Matrix for cell transplantology

FIELD: medicine.

SUBSTANCE: invention refers to cell transplantology and tissue engineering, and describes a matrix, a basic element of which is a flat plate made from a spatially cross-linked hydrophobic polymer containing hydrophilic groups and forming on the plate surface a layer of saturated hydrocarbons having a chain length of 8 to 16 carbon atoms and directed preferentially along the normal line to the plate surface. The spatially cross-linked polymer is formed on substrates by exposing a photopolimerisable composition containing oligourethane methacrylate, a methacrylate monomer having a chain length of 8 to 18 carbon atoms, 2,2-dimethoxy-2-phenylacetophenone and 2,4-ditertbutylorthquinone, to light at a wave length of 320-380 nm.

EFFECT: matrix has a good adhesive capacity; it is biocompatible and bioresorbable, preserves progenitor cells, promotes their differentiation and growth, and is mechanically strong.

5 cl, 1 tbl, 5 ex, 8 dwg

The invention relates to materials used in cell transplantation and tissue engineering to restore the structure or function of organs and tissues. In particular, the present invention can be an implant that can be used when replacing the internal tissues of any part of the soft organs, wound healing, tissue regeneration and recovery of the body in General, especially in the developing and adult nervous system or in other similar areas of therapy.

There are many different types of matrices that are trying to use in cell transplantation. Among them we can distinguish two groups: (1) using fragments of biological tissues and (2) synthetic materials.

To the group (1) include materials patent RU 2265446 IMPLANT FOR the TREATMENT of PARKINSON's DISEASE", EN 2216281 "METHOD PLASTICS DEFECT of the SPINAL cord VASCULAR GRAFT WITH BIOLOGICAL TISSUES", EN 2290939 "CURE FOR SPINAL cord AND BRAIN". The main drawback of all matrices using fragments of biological tissues is that they cause undesirable nonspecific defensive response of macrophages, also ALLO - and xenografts have immunological and infectious risk. A common disadvantage of all types of natural matrices - continuous Kazama (unmanaged) resorption, which slows down the formation of new tissue.

To the group (2) include materials patent RU 2375080 "METHOD of promoting regeneration of NERVE TISSUE BASED ON the use of BIOCOMPATIBLE SLURRIES OR SUSPENSIONS of SILICON NANOCLUSTERS", EN 2394593 "IMPLANTABLE NEUROENDOCRINE SYSTEM, ITS preparation AND METHOD of performing RECONSTRUCTIVE NEUROSURGICAL OPERATION", EN 2198686 "POLYMER HYDROGEL ACRYLAMIDE COPOLYMER THERAPEUTIC APPLICATIONS AND METHOD thereof". The main disadvantage of using suspensions of silicon nanoclusters is that they do not promote functional regeneration of the nervous conductive paths. The silicon nanoclusters are in the tissue of the Central nervous system by artificially generated electrical synapses. Electrical synapses are much less characteristic of the nervous system of adult mammals than chemical. The initiation of the electrical synapse can occur in both directions, in contrast to chemical synapses, and has a very low degree of controllability. United electrical synapses pre - and postsynaptic cells do not possess the property of plasticity, which significantly reduces the efficiency of the brain, due to the inability of the education and formation of dolgovremennoi memory. In addition, the resulting structure of the nanoclusters is not surrounded by the electrically insulating layer, as opposed to individual nerve fibers, which may cause nonspecific process of excitation transfer and simultaneous activation of neurons with opposite functions. The formation of silicon nanoclusters is a nonspecific process, so it is possible a cross join that does not match the physiological pathways of conduction of nerve impulse.

The disadvantage of using polymer hydrogels is their poor biocompatibility. Polymer hydrogels are not a nutrient medium for the filled cells and do not possess biocompatibility, these substances turn into scar element that prevents the growth of axons and engraftment of the transplant. The disadvantage of applying neuroendocrinal system is that the use of artificial prosthesis artery to form conduit restricts the flow of nutrients to the cells, placed in the implant.

The invention is used as a prototype (patent RU 2198686), refers to a polymeric hydrogel for therapeutic applications, acts as filling the space in the material and as a framework that stimulates tissue regeneration, morphogenesis and remodeling in the integrated structure of the body. Received n is biodegradable synthetic matrix of polymeric hydrogel with anisotropic porous structure, with the active area, good adhesion and compatibility with the tissue intended for implantation in the structure of the soft tissue, especially in the nervous system, which gradually becomes a part of the body.

The material is a copolymer of (a) N-substituted methacrylamide or acrylamide, (b) a crosslinking agent and (C) material with the ability to polymerization, selected from the group comprising sugar, sugar derivatives, peptide, connecting tissue, tissue proteins with various molecules such as bone morphogenetic proteins and conjugated polymer with antibodies against derivative of lipid, which has elastic strain capability and has an equilibrium water content of approximately 80%.

The hydrogel is a crosslinked by covalent bonds, non-transparent, heterogeneous material, which preferably has svetlopisno separated structure formed by the polymer particles size of about 1 to 10 μm, preferably from 3 to 5 μm, so as to ensure the formation of a region with a relatively large porosity (macropores), where the hydrogel tends to come in contact with the tissue of the host, and with a relatively small porosity (mesopores), where he seeks to come into contact with the ingrown tissue. Also cells or genetically modi is zirovnice cells can be introduced into the polymer grid. This occurs by the method of gel trap when the temperature is below zero, that is, using cryogenic polymerization, during which the porous matrix, which are immobilized cells and which cells can be reorganized, can grow and/or differentiate for subsequent transplantation. The polymer mixture can be mixed with live cells and combines the physical properties of the polymer matrix taking into account the behavior of the model hydrogel (porosity, stability, the guide surface, permeability) and taking into account biological factors of cells (e.g., growth factor). It is also possible to use three-dimensional culture system that can be used to culture a variety of cells in vitro for a long period of time.

The disadvantages of this polymer is that the method of its production leads to the formation of free radicals, which provokes the appearance of unwanted toxic properties. Also due to the lack of maintaining the form of material transport is impossible without violating formed intercellular contacts.

In addition, the process of the gel trapping of nerve cells, including kryzanowska, may lead to violation of the functional processes of regeneration. Culture of cells derived from embryonal the CSOs and the postnatal mammalian brain, not resistant to cryogenic. In the process of cryopreservation most neurons die, and after removal of the cryopreservation take their place actively dividing elements glial cells. Application closewith agents, such as DMSO (dimethyl sulfoxide), leads to the partial destruction of cell membranes, and because the metabolism of neurons differs from the metabolism of constantly dividing cells, restoring the membrane does not occur, which further increases the number of people killed in the process of cryopreservation of neurons.

The aim of the invention is the creation of a matrix for cell transplantation, which is a synthetic complex with properties of biologically compatible matrix for the creation of tissue-engineered constructs, namely: the absence of cytotoxicity, maintaining adhesion, fixing, proliferation and differentiation is placed on the surface of cells, no effect of inflammation, including immune, sufficient mechanical strength in accordance with the purpose and possible bioresorbability normal biological pathways, for example, or enzymatic hydrolysis.

The technical result is achieved by creating a matrix for cell transplantation, in which the main element is a flat perforated plate, made the from the spatial-linked hydrophobic polymer, containing hydrophilic groups and forming on the surface of the plate layer of saturated hydrocarbons with a chain length from 8 to 18 carbon atoms, oriented mainly along the normal to the surface of the plate. This spatial-cross-linked polymer is formed on the substrate with a hydrophobic surface by exposing light with a wavelength of 320-380 nm photopolymerizable compositions containing the ingredients:

Algorithmically the following structure:

where n is of the order of 35.

One of the monomers methacrylic range in chain length from 8 to 18 carbon atoms following structure:

CH₃-(CH₂)_k-O-C(O)-(CH₃)=CH₂where k is from 7 to 17.

2,2-Dimethoxy-2-phenylacetophenone the following structure:

2,4-Distritbution the following structure:

while the above components are taken in the following ratio, wt.%:

Algorithmically - 15-60,

The monomer methacrylic range in chain length from 8 to 18 carbon atoms - 39-84,

2,2-Dimethoxy-2-phenylacetophenone - 0,4-3,0,

2,4-Distritbution - 0,01-0,06.

A distinctive feature of this method of obtaining a matrix for cell transplantation is that the formation process occurs in one-step scheme, sluchaya any mechanical impact. Any mechanical effect on the polymer, as is known, provokes the formation of free radicals that lead to degradation of the polymer and unwanted toxic reactions. This material has a high resistance in biologically active environments, increased resistance to oxidative processes and the processes of adsorption of proteins on the surface, preventing the formation of coarse connective tissue capsule. This matrix is intended for cultivation of cells in tissues, including neuronal cells. First matrix is made, and then it is cultivated specified groups of cells. The process of growing cell cultures is that this matrix is planted dissociatively cells with the required amount of the nutrient medium. A distinctive feature of the present invention is that the surface of the matrix layer formed from saturated hydrocarbons with a chain length from 8 to 18 carbon atoms, oriented mainly along the normal to the surface of the plate. The hydrophobic ends facing toward the medium and absorb themselves lipids contained in the nutrient medium, so that the hydrophobic ends of the lipids are converted to hydrophobic ends of the matrix and the hydrophilic - out. The hydrophilic ends of the lipids, in turn, AB is orbitouch themselves proteins, contained in the nutrient medium. Thus, the playing surface similar to the surface of the cell membrane. Such a solution allows to provide high adhesion of cultured cells to the matrix, which provides a high density attachment of cells to the substrate. The perforated structure of the basic element of the matrix - plate - allows you to create links between groups of cells on both sides of the plate and facilitates their interaction. Also the matrix for cell transplantation may be in the form of a volume element consisting of a multilayer complex of perforated plates. Further transplantation data structures can be used to replace damaged tissue, plastics defect, as well as stimulating the proliferation and differentiation of tissue that may lead to reducing treatment of various defects of the nervous tissue after injury or surgery.

On the stated matrix were cultivated cells of the cerebral cortex of mouse embryos. In the course of work was cultivated dissociatively neuronal cells of the cerebral cortex (KBP) 18 - day-old mouse embryos in the following stages:

stage 1. The extraction took place culture KBP of the embryo.

stage 2. Culture crust is mechanically grinded with a scalpel, and the cloth on which was escalas in 0.025% trypsin solution.

stage 3. The fabric twice was filtered solution of PBS, culture medium NBM.

stage 4. The fabric was centrifugals and resuspendable in the required amount of the nutrient medium.

stage 5. The suspension was uncovered 50 ál to all types of polymers were stored in this form for 2 hours, then poured a medium NBM1 with the addition of 5% fetal calf serum.

stage 6. The activity of the culture was maintained under conditions of CO₂incubator at a temperature of 35.5°C and a gas mixture containing 5% CO₂.

7 stage. The change of environment was made on the first day of cultivation, in the future 1 every 3 days.

Dissociative cells was achieved by treating the fabric KBP 0.25% trypsin (Invitrogen 25200-056). Cells resuspendable in neuropathology Neurobasal medium^TM(Invitrogen 21103-049) in combination with bioactive Supplement B27 (Invitrogen 17504-044), glutamine (Invitrogen 25030-024), fetal calf serum (Paneco C). Maintaining the viability of the culture was carried out under conditions of CO₂incubator at a temperature of 35.5°C and the gas mixture with 5% CO₂incubator MCO-ES (Sanyo). The structure and dynamics of the cultures was assessed using an inverted microscope DM 1000 (Leica). The composition of neuroglial networks in cultures and the dynamics of spontaneous calcium oscillations, reflecting the state of calcium homeostasis tile is to, forming in vitro neuroglial network, were studied by means of confocal laser microscope (Zeiss LSM510 NLO Duoscan. As fluorescent probes used specific calcium dye Oregon Green 488 VARTA-1 and CA²⁺-insensitive dye Sulforhodamine 101, a selective marker for glial cells. For labeling cells in dissociated cultures were used specific antibodies to surface and intracellular proteins. A characteristic marker of glial cells were glial fibrillar acidic protein (GFAP), and one of the most heavily used markers of neuronal - marker cores of Mature neurons (NeuN). For visualization of the obtained results requires adding a secondary antibody with a fluorescent label. As secondary antibodies we used: antimurine antibodies labeled with a fluorescent dye Alexa-fluo 430 to visualize protein NeuN (Alexa-fluo 430 anti-mouse, Invitrogen A 11063), and antimurine antibodies labeled with a fluorescent dye CY-5 - for visualization of protein GFAP (Cy-5 anti cnk Millipor AP194S).

During the analysis of the dynamics of growth and development of cell culture using an inverted microscope revealed that at the end of the first day of cultivation viable cells attached in large quantities stated on the matrix. On the second day of cultivation, some cells of the Department who is from the polymer, the remaining progenitor cells form neurofibromatosis patterns with the exhaust from the center processes. In the process of dividing progenitor cells and development cords glial cells, along which are the neurons, is the formation of new neurosteroidogenic structures. For the ninth day of cultivation observed the development of the glial network around neurostar. There is the further formation on the surface of the substrate complex interweaving of growing nerve cells and glial cells, intensive processes of growth, differentiation and proliferation of dissociated cells of the nervous system, the connection neurosteroidogenic structures between numerous processes. If we talk about the duration of cultivation, from the time of the first planting has been more than 80 days and culture remain viable, which indicates the absence of toxic properties of this polymer on the substrate. Method of functional neuroimaging characterizing metabolic changes in cells, revealed spontaneous calcium oscillations manifested in changes in the intensity of fluorescence of Oregon Green 488 VARTA-1, which show an active functional state neuroglial networks of cultured cells. By immunohistochemical analysis using specific antibodies proved the existence of the WPPT is erozirovanne neuronal network, cultivated on this polymer. It is shown that neurovirology structure is a collection of neurons and glial cells, rising above the surface of the polymer, on average, 200 μm with numerous bands and developed glial network in the basis of it.

Growth and development of cell culture is illustrated by the following figures:

Figure 1. The first day of the cultivation of viable cells (1) are attached to the matrix in isolation or in small groups.

Figure 2. The perforated structure of the matrix for cell Transplantology.

Figure 3. The second day of cultivation - the remaining progenitor cells form neurofibromatosis structure (2) with the exhaust from the centre of the processes (3).

Figure 4. The formation of new neurosteroidogenic structures (a, B).

Figure 5. By the tenth day of cultivation observed the development of the glial network around neurosteroidogenic structures:

With - neurovirology structure

D - glial network.

6. Connection neurosteroidogenic structures between numerous processes of growing neurons and glial cells (the twenty-seventh day of cultivation). The figure shows that the matrix for cell Transplantology has a perforated structure.

7. - The twenty-fifth day of cultivation.

Fig - Fifty-ninth day of cultivation. N is the matrix for cell Transplantology is the formation of more dense and developed monolayer neuronal and glial networks.

It has been proven that the claimed matrix has good adhesive ability, preserves progenitor cells and promotes their differentiation, stimulates growth, which serves as the basis for the further introduction of this material into clinical practice.

The invention is illustrated by the following examples.

Example 1

The main element of the matrix for cell transplantation - plate - made in the following way. In a reaction flask equipped with stirrer, sequentially enter the components in the following ratio, wt.%:

Algorithmically - 49,55,

Octylacrylate (k=7) - 49,55,

2,2-Dimethoxy-2-phenylacetophenone - 0,89,

2,4-Distritbution of 0.01.

The resulting mixture was stirred at room temperature for 40 min until complete dissolution. After mixing, the composition is filtered and pumped using a vacuum pump at a pressure of 0.5-1 mm Hg to a complete cessation of gas evolution. On a hydrophobic substrate, installed on the edge of the strip forming the thickness of a plate, pour the composition. Then covered with a photomask comprising a transparent pattern on opaque to UV light background (hole perforations correspond to an opaque background, the rest is transparent), and impact. The resulting structure is irradiated with UV light with a wavelength of 320-380 n the side of the photomask during the time, optimal for the playback of the specified geometry. After exposure, the photomask and the substrate to be separated. The resulting polymer plate is washed in a suitable solvent from the residue of the uncured composition and dried to remove the solvent. Because of the use of photochemical technology according to EPR, free radicals are missing, a stable material in biologically active environments.

Example 2

The composition is prepared as in example 1, with the following ratio of components:

Algorithmically - 32,27,

Decylmethacrylate (k=9) - 66,0,

2,2-Dimethoxy-2-phenylacetophenone - 1,7,

2,4-Distritbution - 0,03.

Make a matrix for cell transplantation, as in example 1.

Characteristics are given in table 1.

Example 3

The composition is prepared as in example 1, with the following ratio of components:

Algorithmically - 58,0,

Octadecylammonium (k=17) - 40,5,

2,2-Dimethoxy-2-phenylacetophenone -1,48,

2,4-Distritbution - 0,02.

Make a matrix for cell transplantation, as in example 1.

Characteristics are given in table 1.

Example 4

The composition is prepared as in example 1, with the following ratio of components:

Algorithmically - 9,4,

Hexadecimalscalar (k=15) - 90,576,

2,2-Dimethoxy-2-phenylacetophenone - 0,02,

2,4-Ditertbutyl thenon - of 0.0004.

Make a matrix for cell transplantation, as in example 1.

Characteristics are given in table 1.

Example 5

The composition is prepared as in example 1, with the following ratio of components:

Dodecylmercaptan (k=11) - 99,522,

2,2-Dimethoxy-2-phenylacetophenone - 0,41,

2,4-Distritbution - 0,068.

Make a matrix for cell transplantation, as in example 1.

Characteristics are given in table 1.

From table 1 it follows that in examples 1, 2, 3, in which the ingredients are taken in an amount corresponding to the formula of the invention, the matrix for cell transplantation has a high capacity for mounting planted cells, intensive growth and proliferation cleto is, a large number of cellular structures, the formation of a dense monolayer cellular network. Deviations from the method corresponding to the formula of the invention (examples 4, 5), result in a matrix with low specified characteristics.

Recently increasingly used new methods based on the use of cell transplantation and tissue engineering to restore the structure or function of organs and tissues. Implementation of the claimed matrix for cell transplantation is of great interest to medicine.

1. Matrix for cell transplantation, characterized in that its main element is a flat plate made of a space-crosslinked hydrophobic polymer containing hydrophilic groups, and forming on the surface of the plate layer of saturated hydrocarbons with a chain length from 8 to 18 carbon atoms, oriented along the normal to the surface of the plate.

2. Matrix for cell transplantation according to claim 1, in which the spatial-cross-linked polymer is produced by exposing light with a wavelength of 320-380 nm photopolymerizable compositions containing the ingredients:
Algorithmically the following structure:

where n 35,
One of the monomers methacrylic range in chain length on the 8 to 18 carbon atoms following structure:
CH₃-(CH₂)_k-O-C(O)-(CH₃)=CH₂where k is from 7 to 17,
2,2-Dimethoxy-2-phenylacetophenone the following structure:

2,4-Distritbution the following structure:

while the above components are taken in the following ratio, wt.%:
Algorithmically - 15-60,
The monomer methacrylic range in chain length from 8 to 18 carbon atoms - 39-84,
2,2-Dimethoxy-2-phenylacetophenone - 0,4-3,0,
2,4-Distritbution - 0,01-0,06.

3. Matrix for cell transplantation according to claim 1, in which the spatial-cross-linked polymer is formed on the substrate with a hydrophobic surface.

4. Matrix for cell transplantation according to claim 1, in which the structure of the perforated plate.

5. Matrix for cell transplantation according to claim 1 in the form of a volume element consisting of a multilayer complex of perforated plates.