Method of growing and maintaining non-differentiated hemopoietic stem cells or precursors cells (options), method of preparing conditioned stromal cell medium, method of transplanting non-differentiated hemopoietic stem cells or precursors cells (options)

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

 

Scope and background of the invention

The present invention relates to a method and apparatus for maintaining the life and reproduction of hematopoietic stem cells. More specifically, the present invention relates to porshneva flow bioreactor with three-dimensional filling of stromal cells, intended to sustain and/or reproduction of hematopoietic stem cells and/or for production of conditioned medium to sustain and/or reproduction of hematopoietic stem cells.

Hematopoietic system in mammals consists of a heterogeneous population of cells that functions range from adult cells with limited proliferation to pluripotent stem cells with ample opportunities for proliferation, differentiation and self-renewal (1-3). Hematopoietic stem cells (HSC) are only required for hematopoietic recovery after transplantation and serve as primary targets for gene therapy. Despite the key role of stem cells in the maintenance of the hematopoietic system, their extremely low frequency of occurrence in hematopoietic tissue, and limited ability to support or sustain life, or reproduction of nidiffer tirovannyh stem cells in ex vivo for extended periods of time, not only remains a major limitation to the clinical use of these cells, but also reflects the current unavailability of new regulators of stem cells and the need for them.

It is widely believed that stem cells are closely associated in vivo with discrete niches within the bone marrow (4-6), which provide molecular signals that collectively mediate their differentiation and self-renewal through contact of cells or short-range interactions (7). These niches are part of the "hematopoietic inductive microenvironment" (HIM), consisting of stromal bone marrow cells, such as macrophages, fibroblasts, adipocytes and endothelial cells (8). Stromal cells in the bone marrow maintain the functional integrity of HIM by creating proteins of the extracellular matrix (ECM) and components of the basal membrane, which facilitates the contact of cells (9-11). They also create a variety of soluble or resident cytokines necessary for the controlled differentiation and proliferation of hematopoietic cells (12-14).

With this in mind, will not seem surprising that the development of culture systems for long sustain HSC person focused mainly on the use of pre with the data of the primary monolayers of stromal cells in the bone marrow. They contain long-lived culture of non-irradiated (dexterously culture, 15) or irradiated (16-19) primary stromal cells of human bone marrow, as well as lines of stromal cells of human or mouse (16, 19-24), with added exogenous cytokines or without them. Analysis of the production of HSC first was based on the ability of such cells to the production of myeloid progeny (initiating cells for long-lived culture; LTC-IC) or to generate colonies with "a stone" morphology (cells forming region like boulyjenkov; CAFC) after long-term cultivation (5-7 weeks) on such stromal cells (16, 17). Despite the widespread use of tests based on the LTC-IC and CAFC, it is increasingly clear, however, that they are likely to register very primitive undifferentiated precursor cells than the true hematopoietic stem cells, reproducing population (25, 26).

In a recent analysis of human stem cells are logged repopularize cells SCID (SRC), which colonize the bone marrow of diabetic non-obese individuals (NOD)/SCID mice (27), where they give life to myeloid, lymphoid, erythroid and CD34+ populations of undifferentiated precursor cells of human (28-30). SRC is found exclusively in fractions of hematopoietic cells expressing CD34+38 - surface antigen (31),and their frequency in CB (1/3× 105cells) increases compared to the VM (1/9×105cells) or immobilized PB (1/6×106cells) (32). The most recent studies indicate that SRC living in a subpopulation of cells CD34+/38-/CXCR4+ (33). CXCR4 surface receptor for factor 1 chemokine derived from stromal cells (SDF-1, 34), clearly is the most important for the colonization and establishment of hematopoietic stem cells in human bone marrow of NOD/SCID (33).

Research aimed at long-term maintenance of life/reproduction HSC human stromal cells, mainly based on the analysis of the final phenotype of the CAFC, LTC-1C or CD34+38- (16, 19-24). Rare reports maintenance/reproduction SRC on the cultures of stromal cells fail to identify significant long-term support of life. For example, allogeneic stromal tissue of human bone marrow, as detected induces short-term (7-day) support livelihoods SRC, with subsequent rapid, noticeable reduction (6-fold) activity (26). The failure of long-term life support/reproduction of transplanted human stem cells in the layers of stromal cells can be attributed to several factors related to the cultures of these cells in vitro. One can is to include the use of monolayers of stromal cells, which does not reflect the growth conditions in vivo inside a natural, three-dimensional structure of the bone marrow. Such conditions can reduce the ability of stromal cells to ensure optimal, appropriate as a carrier microenvironment, as well as the ability of stem cells to localize in specific niches and physical interaction with stromal cells and their products. In fact, the proof of the importance of three-dimensional (3D) structures for biological activity of hematopoietic progenitor cells provides excellent growth lines of hematopoietic cells on stromal cells seeded on 3D collagen matrix compared to their proliferation on the monolayers of these cells (35). More importantly, 3D-coated porous tantalum biomaterial, as recently shown, improves short-term maintenance of healthy cells LTCIC or CD34+38 - macaques compared with cells cultured alone or in monolayers stromal bone marrow cells (36). However, the impact of 3D media covered stromal cells has not been studied.

Recent studies have shown that the cell line AFT024 mouse exceeds the stromal tissue of the person maintaining the survival and maintenance (but not breeding) SRC HOLY man within 2-3 weeks (37). This Lin the I, as found, expresses some HIM new genes encoding membrane-bound proteins (21, 38, 39), which can play a major role in the physiology of stem cells. Possible expression of these and other genes stromal cells under conditions that better mimic their 3D microenvironment in the bone marrow, and thus allow them to realize their optimal physiologically functional activity remains to be determined.

Extensive research has shown that avoidant culture of stromal tissue(19, 21, 22, 40, 41) or the environment, air-conditioned stromal tissue (SCM) (21, 42-44), by themselves or together with cytokines, can support ex-vivo maintenance or reproduction of primitive undifferentiated hemopoietic precursor cells. SCM, as also was shown to improve the output and efficiency of transduction of these cells (45, 46). Although these findings again point to the importance of soluble factors, stromal cells, the use of LTC-IC, CAFC or CD34+38 - as of the end points in these analyses may not reflect the impact of SCM on the maintenance/duplication of transplantable HSC. Further, it is unknown whether such SCM obtained from cultures of stromal monolayers of cells, all gene products associated with stromal cells included in the physiology HSC human is A.

Recently, the attention of the intended reproduction of transplanted hematopoietic stem cells ex-vivo, focuses on creating cultures of suspensions with added cytokines (47-53). These studies helped to identify the main cytokines that are relevant to this process, for example those that are in the early stages, such as stem cell factor (SCF), FLT3 ligand and thrombopoietin (SRW). However, the obtained different results, indicating rapid loss (48, 49), maintenance (50-52), but also some rare examples breeding SRC, continued during 2-4 weeks of culture (47, 53). The ability of these cytokines and stromal cells interact in the 3D growth conditions to support life/reproduction SRC, yet to be determined.

Thus, there is an urgent need for a method and device for reproduction and/or sustain ex-vivo transplanted hematopoietic stem cells free from the above limitations, and it would be highly useful if the results obtained were superior to those known in the art.

BRIEF description of the INVENTION

If you can flatten the present invention to practice, the system of piston-flow bioreactor, which imit the plans 3D microenvironment of the bone marrow, and capable of supporting the growth and long-term maintenance of stromal cells. Last planted on a porous inorganic carrier, made in the form of matrices of the non-woven material made of a tough polyester, (54), which makes possible the reproduction of large quantities of cells in a relatively small volume. Structure and packing density of the media has a major influence on the transport of oxygen and nutrition, as well as on the local concentration of the released products stromal cells (e.g., ECM proteins, cytokines, 55). In addition, the ability of stromal cells cultured in this system, to stimulate sustain/reproduction of transplanted hematopoietic stem cells by direct contact of cells, as defined, is far superior to the methods known in this field. Moreover, the ability of the air-conditioned environment stromal cells cultured in this system, to stimulate sustain/ reproduction of transplanted hematopoietic stem cells using the new factors, stromal cells and associated with stromal cells contained therein, as defined, are far superior to the methods known in this field.

Thus, in accordance with one aspect of the present invention, we propose a method of reproduction/ sustain undifferentiated hemopoietic stem cells or precursor cells, which includes a stage (a) obtaining the undifferentiated hemopoietic stem cells or progenitor cells; and (b) sowing undifferentiated hemopoietic stem cells or progenitor cells in the piston flow bioreactor in the form of a stationary phase in which the pre-generated three-dimensional culture of stromal cells on the substrate in the form of a layer, and the substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, thereby contributing to the reproduction/life-support undifferentiated hemopoietic stem cells or precursor cells.

In accordance with other features of the described preferred embodiments the method further includes the extraction of undifferentiated hemopoietic stem cells or progenitor cells.

In accordance with another aspect of the present invention proposes a method of reproduction/sustain undifferentiated hemopoietic stem cells or progenitor cells, which comprises one hundred and the AI (a) obtaining the undifferentiated hemopoietic stem cells or progenitor cells; and (b) culturing the undifferentiated hemopoietic stem cells or progenitor cells in a medium containing air-conditioned environment stromal cells, and conditioned medium of stromal cells derived from piston-flow bioreactor with a stationary phase, which creates a three-dimensional culture of stromal cells on the substrate in the form of a layer, and the substrate includes a matrix of non-woven fibers, forming a physiologically acceptable three-dimensional network of fibers, thereby contributing to the reproduction/life-support undifferentiated hemopoietic stem cells or progenitor cells.

In accordance with another aspect of the present invention proposes a method of making air-conditioned environment stromal cells, suitable for reproduction/sustain undifferentiated hemopoietic stem cells or precursor cells, which includes a stage (a) create a culture of stromal cells in the piston flow bioreactor with a stationary phase on a substrate in the form of a layer, and the substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, thereby reproducing/sustaining life is the activity of undifferentiated hemopoietic stem cells or progenitor cells; and (b) is achieved when the desired density of stromal cells, selection of medium from the piston flow bioreactor with the stationary phase, thereby obtaining the air-conditioned environment stromal cells, suitable for reproduction/sustain undifferentiated hemopoietic stem cells or progenitor cells.

In accordance with another aspect of the present invention proposes a method of transplantation of undifferentiated hemopoietic stem cells or progenitor cells in the recipient, which includes a stage (a) breeding/sustain undifferentiated hemopoietic stem cells or progenitor cells by (i) obtaining the undifferentiated hemopoietic stem cells or progenitor cells; and (ii) sowing undifferentiated hemopoietic stem cells or progenitor cells in a piston flow bioreactor with a stationary phase, where the pre-generated three-dimensional culture of stromal cells on the substrate in the form of a layer, and the substrate comprises a fibrous matrix of the nonwoven material, forming a physiologically acceptable three-dimensional network of fibers, thereby contributing to the reproduction/life-support undifferentiated heme is poetic stem cells or precursor cells; and (b) transplantation of undifferentiated hemopoietic stem cells or progenitor cells obtained in stage (a), the recipient.

In accordance with further features of the described preferred embodiments the method further includes the extraction of undifferentiated hemopoietic stem cells or progenitor cells prior to stage (b).

In accordance with an additional aspect of the present invention proposes a method of transplantation of undifferentiated hemopoietic stem cells or progenitor cells in the recipient, which includes a stage (a) breeding/sustain undifferentiated hemopoietic stem cells or progenitor cells by (i) obtaining the undifferentiated hemopoietic stem cells or progenitor cells; and (ii) culturing the undifferentiated hemopoietic stem cells or progenitor cells in a medium containing air-conditioned environment stromal cells, and conditioned medium stromal cells obtained from a piston-flow bioreactor with a stationary phase, which creates a three-dimensional culture of stromal cells on the substrate in the form of a layer, and the substrate comprises a fibrous m the Trix from non-woven material, forming a physiologically acceptable three-dimensional network of fibers, thereby contributing to the reproduction/life-support undifferentiated hemopoietic stem cells or progenitor cells.

In accordance with another aspect of the present invention offers the piston to a bioreactor containing a container having outlet and inlet and enclosing the substrate in a layer, and the substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, the substrate supports at least 5×106stromal cells per cubic centimeter of substrate.

In accordance with another aspect of the present invention serves piston flow bioreactor containing the above-mentioned piston bioreactor.

In accordance with further features of preferred embodiments of the present invention described below, the undifferentiated hemopoietic stem cells or precursor cells are cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone marrow.

In accordance with further features of the described item is impactfully embodiments of the undifferentiated hemopoietic stem cells or precursor cells and stromal cells culture of stromal cells have a common HLA-antigens.

In accordance with further features of the described preferred embodiments the undifferentiated hemopoietic stem cells or precursor cells and stromal cells culture of stromal cells obtained from one individual.

In accordance with further features of the described preferred embodiments the undifferentiated hemopoietic stem cells or precursor cells and stromal cells culture of stromal cells obtained from different individuals.

In accordance with further features of the described preferred embodiments the undifferentiated hemopoietic stem cells or precursor cells and stromal cells culture of stromal cells from the same species.

In accordance with further features of the described preferred embodiments the undifferentiated hemopoietic stem cells or precursor cells and stromal cells in culture stromal cells are taken from different types.

In accordance with further features of the described preferred embodiments stromal cell culture stromal cells are grown to a density equal to at least 5×106

In accordance with further features of the described preferred embodiments stromal cell culture stromal cells are grown to a density equal to at least 107cells per cubic centimeter of substrate.

In accordance with further features of the described preferred embodiments the stage seeding undifferentiated hemopoietic stem cells or progenitor cells in the piston flow bioreactor with the stationary phase is carried out at a time when the flow in the bioreactor off at least 10 hours after seeding.

In accordance with further features of the described preferred embodiments the fibers form a pore volume, taken as a percentage of the total volume, from 40 to 95%and the pore size is from 10 microns to 100 microns.

In accordance with further features of the described preferred embodiments the matrix is made of fibers selected from the group consisting of planar, non-circular and hollow fibers, and mixtures thereof, and the fiber is from 0.5 microns to 50 microns in diameter or width.

In accordance with further features of the described preferred embodiments the matrix consists of fibers in the form of tapes, with Shi is inu 2 microns. In accordance with further features of the described preferred embodiments the ratio of the width of the fibers to the thickness is at least 2:1.

In accordance with further features of the described preferred embodiments the matrix has a pore volume, taken as a percentage of the total, from 60 to 95%.

In accordance with further features of the described preferred embodiments the matrix has a height of 50-1000 μm.

In accordance with further features of the described preferred embodiments the matrix material is selected from the group consisting of polyesters, polyalkylene, polyphthalamide, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers and fibers of inert metal.

In accordance with further features of the described preferred embodiments the matrix is in a form selected from the group consisting of squares, disks, rings and crosses.

In accordance with further features of the described preferred embodiments the matrix is covered with poly-D-lysine.

The present invention successfully overcomes the shortcomings of the presently known configurations by providing more effective means for rozmnozhennyapelagofil life undifferentiated hemopoietic stem cells.

The embodiment of the method and bioreactor according to the present invention can include performing or completing selected tasks or stages manually, automatically or by a combination of both methods. Moreover, according to actual instrumentation and equipment equipment of preferred embodiments of the method and bioreactor according to the present invention, several selected stages can be implemented using hardware or software on any operating system on any hardware implemented in hardware or a combination thereof. For example, as hardware, a separate stage of the present invention can be implemented as a chip or Board. As software, a separate stage of the present invention can be implemented as a set of program instructions executable on a computer using any appropriate operating system. In any case, the individual stages of the method and bioreactor of the present invention can be described as implemented using a data processor, such as a computing platform for executing the set of instructions.

BRIEF DESCRIPTION of DRAWINGS

The present invention is described only as an example with reference to the accompanying drawings. Now, with specific reference is and the details of the drawings, have in mind that the details are given only by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention, and are presented with the proviso that, as expected, are the most useful and the most understandable description of the principles and conceptual aspects of the present invention. In this regard does not attempt to provide structural details of the present invention in more detail than is necessary for a fundamental understanding of the invention, the description taken together with the drawings, gives the possibility to understand the professionals in this field, exactly how several forms of the present invention can be implemented in practice.

In the drawings:

Figure 1 presents a schematic representation of an exemplary piston-flow bioreactor used in practice when implementing the present invention; 1 - tank environment; 2 - a container for a mixture of gases; 3 - gas filters; 4 - introduction; 5 - piston or container piston flow bioreactor; 6 - flow sensors; 6A - valves for flow; 7 - a container for selection/branches air-conditioned environment; 8 - the container to replace the environment; 9 - peristaltic pump; 10 - point training and writing sample; 11 - the container to replace the environment; 12 - sensor O2; 14 - device for peremeci the project; PH - the pH probe.

Figure 2 demonstrates the maintenance of CAFC using 14F1.1 cells. Cells from umbilical cord blood CD34+ plated at limiting dilution on irradiated 14F1.1 or primary stromal tissue of human bone marrow. Education "a stone" structure is determined 5 weeks later. The results represent the average value of ± RMS dispersion (SD) of 2 independent experiments.

Figure 3 demonstrates the maintenance of LTC-IC using 14F1.1 cells. Cells from umbilical cord blood CD34+ plated at limiting dilution on irradiated 14F1.1 or primary stromal tissue of human bone marrow. The formation of myeloid colonies define 7 weeks later. The ligand, FLT-3 (300 ng/ml), TPO (300 ng/ml) and SCF (100 ng/ml) added at weekly replacement environment. The results represent the average value of ± RMS dispersion of 2 independent experiments.

Figure 4 illustrates the multiplication of cells CD34+38 - 14F1.1 and primary stromal tissue of human bone marrow. CD34+ cells plated on 14F1.1 or stromal tissue of human bone marrow - 70 cells CD34+38-/well. Cytokines add a weekly basis. Culture trypsinized through 7-21 days. CD34+38-determined by FACS analysis. The results represent the average value of ± RMS di is Persia from 2 independent experiments.

On figa-b shows photographs taken with a scanning electron microscope (SEM), media, seeded line stromal cells 14F1.1 after 10 days (figure 5A) or 40 days (figure 5 (b). Magnification: x 150.

On figa-b demonstrates the effect of 3D and 2D air-conditioned environment 14F1.1 on the reproduction of CD34+38-. CD34+ cells were seeded in culture suspensions in the presence of different concentrations of conditioned medium from 14F1.1 and primary stromal tissue of human bone marrow. The number of cells CD34+38 - determined by FACS analysis. The results represent the average value of ± RMS dispersion of 2 independent experiments.

7 shows the maintenance of cells CD34+38 - on the media covered stromal cells. The media covered stromal cells, are removed from the 3D system in 96-well tablets, coated with silicone, followed by the addition of 1.5×104CD34+cell. Controls contain only the media and is equivalent to a medium number of cells 14F1.1 grown in monolayer (2D). Cells harvested at the indicated time points and analyzed by FACS. The results represent the average value of±RMS dispersion of 2 independent experiments.

DESCRIPTION of the PREFERRED EMBODIMENTS

The present invention is SPO the Oba and bioreactor for reproduction/life support hematopoietic stem cells, which can be used for transplantation to a recipient or for other purposes, as described below in more detail. Specifically, the present invention relates to porshneva flow bioreactor with three-dimensional system of stromal cells, intended to sustain and/or reproduction of hematopoietic stem cells and/or for production of conditioned medium to sustain and/or reproduction of hematopoietic stem cells, which can be used in various applications.

Principles and practice according to the present invention can be better understood with reference to the drawings accompanied by their descriptions.

Before explaining at least one embodiment of the present invention in detail it is necessary to understand that the invention is not limited in its application to details of construction and arrangement of the components presented in the following description or illustrated in the drawings. The present invention gives the possibility of other embodiments and may be practiced or played in different ways. In addition, it must be understood that phraseology and terminology used herein, is presented for the purpose of description and should not be construed as limiting.

Current when retegui, to maintain life or reproduction ex-vivo transplanted hematopoietic stem cells (HSC), have only limited success. This paper describes a new three-dimensional (3D) piston flow bioreactor that mimics the microenvironment of the bone marrow, and able to support the growth and long-term maintenance of stromal cells in the bone marrow. Last planted in porous media made of a fibrous matrix of non-woven material consisting of a complex of the polyester, stuffed in a glass column, thereby contributing to the reproduction of large quantities of cells in a relatively small volume. In the examples, see the Examples that follow, the bioreactor is seeded with a line of stromal cells 14F1.1 mouse or, alternatively, primary stromal cells of human bone marrow. To 40 days after sowing media contain 100-fold increased cell density. Density at different levels of the column is the same, indicating homogeneous transport of oxygen and nutrition to cells. Environment, air-conditioned with stromal cells inside the bioreactor (3D SCM), superior SCM from a monolayer of stromal cells (2D), when they support long-term activity of the cells of umbilical cord blood (CB) is the ne CD34+38-. 3D SCM also capable of supporting the proliferation of cells CD34+38-CXCR4+, which are renewable cells (SRC) SCID/NOD. In the presence of cytokines (FLT3 ligand and TPO) 3D SCM accelerates the self-renewal of stem cells and inhibits differentiation, whereas the opposite effect is induced by using 2D SCM+cytokines. Three-dimensional joint culture of stromal and stem cells also demonstrate excellence while maintaining the activity of cells in CD34+38 - on joint cultures on monolayers of stromal cells. These findings demonstrate that 3D piston flow bioreactor provides a suitable system to sustain/reproduction ex vivo HSC person with excellent contact between stromal and stem cells, and possibly by the production of known and/or new regulators of stem cells.

HSC person is a main target for transplantation and gene therapy. Very low frequency of occurrence HSC, as well as the unavailability currently, growth factors able to induce self-renewal of stem cells in the absence of final differentiation, still delivers the main problems in the implementation of such strategies, and the creation of large-scale banks HSC.

Currently used strategies aimed debt is a temporary life-support/reproduction undifferentiated HSC person, still have only limited success. Although recent studies with cultures of suspensions containing cytokines indicate a reproduction SRC, this process is also accompanied by a massive reduction in the content of early hematopoietic progenitor cells (53, 62), which indicates that there is a significant degree of differentiation of stem cells. The ideal system would be such, for example, in which SRC breed, while LTC-IC to remain present in small quantities.

The currently used system for reproduction of hematopoietic cells using perfusion culture suspensions hematopoietic cells themselves (see U.S. patent No. 5646043) or seeded on a monolayer of stromal cells (see U.S. patent No. 5605822). Although the first system shows a very large production commiteeman precursor cells, the second system suffers from the non-physiological nature of the interactions between stromal and stem cells in the monolayer. Additional system for reproduction of stem cells describe the use of air-conditioned environments stromal cells (U.S. patent No. 4536151 and 5437994). However, the latter is obtained from cultures of stromal monolayers of cells, which, as clearly shown in this description are lower in quality and are able on the ti activation of stem cells compared to 3D SCM (see table 3 Examples section). Despite the fact that recently been described (U.S. patent No. 5906940) bioreactor with a stationary phase, which uses covered stromal cells glass beads, these beads do not provide 3D physiological structure and make possible the reproduction of stromal cells per ml in an amount 10 times smaller compared to the media used in the application of the present invention. Comparison of 3D culture of stromal cells from the monolayer clearly demonstrates using the information presented in this description, a relatively superior ability obtained in the 3D conditions SCM or 3D cultures of stromal cells to maintain healthy cells CD34+38- (see figures 6 and 7). Superior 3D effect SCM can be attributed to elevated levels of known cytokines or new regulators of stem cells.

Experiments aimed at the assessment of combination effects 3D SCM and various cytokines (SCF, FLT3 ligand, TPO) on the maintenance/duplication of CD34+38-CXCR4+ (or SRC) (table 3)clearly show the advantage of the 3D effects SCM in the presence of FLT3 ligand and TPO, but not SCF. These data can be attributed to the relative inhibitory effects of 3D SCM on the differentiation of stem cells. These data clearly show that under 3D conditions are produced by new factors, associare what's with stromal cells, which, perhaps, are less active in themselves, but may act synergistically with cytokines. Using the output LTC-IC and commiteeman undifferentiated progenitor cells (GM-CFU), in addition to the release of CD34+, allows you to explore the differentiation of stem cells.

Bioreactor described herein, is unique in that it combines 3D culture of stromal cells with a continuous flow system. Although 3D system stromal-hematopoietic cells without continuous flow environment have been recently described (U.S. patent No. 5541107), the data presented in this description (see, e.g., figure 7)clearly show a decrease in the benefits of 3D cultures of stromal cells in relation to the monolayers in the absence of a continuous stream.

3D piston flow bioreactor described herein, is capable of supporting long-term growth lines stromal cells and primary stromal cells of the bone marrow. The use of stromal cells in the bioreactor is not only essential for creating an excellent contact between stromal and stem cells (with the help of unique niches and interactions cell-cell, cell-ECM), but also for producing stromal cells known and new soluble membrane-bound C is Takenov. Stromal cells may facilitate the procurement of such bioreactors relevant cytokines by using obtained using genetic engineering options, producing cytokines.

Stromal cells in the bioreactor can also be designed in such a way as to serve as lines of packaging cells for retroviruses, making possible the efficient transduction of genetic material in the stem cells inside the bioreactor. The use of different stromal cells in the bioreactor may also make possible the selection of the most suitable substrate for the desensitization of Ph-positive stem cells, the latter known for its lesser ability to stick together with stromal cells (63). Primary stromal cells have the advantage that they allow the creation of "autologous" bioreactors with stromal stem cells that can multiply autologous or even stem cells from umbilical cord blood, and which eliminates the need to remove stromal cells prior to transplantation.

Although the initial experiments by seeding cells in the bioreactor show rather small output cells CD34+38-in the media, the flow velocity after sowing, and the initial number of cells CD34+, seeded in the bioreactor can be easily op is kisirani. Analysis of CD34+38-CXCR4+ in the early periods (1-4 days) after planting is the key to this optimization.

Clear differences from the methods known in this field, in the bioreactor according to the present invention uses the growth matrix, which significantly increases the available contact area for adhesion to stromal cells in order to reproduce the mechanical infrastructure of the bone marrow. For example, for growth of matrix height of 0.5 mm, the increase is at least 5 to 30 times, if we take the projection on the basis of the growth matrix. This increase of about 5-30 times is on one layer, and if the number of such layers, or collected in the service or separated by gaskets or something else, the multiplier in 5-30 times will apply to each such structure. When using the matrix in the form of a layer, preferably of fibrous layers of non-woven materials or layers of foamed polymers with open pores, the preferred thickness of the layer is from about 50 to 1000 microns or more, and are provided with appropriate porosity to enter cells, input supply and removal of by-products from the reservoir. In accordance with a preferred embodiment of the pores have an effective diameter of from 10 μm to 100 μm. Such layers can be obtained from fibers of different thickness, before Occitania the thickness of the fibers or the diameter of the fibers are in the range of about from 0.5 μm to 20 μm, even more preferred fibers have a diameter ranging from 10 μm to 15 μm.

The structure according to the present invention can be supported or even better to be attached to the layer of the porous substrate or grid that provides dimensional stability and physical strength.

Such layers of matrix can also be cut, stamped, or reduced to create particles with a projected area of approximately 0.2 mm2up to 10 mm2with the same order of thickness from about 50 to 1000 microns).

Additional details relating to the manufacture, use and/or benefits of the growth matrix, which is used in the practical implementation of the present invention described in U.S. patent No. 5168085 and especially 5266476, both are included here by reference.

As can easily notice a specialist in this field, the present invention provides a breeding population of undifferentiated hematopoietic stem cells, which can be used in many applications, such as the following, but not limited to: (i) propagation of human stem cells (autologous source or umbilical cord blood) on the stromal tissue of the recipient, without the need for separation of stromal and stem cells prior to transplantation; (ii) the depletion of Volovich cells in Ph+CML with autologous production by interaction between stromal and stem cells; (iii) gene transfer in self-renewing stem cells inside the bioreactor or after selection of the bioreactor; (iv) production of conditioned medium (SCM) 3D stromal cells to maintain life/reproduction ex-vivo undifferentiated hemopoietic stem cells in suspension cultures or in the bioreactor for stem cells; (v) the allocation of new proteins, inducing the self-renewing stem cells in the absence of differentiation, as well as proteins that have additional biological functions; (vi) isolation of RNA 3D stromal cells for cloning new regulators of stromal cells and their associated stem cells, and additional functional gene products stromal cells.

In accordance with one aspect of the present invention proposes a method of reproduction/sustain undifferentiated hemopoietic stem cells or progenitor cells. The method in accordance with this aspect of the present invention is effected by implementing the following steps. First, get the undifferentiated hemopoietic stem cells or precursor cells. Second, the undifferentiated hemopoietic stem cells or precursor cells seeded in a piston flow bioreactor with fixed f is zoé, an example of which is depicted in figure 1 together with the reference numbers, in which three-dimensional culture of stromal cells or from the line of stromal cells or from the culture of primary stromal cells previously created on the substrate in the form of a layer, and the substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, thereby, as further explained above and is explained in the Examples section that follows, contributing to the reproduction/life-support undifferentiated hemopoietic stem cells or progenitor cells.

In this description and in the claims which follows, the phrase "undifferentiated hemopoietic stem cells" refers to commitirovannah hematopoietic cells.

In this description and in the claims which follows, the phrase "precursor cells" refers to comitiorum, immature hematopoietic cells.

As the undifferentiated hemopoietic stem cells or precursor cells that are CD34+cells. Thus, the phrase "obtaining the undifferentiated hemopoietic stem cells or progenitor cells", and the equivalent phrase "get dedifferentiated the E. hematopoietic stem cells or precursor cells", refer to the obtaining or isolated undifferentiated hemopoietic stem cells and/or progenitor cells, or a population of cells CD34+, which contains undifferentiated hemopoietic stem cells and precursor cells.

In this description and in the claims which follows, the terms "multiply" and "reproduction" refers essentially to the growth of undifferentiated cells, that is, to increase the population of cells without differentiation accompanying this increase.

In this description and in the claims which follows, the terms "support activities" and "maintenance" refers, essentially, to update undifferentiated cells, that is, essentially, to the stationary population of cells without differentiation accompanying stationarity.

The term "differentiation" refers to the change [of cells] from relatively generalized [forms] specialized forms in the development process. Differentiation of cells of different cell lines is well documented process and does not require additional description.

The term "ex vivo" refers to cells removed from a living organism and propagated outside the body (for example, in vitro research).

After breeding multiplied nedeff inconvenie hematopoietic stem cells or precursor cells, for example, can be isolated using various affine methods of splitting/tagging, such as (but not limited to the sorting of fluorescently-activated cells and affine separation of the affinity substrate. Affinity molecules that can be used for the implementation of such separation methods include, for example, antibodies against CD34, which bind CD34+cells.

In accordance with another aspect of the present invention provides another method of reproduction/sustain undifferentiated hemopoietic stem cells or progenitor cells. The method in accordance with this aspect of the present invention is effected by implementing the following steps. First, get the undifferentiated hemopoietic stem cells or precursor cells. Second, the undifferentiated hemopoietic stem cells or precursor cells cultured in a medium containing as the sole ingredient or as an additive air-conditioned environment stromal cells, and conditioned medium of stromal cells derived from piston-flow bioreactor with a stationary phase, in which a three-dimensional culture of stromal cells or from the line of stromal cells, or from the cult of the s primary stromal cells is created on the substrate in the form of a layer, and the substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, thereby, as additionally described above and explained in the Examples section that follows, contributing to the reproduction/life-support undifferentiated hemopoietic stem cells or progenitor cells.

In accordance with another aspect of the present invention proposes a method of making air-conditioned environment stromal cells suitable for use in the reproduction/maintenance of the undifferentiated hemopoietic stem cells or progenitor cells. The method in accordance with this aspect of the present invention is effected by implementing the following steps. First, a culture of stromal cells or from the line of stromal cells or from the culture of primary stromal cells in the piston flow bioreactor with a stationary phase on a substrate in the form of a layer, and the substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, thereby contributing to the reproduction/life-support undifferentiated hemopet the ical stem cells or precursor cells. Secondly, when reaching the desired density of stromal cells, say, for example, more than 5 x 106or more than 107cells per cubic centimeter of matrix, the collecting medium from the piston flow bioreactor with the stationary phase, as further described above and explained in the Examples section that follows, getting air-conditioned environment stromal cells, suitable for reproduction/ sustain undifferentiated hemopoietic stem cells or progenitor cells.

In accordance with another aspect of the present invention provides a method of transplantation of undifferentiated hemopoietic stem cells or progenitor cells in the recipient. The method in accordance with this aspect of the present invention is effected by implementing the following steps. First, undifferentiated hemopoietic stem cells or precursor cells multiply/supported using any of the methods described above. Second, the undifferentiated hemopoietic stem cells or precursor cells resulting from the first stage are transplanted to the recipient.

As shown in figure 1, in accordance with another aspect of the present invention, it is proposed the piston bio is eactor, includes the container 5, as a rule, in the form of a column having outlet and inlet and enclosing the substrate in a layer, and the substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, the substrate supports at least 5 x 106stromal cells, preferably at least 107cells, or relating to the line of stromal cells or the culture of primary stromal cells per cubic centimeter of substrate.

In accordance with another aspect of the present invention serves piston flow bioreactor containing the above-mentioned piston bioreactor.

In this respect, it is clear that the substrate can theoretically support up to 5 x 107cells on its cubic centimeter. As soon as the substrate is accumulated a sufficient number of cells, to stop further growth of the cells can be applied to tools such as irradiation, in order to control the exact number of cells supported by the substrate.

Undifferentiated hemopoietic stem cells or precursor cells, which are used as the source for such cells, when implemented methods according to the present invention, can be cleaned is sterile or stand out from the fabric, such as (but not limited to) the cord blood cytokine-mobilized peripheral blood (collected, for example, using leukapheresis) and bone marrow, all of which are known to contain undifferentiated hemopoietic stem cells or precursor cells. How this separation is well known in this field, the most commonly used sorting of fluorescently-activated cells, in which cells first aim by affinity labeling with fluorophore, and then collected.

In accordance with a preferred embodiment of the present invention undifferentiated hemopoietic stem cells or precursor cells and stromal cells culture of stromal cells have a common HLA-antigens. In accordance with another preferred embodiment of the present invention undifferentiated hemopoietic stem cells or precursor cells and stromal cells culture of stromal cells obtained from one individual. Thus, do not require cell division in case of their transplantation to a recipient.

In accordance with another preferred embodiment of the present invention undifferentiated hemopoietic stem cells or precursor cells and stromal cells in culture ctromagnetic are taken from different individuals. For example, the prospective recipient of undifferentiated hemopoietic stem cells or progenitor cells and stromal cells is used to obtain stromal cells, while the undifferentiated hemopoietic stem cells or precursor cells and stromal cells taken from a donor selected in accordance with the HLA compatibility for transplantation of such cells in the recipient. Thus, again, does not require separation of cells prior to transplantation.

In accordance with another embodiment of the present invention undifferentiated hemopoietic stem cells or precursor cells and stromal cells culture of stromal cells from the same species. However, in accordance with another preferred embodiment of the present invention undifferentiated hemopoietic stem cells or precursor cells and stromal cells in culture stromal cells are taken from different types.

In accordance with the preferred in the present embodiment of the present invention stage seeding undifferentiated hemopoietic stem cells or progenitor cells in a piston flow bioreactor with the stationary phase is carried out at a time when the flow in the bioreactor off at least 10 hours is after the sowing, in order to allow the cells to settle on the matrix, covered with stromal cells.

The following descriptions provide insights regarding the preferred substrates that are used in the implementation of the present invention.

Thus, in accordance with one of the embodiments of the fiber substrate to form a pore volume, taken as a percentage of the total volume, from 40 to 95%and the pore size is from 10 microns to 100 microns. In accordance with another embodiment of the matrix forming the substrate is made of fibers selected from the group consisting of planar, non-circular and hollow fibers and mixtures thereof, and the fibers have a diameter or width of from 0.5 microns to 50 microns. In accordance with another embodiment, the matrix consists of fibers in the form of strips having a width of 2 microns. In accordance with another embodiment the ratio of the width of the fibers to the thickness is at least 2:1. In accordance with another embodiment of the matrix forming the substrate has a pore volume, taken as a percentage of the total, from 60 to 95%. In accordance with another embodiment of the matrix has a height of 50-1000 μm, using the content of his material. In accordance with another embodiment of the matrix material forming the substrate is selected from the group consisting of a complex of the polyester, polyalkylene is in, polyphthalamide, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers, inert metals. In accordance with another embodiment of the matrix is in a form selected from the group consisting of squares, rings, discs and spacers. In accordance with another embodiment of the matrix is covered with poly-D-lysine.

Additional objectives, advantages and new circumstances present invention will become apparent to the person skilled in the art upon study of the following examples which are not intended to be limiting. In addition, each of the various embodiments and aspects of the present invention, as outlined above and described below in the claims, finds experimental support in the following examples.

Now will be made reference to the following examples, which together with the above description illustrate the present invention but without limiting it.

Generally, the nomenclature and laboratory procedures used in the present invention include molecular, biochemical, microbiological methods and techniques of recombinant DNA. Such techniques are explained in detail in the literature. Cm. for example, "Molecular Cloning: A. laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology" John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methods described in U.S. patent No. 4666828; 4683202; 4801531; 5192659 and 5272057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); "Current Protocols in Immunology" Volumes I-III J.E. Coligan, ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W.H. Freeman and Co., New York (1980); available immunoassays are widely described in the patent and scientific literature, see, for example, U.S. patents№3791932; 3839153; 3850752; 3850578; 3853987; 3867517; 3879262; 3901654; 3935074; 3984533; 3996345; 4034074; 4098876; 4879219; 5011771 and 5281521; "Oligonucleotide Synthesis", Gait, M.J., ed. (1984); "Nucleic Acid Hybridization" Hames, B.D. and Higgins S.J., eds. (1985); "reduced and Translation" Hames, B.D., and S.J. Higgins, eds. (1984); "Animal Cell Culture" Freshney, R.I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of them are included in this description by reference in their entirety. Other General references are made throughout the description. Procedures in them, as expected, is well known in this field, and they are for the convenience of the reader. All information contained in them, are included in this description by reference.

MATERIALS AND METHODS EXPERIMENTS/p>

Bioreactor: Bioreactor used in accordance with the concept of the present invention will be collected in accordance with the design described in figure 1. Glass parts design and manufacture at the Technion (Israel) and connect using silicone tubing (Degania, Israel). Media centrifuged overnight in phosphate buffered saline (PBS; Beit Ha Emek Industries, Israel) without CA+2and Mg+2with the subsequent removal of PBS and released balances. Each speaker load 10 ml of Packed media. Bioreactor fill PBS-Ca-Mg, seal all exits and the system is subjected to autoclave treatment (120°C, 30 minutes). PBS is removed through the container [8] and the bioreactor in the incubator at 37°To bring into contact with 300 ml of a circulating medium, Dulbecco with high glucose (DMEM; GIBCO BRL)containing 10% heat-inactivated fetal calf serum (FCS; Beit Ha Emek Industries, Israel) and a mixture of penicillin-streptomycin-nystatin (100 M.E. Ter-Minassian/ml:100 μg/ml:1,25 µg/ml; Belt Emek Ha), during the period of 48 hours. Circulating the medium replaced with fresh DMEM containing the same as above + 2 mm L-glutamine (Beit Ha Emek).

Stromal cells: stromal cells are maintained at 37°in DMEM containing 10% FCS, in a fully humidified incubator with 5% CO2in the air. Cells are grown in flasks for tissue culture (Corning) and share with trypsinized and when reaching confluence. Primary cultures of stromal cells of human bone marrow obtained on the basis of aspirated bone marrow of the sternum gematologichesky healthy donors undergoing surgery on the open heart. In General, bone marrow aspirates 3-fold diluted in balanced salt solution Hanks (HBSS; GIBCO BRL) and centrifuged in a density gradient in Ficoll-Pak (Robbins Scientific Corp. Sunnyvale, CA). Mononuclear bone marrow cells (<of 1.077 GM/cm3) is collected, washed 3 times in HBSS and re-suspended in the medium to long-term cultivation (LTC), consisting of DMEM, containing a 12.5% FCS, 12.5% horse serum (Beit Ha Emek), 10-4M β-mercaptoethanol (Merck) and 10-6mol/l of hydrocortisoneodoquinol sodium (Sigma). Cells incubated in 25 ml flasks for culturing tissues (Corning) for 3 days at 37°C (5% CO2), and then at 33°With (under the same conditions) with weekly feeding culture. For each bioreactor use planing cells from individual donors. For 3D surveys and studies on the monolayer culture of primary stromal cells share with trypsinization (0.25% trypsin and EDTA in Puck''s Saline A; Beit Ha Emek) every 10 days, to allow for adequate reproduction of stromal cells. For LTC-IC and CAFC (see below) stromal cells irradiated (1500 cGy), with use of the cation source 137Cs, culture is supported at 33°in the LTC environment.

Sowing stromal cells: Confluent culture lines stromal cells or 5-week primary stromal cells in the bone marrow trypsinized and cells washed 3 times in HBSS, re-suspended in the environment bioreactor (see above), counted and seeded at 106cells/ml in 10 ml volumes using the introduction ([4], figure 1) in 10 ml of media in a glass column bioreactor. Directly after sowing, the circulation is stopped for 16 hours to allow the cells to settle on the media. The growth of stromal cells in the bioreactor track by removing the media and counting of cells by MTT method (56). When stromal cells are merged for further research (preparation of SCM, seeding stem cells) environment replace the LTC environment.

Preparation of conditioned medium stromal cells (SCM): At equivalent densities of cells stromal cells in monolayer and in the bioreactor filled with fresh culture medium LTC. SCM collected after incubation of the cells during the night. For this purpose, the flow in 3D cultures stop for 16 hours and the environment remove immediately from the column before resuming circulation. To analyze the impact of CD34+ cell for producing stromal cells SRC circulation stopped at various online is rwally time (2-7 days) after sowing CD34+ 3D system and the environment are taken from the column, as is described above. SCM centrifuged (1000×g, 10 min), filtered and stored at -20°C. Stromal cells to collect SCM are also grown in the bioreactor in a medium not containing serum, thereby eliminating the uncertain parameters.

Selection of CD34+cell Samples of umbilical cord blood, collected in a sterile environment during childbirth, fractionary in Ficoll-Pak and collect the top fraction (density <of 1.077 g/cm3) mononuclear cells. Cells from individual samples SV harvested, incubated with antibodies to CD34 and allocate using midi MACS (Miltenyl Biotech).

Culture suspensions CD34+cell: the Cells are CD34+ ST (5×105/well) are incubated in 24-hole plates (TPP, Switzerland), 0.5 ml 0-100% SCM, minus or plus 300 ng/ml of each ligand FLT3, SCF, or TRO, together or one at a time. Controls contain the LTC environment, plus or minus cytokines. Cells are incubated at 37°C, With 5% CO2on the air. The culture medium replaced every week. Before sowing and at different points in time (1-3 weeks), the cells are harvested, counted and analyzed for CD34+/38-/CXCR4+ using flow cytometry. At the output of the analysis may also include SRC, CAFC and LTC-IC.

Joint culture of stromal and stem cells: Isolated, collected in a pool of CD34+ cells seeded ST. in equivalent quantities (about 5×105) on a monolayer or in a bioreactor, to the which contain equivalent density confluent stromal cells. When you add in the bioreactor medium flow is halted for 16 hours to allow contact with stromal cells, and resume a speed of 0.1-1.0 ml per minute. Seeded CD34+ cells-stromal cells on the carrier removed for follow-up research in the absence of a replacement environment. Joint cultural support in the LTC environment with cytokines or without them. At different points in time (up to 4 weeks) not adhering cells harvested from supernatants monolayer or from circulating culture medium through the container ([8], figure 1). Adherent cells are harvested with the help of the subsequent trypsinization and contact with the buffer for dissociation on the basis of EDTA (GIBCO BRL), followed by careful epatirovanie cells. To exclude the presence of stromal cells in the resulting suspension, the cells re-suspended in HBSS+10% PCS and subjected to 60-minute procedure adhesion in plastic cups for tissue culture (Corning)at 37°C. Circulating and separated from the media hematopoietic cells washed, counted and examined separately on CD34+/38-/CXCR4+ using flow cytometry. At the output of the analysis may also include SRC, CAFC and LTC-IC.

Flow cytometry: Cells incubated at 4°C for 30 minutes with saturating concentrations of monoclonal antibody anti-CD34+Regs (Beckton-Dickinson), ant the-R4-fluorescein isocyanate (FITC, R&D systems) and - liquiritin (RE, Beckton-Dickinson). Cells are washed twice in a cooled ice PBS containing 5% heat-activated FCS, and re-suspended for three-color flow cytometry on a FACSscan (Beckton-Dickinson).

Tests for LTC-IC and CAFC: freshly isolated CD34+cells, cells isolated from the joint cultures of stromal and stem cells or from suspension cultures, analyze on LTC-IC and CAFC, as described previously (16, 17). Confluent primary stromal cells in the bone marrow trypsinized, irradiated (1500 cGy) and placed in 0.1 ml in 96-well plates (Corning) at 1.5×105/well. Create 24 identical wells/group. Stromal cell filled with 0.1 ml of medium LTC containing serial dilution of the cells are CD34+ (500-5 cells/well), or a serial dilution of cells collected from the different analyses. The culture is incubated directly with 33°C for 5 weeks, with weekly replacement of half of the environment. Tablets centrifuged at 1000 rpm for 10 minutes, supernatant cultures were removed and the remaining cells cover methylcellulose cultures and cytokines for research myeloid precursor cells, as described previously (57). The colony count in the next 14 days and the frequency of LTC-IC is determined in accordance with the reciprocal of the concentration of the investigated cells, which gives 37% negative cultures (16). The research is of CAFC-analysis mainly produce, as described above, except there is no fill methylcellulose and cytokines. The percentage of wells with at least one hematopoietic clone in the dark phase of at least five cells ("bliznakovska" region) under a layer of moulding cells determined at week 6 after sowing of the studied suspensions of cells in serial dilutions.

EXPERIMENTAL RESULTS

System bioreactors, practically used in the present invention, depicted in figure 1. It contains four parallel node piston flow bioreactor [5]. Each node in the bioreactor contains 1 gram of porous media (4 mm in diameter), made of a matrix of non-woven material consisting of a complex of the polyester (58). These media make possible the reproduction of large quantities of cells in a relatively small volume. The structure and degree of packing of the media has a major impact on the transport of oxygen and nutrition, as well as on the local concentration and release products stromal cells (e.g., ECM proteins, cytokines, 59). The bioreactor is kept in incubator at 37°C.

The flow in each bioreactor track [6] and regulate by means of a valve [6A]. Each bioreactor contains a paragraph sample preparation and introduction of [4], which makes possible the sequential seeding of stromal and heme is poetic cells. Culture medium served at pH 7.0 [13] from the reservoir [1]. Tank supply filtered [3] a mixture of gases containing air/CO2/O2[2] in different proportions, in order to maintain 5-40% dissolved oxygen at the outlet of the column, depending on the density of cells in the bioreactor. The share O2corresponds to the level of dissolved O2at the outlet of the bioreactor, which is determined using the sensor [12]. The gas mixture is fed into the tank through the silicone tube. The culture medium passes through the separation container [7], which makes possible the circulating collection, not adherent cells. The recirculation get through the peristaltic pump [9], operating at a speed of 0.1-3 ml/minute. Nodes bioreactors provide additional sites for sample preparation [10] and two containers [8, 11] for continuous replacement of the medium with the speed of 10-50 ml/day. The use of four parallel nodes bioreactors makes it possible, periodic disassembly for purposes such as cell removal, scanning electron microscopy, histology, immunohistochemistry, RNA extraction, and the like.

In one experiment, create a system of bioreactors containing line stromal 14F1.1-mouse cells (24, 60, 61), which, as shown earlier, support the growth commiteeman myeloid cells-preds the relatives of the person (24). This cell line can equally support the CAFC (figure 2), LTC-IC (figure 3) cells and CD34+38- (figure 4) HOLY man, and primary stromal cells from human bone marrow. The results presented in these figures also show that the addition of ligand FLT3+TRO to these crops does not affect LTC-IC, while cytokines significantly increase the yield of cells CAFC and CD34+38-. On the contrary, SCF induces a decrease in both LTC-IC, and the CAFC. When seeded in the bioreactor at 1.5×106cells/10 ml volume of culture, 14F1.1 cells grow and multiply in the media (figure 5). On day 40 after sowing media contain 100-fold increased cell density, i.e. about 1.5×106cells/media 1,5×107cells/ml (table 1).

TABLE 1
The growth kinetics of the 14F1.1 and primary stromal tissue of human bone marrow-media
Time stromal cells on the carrier (days)1014203040
 14F1.1stromal tissue of man14F1.114F1.114F1.1
The upper part1.5×1031.5�D7; 1031×1053.5×1051.3×106
The middle part1×1031.2×1031.3×1052.0×1051.3×106
The lower part1×1031×1037×1042.0×1051.5×106

The MTT assay includes 5 media/definition. The average of 2 independent experiments.

The density of cells on the carriers at different levels of the column is the same, indicating homogeneous transport of oxygen and nutrients to the cells. For these purposes, optimize the cultivation conditions: culture medium (environment Dulbecco with high glucose + 10% fetal calf serum), flow rate (1 ml/min), the frequency of replacement of the medium (once a week), initial planting density (as described above). When the coating device of collagen or poly-L-lysine did not reveal any beneficial impact on the growth rate and the final density 14F1.1 cells. Preliminary data for the primary stromal cells of human bone marrow (table 1) indicate approximately the same density 14F1.1 cells and primary is chromaline cells on days 10 and 14 after seeding, respectively.

In order of analysis functional activity of stromal cells in the bioreactor determine the impact of air-conditioned environment stromal cells (SCM), obtained from the column bioreactor (3D SCM), on the proliferation of cells CD34+38 - suspension cultures, seeded with cells CD34+HOLY man. Activity compare with SCM obtained from monolayers cultures (2D SCM)containing the same concentration of stromal cells. As shown in figure 6, SCM from 14F1.1-cells, as detected, is equally or more capable of maintaining the activity of cells in CD34+38 - HOLY man than SCM of primary stromal cells of the bone marrow. The maximum exposure to SCM 14F1.1 clearly observed at concentrations lower than for SCM primary stromal cells of the bone marrow. Next, 3D SCM, as detected exceeds 2D SCM both types of cells while maintaining cell multiplication CD34+38 - HOLY man. The difference in activities between 2D and 3D SCM becomes more pronounced with increasing duration of cultivation (compare days 14 and 21). Adding 3D SCM 14F1.1 to culture suspension cells CD34+ HOLY man also leads to living cells CD34+38-CXCR4+ (table 2) compared to control cultures containing only one environment.

TABLE 2
The impact of 3D SCM 4F1.1 on the yield of CD34+38-/CD34+38-CXCR4+
The cell surface phenotypeWednesday LTCSCM 14F1.1 (50%)
CD34+38-3701296
CD34+38-CXCR4+038

CD34+ cells HOLY man (8×104/point) were seeded in culture suspensions containing environment LTC or 50% 3D SCM 14F1.1. Cultures are harvested after 7 days and the cells analyzed by FACS. The outputs of CD34+38 - and CD34+38-CXCR4+ are 2800 and 112, respectively.

Table 3 demonstrates the influence of cytokines in cultures of CD34+containing 2D and 3D SCM. The results clearly demonstrate that the 3D SCM superior to 2D SCM while maintaining life as CD34+38-and, more importantly, the subpopulation of CD34+38-CXCR4+ (SRC).

td align="center"> 1820
TABLE 3
The effects of cytokines on the proliferation of cells CD34+38-/CD34+38-CXCR4+ 3D 14F1.1 SCM
The cell surface phenotype2D14F1.1SCM(50%)3D14F1.1 SCM (50%)
 
 itselfligand FLT3+TROSCFitselfligand FLT3+TROSCF
CD34+38-140027204080130
CD34+38-CXCR4460700620930about
CD34+370001780003610001700025000210000

CD34+ cells HOLY man (2,6×105/point) 50% of 2D and 3D SCM 14F1.1, in the absence or in the presence of FLT3 ligand (300 ng/ml), TPO (300 ng/ml) or SCF (50 ng/ml). Cultures are harvested after 7 days and the cells analyzed by FACS. The outputs of CD34+38 - and CD34+38-CXCR4+ are 7900 and 360, respectively.

This may be due to a stronger influence of 2D SCM on the differentiation of cells that is determined by the output of the CD34+cell. SRW+ FLT3 ligand reduces the yield of CD34+38-/CD34+38-CXCR4+ in the presence of 2D SCM, but increase their output in cultures containing 3D SCM. Again, this may be attributed to less differentiation in a 3D system that is determined by using the surface marker CD34+. Both 2D and 3D cultures SCM SCF induces a reduction in the differentiation of cells and a marked reduction of output cells CD34+38-/CD34+38-CXCR4+.

In order analyze interactions between stromal and stem cells in the proposed bioreactor sleep is Ala assess the maintenance/proliferation of cells CD34+38 - on the media covered stromal cells (14F1.1). The last is removed from the bioreactor covered in silicone 96-well plates, followed by the addition of CD34+cell. Controls contain only the media and is equivalent to a carrier of the number 14F1.1 cells in monolayers. As shown in figure 7, the cell survival of CD34+38-increases in the presence of only one carrier, which confirms the beneficial effects of 3D structure on the survival/maintenance of primitive progenitor cells (36). The media covered stromal cells exceed the carriers themselves or monolayers of cells 14F1.1 in stimulating the 7-day survival/ livelihood cells CD34+38-. Long-term cultivation (day 14) leads to an increase in the number of CD34+38 - as in cultured monolayers 14F1.1 and media covered 14F1.1.

In the following experiment, 6×106collected in a pool of cells CD34+ ST (3×105 CD34+38-) seeded in the bioreactor containing 4 columns with non-irradiated covered 14F1.1 media in 350 ml of circulating culture medium. The flow stop for 16 hours, and then resume at normal speed (1 ml/min). After 4 days of culturing, the circulating medium contains 10% of the collected viable cells from the initially seeded cells CD34+38-that is determined using FACS and the aleesa. After 18 days of cultivation circulating medium contains 0.4% of cells CD34+38-, while adhering to the carrier cells make up 3% of the initially seeded populations of CD34+38-.

Although the present invention is described in conjunction with specific embodiments, it is evident that many alternatives, modifications and variations will be apparent to a person skilled in this field. Accordingly assumes coverage of all such alternatives, modifications and variations that are within the essence and scope of the attached claims. All publications cited herein, incorporated by reference in its entirety. Citation or identification of any reference in this application can be considered as an admission that such reference is available as prior art of the present invention.

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1. The method of reproduction/maintaining undifferentiated hemopoietic stem cells or precursor cells, which implies the following stages:

(a) culturing the three-dimensional culture of stromal cells under conditions of flow in the piston flow bioreactor with a stationary phase comprising a piston bioreactor, and the specified piston bioreactor includes a container having an inlet and outlet and enclosing the substrate in a layer, and the specified substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, where the specified three-dimensional culture of stromal cells grown to a density of at least 5×106cells per cubic centimeter of the specified substrate; and

(b) seeding undifferentiated hemopoietic stem cells or progenitor cells in the specified piston flow, biurea the Torah with the stationary phase, includes the specified three-dimensional culture of stromal cells from step (a), at a time when the flow in the specified bioreactor overlap for a predetermined time.

2. The method according to claim 1, where these undifferentiated hemopoietic stem cells or precursor cells are cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone marrow.

3. The method according to claim 1, wherein the specified undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells have a common HLA antigens.

4. The method according to claim 1, where these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in one individual.

5. The method according to claim 1, where these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in different individuals.

6. The method according to claim 1, where these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take the same view.

7. The method according to claim 1, which indicated the s undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in different species.

8. The method according to claim 1, in which the stromal cells of this culture of stromal cells grown to a density of at least 107cells per cubic centimeter of the specified substrate.

9. The method according to claim 1, where these fibers form a pore volume, taken as a percentage of the total volume, from 40 to 95%and the pore size is from 10 to 100 microns.

10. The method according to claim 1, where the specified matrix is made from fibers selected from the group consisting of planar, non-circular and hollow fibers and mixtures thereof, and the said fibers comprise from 0.5 to 50 microns in diameter or width.

11. The method according to claim 1, where the specified matrix consists of fibers in the form of a tape having a width of 2 to 20 μm, and the ratio of the width of the fibers to the thickness is at least 2:1.

12. The method according to claim 1, where the specified matrix has a pore volume, taken as a percentage of the total, from 60 to 95%.

13. The method according to claim 1, where the matrix has a height of 50-1000 μm.

14. The method according to claim 1, where the matrix material is selected from the group consisting of polyesters, polyalkylene, polyfloral-atilano, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers and fibers of inert metal.

15. The method according to claim 1, where the matrix is in a form selected from the group consisting of squares, disks, rings and crosses.

16. The method according to claim 1, where the matrix is in fo the IU disk.

17. The method according to claim 1, where the matrix coated with poly-D-lysine.

18. The method according to claim 1, further comprising the extraction of these undifferentiated hemopoietic stem cells or progenitor cells.

19. The method of reproduction/maintaining undifferentiated hemopoietic stem cells or precursor cells, which implies the following stages:

(a) culturing the three-dimensional culture of stromal cells under conditions of flow in the piston flow bioreactor with a stationary phase comprising a piston bioreactor, and the specified piston bioreactor includes a container having an inlet and outlet and enclosing the substrate in a layer, and the specified substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, where the specified three-dimensional culture of stromal cells grown to a density of at least 5×106cells per cubic centimeter of the specified substrate; and

(b) culturing the undifferentiated hemopoietic stem cells or progenitor cells in a medium containing air-conditioned environment stromal cells, and the specified air-conditioned environment stromal cells derived from the specified piston flow, biurea the Torah with the stationary phase, includes the specified three-dimensional culture of stromal cells from step (a).

20. The method according to claim 19, in which these undifferentiated hemopoietic stem cells or precursor cells are cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone marrow.

21. The method according to claim 19, in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells have a common HLA-antigens.

22. The method according to claim 19, in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in one individual.

23. The method according to claim 19, in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in different individuals.

24. The method according to claim 19, in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take the same view.

25. The method according to claim 19, in which these undifferentiated geopoetics is their stem cells or precursor cells and stromal cells of this culture of stromal cells take in different species.

26. The method according to claim 19, in which the stromal cells of these stromal cell cultures were grown to a density of at least 107cells per cubic centimeter of the specified substrate.

27. The method according to claim 19, wherein these fibers form a pore volume, taken as a percentage of the total volume, from 40 to 95%and the pore size is from 10 to 100 microns.

28. The method according to claim 19, wherein the specified matrix is made from fibers selected from the group consisting of planar, non-circular and hollow fibers and mixtures thereof, and the said fibers comprise from 0.5 to 50 microns in diameter or width.

29. The method according to claim 19, wherein the specified matrix consists of fibers in the form of a tape having a width of 2 to 20 μm, and the ratio of the width of the fibers to the thickness is at least 2:1.

30. The method according to claim 19, wherein the specified matrix has a pore volume, taken as a percentage of the total, from 60 to 95%.

31. The method according to claim 19, wherein the matrix has a height of 50-1000 μm.

32. The method according to claim 19, wherein the matrix material is selected from the group consisting of polyesters, polyalkylene, polyphthalamide, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers and fibers of inert metal.

33. The method according to claim 19, wherein the matrix is in a form selected from the group consisting of squares, disks, number of the TS and crosses.

34. The method according to claim 19, wherein the matrix is in the form of a disk.

35. The method according to claim 19, wherein the matrix cover poly-D-lysine.

36. Method of making air-conditioned environment stromal cells, suitable for reproduction/sustain undifferentiated hemopoietic stem cells or precursor cells, which implies the following stages:

(a) culturing the three-dimensional culture of stromal cells under conditions of flow in the piston flow bioreactor with a stationary phase comprising a piston bioreactor, and the specified piston bioreactor includes a container having an inlet and outlet and enclosing the substrate in a layer, and the specified substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, where the specified three-dimensional culture of stromal cells grown to a density of at least 5×106cells per cubic centimeter of the specified substrate; and

(b) collecting medium from the specified piston-flow bioreactor with the stationary phase, thereby obtaining the air-conditioned environment stromal cells, suitable for reproduction/sustain undifferentiated hemopoietic stem cells or cells-ol is Shesterikov.

37. The method according to p, in which stromal cells of this culture of stromal cells grown to a density of at least 107cells per cubic centimeter of the specified substrate.

38. The method according to p at which these fibers form a pore volume, taken as a percentage of the total volume, from 40 to 95%and the pore size is from 10 to 100 microns.

39. The method according to p at which the specified matrix is made from fibers selected from the group consisting of planar, non-circular and hollow fibers and mixtures thereof, and the said fibers comprise from 0.5 to 50 microns in diameter or width.

40. The method according to p at which the specified matrix consists of fibers in the form of a tape having a width of 2 to 20 μm, and the ratio of the width of the fibers to the thickness is at least 2:1.

41. The method according to p at which the specified matrix has a pore volume, taken as a percentage of the total, from 60 to 95%.

42. The method according to p, wherein the matrix has a height of 50-1000 μm.

43. The method according to p, in which the matrix material is selected from the group consisting of polyesters, polyalkylene, polyphthalamide, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers and fibers of inert metal.

44. The method according to p, in which the matrix is in a form selected from the group consisting of squares, disks, to the EC and crosses.

45. The method according to p, in which the matrix is in the form of a disk.

46. The method according to p, in which the matrix cover poly-D-lysine.

47. The method of transplantation of undifferentiated hemopoietic stem cells or progenitor cells in the recipient, which implies the following stages:

(a) reproduction/maintenance of the undifferentiated hemopoietic stem cells or progenitor cells by

(i) culturing the three-dimensional culture of stromal cells under conditions of flow in the piston flow bioreactor with a stationary phase comprising a piston bioreactor, and the specified piston bioreactor includes a container having an inlet and outlet and enclosing the substrate in a layer, and the specified substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, where the specified three-dimensional culture of stromal cells grown to a density of at least 5×106cells per cubic centimeter of the specified substrate; and

(ii) sowing undifferentiated hemopoietic stem cells or progenitor cells in the specified piston flow bioreactor with a stationary phase that includes the specified three-dimensional stromal culture the cells from step (i), at that time, when a thread in the specified bioreactor overlap to a predetermined time; and

(b) transplantation of these undifferentiated hemopoietic stem cells or progenitor cells obtained in stage (a), the recipient.

48. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells are cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone marrow.

49. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells have a common HLA-antigens.

50. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in one individual.

51. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in different individuals.

52. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells stromlinie cells of this culture of stromal cells take the same view.

53. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in different species.

54. The method according to p, in which stromal cells of this culture of stromal cells grown to a density of at least 107cells per cubic centimeter of the specified substrate.

55. The method according to p at which these fibers form a pore volume, taken as a percentage of the total volume, from 40 to 95%and the pore size is from 10 to 100 microns.

56. The method according to p at which the specified matrix is made from fibers selected from the group consisting of planar, non-circular and hollow fibers and mixtures thereof, and the said fibers comprise from 0.5 to 50 microns in diameter or width.

57. The method according to p at which the specified matrix consists of fibers in the form of a tape having a width of 2 to 20 μm, and the ratio of the width of the fibers to the thickness is at least 2:1.

58. The method according to p at which the specified matrix has a pore volume, taken as a percentage of the total, from 60 to 95%.

59. The method according to p, wherein the matrix has a height of 50-1000 μm.

60. The method according to p, in which the matrix material is selected from the group consisting of polyesters, polyalkylene, polyphthalamide, polyvinyl orida, polystyrene, polysulfones, cellulose acetate, glass fibers and fibers of inert metal.

61. The method according to p, in which the matrix is in a form selected from the group consisting of squares, disks, rings and crosses.

62. The method according to p, in which the matrix is in the form of a disk.

63. The method according to p, in which the matrix cover poly-D-lysine.

64. The method according to p, further comprising the extraction of these undifferentiated hemopoietic stem cells or progenitor cells prior to stage (b).

65. The method of transplantation of undifferentiated hemopoietic stem cells or progenitor cells in the recipient, which implies the following stages:

(a) reproduction/maintenance of the undifferentiated hemopoietic stem cells or progenitor cells by

(i) culturing the three-dimensional culture of stromal cells under conditions of flow in the piston flow bioreactor with a stationary phase comprising a piston bioreactor, and the specified piston bioreactor includes a container having an inlet and outlet and enclosing the substrate in a layer, and the specified substrate comprises a fibrous matrix of non-woven material forming a physiologically acceptable three-dimensional network of fibers, where the specified three-dimensional culture of stromal cells grown to a density of at least 5× 106cells per cubic centimeter of the specified substrate; and

(ii) culturing the undifferentiated hemopoietic stem cells or progenitor cells in a medium containing air-conditioned environment stromal cells, and the specified air-conditioned environment stromal cells derived from the specified piston-flow bioreactor with a stationary phase that includes the specified three-dimensional culture of stromal cells on stage (i);

(b) transplantation of these undifferentiated hemopoietic stem cells or progenitor cells obtained in stage (a), the recipient.

66. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells are cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone marrow.

67. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells have a common HLA-antigens.

68. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of the specified stromal culture the cells take in one individual.

69. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in different individuals.

70. The method according to p, wherein the undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take the same view.

71. The method according to p in which these undifferentiated hemopoietic stem cells or precursor cells and stromal cells of this culture of stromal cells take in different species.

72. The method according to p, in which stromal cells of this culture of stromal cells grown to a density of at least 107cells per cubic centimeter of the specified substrate.

73. The method according to p at which these fibers form a pore volume, taken as a percentage of the total volume, from 40 to 95%and the pore size is from 10 to 100 microns.

74. The method according to p at which the specified matrix is made from fibers selected from the group consisting of planar, non-circular and hollow fibers and mixtures thereof, and the said fibers comprise from 0.5 to 50 microns in diameter or width.

75. The method according to p at which the specified matrix consists of fibers in the form of a tape, have them in width from 2 to 20 μm, moreover, the ratio of the width of the fibers to the thickness is at least 2:1.

76. The method according to p at which the specified matrix has a pore volume, taken as a percentage of the total, from 60 to 95%.

77. The method according to p, wherein the matrix has a height of 50-1000 μm.

78. The method according to p, in which the matrix material is selected from the group consisting of polyesters, polyalkylene, polyphthalamide, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers and fibers of inert metal.

79. The method according to p, in which the matrix is in a form selected from the group consisting of squares, disks, rings and crosses.

80. The method according to p, in which the matrix is in the form of a disk.

81. The method according to p, in which the matrix cover poly-D-lysine.



 

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FIELD: medicine, genetic engineering.

SUBSTANCE: invention relates to applying genetic engineering approaches for treatment of autoimmune diseases, in particular, for treatment of cerebrospinal sclerosis. This is achieved by incorporation of one or some recombinant genes encoding autoantigens that represent a target for autoimmune response. In particular, invention claims a method for designation of gene encoding encephalitogenous epitope of proteolipid protein and expression of gene product in vivo by using the recombinant retroviral vector. Expression and secretion of encephalitogenous epitope improves histopathological and clinical indices in experimental autoimmune encephalomyelitis in mice that is used as a model of cerebrospinal sclerosis. The advantage of invention involves the development of a method for recovery the tolerance in treatment of cerebrospinal sclerosis being without suppression of immune system.

EFFECT: improved and valuable method for treatment.

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EFFECT: valuable biological and medicinal properties of cells.

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FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

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EFFECT: improved preparing methods, valuable medicinal properties of antibody.

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FIELD: organic chemistry, natural compounds, medicine, oncology.

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EFFECT: valuable medicinal properties of compositions.

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FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

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The invention relates to biotechnology and Cryobiology

The invention relates to medicine, namely to cell therapy, and for the culture of cells containing precursor cells osteogenesis of the implant based on it and use it to restore the integrity of the bone

FIELD: cellular biology, medicine.

SUBSTANCE: invention relates to isolating and cryopreserving precursor-cells. Methods involve treatment of human liver tissue for preparing the essentially monocellular suspension containing precursor-cells and cells that are not precursor-cells, a single or more lines of cellular differentiation presenting in the human liver. Invention describes methods involving stage for separating cellular population resulting to reducing amount of cells that are not precursor-cells and providing preparing the separated suspension enriched with precursor-cells expressing one or more markers and associated with a single or more lines of the cellular differentiation. Also, invention describes a method for selection cells from the separated suspension wherein these cells or their progeny, or their more matured forms express one or more markers associated with lines of the cellular differentiation. These markers involve: CD14, CD34, CD38, CD45 and ICAM. Hepatic precursor-cells have diameter size 6-16 mc, they are diploid and show indices: glycoforin A-, CD45-, AFP+++, ALB+, ICAM+ and they comprise subpopulations varying with respect to expression of CD14+, CD34++, CD38++ and CD117++. These cells are useful for carrying out cellular and genetic therapy in liver treatment and for preparing artificial organs also.

EFFECT: valuable biological and medicinal properties of cells.

41 cl, 7 tbl, 13 dwg, 15 ex

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