Cryogenically structured macroporous biopolymer material for biomedical technologies and a method for its production

Abstract

FIELD: biotechnology; regenerative medicine.SUBSTANCE: invention relates to a technology for obtaining biomedical materials in the field of biotechnology and regenerative medicine. A cryogenically structured macroporous biopolymer material for biomedical technologies is proposed, which is a cryogenically structured chemically cross-linked decellularized mammalian tissue; macroporous carriers obtained from it can be used in tissue engineering and in the development of bioconstructs. The material in a dry state has a density from 0.16 to 0.28 g / cm3 and has communicating pores with a cross-section from 25 to 220 µm; and a method for obtaining it.EFFECT: possibility of using a wide range of tissues from different mammals as a starting material; a reduction in the time of the negative effect of pepsin on the proteins of the ECM; the possibility of varying the content of lyophilized decellularized tissue in an acetic acid solution of pepsin; a standardized sterilization regime for the matrix; the possibility of creating TCIs with the inclusion of human cells; and a long-term cultivation of human cells on the claimed carrier.2 cl, 14 dwg, 5 ex

Description

[0001] Field of technology to which the invention relates

[0002] The claimed interdisciplinary invention relates to the technology of producing biomedical materials and to medicine, specifically biotechnology and regenerative medicine, and can be used to restore damaged organs and tissues, both individually and as part of long-lasting specialized biomedical products in the form of tissue-engineered constructs (TECs). The claimed technical solution and the macroporous carriers obtained therefrom can be used in tissue engineering, as well as in the development of bioconstructs for the creation of artificial organs. Furthermore, the invention can find application in pharmacology for the creation of model systems for in vitro drug testing. The claimed material can be used in specialized departments engaged in reconstructive surgery and / or in situ stimulation of regenerative processes in damaged organs and tissues, as well as in specialized laboratories for TEC research.

[0003] Technology Level

[0004] Currently, one of the promising approaches offered by regenerative medicine for restoring the functions of damaged organs and tissues is the implantation of extracellular matrix (ECM), in which cells of one or more types are immobilized on a hydrogel or scaffold carrier [Sevastianov, V. I., Basok, Yu. B. Biomimetics of Extracellular Matrices for Cell and Tissue Engineered Medical Products. / / Cambridge Scholars Publishing: Newcastle upon Tyne, UK, 2023. - P. 339. ISBN: 1-5275-5080-X, ISBN13: 978-1-5275-5080-3]. The choice of material for the manufacture of such a carrier is of great importance, since the material must meet a number of criteria. In particular, the material must be biocompatible, i.e.maintain adhesion and proliferation of the desired cell types long enough to achieve the effect of TEC implantation, be bioresorbable, ultimately being replaced by the body's own tissue, and have the mechanical characteristics necessary for surgical manipulations, as well as the ability to be sterilized without changing the structure and biological properties [Sevastianov, V. I., Basok, Yu. B. Biomimetics of Extracellular Matrices for Cell and Tissue Engineered Medical Products. / / Cambridge Scholars Publishing: Newcastle upon Tyne, UK, 2023. - P. 339. ISBN: 1-5275-5080-X, ISBN13: 978-1-5275-5080-3; Eltom, A., Zhong, G., & Muhammad, A. (2019). Scaffold Techniques and Designs in Tissue Engineering Functions and Purposes: A Review. Advances in Materials Science and Engineering, 2019, 1-13. doi:10.1155 / 2019 / 3429527].To obtain such carriers, a wide range of both synthetic and natural polymers and their derivatives are used, such as alginates, collagen, gelatin, chitosan, hyaluronic acid, and polyesters of bacterial origin [R. Langer, N. A. Peppas. Advances in biomaterials, drug delivery and biotechnology. / / AICHE Journal, 2003, Vol. 49, P. 2990-3006; Biocompatible materials. Ed. by V. I. Sevastyanov, M. P. Kirpichnikov / / Moscow: MIA Publishing House, 2011. 544 p.]. In the case of carriers based on biopolymers, more positive results are usually achieved, since carriers made of synthetic polymers often have insufficient adhesive capacity for different cells or do not provide conditions for their long-term cultivation.

[0005] To improve cell adhesion, the carrier surface is often modified with substances that increase the number of cell binding sites [He X, Lu H, Kawazoe N, Tateishi T, Chen G. A novel cylinder-type poly (L-lactic acid)-collagen hybrid sponge for cartilage tissue engineering. / / Tissue Eng. Part C Methods. 2010 Jun; 16(3): 329-38]. However, this complicates and increases the cost of obtaining the corresponding carriers.

[0006] Since the main task of the carrier is to maintain the proliferation and functional activity of cells, the best results are achieved by creating a cellular microenvironment that is as similar as possible to their natural habitat. Thus, it is known that some of the promising materials for creating cell carriers in the development of TECs are injectable forms of viscoelastic multicomponent hydrogels - biomimetics of the extracellular matrix (ECM), which to one degree or another reproduce its composition. Examples of such biomimetic matrices are hydrogels based on acetic acid extracts of animal tissues or hydrogels based on decellularized animal tissues [VI Sevastianov, YB Basok, LA Kirsanova. A comparison of the capacity of mesenchymal stromal cells for cartilage regeneration depending on collagen-based injectable biomimetic scaffold type. Life (Basel), 2021, V. 11, no. 8, article 756; Patent No. 2433828 C1 Russian Federation, IPC A61K 35 / 12, A61K 38 / 39.Injectable heterogeneous biopolymer hydrogel for substitution and regenerative surgery and the method for producing it: No. 2010141934 / 15: declared 14.10.2010: published 20.11.2011 / / V.I. Sevastyanov, N.V. Perova; applicant Closed Joint-Stock Company "BIOMIR service"; Saldin LT, Cramer M.S., Velankar SS, White LJ, Badylak SF Extracellular matrix hydrogels from decellularized tissues: Structure and function. / / Acta Biomater. 2017; 49: 1-15]. However, the disadvantage of injectable forms of hydrogels is the lack of a macroporous structure and low mechanical strength, which complicates the uniform distribution of cells in the composition of the TEC and requires an additional reinforcing structure to maintain the 3D structure, i.e. complicates the process of forming the corresponding carrier and, as a result, significantly increases its cost.

[0007] To ensure long-term cell culturing, it is preferable to use macroporous supports, which ensure better availability of nutrients and oxygen to the cells [MS Shoichet, Polymer scaffolds for biomaterials applications. / / Macromolecules, 2010, Vol. 43, P. 581-591. CM Murphy, MG Haugh, FJ O'Brien. The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering. / / Biomaterials, 2010, Vol. 31, no. 3, P. 461-466]. However, it is important to ensure that the mechanical properties of the support are sufficient for use in bone tissue engineering; therefore, special techniques are required to combine, on the one hand, the branched macroporous morphology of the formed material, and, on the other hand, to impart the necessary strength characteristics to it.

[0008] In particular, an approach is known to form a macroporous structure for such heterophase carriers using cryogenic structuring techniques, where a three-dimensional polymer network of the polymer phase (i.e., the walls of the macropores) is formed at subzero temperatures, and crystals of the frozen solvent act as a porogen [V.I. Lozinsky, E.S. Vainerman, S.V. Rogozhin. Method for Obtaining Macroporous Polymer Materials. / / USSR A.S. No. 1008214 (1982), B.I. No. 12 (1983); V.I. Lozinsky. Cryogels Based on Natural and Synthetic Polymers: Production, Properties, and Applications. / / Uspekhi Chem., 2002, Vol. 71, Pp. 559-585]. Such cryogenically structured materials have found application as cell carriers in the creation of TEC [A. Kumar (Ed.) "Supermacroporous Cryogels: Biomedical and Biotechnological Applications." / / CRC Press: Boca Raton, FL, USA; Taylor & Francis Group: Abingdon, UK, 2016; 480 p.; H. Omidian, S. D. Chowdhury, N. Babanejad.Cryogels: advancing biomaterials for transformative biomedical applications. / / Pharmaceutics, 2023, V. 15, article 1836.; R.P. Rathna, M. Kulandhaivel Advancements in wound healing: integrating biomolecules, drug delivery carriers, and targeted therapeutics for enhanced tissue repair. / / Archives of Microbiology, 2024, V. 206, article 199].

[0009] For example, a cryogenically structured carrier for three-dimensional cultivation of fibroblasts and myoblasts is known [L. Elowsson, H. Kirsebom, V. Carmignae, M. Durbeej, D. Mattiasson, Porous protein-based scaffolds prepared through freezing as potential scaffolds for tissue engineering. / / J. Mater. Sci., Mater. Med., 2012, Vol. 23, P. 2489-2498]. This macroporous material is obtained by freezing (-12°C for 16 h) a mixture of 5 ml of a 4% aqueous solution of gelatin and 0.01 ml of a 50% aqueous solution of glutaraldehyde. After thawing at room temperature, the resulting cryogel is washed with water to remove soluble impurities, treated with a 0.05 M solution of sodium borohydride in 0.1 M carbonate buffer (pH 9.2) to restore the aldimine groups in the cross-linking sites, and then the samples are washed with water to a neutral pH value.When using the resulting cryogel as a carrier for cultured cells, its disks (1 mm thick) are sterilized in isopropanol, washed, and then inoculated with a cell suspension. A 1% agarose solution is applied on top, forming a gel layer that holds the cells in the carrier. Analysis of cell viability and proliferative activity shows that the results obtained from culturing myocytes are significantly better than those obtained from culturing fibroblasts in the same carriers. The disadvantages of this technical solution include the experimental difficulties in obtaining such gelatin cryogels with reproducible properties, which is associated with the gelation of a mixture of gelatin and a cross-linking agent (glutaraldehyde) even at low positive temperatures. This is a well-known fact: gelation of gelatin solutions with a protein concentration of more than 3-5% by weight occurs upon cooling, and this is further accelerated in the presence of a chemical cross-linker.In this case, if a cross-linked gelatin gel forms before the sample freezes, the crystals of the freezing solvent will destroy the already formed primary network. Therefore, this technical solution is only suitable for forming small-volume gelatin cryogels (0.5 ml), which, under certain conditions, freeze quickly, i.e., before the primary cryogel network has time to form. Testing this known method for producing larger samples revealed the aforementioned shortcomings: even with a frozen sample volume exceeding 1.5 ml, the reproducibility of the method drops sharply, and most of the resulting gels have a non-uniform morphology and are mechanically fragile.

[0010] The closest to the claimed invention in terms of the type of biopolymer precursor used in obtaining a cryogenically structured carrier for TEC is a macroporous spongy material based on decellularized renal tissue ECM, which we take as a prototype [JY Kim, T. Sen, JY Lee, D.-W. Cho. Degradation-controlled tissue extracellular sponge for rapid hemostasis and wound repair after kidney injury. / / Biomaterials, 2024, Vol. 307, article 122524]. The carrier is obtained from decellularized pig kidney tissue by forming a chemically cross-linked cryogel followed by its lyophilization.

[0011] The method for obtaining material according to the prototype includes the following operations:

[0012] a) Preparation of decellularized material. Frozen porcine kidney tissue is cut into 0.4-0.6 mm thick fragments and washed in hot water for 2 hours with stirring at 120 rpm. The washed tissues are treated with 0.5% Triton-X100 solution in 1 M phosphate-buffered saline for 16 hours to remove intracellular components and then incubated at 37°C in a DNase solution (50 units / ml) to remove residual DNA, after which the samples are sterilized with a mixture of 0.1% peracetic acid and 4% ethanol for 1 hour. The resulting sterile decellularized porcine kidney ECM is lyophilized and stored at -20°C until use.

[0013] b) Preparation of cryogenically structured carrier. A solution of 3 wt.% lyophilized powder obtained from decellularized porcine kidney ECM is prepared in 0.5 M acetic acid. Pepsin is added to the solution at a rate of 1 mg of enzyme per 10 mg of powder and incubated for 3 days. Then, a 0.625 M buffer solution of 4-morpholine ethanesulfonic acid monohydrate (MES) is prepared, in which 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are dissolved in EDC / NHS concentration ratios of 12 mM / 3 mM, 24 mM / 6 mM, and 48 mM / 12 mM. Next, the decellularized porcine kidney ECM solution and the EDC / NHS solution are mixed in a 6:1 ratio (by volume), transferred to a 24 mm × 24 mm × 5 mm mold, and frozen at -20°C, -60°C, or -80°C for 24 hours. The samples are thawed and transferred to distilled water to remove excess EDC / NHS and chemical byproducts. The washed samples are then re-frozen at -80°C and lyophilized.The dried porous sponge is sterilized with ultraviolet light.

[0014] The resulting material is used as a hemostatic sponge and also as a carrier for fibroblasts isolated from rat kidney (NRK-49F, ATCC CRL-1570, USA) in TEC. The culture period was 7 days. The viability of linear rat fibroblasts on the seventh day of culture was confirmed using intravital microscopy with Live / Dead™ fluorescent dyes.

[0015] The disadvantages of the prototype include the following points:

[0016] 1) The prototype material lacks the ability to cultivate human cells, which sharply reduces the demand for this well-known carrier, limiting its applicability only to working with the cells of rodents such as rats.

[0017] 2) According to the prototype, it is possible to use only one source of raw material - rat kidney, i.e. the multi-stage prototype method is not sufficiently universal in relation to the original animal tissues for obtaining cryogenically structured biomedical materials.

[0018] 3) The prototype method includes the need for long-term (3 days) harsh enzymatic treatment of biological material at the stage of preparing the decellularized tissue solution, which is undesirable due to damage to the protein structure.

[0019] 4) The prototype, when forming the target material, provides for the use of only a single concentration option (3 wt.%) of the solution of lyophilized decellularized tissue, which limits the possibility of varying the physicochemical properties of carriers for cell cultivation.

[0020] 5) The prototype uses ultraviolet radiation as a sterilization method, which is insufficient for clinical use and is not very effective in the case of materials with a porous sponge structure.

[0021] 6) The prototype method involves removing crystallized solvent (ice) only by lyophilization from frozen samples containing, along with other components, acetic acid, which will lead to corrosion of the materials of expensive sublimation equipment.

[0022] The objective of the present invention is to create an effective cryogenically structured biopolymer material from decellularized mammalian tissues for use as a biocompatible macroporous cell carrier, devoid of the disadvantages of the prototype, and to develop an accessible and inexpensive method for obtaining said material, which can be used in the composition of TEC.

[0023] Disclosure of the essence of the invention

[0024] The problem is solved by the claimed macroporous biopolymer material for biomedical technologies, which is a cryogenically structured chemically cross-linked decellularized mammalian tissue, and the material itself in a dry state has a density of 0.16 to 0.28 g / cm 3and has communicating pores with a cross-section of 25 to 220 µm. Furthermore, this problem is solved by a method for producing such a biopolymer material, which includes preparing a dispersion of decellularized mammalian tissue with a dry matter content of 1 to 5 wt. % in a 0.5 M acetic acid solution with the addition of pepsin at a concentration of 1-10 mg / ml, incubation of the dispersion for 24 hours at room temperature with constant stirring, subsequent freezing of the said dispersion at -15…-35°C, removal of the crystallized solvent either by lyophilization or cryoextraction with cold ethanol, treatment of the resulting macroporous cryostructure with a 0.04-0.12 M ethanol solution of carbodiimide at 5-25°C for 24-36 hours, removal of excess carbodiimide by extraction with ethanol and transfer of the target biopolymer material to 70-96% ethanol before use as a cell carrier.

[0025] The claimed macroporous biopolymer material can be prepared in any geometric shape: in the form of blocks, plates, disks, granules, irregularly shaped particles (obtained by crushing a block), tubes, etc.

[0026] The specific parameters of the claimed material and the method of its production are determined by the following factors:

[0027] 1. Decellularized mammalian tissue.

[0028] It is known [Crapo, PM, Gilbert, TW, Badylak, SF (2011). An overview of tissue and whole organ decellularization processes. / / Biomaterials, 32(12), 3233-3243] that decellularization is a tissue treatment procedure that ensures the destruction of cells with maximum preservation of the ECM, whose proteins, unlike cells, carry an insignificant amount of antigens that cause a reaction of transplant rejection. As a rule, decellularization involves the treatment of the original biological material (tissue of the liver, kidney, blood vessels, brain, pancreas, etc.) with solutions of surfactants, in particular, ionic sodium dodecyl sulfate and non-ionic Triton X-100, in combination with other physical, chemical or biological effects [Crapo, PM, Gilbert, TW, Badylak, SF (2011). An overview of tissue and whole organ decellularization processes. / / Biomaterials, 32(12), 3233-3243; Keane, T. J., Swinehart, I. T., Badylak, S. F. (2015).Methods of tissue decellularization used for the preparation of biologic scaffolds and in vivo relevance. / / Methods (San Diego, Calif J, 84, 25-34]. The claimed technical solution provides for the use of known methods for obtaining preparations of decellularized mammalian tissues, taking into account the specifics of a particular type of original biological material (animal, organ, physical properties of tissue). The dry matter content in an aqueous dispersion of decellularized mammalian tissue, obtained by incubating a sample of the corresponding crushed tissue in a 0.5 M acetic acid solution with the addition of pepsin (pepsin concentration 1-10 mg / ml of solution) for 24 hours at room temperature with constant stirring, then used to obtain the target macroporous biopolymer material, was found experimentally and ranges from 1 to 5 mass. %. At less than 1 mass.If the dry matter concentration in such a dispersion is below 5% by weight, the resulting biopolymer material has low strength and is therefore unsuitable for further use as a cell carrier. When the dry matter content in the dispersion exceeds 5% by weight, the resulting biopolymer material becomes brittle, especially in its dehydrated form, making further handling difficult.

[0029] 2. The method for producing the claimed biopolymer material includes the following stages:

[0030] a) preparation of an initial aqueous dispersion of decellularized mammalian tissue;

[0031] b) freezing the resulting dispersion;

[0032] c) removal of crystallized solvent by lyophilization or cryoextraction;

[0033] d) chemical cross-linking (hardening) of the obtained cryostructurate;

[0034] d) final rinsing of the cryogenically structured macroporous sponge. The modes of these stages are determined by the following factors:

[0035] Freezing temperature regimes were determined experimentally and are determined by the component concentrations, the volume of the frozen liquid, and the sample geometry. According to the claimed technical solution, freezing is carried out at -15 to -35°C. At subzero temperatures above -15°C, due to supercooling effects, the initial biopolymer solution (especially in the case of more concentrated samples) often fails to freeze. Below -35°C, very small ice crystals form, which create insufficiently large pores in the target polymer material.

[0036] According to the claimed invention, the removal of crystallized solvent (ice) from frozen samples is carried out using known techniques - either lyophilization or cryoextraction [V.I. Lozinsky. Principles and methods of cryostructuring polymer systems to create innovative materials for biomedical purposes. In the book "Hybrid nanoforms of bioactive and medicinal substances". Ed. Melnikov M. Ya. and Trakhtenberg L.I., Moscow: Technosfera Publishing House, 2020, Chapter 3, pp. 69-101]. The temperature regime and duration of lyophilization depend on the size of the frozen samples and the operational capabilities of the technological equipment used. In the case of removing crystals of frozen solvent by cryoextraction, the claimed technical solution provides for the use of chilled ethanol, which dissolves ice without thawing it, but does not dissolve the polymer components of the processed material.The temperature of the ethanol extractant corresponds to the temperature of the frozen sample.

[0037] After lyophilization or cryoextraction, the macroporous cryostructured material is tanned via chemical crosslinking to render it water-insoluble and to fix its macroporous morphology. This is achieved by treating the resulting material with a 0.04-0.12 M ethanol solution of alcohol-soluble carbodiimide at 5-25°C for 24-36 hours, followed by removal of excess carbodiimide by ethanol extraction. These processing conditions have also been determined experimentally; they are determined by the volume and geometry of the sample, the concentration of the specific tanning agent, and, to a certain extent, its reactivity.

[0038] After chemical tanning is completed, the resulting material is washed with ethanol to remove excess crosslinking agent and then stored in a 70-96% ethanol environment to prevent contamination.

[0039] Before using the obtained material, it is rehydrated in distilled water for 1 day, packaged and sterilized by gamma irradiation at a dose of 1.5 Mrad.

[0040] It was experimentally found that cryogenically structured biopolymer materials based on decellularized mammalian tissues obtained in accordance with the above-mentioned modes have a dry density of 0.16 to 0.28 g / cm 3 and have communicating pores with a cross-section from 25 to 220 µm, which is optimal for populating the carrier with cells.

[0041] The claimed composition of macroporous carriers for use in TEC, the carriers themselves and the method for their production, as well as the claimed combination of features, were not previously known, that is, the proposed technical solution meets the criterion of “novelty”.

[0042] Technical result achieved by the declared technical solution:

[0043] • the possibility of using a wide range of tissues from different mammals as raw materials;

[0044] • Reduction of the time of negative influence of pepsin on ECM proteins;

[0045] • the ability to vary the content of lyophilized decellularized tissue in an acetic acid solution of pepsin;

[0046] • standardized matrix sterilization mode;

[0047] • the possibility of creating TECs with the inclusion of human cells;

[0048] • long-term cultivation of human cells on the declared carrier.

[0049] Brief description of drawings

[0050] The invention is explained by the following figures.

[0051] Fig. 1. Microstructure of the claimed material (light microscopy in phase contrast mode; scale bar size 100 μm).

[0052] Fig. 2. Microstructure of the material according to scanning electron microscopy data (scale bar size 50 μm).

[0053] Fig. 3. Micrograph of NIH / 3T3 cell culture in contact with cryogenically structured biopolymer material based on decellularized rat spleen tissue (light microscopy; UV ×100).

[0054] Fig. 4. Micrograph of NIH / 3T3 cell culture in contact with cryogenically structured biopolymer material based on decellularized rat spleen tissue (light microscopy; fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0055] Fig. 5. Micrograph of human adipose tissue mesenchymal stromal cells cultured (3 days) on a cryogenically structured biopolymer material based on decellularized pig liver tissue (light microscopy; fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0056] Fig. 6. Micrograph of human adipose tissue mesenchymal stromal cells cultured (14 days) on a cryogenically structured biopolymer material based on decellularized pig liver tissue (light microscopy; fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0057] Fig. 7. Micrograph of human hepatocellular carcinoma HepG2 cells cultured (3 days) on a cryogenically structured biopolymer material based on decellularized pig liver tissue (light microscopy; fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0058] Fig. 8. Micrograph of human hepatocellular carcinoma HepG2 cells cultured (10 days) on a cryogenically structured biopolymer material based on decellularized pig liver tissue (light microscopy: fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0059] Fig. 9. Micrograph of EA.hy926 hybrid culture cells (a hybrid of primary umbilical cord endothelial cells with the A549 carcinoma clone) cultured (7 days) on a cryogenically structured biopolymer material based on decellularized porcine liver tissue (light microscopy; fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0060] Fig. 10. Micrograph of EA.hy926 hybrid culture cells (a hybrid of primary umbilical cord endothelial cells with the A549 carcinoma clone) of pig liver (light microscopy; fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0061] Fig. 11. Micrograph of human chondrocytes cultured (7 days) on a cryogenically structured biopolymer material based on decellularized porcine articular cartilage tissue (light microscopy; fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0062] Fig. 12. Micrograph of human chondrocytes cultured for a long time (30 days) on a cryogenically structured biopolymer material based on decellularized porcine articular cartilage tissue (light microscopy; fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0063] Fig. 13. Micrograph of human chondrocytes cultured for a long time (60 days) on a cryogenically structured biopolymer material based on decellularized porcine articular cartilage tissue (light microscopy; fluorescent staining with Live / Dead™ dyes; green - live cells, red - dead; UV ×100).

[0064] Fig. 14. Growth curve of NIH / 3T3 cell line in the presence of cryogenically structured biopolymer material obtained according to Example 1 after soaking in an ethanol solution (blue color) and undergoing radiation sterilization.

[0065] Implementation of the invention

[0066] The following examples are given to prove the possibility of achieving the specified technical result.

[0067] Example 1. Cryogenically structured biopolymer material obtained using cryoextraction to remove ice from a frozen aqueous dispersion of decellularized porcine liver tissue

[0068] a) Preparation of aqueous dispersion of decellularized pig liver tissue

[0069] Decellularized porcine liver particles are incubated for 24 hours at room temperature with constant stirring in a 0.5 M acetic acid solution with added pepsin (pepsin concentration 1 mg / ml). The resulting aqueous dispersion of decellularized porcine liver tissue has a dry matter content of 1% by weight.

[0070] b) Formation of cryogenically structured biopolymer material

[0071] An aqueous dispersion of decellularized porcine liver tissue is placed in a 2.5 mm thick layer in 35 mm diameter plastic Petri dishes and frozen at -15°C. Ice is then removed from the frozen samples by treating them with ethanol at -15…-20°C for 3 days, replacing the liquid extractant with a fresh portion daily. Next, the resulting macroporous cryostructure is tanned at 5°C for 36 hours to impart water insolubility and fix the macroporous morphology by chemical cross-linking by treatment with a 0.04 M ethanol solution of alcohol-soluble N,N'-diisopropyl carbodiamide, followed by rinsing the biopolymer material from excess cross-linking agent with 70% ethanol, in which environment the material is stored to prevent contamination. Before using the resulting cryogenically structured biopolymer material as a carrier for cell cultivation, it is rehydrated in distilled water, packaged and sterilized by gamma irradiation at a dose of 1.5 Mrad.

[0072] c) Characterization of the obtained biopolymer material.

[0073] The microstructure of the material is analyzed using light and low-vacuum scanning electron microscopy (SEM); in the first case, for a water-swollen sample, in phase contrast mode (Fig. 1), and in the second case, for a dry sample, with lanthanide contrasting of the preparation (Fig. 2).

[0074] According to SEM data, the pore cross-section of the dry material ranges from 35 to 220 μm. The dry density of this cryogenically structured biopolymer material is 0.16 g / cm3. 3 .

[0075] Example 2. Cryogenically structured biopolymer material obtained using cryoextraction to remove ice from a frozen aqueous dispersion of decellularized rat spleen tissue

[0076] a) Preparation of aqueous dispersion of decellularized rat spleen tissue

[0077] Desquamated rat spleen particles are incubated for 24 hours at room temperature with constant stirring in a 0.5 M acetic acid solution supplemented with pepsin (pepsin concentration 10 mg / ml). The resulting aqueous dispersion of decellularized rat spleen tissue contains 5% dry matter.

[0078] b) Formation of cryogenically structured biopolymer material

[0079] An aqueous dispersion of decellularized rat spleen tissue in a 3.0 mm thick layer is placed in 60x60 mm square plastic molds and frozen at -25°C, and then the ice is removed from the frozen samples by treating them with ethanol at -25…-30°C for 5 days with daily replacement of the liquid extractant with a fresh portion. Next, the resulting macroporous cryostructure is tanned by chemical crosslinking at 15°C for 30 hours to make it water-insoluble and to fix the macroporous morphology, using a 0.1 M ethanol solution of alcohol-soluble N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, followed by washing the biopolymer material from excess crosslinking agent with 96% ethanol, in which medium the material is stored to prevent contamination.Before using this cryogenically structured biopolymer material as a cell culture carrier, it is rehydrated in distilled water, packaged and sterilized by gamma irradiation at a dose of 1.5 Mrad.

[0080] c) Characterization of the obtained biopolymer material.

[0081] According to SEM data, the pore cross-section of the dry material is from 30 to 175 μm, and its density is 0.28 g / cm 3 .

[0082] d) Evaluation of the cytotoxicity of the obtained biopolymer material

[0083] To prove the absence of cytotoxic properties in the obtained material, its effect on the culture of linear mouse fibroblasts from NIH ЗТЗ is studied. The study is carried out in accordance with GOST R ISO 10993-1-2011) [Interstate standard GOST ISO 10993-1-2011 "Medical devices. Evaluation of the biological effect of medical devices. Part 4. Study of devices in contact with blood"; GOST ISO 10993-1-2011 "Medical devices. Evaluation of the biological effect of medical devices. Part 5. Cytotoxicity study: in vitro methods"].

[0084] The cells are cultured in a complete growth medium (CGM) (DMEM HG culture medium with GlutaMAX™ supplement; sterile fetal bovine serum tested for cytotoxicity and the absence of mycoplasma - 10%; Gibco® Antibiotic-Antimycotic complex antimicrobial supplement - 1%; HEPES 1 mM. All manipulations are carried out under aseptic conditions. NIH / 3T3 cells are seeded in 6-well flat-bottomed culture plates and incubated at 37°C under standard conditions: a humidified atmosphere containing (5±1)% CO2, until a monolayer with a confluence of 80-85% is formed.

[0085] The cytotoxicity of a cryogenically structured biopolymer material is studied directly using a direct contact method. A sample of the cryogenically structured biopolymer material, cut from a square sample into a disk 8 mm in diameter and 3 mm thick, pre-hydrated in distilled sterile water for 24 hours, is placed on the surface of a cell monolayer in a well of a 6-well plate and incubated under standard conditions. Initial results are recorded after 24 hours. To detect a possible prolonged cytotoxic effect, cell growth is monitored for up to 3 days.

[0086] The negative control is a cell culture medium containing serum, the positive control is a standard solution of zinc in nitric acid Zn 1-2 wt.% HNO3, diluted 1:200 with a solution of 0.9% NaCl for injection.

[0087] The results are assessed using optical microscopy methods in phase contrast modes, as well as using the Live / Dead™ fluorescent dye complex (Invitrogen, USA) to determine cell viability.

[0088] Microphotographs in Figs. 3 and 4 show the state of the cell culture at the points of contact with the test material. The cells have normal morphology, are flattened, and actively proliferating. No lysis zones, balled cells, or cells with altered morphology are observed. Staining with fluorescent dyes of the Live / Dead™ complex demonstrates the absence of dead cells. These results indicate that the resulting cryogenically structured biopolymer material does not exhibit any cytostatic or cytotoxic effects on mammalian cells.

[0089] Example 3. Cryogenically structured biopolymer material obtained using lyophilization to remove ice from a frozen aqueous dispersion of decellularized porcine liver tissue

[0090] a) Preparation of aqueous dispersion of decellularized rat spleen tissue

[0091] Desiccated pig liver particles are incubated for 24 hours at room temperature with constant stirring in a 0.5 M acetic acid solution supplemented with pepsin (pepsin concentration 5 mg / ml). After adjusting the pH of the medium to neutral, an aqueous dispersion of decellularized rat spleen tissue with a dry matter content of 2.5% by weight is obtained.

[0092] b) Formation of cryogenically structured biopolymer material

[0093] An aqueous dispersion of decellularized porcine liver tissue is placed in a 2.0 mm thick layer in 60 mm diameter plastic Petri dishes and frozen at -20°C, followed by lyophilization using an ALPHA 1-2 LD plus sublimation unit (Martin Christ, Germany). The resulting macroporous cryostructure is then tanned at 25°C for 24 h to impart water insolubility and fix the macroporous morphology by chemical cross-linking by treatment with a 0.06 M ethanol solution of alcohol-soluble N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, followed by washing the biopolymer material to remove excess cross-linking agent with 85% ethanol, in which environment the material is stored to prevent contamination. Before using the resulting cryogenically structured biopolymer material as a carrier for cell cultivation, it is rehydrated in distilled water, packaged and sterilized by gamma irradiation at a dose of 1.5 Mrad.

[0094] c) Characterization of the obtained biopolymer material

[0095] According to SEM data, the pore cross-section of the dry material is from 25 to 205 μm, and its density is 0.19 g / cm 3 .

[0096] d) Evaluation of the possibility of using the obtained biopolymer material as a carrier for culturing human cells.

[0097] To prove the possibility of culturing various types of human cells, the resulting cryogenically structured biopolymer material based on decellularized pig liver tissue is populated with various types of cells.

[0098] Human adipose tissue-derived mesenchymal stromal cells (hATMSCs) isolated from human subcutaneous adipose tissue fragments obtained during surgical interventions with the patient’s informed consent according to the standard method [Surguchenko VA, Ponomareva AS, Kirsanova LA, Skaleckij NN, Sevastianov VI The cell-engineered construct of cartilage on the basis of biopolymer hydrogel matrix and human adipose tissue-derived mesenchymal stromal cells (in vitro study). / / J. Biomed. Mat. Soc. Part A 2015; 103(2): 463-470], and PRS are used. Third passage cells were used in the experiment. Model liver cells (hepatocellular carcinoma HepG2 cells) and model endothelial cells of the EA.hy926 line (a hybrid of primary umbilical cord endothelial cells with the A549 carcinoma clone) are also used.For culturing HepG2 cells, Williams medium with the addition of 5% sterile fetal bovine serum, tested for cytotoxicity and the absence of mycoplasma; 1% Gibco® Antibiotic-Antimycotic complex antimicrobial supplement; 1 mM HEPES, 146 mM glutamine is used. For EA.hy926 endothelial cells, DMEM HG medium with the addition of 10% Biosera sterile serum, tested for cytotoxicity and the absence of mycoplasma, 1% Gibco® Antibiotic-Antimycotic complex antimicrobial supplement; 1 mM HEPES, 146 mM glutamine is used.

[0099] The cells are grown in culture flasks until a confluent layer is reached, after which they are washed off using the TrypLe™ reagent (Invitrogen, USA) and a working cell suspension with a concentration of 5×10 is prepared. 5Cells / ml. Samples of the cryogenically structured biopolymer material, prepared in a manner similar to that described in Example 2, are placed in sterile centrifuge tubes, and 1 ml of the cell suspension is added to each tube. The tubes are then shaken on a laboratory shaker in reciprocating mode at 60 rpm for 1 hour to evenly distribute the cells across the sample surface. Next, 4 ml of culture medium is added to each tube, and the samples are incubated under standard conditions. At appropriate observation times, selected based on the specifics of each culture, a portion of the samples are removed and subjected to intravital microscopy with Live / Dead™ fluorescent dyes to visualize the distribution of live and dead cells and their morphology on the matrix surface. For AF MSCs this is day 3 (Fig. 5) and day 14 (Fig. 6), for HepG2 - day 3 (Fig. 7) and day 10 (Fig. 8), for EA.hy926 - day 7 (Fig. 9) and day 16 (Fig. 10).For all cell types, an increase in cell mass was observed, with the formation of continuous cell sheets by the end of the experiment (Fig. 6, Fig. 8, Fig. 10). Cells in all variants had normal morphology, and virtually no dead cells were observed. The cells actively proliferated until the end of the experiment.

[0100] Thus, this example confirms the possibility of cultivating different types of human cells on cryogenically structured biopolymer material.

[0101] Example 4. Cryogenically structured biopolymer material obtained using cryoextraction to remove ice from a frozen aqueous dispersion of decellularized porcine cartilage tissue

[0102] a) Preparation of aqueous dispersion of decellularized porcine cartilage tissue

[0103] Desiccated porcine cartilage particles are incubated for 24 hours at room temperature with constant stirring in a 0.5 M acetic acid solution with added pepsin (pepsin concentration 7.5 mg / ml). The result is an aqueous dispersion of decellularized porcine cartilage tissue with a dry matter content of 3% by weight.

[0104] b) Formation of cryogenically structured biopolymer material

[0105] A 2.7 mm thick aqueous dispersion of decellularized cartilage tissue is placed in 60 mm diameter plastic Petri dishes and frozen at -35°C. Ice is then removed from the frozen samples by treating them with ethanol at -30…-35°C for 7 days, replacing the liquid extractant with a fresh portion daily. Next, to impart water-insolubility and fix the macroporous morphology, the resulting macroporous cryostructure is tanned by chemical cross-linking at 10°C for 27 hours by treatment with an ethanol solution of alcohol-soluble N,N'-diisopropyl carbodiamide (0.2M), followed by rinsing the biopolymer material to remove excess cross-linking agent with 96% ethanol, in which the material is stored to prevent contamination. Before using this cryogenically structured biopolymer material as a cell culture carrier, it is rehydrated in distilled water, packaged and sterilized by gamma irradiation at a dose of 1.5 Mrad.

[0106] c) Characterization of the obtained biopolymer material

[0107] According to SEM data, the pore cross-section of the dry material is from 25 to 155 μm, and its density is 0.18 g / cm 3 .

[0108] d) Evaluation of the possibility of using the obtained biopolymer material to create a tissue-engineered structure

[0109] To confirm the possibility of creating TICs using a cryogenically structured biopolymer material based on decellularized pig cartilage tissue, human chondrocytes are cultured on such a carrier.

[0110] Human primary chondrocytes are isolated from human costal cartilage fragments obtained during surgical procedures with the patient's informed consent. The material is washed in Hank's solution with the addition of a culture antibiotic-antimycotic. The tissue is minced with scissors to obtain a homogeneous mass consisting of fragments of approximately 1 mm in volume. 3, after which they are incubated in a 0.1% solution of type I collagenase at 37°C for 12 hours. After that, the isolated cells are washed from residual collagenase with Hanks' solution, seeded into culture flasks and placed in a CO2 incubator. The culture medium used is DMEM / F12 supplemented with GlutaMAX™; fetal bovine serum, sterile, tested for cytotoxicity and the absence of mycoplasma - 10%; complex antimicrobial supplement Gibco® Antibiotic-Antimycotic - 1%; HEPES 1 mM. Cells of the third passage are used in the experiment.

[0111] Cells are grown in culture flasks until a confluent layer is reached, after which they are washed off using the TrypLe™ reagent (Invitrogen, USA) and a working cell suspension with a concentration of 5×10 is prepared. 5cells / ml. Population of the cryogenically structured biopolymer material with cells is carried out similarly to the methods described in Example 3. TECs were cultured under standard conditions for 60 days. On the 7th day (Fig. 11), 30th day (Fig. 12), and 60th day (Fig. 13), some samples were removed and subjected to intravital microscopy with Live / Dead™ fluorescent dyes. It was shown that over the course of 60 days, the cells proliferate and populate the space of the material. Only a small number of dead cells is observed, not exceeding the natural cell loss.

[0112] Thus, the possibility of long-term cultivation of human cells on this cryogenically structured biopolymer material has been confirmed.

[0113] Example 5. Cryogenically structured biopolymer material obtained using lyophilization to remove ice from a frozen aqueous dispersion of decellularized rabbit liver tissue

[0114] a) Preparation of aqueous dispersion of decellularized rat spleen tissue

[0115] Desiccated rabbit liver particles are incubated for 24 hours at room temperature with constant stirring in a 0.5 M acetic acid solution with added pepsin (pepsin concentration 4 mg / ml). After adjusting the pH of the medium to neutral, an aqueous dispersion of decellularized rat spleen tissue with a dry matter content of 2% by weight is obtained.

[0116] b) Formation of cryogenically structured biopolymer material

[0117] An aqueous dispersion of decellularized rabbit liver tissue is placed in a 2.0 mm thick layer in 35 mm diameter plastic Petri dishes and frozen at -25°C, then lyophilized using an ALPHA 1-2 LD plus sublimation unit (Martin Christ, Germany). The resulting macroporous cryostructure is then tanned at 18°C ​​for 18 h to impart water insolubility and fix the macroporous morphology by chemical cross-linking by treatment with a 0.08 M ethanol solution of alcohol-soluble N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, followed by washing the biopolymer material to remove excess cross-linking agent with 70% ethanol, in which environment the material is stored to prevent contamination. Before using the resulting cryogenically structured biopolymer material as a carrier for cell cultivation, it is rehydrated in distilled water, packaged and sterilized by gamma irradiation at a dose of 1.5 Mrad.

[0118] c) Characterization of the obtained biopolymer material

[0119] According to SEM data, the pore cross-section of the dry material ranges from 30 to 190 µm. The density of this cryogenically structured biopolymer material is 0.17 g / cm3. 3 .

[0120] d) Evaluation of the effect of radiation sterilization on the cytotoxicity of the obtained biopolymer material

[0121] To confirm the absence of a negative impact of radiation sterilization on the cytotoxicity of cryogenically structured biopolymer material, cryogenically structured biopolymer material samples, after radiation sterilization with 1.5 Mrad gamma radiation, were placed on a monolayer of NIH / 3T3 mouse fibroblasts similarly to samples soaked in 96% ethanol for 24 hours and then washed in distilled water instead of gamma irradiation. Cell culturing was carried out for 120 hours.

[0122] At 24, 48, and 120 hours, a test was performed to determine the metabolic activity of the cells using the PrestoBlue™ reagent (Invitrogen, USA) according to the manufacturer's instructions. The graph in Fig. 14 shows that the cryogenically structured biopolymer material did not exhibit a cytotoxic effect on the cells after radiation treatment throughout the experiment.

[0123] Thus, it has been shown that radiation sterilization with a dose of 1.5 Mrad does not degrade the biological properties of the matrix and is acceptable for its production.

[0124] The claimed invention has the following advantages compared to analogues and the prototype:

[0125] 1. Unlike the prototype, the claimed invention allows for the effective use of the proposed biopolymer material for culturing human cells (see examples 3 and 4).

[0126] 2. Unlike analogs and prototype biopolymer materials, the proposed invention allows the use of various types of tissues from different mammals as raw materials, which indicates an increase in the versatility of the approach used in this invention for obtaining a cryogenically structured biopolymer material based on decellularized animal tissues.

[0127] 3. Unlike the biopolymer analog material [He X, Lu H, Kawazoe N, Tateishi T, Chen G. A novel cylinder-type poly (L-lactic acid)-collagen hybrid sponge for cartilage tissue engineering. / / Tissue Eng Part C Methods. 2010 16(3): 329-38], in the claimed technical solution there is no need to modify the carrier surface with substances that increase the number of cell binding sites, which simplifies the production of the material and reduces its cost.

[0128] 4. Unlike the cryogenically structured biopolymer analogue material based on gelatin [L. Elowsson, H. Kirsebom, V. Carmignae, M. Durbeej, D. Mattiasson, Porous protein-based scaffolds prepared through freezing as potential scaffolds for tissue engineering. / / J. Mater. Sci., Mater. Med., 2012, Vol. 23, P. 2489-2498], the claimed material can be obtained in various geometries and volumes, since there is no risk of self-gelling of the initial biopolymer solution when it is cooled before the system freezes. In addition, the claimed technical solution uses low-toxic alcohol-soluble carbodiimide as a cross-linking agent, whereas highly toxic glutaraldehyde is used to obtain the analogue material.

[0129] 5. In the claimed invention, the duration of the negative effect of pepsin on ECM proteins is reduced by 3 times compared to the prototype [JY Kim, T. Sen, JY Lee, D.-W. Cho. Degradation-controlled tissue extracellular sponge for rapid hemostasis and wound repair after kidney injury. / / Biomaterials, 2024, V. 307, article 122524] by reducing the time of enzymatic processing of the original biological material.

[0130] 6. Based on decellularized mammalian tissue, the claimed cryogenically structured biopolymer material allows for long-term (more than 1 month) cell cultivation as a cell carrier.

Claims

1. A cryogenically structured macroporous biopolymer material for biomedical technologies, which is a cryogenically structured chemically cross-linked decellularized mammalian tissue, and the material in a dry state has a density of 0.16 to 0.28 g / cm 3 and has communicating pores with a cross-section from 25 to 220 µm.

2. A method for producing a biopolymer material according to claim 1, comprising preparing a dispersion of decellularized mammalian tissue with a dry matter content of 1 to 5 mass % in a 0.5 M acetic acid solution with the addition of pepsin at a concentration of 1-10 mg / ml, incubating the dispersion for 24 hours at room temperature with constant stirring, then freezing it at -15…-35°C, removing the crystallized solvent either by lyophilization or by cryoextraction with cold ethanol, treating the resulting macroporous cryostructure with a 0.04-0.12 M ethanol solution of carbodiimide at 5-25°C for 24-36 hours, removing excess carbodiimide by extraction with ethanol, and transferring the target biopolymer material to 70-96% ethanol before use.

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