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Macroporous chitosan granules and method of production thereof, a method of culturing cells
Macroporous chitosan granules and method of production thereof, a method of culturing cells
IPC classes for russian patent Macroporous chitosan granules and method of production thereof, a method of culturing cells (RU 2234514):
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The invention relates to macroporous chitosan granules having a relatively large and uniform pore size of 30-150 μm inside and outside, which are distributed from the surface to the area of the nucleus, and the way they are received, which includes the following stages: adding dropwise chitosan solution, the aqueous chitosan solution, or a mixture thereof in the low-temperature organic solvent or liquid nitrogen; regulation of pore size using the method of phase separation due to temperature differences. Macroporous chitosan granules according to the invention make cultivation more efficient than previous substrates, as cells can efficiently be attached to them due to the large area of their surface, cells can log in to them and the cells attached to such substrates, there may be longer due to their three-dimensional structure, so macroporous chitosan granules can be used to study the production of the protein, antibiotics, anticancer agents, polysaccharides, physiologically active substances, hormones, animal or plant hormones, as well as exploring the substitution of metabolic organs, cartilage, or bone. 4 C. and 10 C.p. f-crystals, 8 tab., 3 Il.

BACKGROUND of INVENTION

Recently, active research has been directed to the cell cultures to obtain replacement metabolic tissues such as the liver and pancreas, as well as cartilage and bone. For the effective cultivation of cells necessary matrix for crops, to be able to attach cells to facilitate cell growth and help the cells to maintain their functions, except that they must have biocompatibility, ability to biodegradation, plasticity and must be porous. In particular, the matrix of cells must be porous to accommodate as many cells in a confined space. In this regard, the size and three-dimensional structure have on the present moment as a matrix for cell cultures used a lot of natural and synthetic polymers. For example, to create a three-dimensional porous bone substitutes used a grid of ISC (PGA; polyglycolic acid), which allows many cells to attach to it, except that it promotes rapid tissue regeneration and is excellent for biodegradation (Vunjak-Nonakovi G. et al., Journal of Biotechnology Progress, vol. 14, 193-202, 1998). Du, S. et al. synthesized PNAs (nano-Drug/collagen) (Journal of Biomedical Materials Research, vol. 44, 407-415, 1999). PLLA (poly-L-lactic acid) was successfully used for the cultivation of osteoblasts (Lo et al., Journal of Biomedical Materials Research, vol. 30, 475-484, 1996; Evans G. R. et al., Journal of Biomaterials, vol. 20, 1109-1115, 1999). From PGA and PLLA were made grid or a three-dimensional porous skeleton using the method of casting in a solvent or leaching of the particles, which are cultivated chondrocytes (Freed et al., Journal of Biomedical Materials Research, vol. 27, 11-23, 1993). An attempt was made to cultivate fibroblast cells on a porous matrix made of PEG (polyethylene glycol), paired with fibrinogen (Pandit A. S. et al., Journal of Biomaterials Application, vol. 12, 222-236). Another success in the cultivation of fibroblasts was achieved through the use of tubular ISC formed using a molding plating solution of PLLA or PLGA (poly-D,L-lactic-coglycolide acid) in Hloc necessary mainly porous matrix, to give the ability to easily and evenly attached to them as much as possible the number of cells in a confined space, as well as to facilitate cell growth. However, the above matrices will not sufficiently meet these requirements.

An assumption was made that rusinophobia matrix eliminates the disadvantages of the above matrices. The study of different biocompatible materials with the properties of the growth factor, effective and homeostasis in case of injury, led to the discovery that positively charged granules more effective in stopping bleeding (L. Wu et al., Journal of Surgical Research, vol. 85, 43-50, 1999). Alginate pellets used for the culture of chondrocytes and after 1-2 days of cultivation were added to IL-1(interleukin-lto facilitate the formation of extracellular matrix (Beekman C. et al., Osteoarthritis Cartilage, vol. 5, 330 to 340, 1998).

In addition, they developed other porous matrix for cell cultures of gelatin, collagen, hyaluronic acid, cellulose and glass. Porous gelatin granules will polimerizuet by adding HEMA (2-hydroxyethylmethacrylate) and EDM (etilenglikolevykh) and give them a porosity using repeated cycles of the s cells. The cells are then implanted into the tissue in order to study the replacement of tissue. These granules can vary in size, depending on the materials, but they are unsuitable for use in cell culture because of the small size of their pores, prisoners in the range from 0.7 to 2.6 μm. Matrix in the form of circular pellets have the advantage of placing a large number of cells in a confined space that provides good cell growth and efficient allocation of products. However, the matrices in the form of granules made from alginate or gelatin, there are difficulties in the formation of pores of the desired size and uniform distribution on them and in them. When they are made of collagen or glass granules suffer from the disadvantage that have poor biocompatibility. Therefore, these granules suitable as matrices for adsorption of cells from the point of view of the instability of the cells and the strength of adsorption.

With regard to the effective application in the study of replacement of tissue by implantation of cells needed polymers, which have the ability to attach cells and possess biocompatibility, ability to biodegradation, plasticity and porosity. Superior plasticity in the AI biodegradation. Therefore, synthetic polymers have the ability to have various side effects on the direct implantation of tissue. For these reasons, natural polymers, which are safe and have a wide variety of applications, are being actively investigated.

Chitin, the predecessor of chitosan, is detected at quantifiable levels in the shells of crustaceans, such as crabs and shrimp, and insects, and in the cell walls of the lower fungi, fungi and bacteria. It is a polymer composed of repeating N-acetyl-D-glucosamine units, which are connected to each other through (14)--glycosidic bond. Chitosan, alkaline polysaccharide, obtained by N-dezazetilirovanie chitin with high concentrations of alkali, as you know, is superior in the ability to attach cells, biocompatibility, Biodegradability and plasticity the above-mentioned synthetic polymers.

These advantages have been the reason for that was made many attempts to use chitosan as a matrix for cell culture. For example, structured by glutaraldehyde chitosan and modified fructose chitosan was used as m the new matrix can be obtained by mixing glutaraldehyde or fructose with pure chitosan to improve the attachment of cells and manufactured in the desired shape. However, culturing cells using these modified chitosan matrices can occur only in two-dimensional culture systems, as the cells adsorbed only on the surface of the matrix.

Chitosan films with desired pore sizes were obtained by using different methods of lyophilization and used in tissue engineering (Madihally, S. V. et al., Journal of Biomaterials, vol. 20, 1133-1142, 1999). These chitosan film was very effective to obtain pores of the desired size, but was still limited by the techniques of two-dimensional cultivation of cells.

In addition, it was reported chitosan granules, which were obtained by lyophilization (Tsu-Yang et al., Journal of Industrial Engineering Chemical Research, vol. 36, 3631-3638, 1997). These chitosan granules were modified by glutaraldehyde crosslinking with amino acid residues of chitosan granules, and measurement was made to demonstrate a high degree of adsorption of cadmium ions.

New chitosan granules were also described in the publication, owned by Wolfgang G. et al. from the USPTO, 1999. These authors used a non-magnetic succinic anhydride to obtain chitosan granules with carboxyl groups. They were subjected to interaction with chlorideae, reportedly, can be used for protein purification or for absorption of magnetic materials. Due to the small pore size of the porous chitosan granules are used for adsorption and/or purification of ions of magnetic materials. However, nowhere was detected using porous chitosan granules as matrices for cell culture.

BRIEF description of the INVENTION

In view of the superiority of chitosan on the ability to attach cells, biocompatibility, biodegradation and its plasticity was studied in order to obtain macroporous granules with uniformly distributed large pores, which can safely be cultivated cells. On the way to the invention of deep and intensive research conducted by the authors of the present invention, led to the discovery that the solution of chitosan undergoes phase separation into an organic solvent, which can be obtained macroporous chitosan granules with uniform pores on and inside them.

Therefore, the object of this invention is to overcome the problems encountered in previous decisions, and getting a macroporous chitosan granules that have the same pores inside and outside.

Another Shui surface area, to absorbitivity on her cell.

An additional object of this invention to provide a macroporous chitosan granules, which are the best in their ability to attach cells, biocompatibility and degradiruemosti and, thus, suitable for cell growth, angiogenesis, and diffusion of nutrients.

Another object of this invention is to provide a method of producing macroporous chitosan beads.

And another object of this invention is to provide a method of cultivation of animal and plant cells using macroporous chitosan beads.

BRIEF DESCRIPTION of DRAWINGS

Fig.1 is a SEM photograph which presents a surface of the porous chitosan granules of the present invention before the cells are cultured inside the granules and granules.

Fig.2 is a SEM photograph representing a cross-section of the porous chitosan granules of the present invention before the cells are cultured inside the granules and granules.

Fig.3 is a SEM photograph which presents porous chitosan granules of the present invention after the hepatocytes cultivated inside porous who disclosed or described these macroporous chitosan granules and receive them, it should be clarified that the terminology used here is only for the purpose of describing particular embodiments only and is not intended to limit their invention.

The term “solution of chitosan, which is used here, means an aqueous solution of acetic acid containing chitosan. The term “aqueous solution of chitosan, which is here used, means the solution of water-soluble chitosan in deionized water.

And also the term “chitosan granules or porous chitosan granules”, which is here used, means a porous chitosan particles 1-4 mm with relatively uniform pores, made from a solution of chitosan aqueous solution of chitosan and mixtures thereof.

At the same time, the term "matrix ("matrix" or “matrix for cell culture”, which is used here, means a solid or solid carrier to which cells are attached under cultivation in the environment, in order to multiply.

In one aspect this invention relates to porous chitosan pellets for cell culture, which are excellent for their biocompatibility, Biodegradability, the ability to attach cells and plasticity, with the same large on p is that it is possible to cultivate various kinds of animal and plant cells. Porous chitosan granules of this invention can be used as matrices for the cultivation of animal and plant cells of all kinds, and particularly suitable for culturing hepatocytes, fibroblasts, osteoblasts, epithelial cells and packaging cells for viruses.

As for the pores of the porous chitosan granules of the present invention, the size is preferably in the range 1-500 microns and more preferably in the range of 5-200 μm. The granules preferably range in size from 0.1 to 10 mm and more preferably from 1 to 4 mm,

In another aspect this invention relates to a method for producing a porous chitosan beads. Fabrication of chitosan granules begins with a solution of chitosan, water chitosan or mixtures thereof. As mentioned above, a solution of chitosan is obtained by dissolving chitosan in an aqueous solution of acetic acid, whereas an aqueous solution of chitosan is obtained by dissolving water-soluble chitosan in deionized water. Then the solution is added dropwise in an organic solvent at low temperature or liquid nitrogen to obtain granules. In conclusion, chitosan granules lyophilizer.

Although chitosan is soluble in acid, water is itsan with an average molecular weight, component 5000-1000000.

The preferred average molecular weight water-soluble chitosan lies in the interval 5000-1000000. For dissolving the chitosan solution of acetic acid preferably has a concentration of 0.1-10% by weight. After complete dissolution of chitosan is preferably present in an amount of 0.1-20% by weight in acetic acid solution. When dissolved in deionized water content of water-soluble chitosan preferably ranges in concentration from 0.5 to 1.5% by weight. Higher concentrations result in smaller pore size. Thus, when the concentration of chitosan above 4%, the formation of very small pores, limiting the introduction and growth of cells.

When chitosan is used in conjunction with water-soluble chitosan, chitosan preferably mixed at a weight ratio of 1:9-9:1 with water-soluble chitosan. The higher the proportion of water-soluble chitosan, the more pores by size.

Examples of the organic solvent usable in this invention include chlorocyclohexane, Harpenden, n-hexane, dichloromethane, chloroform and ethyl acetate. These organic solvents having a low melting temperature, although not dissolve chitosan, though the temperature of melting. As shown in the study of the changes of pore size from organic solvents, Harpenden forms pores larger than dichloropentane.

Preferably, the temperature of the organic solvent remained constantly low. If the temperature to be maintained constant, fluctuating, cured porous granules suddenly melt on their surface, so that lose their porosity to such an extent that the three-dimensional structure required for attachment of cells and using the cells to perform their functions, is destroyed. Organic solvents preferably support at a temperature of -5 - -65With, and liquid nitrogen at about -198C. for Example, lower temperatures lead to the formation of pores smaller. On the other hand, if the organic solvent is maintained at too high temperatures, phase separation due to differences in temperature does not occur. The most preferred conditions for the present invention include adding a 1% solution of chitosan in carpentary solvent, maintained at -5 - -25With, and adding 1% aqueous solution of chitosan in horor organic solvents can be used dry ice or ethanol, chilled in the freezer. Alternatively, you can use liquid nitrogen at about -198C.

Thus obtained porous chitosan granules are homogeneous size distribution in the range from 1 to 4 mm in Order to be suitable as matrices for cell culture, porous chitosan granules should allow them to different pre-processing, such as lyophilization, neutralization to remove residual acids and organic solvents, sterilization with ethanol, filling in the culture medium and then the lyophilization.

The following aspect of the invention is a method of culturing animal cells and plants using porous chitosan beads. First, after the prepared porous chitosan granules are immersed in the culture medium, conduct preliminary cultivation for attaching cells to porous chitosan granules. After removal of unbound cells attached cells proliferate, and at this time the old medium replaced with fresh medium. Pre-cultivation for the attachment of cells is preferably carried out within 4-6 hours. Preferably, staatlich conditions the concentration of chitosan and acetic acid and types of temperatures and organic solvents, and was used as a matrix for culturing different types of cells, including hepatocytes, fibroblasts, osteoblasts, endothelial cells, packing cells for viruses.

As a result, the formed pores of different sizes, corresponding to the conditions obtaining. More, the size of the formed pores of the porous chitosan granules were found, is small, when organic solvents support at low temperatures or a solution of chitosan or an aqueous solution of chitosan has a high concentration. That is, the pore size is determined by the temperature at which there is a separation of chitosan solution or an aqueous solution of chitosan, concentration of chitosan solution or an aqueous solution of chitosan. Moreover, in view of an organic solvent affects the predestination of the pore size of chitosan granules. When using chloropentane pore size according to the measurements was greatest. On the other hand, dichloropentane gave in and the smallest pores. In the case of mixtures of chitosan and water soluble chitosan largest pore size is obtained when used a mixture of chitosan and water soluble chitosan in the ratio of 4:6. Comparison of water-soluble chitosan with chitosan at one and Im chitosan and in the formation of larger pores.

Therefore, 1% aqueous chitosan and chloroform at -5 - -25Or 1% chitosan and Harpenden at -5 - -25With can lead to the formation of pores chitosan granules largest size.

In comparison with conventional matrices porous chitosan granules obtained by the method of the present invention, demonstrate the superiority of the adsorption of different types of cells of animals and plants. 2-3 days after they were adsorbiroval on porous chitosan granules, cells were grown inside and outside the granules, as well as on their surface. In addition, it was found that hepatocytes, cultured using matrix according to the invention retain their cellular functions, which is confirmed by various biochemical experiments.

EXAMPLES

A better understanding of this invention may be obtained in light of the following examples, which are provided for illustration, but should not be interpreted as limiting the present invention.

Example 1. Obtain porous granules using a 1% solution of chitosan and chloropentane

A 1% aqueous solution of acetic acid was dissolved chitosan (Fluka, USA) in an amount equal to 1% by weight, after which the solution �="http://img.russianpatents.com/chr/176.gif">And -45 - 65With using ethanol containing dry ice (Sigma, USA), using a 10 ml syringe. Granules, which were formed after 5-10 sec after the additions were separated using a spoon, and frozen at -70C for 1 day, followed by lyophilization in 2-3 days in apparatus for freeze drying (Cole-Parmer Instrument Company, USA). They studied the surface morphology under scanning electron microscope and determined the pore size. The results are presented in table 1 and shown in Fig. 1 and 2.

When porous chitosan granules were obtained using 1% chitosan with the same values for all parameters except temperature organic solvent that can be seen from table 1, were obtained pores smaller at lower temperatures.

Example 2. Obtain porous granules with 1% chitosan and various organic solvents

Chitosan granules were obtained by the method similar to that of example 1, except that used a 1% aqueous solution of acetic acid containing chitosan in the amount of 1% by mass, and various organic solvents, such as Harpenden, n-economu scanning electron microscope and determined the pore size. The results are presented in table 2.

Under the same conditions with the same parameters, except for organic solvents, the average pore size of chitosan granules according to the measurements was lowest when using chloroform and the highest when using chloropentane.

Example 3. Obtaining porous pellets with 2% chitosan and chloropentane

Chitosan granules were obtained by the method similar to that of example 1, except that used a 1% solution of acetic acid containing chitosan in 2% by weight, and Harpenden, maintained at -5 - -15C and -15 - -25C.

Chitosan granules was investigated using scanning electron microscope and determined the pore size. Changes of pore size depending on the concentration of chitosan are presented in table 3.

As can be seen from table 3, chitosan granules are smaller then, when the concentration of chitosan in the solution increases.

Example 4. Obtain porous granules using solutions of 2% chitosan in 1-4% aqueous acetic acid and chloropentane

Chitosan granules recip, and 4% aqueous solution of acetic acid and Harpenden, maintained at -15 - -25C.

The examination under the scanning electron microscope showed that the pore size of the porous chitosan granules enclosed in the range from 10 to 80 μm. Results of observations are presented together with the results from example 3 in table 4. As can be seen from table 4, a higher concentration solution of acetic acid resulted in larger pore sizes.

Example 5. Obtaining porous pellets with 2% chitosan and liquid nitrogen

Chitosan granules were obtained by the method similar to that of example 1, except that used a solution of 2% (wt.) chitosan in 1% aqueous solution of acetic acid and liquid nitrogen.

Research in the scanning electron microscope showed that the pore size of the porous chitosan granules enclosed in the range of 5 to 50 μm. This observation is consistent with the data obtained in example 1, which allows to conclude that lower temperatures correspond to the formation of pores smaller as the temperature of liquid nitrogen is lower than the temperature of the organic solvents.

Example 6. Obtain porous granules to use the measure 1, except that used 2% (wt.) a solution of chitosan in 1% aqueous solution of acetic acid and chlorocyclohexane, maintained at -5 - -15C -15 - -25C and-25-50C. Research in the scanning electron microscope showed that the pore size of the porous chitosan granules enclosed in the range from 10 to 150 μm. Results of observations are presented in table 5.

Under the same conditions for all parameters except temperature organic solvent, as can be seen from table 5, the average pore size of chitosan granules according to the measurements was similar to the pore size, obtained using chloropentane, and was less at lower temperatures.

Example 7. Obtain porous granules using mixtures of chitosan and water soluble chitosan in chloropentane

Chitosan granules were obtained by the method similar to that of example 1, except that used a solution of 1% (wt.) mixtures of chitosan and water soluble chitosan (Jakwang Co. Ltd., Korea) in the ratio 8:2, 6:4, 4:6 2:8, and Harpenden, maintained at 5-25C and-25-45C.

Research in the filigree is according to the proportions in the mixture, and the temperature of the organic solvent shown in table 6.

As can be seen from table 6, a higher content of water-soluble chitosan promotes the formation of pores of larger size, while the temperature chloropentane almost no effect on the pore size. In particular, when using a mixture of chitosan and water soluble chitosan 4:6 shows a larger average pore size of chitosan granules, which according to the measurements ranges from 30 to 120 μm.

Example 8. Obtain porous granules using water-soluble chitosan in chloropentane

Chitosan granules were obtained by the method similar to that of example 1, except that used a 1% (wt.) the solution of water-soluble chitosan in deionized water and Harpenden, maintained at -5 - -25C -25 to -45C and -45 -65C. Research in the scanning electron microscope showed that the pore size of the porous chitosan granules is from 10 to 70 μm. Was measuring pore size of the granules, and the results are presented in table 7.

As can be seen from table 7, chitosan granules obtained from a solution of water-soluble chitosan, have a pore size less than R the str does not undergo a large change depending on temperature, unlike chitosan granules obtained from a solution of chitosan.

Example 9. Obtain porous granules using water-soluble chitosan and various organic solvents

Chitosan granules were obtained by the method similar to that of example 1, except that used a 1% (wt.) the solution of water-soluble chitosan in deionized water and various organic solvents, such as Harpenden, n-hexane, dichloropentane, chloroform and ethyl acetate, maintained at -5 - -25°C. the Study using a scanning electron microscope showed that the pore size of the porous chitosan granules ranges from 20 to 200 μm. In the manufacture of water-soluble chitosan measured pore size of chitosan granules in accordance with the kinds of organic solvents. The results are presented in table 8.

In chitosan granules obtained from the solution of the granules of larger size are formed when the organic solvent is chloroform, than when using other organic solvents. On the other hand, chitosan pellets made from chitosan solution had the largest pore size in use the solution of chitosan or a solution of water-soluble chitosan, impact of organic solvent.

Experimental example 1. Culture of hepatocytes

Porous chitosan granules with a size of 1-4 mm long 50-150 μm obtained in example 1 was neutralized with 5 N sodium hydroxide solution in ethanol to remove any remaining acid and organic solvents, followed by sterilization with 70% ethanol. After entering into the culture medium (DMEM, pH 7.4, Gibco BRL, USA) chitosan granules liofilizirovanny. In the culture medium was loaded lyophilized chitosan granules, which were then attached hepatocytes of rats. To do this, attach conducted preliminary cultivation for 4-6 hours. To delete cells, the remaining unbound, the medium was replaced with fresh. Since that time, the medium was replaced every two or three days for 1-10 days, during which the hepatocytes attached to chitosan granules, were cultured at 37C. When the cells were glomeruli, scanning electron microscope observed cells that were growing in the pores of chitosan granules, as well as on the surface of chitosan granules, as shown in Fig.3.

Experimental example 2. Cell culture NIH3T3

Using NIH3T3 cells, which are fibroblastlike glue the least 1.

The study of scanning electron microscopy showed that the cells firmly attached to the chitosan granules, and grew up in the pores. In addition, fibroblast cells, as it turned out, grew quickly and firmly in contact with each other.

Experimental example 3. Cell culture MSSTS-E1

Using cells MSSTS-E1, which are the cells of osteoblasts (Korean Cell Line Bank in Seoul National University College of Medicine, Seoul, Korea) was subjected to the same procedure as in experimental example 1 with respect to cell culture.

In the scanning electron microscope was observed that these cells are strongly related to chitosan granules and grow well.

Experimental example 4. Cell culture Cho-K1

Using cells Cho-K1, which are epithelial cells (ATSC CCL-61, USA) was subjected to the same procedure as in experimental example 1 with respect to cell culture.

In the scanning electron microscope was observed that these cells are strongly related to chitosan granules, and grow in the pores.

Experimental example 5. Cell culture RT

Using cells RT that are packaging cells (Korean Cell Line Bank in Seoul National University College of Medicine, Seoul, Korea) was subjected to the same snowcrash watched these cells are not only strongly associated with chitosan granules secretion at the same time, the extracellular matrix in large quantities, but also grow rapidly.

INDUSTRIAL APPLICABILITY

As described above, the porous chitosan granules according to the invention have the same pores inside and outside, so that they can be suitable as matrix, which provides a three-dimensional structure that is used to help the cells in the exercise of their functions. In addition, unlike the well-known matrices for cell culture, porous chitosan granules according to the invention achieved excellence in the attachment of cells, biocompatibility and Biodegradability, and in relation to cell growth, angiogenesis, and diffusion of nutrients. With the advantages of porous chitosan granules according to the invention are suitable as matrix for the cultivation of animal and plant cells. In addition, the porous chitosan granules can be used effectively for research on substitution metabolic tissues such as liver and pancreas, or cartilage, or bone, as well as the production of biologically useful substances, including proteins, antibiotics, ProE the invention has been described in an illustrative manner, and it should be clear that the terminology used is intended to describe and not limitation. It may be many modifications and variations of the present invention in light of the above indications. Therefore, it should be clear that within the enclosed formula this invention can be implemented in practice and otherwise than specifically described.

Claims

1. Matrix for use in the cultivation of cells, and the specified matrix formed from a material selected from the group consisting of chitosan, water soluble chitosan, and mixtures thereof, the matrix has the form of porous granules having the same pore inside and outside, and the pore size is concluded within 30 to 150 μm, and the cells that you want to cultivate, are attached to the matrix by means of absorption, and then grow in the inner pores and on the surface.

2. Matrix under item 1, where the cells are animal cells selected from the group consisting of hepatocytes, fibroblastic cells, osteoblastic cells, epithelial cells and packaging cells, or plant cells selected from the group consisting of cells CEL, UV18 To-1.

3. The method of obtaining porous chitosan granules, including the Ana, where water-soluble chitosan dissolved in deionized water, or a mixture thereof; adding dropwise the solution of chitosan aqueous solution, or a mixture thereof in an organic solvent with a low temperature -5 ~ -65With obtaining granules; and lyophilization of chitosan granules.

4. The method according to p. 3, where the chitosan has an average molecular weight 30000-100000 and aqueous chitosan has an average molecular weight 100000-400000.

5. The method according to p. 3, where an aqueous solution of acetic acid has a concentration of 1.0 to 4.0 wt.%.

6. The method according to p. 3, where the solution of chitosan is the concentration of chitosan 0.5 to 2.0 wt.%.

7. The method according to p. 3, where an aqueous solution of chitosan is the concentration of chitosan in 0.5 - 1.54 wt.%.

8. The method according to p. 3, where the mixture has a mass ratio of chitosan solution to aqueous solution of chitosan in the range of 2:8 to 8:2.

9. The method according to p. 3, where the organic solvent is selected from the group of chlorocyclohexane, chloropentane, n-hexane, dichloromethane, chloroform and ethyl acetate.

10. The method according to p. 3, where the organic solvent is maintained at -5 ~ -25C.

11. The method according to p. 10, where the organic solvent is cooled with ethanol, supported -5 - -65With using dry ice or morezmore chitosan, where chitosan is dissolved in an aqueous solution of acetic acid, an aqueous solution of chitosan, where the water-soluble chitosan dissolved in deionized water, or a mixture thereof; adding dropwise the solution of chitosan aqueous solution, or a mixture thereof in an organic solvent with a low temperature -5 ~ -65With obtaining granules; and lyophilization of chitosan granules; neutralization of the porous granules to remove acids and organic solvents with subsequent sterilization of porous granules; conducting porous chitosan beads of pre-cultivation within 4-6 h for the attachment of cells to porous chitosan granules; and periodic refreshment culture medium of cells attached to the chitosan granules.

13. The method according to p. 12, where the cells are cultivated for replacement metabolic tissues, including the liver and pancreas, or cartilage, or bone, and for obtaining biologically applicable substances, including proteins, antibiotics, anti-cancer substances, polysaccharides, biologically active substances and hormones of animals and plants.

14. The method of cultivation of cells, which includes the pre-cultivation in the matrix under item 1 in teeeny pores, as well as on its surface.

 

 
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