together in the form of a mixture to produce complementary components and structures similar to those found in native tissues. For example, the type of cells that produce extracellular matrix, may have other types of cells, mixed with him, for generating such a large number of extracellular matrix, which is not usually produced by the first cell type. Alternatively, the type of cells that produce extracellular matrix, can also be mixed with other types of cells that form specialized tissue structures in the tissue, but not make a substantial contribution to the total education of the matrix aspect of the construct of the cell matrix, for example, in certain skin constructs of the invention.

Although in accordance with this invention can use any type of cells that produce extracellular matrix, the preferred cell types for use in this invention originate from the mesenchyme. Preferred cell types are fibroblasts, stromal cells and other supporting cells of the connective tissue, more preferably dermal fibroblasts in human dermis, to obtain human dermal construct. Cells fibroblasts, as a rule, produce several extracellular matrix proteins, mainly collagen. There are several types of collagens produced by fibroblast and, however, collagen type I is the most common in vivo. Strains of cells human fibroblasts can occur from several sources, including, without limitation, the male foreskin of a newborn, dermis, tendon, lung, umbilical cord, cartilage, urethra, corneal stroma, the mucous membrane of the mouth and intestines. Human cells are not limited to fibroblasts and may include smooth muscle cells, chondrocytes and other cells of the connective tissue of mesenchymal origin. Preferred, but not required, that the origin of the generating matrix of cells used in obtaining tissue construct was carried out on this type of tissue with which they will have similarities or will imitate it after using the invented methods of cultivation. For example, in the embodiment, in which produces a skin construct, the preferred cell generating matrix is a fibroblast, preferably dermal origin.

In another preferred embodiment, fibroblasts, detachable micropreparative from the papillae of the dermis hair follicle, can be used to obtain a matrix separately and in combination with other fibroblasts. In the embodiment, which is produced corneal construct, glue the ka, generating the matrix comes from the corneal stroma. Donor cells can vary depending on the development and age. Cells can be harvested from donor tissues of embryos, newborn or older individuals, including adults. Embryonic precursor cells, for example, stem cells of the mesenchyme, can be used in the invention and induced to differentiate and develop into the desired tissue.

Although human cells are preferred for use in the invention, the cells used in the method are not limited to cells from human sources. You can use cells from other mammalian species, including but not limited to: horse, dog, pig, cow and sheep sources or sources of rodent species, such as mouse or rat. In addition, in the invention it is also possible to use cells that spontaneously chemically or viral transliterowany, recombinant cells or genetically engineered cells. In those variants of implementation, which include the use of several types of cells, you can use a hybrid mixture of normal cells from two or more than two sources, a mixture of normal and genetically modified or transfected cells or a mixture of cells from two or more than two types or tissue history is nicks.

Recombinant or genetically engineered cells can be used to generate the construct in the cell matrix with the aim of creating a tissue construct, which serves as an implant for drug delivery to the patient in need of increased quantities of natural cellular products or therapeutic treatment. Cells can produce or deliver to a patient through an implant products of recombinant cells, growth factors, hormones, peptides or proteins over an extended period of time, or, if necessary, with biological, chemical or thermal signal about the state of the patient. Desirable long-term or short-term expression of the gene product, depending on the instructions on the use of cultured tissue construct. Long-term expression is desirable in the case when cultured tissue construct is implanted to supply the patient's therapeutic products for a long period of time.

On the contrary, short-term expression is desirable in those cases, when cultured tissue construct are transplanted to a patient with a wound in which cells cultured tissue construct activate normal or close to normal healing or reduce the scarification wounded area. After healing tissue is necessary the cost in the gene products of the cultured tissue construct at the site disappears or can disappear. Cells can also be treated by genetic engineering methods for the expression of proteins or various types of extracellular matrix components that are either "normal", but expressed at a high level, or otherwise modified to create a transplanted devices containing extracellular matrix and living cells that are therapeutically useful for better wound healing, facilitated or directed the formation of new blood vessels or minimized the formation of scar or keloid zone. These procedures are well known in the art and described in Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), incorporated in this document by reference. All of the above types of cells included within the definition of "cell generating matrix used in this invention.

The predominant main component of the extracellular matrix produced by fibroblasts, is fibrillar collagen, particularly collagen type I. Fibrillar collagen is a key component in the structure of the cell matrix, but this invention is not limited to matrices consisting only of this protein and protein type. For example, other collagen - like fibrillar and defibrillatory collagen family of these types of collagens, as II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XII, XIV, XV, XVI, XVII, XVIII, XIX, can be obtained by using an appropriate cell type. Similarly, other matrix proteins, which can be produced and deposited using the present method include, without limitation: elastin; proteoglycans, such caldecrin and biglycan; or glycoproteins, for example, tenascin, vitronectin, fibronectin, laminin, thrombospondin I and glycosaminoglycans (GAGS)such as hyaluronic acid (ha).

The cell producing the matrix, cultivated in the vessel, suitable for cell or tissue culture animal, for example, in a Cup for cultivation, flask or rotating the vial, which contribute to the formation of three-dimensional mcanearney patterns. Suitable surface for cell cultivation can be any biologically compatible material to which cells can adhere and which can provide locking means for the generated construct the cell matrix. As surfaces for cell cultivation, you can use materials such as: glass, stainless steel, polymers, including polycarbonate, polystyrene, polyvinyl chloride, polyvinylidene, polydimethylsiloxane, fluorocarbon polymers and fluorinated ethylene-propylene polymer and silicone substrates, including quartz glass, polycrystalline silicon or cu is mnovie crystals. Surface material for growing cells can be chemically treated or modified, to electrostatically charge or cover of biological agents, such as poly-L-lysine or peptides. An example of a peptide coating is an RGD peptide.

While tissue construct of the invention can be grown on a solid surface for growing cells, the preferred surface for the cultivation of cells with pores that connect both the upper and the lower surface of the membrane, giving the opportunity for bilateral contact environment with developing tissue construct or to contact only the bottom from the culture. Bilateral contact allows the environment to be in contact with both the upper and lower surfaces of the developing construct, providing maximum size of the impact of nutrients in the environment. The environment can also contact only with the bottom surface of the formed tissue construct, so that the upper surface can be opened for air, as with the development of cultured skin construct.

The preferred vessel for culturing is such a vessel, which is used to insert media, culture-treated permeable element, such as a porous membrane, which is suspended in the vessel for suspendresume, mA the future environment. Typically, the membrane is fixed at one end of the tubular element or frame, which is injected inside and sostakovic with base, such as a Petri dish or Cup for cultivation, which you can cover. Vessels for cultivation, including the insertion of a carrier with a porous membrane known in the art and are preferred for carrying out the invention are described in several U.S. patents relating to this field, some of which have become commercially available, including, for example, patents№№: 5766937, 5466602, 5366893, 5358871, 5215920, 5026649, 4871674, 4608342, the disclosure of these patents are incorporated into this document by reference. When using these types of vessels for culturing tissue construct is produced on the membrane surface, preferably on the upper front surface, and the culture is in contact with the cellular environment on both upper and lower surfaces.

The pores in the surface of the growth allow the passage of culture medium to provide nutrients to the bottom of the culture through the membrane, thereby allowing the cells to be fed from two sides or only on the lower side. The preferred pore size is the size that is small enough not to allow cells to grow through the membrane, but large enough to ensure that is to ensure the passage of nutrients, contained in the culture medium, the underside of the construct in the cell matrix, for example by capillary action.

Preferred pore sizes are less than 3 microns and in the range from 0.1 micron to 3 microns, more preferably in the range from 0.2 micron to 1 micron, and most preferably use a pore size of 0.4 micron to 0.6 micron.

In the case of dermal fibroblasts are the most preferred material is polycarbonate with a pore size of 0.4 to 0.6 microns. The maximum pore size depends not only on the size of the cells, but also on the ability of the cell to change its shape and to pass through the membrane. It is important that mcanearney construct were glued to the surface, but did not grow or seize the substrate so that it can be removed from it, for example, by peeling with a minimum application load.

The size and shape of the formed tissue construct are determined by the size of the surface vessel or membrane on which it is grown. The substrates may be of round, angular shape, a shape with rounded corners or an irregular shape. The substrates may also be flat or have a contour in the form of a mold to obtain a shaped construct for mating with the wound or the simulation of the physical structure of the nature of the Noah fabric. When calculating on the large surface area of the substrate for growing sow a proportionately larger number of cells on the surface, and requires a greater amount of environment for effective immersion and power cells. When the tissue construct is fully formed, either in the form of a single layer construct the cell matrix, either in the form of a two-layer construct, it is removed by peeling off the membrane substrate before the transplant patient.

Cultured tissue constructs of the invention does not rely on synthetic and absorbable elements, for example, in a mesh elements for the formation of tissue constructs. Mesh elements arrange in the form of woven, knitted or felted material. In systems that use a mesh element, the cells are cultivated and grown on both sides and inside the hollows of the grid enabling grids inside cultured tissue construct. The final construct, formed by methods that include such network relies on it to give physical support and volume. Examples of cultured tissue constructs, which are based on synthetic mesh elements found in U.S. patents№№5580781, 5443950, 5266480, 5032508, 4963489 authored by Naughton, et al.

System for generating cellular matrix layer or may be stationary, l is Bo it can be used perfusion means to culture medium. In a stationary system, the cultural environment is calm and relatively immobile compared to the perfusion system, in which the medium to be in motion. Perfusion environment affects cell viability and increases the development layer of the matrix. To the perfusion media includes, but without limitation: the use of a magnetic stirrer or motorized blade mixer in a Cup for cultivation below the carrier substrate containing a membrane for cultivation, or near it, for mixing environment; the pumping medium into or through the Cup or chamber for cultivation; moderate shaking cups for cultivation on a vibrating or rotating platform; or rotation, if the energy flows in a rotating flask. Other perfusion agents for use in the method of the invention can be determined by a person skilled in the technical field.

The compositions of the culture media suitable for use in the present invention, chosen on the basis of kultiviruemykh types of cells and produced a tissue construct. Used cultural environment and special conditions of cultivation required to activate cell growth, synthesis of matrix and viability will depend on the type of cultured cells.

In some cases, voltage is emer, in the manufacture of bioengineering two-layer skin constructs of the present invention, the medium composition may vary at each stage of manufacture, as needed for different purposes of various additives. In a preferred method, a layer of cellular matrix form under certain conditions, namely, cultivated in environments that have a specific chemical composition. In another preferred method, the tissue construct contains a layer of cellular matrix, comes with a second layer of cells, and have cultivated it, while both types of cells are cultivated in a system with a given cultural environment. Alternatively, the tissue construct contains a layer of cellular matrix produced under certain environmental conditions, and a second layer formed thereon in an uncertain environment. On the contrary, the tissue construct contains a layer of cellular matrix, which can be made in an uncertain environment, and the second layer it under certain conditions.

Preferably the use of cultural media of a certain chemical composition, namely, uncertain environments without organs or tissue extracts of animals, for example, without serum, pituitary extract, hypothalamic extract, placental extract and embryonic EC of the tract or proteins and factors allocated supply cells. In the most preferred embodiment, the environment is free from uncertain components and certain biological components derived from sources inhuman origin. Although adding undefined components are not desirable, they can be added to the culture at any time in accordance with the disclosed methods to successfully manufacture of a tissue construct. During implementation of the invention using sifted cells of human origin, cultivated with the use of chemically defined components, the resulting tissue construct is a human tissue construct a particular composition. You can also add synthetic functional equivalents to replenish the environment of a particular chemical composition within the scope of the definition of "definite chemical composition", to use the preferred method of manufacture. Typically, a specialist in the field of cultivation of the cells will be able to identify suitable natural human, recombinant human, or synthetic equivalents of the well-known animal components to replenish the culture medium of the invention, without conducting thorough research or experimental is the same. The advantages of using such a construct in the clinic is that the problem of unintentional infection of an animal or hybrid species is reduced. Advantages construct a specific chemical composition is that the test does not occur confusing results due to the presence of uncertain components.

The cultural environment consists of the nutrient base, which, in addition, usually Supplement other components. A trained professional can determine the appropriate nutrient base used in the cultivation of animal cells, with a reasonable expectation of successful formation of a tissue construct of the invention. Many of the commercially available nutrient sources suitable for implementing the present invention. These include commercially available nutrient sources that supply inorganic salts, a source of energy, amino acids and b vitamins, such as Needle in a modification of Dulbecco (DMEM), minimal medium (MEM); M; RPMI 1640; environment Dulbecco modification Iscove (EDMEM). Minimal nutrient medium (MEM) and M require additional recharge phospholipid precursors and nonessential amino acids. Commercially available mixtures enriched with vitamins that post is make more amino acids, nucleic acids, cofactors of enzymes, precursors of phospholipids and inorganic salts, are medium ham's F-12 medium ham's F-10, NCTC NCTC 109 and 135. Even when the variation of the concentrations of all nutrient environments provide cells a major nutrient source in the form of glucose, amino acids, vitamins and inorganic ions together with other major components of the environment. The most preferred basic medium of the invention contains a nutrient basis, not containing calcium or Wednesday Needle in the modification of Dulbecco (DMEM) with low calcium content, or, alternatively, DMEM and medium ham's F-12 in a ratio of from 3:1 to 1:3, respectively.

The base environment is filled components, such as amino acids, growth factors and hormones. Defined culture medium for culturing cells of the invention are described in U.S. patent No. 5712163 under the authorship Parenteau and in international publication WO 95/031473, disclosure of which is incorporated in this document by reference. In the art of famous and other environments, for example, those described in Ham and McKeehan, Methods in Enzymology, 58:44-93 (1979), or in other suitable environments of a certain chemical composition, see Bottenstein et al., Methods in Enzymology, 58:94-109 (1979). In a preferred embodiment, the primary environment updated with the following components known to the skilled is the individual in the field of cultivation of animal cells: insulin, the transferrin, triiodothyronine (T3), or one of two such components, nacatamales and on-phosphoryl-ethanolamine, or both of these components, the concentrations and substitutes for additives can be installed by a qualified technician.

Insulin is a polypeptide hormone that activates the uptake of glucose and amino acids to ensure long-lasting effects compared with multiple passes. Replenishment of insulin or insulin-like growth factor (IGF) is necessary for long-term cultivation because of the possible exhaustion of the ability of cells to absorb glucose and amino acids and because of the possible destruction of the cellular phenotype. Insulin can be obtained from the animal, for example, from cows, from human sources or by recombinant methods, as in the case of human recombinant insulin. Therefore, human insulin could be classified machinecheck a specific component that is not originating from the biological source of superhuman origin. Supplement insulin is recommended during serial cultivation, and it is available for environments in a wide range of concentrations. The preferred concentration range is from about 0.1 μg/ml to 500 μg/ml, more preferably from 5 μg/ml to 400 μg/ml and most before occhialino about 375 µg/ml A suitable concentration for supplements of insulin-like growth factor such as IGF-I or IGF-2, can be easily determined by a person skilled in the field of technology for cell types selected for cultivation.

Transferrin is present in the environment for regulation of iron transport. Iron is an essential trace element found in the serum. Because iron in free form can be toxic to cells, it is delivered to cells in serum is associated with transferrin form and its concentration is from 0.05 to 50 μg/ml, more preferably about 5 μg/ml

Triiodothyronine (T3) is the main component and active form of thyroid hormone, which include environment to maintain speeds of cellular metabolism. Triiodothyronine is added to the environment so that its concentration is from approximately 0 to 400 PM, more preferably from 2 to 200 PM and most preferably about 20 PM.

Any two components, such as ethanolamine and o-phosphoryl-ethanolamine, which are phospholipids, or both of these components add for the reason that they serve as important precursors in the metabolic pathway of Inositol and fatty acid metabolism. Replenish lipids, which are usually found in the serum, it is necessary for a medium not containing serum. Ethan is Lamin and on-phosphoryl-ethanolamine feature for environments at concentrations from 10 -6up to 10-2M, more preferably in a concentration of about 1·10-4M

Throughout the cultivation period basic environment additionally replenish other components to induce synthesis and differentiation, or to improve cell growth, such as hydrocortisone, selenium and L-glutamine.

It is shown that hydrocortisone in the culture of keratinocytes activates the phenotype of keratinocytes and therefore enhances the differentiating characteristics, such as content involucrin and keratinocyte transglutaminase (Rubin et al., J. Cell Physiol., 138:208-214 (1986)). Therefore, hydrocortisone is a desirable additive in cases where these characteristics are useful, for example, in the formation of lamellar implants keratinocytes or dermal constructs. Hydrocortisone can be added so that its concentration ranged from 0.01 mg/ml to 4.0 mg/ml, most preferably from 0.4 μg/ml to 16 m kg/ml

Selenium added to medium containing no serum, to replenish the trace elements selenium, which is usually provided by serum. Selenium can be added so that its concentration ranged from 10-9M to 10-7M, most preferably about 5.3·10-8M

The amino acid L-glutamine is present in some nutrient basis and can be added in those cases when it is not in outstay or contained in insufficient quantities. L-glutamine can also add in a stable form, for example, sold under the trademark GlutaMAX-1™ (Gibco BRL, Grand Island, NY). GlutaMAX-1™ is the stable form of the dipeptide L-alanyl-L-glutamine, it can be used interchangeably with L-glutamine and add in equimolar concentrations as a substitute for L-glutamine. Dipeptide ensures the stability of L-glutamine from destruction over time during storage and during aging, which can lead to inaccurate determination of the effective concentration of L-glutamine in the medium. Usually a basic environment Supplement L-glutamine or GlutaMAX-1™ so that its concentration is preferably from 1 mm to 6 mm, more preferably from 2 mm to 5 mm and most preferably 4 mm.

Growth factors, such as growth factor epidermal (EGF), can also be added to the environment as an aid in the establishment of cultures through cell multiplication and sowing. You can use EGF in native or recombinant form. The human form EGF, native or recombinant, preferred for use in the environment in the manufacture of equivalent skin that do not contain biological components of human origin. EGF is an optional component and can be added so that its concentration ranged from 1 to 15 ng/ml, more preferably from 5 to 10 ng/ml/p>

The environment described above, usually prepared as described below. However, it should be understood that the components of the present invention can be prepared and collected using traditional methodology, compatible with their physical properties. In this field it is well known in the replacement of certain components suitable similar or functionally equivalent to the applicable agent for the purpose of availability and economy, while achieving similar results. A naturally occurring growth factors can be replaced recombinant or synthetic growth factors, which have similar characteristics and provide similar results when used for carrying out the invention.

Environment in accordance with the present invention are sterile. Sterile components have become sterility or are in a sterile condition after preparation according to traditional methods, for example, by filtration. Appropriate aseptic procedures were used in the following Examples. First combine medium DMEM and F-12, and then add individual components to populate the environment. Royal solutions of all components can be stored at -20°C, except for the nutrient source that can be stored at 4°C. All uterine solutions prepared with a final concentration of 500X, providing what Uta above. Royal solutions of insulin, transferrin and triiodothyronine (all from Sigma) is prepared as follows: triiodothyronine first dissolved in 1 N hydrochloric acid (HCl) in absolute ethanol in the ratio of 2:1. Insulin is dissolved in dilute HCl (approximate concentration of 0.1 M), and transferrin dissolved in water. Received three solution is then mixed and diluted with water to a concentration of 500X. Ethanolamine and o-phosphoryl-ethanolamine dissolved in water to a concentration of 500X and sterilized by filtration. Progesterone is dissolved in absolute ethanol and diluted with water. Hydrocortisone dissolved in absolute ethanol and diluted in phosphate buffered saline (SFFR). Selenium is dissolved in water to a concentration of 500X and sterilized by filtration. EGF become sterile and dissolved in SFR. Adenine is dissolved difficult, but it can be dissolved in a manner known to a person skilled in the art. Serum albumin can be added to specific components in order to stabilize them in solution, these components are currently produced either from human or animal sources. For example, serum albumin human (HSA) or bovine serum albumin (BSA) can be added during prolonged storage to maintain the activity of progesterone and EGF uterine dissolve the RC. The environment can either be used immediately after preparation or stored at 4°C. When stored EGF should not be added until time of use.

To form the layer of the cell matrix by culturing cells that produce matrix environment contribute additional agents that activate the synthesis and deposition of matrix cells. These complementary agents compatible with the cells, have a high degree of purity and contain no contaminants. The medium used for the formation of a layer of cellular matrix, denoted by the term "matrix-producing environment.

For the preparation of matrix-producing environment the basic environment is filled ascorbate derivatives, such as sodium ascorbate, ascorbic acid or one of its chemically more stable derivatives, such as n-hydrate phosphate magnesium salt of L-ascorbic acid. Ascorbate is added to activate the hydroxylation of Proline and secretion of procollagen, a soluble precursor to the deposited molecules of collagen. It was also shown that ascorbate is an essential cofactor for the posttranslational processing of other enzymes, as well as a positive regulator in the synthesis of collagen type I and III.

Not wishing to be bound by theory, the replenishment of the environment of amino acids included in the synthesis be the Cove, maintains cellular energy, discarding the need for the production of amino acids by cells. The addition of Proline and glycine preferred, because they, as well as gidrauxilirovannaya form of Proline, hydroxyproline, are the main amino acids that make up the collagen structure.

Despite the lack of the necessary matrix-producing environment optional Supplement neutral polymer. Invented constructs the cell matrix can be produced without neutral polymer, but again, not wishing to associate with theory, its presence in the matrix-generating environment may contribute to more consistent processing and deposition of collagen between samples. One preferred neutral polymer is polyethylene glycol (PEG), which has been shown to activate in vitro processing of soluble procollagen produced by cultured cells prior to collagen deposited on the matrix. Grade PEG tissue culture molecular weight in the range from 1000 to 4000, more preferably in the range from 3400 to 3700 preferred in environments invention. The preferred concentration of PEG for use in the method can comprise about 5% weight by volume or less, preferably from 0.01% to 0.5% by mass to volume, b is more preferably from 0.025% to 0.2% by mass to volume most preferably about 0.05% weight by volume. Neutral polymers other crop varieties, for example, dextran, preferably dextran T-40, or polyvinylpyrrolidone (PVP), preferably with a molecular weight in the range of 30000-40000, can also be used at a concentration of about 5% or less, preferably from 0.01% to 0.5%, more preferably from 0.025% to 0.2%, most preferably about 0.05% weight by volume. Another variety of cell culture and cell-compatible agents that enhance processing and deposition of collagen, can be installed by a qualified specialist in the field of cultivation of mammalian cells.

In the case when the cells that produce matrix are confluent, the culture medium Supplement components that are involved in the synthesis, secretion or organization matrix, cells, as mentioned, stimulate the formation of a tissue construct, consisting of cells and matrix synthesized by the cells.

Therefore, the preferred composition of the medium for generating includes: a main mixture with a ratio of 3:1 eagle medium modification of Dulbecco (DMEM) (high glucose, without L-glutamine) and medium ham's F-12 replenished or 4 mm L-glutamine, or equivalent, 5 ng/ml factor in the growth of the epidermis, and 0.4 μg/ml hydrocortisone, 1·1 -4M ethanolamine, 1·10-4M o-phosphoryl-ethanolamine, 5 μg/ml insulin, 5 μg/ml transferrin, 20 PM by triiodothyronine, is 6.78 ng/ml selenium, 50 ng/ml L-ascorbic acid, 0.2 ág/ml L-Proline, and 0.1 mg/ml glycine. To produce culture medium can be added and other pharmacological agents to change the nature, amount and type of secreted extracellular matrix. These agents may include polypeptide growth factors, transcription factors or inorganic salts to activate transcription of collagen. Examples of polypeptide growth factors include transforming growth factor beta 1 (TGF-β1) and tissue plasminogen activator (TPA), each of which, as you know, activates the synthesis of collagen. Raghow et al., Journal of Clinical Investigation, 79:1285-1288 (1987); Pardes et al., Journal of Investigative Dermatology, 100:549 (1993). Example inorganic salts, which stimulates the production of collagen, is a cerium salt. Shivakumar et al., Journal of Molecular and Cellular Cardiology 24:775-780 (1992).

Culture is supported in the incubator to ensure adequate external conditions with controlled temperature, humidity and gas smethods culturing cells. To the preferred conditions include a temperature in the range from 34°C to 38°C, more preferably 37±1°C With a gaseous medium containing 5-10±1% CO2and the relative humidity is (Rh) of about 80-90%.

In a preferred embodiment, the construct of the cell matrix is a dermal construct, composed of dermal fibroblasts and the emitted matrix. Preferably using human dermal fibroblasts obtained in the form of primary cells from the dermis or, more preferably, a sequence of successively passing or parisianne from established cell strains or banks, which were screened to viral or bacterial infection and testing for purity. Cells grown under sufficient conditions in a nutrient medium in order to cause them to multiply to the number suitable for establishing the cells in the substrate for cultivation, which is formed by the construct of the cell matrix. Alternatively, cells from frozen cell strains may be seeded directly on the substrate for cultivation.

After receiving a sufficient number of cells, cells are harvested and seeded on a suitable surface for cultivation, and cultured under appropriate growing conditions to form a slit (confluent layer of cells. In a preferred embodiment, cells are seeded on the porous membrane which is immersed in water to allow the environment contactyou the ü from the bottom of the culture through the pores or directly from above. Preferably, the cells are suspended either in a basic environment, or in the culture medium and seeded on the surface for culturing cells at a density factor of 1·105cells/cm2to 6.6·10 cells/cm2, more preferably from 3·105cells/cm2to 6.6·105cells/cm2and most preferably about 6.6·105cells/cm2(cells per square centimeter of surface area). Culture is cultivated in a culture medium with the aim of establishing a culture and grown to approximately 80%-100% confluence, they chemically induced by replacing the medium on the matrix-generating environment to activate the synthesis and secretion of extracellular matrix. In an alternative method, cells are seeded directly in producing environment to eliminate the need for replacement of the primary environment in producing environment, but this method requires a higher density of sowing.

During culturing fibroblasts organize molecules secreted matrix for the formation of three-dimensional mcanearney patterns, but do not show significant contractile abilities in order to force the resulting construct the cell matrix to spontaneous contractions and peeling from the substrate for cultivation. Replacement media for the latest the matrix is rabatyvaemy environment is conducted every two to three days and after a period of time, for which the allocated matrix increases in thickness and the organization. The time required to create a construct in the cell matrix, depends on the initial density of sowing, cell type, age of the cell lines and the ability of cell lines to the synthesis and secretion of matrix. When the constructs of the invention are fully formed, they are bulky thickness due to the fibrous matrix, produced and organized by the cells; they are unusually merging or overly confluent cell cultures, in which cells can loosely stick to each other. Fibration gives the constructs of cohesion mcanearney properties, not properties similar to conventional crops because they are resistant to physical damage, such as tearing or cracking during routine treatment in a clinical setting. In the manufacture of cultured dermal construct cells will form an organized matrix around themselves on the surface of the cell culture, the thickness of which is preferably at least about 30 microns or more, more preferably the thickness through the surface of the membrane ranges from 60 to 120 microns, however, the resulting thickness of 120 microns and is suitable for use at trial or in clincheck the x applications in need of large thickness.

In a more preferred method, the layer of epithelial cells is applied on one surface, preferably on the upper front surface of the construct of the cell matrix. Epithelial cells may be seeded or cultivated on the construct of the cell matrix for the formation of multi-layered tissue construct. In the most preferred method keratinocytes derived from skin, grown on the cell construct for the formation of skin construct. In other preferred embodiments, the implementation of the cells of the corneal epithelium, also called a corneal keratinocytes, and which may be seeded on the construct of the cell matrix for the formation of corneal construct. Epithelial cells of the mucous membranes of the oral cavity can be grown on the construct of the cell matrix to form the construct of the mucosa. Epithelial cells of the esophagus may be seeded on the construct of the cell matrix to construct the esophageal tissue. Cells uroepithelial from the urinary tract may be seeded on the construct of the cell matrix to construct uroepithelial. Other cells of epithelial origin can be selected for the formation of a tissue construct from which these cells are derived.

Ways zag the training of epidermal cells to dermal construct and methods of their cultivation, including activation of differentiation and keratinization for the formation of a layer of differentiated keratinocytes, known in the art and are described in U.S. patent No. 5712163 under the authorship Parenteau, et al. and in U.S. patent No. 5536656 authored by Kemp, et al, the contents of which are incorporated into this document by reference. Usually for holding epidermolysis construct the cell matrix, the keratinocytes are seeded in the construct of the cell matrix and cultivate it until then, until it forms a layer thickness of one to three cell layers. The keratinocytes are then encouraged to differentiate to form a multi-layered epidermis, and then encourage to keratinization for the formation of the stratum corneum.

In the method of formation of differentiated epidermal layer of the cell strain selected subculturally keratinocytes, and increase the number of their cells. After receiving the required number of cells are separated from the substrate for cultivation, suspenders, counted, diluted, and then seeded on the top surface of the construct of the cell matrix with a density of 4.5·103cells/cm2to 5.0·105cells/cm2, more preferably from 1.0·104cells/cm2to 1.0·105cells/cm2and most preferably about 4.5·104cells/cm2. Constructs then withstand is for 60-90 minutes at 37±1°C, in an atmosphere of 10% CO2to enable keratinocytes to gain a foothold. After keeping the constructs are immersed in the environment for epidermolysis. After a sufficient period of incubation in the culture of the keratinocytes proliferate and sold, forming a merged monolayer along the construct in the cell matrix. After the merger, the composition of cellular environments, replace the medium for differentiation with the intention of inducing cellular differentiation. After the formation of multilayered epithelium use the environment to merge, and the culture is transferred onto the surface section of the air-liquid. For the differentiation and fusion of keratinocyte cells put on dry or malovlazhnom surface section of the air-liquid. Dry or malovlazhnom surface section can be described maxvoice kind of imitation leather with low humidity. Over time, the keratinocytes will discover the main part or all of keratins and other features found in natural skin affected by these conditions.

As mentioned above, the system for generating construct the cell matrix can be used in the formation of corneal construct. Cells of the corneal epithelium can occur from a variety of mammals. Preferred epithelial cell is a cell corneal epithelium of the rabbit and the human and (corneal keratinocyte), but you can use corneal keratinocyte any mammal. You can replace them with other epithelial keratinocytes, for example, those that come from the sclera (outer opaque white part) of the eyes or epidermis, but corneal keratinocytes preferred. In the method of formation of corneal construct an environment removed from the inserts for cultivation (containing the construct cellular matrix) and its environment. Normal cells of the corneal epithelium of the rabbit extend through reseeding, trypsinized to remove them from the substrate for cultivation, suspended in culture medium and seeded on top of the membrane at a density of about 7.2·104up to 1.4·105cells/cm2. Constructs then aged without medium for four hours at 37±1°C, in an atmosphere of 10% CO2to give the ability of epithelial cells to gain a foothold. After keeping the construct is immersed in the medium for storage of the cornea (SMM) (Johnson et al., 1992). Epithelial cells are cultivated up until the construct of the cell matrix is covered with epithelial cells. The fullness of the epithelial layer may be defined in various ways, for example, by staining the cultures with sulfate solution of Nile blue (in phosphate buffered saline solution at a ratio of 1:10000). Approximately h is cut to seven days after coverage of the construct of the cell matrix constructs aseptically transferred to the new tablets for cultivation with sufficient environment to maintain the cornea (SMM), bringing the liquid level accurately to the surface of the construct to maintain the boundary surface in the wet state without dipping the epithelial layer. Constructs incubated at 37±1°C, in an atmosphere of 10% CO2and at a humidity of over 60%, hmm, if necessary, carrying out replacement of environments, usually three times a week.

For differentiation, but not for keratinization layer of epithelial cells, which is necessary for the production of corneal construct, the surface epithelial cells are placed on a wet surface section of the air-liquid. Ways to ensure the wet surface section of the air-liquid are described in U.S. patent No. 5374515 authored by Parenteau. Used in this document, the term "wet surface area" is intended to refer to the surrounding culture, which is regulated so that the surface remains wet, high humidity, but not dry or submerged in water. The exact level of moisture and humidity in the culture medium is not critical, but it should be quite wet and damp in order to avoid the formation of dead skin cells. Wet the surface of the partition can be characterized as an attempt to copy the same moisture level of the human eye.

In an alternative preferred embodiment, the sowing of the second cells that produce matrix, can be initially formed the construct of the cell matrix to obtain the construct in the cell matrix with a greater thickness or a double-layer construct of the cell matrix. The second sowing can be carried out with the same or different type or strain of cells depending on the desired result. The second sowing is carried out in the same conditions using procedures and the matrix-generating medium used in the production of the first layer. One result of the second sowing distinct type of cells is the formation of a matrix with different profiles of the components of the matrix or packing density matrix, influencing wound healing when transplanting construct patient. The first seeding of the cells produces a matrix similar to the reticular layer of the dermis, more densely Packed layer of collagen type I and components of the extracellular matrix. The second seeding cells could develop a matrix similar to the papillary layer of the dermis characterized more loose fibrils of collagen and extracellular matrix. Another result is that the second cell type can produce a medicinal substance that could affect wound healing, for example, improving the engraftment or integration of the implant or mini is sirova or preventing scar formation.

In another preferred embodiment, the mixed population of cells of two or more than two types of cells can be grown together during the formation of the construct of the cell matrix, provided that at least one of the types of cells capable of synthesizing extracellular matrix. The second cell type may be the type that is needed to perform other functions of the tissue or the development of certain features of the tissue construct. For example, if the production of skin construct the cells of the papillae of the dermis or epithelial cells of the epididymis can be grown together with cells that produce matrix, to allow the formation of epithelial appendages or their components. Epidermal appendages, such as structures or components sweat glands or sebaceous glands or structure or components of the hair follicle, can be formed under cultivation with cells that produce matrix. Epithelial cells can be derived from if Yes, the exact structures of the gland or hair, located in the deep dermis, for example, by micropreparative, and flucytocine cells, myoepithelial cells, glandular secretory cells, stem cells of the hair follicle. You can also add other types of cells, which are usually finding is camping in the skin and make it for example, melanocytes, Langerhans cells and Merkel cells. Similarly, cells of the vascular endothelium can work together to cultivate to produce rudimentary components to form a new vascular network. Adipocytes can also be cultivated with cells that produce matrix for the formation of the construct used in reconstructive surgery. As an alternative method of delivery of this second type of cells, the cells can locally be seeded in the form of spots or locations of any number of spots of cells formed on or fully-formed construct cellular matrix or within it with the goal of localized development of these structures. In the case of seeding cells within the construct of the cell matrix, the cells can enter between the upper and lower surfaces, inside the cell matrix, so that the cells were grown, forming a specialized structure and performing its special function. To generate the three-layered tissue construct the first seeding of cells containing the cell type producing or not producing the matrix, is performed on the substrate for cultivation over a period of time sufficient to generate the construct of cell-matrix or cell layer. After formation of the first design, the KTA cell-matrix or cell layer hold the second seeding of cells, contains the type of cells that produce matrix on the upper surface of the first construct cell-matrix or cell layer and cultivated over time under conditions sufficient for the formation of the second construct of the cell matrix on the first construct. The second construct of the cell matrix hold the third seed of the third type of cells and culturing them under conditions sufficient for the formation of the third layer. As an example, for generating three-layered corneal construct cells of the first type can consist of cells of endothelial origin, for example, cells of the corneal endothelium; the second type of cells may include cells derived from connective tissue, such as corneal keratinocytes; and the third type of cells may include cells of epithelial origin, for example, the cells of the corneal epithelium. As another example, a three-layer construct of the skin, the cells of the first sowing may be of vascular origin with the aim of providing components for vascularization, the cells of the second sowing may contain dermal fibroblasts for education construct cellular matrix that performs rollermania construct and cells of the third sowing can be epidermal keratinocytes for the formation of epidermal what about the layer.

Tissue constructs of the invention can be stored at cryogenic temperatures using methods saleclonidine or cryopreservation. Methods saleclonidine tissue constructs are described in U.S. patent No. 5518878, and methods of cryopreservation are described in U.S. patent No. 5689961 and 5891617 and in international publication WO 96/24018, disclosure of which is incorporated in this document by reference.

From: Cultured tissue constructs gel containing a mixture of collagen solution with an agent that causes contraction

In another embodiment of the present invention cultured tissue constructs contain a gel mixture comprising a solution of collagen and the agent causing the failure.

This cultured tissue construct is produced by the formation of hydrated collagen mesh, in vitro, which is disclosed in U.S. patent No. 4485096 authored by Bell, which is included in this document by reference in its General form. This grid is subjected to contraction in kultivirovanii tissue construct of the agent causing the contraction included in it. Examples of agents that cause contraction, cells are fibroblasts and platelets.

Equivalent to the skin can be formed from the substrate of living connective tissue by seeding it cells keratinocytes and ensure R is a hundred. This equivalent skin clearly different from the previously described artificial skin types because of its basic organization similar to the organization of the skin and its constituent living cells can even be donated to a possible recipient of the implant.

Equivalent glands/organs or equivalent small vessels can be formed from a subject to contraction of hydrated grids collagen described in this document.

Thus, it can be seen that the cultured tissue construct produced according to this invention enables the formation of a living tissue equivalent of the glands and organs of many types, and functions. Such equivalents can even produce and store as inventory until such time as is necessary in their use.

One of the main advantages of such cultured tissue constructs is possible their use in the body of the host that is different from the donor cells used to generate the cultured tissue constructs, without any serious problems of exclusion, which would be expected. This is due to selection against cells responsible for rejection by the immune system of the recipient, which occurs when cells used for production of living tissue, proliferate under izaberete the Oia. In addition, certain cells lose their ability to stimulate the exclusion in that case, when they remain in tissue culture under certain conditions according to the present study reports.

Hydrated collagen mesh can be prepared using the collagen of tendons of the tail of the rat, and the collagen of the skin of a calf. Appropriate may also be other sources of collagen, including already used the skin of a human embryo. Solutions of collagen prepare and maintain at slightly acidic conditions. The lattice formed by the addition of fibroblast cells with a nutrient medium and the substrate, which raises the pH sufficient to precipitate the collagen fibrils of the solution. Preparation of hydrated collagen meshes is described in more detail in the following references, details of which are included by reference: Elsdale, T. and Bard, J., "Collagen Substrata For Studies On Cell Behavior," J. Cell Biol. 54, 626-637 (1972); Ehrmann, R.L and Gey, G.O., "The Growth of Cells on A Transparent Gel of Reconstituted Rat-Tail Collagen", J. Natl. Cancer Inst., 16, 1375-1403 (1956); Emermann, J. T. and Pitelka, D.R., "Hormonal Effects on Intracellular and Secreted Casein in Cultures of Mouse Mammary Epithelial Cells on Floating Collagen Membranes", In Vitro, 13, 316-328 (1977); Michalopoulous, G. and Pitot, H.C, "Primary Culture of Parenchymal Liver Cells on Collagen Membranes", Exp. Cell Res. 94, 70-78 (1975); Gey, G.O., Svotelis, M., Foard, M. and Bang, F.., "Long-Term Growth of Chicken Fibroblasts On A Collagen Substrate", Exp. Cell Res., 84, 63-71 (1974); and Hillis, W.D. and Band, F.., "The Cultivation of Human Embryonic Live Cells", Exp. Cell Res., 26, 9-36 (1962).

Cells fibroblasts actually used as an agent that causes contraction in the experiments described in this document was a foreskin fibroblasts human and dermal fibroblasts Guinea pigs. We also used fibroblasts from other sources, and it is assumed that, in fact, fibroblasts from any vertebrate animal could be suitable for contractions gidratirovannuyu grids. A convenient method for the simultaneous formation of the grid and seeding cells on it includes the neutralization of the acidic solution of collagen, supported in a Cup for culturing a nutrient medium containing cells fibroblasts. During neutralization, the collagen fibrils are deposited from a solution with the formation of grid cells, fibroblasts, homogeneous distributed on it. Cells and collagen mesh then support under conditions which allow the cells to attach to the collagen mesh and contrast it to the original size, thereby providing a living tissue.

The introduction of fibroblast cells in hydrated collagen causes the contraction of the grid as the displacement of the captured water. If the surface on which is formed a grid that is not wetted, for example, if a hydrophobic plate, resulting in R is the result, the fabric has a regular geometry. In the cups for the cultivation of tissues, some cells migrate from the grid to the surface of the Cup, and therefore the contraction of the grid is not always regular. When using non-wetted surface, such as a bacteriological Petri dish, the grid is saved as a perfect disk decreasing its radius cells.

Cells fibroblasts are homogeneous distributed state throughout the collagen mesh, not only on its surface, thereby mimicking the dermal layer of human and other mammals.

In the absence of cell radius grids are not subject to change. For example, an air-conditioned environment, prepared by cultivation of 1·106cells, foreskin fibroblasts person for five days in a nutrient medium did not cause contractions in the absence of cells.

Collagen mesh, obtained by contraction with cells similar to the skin or dermis; even under partial compression, they have acceptable texture, they're easy to apply. When the first production of the cells of the grid are almost transparent, but gradually become opaque as the displacement of water and reduction of diameter. After 20-30-fold decrease of the area of the grid they have kauchukopodobnoe consistency, light pink color and can be stretched to some extent without rupture and the and strain.

The initial mesh diameter is determined by the quantity of materials used and the plate on which they are formed. Thus, the maximum plasma density is an arbitrary parameter, but is associated with the cell count and protein concentration.

Although the majority obtained by the contraction of hydrated collagen meshes have the form of plates, can be formed and other forms. Tube, for example, can be formed by contraction of the grid in annular form, or in a suitable form to make a glove for the skin.

Keratinocytes of human skin, obtained by biopsy, precipitated received by the contraction of hydrated collagen meshes. The same make and keratinocytes, cultured in vitro. Seeding keratinocytes can be performed during the formation of the gel matrix, at any time during the period of contraction of the grid or at any time after the compression. Within three days after seeding suspensions of dissociated keratinocyte cells formed a continuous layer on the surface of the grid, and the process of keratinization began to lead to the formation of the cornified layer, which would prevent the loss of tissue fluids.

There are other cellular agents that cause contraction, addition of fibroblast cells. These include smooth muscle cells, cells poperechnom the lostae muscle and cardiac muscle cells.

Compression grid agent causing the contraction, such as cells, fibroblasts or platelets, makes collagen mesh in equivalent tissues with relatively high tensile strength compared to the strength of the collagen mesh, molded without an agent that causes contraction, in the case when both mesh survive in conditions of 100% relative humidity. Collagen mesh, molded without an agent that causes contraction, it has a texture similar to fresh gelatin, or falling apart during operation. Mesh obtained by contraction with platelets or cells, can be used to stretch or overlay in the form of a seam without damage.

Tensile strength was tested by determining the maximum weight in a given time, which can be hung on the grids obtained by contraction. In one example, the net volume of 5 ml was formed in a Cup with a diameter of 5.3 cm with contraction up to a diameter of about 2 cm cells, fibroblasts, supported 3.5 grams for 7 minutes. Another grid with the same volume of 5 ml in a Cup with a diameter of 5.3 cm was dropped from a height of 0.23 cm to cm 0,09 without changing the diameter by platelets and supported 11 grams for 10 minutes.

It is observed that the tensile strength and other properties are a function of many parameters, including the types and amounts of collagen, use the th agent, causing the contraction, and other additives used. Described in this document were used, for example, collagen type I. However, it is known that collagen type III gives the skin and blood vessels additional tensile strength, and therefore one would expect that the use of collagen type III collagen meshes described in this document, will increase their tensile strength. Similarly, it was found that the addition of glycosaminoglycans, for example, hyaluronic acid, chondroitin 4-sulfate and dermatan sulfate, improves the tensile strength and water-retaining properties.

Antibiotics such as penicillin, streptomycin and Fungizone can also be added, if desired, to prevent microbial contamination.

Although much of the work described in this document, refers to the formation of skin equivalents by cultivation of keratinocytes on collagen meshes obtained by contraction, these nets or they could grow other types of cells. Examples of such cells are cells of smooth and striated muscle, cartilage cells, bone cells, pancreatic cells, liver cells, etc.

We developed certain methods and devices for manufacturing plates of controlled sizes and/or different forms of contractorowned to ragenovich grids. In the case of use as the Contracting agent of fibroblast cells, unlimited collagen mesh usually undergo contraction in all directions. However, the plate, the boundaries of which remain fixed, is compressed only in thickness.

Fixture suitable for limiting boundaries, can be made from a sheet of stainless steel mesh of any form. The desired form for casting cut from the center of the stainless steel mesh, after which the surplus of the plate are cut off, leaving the edge of the grid, approximately the size of half an inch, around the form. Thus, form the skeleton stainless steel mesh that can be placed in a ditch, covered with non-stick material such as Teflon™ polytetrafluoroethylene, then enter the components used for the formation of the grid. After casting components and education grid it fill the cavity of a steel grid to which it is secured. As the cellular elements of the grid condense it by tightening together of the collagen fibrils, the volume mesh is reduced, but since the perimeter remains fixed reducing parameter is thickness. As the process grid loses fluid.

The particular advantage of the steel frame is that the final size of the equivalent tissue precisely equal to the size of the internal parameter is in the frame, and in particular the net gains additional strength due to the orientation of cells, set the bounding frame. In addition, since the mesh sizes do not change in width or length in the case when it is cast in a rectangular frame, even after it is cut out of the frame, there is a possibility for the application of epidermal component equivalent skin maternally equivalent later than one day after casting the grid, thereby reducing at least four days from the time required for preparation of the implant equivalent to the skin from the biopsy provided by the patient.

Grid components can be poured into the cell to cover the bounding mesh. After the grid is installed, it is fixed in the grid, so that when the contraction length and its width does not change. Is only reduced thickness. The final size of the thickness is a function of (1) initial mesh, (2) cell concentration, and (3) the content of collagen. The presence of proteoglycans, for example, hyaluronic acid and chondroitin sulphate, leads to increased contractions and reduces the thickness of the grid. A rectangular grid that holds the grid or dermal equivalent, which is seeded with epidermal cells, can be applied in solid form to the wound, needing skin. While it is on m is CTE, it can be cut on the inner perimeter of the grid immediately or after some time after its presence will contribute to maintaining the integrity of the implant.

The following describes another technique or method that is useful for anchoring the epidermis to the dermis equivalent drugs equivalent skin. Cast at the beginning of the dermal equivalent and a plastic plate, for example, Teflon™, polytetrafluoroethylene, skipped through which the ends of the needles and which allow him to remain in the normal template, put on a fresh casting. Plastic plates and the ends of the needles are removed after 1-4 days after sowing casting epidermal cells. This leads to the formation of the recesses, in which flow of epidermal cells, thus providing a large contact area between the epidermis and the dermal equivalent.

The cells of the follicle and granularity that produce by the method of enzymatic dissociation, may be seeded with suspensions of epidermal cells to fill such holes.

The main advantage of living tissue, described in this document, is the absence of rejection, which occurs when the receiver is different from the donor cells used in the production of equivalent tissue. For example, the implants of equivalents of the skin is made of cells, other than cells Retz is pienta implant, now made for animals. Implants equivalents leather, made as described above, but collected from the cells of the female line rats, Sprague-Dawley, transplanted male species rats Fischer and left in place for different periods of time. We can conclude that the implants equivalents of any kind that are made without specialized immune cells that are present everywhere in native tissues, would not be rejected because of antigenic determinants responsible for the rejection of the implant, is not expressed on the surfaces of cells included in the tissue equivalent. Their absence makes it impossible for the perception of foreign cells by the immune cells of the host body. This provides the ability to replace or add those types of cells, tissues or organs that need the recipient, because he himself they are lacking or missing.

D: Cultured tissue constructs containing collagen gel, layered on collagen gel:

Kultivirovaniya constructs of this variant implementation, although similar in both methods of preparation and methods of use with constructs containing gidratirovannuyu the net, in addition, contain a layer of collagen.

It has been discovered that the call is Genova mesh, cast on acellular, hydrated collagen gel in contact with the permeable element is not exposed to significant radial or transverse contraction and contrasts in thickness, eliminating thus the need to strengthen the collagen mesh, for example, stainless steel frame, for regulating the radial or transverse contraction. As an example, collagen mesh with a diameter of 24 mm, molded by the above method, but without fixing means could be contrasted radially to a diameter of 5 mm or less. On the contrary, collagen mesh with a diameter of 24 mm, cast on acellular, hydrated collagen gel, in contact with the permeable element will usually be contrasted radially to a diameter of about 15 mm Remove fixing means for regulating the transverse/radial contraction gives advantages from the standpoint of cost and ease of fabrication of tissue equivalents. It should be understood that such securing means can be used if desired in combination with gidratirovannym collagen gel in contact with the permeable element.

One method of obtaining tissue equivalents of the present invention includes:

(a) formation of a mixture containing collagen and at least one agent that causes contraction; and (b) on Esenia mixture, obtained in stage (a) for acellular, hydrated collagen gel in contact with the permeable element, and curing the mixture and gel under conditions which allow the formation of equivalent tissue.

In some embodiments, implementation of the present invention, one or more shock-absorbing elements, including, but without limitation, fiber gaskets, gaskets made of cotton and gels, agarose, used in combination with collagen gel, as described above. It was found that such absorbing elements, provide a firm and level of physical support and activate uniform contact between the equivalent tissue and environment for cell culture. Typically, the absorbent element is located near the surface of the collagen gel in front of hydrated collagen mesh. If absorbing element itself is a gel, such as agarose, the gel can be granted together with a nutrient medium to provide equivalent tissue nutrient.

Was observed following experimental endpoints equivalents fabric, equipped or not equipped gidratirovannym collagen gel and/or absorbing element, during the different phases of development of skin tissue equivalent:

1. The utilization of glucose;

2. The degree and quality of stratification or keratinization of the epidermis;

p> 3. the pH of the medium.

The observed pH environments obtained from tissue equivalents, supported together with the absorbent(s) element(s)were equally higher and closer to physiological pH than in environments derived from tissue equivalents that are supported in the absence of such elements. In addition, it was observed that the utilization of glucose, as a rule, was lower in tissue equivalents, supported with an absorbing element.

Surprisingly, it was found that Mature keratinization is activated in the skin tissue equivalents made with absorbing(x) item(s), in comparison with the control skin equivalents produced without such elements. Although the mechanism by which these absorbing elements can affect the differentiation of the epidermis, is unknown, but it is assumed that these elements can act as diffusion barriers, such as barriers permeability, and can filter the environment and/or to keep the products highlight the cells in the immediate vicinity of the tissue equivalents.

Living skin equivalents of the present invention prepared as described above, except that in accordance with this embodiment gidratirovannuyu collagen mesh cast on acellular, collagen gel.

One way of generating equivalent to the Noah fabric in accordance with the present invention includes:

(a) formation of a mixture containing collagen and at least one agent that causes contraction;

(b) application environment, obtained in stage (a), acellular, hydrated collagen gel in contact with the permeable element, and curing the mixture and gel under conditions which allow the formation of equivalent tissue; and

(c) seeding equivalent tissue obtained at stage (b) keratinocytes.

As the original, is one of the traditional Protocol casting tissue equivalents of the present invention, includes rapid mixing of the acidic solution of collagen having a pH of about 3-4, with a nutrient medium, bringing the pH of the final solution, if necessary, to a value of about 6.6-7,8, the addition of fibroblast cells, transferring the resulting mixture ("cast mixture") in a suitable mold or fixture for casting containing hydrated collagen gel, and then, the temperature is preferably in the range from 35°C to 38°C. it is Most convenient to bring the pH to combine the ingredients molded mixture at the same time. However, these stages can be performed in any desired order, provided that the stages completed so that it becomes possible to transfer molded mixture in the form for proper installation. The collagen fibrils are deposited from the cast mixture in the heated solution, raising the pH for the formation of hydrated collagen gel, which is subjected to contraction agent causing the contraction, and have on hydrated collagen gel.

Although ways of making a living tissue provided by this embodiment, it is applicable for the manufacture of tissue equivalents in General, these methods will be illustrated in connection with the production of skin equivalents for use in applications for skin grafts and test systems, including the skin equivalents.

Regarding the drawings, Figures 16-18 illustrate one implementation of the apparatus for determining the interaction of the skin and one or more agents using equivalents of skin tissue in accordance with the present invention, in which multiple containers 10, 22 placed in them by the tissue equivalents of the present invention provide together with the base or holder. The apparatus shown in Figures 16-18, also provide the funds, along with the cover 2. In some embodiments, the implementation coating (not shown) ensure that each container 10, 20. The coating is chosen from any biocompatible material that can support the seal on the container. Acceptable covering materials are foil or protective film, which can be glued to the apparatus by means of glue or by heating. In particular in practice of the present invention is suitable termoklina polyester film.

Containers for tissue equivalents include external container, the container 10 and the inner container 20. The inner container 20 provides together with the brace 50 to provide the means of installation of the inner container 20 into the outer container 10, thus defining the outer region 14 and the inner region 22. The inner container 20 together provide the equivalent of skin tissue 26, 28 placed on hydrated collagen gel 25, which is in turn located near the permeable element 24. Permeable element is hermetically attached to the inner container 20 with the formation of its bottom surface. The equivalent skin tissue contains two layers 26, 28, with a layer 28 contains epidermal layer, and the layer 26 contains the dermal layer. In some embodiments, the implementation of the sealing element 30 obespechivayushchaya between the inner wall of the inner container 20 and the equivalent skin 26, 28 and covers the perimeter of hydrated collagen gel 25 in that case, if the outer boundary of equivalent tissue 26, 28 is located inside of hydrated collagen gel 25. In variants of the implementation shown in the Figures, the container 10 provides a hole 21, which opens access to the outside area 14.

The apparatus depicted in Fig, in addition, provide an absorbing element 32. To the ome this, in other embodiments, the external camera 14 can contain a gel that is not shown and is located inside it.

Figures 19-21 illustrate another variant of implementation of the apparatus for determining the interaction of tissue and one or more agents using tissue equivalents in accordance with the present invention. Elements similar to elements in the other described embodiments implement denoted by the same numbers. In this embodiment, the outer wall 10 is square and is provided with raised sections 60 in the lower part, on which you can position the inner container 20. The outer wall 10, being extended upwards from the lower part of the machine, is formed as a pocket in the upper part of the device.

The outer wall 10 to provide a nourishing environment for equivalent tissue. Such environments are known in the art. The preferred serum-free nutrient medium are disclosed in co-pending application for U.S. patent, under serial No. 361041. The volume of medium should be chosen to fill the outer container 14 to a suitable level, so that was not created by the pressure head, which could displace the environment through permeable element 24, hydrated collagen gel 25, the equivalent of the fabric 26, 28 in the inner container 20.

In some variations the tah implementation of the present invention, the outer container 10 provides an absorbing element 32, which is located in the outer container 10, so that contact with the outer surface of the permeable element 24. An absorbent element 32 must be compatible with living tissue equivalents. For the preferred materials for absorbing element includes cotton, polyester and viscose. Particularly preferred absorbent material is cotton. In General, prefer to absorbing element was free of additives such as detergents.

In other embodiments, implementation of the present invention, the outer wall 10 to provide a gel (not shown), such as agarose, which sposobstvuyushchie. Because agarose and gel can add up to a level just below the hole 21, the equivalent tissue becomes available higher amount of nutrient medium, and the nutrients supplied by the equivalents of the fabric 26, 28 are exhausted not quick. The use of such gels provides benefits during storage and transportation apparatus of the present invention from the point of view of minimizing leakage environments and possible contamination of equivalent tissue due to leakage.

The outer 10 and inner 20 containers can be made of any desired material which does not react or does not have unwanted environmenta what I components analysis, including the equivalent tissue. For example, the molded mixture for the equivalent of living tissue does not stick to the walls of the inner container during contractions, so as not to interfere with the education of equivalent tissue.

It was found that the methods used for sterilization of the inner container 20 can impart stickiness to the equivalent tissue. For example, the resulting fabric will stick to the polystyrene, one of the preferred materials for the inner container, when it is sterilized by electron beam, not when it is sterilized with ethylene oxide. On the contrary, K-RESIN ® styrene butadiene polymer alloy of polystyrene and butadiene, which in particular is the preferred material for the inner container can be sterilized by electron beam without causing bonding of the resulting equivalent tissue. (K-RESIN ® styrene butadiene polymer is a trademark of Phillips Petroleum company.) In some embodiments, the implementation is desirable that the containers were made so that the equivalent fabric was see through container, for example, through the container wall or through a window in the container. The preferred material for the container 10 are polystyrene and PETG.

The inner container 20 may be of any shape or size that will fit the size and shape desired is quivalent tissue. The dimensions of the container again depends on the size and shape of the desired equivalent material and the desired volume for analysis. For example, a container with an outer diameter of about 25 mm and a volume of about 5 ml suitable for implementing the present invention in practice. In the embodiment shown in Figures 16-18, numerous containers provide the base or holder.

Permeable element 24 must have sufficient strength to maintain acellular, hydrated collagen gel 25 and equivalent tissue 26, 28. Porous membranes are suitable in the practice of the present invention. The pore size of such membranes choose to provide attaching an acellular, hydrated collagen gel 25. The preferred membranes are hydrophilic membrane with a thickness of about 1-10 mm and the diameter of pores in the range from 1 to 10 μm. For the preferred materials for the permeable element 24 is polycarbonate. Particularly preferred permeable element is a polycarbonate membrane, preferably free of wetting agents, commercially available from Nuclepore and having a pore size of about 3-10 microns.

Sealing means 30 can be manufactured from any material which is inert in the conditions of the analysis and used for tissue equivalents, and about who has a good seal between the inner container 20 and the equivalent tissue. For the preferred materials include polyethylene, TEFLON™ polytetrafluoroethylene (PTFE), a trademark of E.I. DuPont de Nemours and Company, polycarbonate and nylon. Sealing means is particularly suitable in the case when you want to save the contents of the outer container separately from any solution or substance deposited on the epidermis 25, for example, when measuring diffusion or permeability of a substance through the equivalent of a tissue.

As the equivalent tissue and acellular, hydrated collagen gel in accordance with the present invention can be prepared using the collagen extracted from the skin and tendons, including the tendons of the tail of the rat, the collagen of calf skin and tendon of extensor calf. Other sources of collagen could be suitable. Particularly preferred collagen composition that is derived from the tendon of the common digital rastiagivala calf, and ways of removing such collagen compositions are disclosed in co-pending application for U.S. patent, under serial No. 07/407465 registered 02.09.1994, the disclosure of which are incorporated into this document by reference.

In one method of the present invention acellular, hydrated collagen gel 25 is prepared from collagen compositions containing collagen in a concentration of 0.5-2.0 mg/ml, preferably the Colo 0.9 to 1.1 mg/ml and nourishing environment. This collagen composition is added into the inner container 20 and incubated under conditions which allow the installation of collagen composition and the formation of acellular, hydrated collagen gel of suitable size, typically about 1-5 mm in width, the preferred range of the width is approximately 2 to 3 mm, an Acellular, hydrated collagen gel 25 is preferably thick enough so that the portion of the gel remained acellular as the movement of cells from the equivalent tissue acellular, hydrated collagen gel and thin enough to make the equivalent tissue was not removed unwanted way from the nutrient source, provided in the outer container 10.

Next cast dermal equivalent acellular, hydrated collagen gel, using methods in accordance with the patents and the procedure described below. Cast mixture containing collagen and fibroblasts, add in the inner container 20 above cell-free, gidratirovannym collagen gel 25 and incubated under conditions which promote the formation of equivalent tissue. As education equivalent tissue on acellular, hydrated collagen gel 25, he contrasts radially. However, acellular, hydrated collagen gel 25 predot await excessive radial contraction of equivalent tissue without the use of mechanical restraints, such as textured metals and plastics or securing hooks and loops of VELCRO™is a trademark of Velcro Corporation.

As a rule, the parties to the equivalent fabric 26 is inclined toward the outer border of hydrated collagen gel 25 for the formation of the Mesa structure, as shown in Figures 6 and 18, at 52. Equivalent tissue 26 is now seeded with epithelial cells for the formation of the epidermal layer 28. The epidermal cells are seeded in culture medium at a concentration of about 0.3·10630·106cells/ml. Volume sown epidermal cells will depend on the size of the Mesa structure.

The concentration of collagen, the number of cells and volume of the molded mixture can be adjusted to optimize the diameter and thickness of the living tissue equivalent. Cast mixture contains cells at a concentration of about 1.25·104-5·104cells/ml collagen at a concentration of about 0.5-2.0 mg/ml in a nutrient medium. The preferred concentration of the cells is about 2.5·104cells/ml Was found that the ratio of the volume of the molded mixture for equivalent tissue to the volume of the molded mixture for acellular, hydrated collagen gel has an impact on the viability and differentiation of cells. Suitable volume ratio is molded of a mixture of equivalent tissue kototoi mixture of collagen gel composition is Aut from 3:1 to 1:3. The preferred ratio at which the concentration of cells in collagen grid is 2.5·104cells/ml, is 3:1.

The invention, moreover, will be explained in the following examples, which by nature are purely illustrative, and not intended to be used to limit the scope of the invention.

The materials used in the following examples, obtained from the sources described in the examples or made in accordance with these publications. For Examples used sterile procedures. Equivalents tissue incubated in an atmosphere of 10% CO2in the incubator, and throughout the experiment using sterile procedures.

The following examples are provided for better understanding of the practice of the present invention, and should not be interpreted in any way as limiting the scope of the present invention. Specialists in the art will recognize that it is possible to make various modifications of the methods described in this document without deviating from the essence and scope of the present invention.

Examples

Example 1: Formation of the collagen matrix by fibroblasts of the foreskin of a newborn human

The fibroblasts of the foreskin of a newborn man (produced by Organogenesis, Inc. Canton, MA) is seeded at number 5·105 cells/162 cm 2bulb for processing tissue culture (Costar Corp., Cambridge, MA, catalog No. 3150) and grown in a nutrient medium. Culture medium consists of: eagle medium modification of Dulbecco (DMEM) (high glucose, without L-glutamine, BioWhittaker, Walkersville, MD), which contribute 10% serum of newborn calves (NBCS) (HyClone Laboratories, Inc., Logan, Utah) and 4 mm L-glutamine (BioWhittaker, Walkersville, MD). Cells incubated in the incubator at 37±1 C with an atmosphere of 10±1% CO2. Wednesday replaced with a fresh medium every two to three days. After 8 days of culturing cells grow to confluence, namely, the Packed cells form a monolayer along the bottom of the flask with the tissue culture medium is sucked off from the flasks with culture. For rinsing the monolayer at the bottom of each flask with culture add sterile-filtered phosphate buffered saline, and then suck it from the flasks. Cell release from the flask by adding to each flask 5 ml of a mixture of trypsin, disodium salt EDTA and glutamine (BioWhittaker, Walkersville, MD) and moderate shaking to ensure complete coverage of the monolayer. Culture returned to the incubator. Immediately after release of cells added to each flask 5 ml SBTI (soybean trypsin inhibitor) and mix it with the suspension to termination of trypsin-disodium salt of EDTA. The cell is Uspenskiy removed from the flasks and evenly divided between the sterile, conical centrifuge tubes. Cells are harvested by centrifugation at approximately 800-1000 grams for 5 minutes.

Cells resuspended in fresh medium to a concentration of 3.0·106cells/ml, and seeded in the inserts for processing tissue culture with pore size of 0.4 micron and a diameter of 24 mm (TRANSWELL®, Corning Costar) in a six-cell cuvette at a density of 3.0·106cells/insert (6,6·105cells/cm2). Cells incubated in the incubator at 37±1°C with an atmosphere of 10±1% CO2and fed fresh medium for generating every 2-3 days for 21 days. Environment for the formation contains: 3:1 basic mixture of DMEM medium and medium ham's F-12 (Quality Biologies Gaithersburg, MD), 4 mm GlutaMAX-1™ (Gibco BRL, Grand Island, NY) and additives with the final concentration: 5 ng/ml human recombinant growth factor epidermis (Upstate Biotechnology, Lake Placid, NY), 2% serum of newborn calves (Hyclone, Logan, Utah), and 0.4 μg/ml hydrocortisone (Sigma St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY ACS grade), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee, WI), 50 ng/ml L-ascorbic acid (WAKO Chemicals USA, lnc. #013-12061), 0.2 ág/ml L-Proline (Sigma, St. Louis, MO), 0.1 mg/ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) with a molecular mass of 3400-3700 (type CL the exact culture) (Sigma, St. Louis, MO).

Samples for histological assay, 7, 14 and 21 days and fixed in formalin, and then immersed in paraffin. Fixed with formalin samples immersed in paraffin and section size of 5 micrometers stained with hematoxylin and eosin (H&E) according to methods known in the art. Using stained with hematoxylin and eosin preparations, carry out thickness measurements on ten randomly selected microscopic fields using a 10X eyepiece supplied with ocular scale 10 mm/100 micrometers.

The results for two different cell strains of human dermal fibroblasts are collected in table 1, which shows the thickness of the construct in the cell matrix as its development.

Table 1
Thickness (microns)
Day 0Day 7Day 14Day 21
W average (n=3)030,33±2,6163,33±4,4084,00±4,67
W average (n=4)0 42,00±5,1463,85±4,5076,25±8,84

The samples are subjected to analysis for the concentration of collagen on days 7, 14 and 21 days. The collagen content assessed by the colorimetric method of analysis of the content of hydroxyproline known in the art (Woessner, 1961). At the same time determine the number of cells. Table 2 is a summary of data on concentrations of collagen, and table 3 is a summary of the data cells of the cell matrix constructs produced from two different cell strains (B and V) using the above methods.

Table 2
Collagen (µg/cm2)
Day 0Day 7Day 14Day 21
W average (n=3)093,69±22,73241,66±21,08396,30±29,38
W average (n=3)0107,14±17,16301,93±23,91457,51±25,00
Table 3
Cells (cells/cm2)
Day 0Day 7Day 14Day 21
W average (n=3)6,6×10511,8±4,4×10511,4±1,7×10513,9±1,2×105
B156 average (n=3)6,6×10513,1±0,5×10514,0±2,1×10517,1±1,7×105

Samples of the dermal matrix derived from human cells, analyzed at 7, 14 and 21 days by electrophoresis SDS page-ordinator with the delayed decline to determine collagen composition with detection in samples of alpha bands of collagen type I and type III.

Biochemical characteristics of the dermal matrix is determined using immunohistochemistry methods. Identification of fibronectin spend on fixed paraffin sections, using enzymatic historicana system streptavidin-Biotin (Zymed Laboratories Inc., South San Francisco, A). The presence of tenascin define primary staining anti-Tenancingo antibodies (Dako, Carpintheria, CA) followed by staining protivorechivogo antibodies labeled with horseradish peroxidase (Calbiochem) as a secondary antibody. Samples visualize by applying diaminobenzene (Sigma St. Louis, MO) and contrast dye dye Nuclear fast red.

Quantification of glycosaminoglycans and collagen (GAG) carried out on samples 21 days, using a previously described method (Farndale, 1986). The analysis shows the presence of 0.44 grams GAG on cm2in the sample of the dermal matrix derived from human cells and selected for 21 days after sowing.

Example 2: a full-layer skin construct

Using dermal construct formed by the method described in example 1, normal epidermal keratinocytes foreskin damaged man (produced by Organogenesis, Inc. Canton, MA) placed on the construct of the cell matrix for the formation of the epidermal layer of the skin construct. Wednesday aseptically removed from the inserts for cultivation and its environment.

Normal human epidermal keratinocytes to multiply passage 4 from the frozen cell strain subculture to merge. Cells then release of the cups for cultivation, using trypsin-disodium salt of EDTA, the joint is both, centrifuged to education in vitro cellular precipitate, resuspended in the environment for epidermolysis, counted and seeded on top of the membrane at a density of 4.5·104cells/cm2. The constructs are then incubated for 90 minutes at 37±1 C, in an atmosphere of 10% CO2to enable keratinocytes to gain a foothold. After keeping the constructs are immersed in the environment for epidermolysis. The environment for epidermolysis consists of: basic mixture with a ratio of 3:1 eagle Medium modification of Dulbecco (DMEM) (high glucose, without L-glutamine (BioWhittaker, Walkersville, MD) and medium ham's F-12 (Quality Biologies Gaithersburg, MD), which fill up the following additives with final concentrations: of 0.4 μg/ml hydrocortisone (Sigma St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO), is 6.78 ng/ml selenium (Aldrich), is 24.4 μg/ml adenine (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 4 mm L-glutamine (BioWhittaker, Walkersville, MD), 0,3% chelated serum of newborn calves (Hyclone, Logan, Utah), 0,628 ng/ml progesterone (Amersham, Arlington Heights, IL), 50 μg/ml sodium salt of L-ascorbic acid (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 10 ng/ml of growth factor epidermal (Life Technologies Inc., MD) and 50 μg/ml gentamicin sulfate (Amersham, Arlington Heights, IL). Constructs cultivate the environment for epidermolysis for 2 days at 37±1°C, in an atmosphere of 10% CO2.

Two days later, the construct is immersed in a medium consisting of: a mixture with a ratio of 3:1 eagle medium modification of Dulbecco (DMEM) (high glucose, without L-glutamine, BioWhittaker, Walkersville, MD), environment Hamar-12 (Quality Biologies, Gaithersburg, MD), which fill up the following additives with final concentrations: of 0.4 μg/ml hydrocortisone (Sigma, St. Louis, MO), 1·10-4ethanolamine (Fluka, Ronkonkoma, NY), 1·10-4on-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), is 24.4 μg/ml adenine (Sigma Aldrich Fine Chemicals Company), 4 mm L-glutamine (BioWhittaker, Walkersville, MD), 0,3% chelated serum of newborn calves (BioWhittaker, Walkersville, MD), 0,628 ng/ml progesterone (Amersham, Arlington Heights, IL), 50 μg/ml sodium ascorbate, 265 ág/ml calcium chloride (Mallinckrodt, Chesterfield, MO) and 50 µg/ml gentamicin sulfate (Amersham, Arlington Heights, IL). The construct again incubated at 37±1°C, in an atmosphere of 10% CO2for 2 days.

Two days later, the medium containing the construct, aseptically transferred into a new cell for cultivation with sufficient environment for keratinization, 9 ml, before reaching the liquid level, almost reaching the surface of the membrane carrier, to maintain a dry surface section, to allow for layering epitheliale the layer. Constructs incubated in media at 37±1°C, in an atmosphere of 10% CO2and low humidity, replacing the medium every 2-3 days for 7 days. This environment consists of: a mixture with a ratio of 1:1 environment the Needle in the modification of Dulbecco (DMEM) (high glucose, without L-glutamine BioWhittaker, Wallcersville, MD), medium ham's F-12 (Quality Biologies, Gaithersburg, MD), which fill up the following additives with final concentrations: of 0.4 μg/ml hydrocortisone (Sigma, St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO), is 6.78 ng/ml selenium (Aldrich), is 24.4 μg/ml adenine (Sigma Aldrich Fine Chemicals Company), 4 mm L-glutamine (BioWhittaker, Walkersville, MD), 2% serum of newborn calves (BioWhittaker, Walkersville, MD), 50 μg/ml sodium ascorbate and 50 μg/ml gentamicin sulfate (Amersham, Arlington Heights, IL). Construct fuel through 7 days for more than 10 days, replacing a supportive environment every 2-3 days. This supportive environment consists of: a mixture with a ratio of 1:1 environment the Needle in the modification of Dulbecco (DMEM) (high glucose, without L-glutamine, BioWhittaker, Walkersville, MD), medium ham's F-12 (Quality Biologies Gaithersburg, MD) with the following additives: a 0.4 μg/ml hydrocortisone (Sigma St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 is kg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), is 24.4 μg/ml adenine (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 4 mm L-glutamine (BioWhittaker, Walkersville, MD), 1% serum of newborn calves (BioWhittaker, Walkersville, MD) and 50 μg/ml gentamicin sulfate (Amersham, Arlington Heights, IL).

The final samples processed by hematoxylin and eosin as described in example 1 to determine the macroscopic picture under a light microscope. The resulting construct consists of the lower (dermal) layer, which consists of fibroblasts, surrounded by a matrix with features, as described in example 1, and completely covered with multi-layered, layered and well-differentsirovannym layer of keratinocytes, which are the basal layer, advasary layer, granular layer and stratum corneum, similar to the skin layer in situ. Skin construct has a well-developed basal membrane, located at the dermal-epidermal junction, which is manifested by the method of transmission electron microscopy (TEM). The basal membrane is thickest around polydesmida bearing retainer fibrils that are composed of collagen type VII, as seen by TEM. As expected, these locking fibrils can easily be seen emerging from the basal membrane and exciting of fibril what s collagen. Shows the presence of laminin-glycoprotein basal membrane by previously described immunoassay techniques based avidin and Biotin (Guesdon, 1979).

Example 3: In vitro formation of the collagen matrix by fibroblasts of the foreskin of a newborn human in the environment of specific chemical composition

The fibroblasts of the foreskin of a newborn human increase, using the procedure described in example 1. Cells then resuspended to a concentration of 3·106cells/ml and seeded on the membrane inserts for processing tissue culture with pore size of 0.4 micron and a diameter of 24 mm, six-cell cuvette at a density of 3.0·106cells/TW (6,6·105cells/cm2). These cells then support as in example 1, the serum of newborn calves produced from the environment over the entire area. More specifically, the environment includes: a main mixture with a ratio of 3:1 DMEM medium, medium ham's F-12 (Quality Biologies, Gaithersburg, MD), 4 mm GlutaMAX (Gibco BRL, Grand Island, NY) and additives: 5 ng/ml human recombinant growth factor epidermis (Upstnpne Biotechnology, Lake Placid, NY), and 0.4 μg/ml hydrocortisone (Sigma, St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY catalog No. 02400 ACS grade), 1·10 M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Company, player, Milwaukee, WI), 50 ng/ml L-ascorbic acid (WAKO Chemicals USA, Inc.), 0.2 ág/ml L-Proline (Sigma, St. Louis, MO), 0.1 mg/ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO). The concentration of collagen and the number of cells in the samples tested for 7, 14 and 21 days, using the described methodology. The results are collected in tables 4 (number of cells) and 5 (concentration of collagen). Samples are also fixed with formalin and subjected to staining with hematoxylin and eosin for analysis in the light microscope as described in example 1. Histological evaluation shows that the constructs grown in a particular environment, similar to those grown in the presence of 2% serum of newborn calves. Samples are also positively stained with fibronectin according to the method described in example 1.

Table 4
Collagen (µg/cm2)
Day 0Day 7Day 14Day 21
The average amount of collagen in each construct (n=3)0107,63±21,96329,85±27,63465,83±49,46

Table 5
Cells (cells/cm2)
Day 0Day 7Day 14Day 21
The average number of cells in each construct (n=3)6,6×1057,8±2.2 x 1059,6±2,5×1051,19±2,1×105

In addition to the endogenous-produced fibrillar collagen, construct the cell matrix are also present decorin and glycosaminoglycan.

Example 4: a full-layer skin construct, formed with the specific chemical composition

Using a 25-day dermal construct, educated human dermal fibroblasts under chemically defined conditions similar to the method described in example 3, normal epidermal keratinocytes of the foreskin of a newborn human is seeded on the top surface of the construct of the cell matrix for the formation of the epidermal layer of the skin construct.

Wednesday aseptically removed from the inserts for cultivation and its environment. Normal human epidermal the nye keratinocytes to multiply passage 4 from the frozen cell strain subculture to merge. Cells then release of the cups for cultivation, using trypsin-disodium salt of EDTA are pooled, centrifuged for formation of cellular precipitate in the test tube, resuspended in the environment for epidermolysis, counted and seeded on top of the membrane at a density of 4.5·104cells/cm2. The constructs are then incubated for 90 minutes at 37±1°C, in an atmosphere of 10% CO2to enable keratinocytes to gain a foothold. After keeping the constructs are immersed in the environment for epidermolysis. The environment for epidermolysis consists of: basic mixture with a ratio of 3:1 eagle medium modification of Dulbecco (DMEM) (not containing glucose and calcium, BioWhittaker, Walkersville, MD) and medium ham's F-12 (Quality Biologies Gaithersburg, MD), which fill up the following additives: a 0.4 μg/ml hydrocortisone (Sigma St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO), is 6.78 ng/ml selenium (Aldrich), is 24.4 μg/ml adenine (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 4 mm L-glutamine (BioWhittaker, Walkersville, MD), 50 μg/ml sodium salt of L-ascorbic acid (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 16 μm linoleic acid (Sigma, St. Louis, MO), 1 μm tocopherol acetate (Sigma, St. Louis, MO) and 50 μg/ml gentamicin sulfate (Amersham, Arlington Heights, IL). Constructs cultured in cf is de to epidermolysis for 2 days at 37±1°C, in an atmosphere of 10±1% CO2.

Two days later, the medium replaced with fresh medium with the above composition and return to the incubator with a temperature of 37±1°C and an atmosphere of 10±1% CO2, soaking for 2 days. Two days later, the medium containing the construct, aseptically transferred into a new cell for cultivation with enough media to reach the level of the liquid, almost reaching the surface of the membrane carrier to support an emerging construct in the surface section of the air-liquid. The air contacting with the upper surface of the resulting epidermal layer, allows you to feel the epithelial layer. Constructs incubated at 37±1°C, in an atmosphere of 10% CO2and low humidity in the environment, changing it every 2-3 days for 7 days. These environments contain a mixture with a ratio of 1:1 environment the Needle in the modification of Dulbecco (DMEM) (does not contain glucose and calcium, BioWhittaker, Walkersville, MD), medium ham's F-12 (Quality Biologies, Gaithersburg, MD), which replenish additives: 0.4 µ g/ml hydrocortisone (Sigma, St. Louis, MO), 5·10-4M ethanolamine (Fluka, Ronkonkoma, NY), 5·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO), is 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Company), is 24.4 μg/ml adenine (Sigma Aldrich Fine Chemicals Company), 4 mm L-glutamine (BioWhittaker, Walkersville, MD), to 2.65 mg/ml calcium chloride (Mllinckrodt, Chesterfield, MO), 16 μm linoleic acid (Sigma, St. Louis, MO), 1 μm tocopherol acetate (Sigma, St. Louis, MO), 1.25 mm serine (Sigma, St. Louis, MO), 0.64 mm choline chloride (Sigma, St. Louis, MO) and 50 μg/ml gentamicin sulfate (Amersham, Arlington Heights, IL). Culture fed every 2-3 days for 14 days.

The samples, in triplicate, processed with hematoxylin and eosin as described in example 1, to 10, 12 and 14 days after the construct rises to the surface section of the air-liquid, to determine the macroscopic picture under a light microscope. The resulting construct consists of the lower (dermal) layer, which consists of fibroblasts, surrounded by a matrix that has the characteristics described in example 3, and cover with a layer of stratified and differentiated keratinocytes.

Example 5: In vitro formation of the collagen matrix human fibroblasts Achilles tendon

Constructs the cell matrix form, using the same method as described in example 1, replacing the fibroblasts of the foreskin of a newborn human fibroblasts Achilles tendon person (HATF). After 21 days exposure to generate the samples subjected to staining with hematoxylin and eosin and determine their thickness, using the procedure described in example 1. The resulting construct is rendered the AK Dane-like construct of the cell matrix with a thickness of 75.00±27,58 microns (n=2). Endogenous-produced fibrillar collagen, decorin and glycosaminoglycan also present in the construct.

Example 6: In vitro formation of the collagen matrix transfitsirovannykh fibroblasts foreskin of a newborn human

Transfetsirovannyh dermal fibroblasts produce, using the following methodology. Thaw one vial jCRJP-43 virus producers platelet-derived growth factor (PDGF) (Morgan, J, et al.), and seeded with cells at a density of 2·106cells/162 cm2flasks (Corning Costar, Cambridge, MA). These flasks served medium, and incubated them in the incubator at 37±1°C with an atmosphere of 10±1% CO2. Culture medium consists of: eagle medium modification of Dulbecco (DMEM) (high glucose, without L-glutamine, BioWhittaker, Walkersville, MD), which contribute 10% serum of newborn calves (HyClone Laboratories, Inc., Logan, Utah) and 4 mm L-glutamine (BioWhittaker, Walkersville, MD). On the same day, thaw 1 vial fibroblasts foreskin of a newborn human (HDFB 156) and apply a thin layer at a density of 1.5·106cells/162 cm2flasks (Corning Costar, Cambridge, MA). After three days jCRIP PDGF-43 virus producers fed fresh nutrient medium. HDFB156 energize the above-mentioned culture medium in combination with 8 µg/ml polybrene (Sigma, St. Louis, MO). The next day cells HDFB156 infect as follows. the fleecing old environment from jCRIP PDGF-43 virus producers, and filtered through a filter with pore size of 0.45 microns. Add 8 µg/ml polybrene to this filtered wastewater environment. Then the empty environment is placed on HDF. In the next two days HDF fed fresh nutrient medium. Through the day HDF Passepartout from P5 to P6 and seeded at a density of 2.5·106cells/162 cm2flasks (Corning Costar, Cambridge, MA). Cells Passepartout as follows: sucked off the empty environment, then the flask is rinsed with saline, buffered with phosphate to remove any residual serum of newborn calves. Cell release from the flask by adding 5 ml of trypsin-disodium salt of EDTA in each flask and moderate shaking to ensure complete coverage of the monolayer. Culture returned to the incubator. Immediately after release of the cells in each flask add 5 ml SBTI (soybean trypsin inhibitor) and mixed with the suspension to termination of trypsin-disodium salt of EDTA. The suspension of cells, trypsin and SBTI removed from the flasks and evenly divided between sterile, conical centrifuge tubes. Cells are harvested by centrifugation at approximately 800-1000 grams for 5 minutes. Cells resuspended environment for growing to be sown at a density specified above. Two days later, cells are fed with fresh medium to grow the project. The next day, cells are harvested as described above and diluted to a density of 1.5·106cells/ml of medium for the cultivation containing 10% serum of newborn calves (NBCS) and 10% dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO). Cells are then frozen in 1 ml Cronulla at a temperature of about -80°C.

For the formation of the collagen matrix in this example, use the same technique as described in examples 1 and 3, replacing the fibroblasts of the foreskin of a newborn human fibroblasts foreskin of a newborn human, transformed to produce high levels of platelet-derived growth factor (PDGF), as described above. The samples selected for staining with hematoxylin and eosin as described above, at 18 days after sowing. Samples also stained using the avidin-biotinamide methods for determining the presence of fibronectin indicated in example 10. Samples taken at 18 days after sowing for staining with hematoxylin and eosin as described in example 1, they exhibit a macroscopic picture of the cell matrix similar to that described in example 1, with the measured thickness is 123,6 microns (N=1). PDGF output transfected cells in the construct of the cell matrix during the cultivation period (18 day) according to the ELISA analysis of 100 ng/ml, while the release of PDGF, in the context of the role is not found.

Example 7: Using dermal construct as an implantable material

Constructs cellular matrix get according to the methods described in example 1, using dermal fibroblasts derived from foreskin of a newborn, and transplanted on wounds complete excision created on naked Nude mice. Mice make the connection according to the methods described Parenteau, et al. (1996), the disclosure of which is included in this document. The implants tested for 14, 28 and 56 days for signs of bonding with the surface of the wound, the evidence of contraction of the wound, square loss of the implant and the presence of vascularization (color). Implantation area of taking pictures while they are on mice in General form. Several mice killed at certain points in time, excised and implanted areas and their surroundings together with edges around the mouse skin, at least until the fleshy layer of tissue. The connection between the implant and the mouse skin retain in each sample. Explantion tissue samples are then fixed in 10% formalin, buffered with phosphate, and subjected to fixation in methanol. Fixed with formalin samples subjected to staining with hematoxylin and eosin according to the procedure described in example 1. Implants have the ability to integrate with mouse skin, noting the minimum the contract. Within 14 days after transplantation the epidermis of mice completely migrate over the implant. Using stained with hematoxylin and eosin sample vessels become visible on the inside of the implant on day 14 and continuing throughout the experiment. Through makroskopicheskogo observation stained with hematoxylin and eosin samples determines that the implant continues to exist and remains healthy, looking containing living cells, without macroscopic violations of the matrix and so on, throughout the period of the experiment.

Example 8: Using full-thickness skin construct as implant skin

Two-layer skin constructs receive, as described in example 2, using dermal fibroblasts derived from foreskin of a newborn human, Vermalen layer and human keratinocytes derived from great foreskin of a newborn, in the epidermal layer. Skin constructs can manually rip-off of the membrane, to operate without carrier support and placed on the implanted area. Two-layer skin constructs are transplanted in complete wound excision Nude naked mice according to the methods described in Parenteau, et al. (1996), the disclosure of which is included in this document. The time points for sampling are 7, 14, 28, 56, and 184 days after transplantation. The field pulse is antali photographed, while they are in the General form in mice. Several mice killed at certain points in time, excised and implanted areas and their surroundings together with edges around the mouse skin, at least until the fleshy layer of tissue. Retain the connection between the implant and the mouse skin in each sample. Explantion tissue samples are then fixed in 10% formalin, buffered with phosphate, and subjected to fixation in methanol. Fixed with formalin samples subjected to staining with hematoxylin and eosin according to the procedure described in example 1.

Implants integrate with the tissue of the host body within 7 days, as seen by macroscopic observation, as well as by histological manifestations. By staining with hematoxylin and eosin vessels are visualized sprouted into the implant from the host tissue within 7 days of transplantation. The implants remain healthy and continue to exist throughout the experiment, while there is minimal contraction. Using staining protivoallergicheskim involucrin shows the persistence of human epidermal cells throughout the implantation period.

Example 9: In vitro formation of corneal matrix by human keratinocytes

Cells corneal keratinocytes (manufactured by Organogenesis, Inc. Canton, is A) used in the formation of stromal construct of the cornea. Confluent cultures of human keratinocytes release from the substrate for cultivation, using trypsin-dinitroethylene. After the release of the use of soybean trypsin inhibitor to neutralize the trypsin-disodium salt of EDTA, the cell suspension is centrifuged, the supernatant discarded, and the cells then resuspended in basic medium to a concentration of 3·106cells/ml Cells seeded on the membrane inserts for processing tissue culture with pore size of 0.4 micron and a diameter of 24 mm, six-cell cuvette at a density of 3.0·10 cells/TW (6,6·105cells/cm2). These cultures support throughout the night in the medium for planting. Medium for inoculation consists of: basic mixture with a ratio of 3:1 eagle medium modification of Dulbecco (DMEM) and medium ham's F-12 (Quality Biologies Gaithersburg, MDcat.), 4 mm GlutaMAX (Gibco BRL, Grand Island, NY) and the following additives: 5 ng/ml human recombinant growth factor epidermal (EGF) (Upstate Biotechnology, Lake Placid, NY), and 0.4 μg/ml hydrocortisone (Sigma St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI). After this culture fed with fresh medium to produce. Environment for the formation consists of the: basic mixture with a ratio of 3:1 DMEM medium, medium ham's F-12 (Quality Biologies Gaithersburg, MD), 4 mm GlutaMAX (Gibco BRL, Grand Island, NY) and the following additives: 5 ng/ml human recombinant growth factor epidermis (Upstate Biotechnology, Lake Placid, NY), 2% serum of newborn calves (Hyclone, Logan, Utah), and 0.4 μg/ml hydrocortisone (Sigma,St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY ACS grade), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee, WI), 50 ng/ml L-ascorbic acid (WAKO pure chemical company), 0.2 ág/ml L-Proline (Sigma, St. Louis, MO), 0.1 mg/ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO, variety of cell culture).

Cells incubated in the incubator at 37±1°C with an atmosphere of 10%±1% CO2and fed fresh medium for generating every 2-3 days for 20 days (for a total of over 21 days of cultivation). After 21 days of culturing keratinocytes are deposited as a layer of matrix thickness of about 40 microns, as measured by the method described in example 1. Endogenous-produced fibrillar collagen, decorin and glycosaminoglycan also present in the construct of the cell matrix.

Example 10: In vitro formation of the collagen matrix by fibroblasts of the foreskin of a newborn human, sown in the environment to generate the

Fibroblasts kr is ina the flesh of a newborn human (production Organogenesis, Inc. Canton, MA) seeded in quantities of 1·105cells on carriers for processing tissue culture with pore size of 0.4 micron and a diameter of 24 mm, six-cell cuvette (TRANSWELL®, Costar Corp.Cambridge, MA) and grown in a nutrient medium. Culture medium consists of eagle medium modification of Dulbecco (DMEM) (high glucose, without L-glutamine, BioWhittaker, Walkersville, MD), which contribute 10% serum of newborn calves (HyClone Laboratories, Inc., Logan, Utah) and 4 mm L-glutamine (BioWhittaker, Walkersville, MD). Cells incubated in the incubator at 37±1°C with an atmosphere of 10±1% CO2. Wednesday, replace every two or three days. After 9 days of cultivation medium is sucked off from a Cup for the cultivation and replaced by the environment to produce. Cells incubated in the incubator at 37±1°C with an atmosphere of 10±1% CO2and fed fresh medium for generating every 2-3 days for 21 days. Environment for the formation consists of: basic mixture with a ratio of 3:1 DMEM medium, medium ham's F-12 (Quality Biologies, Gaithersburg, MD), 4 mm GlutaMAX (Gibco BRL, Grand Island, NY) and the following additives: 5 ng/ml human recombinant growth factor epidermis (Upstate Biotechnology, Lake Placid, NY), 2% serum of newborn calves (Hyclone, Logan, Utah), and 0.4 μg/ml hydrocortisone (Sigma St. Louis, MO), 1·10-4Mutanolysin(Fluka, Ronkonkoma, NYACS grade), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml Shuttle service is Jn (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee, WI), 50 ng/ml L-ascorbic acid (WAKO Pure Chemical Company), 0.2 ág/ml L-Proline (Sigma, St. Louis, MO), 0.1 mg/ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO, variety of cell culture).

Samples taken on day 21 and fixed in formalin, then dipped in paraffin. Fixed with formalin samples immersed in paraffin, and stained with hematoxylin and eosin section size of 5 μm according to the procedures routinely used in the art. Using stained with hematoxylin and eosin preparations, conduct measurements at ten randomly selected microscopic fields using a 10x eyepiece (Olympus America Inc., Melville, NY)equipped with ocular scale 10 mm/100 micrometer (Olympus America Inc., Melville, NY). The constructs created in this sposobem, the structure and biochemical composition similar to the constructs created in example 1, and have a thickness in 82,00±of 7.64 microns.

Example 11: In vitro formation of the collagen matrix dermal fibroblasts pigs

Dermal fibroblasts pigs (manufactured by Organogenesis, Inc. Canton, MA) seeded at a density of 5·105cells/162 cm2bulb for processing tissue culture (Costar Corp., Cambridge, MA. catalog No. 3150) and grown in a nutrient medium as described below. Nutrient medium sostoi is from eagle medium modification of Dulbecco (DMEM) (high glucose, without L-glutamine, BioWhittaker, Walkersville, MD), which contribute 10% fetal calf serum (HyClone Laboratories, Inc., Logan, Utah) and 4 mm L-glutamine (BioWhittaker, Walkersville, MD). Cells incubated in the incubator at 37±1°C with an atmosphere of 10%±1% CO2. Wednesday, replace every two or three days. At the confluence, where the cells form a Packed layer at the bottom of the bulb with a tissue culture medium is sucked off from a Cup for cultivation. For propulsive monolayer to it add sterile-filtered phosphate buffered saline, and then sucked off from a Cup. Cell release from the flask by adding to each of kolob 5 ml of a mixture of trypsin, disodium salt EDTA and glutamine (BioWhittaker, Walkersville, MD) and careful shaking to ensure complete coverage of the monolayer. Culture returned to the incubator. Immediately after release of the cells in each flask add 5 ml SBTI (soybean trypsin inhibitor) and mixed with the cell suspension to termination of trypsin-disodium salt of EDTA. The suspension is removed from the flasks and evenly divided between sterile, conical centrifuge tubes. Cells are harvested by centrifugation at approximately 800-1000 grams for 5 minutes. Cells resuspended and diluted to a concentration of 3·106cells/ml, and seeded on the membrane inserts for printing handling the key tissue culture with pore size of 0.4 micron and a diameter of 24 mm, six-cell cuvette at a density of 3.0·10 6cells/TW (6,6·105cells/cm2). Cells support throughout the night in the medium for planting. Medium for inoculation consists of: basic mixture with a ratio of 3:1 DMEM medium, medium ham's F-12 (Quality Biologies, Gaithersburg, MD), 4 mm GlutaMAX (Gibco BRL, Grand Island, NY) and the following additives: 5 ng/ml human recombinant growth factor epidermis (Upstate Biotechnology, Lake Placid, NY), and 0.4 μg/ml hydrocortisone (Sigma St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NYACS grade), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee, WI), 50 ng/ml L-ascorbic acid (WAKO Pure Chemical Company), 0.2 ág/ml L-Proline (Sigma, St. Louis, MO) and 0.1 mg/ml glycine (Sigma, St. Louis, MO). Cells incubated in the incubator at 37±1°C with an atmosphere of 10±1% CO2and fed fresh medium for generating every 2-3 days for 7 days. Environment for the formation consists of: basic mixture with a ratio of 3:1 DMEM medium, medium ham's F-12 (Quality Biologies, Gaithersburg, MD), 4 mm GlutaMAX (Gibco BRL, Grand Island, NY) and the following additives: 5 ng/ml human recombinant growth factor epidermis (Upstate Biotechnology, Lake Placid, NY), 2% serum of newborn calves (Hyclone, Logan, Utah), and 0.4 μg/ml hydrocortisone (Sigma St. Louis, MO), 1·10-4Mutanolysin (Fluka, Ronkonkoma, NY ACS grade), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis), 5 μg/ml insulin (Sigma, St. Lois, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee, WI), 50 ng/ml L-ascorbic acid (WAKO Pure Chemical Company), 0.2 ág/ml L-Proline (Sigma, St. Louis, MO), 0.1 mg/ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO) grade a cell culture). After 7 days of medium substitute environment to deliver without serum of newborn calves. This environment serves to fresh cells every 2-3 days for more than 20 days, a total of over 28 days of cultivation.

Samples taken on day 21 and fixed in formalin, then dipped in paraffin. Fixed with formalin samples immersed in paraffin, and stained with hematoxylin and eosin (H&E) section 5 microns according to the methods commonly used in the art. Using stained with hematoxylin and eosin preparations, conduct measurements at ten randomly selected microscopic fields using a 10x eyepiece (Olympus America Inc., Melville, NY)equipped with ocular scale 10 mm/100 micrometer (Olympus America Inc., Melville, NY). The samples show a structure consisting of cells and matrix with a thickness of 71,20±to 9.57 microns. In addition to the endogenous-produced fibrillar collagen, construct the cell matrix are also present decorin and glycosaminoglycan.

Example 12: In vitro formation dwahlin the th skin construct, containing cells of the papillae of the dermis

Cellular matrix are manufactured according to the method described in example 1, using fibroblasts foreskin of a newborn human as the first type of cells that produce matrix. Cellular matrix seeded with locally drops cells of the papillae of the dermis as a second cell population, which in turn seeded with keratinocytes as a third cell population to form a continuous epidermal layer over the cellular matrix and cells of the papillae of the dermis.

First, construct the cell matrix form, using dermal fibroblasts (HDF), derived from the foreskin of a newborn human. HDF propagated by sowing them at a density of 5·105cells/162 cm2bulb for processing tissue culture (Costar Corp., Cambridge, MA) in a nutrient medium consisting of eagle medium modification of Dulbecco (DMEM) (high glucose, without L-glutamine, BioWhittaker, Walkersville, MD), which contribute 10% serum of newborn calves (NBCS) (HyClone Laboratories, Inc., Logan, Utah) and 4 mm L-glutamine (BioWhittaker, Walkersville, MD). After the merger HDF release from a Cup, using trypsin-disodium salt of EDTA, resuspended in fresh medium to a concentration of 3.0·106cells/ml and seeded in the inserts for processing tissue culture with pore size of 0.4 micron and diameter is ω 24 mm (TRANSWELL®, Corning Costar) in a six-cell cuvette at a density of 3.0·106cells/insert (6,6·105cells/cm2). HDF cultures incubated in the incubator at 37±1°C with an atmosphere of 10±1% CO2and fed fresh medium for generating every 2-3 days for 23 days according to the method described in example 1.

After the formation of the construct cellular matrix it is seeded with the drops of the cells of the papillae of the dermis as a second cell population. The cells of the papillae of the dermis represent a discrete population of specialized fibroblasts surrounded by the bulb of the hair follicle and plays an important role in hair growth. Papillae of the dermis can be separated by means located in the hair follicle and to cultivate in vitro, using the method previously described by Messenger, A.G. The Culture of Dermal Papilla Cells from Human Hair Follicles. Br. J. Dermatol. 110: 685-9 (1984), a method which is included in this document. After culture of the cells of the papillae of the dermis reaches confluence, these cells form aggregates that can be transferred to flasks for cultivation for re-formation of new aggregates. Papillae of the dermis is separated from skin biopsies obtained from pigs aged 4 weeks. Cells of the papillae of the dermis (PDP), were cultured in DMEM containing 20% NBCS, up to passage 8. After 3 weeks of cultivation PDP cells re-education is comfort dermal masocco-shaped structures or units, each of which has approximately the diameter from 90 to 210 microns. The units are then removed from the plate for cultivation by vigorous pipetting in their environment, and then seeded with human collagen matrix at a density of 200 units cm2. Units stump Viru are shipped and throughout an additional 15 days in the environment of DM EATEN, containing 20% NBCS, when this old medium replaced with fresh medium every 2-3 days.

The cell culture matrix containing cells of the papillae of the dermis on the matrix seeded with keratinocytes and cultured for continuous education of the epidermal layer over the cellular matrix and papillae of the dermis. Get two different construct: first, with human keratinocytes, the second pig keratinocytes. Normal epidermal keratinocytes isolated from the foreskin of a newborn man (NER) or keratinocytes pigs (PEP), using the growth of explants to establish primary cultures. These cells then stump Viru Ute and extend up to passage 3 in the case of swine strain or until passage 4 in the case of the human strain. After about 5-6 days of culturing cells release of cups for cultivation, using trypsin-disodium salt of EDTA are pooled, centrifuged for formation of cellular precipitate in the test tube, resuspending the midst for epidermolysis, counted and seeded on top of the membrane at a density of 4.5·104cells/cm2for NER cells at a density of 1.6 x 105cells/cm2for PEP cells. Epidermiology culture cultivated for 12 days, as described previously in example 2.

The final samples processed with hematoxylin and eosin for analysis by light microscopy. The resulting skin constructs are the basic morphological organization, similar to the skin: the dermal layer is composed of fibroblasts, surrounded endogenous-produced matrix, including endogenous-produced fibrillar collagen, decorin and glycosaminoglycan localized area of cells of the papillae of the dermis and continuous, stratified layer of keratinocytes in parallel to construct the cellular matrix and papillae of the dermis. In both tissue constructs coated with either human or pig keratinocytes, papillae of the dermis supports Packed structure, which causes a small ripple layered epithelium. Differentiated epithelial cells are often located close to the cells of the papillae of the dermis.

Example 13: Determination of hyaluroni acid by the method of the sandwich ELISA

Hyaluronic acid (ha) define constructs in the cell matrix, apartmenthomeliving fibroblasts in with the food, containing serum, according to the methods of Examples 1 and 3, respectively.

Constructs the cell matrix form on a circular discs with a diameter of 75 mm, including the porous membrane (TRANSWELL®, CorningCostar). Extracts from the constructs of the cell matrix is obtained by adding 10 ml of ammonium acetate buffer and 0.5 mg/ml proteinase K in the test tube containing the construct of the cell matrix. The mixture was kept at 60°C over night. After completion of the cleavage mixture rotate, and the supernatant extract is transferred into a separate tube for analysis of hyaluronic acid. 96-cell plate cover 50 ál binding protein GC, with a concentration of 20 μg/ml in 0.1 M solution of NaHCO3and stored over night at 4°C. the Plate is then washed three times of 0.85% NaCl containing 0.05% Tween 20. In each cell add 250 μl of blocking solution (phosphate-sodium buffer, 10 mmol, pH 7.4, containing 3% BSA and 0.9% NaCl, SFR±3% BSA) and the plate incubated at room temperature for 2 hours Then the plate is washed three times of 0.85% NaCl containing 0.05% Tween 20. Then to the plate add 50 ál of the standard solutions and extracts of GK obtained in both experimental conditions, including different dilution of these conditions. The plate is incubated at room temperature (about 20°C) for 2 hours and Then the plate was washed with Teresa of 0.85% NaCl, containing 0.05% Tween 20, and to each cell add 50 ál biotinylated Ledger (dilution 1:2000), and then incubated for 2 h at room temperature. The plate is then washed three times of 0.85% NaCl containing 0.05% Tween 20, and then added to each well 50 µl of HRP-avidin D (dilution 1:3000). The plate is then incubated for 45 minutes at room temperature. Then the plate is washed three times of 0.85% NaCl containing 0.05% Tween 20, and in each of the wells add 100 ál of a solution of ortho-phenyldiamine substrate. The plate is incubated at 37°C for 10 minutes. The reaction is stopped by adding 50 μl 1 M HCl. Finally, using a spectrophotometer to read the tablet, read the absorbance at 492 nm and record the readings.

Measurements of absorption average and is converted into quantitative units. In each round the construct cellular matrix (75 mm in diameter)formed in media containing serum, contains 200 µg by hyaluroni acid, while in the constructs formed in the environment of specific chemical composition contains 1.5 mg of hyaluroni acid.

Example 14: a Physical test and mechanical properties produced by the construct in the cell matrix

The mechanical properties of tissue constructs of example 1 (construct cellular matrix), example 2 (construct cell is about matrix with a layer of keratinocytes on it) and example 3 (construct cellular matrix, formed in the environment of a particular composition) opredelyaytes by means of tests on the swelling of the membrane. These tests are similar to the methods of analysis used in the clinic (for example, Dermaflex®, Cyberderm, Inc., Media, PA and Cutameter®, Courage Khazaka, Cologne, Germany), but include the application of high pressure, is able to break the membrane. An exemplary construct of the cell matrix is exactly placed on the polycarbonate block over a cylindrical cell with a diameter of 10 mm, filled normostenicheskim saline. The metal plate with a circular hole corresponding to the diameter of cylindrical cells placed above the sample and press to the block. The samples are then filled by pouring the saline solution in the cell syringe pump. Determine the final pressure at the pressure sensor. The pressure increases until the destruction of the device to determine the tensile strength, which averages 439,02 mm Hg to construct the cell matrix generated by the method of example 1; 998.52 mm Hg for samples construct the cell matrix with a layer of keratinocytes generated by the method of example 2; and 1542,26 mm Hg for samples construct the cell matrix formed in the environment of a particular composition and generated according to the method of example 3.

To determine the melting temperature of the dermal matrix samples (construct cellular matrix, selected on day 21, prepared according to the method described in example 1. The temperature of denaturation of the samples was determined on a differential scanning calorimeter Mettler Toledo (Highston, NJ) (No. DSC product DSC12E). For our purposes, the melting temperature is determined by heating the sample from 45 to 80°C at a rate of 1°C per minute. The average temperature of denaturation for samples is of 60.8±1,2°C (n=3).

The holding capacity of suture material and tensile strength epidermalnogo matrix created according to the methods described in examples 1 (construct cellular matrix) and 3 (construct cellular matrix formed in the environment of a particular composition) is measured to determine the strength of the fusion construct under certain clinical conditions. The strength retention of the suture human dermal matrix by the age of 21 day determined according to the method described in the American national edition of the standards for implantable vascular prosthesis (Instruments, 1986), using a system for testing Mini-Bionex 858 (MTS systems Corporation, Minneapolis, Minn.)

For samples of example 1 (construct cellular matrix) specific tensile strength is 365 N/m; for the samples obtained according to example 2 (a construct of the cell matrix with a layer of keratinocytes), the tensile strength is 2720 N/m

Holding JV is the ability of suture material for samples, obtained according to example 1 is 0.14 N; for those that obtained according to example 2, 0,22 N.

The constructs generated as described in examples 1, 2 and 3, the diameter of 24 mm and 75 mm Constructs produced according to the methods of cultivation of all 3 ways, represent a cohesive mcanearney patterns that are easy syraucse with membranes with a minimum of strain, they "syraucse" and can physically handle and manipulate during use and test without damage.

Example 15: In vitro formation of the collagen matrix by fibroblasts of the foreskin of a newborn human in the environment of specific chemical composition

The fibroblasts of the foreskin of a newborn human expand according to the method described in example 1. Cells then resuspended to a concentration of 3·106cells/ml and seeded on the membrane inserts for processing tissue culture with pore size of 0.4 micron and a diameter of 24 mm, six-cell cuvette at a density of 3.0·106cells/TW (6,6·105cells/cm2). Cells throughout this example are cultivated in the environment of specific chemical composition.

The environment contains: the basic mixture with a ratio of 3:1 DMEM medium, medium ham's F-12 (Quality Biologies, Gaithersburg, MD), 4 mm GlutaMAX (Gibco BRL, Grand Island, NY) and the following additives: 5 ng/ml human being is a recombinant growth factor, epidermal (Upstate Biotechnology, Lake Placid, NY), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY catalog No. 02400 ACS grade), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 50 ng/ml L-ascorbic acid (WAKO Chemicals USA, Inc.), 0.2 ág/ml L-Proline (Sigma, St. Louis, MO), 0.1 mg/ml glycine (Sigma, St. Louis, MO). Quiteunusual primary environment add other components in the following environments:

1. 5 µg/ml insulin (Sigma, St. Louis, MO), and 0.4 μg/ml hydrocortisone (Sigma, St. Louis, MO), 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO).

2. 5 µg/ml insulin (Sigma, St. Louis, MO), and 0.4 μg/ml hydrocortisone (Sigma, St. Louis, MO).

3. 375 μg/ml insulin (Sigma, St. Louis, MO), 6 μg/ml hydrocortisone (Sigma, St. Louis, MO).

Samples fixed with formalin and subjected to staining with hematoxylin and eosin for examination under a light microscope. Visual histological assessment demonstrates that the environment is 2, not containing PEG, shows relatively similar to the matrix medium 1 containing PEG. Biochemical analysis of collagen in the construct shows almost the same amount of collagen in both cases: 168,7±7,98 g/cm2for environment 1 with PEG compared to 170,88±9,07 µg/cm2for medium 2 without PEG. Wednesday 3, containing large amounts of insulin and hydrocortisone, shows a higher expression of matrix, including collagen to generate the n, at an earlier point in time compared to the other two environments. In addition to the endogenous-produced fibrillar collagen in the cell matrix constructs in all environments also present decorin and glycosaminoglycan. The porous membrane of cultured dermal construct formed with the medium 2 of this example looks like in the form of a thin transparent strip below the construct, you can see that the cells grow on the membrane surface and not gebaude when integrating the membrane matrix.

Cultured dermal construct formed by the method with the environment 2 this example on day 21 shows the alignment of the fibers of endogenous collagen between fibroblasts and showing the distribution and packing of the fibrils.

In all environments this example, educated cultured dermal constructs contain dermal fibroblasts and endogenous-produced matrix. In all cases there is a fully formed collagen fibrils are Packed in the organization, distributed between the cells. These fibrous quality, thickness and cohesive integrity give the construct of considerable strength, allowing you to easily remove it from the cell membrane and to deal with construction when transferring a patient with graft or implant.

Example 16: Pornoslon the th skin construct

Using 21-day dermal construct, formed by dermal fibroblasts in chemically defined conditions on the way to environment 2 (without PEG), as described above in example 15, normal epidermal keratinocytes of the foreskin of a newborn human is seeded on the top surface of the construct of the cell matrix for the formation of the epidermal layer of the skin construct.

Wednesday aseptically removed from the inserts for cultivation and its environment. Normal human epidermal keratinocytes to multiply passage 4 from the frozen cell strain subculture to merge. Cells then release of the cups for cultivation, using trypsin-disodium salt of EDTA are pooled, centrifuged for formation of cellular precipitate in the test tube, resuspended in the environment for epidermolysis, counted and seeded on top of the membrane at a density of 4.5·104cells/cm2. The constructs are then incubated for 90 minutes at 37±1°C, in an atmosphere of 10% CO2to enable keratinocytes to gain a foothold. After keeping the constructs are immersed in the environment for epidermolysis. The environment for epidermolysis consists of: basic mixture with a ratio of 3:1 eagle medium modification of Dulbecco (DMEM) (not containing glucose and calcium, BioWhittaker, Walkersville, MD) and medium ham's F-12 (Quality iologies Gaithersburg, MD), which fill up the following additives: a 0.4 μg/ml hydrocortisone (Sigma St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO), is 6.78 ng/ml selenium (Aldrich), is 24.4 μg/ml adenine (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 4 mm L-glutamine (BioWhittaker, Walkersville, MD), 50 μg/ml Sodium salt of L-ascorbic acid (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 16 μm linoleic acid (Sigma, St. Louis, MO), 1 μm tocopherol acetate (Sigma, St. Louis, MO) and 50 μg/ml gentamicin sulfate (Amersham, Arlington Heights, IL). Constructs cultured in the medium for epidermolysis for 2 days at 37±1°C, in an atmosphere of 10±1% CO2.

Two days later, the medium replaced with fresh medium with the above composition and return to the incubator with a temperature of 37±1°C and an atmosphere of 10±1% CO2by keeping it on for 2 days. Two days later, the medium containing the construct, aseptically transferred into a new cell for cultivation with sufficient environment to achieve the liquid level, almost reaching the surface of the membrane carrier to support the development of the construct at the interface of the air-liquid. The air contacting with the upper surface forming the epidermal layer, allows you to feel the epithelial layer. Constructs incubated in media at 37±1°is in the atmosphere of 10% CO 2and when humidity is low, replacing the medium every 2-3 days for 7 days. This environment contains a mixture with a ratio of 1:1 environment the Needle in the modification of Dulbecco (DMEM) (not containing glucose and calcium, BioWhittaker, Walkersville, MD), medium ham's F-12 (Quality Biologies, Gaithersburg, MD), which fill up the following additives: a 0.4 μg/ml hydrocortisone (Sigma, St. Louis, MO), 5·10-4M ethanolamine (Fluka, Ronkonkoma, NY), 5·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO), is 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Company), is 24.4 μg/ml adenine (Sigma Aldrich Fine Chemicals Company), 4 PM L-glutamine (BioWhittaker, Walkersville, MD), to 2.65 mg/ml calcium chloride (Maflinckrodt, Chesterfield, MO), 16 μm linoleic acid (Sigma, St. Louis, MO), 1 μm tocopherol acetate (Sigma, St. Louis, MO), 1.25 mm serine (Sigma, St. Louis, MO), 0.64 mm choline chloride (Sigma, St. Louis, MO) and 50 μg/ml gentamicin sulfate (Amcrs GCF, Arlington Heights, IL). Culture fed every 2-3 days for 14 days.

The samples, in triplicate, subjected to 10, 12 and 14 days to staining with hematoxylin and eosin as described in example 1, after lifting to construct the boundary surface of the air-liquid to determine the macroscopic picture under a light microscope. The resulting construct is a double-layered skin construct, consisting of the lower dermal layer, which consists of the dermis is lnyh fibroblasts, surrounded by a matrix covered the upper epidermal layer of stratified and differentiated keratinocytes.

Example 17: the Formation of the collagen matrix of the buccal fibroblasts

Aluminophosphate is getting construct the cellular matrix of the buccal fibroblasts secreted from human tissue cheeks. Buccal fibroblasts cultured in flasks T-150 in the environment DM EM containing 10% environment NBCS. After 7 days for an additional increase in the number of cells buccal cells collected and passedout in nine flasks in the amount of 4.0·106cells in DMEM containing 10% environment NBCS, and cultured to confluence, at which time the cells are harvested.

To collect the cells, the medium is sucked off from the flasks with culture. For propulsive monolayer add sterile-filtered saline solution, phosphate buffered in the lower part of each flask with culture, and then suck it from the flasks. Cell release from the flask by adding to each flask 5 ml of a mixture of trypsin, disodium salt EDTA and glutamine (BioWhittaker, Walkersville, MD) and careful shaking to ensure complete coverage of the monolayer. Culture returned to the incubator. Immediately after release of cells added to each flask 5 ml SBTI (soybean trypsin inhibitor) and mixed with the suspension to prekrseni the action of the trypsin-disodium salt of EDTA. The cell suspension is removed from the flasks and evenly divided between sterile, conical centrifuge tubes. Cells are harvested by centrifugation at approximately 800-1000 grams for 5 minutes.

Cells resuspended in fresh medium to a concentration of 3.0·106cells/ml and seeded in the inserts for processing tissue culture with pore size of 0.4 micron and a diameter of 24 mm (TRANS WELL®, Corning Costar) in a six-cell cuvette at a density of 3.0·106cells/insert (6,6·105cells/cm2). Cells incubated in the incubator at 37±1°C with an atmosphere of 10±1% CO2and fueling environment, comprising: a main mixture with a ratio of 3:1 DMEM medium, medium ham's F-12 (Quality Biologies, Gaithersburg, MD), 4 mm GlutaMAX (Gibco BRL, Grand Island, NY) and the following additives: 5 ng/ml human recombinant growth factor epidermis (Upstate Biotechnology, Lake Placid, NY), and 0.4 μg/ml hydrocortisone (Sigma, St. Louis, MO), 1·10-4M ethanolamine (Fluka, Ronkonkoma, NY catalog No. 02400 ACS grade), 1·10-4M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg/ml insulin (Sigma, St. Louis, MO), 5 μg/ml transferrin (Sigma, St. Louis, MO), 20 PM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng/ml selenium (Sigma Aldrich Fine Chemicals Company, Milwaukee, WI), 50 ng/ml L-ascorbic acid (WAKO Chemicals USA, Inc.), 0.2 ág/ml L-Proline (Sigma, St. Louis, MO), 0.1 mg/ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO).

At 1 day after sowing Wednesday, replace the medium on the I formation, does not contain serum, by replacing every 2-3 days for 21 days. On day 21 samples fixed in formalin for histology. Using three samples for the analysis of the formation of protein and collagen.

The production of collagen for constructs with a diameter of 24 mm, an average of 519 ág after 21 days of cultivation. The total production of the protein construct with a diameter of 24 mm, an average of 210 µg construct after 21 days of cultivation. Morphologically construct the cellular matrix of the buccal fibroblasts, cultured tissue construct of oral connective tissue, shows buccal fibroblasts surrounded by matrix, while physically construct has a physical volume and integrity.

Example 18: Preparation of equivalent tissue by contraction of collagen nets seeded with cells fibroblasts

The crude solution of collagen is prepared as follows. Frozen tails of rats from rats weighing 450 g of thawed in 70% EtOH for 20 minutes. Tendon ligaments are excised in 70% EtOH in a fume hood with laminar flow. Individual tendon is removed from the tendon sheath, crushed and placed in a diluted acetic acid (1:1000), using it in the amount of 250 ml per tail. This solution is left to stand for 48 h at 4°C, crushed su is agile swell, occupying the entire volume. This viscous solution was centrifuged with a speed of 23,000 rpm in a Beckman ultracentrifuge L with rotor SW25 for 1 h the Supernatant assign channel and stored at 4°C in the form of a crude solution of collagen (Protein).

The purified solution of collagen is prepared by mixing a crude solution of collagen with 0.1 M NaOH in the ratio of 6:1 for the neutralization of acetic acid, under which the collagen is deposited. This solution is centrifuged with a speed of 1500 rpm for 5 minutes in a clinical centrifuge. The supernatant discarded, and introduce an equal volume of the fresh acetic acid (1:1000) for re-dissolution of collagen. This solution is stored at 4°C in the form of a purified solution of collagen (a Protein "R").

The protein concentration determined by the method of Lowry et al. Cm. Lowry, O., Rosebrough, N.J., Farr, N.J. and Randall, R.J., J. Biol. Chem., 193, 265-275 (1951) and Waddel, W.J., J. Lab & Clin. Med. 48, 311-314 (1956).

Protein mesh prepared in 60 mm bacteriological cups Falcon's Twister, which fibroblasts pasted badly. Each Cup contains 1.0 ml of medium McCoy's 5A with a concentration of 5X, 1.0 ml fetal calf serum, 0.25 ml of 0.1 M NaOH, 1.5 ml solution of collagen and 1.0 ml of fibroblasts suspended in the environment McCoy with a concentration of 1X. These cups are initially fill the above medium volume McCoy, serum and NaOH, and then take in one hundred the ONU as long until you are willing suspension of fibroblasts. While adding a solution of collagen and fibroblasts speed is an important parameter because the gel begins to set immediately. Cups placed in the incubator at 37°C in atmosphere of 5% CO2and at 100% humidity. Gels, including fibroblasts, fully installed in 10 minutes.

Used by the fibroblasts are foreskin fibroblasts human strain 1519 obtained from the repository of genetic human cells at the Institute for medical research in Camden, new Jersey. These cells grow and maintain in a modified medium McCoy's 5A with 20% serum, penicillin and streptomycin. Culture free from Mycoplasma. Center for cell cultivation Massachusetts Institute of technology prepares and freezes cells every tenth level of population doubling (PDL).

To measure the diameters of the mesh cups are placed on the upper part of the transparent metric ruler on a dark background. Optimum visibility of the edges of the gel get through a brilliant white light, horizontal against the edge of the Cup. Compressed gels are disks of the correct form; they show very slight differences in diameter at various points. The average size of the main and secondary axes accept as diameter.

When is EP 19: the Measurement of the contraction of hydrated collagen grid cells fibroblasts

Determine the contraction of hydrated collagen mesh, obtained according to the methods of Example 18 and containing 570 mg/ml of Protein C by 7,5·106of foreskin fibroblasts human strain 1519, 19th PDL. Wednesday in the Cup is replaced by the first, fourth and eighth days. The data obtained are graphically presented in figure 3, which indicates a 112-fold lower square of the grid for a period of less than seven days. During the first day is a seven-fold reduction in the area.

Example 20: the Contraction of hydrated collagen meshes with different concentrations of protein

The effect of the concentration of protein in the hydrated collagen meshes on their contraction is determined as follows. Three hydrated collagen mesh prepared according to the methods of Example 18, except that each grid contains different concentrations of Protein "R". Use foreskin fibroblasts human strain 1519 19th PDL, and environment change on the fourth day.

The data obtained are graphically presented in figure 4, where you can see that the rate of contraction of the grid varies with the concentration of protein in the gel. Size of the grid decreases with time.

Example 21: the Influence of cell number on the contraction of hydrated collagen meshes

Effect of the number of cells on the contraction of hydrated collagen meshes determined by the t in the following way. Several of hydrated collagen meshes containing 720 mg/ml Protein "R", prepared by the method of Example 18. Use foreskin fibroblasts human strain 1519, and the environment in every culture change on the third, seventh and tenth days.

Use control samples to which no added cells. In addition, spend four series of experiments in which add different numbers of cells. The data obtained are graphically presented in Figure 5, where each point shows the average contraction of three or four grids. The deviation is not shown, because they are very small (less than ±1.0 mm).

As can be seen, the number of cells influences the speed of the contraction of the mesh, but this difference in contraction becomes less significant, because the velocity is a function of time. The diameters of the grid reaches the General small size for concentrations above a certain minimum value. Below the minimum value, it is evident that the relationship between the speed of the contraction of the grid and the number of cells is not straight. In lattices from 8.1·104cell contraction does not occur within 24 hours. These sparsely populated grid is very behind the more densely populated grids during experiments.

Example 22: the Ability of cells with different levels of PDL to cause contraction gidratirovana what s collagen meshes

Reducing the ability of cells at various levels of doubling populations (PDL) in hydrated collagen meshes, i.e. cells that undergo cell division in different amounts, determined in the following way. Culture consists of hydrated collagen meshes obtained according to the methods of Example 18 and containing 720 mg/ml Protein "R". Environment change on the third, seventh and tenth days.

Control cultures did not contain cells. In addition, conduct a series of experiments with cells of different levels of PDL.

The collected data are graphically presented in Figure 5, where each point represents an average value of the contraction for three or four grids. Deviation is not more than ±1,0 mm As can be seen, the efficiency of the cells demonstrate 35th PDL, as well as cells of the 19th PDL, but the cells of the 50th PDL is not able to cause contraction of the mesh with comparable speed.

Example 23: the Influence of cytochalasin In the ability of cells to cause contraction of hydrated collagen mesh

The effect of the inhibitor of cytochalasin In the ability of cells to cause contraction of hydrated collagen mesh is determined as follows. Hydrated collagen mesh prepared in Example 18, of a Protein content "C" which is 570 µg/ml Concentration of fibroblast cells (foreskin man, strain 1519, 19-the th PDL) in cultures is 5.0·10 5. In each culture type of 10.0 μg/ml of cytochalasin In, and change the environment at the fourth and eighth days.

The obtained data are presented graphically in Fig.6, and as you can see, the concentration of cytochalasin In completely blocks the contraction of the mesh, even when using relatively high concentrations of cells.

Example 24: the Impact of colcemid on the contraction of the collagen mesh

Effect of inhibitor colcemid on the contraction of the protein of the grid is determined as follows. Culture containing hydrated collagen mesh, prepared according to the methods of Example 18, which contains 570 µg/ml Protein "C". In every culture, except for controls that do not contain colcemid add to 0.36 µg/ml colcemid. Add the same number of cells as the test cultures and control cultures. The obtained data are presented graphically in Fig.7. As can be seen, the cells of the 45th PDL superior to cells of the 19th PDL, whereas untreated cells 45th PDL lag behind from untreated cells 19th PDL. It is clear that colcemid can be used to regulate the speed and degree of contraction of the grid.

Example 25: the Effect of cytosine arabinoside on the contraction of the collagen mesh cells at various levels PDL

The impact of 1.0 μg/ml of cytosine arabinoside on the contraction of the protein grid cells different from the anti-shudder performance PDL checked as follows. Prepare culture containing hydrated collagen mesh, which contain Protein C at a concentration of 570 ág/ml foreskin Fibroblasts human strain 1519 19th PDL or 47th PDL add as controls, not containing cytosine arabinoside, and to experience cultures containing cytosine arabinoside. The obtained data are presented graphically on Fig, where you can see that the cells of the 47th PDL exceed cells lower level PDL, even if their number is less. In these experiments, cytosine arabinoside used to block DNA synthesis and thereby to save the number of cells in the grid at a constant level.

Example 26: Education equivalent to the skin when using foreskin fibroblasts and keratinocytes

Gidratirovannuyu collagen mesh prepared according to the methods of Example 18, which contains Protein C at a concentration of 500 µg/ml foreskin Fibroblasts person obtained in the biopsy removed from the plate for culturing EDTA and trypsin. A suspension of single cells centrifuged for pelleting cells, then cells resuspended in culture medium, and then precipitated on top of hydrated matrix within seven days after the introduction of fibroblast cells. For three days, the keratinocytes are attached to the substrate grid, and the process is keratinization begins to lead to the formation of opaque cornea. Histological observations carried out by electron microscopy.

Example 27: In vivo studies of equivalent skin using fibroblasts and keratinocytes of the skin of the Guinea pig

Select a skin biopsy from Guinea pigs, and surgically separate the dermis from the epidermis. The dermis decompose the enzymatic components of the cells that were seeded on plates for culturing tissues and enable them to undergo proliferation. Cells from each experimental animal grown in separate cups, so that their identity is preserved. Tissue produced in vitro by contraction of hydrated collagen meshes according to the methods of Example 18, except for using fibroblasts from Guinea pigs. Some of the nets after the contraction posevayut according to Example 26 epidermal cells or keratinocytes taken from the second biopsy, so keratinocytes and fibroblasts in each implant originate from the animal, which becomes the recipient of the implant.

Implants these skin equivalents are made for the backs of experimental animals (Guinea pigs). It was found that such implants are fully integrated at all levels in one week. They become vascularsurgery lower; at the level of the dermis, the collagen fibrils of the implant perepletayas is with fibrils surrounding tissue of the host body. In histological sections of the implants can be distinguished by the density of fibroblast cells and by reducing the double refraction in them, compared to a section of the surrounding skin, when viewed through a polarizing microscope. Even those implants that are highly secured epidermis completely covered by a layer of epidermal cells (keratinocytes), so that many cells are deep. The layer is continuous with the layer adjacent the skin of the host body. It is also clear that the contraction of wounds in the dermis is inhibited by the presence of the implant equivalent to the skin, just like when making autograft.

Example 28: In vivo studies equivalent of the skin using skin fibroblasts, dermal and epidermal cells of the rat

The formation of the dermal equivalent

A small biopsy of a possible recipient of the implant is cut 1.0 mm2the fragments. Fibroblasts grown from fragments and sown in a Cup Suite for culturing tissue, and after 4-7 days, the fragments are removed and discarded or transferred to a new plate. The cells on one or more plates give the opportunity to breed until then, until they become almost merged, at this time, the cells are removed by processing with trypsin, washed, and then multiply in flasks for culturing tissue. For each Quad is Atego centimeter dermal equivalent to about 5·10 4cells, and a corresponding number of cells tide Ute by removing them from the substrate with trypsin, after which they are suspended and combined with a solution of collagen, serum, rat and environment for culturing tissue. Collagen is produced by extraction of the tendons of the tail of the rat in 0.02 M acetic acid and purification by centrifugation. When collagen is stored at acidic pH and protein concentration of 1.5 mg/ml collagen is a viscous, slightly opalescent solution. It consists only of collagen type I, but does not contain contaminating proteins detected by electrophoresis on polyacrylamide gel with the addition of LTOs. At that moment, when collagen is combined with the cells and other ingredients, the pH is brought to a value of 7.2 with NaOH solution, which induces collagen to come out of solution in the form of fibrils. As this process forms a gel or mesh, which delayed the liquid. Cells in the grid are distributed more or less evenly. Through a process of active contraction of the collagen fibrils cells in the grid turns into a fabric with a dense texture. The result is the loss of the swelling fluid and multiple reduction of the initial mesh. The result is a fabric which is dermal equivalent (DE).

Add epidermis is and

Epidermal cells isolated from biopsy fragments with trypsin, distributed in the form of suspension on the dermal equivalent. 2-4 day epidermal cells form a continuous plate which covers the dermal layer. During this time, the slit plate cells begins to differentiate. Become visible desmosomal merger, the latters and keragialin granules, and proceeding the process of keratinization leads to the formation of an impermeable stratum corneum. If possible, the total process could occur in vitro at TE. However, in this work it is considered that the live two-layer fabric ready for transplant as soon as the epidermal cells will form the slit plate, which serves as the equivalent of the skin (EC).

The skin equivalents are transplanted to a large number of rats, obtaining the following results. Firstly, 3-5 days after implantation begins vascularization, it flows quickly without the occurrence of necrosis or ischemia. Secondly, with few exceptions, the implant inhibits the contraction of the wound. After fabrication of the implant border of normal skin adjacent to the implant, tattoo to identify the limits of implants, this procedure contribute to the observation time for the size of the implant. Exceptions to suppress counter the functions of the wound can occur because of lack of initial coverage of the dermal equivalent of the epidermis, displacement of the implant animals or education nonconforming grid, which depends on the quality of the used method of preparation of collagen.

In one study, thirty-one of the implant measured during the removal of the bandage (9-14 days), seven suppress the contraction of the wound completely, fifteen blocking the contraction of the wound by 75% or more, and twenty-three blocks by 50% or more.

In another study, fifty-two implants, wound contraction was blocked, at least 75-80% of the implants.

Although several of the implants were observed decrease in size over time, most of them are stabilized on the sixtieth day, and none of them rejected. Implants large size (approximately 8-12 cm) with good epidermal coverings effectively block the contraction of the wound (75% or more) and can be used as a good replacement for the burnt skin.

Implants last for long periods of time, the longest of them remains in place for two years.

In addition, the matrix of the dermal equivalent well vascularized, it will undergo a significant renovation during the first few months after transplantation. Changes in its structure are estimated by studies of double refraction histological sections, and the shows the matrix shows birefringence through one week after transplantation, this phenomenon is not observed in the granulation tissue of comparable age. As the birefringence increases over time in intensity, the sample, as a rule, is not a sample configuration chess weave typical of normal dermis. However, after ten weeks in the transition region where the implant meets with normal tissue, chess model begins to develop.

In vivo, hypertrophy of the epidermis, even after ten weeks after transplantation was significantly better than in adjacent normal tissue. Tongues or pins, or epidermis look penetrating the dermal equivalent. Externally, the same for seven months of the epidermis looks slightly scaly, and the implant has a reddish hue. After about three months, the implants become smooth and acquire a pinkish color, like normal skin white rat, but they have no hair. A certain degree of cheshuichatoe is maintained even up to seven months, which may be due to the lack of sebaceous glands.

Example 29: Preparation of equivalent tissue by contraction of the collagen mesh platelets

Outdated platelet concentrates obtained from the blood center of the red cross in Boston, Massachusetts the Collagen of tendons of the tail of the rat and pig extracted that to, as described in Example 18; pig skin collagen delivered by Dr. Paul Ehrlich from the Burn Institute Shriners in Boston; telogen, bovine collagen, derived from Collagen Corporation, Palo Alto, Calif. The concentration of collagen determined using the modified method Waddel. Cm. Waddel, W.J., J. LabandClin. Med. 48, 311-14 (1956). Use the formula: μg/ml=0, D. 215-0,D. 225/64 .6.

Concentratable with a concentration of 60 received from the red cross, optionally concentrated by centrifugation for 50 minutes at 500 rpm in a centrifuge IEC PR-2 253 rotor at 4°C to remove red blood cells. The supernatant is then centrifuged additionally for 50 minutes with a speed of 1500 revolutions per minute, using the same rotor for concentration of platelets. The supernatant assign channel and used for resuspendable obtained granules of platelets. The absolute concentration of platelets varies from one experiment to another, depending on the concentration of blood platelets donors, but all concentrations based on the use of a single sample or the combined samples of the concentrate and then dilute. The concentration of platelets 1x equivalent concentration of platelets in 1.0 ml of blood.

Trombotsitnoy nets cast by the Association in the following order: 2,3 ml of DMEM medium the concentration of 1,h (Flow Laboratories), 1.5 ml of collagen tendons of the tail of the rat, 0.25 ml of 0.1 N NaOH (concentration of NaOH needed to neutralize the collagen varies slightly depending on the acidity of the used preparation of collagen) and 0.45 ml SFR (Flow Laboratories). This mixture is poured into 60 mm Petri dish Falcon 0007, adding 0.5 ml of 5x concentrated platelets small drops on the surface of the Cup. Alternatively, all ingredients together in a separate bowl and pour into an empty Petri dish. The resulting grid is maintained at 37°C in an atmosphere of 90% air/10% CO2with 100% humidity. The thickness of the grid adjust by increasing or decreasing the total fill volume mesh. With different ways of casting not observe differences in the speed of contraction of the grid.

The rate of contraction is determined by the withdrawal of fluids net, beginning in 1 ml pipette, the pipette larger, and then recording the volume of the withdrawn liquid, and then withdrawn liquid back into the Cup. These measurements are repeated every hour up to a maximum of contraction of the grid. In grids with a concentration of platelets 1x or lower edge of the first grid is separated from the Cup with a scalpel to release the trapped fluid under the net to measure. Because the grid will allocate additional liquid at break, be careful to avoid surface is idania grid in order to measure liquids that are released only on the contraction of the grid. Experiments are also conducted to determine the effect of adding thrombin (Sigma Chemicals) by applying spots of thrombin and platelet concentrate in a Petri dish in the form of discrete drops before adding the United DMEM solution, collagen, NaOH and SFR.

Fig.9 is a graph of the received data, which shows the effect of thrombin on the contraction under the action of platelet-collagen meshes in the case when the concentration is 4.0 units/ml

Figure 10 is a graph illustrating the contraction of the mesh, obtained as a function of the concentration of platelets and the presence of trobman at a concentration of 4.0 units/ml

As can be seen, the reaction is quite fast. When the concentration of platelets 1x is a selection of 80-90% of the liquid at the third hour after casting the grid. After six hours of reaction basically proceeds in full, and a balance is achieved; namely, not see additional fluid under conditions of 100% humidity. At this point, 20-80% of the total initial volume of fluid released from the grid, with the exact number depends on the concentration of platelets and collagen and other environment settings for casting. The process of contraction of the grid platelets results in the formation of equivalent TC is neither with relatively high tensile strength, compared with the tensile strength of the collagen mesh, molded without platelets.

When the cast mixture with a sufficient quantity of platelets include thrombin, the equivalent of the fabric is formed more quickly, which can be seen in Figure 2. Equivalent fabrics, containing thrombin, has properties somewhat different from the properties of the equivalent cast without thrombin, its tensile strength is slightly smaller and the fluid in it below. Tensile strength is measured by binding of gram-equivalents balance for tissue after casting and measuring the time gap.

The rate of platelet contraction of nets associated with the concentration of platelets. The increase in the concentration of trombotsitov increases the speed of the contraction of the grid and leads to the formation of a grid with a proportionally smaller amount of fluid that can be seen on Figure 10,

Example 30: Effect of concentration and type of protein on the platelet contraction

The effect of concentration and source of protein on the plasma density grids platelets is determined by use of collagen from different sources in different concentrations, but otherwise following the procedures of Example 29. The resulting data are graphically presented in Figures 11-14.

As can be seen, increasing the collagen content in the environment for casting reduces the speed of contraction and the amount of liquid. These R the results correspond generally to those results, which is obtained by using fibroblasts as an agent that causes contraction.

Collagen from four other sources works in the same way, except that telogen gives a much lower resistance to contraction, compared with collagen from three other sources.

Example 31: the Effect of inhibitors of cytochalasin In and colcemid on the contraction of collagen meshes with platelets

Influence of inhibitors of cytochalasin In and colcemid on the ability of platelets to cause contraction of hydrated collagen meshes determined by following the General procedures of Example 29, except as follows.

Cytochalasin (Sigma Chemicals) and colcemid (CIBA Pharmaceutical Company) are added directly to the concentrated DMEM to obtain a final concentration in the net volume of 5 ml, comprising 10 μg/ml and 0.5 μg/ml, respectively. The obtained data are presented graphically on Fig.

As can be seen in the nets, cast together with platelets in the presence of cytochalasin In, the contraction does not occur, on the other hand, in nets, cast with colcemid not seen a noticeable effect on contraction; this is expected, since we know that colcemid does not influence the ability of human platelets to cause contraction of fibrin clots. Cytochalasin, on the other hand, stabilizethe discoid shape of the platelet and prevents the formation of pseudopodia.

Example 32: Rabbit implant

Equivalents fabric formed of hydrated collagen meshes subjected to contraction in platelets, and suitable for transplantation on the ears of rabbits prepared as follows. Platelets collected from 10 ml of rabbit blood, taken from a possible recipient of the implant through a puncture in the heart, and concentrated by differential centrifugation as described in Example 29. Cast gels rabbit platelets, also following the procedures of Example 29. After the contraction of the mesh seeded with epidermal cells, derived from a biopsy of the same rabbit, following the methods of Example 28.

Shot rabbit is prepared for transplantation by clipping the hair at the site of implantation. Lead round the surface of the implant and tattoo marks are placed directly outside of the edge of the shape. The plot was washed with 70% ethanol, and remove all the fabric from the bottom of the fascia above the cartilage. Equivalent tissue is separated from the Cup and placed on the surface of the implant so that the edge of the implant slightly overlapping the edge surface of the implant. The implant is covered with a gauze bandage type "tulle gras cooked in application of Telfa pads with vaseline. Telfa pads soaked in saline Earl, put on a blindfold "tulle gras". Foam gasket used in the t in the ear to maintain its shape, and ear three-inch bandage tape Elastoplast. To prevent the removal of the bandages rabbit both ears wrapped together then Elastoplast. Bandages will be removed after seven days after transplantation. Rabbit supply collar Elizabeth to prevent scratches over the next two days.

The implanted area with a border of normal skin cut in a single piece from rabbit ear and immersed in 10% formalin solution in 0.03 M phosphate buffer. Skin fix over night, rinsed with distilled water, dehydrated in ethanol, distilled amylacetate, and then toluene, and embedded in Paraplast. Section seven micron cut by a rotary microtome, and stained these sections with hematoxylin and eosin.

Implants root inhibiting wound contraction in all experiments. Macroscopically, the implants look whiter than the surrounding skin, while their surface is slightly scaly and no hair. Even after six weeks in the center still has a small cork.

The implant is located on the site within six weeks, evaluate histologically in part to determine the extent of penetration into the implant fibroblasts from the surrounding tissue. The density of fibroblasts in the implant significantly higher density in the surrounding tissue. The implant is distinct from the he fabric at a reduced level double refraction which, as noted, observed under a light microscope. The vascularization of the implant seems to be higher than in surrounding tissue. The implant, in addition, distinct from the surrounding tissue by the absence of second derivatives, namely, hair follicles and sebaceous glands. The epidermal layer of the implant markedly hypertrophied.

Example 33: In Vivo studies exclusion equivalent of the skin in rats

Education equivalent to the skin

Small pieces of skin are removed from the backs of female rats Fischer 344 (Charles River Breeding Labs.). These biopsies are cleaned from foreign fabric, cut into 2-3 mm3pieces and dried on the bottom surface of the cups Suites for the cultivation of tissues. Fibroblasts grown from biopsies, cultured in a minimal nutrient medium Needle in the modification of Dulbecco (DMEM)supplied with 10% fetal calf serum, penicillin, streptomycin and Fungizone (Flow Laboratories) in an atmosphere of 10% CO2at 37°C.

Collagen mesh is prepared by following the procedures of Example 28. Briefly, 2-8·105of fibroblast cells unite in 100 mm plastic Cup with concentrated DMEM and 10% fetal calf serum, antibiotics and 6 mg of collagen tail of the rat in acetic acid, diluted with water in ratio of 1:1000. During the week fibroblasts causes contraction of the collagen mesh, and the suspension is epidermal to etok, separated from fresh biopsy layer on the surface of the grid. Choleragen in the number of 10-10 M, the growth factor of the epidermis in the amount of 20 μg/ml and hydrocortisone in an amount of 0.4 μg/ml is added to the medium to activate the growth of epidermal cells, and cups move in an atmosphere of 5% CO2. During the week of epidermal cells form the slit plate on the surface of the collagen mesh, then the equivalent skin ready for transplant.

Testing allografts grid prepared with cells from female rats of Sprague-Dawley and implanted male individuals of Fischer rats.

The transplantation procedure

Male rats of the Fischer weighing approximately 350-400 g anastasimatarion sodium. Prepare implanted surface of approximately the same size as the grid, by deleting the full layer of skin from the backs of each animal. The grating is placed on the wound and cover with Telfa pad soaked in vaseline, and a Telfa pad soaked in saline Earl. These bandages cover, and binds the body with several layers of Elastoplast. Intervals from 9 days to 13 months after the initial application of the implant animals again anaesthetize and cut off the entire implant. Half of the implant is fixed in formalin, buffered with phosphate, dehydrated in ethanol and insert in paraffin. C is stralow part of the other half of the implant is cleaned from the underlying fat tissue, cut into 2-3 mm3pieces and placed in tissue culture, to give the opportunity to grow settled fibroblasts.

Preparation of karyotypes

The population of fibroblasts that have sprouted from the implant, subcultured to obtain three to six flasks T-150 with rapidly dividing cells. Fibroblasts treated with colchicine at 2 µg/ml for four hours, removed from the flasks, placed in a hypotonic environment for swelling (1 part medium DMEM into 3 parts distilled H2O), dried in air on slides and stained with acetoorcein. Photographed at each point in time from twenty to thirty full chromosomal distributions, and prepare karyotypes to determine the percentage of female cells in the total population of fibroblasts.

Implants with cells of the female Sprague-Dawley settle down and held organisms male Fischer, which is determined by the presence of female cells in the implant after 1-2 months.

Example 34: Preparation of systems for validation of equivalent skin tissue

A. Apparatus similar to that shown in Fig use when carrying out the following work. The coating is removed to perform the operation, but otherwise keeping on the site to maintain sterility. The following information related to the device:

The outer container 10:
diameter 38 mm
capacity 35 ml
The inner container 20:
ø 24 mm
capacity 4 ml
Permeable element 24:
Polycarbonate membrane
Nuclepore,
The pore size of 3 μm, a thickness of 5 μm.

Century Acellular, hydrated collagen gel is formed on the permeable element 24 as follows:

(1) Pre-cooked mixture:
10X MEM of 16.2 ml
L-glutamine (200 mm) 1.6 ml
Gentamicin (50 mg/ml) 0.2 ml
The rest of bovine serum 18,0 ml
Sodium bicarbonate (71,2 mg/ml) 5.0 ml

Royal solutions are mixed at 37°C, combining the above sequence, and stored at 4°C for approximately 30 min in a 50 ml tube (without gas). (2) 27.8 g of 1 mg/ml solution of collagen (extracted by acid from the tendon of the common digital rastiagivala calf) 0.05% (by volume) acetic acid is weighed into a 50 ml tube and stored at 4°C for 30 minutes (3) Type of 8.2 ml of a previously prepared mixture described above, and 4 ml of complete DMEM medium (containing 10% FBS, 4 mm L-glutamine, 50 μg/ml gentamicin), and pipeinput 1 ml aliquots into the inner container 20 and give them the opportunity to thicken in a fume hood.

C. the tissue Equivalents fill in dermal fibroblasts and seeded with epidermal (epithelial) cells as described below. General description of methods and reagents can also be found in patents and jointly consider the application under serial No. 07/361041 registered June 5, 1989.

(1) the Molded mixture:

of 8.2 ml of a previously prepared mixture described above is added to 27.8 g of 1 mg/ml solution of collagen in 0.05% (by volume) acetic acid as described for stage And(2) above, and combine 4 ml dermal fibroblasts (2,5·105cells/ml). 3 ml aliquots will pipeinput in the container 20 above cell-free, gidratirovannym collagen gel formed at the stage In(2) above, and give them a chance to thicken. to 4.5 ml of complete DMEM medium add in the external container 20, and then incubated at 36°C in an atmosphere of 10% CO2usually for 4-8 days.

Use the following environment:
ComponentsmSBM
Hydrocortisone1.1 µm
Insulin5 m kg/ml
Transferrin5 m kg/ml
Trijodthyronin20 PM
Ethanolamine1·10-4M
o-phosphorylethanolamine1·10-4M
Adenine0.18 mm
Progesterone2·10-9M
Selenium5,26·10-8M
Bovine serum0,3%
Facto the growth of the epidermis 10 ng/ml
Not containing calciumDMEM75%
Wednesday ham F-1225%

The other two environments are identical in mSBM, except as stated below:

cSBMmainSBM
Progesterone00
Telicia serum2,0%1%
Growth factor1 ng/ml1 ng/ml
epidermis
Not containing calcium50%50%
DMEM
Wednesday ham F-1250%50%

In some cases, the medium was added calcium chloride in the amount of 1.8 mm. This is denoted as "medium+calcium, for example, mSBM+calcium.

D. Epidermolysis begins on the 6th day after the ex is Cai equivalent tissue.

(1) Epidermolysis

The environment above the stage To take out both the inner 20 and outer 10 containers. 50 μl of a suspension of epidermal cells of 3.33·106cells/ml) is placed at the equivalent of a fabric formed according to the above stage C. Then the container is maintained at 36°C in an atmosphere of 10% CO2for 4 hours. Then add to 12.0 ml mSBM the external camera and 4 ml in the cell. After that, the device will return in the same incubator.

(2) Differentiation

2 days after epidermolysis medium is removed and replaced by the environment "mSBM+calcium".

(3) Air transportation

5 days after epidermolysis carry out the following procedure. The environment is collected from internal and external cameras. The inner container 20 is removed, install the cotton pads soaked in the two volumes of the environment "cSBM+calcium", at the bottom of the outer chamber 10, and added to 9.0 ml of medium "cSBM+calcium". The inner container 20 is then replaced, and the apparatus is maintained at 35,5°C in an atmosphere of 10% CO2.

(4) Maintenance

Every 4 days, the medium is removed and replaced with fresh medium "mainSBM + calcium".

Example 35: Preparation of test systems, including agarose

2% solution of agarose in water is sterilized in an autoclave, cooled to 40°C and mixed with an equal volume of double-concentrated mainSBM. Absorbing cotton strips at the DKI removed fill in 13 ml of the mixture in the outer region 14, giving you the ability to set a temperature of 36°C (10% CO2), and the apparatus returned to the incubator for storage prior to shipment.

It should be understood that the examples and embodiments of described in this document are solely for the purpose of illustration, and that various modifications or changes in light of the fact that they can be offered by experts in the field of technology include the essence and content of this application and the scope of the approved claims.

Example 36: the effectiveness of the cultured tissue construct for the treatment of the notches in the gums in the mouth

The primary goal was to determine whether cultured tissue construct to ensure the functional area of the fixed portion of the gums, comparable with three autogenous grafts.

During this study involve up to 25 subjects. The age of the subjects is, at least, 18-70 years. The first three subjects are used to determine the methods of surgical treatment of the material, these subjects are not included in the statistical analysis. The following involve patients after four weeks of tracking for these three patients. The involved actors has at least two non-adjacent teeth with insufficient stationary part of what was, for which you want to transplant tissue. Two selected teeth must be located in contralateral quadrants. During the transplant root coverage is not indicated, or it is not needed. Subjects administered either cultured tissue construct of the invention or a free autologous transplant. The implants evaluated clinically to determine changes in the fixed part of the gums, and at least three subjects selected a small biopsy for histological evaluation and comparison of both grafts. Variables secondary actions are changes to the baseline width of keratinized tissue, depth of excavation, the source of inflammation, comparison of color and texture translational tissue from adjacent tissue, resistance to muscle stretch oral clinical level of attachment, depth sensing and discomfort of the subject.

The study used a comparison of the observed treatment, treatment of random character between the two entities. Assessment of the quantity and quality of the fixed part of the gums generated in cultured tissue construct and free autogenous grafts, spend one week and at 1, 3 and 6 months after surgery. Primary endpoint occurs at 6 months. All postoperative evaluations are conducted by the coordinator of the research is for, which posidonian on the performed surgical procedures. Color, texture, inflammation and resistance to muscle stretch independently noted the coordinator, calibrating the study.

Although the above invention is described in some detail by way of illustration and Examples for clarity and understanding, the specialist in the art it will be obvious that within the scope of the attached claims you can make certain changes and modifications.

1. The treatment condition of the oral cavity of the patient, including the implantation of the cultured tissue construct the fabric of the oral cavity of the patient, in which the aforementioned cultured tissue construct includes a layer of extracellular matrix produced from dermal cells, fibroblasts, cultured without exogenous components of the matrix or supporting scaffold.

2. The method according to claim 1, wherein treating the condition of the oral cavity selected from the group consisting of a notch in the gums, loss of the interdental papilla, the failure of the alveolar ridge, damaged oral implant defect of the separation zone of the roots of the teeth and resection of oral and maxillofacial tumors.

3. The method according to claim 1, in which the extracellular matrix using extracellular matrix, which contains the ASCII decorin, glycosaminoglycans and fibrillar collagen with packing of fibrils and fibrillar bundles shifted by a quarter period characterized by iscertainly equal to 67 nm.

4. The method according to claim 1, in which the use of extracellular matrix containing cultured cells fibroblasts from tissue selected from the group consisting of male foreskin of a newborn, dermis, tendon, lung, urinary tract, umbilical cord, corneal stroma and intestines.

5. The method according to claim 1, in which the use of extracellular matrix containing cultured cells fibroblasts from the dermis.

6. The method according to claim 1, in which use cells of fibroblasts genetically modified to enhance production of extracellular matrix components.

7. The method according to claim 6, in which the cells of fibroblasts genetically modified to enhance production of a component of the extracellular matrix selected from a growth factor, hormone, peptide and protein.

8. The treatment condition of the oral cavity of a patient, comprising implanting the tissue of the oral cavity of the patient cultured tissue construct, comprising a first layer of extracellular matrix and the second layer of cells located on said first layer, in which the mentioned first layer is produced from dermal cells, fibroblasts, cultured be the use of exogenous components of the matrix or supporting scaffold, as mentioned, the second layer contains epithelial cells.

9. The method according to claim 8, in which are treated the condition of the oral cavity selected from the group consisting of waste gums from the base of the teeth, loss of the interdental papilla, the failure of the alveolar ridge defect of the separation zone of the roots of the teeth and resection of oral and maxillofacial tumors.

10. The method according to claim 8, in which using cultured tissue construct, the extracellular matrix which contains fibrillar collagen, decorin and glycosaminoglycans, thus choose fibrillar collagen with packing of fibrils and fibrillar bundles shifted by a quarter period characterized by iscertainly equal to 67 nm.

11. The method according to claim 8, in which using cultured tissue construct, the second layer, whose epithelial cells contains keratinocytes, cells of the corneal epithelium, the epithelial cells of oral mucosa, epithelial cells of the esophagus or cells uroepithelial.

12. The method according to claim 8, in which using cultured tissue construct, the first layer which contains the cultured cells fibroblasts from tissue selected from the group consisting of male foreskin of a newborn, dermis, tendon, lung, cartilage, urethra, corneal stroma, the mucous membranes of the mouth and intestines.

1. The method according to claim 8, in which the cultured tissue construct using cultured tissue construct, the extracellular matrix which further comprises fibronectin and tenascin.

14. The method according to claim 8, in which using cultured tissue construct, cells, fibroblasts and epithelial cells which originate from human tissues.

15. The method according to claim 8, in which the epithelial cells using cells keratinocytes in the epidermal cell layer.

16. The method according to item 15, in which the said layer of epidermal cells is layered, stratified and differentiated, but he has a basal layer, advasary layer, granular layer and stratum corneum, thereby mentioned cultured construct includes the basement membrane adjacent to the first layer.

17. The method according to claim 8, in which use cells of fibroblasts genetically modified to enhance production of extracellular matrix components.

18. The method according to 17, in which the cells of fibroblasts genetically modified to enhance production of a component of the extracellular matrix selected from a growth factor, hormone, peptide and protein.

19. The treatment condition of the oral cavity of a patient, comprising implanting the tissue of the oral cavity of the patient cultured TC is newago construct, includes gel mixture solution of collagen and agent, causing the failure.

20. The method according to claim 19, in which are treated the condition of the oral cavity selected from the group consisting of waste gums from the base of the teeth, loss of the interdental papilla, the failure of the alveolar ridge defect of the separation zone of the roots of the teeth and resection of oral and maxillofacial tumors.

21. The method according to claim 19, in which using cultured tissue construct containing as an agent that causes contraction, cell fibroblasts.

22. The method according to item 21, in which using cultured tissue construct containing as an agent that causes contraction, cell fibroblasts from human tissues.

23. The method according to item 21, in which using cultured tissue construct containing as an agent that causes contraction, cell fibroblasts from tissue selected from the group consisting of male foreskin of a newborn, dermis, tendon, lung, urinary tract, umbilical cord, corneal stroma and intestines.

24. The method according to item 21, which use cells of fibroblasts genetically modified to enhance production of extracellular matrix components.

25. The method according to paragraph 24, in which the cells of fibroblasts genetically modified to enhance production of a component of the extracellular matrix, is selected from a growth factor, hormone, peptide and protein.

26. The treatment condition of the oral cavity of a patient, comprising implanting the tissue of the oral cavity of the patient cultured tissue construct, comprising a first layer containing a cell-free collagen gel, and located on the first layer a second layer containing a second collagen gel, which contains collagen and the agent causing the failure.

27. The method according to p treating the condition of the oral cavity selected from the group consisting of waste gums from the base of the teeth, loss of the interdental papilla, the failure of the alveolar ridge defect of the separation zone of the roots of the teeth and resection of oral and maxillofacial tumors.

28. The method according to p that uses cultured tissue construct, the second collagen gel which as an agent that causes contraction, contains cells fibroblasts.

29. The method according to p, which referred to the collagen gel as an agent that causes contraction, contains cells fibroblasts from tissue selected from the group consisting of male foreskin of a newborn, dermis, tendon, lung, urinary tract, umbilical cord, corneal stroma and intestines.

30. The method according to p, in which the second layer contains epidermal cells.

31. The method according to p, which mentioned collagenoma the gel as agent, causing the contraction, contains cells of fibroblasts from human tissues.

32. The method according to p, which use cells of fibroblasts genetically modified to enhance production of extracellular matrix components.

33. The method according to p, in which the cells of fibroblasts genetically modified to enhance production of a component of the extracellular matrix selected from a growth factor, hormone, peptide and protein.



 

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EFFECT: agent possessing antimicrobial action and representing robinia (Robinia pseudaacacia L) seed essence prepared by collecting the seeds in October and extracting them three times in petroleum benzene under certain conditions.

2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely preparing an agent of hindu lotus seed extract (Nelumbo nucifera) possessing anxiolytic and antidepressive action. The agent possessing anxiolytic and antidepressive action prepared by extraction of hindu lotus seed extract (Nelumbo nucifera) in 50% ethanol in a Soxhlet extraction apparatus in the specific proportions.

EFFECT: agent possess the effective anxiolytic and antidepressive action.

3 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a method for preparing an enterosorbent. The method for preparing the enterosorbent involving milling birch bark, activating in evaporated water under certain conditions, processing in 0.5-2.0% alkali for 30-60 min, water flushing, nautralising, condensing and drying to the air-dry condition.

EFFECT: enterosorbent prepared by the method described above is characterised by high methylene blue sorption capacity and gelatine sorption capacity.

1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: declared invention refers to veterinary science, and aims at treating gastrointestinal diseases in calves. A method involves using the subcutaneous injections of convascelent's blood serum containing rota- and coronavirus antihemagglutinin in titres 1:128 and 1:64 respectively. The serum is introduced in a dose of 10.0 ml once a day for 9 days every 48 hours. That is combined with administering an infusion of a milled mixture of equal portions of purple Echinacea herb and blossom clusters (Echinazea purpurea), coltsfoot herb (Tussilago farfara), tillet blossom (Tilia cordata) and liquorice root (Glycyrrhiza glabra) prepared with the relation of the raw material to extractant that is 70% alcohol - 1:10 and kept at temperature 15-20°C for 7 days in the form of 7-8% aqueous solution in a dose of 3.5-4.5 ml/kg of animal's body weight every 12 hours to the clinical recovery.

EFFECT: use of the declared invention provides the higher body resistance and faster recovery of the animal's body micro- and macroelement balance.

5 tbl, 3 ex

Immunomodulator // 2497514

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry, and represents an immunomodulator for treating chronic hepatitis, hepatic cancer, lymphatic sarcoma, chronic leukemia, and for improving the functions of liver and blood-forming organs, for enhancing the immunobiological body characteristics, prepared by mixing 1000 ml of an aqueous infusion of sandy everlasting blossom, pepper mint herb and chicory herb with 50 ml of bovine serum containing leukaemia oncovirus antibodies, 20 ml of wild rosemary infusion, 40 g of ascorbic acid, 2 g of sorbic acid, 0.2 g of folic acid until the ingredients are dissolved completely, with adding 60 g of liver powder, 30 g of lymphatic node powder, 30 g of young bovine spleen powder; the prepared solution is settled at room temperature for 24 hours, then kept at a boiling water bath for 30 minutes and cooled for 6-8 hours at room temperature; the settled solution is filtered, wherein the aqueous herbal solution is prepared by mixing equal proportions of the separately prepared aqueous infusions of 40 g of pepper mint herb in 1000 ml of water, 30 g of sandy everlasting blossom in 1000 ml of water and 30 g of chicory herb in 1000 ml of water, while the wild rosemary infusion is prepared by infusing 60 g of ground wild rosemary blossom in 1000 ml of 70% purified ethanol.

EFFECT: invention provides creating the high-efficacy agent and reducing the length of treatment.

FIELD: chemistry.

SUBSTANCE: claimed invention relates to two-component hair dye, which includes first component, containing alkaline agent, second component, containing hydrogen peroxide, as well as non-aerosol vessel-foaming agent, intended for supply of liquid mixture of first component and second component in form of foam. Said liquid mixture contain anionic SAS, selected from components (A1)-(A3) or components (A4) and (A5), where component (A1) is represented by anionic SAS of carboxylate class, component (A2) is represented by anionic SAS of sulfonate class, component (A3) is represented by anionic SAS of phosphate class, component (A4) is represented by ionic SAS, and component (A5) is represented by alkanolamide of fatty acid. Also claimed invention relates to method of hair dyeing.

EFFECT: invention has good discoloration and dyeing capacities.

22 cl, 4 tbl, 22 ex

FIELD: medicine.

SUBSTANCE: invention refers to production of oral hygiene products. The presented oral care composition contains amorphous quartz having the particle size characterised by the fact that D90 makes less than 50 mcm, and one or more active substance is an antibacterial agent, and specified in a group consisting of cationic antibacterial agents specified in a group consisting of chlorhexidine, cetylpyridinium chloride, bisguanides, benzalkonium chloride, benzethonium chloride; metal salts specified in a group consisting of bivalent stanum salts, zinc salts, copper salts; and combinations thereof.

EFFECT: using amorphous quartz having good compatibility with the above antibacterial properties as a part of the product, particularly a tooth paste, provides storage-stable and high availability of the antibacterial product alongside with the effective and safe cleansing and polishing of the dental tissue.

8 cl, 2 ex, 30 dwg

FIELD: medicine.

SUBSTANCE: invention refers to using a tracer agent for magnetic particle imaging (MPI) for the purpose of visual monitoring of a biocompatible product. The biocompatible product contains the tracer agent for magnetic particle imaging (MPI) by imaging technique using magnetic particles. The biocompatible product represents a microcontainer comprising a solid or semi-solid material applicable for cell culture support. The invention also refers to a method for visual monitoring of the biocompatible product. The declared method involves the stages of providing the biocompatible product containing the tracer agent for MPI, introducing the biocompatible product into a target area, and detecting signals generated by the tracer agent for MPI by imaging technique using magnetic particles.

EFFECT: invention provides monitoring of the targeted cell or active agent delivery into the target area.

10 cl, 2 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to transplantology, and may be used for treating graft-versus-host disease (GVHD). That is ensured by introducing pluripotent cells, other than embryo stem cells, embryo germ-line cells, germ-line cells into an individual. The cells may differentiate in one cell type of any of at least two embyo lines - endodermal, ectodermal and mesodermal. The cells express telomerase; they are allogenic for the individual and promote the negative immune response. The group of inventions also refers to the above cells which are grown and processed during 10 to 40 cycles of cell duplication in a culture, and then specific markers (oct-3/4, rex-1 and rox-1) are expressed.

EFFECT: method enables using the pluripotent mature precursor cells additionally for immune suppression for transplantation.

21 cl, 10 dwg, 12 ex

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