Method of producing three-dimensional matrices for tissue-like structures from animal cells

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to a method of producing three-dimensional matrices for tissue-like structures from animal cells. The method involves covalent bonding of histons with the surface of pre-activated biocompatible polymer microspheres made from crystalline dextran. The microspheres with covalently bonded histons are then deposited by centrifuging. Microspheres containing 160-200 mcg protein per 1.0 g are deposited on a substrate surface in amount of 0.5-1.0 mg per 1.0 cm2 and then dried at room temperature. Further, the substrate is washed with a solution at pH 7.5 to remove material which is not bonded to the substrate. The layer of microspheres obtained on the surface of the substrate on which cells are deposited is used as a base for obtaining tissue-like cell structures.

EFFECT: invention increases reliability of the structure and stability of the protein layer of the three-dimensional matrix, simplifies and reduces the cost of the method of producing three-dimensional matrices for tissue-like structures from animal cells.

6 cl, 11 dwg, 6 ex

 

The invention relates to medicine, in particular to Biomedicine, which includes principles and methods of cell biology and cellular engineering, i.e. the cultivation of cells on substrates modified with microstructures containing polymer microspheres and natural cationic proteins, namely the histones from the tissues of the thymus gland of calves, and such substrates can be used for cultivation-dependent attachment of cells to obtain three-dimensional tissue-like structures.

Create tissue-like cellular structures necessary to restore the integrity of tissues and organs by their implantation in the damage zone. When using different modifier substrates biologically active molecules create a matrix, which are complex cellular composition, specific tissue. Along with this, the spatial structure of the matrix is of great importance for the correct installation of deposited cells, entering the formed fabric. The coating of the substrate microspheres with immobilized on their surface histones may contribute to this process.

To explain the properties of proteins, including histones used in the invention, the applicant considers it appropriate to give them the characteristic in the Appendix to this description (Application).

In nastoyascheevremya developed a special technology for constructing the surface of the substrate with a certain microcephala (1). Such surfaces are used, for example, when the co-cultivation of cells for the purposes of tissue engineering (2).

To construct the culture surface with a certain microcephala use the technique of photolithography to create the desired surface topography containing adhesive and non-adhesive area for cells (3, 4). It is shown that on the surface of the substrate the rate of proliferation and survival of cells correlate with the availability, quantity and distribution of adhesive Islands, and not with the actual adhesive area. For application of this method requires special preparation of the substrate.

Along with the topographic surface modification is widely used in various polymeric materials and compositions for coating the surface of the substrate for the purpose of immobilization of proteins and peptides, which contribute to the highly specific interaction of the cells with the ligand on the media.

Known composition (5) of coating, including water-soluble polymer chemically bonded with adhesive to cells by the peptide, which is meant to cover the surface of the culture vessel or media for cultivation of animal cells in suspension in serum-free medium. However, it does not provide reliable structure.

Known composition (6), containing an adhesive for tile is to proteins, covalently associated with the surface of the substrate for cell culturing to improve cell attachment and stabilization of cell growth. However, such a composition costly.

Known composition (7) for coating substrates by growth factors to stimulate the growth of eukaryotic cells. However, this structure does not allow to obtain stable protein layer.

Known composition (8) for coatings containing poly-L-lysine salt brine to cover the surface of substrates such as plastic, glass or microporous fibers. This method, like the method (7), it is not possible to obtain a stable protein layer, which is a necessary condition for the creation of tissue-like structures.

There is a method of coating the culture surface is positively charged proteins (9), which is the closest to the technical essence of the present invention and is selected as a prototype. In the known method on the surface of the culture vessel put the total histone fabric of calf thymus (Sigma IIA). Such a culture substrate surface containing adsorbed proteins, cell clone in an environment with a minimal amount of serum.

The disadvantages of this method are the unreliability of the structure and instability of the protein layer that prevents efficiently and reliably in order to izdavati tissue-like structure. These drawbacks are due to the fact that the use of the substrate with the adsorbed protein layer does not provide steric matching adhesive proteins and adhesion receptors on the cell surface and there is a possibility of detaching from the substrate adsorbed caps and their transition into the culture medium and the internalization of histone cells attached to the substrate.

The present invention is devoid of the above disadvantages by implementing the topographic surface modification when applied to the biocompatible polymer microspheres of crystallized dextran with a diameter of not more than 1.0 μm with an immobilized on their surface adhesive for cells positively charged histones.

The technical result of the invention is to improve the reliability of the structure and stability of the protein layer of the three-dimensional matrix, simpler and cheaper way of obtaining on the surface of the substrate, which was then used as the basis for tissue-like cellular structures due to adsorption of biocompatible polymeric microspheres of crystallized dextran with a diameter of not more than 1.0 μm, immobilized on their surface histones on the surface of the substrate, providing a more effective attachment of cells to the microspheres and OC has the advantages due, mainly, to the fact that:

physical adsorption of the microspheres is less complicated process than chemical surface modification;

- obtain microspheres with surface modified cheaper and can have industrial scale compared to chemical attach proteins to the surface;

- requires a small amount of microspheres to cover a sufficiently large surface of the substrate in comparison with traditional spherical Mironosetsky, whose diameter is from 100 to 250 microns.

- prevents the transition of the foreign protein from the substrate to the culture medium, which eliminates the possibility of inhibiting the growth of cells.

the microspheres can be used for topographic modification of the substrate surface to create a three-dimensional matrices, which are coated with the cells are the basis for the formation of tissue-like cellular structures, and can also be used for co-cultivation of cells for the purposes of tissue engineering and as models for large-scale in vitro screening of drugs and biologically active substances.

The technical result is achieved by the known method of cloning cells, which includes known and in common with the claimed new way signs, use immobilization of histones from the tissues of the thymus gland of calves on the poverhnosti substrate, the adsorption of histones on the surface of the substrate, removing readsorbing protein, the ability of the attachment, rasplastyvanija, the morphological status and growth rate of the cells during cultivation on a modified histone substrate surface, in the proposed method, the surface of the substrate topographically modified microspheres coated with histones, and used the histone pre-covalently associated with the surface of the biocompatible polymeric microspheres of crystallized dextran with a diameter of not more than 1.0 μm, and before covalent binding of histones carry out the activation of the microspheres, which is carried out by adding to the aqueous suspension of microspheres cross-linking agent, which is used as brazian at a concentration of not more than 0.42 mol/l, at a temperature of not more than 4°C and the incubation time is not more than 2 minutes, then activated microspheres precipitated by centrifugation and the precipitate was washed with distilled water and again centrifuged, followed by re-suspending the beads in a solution of histone at a weight ratio of protein microspheres of 1:100, and the reaction of covalent binding is carried out at pH 7.5 to 8.0, a temperature of not more than 4°C, the incubation time is not more than 2 h, and then the microspheres with covalently bound histones precipitated zentrifugenbau the Institute of economy and management, then the microspheres containing from 160 to 200 μg of protein per 1.0 g, applied to the surface of the substrate in an amount from 0.5 to 1.0 mg 1.0 cm2and dried at room temperature, then washed with a buffer solution of pH 7.5 to remove not associated with the substrate material and the layer of microspheres on the surface of the substrate with the applied cells are used as the basis for tissue-like cellular structures. In addition, the required amount of immobilized protein determine amino acid analysis. Thus for the optimal number of immobilized histone take 180 µg of protein per 1.0 g of the microspheres. For immobilization choose a combination of different types of histones and their covalent conjugates. In particular, choose total histone, core histones and their covalent conjugates. The cells are cultivated in serum-free medium with growth supplements.

In the present invention for topographic modification of the surface of the culture vessels used biocompatible polymer microspheres prepared from crystallized dextran with a diameter of not more than 1.0 μm, and positively charged proteins selected from the group consisting of histone H1, H2A, as H2B, H3, H4, combinations of different types of histones, cross-stitched conjugates of different types of histones.

Sawlani the method was tested in the laboratory at the Saint Petersburg state University and the Institute of Cytology RAS, St. Petersburg. The results of these studies are explained in the following examples (a more detailed explanation of laboratory tests with the description of the attached figures is given in the Appendix to this description):

Example 1.

Getting histones and microspheres of crystallized dextran with a diameter of not more than 1.0 μm and a chemical linking of histones with microspheres is as follows. Preparations of total histone sulfuric acid from tissue of the thymus calves receive a known manner (10). Preparations of core histones containing in its composition histones H2A, as H2B, H3, H4, derived from a total of histone (11). Preparations of individual histone fractions were obtained when the chromatographic separation of the drug core histones (11).

Electrophoretic analysis of drugs of histones is carried out in polyacrylamide gel (PAG) in the presence of sodium dodecyl sulfate (LTOs) by the method of Thomas and Kornberg (12). Electrophoresis in polyacrylamide gel in the presence of LTOs at high pH value system makes it possible to fractionate proteins based on their molecular weight. In the presence of LTOs fraction of histones can be divided in the order of their increasing mobility: H1, H3, as H2B, H2A, and H4. Soluble covalent conjugates of total and core histones were obtained when using the method of cross-linking (13). To obtain soluble Cova is entih conjugates of total histone is as follows.

Total histone kongugiruut when using the method of cross-linking. The total sample of histone in the amount of 20 mg dissolved in phosphate-saline buffer solution of pH 7.5 to a final concentration of 2 mg/ml and cialiswhat against 0.2 M sodium tetraborate, acidified to a pH of 9.0 with concentrated hydrochloric acid. Dialysis performed for 18 h at 4°C. the conjugation Reaction carried out using as a crosslinking agent dimethylsuberimidate hydrochloride to a final concentration of 5 mg/ml and the reaction is carried out for 30 min at 23°C while stirring the solution on a magnetic stirrer. The reaction is stopped by adding equal volume of 1.0 M monoethanolamine, pH 8.0. The solution is incubated for 2 h at 23°C, subjected to dialysis against distilled water, as described above, frozen and dried by sublimation.

Cross-stitched conjugates of total histone analyzed by electrophoresis in LTO - 15% PAG, pH 8.8.

Electrophoretic analysis of cross-stitched conjugates of total histone shows that covalent conjugates of total histone contain dimers and oligomers of histones.

Traditional spherical microneedle obtained on the basis of polystyrene, Sephadex, polyacrylamide, collagen and gelatin, are used for the esmerine adhesive cells in suspension and as a component of a structural matrix for the attachment of the cells in the monolayer culture. Such spherical microsites, in particular, are cross-stitched dextranase granules coated with collagen (Cytodex 3; Pharmacia, Uppsala, Sweden) with a diameter of from 100 to 250 microns (14). The cell growth on the surface is the same as in the monolayer, which is modified only by the radius of curvature of the spherical surfaces of different diameters. Microneedle on the basis of cross-cross-linked dextran have large pores and is prone to compression, which complicates the collection of products that are secreted into the culture medium (15).

In addition, covalent cross-linking of chains of dextran significantly alters the structure and chemical stability of microsetella. Therefore, cross-stitched biodegradable microneedle in their decay to form products that are toxic to healthy cells. Spherical micronesica, surface area which is considerably larger than the proportion of cells that do not provide steric match for the adhesive interaction of cells proteins with cell adhesion receptors. Obtaining microspheres on the basis of crystal dextran carried out according to the method of Schroeder (16). This method is based on the crystallization of proteins in emulsion medium under the action of the precipitator - acetone. To obtain microspheres of 500 mg of dextran with molecular weight of 500,000 or 500 mg of dextran with mo is collaroy weight 10000 dissolved in 0.5 ml of water and add 5 ml of emulsion medium (cottonseed oil). The emulsion is treated with ultrasound at a power of 50 W for 30 sec. Then a homogeneous suspension in portions contribute in acetone (300 ml)containing 0.1% tween-80, with stirring on a magnetic stirrer. The obtained microspheres are washed with acetone (3×50 ml) and distilled water (4×50 ml) by centrifugation and dried at room temperature.

Samples of microspheres of crystallized dextran analyzed using electron microscopy when they are applied to the glass surface.

Standard samples of glass is treated with a mixture of ethanol-ether (in equal volumes) for 1 h and then maintained at 500°C for 1 h

The glass surface is covered with an aqueous suspension of microspheres and dried at room temperature.

The results of electron microscopy are presented in figure 1-3. It is shown that the microspheres have an average diameter of 1.0 μm to form a uniform layer on the surface of the glass. The layer of microspheres on the surface of the glass resistant to culture medium during long-term storage.

In a preferred embodiment of the present invention topographic modification of the surface of the culture vessels conducting polymer microspheres of crystallized dextran diameter of 1.0 μm, covalently associated with total histone.

Covalence linking the amount of the aqueous histone with microspheres is as follows.

The sample microspheres of crystallized dextran 100 mg washed with acetone and precipitated by centrifugation. After centrifugation microspheres suspension in 1.0 ml of distilled water and placed in an ice bath. Before immobilization of histone on the surface of the microspheres hold preliminary activation of the microspheres cross-linking agent, which selects brazian. Activation is carried out in the presence of triethylamine to bind released during activation Hydrobromic acid. To a suspension of microspheres added 50 mg of bromine cyan to a final concentration of 0.42 mol/l and 0.1 ml of triethylamine to a final concentration of 0.64 mol/l) under vigorous stirring and incubated for two minutes. The activated microspheres washed with acetone six times (6×12 ml) and twice with distilled water (2×12 ml) by centrifugation. After the last centrifugation to the activated microspheres add 1.0 ml of 0.1% solution of total histone in phosphate-buffered saline pH 7.5. The secondary suspension of microspheres in a weight ratio of protein microspheres of 1:100, incubated at 4°C for 2 h to bind histone proteins with activated microspheres. Microspheres with immobilized protein precipitated from the suspension by repeated centrifugation and washed distilleria the Oh water six times (6×12 ml), frozen and dried by sublimation.

Covalent binding is caused by the reaction between the ε-amino group of lysine residues of histones and active groups in the chains of polysaccharides.

Determining the amount of protein associated with the microspheres is carried out using amino acid analysis (17). Dextranase microspheres (50 mg), covalently associated with total histone, dried to constant weight at 50°C. as a standard drug use total histone (10 mg). Both samples hydrolyzing 6 N. chloride-hydrogen acid in a sealed tube for 24 h at 110°C. After drying the samples to constant weight spend amino acid analysis and calculate the amount of protein.

The amount of protein is evaluated on the basis of the data content in the hydrolysates resistant to hydrolysis amino acids such as aspartic acid, glycine, glutamic acid.

For example, in the analyzed samples according to amino acid analysis of the content of aspartic acid 200 mcg 10 mg sample of the total histone and 0.18 μg to 50 mg of sample dextranomer microspheres. Therefore, in the sample of microspheres by weight 50 mg contains 9 µg of total histone. Hence the number of covalently linked to total histone with the surface of the microspheres of crystallized dextran diameter not bol is e of 1.0 μm is 180 µg of protein per 1.0 g of the microspheres, or 5.73 picomole 1.0 cm 2the surface of the microspheres (design value).

The calculation of the number of total histone, covalently linked to 1.0 cm2the surface of the microspheres, carried out as follows. The density of the microspheres of crystallized dextran close to 1.0 g 1.0 cm3with a diameter of 1.0 μm (0,0001 cm), hence the number of microspheres in 1.0 cm3is 1012units. The area of the sphere is expressed by the formula: S.=4πR2hence the surface area of each of the microspheres is equal to 314·10-10cm2and the area of all microspheres in 1.0 cm3is 314·102cm2. The total amount of histone 1.0 cm3is 180 mcg (180000 ng), and in terms of 1.0 cm2this value is 573 mg (180000: 314·102=573 ng) or 5.73 picomole total histone with a molecular mass of 100 kDa.

The cultivation is performed on topographically modified microspheres covalently bound to histones, the surface of the culture vessels. Use 6-hole culture vessels company Corning Costar. Catalogue No. 3506. To do this, on the surface of the culture vessel with a diameter of 35 mm put 10-20-fold excess of microspheres, which ranges from 1.5 to 2.0 mg per 1.0 cm2aqueous suspension of microspheres containing microspheres of crystallized dextran with a diameter of about 1.0 μm, covalently associated with total GIS is one.

To calculate the volumetric quantity of microspheres required for formation of a layer thickness of 1.0 μm on the surface of the substrate area of 10 cm2is 10-3cm3. Given that the volume of a ball (ideal microspheres) is 52% of the volume of the cube described, and the density of the microspheres is close to 1.0 g 1.0 cm3then amount to a value of 10-3cm3will accommodate up to 520 mg of microspheres or 52 μg to 1.0 cm2.

Culture vessels kept in a laminar box over night for complete drying of the substrate and then washed with phosphate-saline buffer pH 7.5 to remove not associated with the substrate material and re-dried as described above. On the surface of the substrate is formed of a homogeneous layer of microspheres, which has increased resistance to the effect of culture medium.

Example 2.

In this embodiment of the present invention for topographic modification of the surface of the culture vessels used cross-stitched conjugates of total histone, covalently associated with a polymeric microspheres of crystallized dextran with a diameter of not more than 1.0 μm. Obtain microspheres of crystallized dextran with a diameter of not more than 1.0 μm is carried out, as in example 1. Obtaining cross-stitched conjugates of total histone carried out on the surface of the mi is roster and is as follows. The sample of the microspheres in an amount of 10 mg initially covalently associated with total histone add 2.5 mg of dimethylsuberimidate hydrochloride as a cross-linking agent. The reaction of covalent binding carried out for 30 min at 23°C under stirring suspension on a magnetic stirrer. The reaction of covalent binding initiate bringing the pH of the suspension to 9.1 by adding 0.2 ml of a saturated solution of sodium tetraborate. The reaction of covalent binding is stopped by the addition of equal volume of 1.0 M monoethanolamine, pH 8.0. The suspension is incubated for 2 h at 23°C and then subjected to dialysis against distilled water for 18 h, frozen and dried by sublimation.

Covalent binding is caused by the reaction between the ε-amino lysine residues in the molecules primarily associated with the microspheres of histones and again added histones to a suspension of microspheres.

The amount of protein covalently associated with the microspheres, determined as described in example 1. The amount of covalently cross-linked crosslinked conjugates of total histone with the surface of the microspheres of crystallized dextran with a diameter of not more than 1.0 μm is 200 micrograms of protein per 1.0 g of the microspheres.

Topographic modification of the surface of the culture vessels polymeric microspheres Crist is Litvinova dextran with a diameter of not more than 1.0 μm, covalently linked with a cross-linked conjugates of total histone, carried out as described in example 1.

Example 3.

In this series of experiments evaluated the ability of cells to rasplachivatsa on substrates coated with different types of histones. To do this, analyze changes in the structure of actin cytoskeleton and shape of cells spreading on substrates coated with histones different types. Use a permanent cell line kidney of a human embryo (SOME 293) and a permanent cell line of embryonic mouse fibroblasts (3T3/BALB clone A31), derived from the Russian cell culture collection (Institute of Cytology. Russian Academy of Sciences, St. Petersburg). Cells cultured in the medium Dulbecco (DMEM, Biolot) supplemented with 10%fetal bovine serum (Biolot).

Analyze the following types of histone proteins: H1, H2B, H3, H2A+N4, total histone, core histones, cross-stitched conjugates of total histone, cross-stitched conjugates of core histones.

Staining of actin cytoskeleton carried out as follows. On siliconized glass (Reppel-Silane, Pharmacia) put a solution of protein (histone proteins dissolved in distilled water to a concentration of 20 µg/ml) and incubated for 18 h at 4°C. and then incubated for 1 h at 37°C in a 2% solution of bovine serum is Lumina (BSA) in phosphate-buffered saline pH 7.5 (FSB), to exclude nonspecific binding of cells with the substrate. On the substrate is applied to the cells (100 μl, 105cells in 1.0 ml) and incubated for 1 h in CO2-incubator. Adherent cells fixed for 10 min in 4%formaldehyde solution in the FSB, incubated for 10 min at room temperature with 0.1%solution of Triton X-100 FSB and then stained with rhodamine-phalloidin for 10 min at 37°C. the Preparations in propylgallate analyzed by microscope Opton ICM (Zeiss).

Cells exhibit a different structure of the cytoskeleton after rasplastyvanija on substrates coated with different types of histones, which are depicted on figa-W; figa-d; figa-,

After 4 h incubation, the cells of the line SOME 293 attached to the substrate, but not rasplachivayutsya. Under a fluorescent microscope shows diffuse staining of actin. After 20 h of incubation, the degree rasplastyvanija cells depends on the type histone protein covering the substrate (figa-W). All the studied histone proteins can be divided into three groups according to their ability of cells to rasplachivatsa on substrates coated with histones different types.

The first group. This group represents the histone H1. It is shown that raspletanie cells on the substrate, coated with histone H1, is very weak, cells do not elongate, actin oligomers without any regular p is ctory. These results demonstrate that histone H1 is not a suitable substrate for cell culture SOME 293 and 3T3/BALB.

The second group. This group consists of total histone, core histones and histone H2A. It is shown that raspletanie cells better than in the first group. The polarized cells, actin is concentrated at the ends of elongated cells, however, the formation of structures containing actin, is not observed. Total histone, core histones and histone H2A that make up this group can be the best substrates for cell culture lines SOME 293 and 3T3/BALB. The lack of an organized structure of the cytoskeleton can suppress cell proliferation. From literature data it is known that the formation of stress fibers is an important factor for the stimulation of cell proliferation.

The third group. This group consists of a cross-stitched conjugates of total histone and histone H2B as well as. Cells of SOME 293 and 3T3/BALB well spread on these substrates, is highly polarized, have a wide lamella on the leading edge and a lot of microvilli and filopodia, oriented in different directions. Long processes of individual cells tend to communicate with other cells spreading on the substrate. Observed the formation of fibrillar actin structures. These results suggest that cross-stitched conjugate the total histone and histone H2B as well as, who make up this group are the best substrates for cell culture SOME 293 and 3T3/BALB. It is well known that histone differ on the total content of the hydrophobic and hydrophilic groups. The greatest number of hydrophilic groups is histone H2B as well as (18). It is quite likely that histone H2B as well as contained in the total histone, is responsible for the interaction of cells with total histone and stimulates proliferation.

It is known that cells already after 30 min of incubation on the substrate begin to produce their own extracellular matrix and to use these proteins to attach and rasplastyvanija. Therefore, in a separate series of experiments exploring attachment and raspletanie cells on substrates coated with histones in the presence of an inhibitor of protein synthesis cycloheximide. Cells of SOME 293 spreading on substrates coated with histones different types, in the presence of cycloheximide, presented at figa-D. As already described experiments, the cells spread on cross-crosslinked conjugates of total histone and histone H2B as well as demonstrate the greatest raspletanie and the best structure of the actin cytoskeleton. The cells on these substrates demonstrate cell phenotype and form lamellipodia. In the presence of cycloheximide cells use to attach and rasplastyvanija only what about the histone substrate, not own extracellular matrix. Thus, the results of the above experiments show that cells can use to attach, rasplastyvanija and growth substrates, coated with histones.

Example 4.

In this series of experiments evaluate the pinning and raspletanie adhesive cells on topographically modified microspheres covalently bound to histones, the surface of the culture vessels. Use 6-hole culture vessels company Corning Costar.

For this purpose, the surface of the 6-hole culture vessels topographically modify polymeric microspheres of crystallized dextran with a diameter of not more than 1.0 μm, covalently linked to the positively charged histones.

In these experiments investigate the following types of substrates.

The substrate 1. The surface of the culture vessels coated with microspheres covalently associated with total histone.

Substrate 2. The surface of the culture vessels coated with microspheres covalently linked with a cross-linked conjugates of total histone.

The substrate 3. The surface of the culture vessels coated with microspheres covalently associated with the core histones.

The substrate 4. The surface of the culture vessels coated with microspheres covalently associated with cross-stitched conjugate core histones.

As a control of use not covered by the microspheres surface of the culture vessels.

Use the following cell culture.

(a) a Continuous cell line kidney of a human embryo (SOME 293).

Cell line SOME 293 is normally maintained in an environment of DMEM (Wednesday NEEDLE in the modification Dulbecco), which contains 10% fetal bovine serum.

In each well of a 6-hole culture vessels containing DMEM and fetal bovine serum, cells contribute SOME 293. Cells cultivated in CO2-incubator at 37°C for 10-14 days. Cells removed using trypsin and assess the number of cells.

In each well of a 6-hole culture vessels contribute 5×105cells of SOME 293 and incubated for 4.5 h in serum-free medium and 24 h with the addition of 10% fetal bovine serum at 37°C in CO2-incubator. After incubation, the cells fixed with methanol and stained crystal violet.

Attaching and raspletanie cells analyzed under an inverted microscope (HC. 10×10). The number of cells SOME 293 attached to the substrate coated microspheres covalently associated with combinations of different types of histones, the same in all variants of the experiments and in control. Cells attached to the microspheres through 4.5 hours is Not detected significant differences in the extent of picral the deposits of cells to the investigated various substrates (substrates 1-4). Raspletanie cells on the substrate begins after 4 h of incubation. After 24 h incubation in the presence of serum, cells are well rasplachivayutsya.

Morphological analysis dyed spread cells using an inverted microscope shows that the nature of rasplastyvanija cells on the surface of the substrate coated microspheres of crystallized dextran with a diameter of not more than 1.0 μm, covalently associated with histones differs from rasplastyvanija cells on the control surface of the substrate and has its own characteristics.

On a homogeneous layer of microspheres, where the microspheres are located at an optimal distance from each other, the cells in the process of attaching to multiple microspheres are functional stretching with the formation of long processes that connect cells to each other, forming a three-dimensional seteobraznyh cellular structures (figa-g).

The distribution of the microspheres on the surface of the substrate is taken for optimal if the maximum distance between the microspheres does not exceed the size of the spread cells.

On the monolayer of microspheres cells in the process of attaching interact with a large number of microspheres and well rasplachivayutsya, but without the formation of long processes. Cells communicate among themselves and unite in dense layers, forming Terrasny cellular structure.

On the control surface of the substrate cells in the process rasplastyvanija also form large clusters and network, but a single spread cells there is much more.

(b) Continuous cell line of embryonic mouse fibroblasts (BALB/3T3 clone A31).

In this series of experiments uses the same substrates as in section (a).

There is the same number of adherent cells T BALB the substrate in all variants of the experiments. The greatest number of cells attached to the microspheres through 4.5 h (figa-in). Raspletanie T BALB cells on substrates begins after 4.5 h and is better than SOME of 293 cells. After 24 h incubation in the presence of serum blood cells are well rasplachivayutsya. Cells in the process of attaching to multiple microspheres located at an optimal distance from each other, form long processes that connect cells to each other, forming a three-dimensional seteobraznyh cellular structure. Raspletanie on the microspheres better than in the control.

Example 5.

In this series of experiments evaluate the morphological state of the cells after long-term cultivation on topographically modified microspheres covalently associated with total histone, the surface of the culture vessels.

In this series of experiments using 6-l of the night culture vessels, topographically modified microspheres of crystallized dextran with a diameter of not more than 1.0 μm, covalently associated with total histone. In experiments using cell line SOME kidney 293 human embryo.

The cultivation is carried out, as described in example 4. Cells cultured in medium with serum and serum-free environment.

To assess the condition of the cells of SOME 293 for 6 days of cultivation, the cells fixed with methanol and stained crystal violet. Analyze drugs obtained after 1 and 6 days of cultivation in medium with 10% fetal bovine serum (option 1) and in serum-free medium with growth supplements (option 2). After 1 day of culturing see good raspletanie cells on the tested substrates and the formation of contacts between neighboring cells (figa-d). Between spread cells see a lot of free microspheres without cells. After 6 days of cultivation, the number of spread cells is significantly increased in both versions. They grow in colonies form large clusters and merged into a dense layer of cells forming the seteobraznyh cellular structure. At this stage of cultivation there is very little free microspheres. The culture growth is accompanied by formation of monoclonal cells in the cluster is. Significant differences between cells cultured in medium with serum and in serum-free environment, do not watch. Control surface cells also form large clusters and network, but a single spread cells is much greater than in the advanced options.

Example 6.

In this series of experiments evaluate the growth rate of adhesive cells on topographically modified microspheres covalently bound to histones, the surface of the culture vessels.

For this 6-hole culture vessels coated with microspheres covalently associated with total histone.

As shown in example 4, on the surface of the substrate cells are well attached and rasplachivayutsya.

In this study, using two cell lines: continuous cell line kidney of a human embryo (SOME 293) and a constant line of embryonic mouse fibroblasts (3T3/BALB clone A31). In this series of experiments, the cultivation is conducted in a medium containing serum, and the medium does not contain serum.

The cultivation is usually carried out in a medium containing serum. Serum contains various components that allow the cells to adhesivity and grow on the surface of the substrate. However, it should be noted that serum contains in its composition is a mixture of numerous substances with physiological activity, which are released from the blood cells and endothelial cells of blood vessels. Therefore, the analysis and use of the products of cultured cells in a medium containing serum, require the use of certain methods of cleaning them. Therefore, the creation of a serum-free method of cultivation adhesive cells of humans and animals is required.

A. Continuous cell line kidney of a human embryo (SOME 293).

(a) Culturing cells in a medium with serum or in serum-free medium.

Cells plated on the surface of the culture vessels coated microspheres covalently associated with total histone, and cultured in medium with serum or in serum-free medium with growth supplements.

The cell suspension (3×105cells on the hole diameter 35 mm) plated on these substrates for 4 h in serum-free medium, DMEM (Wednesday NEEDLE in the modification Dulbecco). During this time a large portion of the cells attached to the substrate. Then replaced with serum-free medium or medium with 10% fetal bovine serum or serum-free medium with growth supplements (transferrin, 10 μg/ml insulin, 25 μg/ml, hydrocortisone 0.5 μg/ml) and retinoic acid 0.1 ág/ml).

Cells cultured in these media for 6 days at 37°C and 5% CO2. The index of cell proliferation determined after 1, 3 and 6 of the ut. Each option are repeated 3 times. Thus, the proliferation index determined in the following ways:

- cultivation of cells on the surface of the substrate in the medium with 10% fetal bovine serum (control);

- cultivation of cells on the surface of the substrate coated microspheres covalently associated with total histone in medium with 10% fetal bovine serum;

- cultivation of cells on the surface of the substrate coated microspheres covalently associated with total histone, in serum-free medium with growth supplements.

Morphological analysis of cells at different stages of cultivation in these conditions hold when using the microscope fixed and painted crystal purple drugs. The obtained results show that under cultivation for 6 days cells are well rasplachivayutsya and proliferate on the surface of the coated microspheres covalently linked to total histones and surface control. The most intensive growth was observed after 3-6 days of cultivation (figure 10). At all investigated time the number of cells on the tested substrates is lower than in the control substrate, but the proliferation index is almost the same. The observed fewer cells on the experimental substrates is explained by the stronger is the interaction with the substrate and therefore lack of efficiency of actions, usually used for the treatment of cell reagent (Trypsin/EDTA). These experiments show that the rate of growth of the same cells in medium with 10% fetal bovine serum and in serum-free medium with growth supplements.

(b) Culturing in medium with serum.

In these experiments investigate the following types of substrates:

- cultivation of cells on the surface of the substrate (control);

- cultivation of cells on the surface of the substrate coated microspheres covalently associated with total histone;

- cultivation of cells on the surface of the substrate coated microspheres covalently linked with a cross-linked conjugates of total histone.

Cell suspension (3,0×105cells on the hole diameter 35 mm) plated on these substrates for 4 h in serum-free medium, DMEM. Then serum-free medium is replaced with medium with 10% fetal bovine serum. Cells cultured in this medium for 11 days at 37°C and 5% CO2. The proliferation index determined after 2, 4, 7, 11 days after seeding cells on these surfaces. Each option are repeated 3 times. In vivo morphological analysis of cells at different stages of cultivation in these conditions is performed under an inverted microscope (HC. 10×10). The analysis of cells within 11 with the t demonstrate the cells rasplachivayutsya as microspheres covalently associated with total histone, and cross-linked conjugates of total histone. Cells proliferate with almost the same speed during the first 7 days of cultivation (11). From 7 to 11 days, the index of proliferation of control cells does not change, i.e. the growth curve reaches a plateau, there comes a stage of saturation. In contrast, cells on the microspheres covalently associated with total histone, just beginning to enter the stage of saturation. The most intensive growth of cells from 4 to 11 days observed when culturing them on the microspheres covalently linked with a cross-linked conjugates of total histone. A longer lag-phase during the first 4 days is associated with a relatively low seed concentration of cells and the need for conditioning of the substrate proteins of the extracellular matrix. These experiments demonstrate that microspheres covalently associated with total histone or cross-linked conjugates of total histone provide intensive growth of SOME cells 293 in culture.

B. Continuous line of embryonic mouse fibroblasts (3T3/BALB clone A31).

In these experiments investigate the proliferation of cells during their cultivation on substrates coated microspheres covalently associated with total histone or p is requestno crosslinked conjugates of total histone.

The cell suspension (3×105cells on the hole diameter 35 mm) plated on these substrates for 4 h in serum-free medium, DMEM. During this time a large portion of the cells attached to the substrate. Then serum-free medium replace medium with 10% fetal bovine serum. The number of cells evaluated at 2 and 3 days of cultivation. During this time, there has been very intensive proliferation, and after 3 days formed a complete monolayer of cells. The growth of cells on the microspheres covalently associated with total histone, intense (an index of cell proliferation in two days - 5)than on the microspheres covalently linked with a cross-linked conjugates of total histone (an index of cell proliferation after 2 days - 3). The final number of cells after 3 days was 7 times higher than originally sown.

This invention can be used to create substitutes tissue for implantation and during co-cultivation of cells for the purposes of tissue engineering, as well as models for large-scale in vitro screening of drugs and biologically active substances.

The list of used literature.

1. Nakanishi J, Takarada T, Yamaguchi K., Maeda M. 2008. Recent advances in cell micropatterning techniques for bioanalytical and biomedical sciences. Anal Sci. 24:67-72.

2. Khademhosseini A, Suh KY, Yang JM, Eng G, Yeh J, Levenberg S, Langer R. 2004. Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cell cocultures. Biomaterials. 25: 3583-92.

3. .Y.Suh, J.Seong, A.Khademhosseini, P.E.Laibinis and R.Langer. 2004. A simple soft lithographic route to fabrication of poly(ethylene glycol) microstroctures for protein and cell patterning. Biomaterials. 25: 557-563.

4. Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. 1997. Geometric control of cell life and death. Science. 276(5317): 1425-8.

5. Katsuen S., Ohshima K, Kawamura S, Yamamoto R., Nishino T. 1997. Coating composition for culturing animal cells and method for culturing of the cells in serum-free condition. U.S. Patent 5,643,561.

6. Clapper DL, Hu WS, 1996. Cell culture support containing a cell adhesion factor and a positively-charged molecule. U.S. Patent 5, 512,474.

7. Cima LG, Merrill EW, Kuhl PR. 2000. Cell growth substrates with tethered cell growth effector molecules. U.S. Patent 6,045, 818.

8. Swiderek MS, Mannuzza FJ, Ilsley SR, Myles A. 1999. Preparation of a cell culture substrate coated with poly-D-lysine. U.S. Patent 5,932,473.

9. McKeehan WL, Ham RG. 1976. Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers. J Cell Biol. 71(3):727-734 (prototype).

10. Gorukhina O.A., Miglinieks BTW, Crieve M.A. 1981. The method of obtaining the total histone from animal products. Patent for invention No. 843915. Of The Russian Federation.

11. Gorukhina O.A., mühlberg A.A., Crieve M.A., Tishkin I.E. 1989. The purification method of the preparation of histone H4 from the tissue of the thymus teletraan for invention №1319352. Of The Russian Federation.

12. Thomas JO, Kornberg RD. 1975. An octamer of histones in chromatin and free in solution. Proc Natl Acad Sci USA. 72(7):2626-2630.

13. Gorukhina O.A. 2001. The complex nature of cationic proteins and how to obtain it. Patent for invention No. 2176517. Of The Russian Federation.

14. Bardouille, Lehmann J, Heimann P, Jockusch H. 2001. Growth and differentiation of permanent and secondary mouse myogenic cell lines on microcarriers. Appi Environ Bioechnol. 55: 556-62.

15. Bulter, M. 1987. Growth limitations in microcarder cultures. Adv.Biochem.Eng.Biotech. 4:57-84.

16. Schröder U. 1985. Crystallized carbohydrate spheres for slow release and targeting. Methods Enzymol. 112:116-128.

17. Lundblad R.L. 1995. Techniques in protein modification. CRC Press.

18. Dobretsov G., Gorukhina O.A., borschevskaya T.A. 1969. The state of the polar groups and the aggregation of histones in aqueous solutions. Biochemistry. 34:806-809.

Application

To the description of patent application "a method of obtaining a tissue-like structures of the cells of animal origin for use when creating tissue substitutes" (applicant Federal state educational institution of higher professional education " Saint-Petersburg state University)

It is known that histones is cationic (basic) proteins contained in the cell nuclei of all tissues of animals and plants. In the cell core histones are largely responsible for the compaction of DNA. Histones easily form complexes with nonhistone proteins in the nucleus and cytoplasm, as well as among themselves.

In total there are five classes of histones, which differ in content in the molecule of the basic amino acids lysine and arginine (H1, H2A, as H2B, H3 and H4). Histones heterogeneous and molecular weight. The highest molecular weight has a histone H1 - 21500 Yes, the next largest histones H3, H2A, H2B as well as, N4 - 15320, 14000, 13774, 11280 Yeah, respectively (1).

the General characteristic feature of eukaryotic cells is the receipt of a protein to a specific compartment of the cell. However, there is the observation that some proteins are also found in other compartments of the cell and can go in the extracellular space in addition to their traditional places of localization, i.e. meet mnogokomponentnye isoforms of protein (2-3).

One example mnogokomponentnoi localization of proteins is the presence of nuclear proteins, such as histone and nonhistone chromosomal proteins with high electrophoretic mobility HMG (high mobility group) in the extracellular space (4-7). With the release of histones in the intercellular environment they can exhibit biological activity, causing various biochemical and functional changes in cells.

Exogenous histones are known as antimicrobial and antiviral agents (8, 9).

In addition, exogenous histones are known as bioactive agents, which due to their high positive charge can increase the permeability of biological membranes and penetrate through them (10-13).

Therefore, exogenous histones attract attention in terms of practical realization of useful properties of histones in biotechnology, in particular, in two ways:

from the point of view of realization of the useful properties of histones in Biomedicine [for example, in the patent RU No. 2045278 described potentiating effect of total histone TB action ison the azide in the treatment of experimental tuberculosis in animals (14); in patents US 6,884,423; US 4,818,763 and US 5,182,257 described using histone H1 and dimers of histones H2A:H2B as well as for therapeutic purposes(9, 15, 16)];

from the point of view of the use of natural polymers, containing in its structure a large number of basic groups in the technology of cultivation and cloning of cells, it is especially promising from the point of view of its wide practical implementation.

Tissue culture is a method or process of reproduction and/or maintenance metabolism of tissues or individual cells obtained from an organism in an artificial nutrient medium. For the proliferation of most types of normal cells requires their prior attachment to the surface of a solid substrate.

The nature of the substrate is determined mainly by the type of cells used and the nature of the research. Almost have received widespread currently, polystyrene, specially treated so as to increase the wettability and to give the surface a negative charge. In special cases (culture of neurons, muscle cells, some epithelial culture plastic surface pre-coated with gelatin, collagen or polylysine for giving it a positive charge. The cells of most cultures, including primary, attach to glass or plastic sub is a waste, breed prior to the formation of monolayer.

Adhesion of cells to the surface of the substrate is a multistage process. Interaction-dependent attachment of cells to the adhesive surface includes stages that precede proliferation: the contact of cells with the substrate, attaching the cells to the substrate, the formation of adhesive contacts and raspletanie cells.

When culturing cells the interaction between cells and substrate mediated by proteins, which were either premobilization on the material surface or adsorbed from the culture medium or secreted by the cells during cultivation.

Extracellular matrix proteins nonspecific adsorbed on the surface of the substrate, and the cells interact with the material surface, not directly, but through the interaction with adsorbed proteins of the extracellular matrix such as fibronectin, laminin and collagen (17).

Adaptation of cells to the substrate is manifested in the formation of a strong adhesive contacts (from 1.0 μm2and above), where the molecular interaction between adhesion receptors and proteins of the extracellular matrix (ligands) with the formation of focal contacts (18, 19).

Control of cell adhesion is determined by specific interactions between receptors on the cell surface is ti and their ligands.

The interaction between the cell and the extracellular matrix is mediated by surface receptors and proteoglycans, which interact with proteins of the extracellular matrix such as collagen, fibronectin, vitronectin and laminin. These proteins have specific binding domains of the receptors (20, 21).

For example, fibronectin has a domain that contains a specific sequence of three amino acid residues (Arg-Gly-Asp (RGD) RGD triplet, where R is agenin, G is glycine, D - aspartic acid, which are recognized by integrins (22-24).

Another class of domains in proteins adhesive for cells known as heparin-binding domain (the term got its name due to the fact that the chromatography based on affinity to heparin, is used for protein purification). These domains are associated with cell surface proteoglycans containing sulfated glycosaminoglycans, such as chondroitin sulfate and heparansulfate (20).

Peptide sequence in heparin-binding proteins, which binds to cell surface proteoglycans, enriched cationic residues of basic amino acids such as arginine, lysine, also contains hydrophobic amino acid residues: alanine, isoleucine, leucine, Proline and valine (25-27).

Proteins that contain heparin-binding to the ENES, also used to cover the surface of the substrate.

It is known that histones along with clusters of cationic amino acids (poly-domains) contain heparin-binding domains (27).

Cell adhesion is controlled not only by biochemical signals, as described above. For the attachment of cells to the substrate of the impact of nonspecific factors. These include such factors as the surface topography of the substrate (19).

Such substrates similar three-dimensional matrices, which are more widely known in the literature as scaffold (28, 29). Such matrices are used as substrates for tissue engineering to create complex compositions cells to form tissue-like structures. Cells attach, proliferate and differentiate into such a three-dimensional matrix.

One option for obtaining a biocompatible non-toxic for the cell carriers is the preparation of the microspheres on the basis of crystallized dextran (30). Due to the crystalline structure of such microspheres with a diameter of not more than 1.0 μm have very small pores and is not subject to compression. Figure 1-3 shows the microspheres of crystallized dextran according to electron microscopy, get that way Schroeder (30). For immobilization of histones on the surface of the microspheres hold the eakly covalent binding (examples 1, 2).

The obtained microspheres are used for topographic modification of the substrate (example 1). The calculation of the required number of microspheres to cover the surface of the substrate is illustrated in example 1. The distribution of the microspheres on the surface of the substrate is taken for optimal if the maximum distance between the microspheres does not exceed the size of the spread cells. The results of comparative studies about the ability of cells to attach and rasplachivatsa on substrates coated with different types of histone proteins, shown in example 3. Figs.4-6 presents the analysis of changes in the structure of actin cytoskeleton and shape of cells spreading on substrates coated with histones different types. Figure 4 shows the organization of the actin cytoskeleton permanent cell line kidney of a human embryo (SOME 293), spread on substrates coated with different types of histone proteins (×100), where a is the total histone; b - cross-stitched conjugates of total histone; core histones; g - histone H2B as well as; d - histone H1; e - histone H2A; W - histone H3. Figure 5 shows the organization of the actin cytoskeleton permanent cell line kidney of a human embryo (SOME 293), spread on substrates coated with different types of histone proteins in the presence of cycloheximide, where a is the total histone; b - cross-stitched conjugates of total histone; in - core-histones; g - histone H2B as well as; d - histone H1. The organization of the actin cytoskeleton permanent cell line of embryonic mouse fibroblasts (3T3/BALB clone A31), spread on substrates coated with different types of histone proteins depicted in Fig.6, where a is the total histone; b - cross-stitched conjugates of total histone; core histones; g - histone H1.

It is shown that the histone H2B as well as, total histone, core histones, cross-stitched conjugates of total histone and cross-stitched conjugates of core histones are the best substrates for cell culture.

Topographically modified microspheres covalently associated with total histone, the surface of the substrate is used for the analysis of the processes of attachment, rasplastyvanija and the growth rate of adhesive cells of animal origin. As the substrate used, the surface of a 6-hole culture vessels company Corning Costar. Cells of animal origin are cultivated on such a topographically modified homogeneous layer of microspheres covalently associated with total histone, surface environment syvorotkoj or in serum-free medium with growth supplements. On topographically modified homogeneous layer of microspheres covalently associated with total histone, the surface of the substrate causing cells to p is Amnesty from 3×10 4up to 5×1041.0 cm2.

The results of comparative studies on the characteristics of the substrates coated microspheres covalently associated with total histone, core histones and their cross-stitched covalent conjugates with respect to their effectiveness for attaching and rasplastyvanija cells in the presence of serum or in serum-free medium with growth supplements are given in examples 4 and 5. 7 shows the attachment and raspletanie permanent cell line kidney of a human embryo (SOME 293) on the substrate, the coated microspheres covalently associated with different combinations of histone proteins. Where:

(7a) the Substrate is coated microspheres covalently associated with total histone. The figure is seen a homogeneous monolayer of microspheres (small dark spots). The cells are well spread out, but without long processes. The cells are in contact with each other and United in a dense layer of cells.

(7b) the Substrate is coated microspheres covalently linked with a cross-linked conjugates of total histone. In the figure you can see a smooth layer of microspheres, located at an optimal distance from each other. Cells form long bones and joint in a well developed network.

(7b) the Substrate is coated microspheres covalently associated with the core histones. N the figure is seen a homogeneous monolayer of microspheres. The cells are well spread out, but without long processes. The cells are in contact with each other and United in a dense layer of cells.

(7G) Substrate coated microspheres covalently linked with a cross-linked conjugates of core histones. In the figure you can see a smooth layer of microspheres, located at an optimal distance from each other. Cells form long bones and joint in a well developed network.

On Fig shown attaching and raspletanie constant line of embryonic mouse fibroblasts (3T3/BALB clone A31) on the substrate, the coated microspheres covalently associated with different types of histone proteins. Where:

(8A) the Substrate is coated microspheres covalently associated with the core histones. The figure is seen a homogeneous monolayer of microspheres (small dark spots). The cells are well spread out, but without long processes. The cells are in contact with each other and United in a dense layer of cells.

(8b) the Substrate is coated microspheres covalently linked with a cross-linked conjugates of total histone. In the figure you can see a smooth layer of microspheres, located at an optimal distance from each other. Cells form long bones and joint in a well developed network.

(8b) the Substrate is coated microspheres covalently associated with total histone. On the figures is visible homogeneous monolayer of microspheres. The cells are well spread out, but without long processes. The cells are in contact with each other and United in a dense layer of cells.

Morphological analysis dyed spread cells using an inverted microscope shows that the nature of rasplastyvanija cells on the surface of the substrate coated microspheres of crystallized dextran with a diameter of not more than 1.0 μm, covalently associated with histones differs from rasplastyvanija cells on the control surface of the substrate and has its own characteristics.

On a homogeneous layer of microspheres, where the microspheres are located at an optimal distance from each other, the cells in the process of attaching to multiple microspheres are functional stretching with the formation of long processes that connect cells to each other, forming a three-dimensional seteobraznyh cellular structure.

On the monolayer of microspheres cells in the process of attaching interact with a large number of microspheres and well rasplachivayutsya, but without the formation of long processes. Cells communicate among themselves and unite in dense layers, forming seteobraznyh cellular structure.

On the control surface of the substrate cells in the process rasplastyvanija also form large clusters and network, but a single spread cells have been much the more.

In another series of experiments evaluate the morphological condition of the cells of SOME 293 after long-term cultivation on topographically modified microspheres covalently associated with total histone, the surface of the culture vessels. The results of these experiments are given in example 5. Figure 9 shows a state of permanent cell line kidney of a human embryo (SOME 293) during long-term cultivation on substrate-coated microspheres covalently associated with total histone. Where:

(9a) Cells after 1 day of cultivation in a medium containing 10% fetal serum of calves. The figure is seen a homogeneous monolayer of microspheres. The cells are well spread and are in contact with each other. Between the proportion of cells there is a lot of free of microspheres.

(9b) Cells after 1 day of cultivation in serum-free medium with growth supplements. The cells are well spread and are in contact with each other. Between the proportion of cells there is a lot of free of microspheres.

(9V) Cells after 6 days of cultivation in a medium containing 10% fetal serum of calves. The figure shows that the number of spread cells significantly increased. Cells grow in colonies with the formation of clusters and combined in a well developed network. Free microspheres not OBS is given.

(9g) Cells after 6 days of cultivation in serum-free medium with growth supplements. The figure shows that the number of spread cells significantly increased. Cells grow in colonies with the formation of clusters and combined in a well developed network. Free microspheres not observed.

(9D) Cells after 6 days of cultivation in a medium containing 10% fetal serum of calves. The figure shows that the number of spread cells significantly increased. Cells grow in colonies with the formation of clusters and combined in a well developed network. Free microspheres not observed.

In a separate series of experiments investigating the growth rate of adhesive cells on topographically modified microspheres covalently bound to histones, the surface of the culture vessels (example 6). Figure 10 shows the dependence of cell growth from the time of culturing cells on a substrate coated microspheres covalently associated with total histone. The cultivation of permanent cell line kidney of a human embryo (SOME 293) is carried out in a medium containing serum (medium 1), or in serum-free medium with growth supplements (medium 2). The index of proliferation pending against time of incubation. The proliferation index is the ratio of the number of proliferating cells in the test day of cultivation) cobsea the number of seeded cells.

Figure 11 shows the dependence of cell growth from the time of culturing cells on a substrate coated microspheres covalently linked or cross-linked conjugates of total histone (substrate 1), or with total histone (substrate 2). The cultivation of permanent cell line kidney of a human embryo (SOME 293) is carried out in a medium containing serum.

The results showed that under cultivation for 6 days, the cells proliferate at the same rate as tested on both substrates, and on the control surface. The growth rate of cells on the test substrates are the same as in the medium with serum and serum-free medium with growth supplements.

On topographically modified microspheres covalently bound to histones, the surface of the substrate is formed of a three-dimensional matrix, which is deposited cells is the basis for the formation of tissue-like cellular structures when cultivating cells of animal origin. On the surface of the substrate cells grow in monolayer culture, but when topographically steric accordance adhesive for cells, proteins and adhesion receptors, which come into direct electrostatic interaction. Cells attached to the microspheres with a diameter of not more than 1.0 μm, the horse which surface corresponds to the surface area of focal contacts. Focal contacts are formed between the adhesive to cells, histones, having in its composition a heparin-binding domains, and proteoglycans on the cell surface that act as receptors for histones.

When culturing cells on topographically modified microspheres the surface of the substrate nature rasplastyvanija cells has its own characteristics. Individual cells in the process of attaching interact with multiple microspheres located at an optimal distance from each other that provides functional stretching of the cells with the formation of long processes by which cells are combined with each other, forming a three-dimensional seteobraznyh cellular structure. Such tissue-like cellular structures are intended for use when creating substitutes tissue from cells of animal origin.

Attachment of cells to topographically modified the surface of the culture vessels, as described in the present invention, can be either a receptor-mediated, that is, to be due to factors attaching to cells, and/or dependent on a charge.

All this allows to consider the histones as a new group of adhesive to cells, proteins, along with the known adhesive for cells, proteins, such as fibronectin, Lamy is in and collagen.

The list of used literature

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2. Smalheiser NR. 1996. Proteins in unexpected locations. Mol Biol Cell. 7(7): 1003-1014.

3. Jeffery CJ. 2003. Moonlighting proteins: old proteins learning new tricks. Trends Genet. 19(8):415-417.

4. Zlatanova JS, Srebreva LN, Banchev TV, Tasheva W, Tsanev RG. 1990. Cytoplasmic pool of histone H1 in mammalian cells. J Cell Sci. 96 (Pt 3):461-468.

5. Brix K, Summa W, Lottspeich F, Herzog V.1998. Extracellularly occurring histone H1 mediates the binding of increase to the cell surface of mouse macrophages. J Clin Invest. 102(2):283-293.

6. Henriquez JP, Casar JC, Fuentealba L, Carey DJ, Brandan E. 2002. Increasing interest among matrix histone H1 binds to perlecan, is present in regenerating skeletal muscle and stimulates myoblast proliferation. J Cell Sci. 115:2041-2051.

7. Müller S, Scaffidi P, Degryse B, Bonaldi T, Ronfani L, Agresti A, Beltrame M, Bianchi ME. 2001. The double life of HMGB1 chromatin protein: architectural factor and increasing interest among signal. EMBO J. 20(16):4337-4340.

8. Ashmarin I.P., Zhdan-Pushkina, S.M., Kokryakov VI, Samedov INC., Antonov S. 1972. Antibacterial and antiviral functions of the main cell proteins and prospects of their practical usage. WPI. Acad. Of Sciences of the USSR. Ser. Biol. No. 4: 502-508.

9. Class R. Zeppezauer M. 2005. Antimicrobial histone H1 compositions, kits, and methods of use thereof. U.S. Patent 6,884,423.

10. Gorukhina O.A. 1999. The method of obtaining soluble covalent conjugates. Patent for invention No. 2127606. Of The Russian Federation.

11. Gorukhina O.A., Ilyuk RD, Mishchenko, I. 2000. A comparative study of receipt of exogenous histone in the parenchyma of rat brain. Bull. the experts. Biol. the honey. 130(7):63-66.

12. Mischenko V.A., Gorukhina O.A. 1996. Structure, permeability heme is encephalocele barrier and perspectives delivery through him medicines. Journe. neuropathol. and the psychiatrist. 96(4): 116-120.

13. Gorukhina O.A. 2004. Prospects of application of cationic proteins for transport of drugs into the brain tissue. Nervous system. Issue 37:168-175. Ed. S.-Petersburg University.

14. Ashmarin I.P., Perelman AU, Gorukhina O.A., B. I. Vishnevsky, Vavilin GI, Gaber IA 1995. Means potentiate the anti-TB activity of isoniazid. Patent for invention No. 2045278. Of The Russian Federation.

15. Rusch V, Reichhart R., Zeppezauer, M., Jornvall H. 1989. Biologically active substance with hormonal properties, production process thereof and utilization of histones for medical purposes. U.S. Patent 4,818,763.

16. Zeppezauer M., Reichhart R. 1993. Use of pure histones HI and H2A:H2B dimmers in therapeutic methods. U.S. Patent 5,182,257.

17. Kleinman HK, Luckenbill-Edds L, Cannon FW, Sephel GC. 1987. Use of increasing interest among matrix components for cell culture. Anal Biochem. 166(1):1-13.

18. Vitte J, Benoliel AM, Pierres A, Bongrand P. 2004. Is there a predictable relationship between surface physical-chemical properties and cell behaviour at the interface? Eur Cell Mater. 7:52-63; discussion 63.

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1. A method of obtaining three-dimensional matrices for tissue-like structures of the cells of animal origin, including immobilization of histones from the tissues of the thymus gland of calves on the surface of the substrate, the adsorption of histones on the surface of the substrate, removing readsorbing protein, the ability of the attachment, rasplastyvanija, the morphological status and growth rate of the cells during cultivation on a modified histones cart stand of the substrate, characterized in that the surface of the substrate topographically modified microspheres coated with histones, and used the histone pre-covalently associated with the surface of the biocompatible polymeric microspheres of crystallized dextran with a diameter of not more than 1.0 μm, and before Kowal is ntim binding of histones are activated microspheres, which is carried out by adding to the aqueous suspension of microspheres cross-linking agent, which is used as brazian at a concentration of not more than 0.42 mol/l, at a temperature not exceeding 4°C and the incubation time is not more than 2 minutes, then activated microspheres precipitated by centrifugation, and the precipitate was washed with distilled water and again centrifuged, followed by re-suspending the beads in a solution of histone at a weight ratio of protein microspheres equal to 1:100, and the reaction of covalent binding is carried out at pH 7.5 to 8.0, a temperature of not more than 4°C, the incubation time is not more than 2 hours then the microspheres with covalently bound histones precipitated by centrifugation, after which the microspheres containing from 160 to 200 μg of protein per 1.0 g, applied to the surface of the substrate in an amount from 0.5 to 1.0 mg 1.0 cm2and dried at room temperature, then washed with a buffer solution of pH 7.5 to remove unbound with the substrate material and the layer of microspheres on the surface of the substrate with the applied cells are used as the basis for tissue-like cellular structures.

2. The method according to claim 1, characterized in that the required number of covalently linked protein determine amino acid analysis.

3. The method according to claim 2, characterized in that for optimal if estvo covalently linked histone take 180 µg of protein per 1.0 g of the microspheres.

4. The method according to claim 1, characterized in that the covalent binding of select combinations of different types of histones and their covalent conjugates.

5. The method according to claim 4, characterized in that choose total histone, bovine histones and their covalent conjugates.

6. The method according to claim 1, characterized in that the cells are cultivated in serum-free medium with growth supplements.



 

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FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and cell biology. It has been established that sensitivity of mouse embryonic stem cells to clinostatting increases depending on cell development stages: from embryonic stem cell colonies to embryoid bodies. It has been shown that embryoid body clinostatting leads to slow down of the beginning of cardiomyocyte differentiation and significant reduction of the amount of cardiomyocytes cut. The invention can also be used in space medicine.

EFFECT: delay of neuronal differentiation on later differentiation stages.

2 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: there is offered a method for preparing a soft tissue filler composition for injection to relief or treat skin damages caused by mechanical or physiological reasons, including the stages as follows: 1) digestion of autologous dermal tissue recovered from autologous skin of a patient by processing with pancreatine/EDTA solution, and cell isolation; 2) cultivation and proliferation of the recovered dermal cells by serum-free cultivation in vitro in a medium containing a growth factor and activation factor for preparing autologous cellular culture material of dermal nature containing dermal fibroblastic stem cells, dermal fibroblastic "transitional" dividing cells, dermal fibroblasts and collagen; 3) centrifugation of autologous cellular culture material of dermal nature for separation of autologous cellular sediment of dermal nature; and 4) slurrying of autologous cellular culture material of dermal nature in glucose solution for injection or any solution for injection to prepare suspension for injection. There is offered a composition prepared by specified method which contains 1×107 to 8×107 cells/ml of autologous cells of dermal origins and 10 to 100 mg/ml of collagen as an effective ingredient.

EFFECT: invention provides therapeutic effect over a short period of time and maintains it for a long time.

13 cl, 7 ex, 8 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line ILG has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The ILG cell line is deposited in the Special collection of cell cultures of vertebrates of the Russian collection of cell cultures under number RKKK (P) 697D. All cells of the ILG line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line 31G has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The 31G cell line is deposited in the Special collection of cell cultures of vertebrates of the Russian collection of cell cultures under number RKKK (P) 698D. All cells of the 31G line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line IG has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The IG cell line is deposited in the Special collection of cell cultures of cage vertebrates of the Russian collection of cell cultures under number RKKK (P) 700D. All cells of the IG line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line 26G has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The 26G cell line is deposited in the Special collection of cell cultures of cage vertebrates of the Russian collection of cell cultures under number RKKK (P) 701D. All cells of the 26G line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line PG has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The PG cell line is deposited in the Special collection of cell cultures of cage vertebrates of the Russian collection of cell cultures under number RKKK (P) 702D. All cells of the PG line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to media for growing cultures of autologous lymphocytes and can be used for treating and preventing pyoinflammatory complications in patients. The medium for growing a culture of autologous lymphocytes based on the RPMI - 1640 (Roswell Park Memorial Institute) culture medium contains 10% phytohemagglutinin solution and blood plasma, obtained from any central or peripheral vein of a patient, for whom the culture of autologous lymphocytes is grown, with the following ratio of components in vol. %: blood plasma of the patient - 0.75-1.2; 10% phytohemagglutinin solution in the RPMI-1640 culture medium - 0.008-0.013; RPMI - 1640 culture medium - the rest up to 100%.

EFFECT: invention provides high reliability of preventing allergic reactions of the body of the patient, increases content of lymphocytes in the culture of autologous lymphocytes, prevents occurrence of foreign proteins and provides the required sterility due to prevention of bacteria germination.

3 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: method for combined immunobiological analysis of cells using a biochip involves incubation of the biochip which contains immobilised antibodies, with suspension of cells, washing the biochip from non-bonded cells, determination of coexpression of antigens on the bonded cells. The obtained result is assessed by determining presence of bonded cells in the region of the stain of the biochip and bonding density of cells and interpretation of the obtained result. Coexpression of antigens on cells bonded to the biochip is determined by carrying out one or more immunocytochemical reactions. When reading out the result, morphological analysis of cells bonded to the biochip is also carried out and presence and character of colouring of cells and their components with the reaction product are determined.

EFFECT: use of the disclosed method provides high reliability and information content of analysis.

9 cl, 6 dwg, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: method for combined immunobiological analysis of cells using a biochip involves incubation of the biochip which contains immobilised antibodies, with suspension of cells, washing the biochip from non-bonded cells, determination of coexpression of antigens on the bonded cells. The obtained result is assessed by determining presence of bonded cells in the region of the stain of the biochip and bonding density of cells and interpretation of the obtained result. Coexpression of antigens on cells bonded to the biochip is determined by carrying out one or more immunocytochemical reactions. When reading out the result, morphological analysis of cells bonded to the biochip is also carried out and presence and character of colouring of cells and their components with the reaction product are determined.

EFFECT: use of the disclosed method provides high reliability and information content of analysis.

9 cl, 6 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: method of obtaining a derivative of hydroxyalkyl starch, where the given derivative has a structure, corresponding to formula (I), involves reaction of the hydroxyalkyl starch with formula (I) through its oxidised-reduced end, or a derivative of hydroxyalkyl starch, obtained from reaction of hydroxyalkyl starch of formula (I) through its oxidised-reduced end with compound (D), where compound (D) consists of at least one functional group Z1, capable of reacting with the oxidised-reduced end of hydroxyalkyl starch, and at least one functional group W, with compound (L), consisting of at least one functional group Z1, capable of reacting with the indicated hydroxyalkyl starch, or at least one functional group Z2, capable of reacting with functional group W, contained in the given derivative of hydroxyalkyl starch, and at least one functional group X, capable of reacting with functional group Y in another compound (M), where the given functional group Y is chosen from a group, consisting of an aldehyde group, keto group, hemiacetal group, acetal group and a thio group, where formation of a bond between compound (L) and hydroxyalkyl starch or compound (D) and hydroxyalkyl starch is achieved through reaction of functional group Z1 with the oxidised-reduced end of hydroxyalkyl starch, and where functional group Z1 contains an -NH structure.

EFFECT: invention also relates to derivatives of hydroxyalkyl starch, as well as to pharmaceutical compositions, containing derivatives of hydroxyalkyl starch.

54 cl, 26 dwg, 3 tbl

FIELD: chemistry.

SUBSTANCE: invention concerns process of obtaining hydroxyl starch derivative which includes interaction of the reducing end of hydroxyalkyl starch represented by the formula (I), left unoxidised before this reaction, with a compound of formula (II) R'-NH-R", where R1 R2 and R3 independently denote hydrogen or linear or branched hydroxyalkyl group while R' or R" or R' and R" include at least one functional group X capable of interaction with at least one more compound before or after the reaction of (I) and (II). The invention also concerns hydroxyl starch derivative obtained in this process, and production process for hydroxyl starch derivative and polypeptide, a pharmaceutical composition incorporating the said hydroxyl starch derivatives.

EFFECT: improvement of compound production.

77 cl, 21 dwg, 3 tbl

FIELD: immunochemistry, sorbents.

SUBSTANCE: method for preparing immunosorbent involves modifying aerosil with chitosan solution in acetic acid in common with iron formate followed by oxidation and immobilization of the protein ligand. Invention can be used in diagnosis for detection of specific response antigen-antibody with using immunoenzyme analysis and immunofluorescence reaction.

EFFECT: improved preparing method.

1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to chemistry of polymers, biochemistry and medicine, and specifically to a method of preparing glucose-sensitive polymer hydrogels used as carriers for controlled secretion of insulin. The glucose-sensitive polymer hydrogels are obtained from reaction of a glucose derivative with concanavalin A. The reaction is carried out through copolymerisation in an aqueous solution under the effect of a redox initiator of 0.1-2.0 wt % N-(2-D-glucose)acrylamide, 1-5 wt % acrylamide and 0.01-0.075 wt % N,N-methylenebisacrylamide in the presence of 5-20 wt % concanavalin A.

EFFECT: obtaining polymer hydrogels which can be insulin carriers, with no other components of the hydrogel being released into the surrounding medium.

2 tbl

FIELD: medicine.

SUBSTANCE: offered is method of blood substitute production and related installation for method implementation. Method of blood substitute production includes production of deoxygenated haemoglobin, its polymerisation and purification. Production of deoxygenated haemoglobin includes haemolysis of water addition to erythrocytic mass, stroma separation, non-heme protein precipitation and removal from produced haemoglobin solution. Polymerisation includes processing of produced deoxygenated haemoglobin with modified glutaric aldehyde and restoration with sodium borane, with purification including ultra filtration. Deoxygenated haemoglobin is produced using leukocyte-free erythrocytic mixture. Non-heme proteins are precipitated by concentrated sodium chloride solution added to haemoglobin solution. Removal of non-heme proteins is followed with ultra filtration concentration of haemoglobin solution. Haemoglobin is produced in polymeric disposable containers, while deoxygenation and polymerisation are carried out in gas vortex reactor with nitrogen atmosphere within 1-6 hours each. Diafiltration purification is performed in polymeric disposable containers on shutoff dampers to produce end product molecular weight within 100 kDa to 450 kDa. Method allows for simplified production of polyhaemoglobin with lowered cost and higher outcome. Related installation for method implementation includes series haemoglobin production area, haemoglobin polymerisation reactor and end-product purification system. Haemoglobin production area contains series haemolysis tank with filtration manifold for stroma separation, non-heme protein precipitation tank with filtration manifold for removal of precipitated non-heme proteins. End product purification system contains ultra filtration tanks and units with shutoff dampers. All tanks within haemoglobin production area are polymeric disposable containers. Non-heme protein precipitation tank is connected to the tank for concentrated solution of sodium chloride. Polymerisation reactor is designed as gas vortex unit. End product purification system tanks are polymeric disposable containers. Haemoglobin production area, haemoglobin polymerisation reactor and end product purification system, as well as all tanks and units are interconnected by means of sterile rapid-action coupling.

EFFECT: allows for reduced material consumption of installation with higher productivity, sterile conditions of technological process.

6 cl, 1 dwg

FIELD: biochemistry, biophysic, medicine diagnosis.

SUBSTANCE: claimed method includes application of protein films on solid substrate and is based on using of cellulose mixed ester, namely cellulose acetopyvalinate as immobilizing layer for protein molecules which increases ordering ratio of protein molecules and enhances protein film filling density. Said films are useful as model of cell membrane in drug investigations.

EFFECT: method of increased efficiency.

4 ex, 5 dwg

FIELD: molecular biology, bioorganic chemistry.

SUBSTANCE: invention relates to development of a method for preparing gel biochips with backing from unmodified polymeric materials. Invention proposes using some unmodified polymeric materials used without their preliminary modification for preparing biochips backing that is designated for immobilization of hydrogels on its surface. Also, invention proposes a biochip prepared on backing made of unmodified polymeric materials, method for preparing biochip and method for immobilization of hydrogels on backings made of unmodified polymeric materials.

EFFECT: improved preparing method.

55 cl, 2 dwg, 2 ex

FIELD: medicine, pharmacology and immunology.

SUBSTANCE: claimed vaccine includes polyanion matrix and peptide and Norbornene pharmacophor chemically bonded thereto. As polymer matrix vaccine contains polyanion matrix and as peptide it contains peptide mimetic of chimerical domain representing peptide fragment of p17/18/55 HIV-1/2 structural protein with CD4 cell receptor. Said vaccine has anti-viral, immunomodulating action and provide address delivery of complex vaccine preparation to site of HIV-1/2 infection and suppresses specific viral infection.

EFFECT: vaccine for prophylaxis and treatment HIV infection and AIDS.

1 dwg, 2 tbl, 3 ex

FIELD: immunology, biotechnology.

SUBSTANCE: invention describes murine antibody and its humanized variant (CDP870) showing specificity to human tumor necrosis factor-alpha. Amino acid sequence is given in the description. Also, invention describes compounds showing affinity with respect to human tumor necrosis factor-alpha based on humanized antibody wherein lysylmaleimide group bound covalently with one or some methoxypoly(ethylene glycol) molecules by lysyl residue is joined to one of cysteine residues by C-end of heavy chain of the humanized antibody. Invention discloses DNA sequences encoding antibodies showing specificity to human tumor necrosis factor-alpha and variants if expression vectors involving indicated DNAs. Also, invention describes variants of a method for preparing a host-cell using expression vectors and variants of a method for preparing antibodies based on prepared host-cells. Invention discloses therapeutic compositions used in treatment of pathology mediated by tumor necrosis factor-alpha based on antibodies. Invention provides providing antibodies showing high affinity: 0.85 x 10-10 M for murine antibodies and 0.5 x 10-10 M for its humanized variant and low immunogenicity for human for humanized antibodies. Part of patients with improved ACR20 in administration of 5 and 20 mg/kg of CDP870 is 75% and 75% in 8 weeks, respectively. Half-life value of CDP870 in plasma is 14 days.

EFFECT: valuable biological and medicinal properties of antibodies.

58 cl, 24 dwg, 6 tbl, 1 ex

FIELD: medical engineering.

SUBSTANCE: device has substrate having polymeric working layer on it, produced from copolymer based on methacrylic acid derivatives with biological macromolecules (probes) immobilized thereon. The substrate is manufactured from activated or not activated glass, metal or polymer material. The working layer has macroporous monolithic copolymer glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass with affine biological probes immobilized thereon. Probe-copolymer proportion is 2-10 mg/g of copolymer, for protein, 1-20 mg/g of copolymer for peptide and for oligonucleotide, nucleic acid - 0.5-3 mg/g of copolymer, pore radius of 0.4-1.5 mcm, it has thickness of 50-700 microns and is manufactured as continuous or discrete microcellular layer. The method for manufacturing biochip involves preparing substrate, producing working layer by monomer copolymerization on methacrylic acid derivatives base, immobilizing biological macromolecules - probes on forming copolymer, washing, drying the received biochip. Radical copolymerization of glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass is carried out for producing working layer with photo-or thermal initiation in poregenic solvent medium being applied. Proportion of the sum of monomer volumes to solvent volume being equal to 6:9, initiator concentration in reactionary medium being equal to 0.2-1.0% by weight, given reaction mixture is placed on substrate as continuous or discrete layer. Macroporous monolithic continuous or discrete microcellular layer is formed as a result of copolymerization on the substrate. Then, covalent immobilization of biological macromolecules is carried out in the layer pores or their direct synthesis on formed copolymer with its native or modified epoxy groups being used. Biological affine probe is produced. The probe is introduced into copolymer in quantity of 2-10 mg/g of copolymer for fiber, for peptide - 1-20 mg/g of copolymer and for oligonucleotide or nucleic acid - 0.5-3 mg/g of copolymer.

EFFECT: manufacturing reusable biochip with predetermined controllable and reproduced quality.

17 cl

FIELD: biochemistry.

SUBSTANCE: method relates to new cyclopeptides of general formula cyclo(R1-Arg-Ile-Lys-Pro-His-R2) selected from group containing: P11: cyclo(DPhe-Pro-Gln-Ile-Met-Arg-Ile-Lys-Pro-His-Gln-Gly-Gln-His-Ile-Gly-Glu) (SEQ ID NO:5), P16: cyclo(Arg-Ile-Lys-Pro-His-Gln-Gly (SEQ ID NO:8), P17: cyclo(Pro-Arg-Ile-Lys-Pro-His-Gln-Gly) (SEQ ID NO:9), P19: cyclo(Gln-Ile-Met-Arg-Ile-Lys-Pro-His-Gln-Gly-Gln-His-Ile-Gly-Glu) (SEQ ID NO:10), P20: cyclo(Dphe-Pro-Gln-Ile-Met-Arg-Ile-Lys-Pro-His-Gln-Gly-Gln-His-Ile-Gly) (SEQ ID NO:11), P23: cyclo(DPhe-Pro-Arg-Ile-Lys-Pro-His-Gln) (SEQ ID NO:13), P24: cyclo(Gly-Arg-Ile-Lys-Pro-His) (SEQ ID NO:25), as well as P11, P20 and P23 with DPhe substituted by DTyr. Cyclopeptides are useful in systems for angiogenesis inhibition. System includes substrate with cyclopeptides attached by organic spacer arm optionally containing group cleavable by any fermentation system.

EFFECT: angiogenesis inhibiting cyclopeptides.

23 cl, 4 dwg, 2 tbl, 2 ex

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