Interstitial material for cell encapsulation and its manufacturing method and application

Abstract

This application provides a cell encapsulation stromal material and its manufacturing method and application. The material is prepared by cutting, low-temperature microparticulation grinding, enzymatic decomposition, cell component removal, virus inactivation, freeze-drying and radiation sterilization using cartilage from mammals such as pigs, cows, sheep, horses and deer. The method is not for diagnostic purposes, and the stromal material is used for 3D bioprinting alone or with encapsulated cells. The main components of the material are type II collagen, chondroitin sulfate and hyaluronic acid, and have the advantages of no crosslinking toxicity, low immunogenicity, good biocompatibility and can be used for human implantation. In terms of structure, it has a good surface topology structure, provides a tissue microenvironment similar to that in the body for in vitro cell culture, and can be used as a microcarrier for cell culture, as well as promote the formation of cell mass, and can encapsulate cells in the gel-state stromal during the process of cell storage and transportation, protect cells from the effects of adverse environments and improve the viability of cells.

Description

[Technical field]

[0001] This application claims the rights of Chinese invention patent application number CN202111598820.1, filed on December 24, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] The present application belongs to the field of bioengineering technology, and specifically relates to an interstitial material for cell encapsulation and its preparation method and application. [Background technology]

[0003] Cell therapy refers to a treatment method in which normal cells or cells modified by biotechnology are amplified in vitro and then transplanted or introduced into the patient's body. The newly introduced cells can replace damaged cells and reconstruct tissue structure and function (stem cell therapy technology), or have stronger immune killing function (immune cell therapy technology), thereby achieving the purpose of disease treatment. Stem cell therapy technology utilizes the self-renewal, multi-differentiation and high proliferation characteristics of stem cells to transplant healthy stem cells into the human body to achieve the purpose of repairing lesions or reconstructing normal tissue function. Immune cell therapy technology collects immune cells from the human body, modifies them in vitro, cultures and amplifies them, and then returns them to the human body to kill pathogens, cancer cells and mutant cells with enhanced target cytotoxicity, activating and strengthening the body's immune capabilities. With the rapid development of molecular biology, stem cell biology, tissue engineering and regenerative medicine, the role of cell therapy has become increasingly important in clinical practice. Currently, there are many types of cells used in clinical treatment, including bone marrow stem cells, hematopoietic stem cells, neural stem cells, skin stem cells, pancreatic islet stem cells, adipose stem cells, and multiple types of immune cells such as DC, CIK, NK, CD3AK, and γδT. These are widely used in the treatment of various clinical diseases, including blood diseases, organ transplants, cardiovascular diseases, liver diseases, nervous system diseases, cartilage and bone tissue diseases, tissue damage, and malignant tumor diseases.

[0004] Cell culture and proliferation, preservation and transportation are essential parts of the realization of cell therapy. Currently, there are two main methods for cell proliferation. One is in vitro 2D culture, in which cells are passaged and expanded when they reach a confluent state in cell adhesion proliferation. The disadvantage of this method is that the efficiency of in vitro 2D culture is low and it is unfavorable to the maintenance of cell phenotype. The other is in vitro 3D culture, in which cells are immobilized on a suitable carrier and combined with a bioreactor to achieve high-density cell culture and production, for example, using porous microcarrier cell immobilization technology. There are several types of materials that can be used to manufacture porous microcarriers, including polymeric synthetic materials with good biocompatibility, and purified or structurally modified natural materials (e.g., starch, cellulose, chitosan, sodium alginate, collagen, gelatin, etc.). Cell cluster culture technology can also be used to realize carrier-free immobilization of cells, that is, cell growth is monitored by controlling the formation and depolymerization of cell clusters during the cell culture process to achieve high-efficiency cell culture.

[0005] Extracellular matrix (ECM) is a biological macromolecule material that exists between cells and is secreted by cells, including collagen, elastin, proteoglycan, glycosaminoglycan, and cytokines. The extracellular matrix has a complex spatial network structure and is a microenvironment for cell growth and activity. The interaction between cells and the extracellular matrix has a regulating effect on the functions and behaviors of cells (e.g., adhesion, growth, proliferation, differentiation, migration, intercellular signal transmission, etc.). Therefore, the extracellular matrix material may be used to manufacture microcarriers for cell immobilization, may be used as an active material to promote the formation of cell mass, and may be used as a cell encapsulation stroma (CES) for encapsulating or protecting cells during cell storage and transportation. Patent CN105288737A discloses a tissue engineering cartilage composite scaffold based on cartilage extracellular matrix and a manufacturing method thereof, which relates to using cartilage extracellular matrix particles to rapidly amplify cartilage seed cells and induce the differentiation of stem cells into chondrocytes. The specific manufacturing method of the invention includes the following steps 1) to 3). 1) Fresh articular cartilage is crushed and sieved by low-temperature wet method to obtain cartilage particles with diameters of 100-500 μm, which are then decellularized to produce and obtain cartilage extracellular matrix microcarriers. 2) Cartilage extracellular matrix microcarriers and cartilage seed cells are placed in a bioreactor to be co-cultured, and cartilage microtissues are formed by rapid amplification of chondrocytes or induction of differentiation of stem cells into chondrocytes. 3) Cartilage microtissues are filled into the gaps of a three-dimensional porous scaffold of a water-containing gel precursor, and the cartilage microtissues and the three-dimensional porous scaffold are bonded by a coagulant to obtain a tissue-engineered cartilage composite scaffold by co-culture. The use of extracellular matrix particles or hydrogels in cell co-culture and promotion of specific differentiation of stem cells has been extensively studied. Summary of the Invention

[0006] The following is a summary of the subject matter described herein. This summary is not intended to limit the scope of protection of the claims.

[0007] In a first aspect, the present application provides a method for producing an interstitial material for cell encapsulation, comprising: (1) removing surface tissue and fascia from an in vitro mammalian cartilage material and dividing it into slices; (2) a step of disinfecting the surface of the cartilage tissue slices with a disinfectant, washing and freeze-drying; (3) subjecting the dried and frozen cartilage tissue slices to low-temperature microparticulation grinding with liquid nitrogen to produce a microparticulate material; (4) degreasing the particulate material with isopropyl alcohol in a solid-liquid ratio of 1:5 to 1:20, soaking, centrifuging, discarding the waste liquid, and obtaining a first precipitate; (5) treating the first precipitate by soaking in a saline solution or a phosphate buffer solution at a solid-liquid ratio of 1:5 to 1:20, centrifuging, discarding the waste liquid, and obtaining a second precipitate; (6) treating the second precipitate with a buffered deoxyribonuclease solution at a solid-liquid ratio of 1:5 to 1:20, centrifuging, discarding the waste liquid, and obtaining a third precipitate; (7) treating the third precipitate by soaking in an alcalase solution at a solid-liquid ratio of 1:5 to 1:10, centrifuging, and discarding the waste liquid to obtain a fourth precipitate; (8) adding a detergent to the fourth precipitate in a solid-liquid ratio of 1:5 to 1:20, treating the fourth precipitate by mixing with a phosphate buffer, centrifuging, and discarding the waste liquid to obtain a fifth precipitate; (9) soaking the fifth precipitate with a disodium ethylenediaminetetraacetate solution in a solid-liquid ratio of 1:5 to 1:20, washing, centrifuging, discarding the waste liquid, replacing the solution, washing again, centrifuging again, discarding the waste liquid, and obtaining a sixth precipitate; (10) treating the sixth precipitate by soaking in a saline solution or a phosphate buffer solution in a solid-liquid ratio of 1:5 to 1:20, centrifuging, discarding the waste liquid, replacing the solution and repeating the procedure to obtain a seventh precipitate; (11) adding a mixture of sodium phosphate and peracetic acid to the seventh precipitate in a solid-liquid ratio of 1:5 to 1:10 to inactivate the viruses, centrifuging, discarding the waste liquid, and obtaining an eighth precipitate; (12) treating the eighth precipitate with saline or phosphate buffer in a solid-liquid ratio of 1:5 to 1:20, centrifuging, and discarding the waste liquid to obtain a ninth precipitate; and (13) washing the ninth precipitate by adding sterile water in a solid-liquid ratio of 1:5 to 1:20, freeze-drying, and sterilizing the same by gamma radiation or electron beam after freeze-drying.

[0008] In a second aspect, the present application provides a stromal material for cell encapsulation according to the above method of manufacture.

[0009] In a third aspect, the present application provides applications of said interstitial material for cell encapsulation, i.e. for promoting the formation of cell masses in cell culture processes, for use as an interstitial material for encapsulating or protecting cells during cell storage and transportation, or for use in 3D bioprinting, or for use in the field of aesthetic medicine. [Brief description of the drawings]

[0010] [Figure 1] FIG. 2 is a scanning electron microscope image of cartilage matrix microparticles after alcalase treatment and decellularization in Example 1 of the present application. [Diagram 2] 1 shows the suspension stability of cartilage matrix microparticles after Alcalase treatment and decellularization in Example 1 of the present application (left), and the suspension stability of cartilage matrix microparticles that have only been decellularized without being treated with Alcalase (right). [Diagram 3] FIG. 2 is a differential scanning calorimetry diagram of untreated pig ear cartilage raw material (top) and cartilage matrix particles after Alcalase treatment and decellularization (bottom) in Example 1 of the present application. [Figure 4] FIG. 1 is a diagram showing the co-culture of L929 cells with cartilage matrix microparticles after Alcalase treatment and decellularization in Example 1 of the present application. [Diagram 5] FIG. 1 shows the cell growth status in the microparticles 8 hours and 8 days after co-culture of umbilical cord mesenchymal stem cells and cartilage matrix microparticles in Example 1 of the present application. [Figure 6]FIG. 2 is a graph showing the temperature response characteristics of cartilage matrix microparticles after alcalase treatment and decellularization in Example 1 of the present application. [Figure 7] FIG. 1 is a graph showing the effect of inducing adipose tissue formation by subcutaneously implanting cartilage matrix microparticles after Alcalase treatment and decellularization in Example 1 of the present application into rats. [Figure 8] FIG. 1 shows tissue sections of pig ear cartilage in Example 3 of the present application after treatment with alcalase for 5 hours (left) and 18 hours (right). [Figure 9] 1 shows the general morphology of cartilage matrix material microparticles of different particle sizes in Example 5 of the present application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In an embodiment of a first aspect, the present application provides a method for producing an interstitial material for cell encapsulation, comprising: (1) removing surface tissue and fascia from an in vitro mammalian cartilage material and dividing it into slices; (2) a step of disinfecting the surface of the cartilage tissue slices with a disinfectant, washing and freeze-drying; (3) subjecting the dried and frozen cartilage tissue slices to low-temperature microparticulation grinding with liquid nitrogen to produce a microparticulate material; (4) degreasing the particulate material with isopropyl alcohol in a solid-liquid ratio of 1:5 to 1:20, soaking, centrifuging, discarding the waste liquid, and obtaining a first precipitate; (5) treating the first precipitate by soaking in a saline solution or a phosphate buffer solution at a solid-liquid ratio of 1:5 to 1:20, centrifuging, discarding the waste liquid, and obtaining a second precipitate; (6) treating the second precipitate with a buffered deoxyribonuclease solution at a solid-liquid ratio of 1:5 to 1:20, centrifuging, discarding the waste liquid, and obtaining a third precipitate; (7) treating the third precipitate by soaking in an alcalase solution at a solid-liquid ratio of 1:5 to 1:10, centrifuging, and discarding the waste liquid to obtain a fourth precipitate; (8) adding a detergent to the fourth precipitate in a solid-liquid ratio of 1:5 to 1:20, treating the fourth precipitate by mixing with a phosphate buffer, centrifuging, and discarding the waste liquid to obtain a fifth precipitate; (9) soaking the fifth precipitate with a disodium ethylenediaminetetraacetate solution in a solid-liquid ratio of 1:5 to 1:20, washing, centrifuging, discarding the waste liquid, replacing the solution, washing again, centrifuging again, discarding the waste liquid, and obtaining a sixth precipitate; (10) treating the sixth precipitate by soaking in a saline solution or a phosphate buffer solution in a solid-liquid ratio of 1:5 to 1:20, centrifuging, discarding the waste liquid, replacing the solution and repeating the procedure to obtain a seventh precipitate; (11) adding a mixture of sodium phosphate and peracetic acid to the seventh precipitate in a solid-liquid ratio of 1:5 to 1:10 to inactivate the viruses, centrifuging, discarding the waste liquid, and obtaining an eighth precipitate; (12) treating the eighth precipitate with saline or phosphate buffer in a solid-liquid ratio of 1:5 to 1:20, centrifuging, and discarding the waste liquid to obtain a ninth precipitate; and (13) washing the ninth precipitate by adding sterile water in a solid-liquid ratio of 1:5 to 1:20, freeze-drying, and sterilizing the same by gamma radiation or electron beam after freeze-drying.

[0012] Preferably, in step (1), the mammal is one or more selected from the group consisting of pigs, cows, sheep, horses and deer. Preferably, in step (1), the cartilage material is one or more selected from elastic cartilage and hyaline cartilage.

[0013] Preferably, the elastic cartilage is ear cartilage. Preferably, the hyaline cartilage is one or more selected from articular cartilage, costal cartilage, scapular cartilage and meniscus.

[0014] Preferably, in step (2), the disinfectant is one or more selected from 0.1-0.5% w / v sodium hypochlorite, 0.5-2.0% w / v sodium carbonate, and 50-70% w / v alcohol solution. Preferably, the alcohol solution is one or more selected from ethanol or isopropyl alcohol.

[0015] Preferably, in step (3), the microparticle material has an average particle size based on particle number of 2 to 20 μm, and an average particle size based on particle volume of 20 to 200 μm.

[0016] Preferably, in step (4), the content of isopropyl alcohol is 50 to 70% w / v, the immersion time is 30 to 60 minutes, the centrifuge speed is 500 to 1500 xg, and the centrifuge time is 5 to 20 minutes.

[0017] Preferably, in step (5), the immersion time is 30 to 60 minutes, the centrifugation speed is 500 to 1500 xg, and the centrifugation time is 5 to 20 minutes.

[0018] Preferably, in step (6), the buffer solution of the deoxyribonuclease is one or more selected from a hydroxyethylpiperazine ethanesulfonic acid buffer solution and a tris(hydroxymethylaminomethane hydrochloride) buffer solution, the concentration of the hydroxyethylpiperazine ethanesulfonic acid buffer solution is 5 to 100 mmol / L, and the activity of deoxyribonuclease in the deoxyribonuclease solution is 50 to 250 U / L. Preferably, in step (6), the soaking time is 8 to 12 hours, the centrifugation speed is 500 to 1500 xg, and the centrifugation time is 5 to 20 minutes.

[0019] Preferably, in step (7), the concentration of alcalase in the alcalase solution is 0.02 to 0.2% w / v. Preferably, in step (7), the immersion time is 30 to 120 minutes, the centrifugation speed is 500 to 1500 xg, and the centrifugation time is 5 to 20 minutes.

[0020] Preferably, in step (8), the detergent is one or more selected from 0.5 to 2% w / v sodium deoxycholate, Triton X-100, and sodium dodecyl sulfate. Preferably, in step (8), the soaking time is 8 to 16 hours, the centrifugation speed is 500 to 1500 xg, and the centrifugation time is 5 to 20 minutes.

[0021] Preferably, in step (9), the concentration of disodium ethylenediaminetetraacetate in the disodium ethylenediaminetetraacetate solution is 5 to 50 mmol / L, and is mixed into a 10 mmol / L sodium phosphate buffer solution. Preferably, in step (9), the washing time is 2 to 4 hours, the centrifugation speed is 500 to 1500 xg, and the washing time is 5 to 20 minutes. Preferably, in step (9), the re-washing time is 12 to 24 hours, the re-centrifugation speed is 500 to 1500 xg, and the time is 5 to 20 minutes.

[0022] Preferably, in step (10), the immersion time is 30 to 120 minutes, the centrifugation speed is 500 to 1500 xg, and the centrifugation time is 5 to 20 minutes.

[0023] Preferably, in step (11), the time for virus inactivation treatment is 30 to 60 minutes, the centrifugation speed is 500 to 1500 xg, and the time is 5 to 20 minutes.

[0024] Preferably, in step (12), the soaking time is 30 to 120 minutes, the centrifugation speed is 500 to 1500 xg, and the centrifugation time is 5 to 20 minutes.

[0025] Preferably, in steps (5), (8), (10) and (12), the phosphate buffer is a sodium phosphate buffer.

[0026] In an embodiment of the present application, the manufacturing method may be a method not intended for the diagnosis or treatment of a disease.

[0027] In the embodiments of the present application, unless otherwise specified, "% w / v" is "g / 100 mL".

[0028] In an embodiment of the second aspect, the present application provides a stromal material for cell encapsulation obtainable by the above-mentioned manufacturing method. Preferably, the main components of the cell encapsulation stromal material are type II collagen and glycosaminoglycans. Preferably, the main components of the cell encapsulation stromal material are type II collagen, proteoglycans and glycosaminoglycans. Preferably, the glycosaminoglycans include chondroitin sulfate and hyaluronic acid.

[0029] In some embodiments of the second aspect, the cell encapsulation stromal material comprises type II collagen, chondroitin sulfate, and hyaluronic acid. In some embodiments of the second aspect, the cell encapsulation stromal material comprises type II collagen, chondroitin sulfate, and hyaluronic acid, the sum of which is at least 85% w / w of the dry weight of the cell encapsulation stromal material, preferably at least 90% w / w of the dry weight of the cell encapsulation stromal material, and more preferably at least 96.5±0.5% w / w of the dry weight of the cell encapsulation stromal material.

[0030] In some embodiments of the second aspect, the cell encapsulation stromal material contains at least 75% type II collagen w / w, less than 10% elastin w / w, and a glycosaminoglycan content of 1%-10% w / w, wherein the glycosaminoglycans include chondroitin sulfate and hyaluronic acid.

[0031] In some embodiments of the second aspect, the cell encapsulation stromal material comprises type II collagen, chondroitin sulfate, and hyaluronic acid, the type II collagen, chondroitin sulfate, and hyaluronic acid having a content of 92.4±0.4% w / w dry weight, 2.5±0.09% w / w dry weight, and 1.6±0.04% w / w dry weight, respectively, or 92.4±0.4% w / w dry weight, 2.5±0.1% w / w dry weight, and 1.6±0.0% w / w dry weight, respectively, or alternatively 92% w / w or more (dry weight), 2% w / w or more (dry weight), and 1% w / w or more (dry weight).

[0032] In some embodiments of the second aspect, the residual DNA content in the cell encapsulation stromal material is less than 20 ng / mg, preferably not more than 5 ng / mg, more preferably not more than 2.7±0.6 ng / mg. In some embodiments of the second aspect, the number of α-Gal antigen epitopes in the cell encapsulation stromal material is 2.2×10 12 ±3.0×10 11 Low, or 5.0 x 10 pieces / g dry weight 12 Lower than pieces / g dry weight. In some embodiments of the second aspect, the cell encapsulation stromal material comprises between 83.5±1.5% w / w and 90.5±1.5% w / w type II collagen.

[0033] In a third aspect, the present application provides the use of said stromal material for cell encapsulation in promoting the formation of cell masses in cell culture processes, as a stromal material for encapsulating or protecting cells in the process of cell storage and transportation, or for application in 3D bioprinting, or for filling and inducing autologous adipogenesis in the field of aesthetic medicine.

[0034] In some embodiments of the third aspect, co-culture of the cell encapsulation stromal material with cells does not show significant cytotoxicity, and a large number of cells adhere to the surface of the stromal material, showing good performance in supporting stem cell adhesion and proliferation, and the stromal material can be used as a microcarrier for cell culture and can also promote the formation of cell clusters. In some embodiments of the third aspect, the cell encapsulation stromal material can encapsulate cells by self-assembly, protecting the cells from adverse environmental influences during cell preservation and transportation, thereby improving cell viability. In some embodiments of the third aspect, the cell encapsulation stromal material has a protective effect on the cells in adverse conditions. In some embodiments of the third aspect, the cell encapsulation stromal material may be used as a tissue scaffold material and applied in tissue regeneration repair by intracorporeal injection, or may be applied in the field of aesthetic medicine for filling and autologous adipogenesis induction.

[0035] Conventional cell culture microcarrier materials are mainly produced by chemical crosslinking of polymer synthetic materials or purified or structurally modified natural materials (e.g., starch, cellulose, chitosan, sodium alginate, collagen, gelatin, etc.). In contrast, the present application uses mammalian (e.g., pig, cow, sheep, horse, deer, etc.) cartilage as the raw material, and the main components of the cell encapsulation stromal material produced are natural type II collagen and proteoglycan (chondroitin sulfate and hyaluronic acid), which has the advantages of being non-toxic like crosslinkers, low immunogenicity, excellent biocompatibility, and usable for implantation in the human body.

[0036] The cell encapsulation interstitial microparticle material produced based on the cartilage extracellular matrix of the present application has a good surface topology structure in terms of structure, provides a tissue microenvironment similar to that in the body for in vitro cell culture, and can be used as a microcarrier for cell culture, as well as promote the formation of cell clusters, and has a reversible temperature response property in terms of function, exhibiting a state similar to that of a colloidal solution at 35°C or higher and a gel state at a relatively low temperature, and encapsulating cells by self-assembly when converted from a colloid to a gel. By utilizing the property of encapsulating cells by self-assembly when converted from a colloid to a gel, cells can be encapsulated in the interstitium in a gel state during the process of cell storage and transportation, protecting the cells from the effects of an adverse environment and improving the cell viability.

[0037] In the prior art, cartilage cell matrix microparticles or hydrogels are prepared by grinding cartilage materials, and in acid-alkali treatment or grinding with a normal grinder (e.g., patents CN106075584A and CN112316211A), the thermal effect in the grinding process causes protein modification or denaturation in the material, reducing the stability of the material. On the other hand, the interstitial microparticle material for cell encapsulation prepared based on the cartilage extracellular matrix of the present application has a thermal stability of 40°C or higher, and can be used in 3D bioprinting alone or together with encapsulated cells to form stable tissue and organoid structures.

[0038] The method for producing stromal material for cell encapsulation based on cartilage extracellular matrix of the present application utilizes the simplicity and high effectiveness of alcalase, which is advantageous for large-scale production. Mammalian cartilage is dense, and the decellularization process takes a long time, making it difficult to achieve complete and thorough removal of cellular components. Achieving thorough cell removal by multiple repeated decellularization treatments and extending the decellularization cycle not only takes a long time, but also causes disadvantages such as reduced stability of cartilage extracellular matrix and large loss of constituent components. Alcalase treatment can also reduce α-Gal antigen epitopes in xenogeneic tissues.

[0039] Specific embodiments The present application provides a cell encapsulation stromal material and its manufacturing method and application, which can be used to promote the formation of cell masses in cell culture processes, and can encapsulate and depolymerize cells by self-assembly due to reversible temperature response properties, and can be used to encapsulate and protect cells in the process of cell storage and transportation. The material is a composite natural biomaterial manufactured from cartilage of mammals (e.g., pigs, cows, sheep, horses, deer, etc.), and its main components are type II collagen and glycosaminoglycans (chondroitin sulfate and hyaluronic acid). The type II collagen of the cell encapsulation stromal material of the present application is 75% w / w or more, elastin is less than 10% w / w, and the glycosaminoglycans (chondroitin sulfate and hyaluronic acid) content is 1% to 10% w / w.

[0040] As an option, the method for producing a stromal material for cell encapsulation based on the animal cartilage material of the present application is a method not intended for the diagnosis or treatment of a disease, and includes the following steps (1) to (14).

[0041] In step (1), cartilage material is collected from a mammal (e.g., pig, cow, sheep, horse, deer, etc.) and includes elastic cartilage such as ear cartilage, articular cartilage, costal cartilage, scapular cartilage, and meniscus, as well as hyaline cartilage. Excess tissue and fascia on the cartilage surface are removed, and the cartilage tissue is cut into slices having a thickness of 2 mm or less.

[0042] In step (2), the surface of the cartilage tissue slice is disinfected with a disinfectant, washed with purified water after disinfection, and freeze-dried to a water content of less than 5% w / w after washing. The disinfectant is one or more selected from 0.1-0.5% w / v sodium hypochlorite, 0.5-2.0% w / v sodium carbonate, and 50-70% w / v alcohol (or isopropyl alcohol).

[0043] In step (3), the dried and frozen cartilage tissue slices are subjected to low-temperature liquid nitrogen microparticle grinding to produce a microparticle material having an average particle size based on particle number of 2 to 20 μm and an average particle size based on particle volume of 20 to 200 μm.

[0044] In step (4), the microparticle material is degreased with 50-70% w / v isopropyl alcohol (IPA) at a solid-liquid ratio of 1:5 to 1:20. The microparticles are soaked for 30-60 min, then centrifuged at 500-1500 x g for 5-20 min, the waste liquid is discarded, and the first precipitate is obtained.

[0045] In step (5), the first precipitate is treated by adding 0.9% w / v saline or 10 mmol / L phosphate buffer (pH=7.4) at a solid-liquid ratio of 1:5 to 1:20, soaking for 30 to 60 min, centrifuging at 500 to 1500 xg for 5 to 20 min, discarding the waste liquid, and washing once again to obtain a second precipitate.

[0046] In step (6), the second precipitate is soaked in a buffer solution of deoxyribonuclease at a solid-liquid ratio of 1:5 to 1:20, the buffer solution of deoxyribonuclease being one or more selected from hydroxyethylpiperazine ethanesulfonic acid buffer and trishydroxymethylaminomethane hydrochloride buffer, the concentration of the hydroxyethylpiperazine ethanesulfonic acid buffer being 5 to 100 mmol / L, the pH being 7.6, and the activity of deoxyribonuclease in the deoxyribonuclease solution being 50 to 250 U / L. After soaking for 8 to 12 hours, the precipitate is centrifuged at 500 to 1500 xg for 5 to 20 minutes, and the waste liquid is discarded to obtain a third precipitate.

[0047] In step (7), the third precipitate is soaked in an alcalase solution at a solid-liquid ratio of 1:5 to 1:10, the concentration of alcalase in the solution being 0.02 to 0.2% w / v, and the precipitate is soaked for 30 to 120 minutes, then centrifuged at 500 to 1500 xg for 5 to 20 minutes, the waste liquid is discarded, and a fourth precipitate is obtained.

[0048] In step (8), a detergent is added to the fourth precipitate in a solid-liquid ratio of 1:5 to 1:20, the detergent in the solution being one or more selected from 0.5 to 2% w / v sodium deoxycholate, Triton X-100, and sodium dodecyl sulfate, formulated in a 10 mmol / L phosphate buffer solution, pH=7.4, and treated by soaking for 8 to 16 hours, followed by centrifugation at 500 to 1500×g for 5 to 20 minutes, discarding the waste liquid, and obtaining a fifth precipitate.

[0049] In step (9), the fifth precipitate is soaked in disodium ethylenediaminetetraacetate (EDTA) solution with a solid-liquid ratio of 1:5 to 1:20, the concentration of disodium ethylenediaminetetraacetate is 5 to 50 mmol / L, and the solution is mixed with a phosphate buffer solution of 10 mmol / L, and the pH of the solution is 7.4. After washing for 2 to 4 hours, the precipitate is centrifuged at 500 to 1500 x g for 5 to 20 minutes, the waste liquid is discarded, the solution is replaced and the precipitate is washed again for 12 to 24 hours, and the waste liquid is discarded, to obtain a sixth precipitate.

[0050] In step (10), the sixth precipitate is treated with 0.9% w / v saline or 10 mmol / L phosphate buffer (pH=7.4) at a solid-liquid ratio of 1:5 to 1:20 by soaking for 30 to 120 min, centrifuging at 500 to 1500 x g for 5 to 20 min, discarding the waste liquid, replacing the solution, soaking again for 30 to 120 min, centrifuging at 500 to 1500 x g for 5 to 20 min, discarding the waste liquid, and obtaining a seventh precipitate.

[0051] In step (11), a mixture containing 2-10% w / v sodium phosphate and 0.05-0.5% w / v peracetic acid is added to the seventh precipitate at a solid-liquid ratio of 1:5-1:10 to inactivate the viruses for 30-60 min, followed by centrifugation at 500-1500 x g for 5-20 min, discarding the waste liquid, and obtaining an eighth precipitate.

[0052] In step (12), the eighth precipitate is treated by adding 0.9% w / v saline or 10 mmol / L phosphate buffer (pH=7.4) at a solid-liquid ratio of 1:5 to 1:20, soaking for 30 to 120 min, centrifuging at 500 to 1500 xg for 5 to 20 min, discarding the waste liquid, and obtaining a ninth precipitate.

[0053] In step (13), the ninth precipitate is washed with sterile water at a solid-liquid ratio of 1:5 to 1:20 for 30 to 60 min, and the concentration of the treated microparticle sample is adjusted to 3 to 6% w / w, and then freeze-dried to a water content lower than 5% w / w.

[0054] In step (14), after freeze-drying, the mixture is sterilized by gamma rays or electron beams, and the sterilization dose is 15 to 35 kGy.

[0055] In steps (5), (8), (10) and (12), the phosphate buffer is a sodium phosphate buffer.

[0056] In the process for producing the stromal material for cell encapsulation according to the present invention, decellularization treatment with Alcalase may be performed first, and then the cartilage tissue may be pulverized.

[0057] The interstitial material for cell encapsulation of the present application is obtained by the above-mentioned production method.

[0058] The main components of the stromal material for cell encapsulation are type II collagen and glycosaminoglycans (chondroitin sulfate and hyaluronic acid).

[0059] The present stromal material for cell encapsulation is applicable to 3D bioprinting, as well as for filling and autologous lipogenesis induction in the field of aesthetic medicine.

[0060] The technical contents of the present application will be further described below with reference to examples. The following examples are for illustrative purposes only and are not intended to be limiting, and the following examples cannot limit the scope of protection of the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from the market unless otherwise specified. EXAMPLES

[0061] Example 1 The method for producing a stromal material for cell encapsulation in this example is, as an option, a method not intended for the diagnosis or treatment of a disease, and includes the following steps (1) to (7).

[0062] In step (1), pig ear cartilage is collected from a freshly slaughtered 6-month-old pig, the skin, connective tissue and fascia on the surface of the cartilage are removed, the cartilage tissue is cut into small chunks with a thickness of 2 mm or less, the surface is disinfected with 0.2% w / v sodium hypochlorite for 10 min, washed thoroughly with purified water and freeze-dried.

[0063] In step (2), the freeze-dried cartilage is placed in a 200 mL grinding bottle, the sealed container with the cartilage sample is immersed in liquid nitrogen, and the sample is rapidly ground by electromagnetic collider grinding technique using a SPEX 6875 freezer mill in an environment of -196 °C. After temperature recovery, a 100 μm mesh is used to select small-diameter cartilage microparticles. Measured using a laser diffraction particle size distribution measuring device, the particle sizes distributed based on the particle number are Dx (10) = 1.8 μm, Dx (50) = 2.9 μm, Dx (90) = 6.0 μm, with an average particle size of 2 to 10 μm, and the particle sizes distributed based on the particle volume are Dv (10) = 15 μm, Dv (50) = 67 μm, Dv (90) = 144 μm, with an average particle size of 30 to 100 μm.

[0064] In step (3), the cartilage microparticles are soaked in a 70% w / v isopropyl alcohol solution to degrease them, with a solid-liquid ratio of 1:5 (i.e., 5L of 70% w / v isopropyl alcohol solution is added per 1L of cartilage microparticles), and after 1h of degreasing, they are centrifuged at 500xg for 5min, the supernatant waste liquid is discarded, and a first precipitate is obtained. In a solid-liquid ratio of 1:5, 10mmol / L sodium phosphate buffer (pH=7.4) is added, and after 30min of soaking, they are centrifuged at 500xg for 5min, the supernatant waste liquid is discarded, and a second precipitate is obtained, which is washed once with sodium phosphate buffer.

[0065] In step (4), the cartilage microparticles after defatting are soaked in a buffer solution of DNase solution, the concentration of hydroxyethylpiperazineethanesulfonic acid buffer in the buffer solution of DNase solution is 10mmol / L, the activity of DNase in the DNase solution is 50U / L, pH=7.6, solid-liquid ratio is 1:5, after soaking for 8h, centrifuged at 500xg for 5min, waste liquid is discarded, and a third precipitate is obtained. Then, alcalase solution is added to the third precipitate in a solid-liquid ratio of 1:5, the concentration of alcalase in the solution is 0.05%w / v, mixed with 10mmol / L sodium phosphate buffer, pH=7.4, treated with alcalase for 120min, centrifuged at 500xg for 5min, and the supernatant waste liquid is discarded. After treating with alcalase, add sodium deoxycholate solution to the fourth precipitate in a solid-liquid ratio of 1:5, the concentration of sodium deoxycholate is 1.0% w / v, formulated in 10 mmol / L sodium phosphate buffer, pH=7.4, and after treating for 12 h, centrifuge at 500×g for 5 min, discard the supernatant waste liquid, and obtain the fifth precipitate.

[0066] In step (5), the material that has been degreased, enzyme-treated and washed with detergent is added to an EDTA solution, the concentration of EDTA is 10 mmol / L, it is mixed with 10 mmol / L sodium phosphate buffer, pH=7.4, the solid-liquid ratio is 1:5, it is soaked for 2 hours and washed, centrifuged at 500xg for 5 minutes, the supernatant waste liquid is discarded, the sixth precipitate is obtained, it is repeatedly washed with EDTA solution once, it is then soaked for 2 hours and washed with 10 mmol / L sodium phosphate buffer (pH=7.4) for 2 hours and centrifuged at 500xg for 5 minutes, the supernatant waste liquid is discarded, the seventh precipitate is obtained, it is repeatedly washed with sodium phosphate solution once.

[0067] In step (6), the washed material is added to a mixture of peracetic acid for virus inactivation, a mixture of 2% w / v sodium phosphate and 0.2% w / v peracetic acid is added in a solid-liquid ratio of 1:5, the virus inactivation is performed for 60 min, then centrifuged at 500 x g for 5 min, the supernatant waste is discarded, and the 8th precipitate is obtained. Then, 0.9% w / v saline is added in a solid-liquid ratio of 1:5, the material is soaked for 60 min, centrifuged at 500 x g for 5 min, the supernatant waste is discarded, and the 9th precipitate is obtained.

[0068] In step (7), the virus-inactivated material is washed twice with sterile purified water, the solid-liquid ratio is 1:5, and the ninth precipitate is washed by adding sterile water, each time for 30 min, followed by centrifugation at 500 x g for 5 min, and the supernatant waste liquid is discarded. The dry weight concentration of the treated microparticle sample is adjusted to 3% w / v and then freeze-dried. After freeze-drying, it is sterilized by gamma radiation, and the irradiation dose is 17.1 kGy. A stromal material for cell encapsulation based on cartilage material is produced and obtained.

[0069] The residual DNA content of the cartilage matrix microparticle material produced by the above method was 2.7±0.6ng / mg dry weight, which was 99.6% lower than that of the untreated freeze-dried cartilage raw material (679.8±7.3ng / mg dry weight). The collagen content of the treated cartilage matrix microparticle material was 92.4±0.4% w / w dry weight, and that of the untreated freeze-dried cartilage raw material was 50.9±5.2% w / w dry weight, indicating that other tissue proteins were effectively removed by the alcalase treatment and decellularization process. The chondroitin sulfate content of the treated cartilage matrix microparticle material was 25.5±0.9ug / mg dry weight (2.55±0.09% w / w dry weight), and that of the untreated freeze-dried cartilage raw material was 80.5±4.1ug / mg dry weight (8.05±0.41% w / w dry weight). The hyaluronic acid content of the processed cartilage matrix microparticle material is 16.6±0.4ug / mg dry weight (1.66±0.04% w / w dry weight), and the hyaluronic acid content of the unprocessed freeze-dried cartilage raw material is 136.6±7.3ug / mg dry weight (13.66±0.73% w / w dry weight).The component analysis showed that the processed cartilage matrix microparticle material is a natural composite material of collagen, chondroitin sulfate and hyaluronic acid, and the sum of the above three is 96.5±0.53% w / w dry weight.

[0070] The cartilage matrix microparticles produced by the above method were rehydrated with 0.9% w / v sodium chloride solution and then measured using a laser diffraction particle size distribution analyzer. The produced cartilage matrix material had particle sizes distributed by number Dx(10) = 2.8 μm, Dx(50) = 4.0 μm, Dx(90) = 8.7 μm, with a mean particle size of 3-8 μm, particle sizes distributed by volume Dv(10) = 13 μm, Dv(50) = 80 μm, Dv(90) = 400 μm, with a mean particle size of 40-200 μm. The particle size of the cartilage matrix microparticle material that underwent Alcalase treatment and decellularization treatment increased.

[0071] For the cartilage matrix microparticle material produced by the above method, the number of α-Gal antigen epitopes was quantitatively detected by referring to the method in the standard "Measurement of Residual α-Gal Antigen in Animal-Derived Scaffold Materials for Tissue Engineering Medical Device Products" (YY / T 1561-2017). The number of α-Gal antigen epitopes in untreated pig ear cartilage raw material was 5.4 × 10 13 ±5.2×10 12 The number of α-Gal antigen epitopes in the processed cartilage matrix particle material was 2.2 × 10 12 ±3.0×10 11 The number of α-Gal antigen epitopes was as low as 1 / g dry weight of material, and 95.9% of the epitopes were removed by Alcalase treatment and decellularization.

[0072] The surface topological structure of cartilage matrix particles has an important effect on responsive behaviors such as cell attachment and proliferation, and also has important physiological effects on cell morphology, phenotype, and cell movement. The surface structure of the cartilage matrix microparticles produced by the Alcalase treatment and decellularization process of this example was observed using an ultra-high resolution scanning electron microscope (SU8000), and the results showed that the cartilage matrix microparticles have a three-dimensional porous, uneven, rough surface structure and an extremely large surface area (Figure 1).

[0073] The cartilage matrix microparticle material produced by the above method was mixed into a 4% w / v suspension in 0.9% w / v sodium chloride solution (physiological saline), and after leaving it for 5 days, there was no precipitation or separation, and it had good suspension stability (Figure 2). If only decellularization was performed without alcalase treatment, precipitation occurred in the microparticle material, indicating that the suspension stability was poor. The improvement in suspension stability of cartilage matrix microparticles after alcalase treatment and decellularization was related to the increase in the particle size of the cartilage matrix particles after treatment and reduced the density of the matrix particles.

[0074] Using a differential scanning calorimeter (DSC), the thermal stability of the cartilage raw material rehydrated in saline and the produced cartilage matrix particle material were compared (Figure 3). As a result, the thermal stability of the cartilage matrix particles produced by the enzyme treatment and decellularization method of the present application was good, and at 40°C or higher, it was almost similar to that of the untreated cartilage raw material, indicating that it was not destroyed during the production process. Due to the increase in collagen content, the enthalpy during denaturation of the produced cartilage matrix particle protein was significantly improved. In addition, DSC measurement was performed after storing the cartilage matrix microparticles at 37°C for 24 hours, and no change occurred in the differential scanning calorimetry analysis diagram, which showed the thermal stability of the cartilage matrix particles.

[0075] To verify whether the cartilage matrix microparticles that had undergone Alcalase treatment and decellularization have good biosafety, L929 cells were co-cultured with the cartilage matrix microparticles for 48 hours. As a result, the cells exhibited a relatively significant aggregation growth phenomenon, indicating that the material has good safety and exhibits good biocompatibility to a certain extent (Figure 4).

[0076] To further verify the ability of cartilage matrix microparticles that had undergone Alcalase treatment and decellularization to support cell attachment and proliferation, human umbilical cord mesenchymal stem cells were co-cultured with cartilage matrix microparticles to observe their cell attachment and cytotoxicity. The experimental results showed that the co-culture of the material did not show significant cytotoxicity, and a large number of cells were attached to the surface of the cartilage matrix microparticles, demonstrating good performance in supporting stem cell attachment and proliferation (Figure 5). Cartilage-based matrix materials can be used as microcarriers for cell culture and can also promote the formation of cell clusters.

[0077] The cartilage matrix microparticle material produced by the above method has reversible temperature response characteristics in terms of function (Figure 6). At 37°C, it exhibits a state similar to a colloidal solution and has fluidity, and when the temperature is lowered, it exhibits a gel state and does not have fluidity, and can encapsulate cells by self-assembly when it is converted from a colloid to a gel. By utilizing this property of encapsulating cells by self-assembly when it is converted from a colloid to a gel, it is possible to protect cells from the influence of adverse environments during the process of cell storage and transportation, and to improve the cell viability. Since the temperature response characteristics of the cartilage matrix microparticles are reversible, cells can depolymerize when the temperature rises to 37°C.

[0078] After co-cultivating L929 cells and 2.5% cartilage matrix microparticles at 37°C for 48h, the cell suspension was transferred to a 25°C thermostatic metal bath and a 4°C refrigerator for storage to verify the protective effect of cartilage matrix gel on cells in adverse conditions. On the first and fourth days after transfer to a low-temperature environment, the activity of L929 cells was detected by CCK-8 kit. The cell activity on the first and fourth days in the 25°C thermostatic metal bath was 0.424±0.008 and 0.459±0.022, respectively, indicating no change in cell activity. The cell activity on the first and fourth days in the 4°C refrigerator was 0.410±0.012 and 0.344±0.020, respectively, indicating a slight decrease in cell activity.

[0079] The cartilage matrix microparticle material produced by the above method was injected into the dorsal subcutaneous layer of a rat, and it functioned well as a scaffold to induce the formation of a large amount of adipose tissue (Figure 7). Three weeks after implantation, an inflammatory reaction was observed, but the degree of inflammation decreased over time, and there was no chronic inflammatory reaction. After six weeks, neovascularization occurred in the area of ​​the cartilage matrix microparticles, and the cartilage matrix microparticles degraded to a certain extent over time. After ten weeks, adipocytes began to invade and grow, and by 16 weeks, the area was completely filled with adipocytes. For this reason, it can also be used for filling and autologous lipogenesis induction in the field of aesthetic medicine.

[0080] Example 2 The method for producing a cartilage matrix material in this example is not intended for the diagnosis or treatment of a disease, and the other steps are completely the same as those in Example 1 except for one step below, which is different from Example 1. In step (4), after immersion in a deoxyribonuclease solution, a sodium deoxycholate solution is directly added and treated without going through an alcalase solution treatment.

[0081] The cartilage matrix microparticles produced by the method of Example 2 are significantly different from those produced by the method of Example 1 (Table 1). The residual DNA content of the cartilage matrix microparticles treated by the method of Example 1 is relatively low, and the number of remaining α-Gal antigen epitopes is small. The residual DNA content of the cartilage matrix microparticles treated by the method of Example 2 is relatively high, and the number of remaining α-Gal antigen epitopes is relatively high. The total content of collagen, chondroitin sulfate, and hyaluronic acid in the cartilage matrix microparticle material treated by the method of Example 1 is 96.5±0.53% w / w dry weight, while the total content of collagen, chondroitin sulfate, and hyaluronic acid in the cartilage matrix microparticle material treated by the method of Example 2 is only 75.1±5.2% w / w dry weight. The detection results show that the effect of alcalase treatment is significant.

[0082] [Table 1]

[0083] Example 3 The method for producing a stromal material for cell encapsulation in this example is a method not intended for the diagnosis or treatment of a disease, and includes the following steps (1) to (3).

[0084] In step (1), freshly mashed 6-month-old pig ear cartilage is cleaned of other tissue, and the cartilage, approximately 2 mm thick, is cut into small blocks measuring 2 cm x 3 cm. A 1:5 solid-liquid ratio is added to the cartilage for disinfection for 10 min, after which the waste liquid is discarded. 70% w / v isopropyl alcohol is added for 60 min to degrease the cartilage, after which the waste liquid is discarded, and the cartilage is then immersed in a 0.125% w / v alcalase solution for 18 h.

[0085] In step (2), the material after the Alcalase treatment is subjected to a virus inactivation treatment using a mixture of peracetic acid. A mixture containing 2% w / v sodium phosphate and 0.15% w / v peracetic acid is added in a solid-liquid ratio of 1:5. After the virus inactivation treatment is performed for 120 minutes, the waste liquid is discarded, and then an EDTA solution is added. The concentration of the EDTA solution is 10 mmol / L, and it is formulated with a 10 mmol / L sodium phosphate buffer solution, pH=7.4, and the solid-liquid ratio is 1:5. The material is soaked for 24 hours and washed, and the waste liquid is discarded. The EDTA solution is then repeatedly used for washing for 24 hours.

[0086] In step (3), the sections are washed with saline for 24 hours.

[0087] In the process of preparing cartilage decellularized matrix material by the method of Example 3, samples were taken after 5 hours of treatment with alcalase to detect the progress of decellularization. After 5 hours of treatment with alcalase, there is a cell-containing area in the middle of the cartilage tissue similar to untreated cartilage, but after 18 hours of treatment with alcalase, the entire cartilage is completely changed, and the remaining DNA content is 6.79±0.65 ng / mg dry weight. Histological sections of pig ear cartilage after 5 hours (left) and 18 hours (right) of treatment with alcalase are shown in FIG. 8. The collagen content of the cartilage matrix after alcalase treatment is 76.5±6.5% w / w dry weight, which indicates that cartilage decellularization can be effectively performed by alcalase treatment, but the efficiency is significantly lower when using intact cartilage compared to the microparticulated material (Example 1).

[0088] Example 4 The method for producing a stromal material for cell encapsulation in this example is a method not intended for the diagnosis or treatment of a disease, and includes the following steps (1) to (11).

[0089] In step (1), freshly crushed costal cartilage from a 22-month-old cow is removed of other tissues, the cartilage tissue is cut into slices with a thickness of 2 mm or less, the cartilage tissue slices are degreased and disinfected by adding 50% w / v isopropyl alcohol in a solid-liquid ratio of 1:5, soaked for 30 min, and then centrifuged at 1500 x g for 20 min, the waste liquid is discarded, and a first precipitate is obtained.

[0090] In step (2), the cartilage tissue slices treated with isopropyl alcohol are added to an alcalase solution at a solid-liquid ratio of 1:10, the concentration of alcalase in the solution is 0.2% w / v, and the solution is mixed with a 10 mmol / L sodium phosphate buffer solution at pH=7.4. After treatment for 30 min, the solution is centrifuged at 1500×g for 20 min, the waste liquid is discarded, and a second precipitate is obtained.

[0091] In step (3), the cartilage tissue slices treated with alcalase are added to Triton X-100 in a solid-liquid ratio of 1:20, and then mixed with 10 mmol / L sodium phosphate buffer, pH=7.4. After 16 h treatment, the slices are centrifuged at 1500×g for 20 min, the waste liquid is discarded, and the third precipitate is obtained.

[0092] In step (4), the washed and soaked cartilage tissue slices are processed in 0.9% w / v saline with a solid-liquid ratio of 1:5 for 30 min, centrifuged at 1500 x g for 20 min, the waste liquid discarded, the solution replaced, soaked again for 30 min, centrifuged at 1500 x g for 20 min, the waste liquid discarded, and a fourth precipitate is obtained.

[0093] In step (5), the washed cartilage tissue slices are immersed in a buffer solution of DNase solution with a solid-liquid ratio of 1:20, the buffer solution contains 5 mmol / L hydroxyethylpiperazine ethanesulfonic acid buffer, the DNase activity in the DNase solution is 250 U / L, and the pH is 7.6. After treatment for 12 hours, the slices are centrifuged at 1500xg for 20 minutes, the waste liquid is discarded, and the fifth precipitate is obtained.

[0094] In step (6), the DNase-treated cartilage tissue slices are treated in a solid-liquid ratio of 1:20 by soaking in 10 mmol / L sodium phosphate buffer (pH=7.4) for 120 min, centrifuged at 1500 x g for 5 min, the waste liquid is discarded, the solution is replaced and soaked again for 120 min, centrifuged at 1500 x g for 5 min, the waste liquid is discarded, and the sixth precipitate is obtained.

[0095] In step (7), the virus is inactivated in a mixture containing 10% w / v sodium chloride and 0.5% w / v peracetic acid in a solid-liquid ratio of 1:10 for 30 min, centrifuged at 1500 x g for 20 min, the waste liquid is discarded, and the seventh precipitate is obtained.

[0096] In step (8), the cartilage tissue slices that have undergone virus inactivation treatment are immersed in an EDTA solution with a solid-liquid ratio of 1:20, the concentration of the EDTA solution is 50 mmol / L, the solution pH is 7.4, and after washing for 4 hours, they are centrifuged at 1500 x g for 20 minutes, the waste liquid is discarded, and the EDTA solution is repeatedly used to wash once, centrifuged at 1500 x g for 20 minutes, the waste liquid is discarded, and the eighth precipitate is obtained.

[0097] In step (9), the treated cartilage tissue slices are washed in sterile water at a solid-to-liquid ratio of 1:20 for 60 min, and the washed cartilage tissue slices are freeze-dried to a water content of less than 5% w / w.

[0098] In step (10), the freeze-dried cartilage tissue slices are placed in a 200 mL grinding bottle, the sealed container with the cartilage sample is immersed in liquid nitrogen, and the sample is rapidly ground by electromagnetic collider grinding technique using a SPEX 6875 freezer mill in an environment of -196 ° C. After temperature recovery, a 100 μm mesh is used to select small-sized cartilage particles.

[0099] In step (11), the particulate material is sterilized by gamma radiation or electron beam to prepare interstitial material for cell encapsulation, the sterilization dose is 15 kGy.

[0100] Cartilage matrix particulate material produced by the method of Example 4 above and cartilage matrix particulates produced by the method of Example 1 have similar characteristics.

[0101] Example 5 The method for producing a stromal material for cell encapsulation in this example includes the following steps (1) to (8).

[0102] In step (1), freshly mashed 6-month-old pig ear cartilage is stripped of other tissues, degreased with 70% w / v isopropyl alcohol for 60 min, the waste liquid is discarded, and the cartilage is thoroughly washed with purified water and freeze-dried.

[0103] In step (2), the freeze-dried cartilage is placed in a sealed container and immersed in liquid nitrogen, and then low-temperature polished using a freezer mill in an environment of -196°C (see Example 1). The cartilage is then sieved by particle size using a screen mesh to obtain cartilage microparticles of different particle sizes (<0.1 mm, 0.1-0.2 mm, 0.2-0.3 mm, 0.3-0.4 mm).

[0104] In step (3), cartilage microparticles of different particle sizes are immersed in a buffer solution containing a deoxyribonuclease solution, and 12 mL of the solution is added per 1 gram of dry weight of the cartilage microparticles. The buffer solution contains 5 mmol / L hydroxyethylpiperazine ethanesulfonic acid buffer, and the deoxyribonuclease activity in the deoxyribonuclease solution is 500 U / L (pH=7.6). After treatment at 37°C for 16 hours, the mixture is centrifuged at 1500xg for 20 minutes and the waste liquid is discarded.

[0105] In step (4), 6 mL of 0.05% w / v alcalase is added per gram dry weight of cartilage microparticles, treated at 37° C. for 2 hours, centrifuged at 1500×g for 20 minutes, and the waste liquid is discarded.

[0106] In step (5), 6 mL of 0.5% w / v sodium deoxycholate (SDOC) is added per gram dry weight of cartilage microparticles, the mixture is treated at room temperature for 6 h, centrifuged at 1500 x g for 20 min, and the waste liquid is discarded.

[0107] In step (6), use a solution of penicillin / streptomycin-containing phosphate buffer (pH = 7.0, containing 100 U penicillin / streptomycin / mL) to wash four times at room temperature, for 8 h each time, and then centrifuge at 1500 x g for 20 min to collect the precipitated cartilage matrix microparticles.

[0108] In step (7), the cartilage matrix microparticles are diluted with 0.9% w / v sodium chloride solution to a concentration of 5% w / w.

[0109] In step (8), terminal disinfection is performed by gamma ray irradiation (19.03 kGy to 20.08 kGy), and the waste is sampled and analyzed.

[0110] Cartilage stromal materials made from cartilage microparticles of different particle sizes had different degrees of aggregation. The degree of aggregation of the stromal materials increased with the increase in particle size of the cartilage microparticles (Figure 9). Only when the particle size was <0.1 mm, good dispersibility was observed, and when the particle size was >0.3 mm, significant aggregation and interweaving occurred.

[0111] The DNA content of cartilage microparticles before decellularization is about 411ng / mg, and the DNA content after decellularization is lower than 20ng / mg. The collagen content in the cartilage matrix is ​​83.5±1.2%w / w~90.5±1.5%w / w, and the larger the particle size, the higher the collagen content. The elastin content in the cartilage matrix is ​​1.6~4.0%w / w. The chondroitin sulfate content in the cartilage decellularized matrix microcarrier was measured using a sulfated glycosaminoglycan (sGAG) assay kit, and the chondroitin sulfate content was 7.1±0.7%w / w.

[0112] Cartilage matrix particulate material produced by the method of Example 5 above and cartilage matrix particulates produced by the method of Example 1 have similar characteristics.

[0113] The above description of the embodiments is for those skilled in the art to understand and use the present application. Those skilled in the art can make various modifications to these embodiments and apply the general principles described to other embodiments without any creative effort. Therefore, the present application is not limited to the above embodiments. Any improvements and modifications made by those skilled in the art based on the principles of the present application without departing from the scope of the present application are included in the protection scope of the present application.

Claims

1. 1. A method for producing a stromal material for cell encapsulation, comprising: (1) removing surface tissue and fascia from an in vitro mammalian cartilage material and cutting it into thin slices; (2) a step of disinfecting the surface of the cartilage tissue slices with a disinfectant, washing and freeze-drying; (3) subjecting the dried and frozen cartilage tissue slices to liquid nitrogen low-temperature microparticulation grinding to produce a microparticulate material; (4) degreasing the particulate material with isopropyl alcohol in a solid-liquid ratio of 1:5 to 1:20, centrifuging after soaking, and discarding the waste liquid to obtain a first precipitate; (5) treating the first precipitate by soaking in a saline or phosphate buffer solution at a solid-liquid ratio of 1:5 to 1:20, centrifuging, and discarding the waste liquid to obtain a second precipitate; (6) treating the second precipitate with a buffered deoxyribonuclease solution in a solid-liquid ratio of 1:5 to 1:20, centrifuging, discarding the waste liquid, and obtaining a third precipitate; (7) treating the third precipitate by soaking in an alcalase solution at a solid-liquid ratio of 1:5 to 1:10, centrifuging, and discarding the waste liquid to obtain a fourth precipitate; (8) adding a detergent to the fourth precipitate in a solid-liquid ratio of 1:5 to 1:20, treating the fourth precipitate by blending with a phosphate buffer, centrifuging, and discarding the waste liquid to obtain a fifth precipitate; (9) soaking the fifth precipitate with disodium ethylenediaminetetraacetate solution at a solid-liquid ratio of 1:5 to 1:20, washing, centrifuging, discarding the waste liquid, exchanging the solution, washing again, centrifuging again, discarding the waste liquid, to obtain a sixth precipitate; (10) treating the sixth precipitate by soaking in a saline solution or a phosphate buffer solution in a solid-liquid ratio of 1:5 to 1:20, centrifuging, discarding the waste liquid, replacing the solution and repeating the procedure to obtain a seventh precipitate; (11) adding a mixture of sodium phosphate and peracetic acid to the seventh precipitate in a solid-liquid ratio of 1:5 to 1:10 to inactivate the viruses, followed by centrifugation and discarding the waste liquid to obtain an eighth precipitate; (12) treating the eighth precipitate by soaking in saline or phosphate buffer at a solid-liquid ratio of 1:5 to 1:20, centrifuging, and discarding the waste liquid to obtain a ninth precipitate; and (13) washing the ninth precipitate with sterile water in a solid-liquid ratio of 1:5 to 1:20, freeze-drying, and sterilizing the freeze-dryed precipitate with gamma radiation or electron beam.

2. In step (1), the mammal is one or more selected from the group consisting of pigs, cows, sheep, horses, and deer; Preferably, in step (1), the cartilage material is one or more selected from elastic cartilage and hyaline cartilage; Preferably, the elastic cartilage is ear cartilage; The method for producing a stromal material for cell encapsulation according to claim 1, wherein the hyaline cartilage is preferably at least one selected from the group consisting of articular cartilage, costal cartilage, scapular cartilage and meniscus.

3. In step (2), the disinfectant is at least one selected from 0.1-0.5% w / v sodium hypochlorite, 0.5-2.0% w / v sodium carbonate, and 50-70% w / v alcohol solution; Preferably, the alcohol solution is one or more selected from ethanol or isopropyl alcohol; The method for producing an interstitial material for cell encapsulation according to claim 1, characterized in that in step (3), the average particle size based on the particle number of the microparticle material is 2 to 20 μm, and the average particle size based on the particle volume of the microparticle material is 20 to 200 μm.

4. In step (4), the content of the isopropyl alcohol is 50 to 70% w / v, the soaking time is 30 to 60 min, the rotation speed of the centrifuge is 500 to 1500 x g, and the time is 5 to 20 min; Preferably, in step (5), the immersion time is 30 to 60 min, the centrifuge speed is 500 to 1500 xg, and the centrifuge time is 5 to 20 min; Preferably, in step (6), the buffer solution of the deoxyribonuclease is one or more selected from a hydroxyethylpiperazineethanesulfonic acid buffer solution and a trishydroxymethylaminomethane hydrochloride buffer solution, and the activity of deoxyribonuclease in the deoxyribonuclease solution is 50 to 250 U / L; Preferably, the concentration of the hydroxyethylpiperazine ethanesulfonic acid buffer is 5 to 100 mmol / L; The method for producing an interstitial material for cell encapsulation according to claim 1, characterized in that in step (6), the soaking time is 8 to 12 hours, the rotation speed of the centrifuge is 500 to 1500 xg, and the time is 5 to 20 minutes.

5. In step (7), the concentration of alcalase in the alcalase solution is 0.02 to 0.2% w / v; Preferably, in step (7), the immersion time is 30 to 120 min, the centrifuge speed is 500 to 1500 x g, and the centrifuge time is 5 to 20 min; Preferably, in step (8), the detergent is one or more selected from sodium deoxycholate, Triton X-100, and sodium dodecyl sulfate; The method for producing an interstitial material for cell encapsulation according to claim 1, characterized in that in step (8), the soaking time is 8 to 16 hours, the rotation speed of the centrifuge is 500 to 1500 xg, and the time is 5 to 20 minutes.

6. In step (9), the concentration of disodium ethylenediaminetetraacetate in the disodium ethylenediaminetetraacetate solution is 5 to 50 mmol / L, and is mixed with a 10 mmol / L sodium phosphate buffer; Preferably, in step (9), the washing time is 2 to 4 h, the centrifugation speed is 500 to 1500 x g, and the time is 5 to 20 min; Preferably, in step (9), the re-washing time is 12 to 24 h, the re-centrifugation speed is 500 to 1500 x g, and the time is 5 to 20 min; The method for producing an interstitial material for cell encapsulation according to claim 1, characterized in that in step (10), the soaking time is 30 to 120 min, the rotation speed of the centrifuge is 500 to 1500 xg, and the time is 5 to 20 min.

7. In step (11), the time for the virus inactivation treatment is 30 to 60 minutes, the rotation speed of the centrifuge is 500 to 1500 x g, and the time is 5 to 20 minutes; The method for producing an interstitial material for cell encapsulation according to claim 1, characterized in that in step (12), the soaking time is 30 to 120 min, the rotation speed of the centrifuge is 500 to 1500 xg, and the time is 5 to 20 min.

8. 8. The method for producing an interstitial material for cell encapsulation according to claim 1, wherein in steps (5), (8), (10) and (12), the phosphate buffer is a sodium phosphate buffer.

9. A stromal material for cell encapsulation, obtained by the method according to any one of claims 1 to 8, Preferably, the main components of the cell encapsulation stromal material are type II collagen and glycosaminoglycan; Preferably, the glycosaminoglycan comprises chondroitin sulfate and hyaluronic acid.

10. The cell encapsulation stromal material contains type II collagen, chondroitin sulfate, and hyaluronic acid, and the sum of the type II collagen, chondroitin sulfate, and hyaluronic acid is 85% w / w or more by dry weight, preferably 90% w / w or more by dry weight, of the cell encapsulation stromal material; Preferably, the type II collagen is at least 75% w / w, the elastin is less than 10% w / w, and the glycosaminoglycan content is between 1% and 10% w / w, said glycosaminoglycans including chondroitin sulfate and hyaluronic acid; Preferably, the content of type II collagen, chondroitin sulfate and hyaluronic acid is 92% w / w or more (dry weight), 2% w / w or more (dry weight) and 1% w / w or more (dry weight), respectively; Preferably, the residual DNA content in the cell encapsulation stromal material is less than 20 ng / mg, preferably not more than 5 ng / mg; Preferably, the number of α-Gal antigen epitopes in the cell encapsulation stromal material is 5.0×10 12 The interstitial material for cell encapsulation according to claim 9, characterized in that the cell encapsulation density is less than 10 ...

11. 11. Application of the stromal material for cell encapsulation according to claim 9 or 10 in 3D bioprinting.

12. 11. Application of the cell encapsulation stromal material according to claim 9 or 10 in the encapsulation or protection or encapsulation and protection of cells.

13. 11. The application of the stromal material for cell encapsulation according to claim 9 or 10 in tissue regeneration and repair by injection into the body, or in filling and autologous lipogenesis induction in the field of aesthetic medicine.

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