Interstitial materials for cell encapsulation and their manufacturing methods and applications

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 in their entirety.

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

[0003] Cell therapy refers to a treatment method in which normal cells or cells modified through biotechnology are expanded 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 enhanced immune-killing functions (immune cell therapy technology), thereby achieving the goal of treating disease. Stem cell therapy utilizes the self-renewal, pluripotency, and high proliferation capacity of stem cells to transplant healthy stem cells into the body to repair lesions or restore normal tissue function. Immune cell therapy involves collecting immune cells from the body, modifying and expanding them in vitro, and then returning them to the body. Their enhanced targeted cytotoxicity allows them to kill pathogens, cancer cells, and mutant cells, thereby activating and strengthening the body's immune system. With the rapid development of fields such as molecular biology, stem cell biology, tissue engineering, and regenerative medicine, cell therapy has become increasingly important in clinical practice. Currently, many types of cells are 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 cells 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, proliferation, preservation, and transportation are essential components of cell therapy. Currently, two methods are used for cell expansion. One is in vitro 2D culture, in which cells are propagated by passage when they reach confluence. The drawbacks of this method are the low efficiency of in vitro 2D culture and the poor 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, porous microcarrier cell immobilization technology is used. Various materials 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 achieve carrier-free immobilization of cells. This involves controlling the formation and depolymerization of cell clusters during the cell culture process to monitor cell growth and achieve efficient cell culture.

[0005] The extracellular matrix (ECM) is a biological macromolecule present between cells and secreted by cells, including collagen, elastin, proteoglycans, glycosaminoglycans, and cytokines. The extracellular matrix (ECM) has a complex spatial network structure and serves as a microenvironment for cell growth and activity. Interactions between cells and the ECM have a regulatory effect on cellular functions and behaviors (e.g., adhesion, growth, proliferation, differentiation, migration, intercellular signaling, etc.). Therefore, ECM materials can be used to fabricate microcarriers for cell immobilization, as active materials for promoting cell cluster formation, or as cell encapsulation stroma (CES) for cell encapsulation or protection during cell storage and transportation. Patent CN105288737A discloses a tissue-engineered cartilage composite scaffold based on cartilage extracellular matrix and a method for fabricating the same. The invention relates to the use of cartilage extracellular matrix particles to rapidly expand chondrocyte seed cells and induce stem cell differentiation into chondrocytes. The specific fabrication method of this invention includes the following steps 1) to 3). 1) Fresh articular cartilage is crushed and sieved using a low-temperature wet method to obtain cartilage particles with diameters of 100 to 500 μm. After decellularization, cartilage extracellular matrix microcarriers are produced and obtained. 2) Cartilage extracellular matrix microcarriers and cartilage seed cells are placed in a bioreactor and co-cultured to form cartilage microtissues by rapidly amplifying chondrocytes or inducing stem cell differentiation into chondrocytes. 3) Cartilage microtissues are filled into the gaps of a three-dimensional porous scaffold made of a water-containing gel precursor. The cartilage microtissues and the three-dimensional porous scaffold are then bonded using a coagulant, resulting in a tissue-engineered cartilage composite scaffold. The use of extracellular matrix particles or hydrogels for cell co-culture and promoting specific stem cell differentiation has been extensively studied. Summary of the Invention

[0006] The following is a summary of the subject matter described herein, which does not 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 them, and freeze-drying them; (3) a step of subjecting the dried and frozen cartilage tissue slices to low-temperature micro-grinding with liquid nitrogen to produce a micro-particle material; Particulate material in isopropyl alcohol at a solid-to-liquid ratio of 1:5 to 1:20. Soaking More degreasing treatment , far Step (4) of centrifugation, discarding the waste liquid, and obtaining a first precipitate; (5) adding physiological saline or phosphate buffer to the first precipitate at a solid-liquid ratio of 1:5 to 1:20, soaking the first precipitate, centrifuging the solution, and discarding the waste liquid to obtain a second precipitate; Add deoxyribonuclease solution to the second precipitate at a solid-to-liquid ratio of 1:5 to 1:20. Liquid Step (6) of adding, soaking, treating, centrifuging, discarding the waste liquid, and obtaining a third precipitate; (7) adding an alcalase solution to the third precipitate at a solid-liquid ratio of 1:5 to 1:10, soaking the solution, centrifuging the solution, 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 it by blending it with a phosphate buffer solution, centrifuging it, and discarding the waste liquid to obtain a fifth precipitate; Add disodium ethylenediaminetetraacetic acid solution to the fifth precipitate in a solid-liquid ratio of 1:5 to 1:20, soak, wash, centrifuge, and discard the waste liquid; Add disodium ethylenediaminetetraacetate solution again Step (9) of washing again, centrifuging again, discarding the waste liquid, and obtaining a sixth precipitate; The sixth precipitate is treated by soaking in saline or phosphate buffer at a solid-liquid ratio of 1:5 to 1:20, centrifuged, and the waste liquid is discarded; Add saline or phosphate buffer again. Step (10) of repeating the procedure to obtain a seventh precipitate; (11) adding a mixture of sodium phosphate and peracetic acid to the seventh precipitate at a solid-liquid ratio of 1:5 to 1:10 to inactivate the virus, centrifuging, and discarding the waste liquid to obtain an eighth precipitate; (12) adding physiological saline or phosphate buffer to the eighth precipitate at a solid-liquid ratio of 1:5 to 1:20, soaking the precipitate, centrifuging the precipitate, and discarding the waste liquid to obtain a ninth precipitate; and (13) adding sterile water to the ninth precipitate at a solid-liquid ratio of 1:5 to 1:20 to wash it, freeze-dry it, and sterilizing it with gamma rays or electron beams 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 the above-mentioned cell encapsulation stromal material, i.e., use in promoting the formation of cell clusters in cell culture processes, use as an interstitial material for encapsulating or protecting cells in the process of cell preservation and transportation, or application in 3D bioprinting, or application in the field of aesthetic medicine. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a scanning electron microscope image of cartilage matrix microparticles after Alcalase treatment and decellularization in Example 1 of the present application. [Figure 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 Alcalase treatment (right). [Figure 3] FIG. 1 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. [Figure 5]FIG. 1 shows the cell growth status on the microparticles after 8 hours and 8 days of co-culture of umbilical cord mesenchymal stem cells and cartilage matrix microparticles in Example 1 of the present application. [Figure 6] FIG. 1 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 INVENTION

[0011] In an embodiment of the 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 them, and freeze-drying them; (3) a step of subjecting the dried and frozen cartilage tissue slices to low-temperature micro-grinding with liquid nitrogen to produce a micro-particle material; Particulate material in isopropyl alcohol at a solid-to-liquid ratio of 1:5 to 1:20. Soaking More degreasing treatment , far Step (4) of centrifugation, discarding the waste liquid, and obtaining a first precipitate; (5) adding physiological saline or phosphate buffer to the first precipitate at a solid-liquid ratio of 1:5 to 1:20, soaking the first precipitate, centrifuging the solution, and discarding the waste liquid to obtain a second precipitate; Add deoxyribonuclease solution to the second precipitate at a solid-to-liquid ratio of 1:5 to 1:20. Liquid Step (6) of adding, soaking, treating, centrifuging, discarding the waste liquid, and obtaining a third precipitate; (7) adding an alcalase solution to the third precipitate at a solid-liquid ratio of 1:5 to 1:10, soaking the solution, centrifuging the solution, 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 it by blending it with a phosphate buffer solution, centrifuging it, and discarding the waste liquid to obtain a fifth precipitate; Add disodium ethylenediaminetetraacetic acid solution to the fifth precipitate in a solid-liquid ratio of 1:5 to 1:20, soak, wash, centrifuge, and discard the waste liquid; Add disodium ethylenediaminetetraacetate solution again Step (9) of washing again, centrifuging again, discarding the waste liquid, and obtaining a sixth precipitate; The sixth precipitate is treated by soaking in saline or phosphate buffer at a solid-liquid ratio of 1:5 to 1:20, centrifuged, and the waste liquid is discarded; Add saline or phosphate buffer again. Step (10) of repeating the procedure to obtain a seventh precipitate; (11) adding a mixture of sodium phosphate and peracetic acid to the seventh precipitate at a solid-liquid ratio of 1:5 to 1:10 to inactivate the virus, centrifuging, and discarding the waste liquid to obtain an eighth precipitate; (12) adding physiological saline or phosphate buffer to the eighth precipitate at a solid-liquid ratio of 1:5 to 1:20, soaking the precipitate, centrifuging the precipitate, and discarding the waste liquid to obtain a ninth precipitate; and (13) adding sterile water to the ninth precipitate at a solid-liquid ratio of 1:5 to 1:20 to wash it, freeze-dry it, and sterilizing it with gamma rays or electron beams after freeze-drying.

[0012] Preferably, in step (1), the mammal is one or more species selected from 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 to 0.5% w / v sodium hypochlorite, 0.5 to 2.0% w / v sodium carbonate, and 50 to 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 centrifugation speed is 500 to 1500 xg, and the centrifugation 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×g, and the centrifugation time is 5 to 20 minutes.

[0018] Preferably, in step (6), a deoxyribonuclease lysis The liquid is One or more selected from hydroxyethylpiperazine ethanesulfonic acid buffer solution and trishydroxymethylaminomethane hydrochloride buffer solution Formulated in a 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×g, 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×g, 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 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 with 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×g, and the 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×g, and the time is 5 to 20 minutes.

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

[0023] Preferably, in step (11), the virus inactivation treatment lasts for 30 to 60 minutes, the centrifugation speed is 500 to 1500×g, and the treatment 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×g, 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 that is not intended for the diagnosis or treatment of a disease.

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

[0028] In an embodiment of the second aspect, the present application provides a stromal material for cell encapsulation obtainable by the above 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, wherein the sum of the type II collagen, chondroitin sulfate, and hyaluronic acid is 85% w / w or more, preferably 90% w / w or more, and more preferably 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, wherein the content of type II collagen, chondroitin sulfate, and hyaluronic acid is 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 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 of 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 pieces / g dry weight or as low as 5.0 x 10 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 the above-mentioned 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 application in filling and autologous lipogenesis induction 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, demonstrating 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 interstitial material provides protection to 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 to tissue regeneration repair by intracorporeal injection, or may be applied to filler and autologous adipogenesis induction in the field of aesthetic medicine.

[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.). However, in this application, the main components of the cell encapsulation stromal material produced from mammalian (e.g., pig, cow, sheep, horse, deer, etc.) cartilage are natural type II collagen and proteoglycans (chondroitin sulfate and hyaluronic acid), which have the advantages of being non-toxic like crosslinkers, low immunogenicity, excellent biocompatibility, and usable for implantation in the human body.

[0036] The interstitial particulate material for cell encapsulation manufactured based on the cartilage extracellular matrix of the present application has a favorable surface topology, providing in vitro cell culture with a tissue microenvironment similar to that found in the body, enabling it to function as a cell culture microcarrier and promote the formation of cell clusters. Functionally, it has reversible temperature-responsive properties, exhibiting a colloidal solution-like state at temperatures above 35°C and a gel state at relatively low temperatures, encapsulating cells through self-assembly during the colloid-gel transformation. By utilizing this property of encapsulating cells through self-assembly during the colloid-gel transformation, cells can be encapsulated in the gel-state interstitial material during cell storage and transportation, protecting them from adverse environmental influences and improving cell viability.

[0037] In conventional techniques, chondrocyte matrix microparticles or hydrogels are produced by grinding cartilage material. However, when using acid-alkali treatment or conventional grinding (e.g., Patents CN106075584A and CN112316211A), the thermal effects during the grinding process cause protein modification or denaturation in the material, reducing its stability. In contrast, the interstitial microparticle material for cell encapsulation produced 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 with encapsulated cells to form stable tissues and organoid structures.

[0038] The present method for producing a stromal material for cell encapsulation based on cartilage extracellular matrix utilizes the simplicity and high efficacy of alcalase, making it advantageous for scalable production. Mammalian cartilage is dense, making the decellularization process time-consuming and difficult to achieve complete and thorough removal of cellular components. Achieving thorough cell removal through multiple repeated decellularization treatments and extended decellularization cycles not only takes a long time, but also reduces the stability of the cartilage extracellular matrix and results in the loss of a large amount of constituent components. Alcalase treatment can also reduce α-Gal antigen epitopes in heterologous tissues.

[0039] Specific Embodiments This application provides a cell encapsulation stromal material and its manufacturing method and applications. It can be used to promote cell cluster formation during cell culture, and its reversible temperature-responsive properties allow cells to self-assemble and depolymerize, encapsulating and protecting cells during cell storage and transportation. This material is a composite natural biomaterial manufactured from mammalian (e.g., porcine, bovine, ovine, equine, deer, etc.) cartilage, and its main components are type II collagen and glycosaminoglycans (chondroitin sulfate and hyaluronic acid). The cell encapsulation stromal material contains at least 75% type II collagen (w / w), less than 10% elastin (w / w), and 1% to 10% glycosaminoglycan (chondroitin sulfate and hyaluronic acid) content.

[0040] As an option for the method for producing a stromal material for cell encapsulation based on the animal cartilage material of the present application, the method 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 mammals (e.g., pigs, cows, sheep, horses, deer, etc.), including elastic cartilage such as ear cartilage, articular cartilage, costal cartilage, scapular cartilage, and meniscus, and hyaline cartilage. Excess tissue and fascia on the cartilage surface are removed, and the cartilage tissue is sliced ​​into slices with a thickness of 2 mm or less.

[0042] In step (2), the cartilage tissue slices are surface disinfected with a disinfectant, washed with purified water, 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 micro-particle grinding to produce a micro-particle 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 particulate material is dissolved in 50 to 70% w / v isopropyl alcohol (IPA). Soak for 30 to 60 minutes. It is degreased more and processed at a solid-liquid ratio of 1:5 to 1:20. 、 Centrifuge at 500–1500 x g for 5–20 min, discard the waste liquid, and obtain the first precipitate.

[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 minutes, centrifuging at 500 to 1500 xg for 5 to 20 minutes, discarding the waste liquid, and washing once again to obtain a second precipitate.

[0046] In step (6), the second precipitate is added with a deoxyribonuclease solution at a solid-liquid ratio of 1:5 to 1:20. Liquid Add and soak, and deoxyribonuclease solution The liquid is One or more selected from hydroxyethylpiperazine ethanesulfonic acid buffer solution and trishydroxymethylaminomethane hydrochloride buffer solution Buffer solution The concentration of the hydroxyethylpiperazine ethanesulfonic acid buffer solution is 5-100 mmol / L, pH=7.6, and the deoxyribonuclease activity in the deoxyribonuclease solution is 50-250 U / L. After soaking for 8-12 hours, the solution is centrifuged at 500-1500 xg for 5-20 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 after soaking for 30 to 120 minutes, the precipitate is 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 at a solid-liquid ratio of 1:5 to 1:20, and the detergent in the solution is one or more selected from 0.5 to 2% w / v sodium deoxycholate, Triton X-100, and sodium dodecyl sulfate, and the detergent is formulated in a 10 mmol / L phosphate buffer solution at pH 7.4. The fourth precipitate is soaked for 8 to 16 hours, and then centrifuged at 500 to 1500 xg for 5 to 20 minutes. The waste liquid is discarded, and a fifth precipitate is obtained.

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

[0050] In step (10), the sixth 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 minutes, centrifuging at 500 to 1500×g for 5 to 20 minutes, and discarding the waste liquid; Add 0.9% w / v saline or 10 mmol / L phosphate buffer (pH = 7.4) again. Soak again for 30 to 120 min, then centrifuge at 500 to 1500 x g for 5 to 20 min. Discard the waste liquid and obtain the seventh precipitate.

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

[0052] In step (12), the eighth 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, soaked for 30 to 120 minutes, centrifuged at 500 to 1500 xg for 5 to 20 minutes, and the waste liquid is discarded to obtain 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 minutes, and the concentration of the treated microparticle sample is adjusted to 3 to 6% w / w, followed by freeze-drying to reduce the water content to less than 5% w / w.

[0054] In step (14), the freeze-dried product 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 of the present invention, decellularization treatment with Alcalase may be performed first, and then the cartilage tissue may be pulverized.

[0057] The stromal material for cell encapsulation of the present invention is obtained by the above-described 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 cell encapsulation stromal material of the present application is applicable to 3D bioprinting, as well as for filling and autologous lipogenesis induction in the field of aesthetic medicine.

[0060] The technical content of the present application will be further explained 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. [Example]

[0061] Example 1 The method for producing a stromal material for cell encapsulation in this example is an optional 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 mashed 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 minutes, then thoroughly washed with purified water and freeze-dried.

[0063] In step (2), the freeze-dried cartilage was placed in a 200 mL grinding bottle. The sealed container containing the cartilage sample was immersed in liquid nitrogen. The sample was rapidly ground using an SPEX 6875 freezer mill at -196 °C using electromagnetic collider grinding techniques. After temperature recovery, small cartilage particles were selected using a 100 μm mesh. Measurements using a laser diffraction particle size distribution analyzer revealed particle number distributions of Dx(10) = 1.8 μm, Dx(50) = 2.9 μm, and Dx(90) = 6.0 μm, with an average particle size ranging from 2 to 10 μm. Particle volume distributions of Dv(10) = 15 μm, Dv(50) = 67 μm, and Dv(90) = 144 μm, with an average particle size ranging from 30 to 100 μm.

[0064] In step (3), the cartilage microparticles were immersed in 70% w / v isopropyl alcohol solution at a solid-to-liquid ratio of 1:5 (i.e., 5 L of 70% w / v isopropyl alcohol was added per 1 L of cartilage microparticles). After 1 h of immersion, the microparticles were centrifuged at 500 x g for 5 min, and the supernatant was discarded to obtain the first precipitate. Then, at a solid-to-liquid ratio of 1:5, 10 mmol / L sodium phosphate buffer (pH = 7.4) was added. The microparticles were immersed for 30 min, and the microparticles were centrifuged at 500 x g for 5 min. The supernatant was discarded to obtain the second precipitate, which was then washed once with sodium phosphate buffer.

[0065] In step (4), the defatted cartilage microparticles are dissolved in deoxyribonuclease. In liquid Put it in and let it soak, and deoxyribonuclease solution In liquid The concentration of the hydroxyethylpiperazine ethanesulfonic acid buffer solution in , DeThe RNase activity was 50 U / L, pH 7.6, solid-liquid ratio 1:5, and after 8 hours of soaking, it was centrifuged at 500 x g for 5 minutes, discarding the waste liquid to obtain the third precipitate. Then, Alcalase solution was added to the third precipitate at a solid-liquid ratio of 1:5. The solution had an Alcalase concentration of 0.05% w / v and was formulated in 10 mmol / L sodium phosphate buffer, pH 7.4. After 120 minutes of Alcalase treatment, it was centrifuged at 500 x g for 5 minutes, and the supernatant was discarded. A fourth precipitate is obtained. After treating with alcalase, add sodium deoxycholate solution to the fourth precipitate at a solid-liquid ratio of 1:5. The sodium deoxycholate concentration is 1.0% w / v, and the solution is formulated in 10 mmol / L sodium phosphate buffer, pH 7.4. After treating for 12 hours, the mixture is centrifuged at 500 x g for 5 minutes, and the supernatant liquid is discarded to obtain the fifth precipitate.

[0066] In step (5), the degreased, enzyme-treated, and washed material is added to an EDTA solution (10 mmol / L EDTA concentration) formulated with 10 mmol / L sodium phosphate buffer, pH 7.4, and a solid-to-liquid ratio of 1:5. The material is soaked for 2 hours and washed, centrifuged at 500 x g for 5 minutes, the supernatant liquid is discarded, and a sixth precipitate is obtained. The material is then washed once more with EDTA solution. The material is then soaked for 2 hours and washed with 10 mmol / L sodium phosphate buffer (pH 7.4), centrifuged at 500 x g for 5 minutes, the supernatant liquid is discarded, and a seventh precipitate is obtained. The seventh precipitate is then washed once more with sodium phosphate solution.

[0067] In step (6), the washed material is added to a mixture of peracetic acid to inactivate the virus, a mixture of 2% w / v sodium phosphate and 0.2% w / v peracetic acid at a solid-to-liquid ratio of 1:5 is added, and the virus is inactivated for 60 minutes. After that, the mixture is centrifuged at 500 x g for 5 minutes, and the supernatant is discarded to obtain the eighth precipitate. Then, 0.9% w / v saline at a solid-to-liquid ratio of 1:5 is added, and the mixture is soaked for 60 minutes. After that, the mixture is centrifuged at 500 x g for 5 minutes, and the supernatant is discarded to obtain the ninth precipitate.

[0068] In step (7), 9th Precipitation The 9th precipitate was washed twice with sterile purified water at a solid-to-liquid ratio of 1:5, and then washed with sterile water for 30 minutes each time. It was then centrifuged at 500 x g for 5 minutes, and the supernatant was discarded. The processed microparticle sample was adjusted to a dry weight concentration of 3% w / v and freeze-dried. After freeze-drying, it was sterilized by gamma irradiation at a dose of 17.1 kGy. A cartilage-based stromal material for cell encapsulation was produced and obtained.

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

[0070] The cartilage matrix microparticles produced by the above method were rehydrated in 0.9% w / v sodium chloride solution and then measured using a laser diffraction particle size analyzer. The resulting cartilage matrix material had a particle size distribution based on 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. The particle size distribution based on particle 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 microparticles increased after Alcalase treatment and decellularization.

[0071] The number of α-Gal antigen epitopes in the cartilage matrix microparticle material produced by the above method was quantitatively detected using the method in the standard "Measurement of Residual α-Gal Antigen in Animal-Derived Scaffold Materials for Tissue-Engineered 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 x 10 per gram of dry weight material. 12 ±3.0×10 11 The number of α-Gal antigen epitopes was as low as 95.9% / gram dry weight of material, and 95.9% of the α-Gal antigen epitopes were removed by Alcalase treatment and decellularization.

[0072] The surface topological structure of cartilage matrix particles has a significant impact on responses 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 in this example was observed using an ultra-high-resolution scanning electron microscope (SU8000). 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 formulated into a 4% w / v suspension in 0.9% w / v sodium chloride solution (physiological saline). After standing for 5 days, no precipitation or separation occurred, demonstrating good suspension stability (Figure 2). If decellularization alone is performed without alcalase treatment, precipitation occurs in the microparticle material, indicating poor suspension stability. The improved suspension stability of cartilage matrix microparticles after alcalase treatment and decellularization is related to an increase in the particle size of the cartilage matrix particles after treatment, and a reduction in the density of the matrix particles.

[0074] Using a differential scanning calorimeter (DSC), we compared the thermal stability of cartilage raw material rehydrated in saline with that of the produced cartilage matrix particle material (Figure 3). The results showed that the cartilage matrix particles produced using the present enzyme treatment and decellularization method had good thermal stability, nearly identical to that of untreated cartilage raw material at temperatures above 40°C, indicating that they were not destroyed during the production process. The increased collagen content significantly increased the enthalpy of denaturation of the produced cartilage matrix particle protein. Furthermore, DSC measurements were performed on cartilage matrix microparticles after storing them at 37°C for 24 hours, and no change occurred in the differential scanning calorimetry analysis pattern, demonstrating the thermal stability of the cartilage matrix particles.

[0075] To verify whether 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 treated with Alcalase and decellularized to support cell attachment and proliferation, we co-cultured human umbilical cord mesenchymal stem cells with the cartilage matrix microparticles and observed their cell attachment and cytotoxicity. The experimental results showed that the co-culture of the material did not exhibit significant cytotoxicity, and a large number of cells adhered to the surface of the cartilage matrix microparticles, demonstrating its excellent ability to support 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 possesses reversible temperature-responsive properties (Figure 6). At 37°C, it exhibits a colloidal solution-like state and is fluid; upon temperature reduction, it exhibits a gel state and is non-fluid. It is capable of encapsulating cells through self-assembly during the colloid-gel transformation. This ability to encapsulate cells through self-assembly during the colloid-gel transformation can be utilized to protect cells from adverse environmental influences during cell storage and transportation, improving cell viability. Because the temperature-responsive properties of cartilage matrix microparticles are reversible, cells can depolymerize when the temperature rises to 37°C.

[0078] L929 cells and 2.5% soft tissue Bone After co-culture with the TRIXX microparticles at 37°C for 48 hours, 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 the cartilage matrix gel on cells in adverse conditions. L929 cell activity was measured using a CCK-8 kit on days 1 and 4 after transfer to the cold environment. Cell activity on days 1 and 4 in the 25°C thermostatic metal bath was 0.424±0.008 and 0.459±0.022, respectively, showing no change in cell activity. Cell activity on days 1 and 4 in the 4°C refrigerator was 0.410±0.012 and 0.344±0.020, respectively, showing a slight decrease in cell activity.

[0079] When the cartilage matrix microparticle material prepared by the above method was injected subcutaneously into the dorsal skin of rats, it functioned well as a scaffold and was able to induce the formation of large amounts of adipose tissue (Figure 7). Three weeks after implantation, an inflammatory response was observed, but the degree of inflammation decreased over time, and no chronic inflammatory response was observed. By six weeks, neovascularization had occurred in the area of ​​the cartilage matrix microparticles, and the cartilage matrix microparticles degraded to a certain extent over time. By ten weeks, adipocyte infiltration and growth had occurred, and by 16 weeks, the area was completely filled with adipocytes. Therefore, this method is applicable to fillers 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 disease, and the other steps are completely the same as in Example 1 except for one step: In step (4), after immersion in a deoxyribonuclease solution, a sodium deoxycholate solution is added directly for treatment without going through an alcalase solution treatment.

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

[0082] [Table 1]

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

[0084] In step (1), freshly mashed ear cartilage from a 6-month-old pig is removed from other tissues, and the cartilage, approximately 2 mm thick, is cut into small 2 cm x 3 cm chunks. The cartilage is then disinfected with 0.5% w / v sodium hypochlorite solution at a solid-liquid ratio of 1:5 for 10 minutes, after which the waste solution is discarded. 70% w / v isopropyl alcohol is added for 60 minutes, after which the waste solution is discarded, and the cartilage is then soaked in 0.125% w / v alcalase solution for 18 hours.

[0085] In step (2), the material after Alcalase treatment is subjected to 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 at a solid-liquid ratio of 1:5. After virus inactivation treatment for 120 minutes, the waste liquid is discarded. Then, EDTA solution is added. The concentration of the EDTA solution is 10 mmol / L, and it is formulated with 10 mmol / L sodium phosphate buffer, pH=7.4, and the solid-liquid ratio is 1:5. The material is soaked for 24 hours and washed. The waste liquid is discarded, and the EDTA solution is repeatedly used for 24 hours of washing.

[0086] In step (3), the tissue is washed with physiological saline for 24 hours.

[0087] During the preparation of cartilage decellularized matrix material using the method of Example 3, samples were taken after 5 hours of treatment with alcalase to monitor the progress of decellularization. After 5 hours of treatment with alcalase, cell-containing regions similar to those in untreated cartilage were present in the center of the cartilage tissue. However, after 18 hours of treatment with alcalase, the entire cartilage was completely transformed, with a remaining DNA content of 6.79 ± 0.65 ng / mg dry weight. Figure 8 shows histological sections of pig ear cartilage treated with alcalase for 5 hours (left) and 18 hours (right). The collagen content of the cartilage matrix after alcalase treatment was 76.5 ± 6.5% w / w dry weight. This indicates that alcalase treatment can effectively decellularize cartilage, 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 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 from other tissues, and 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 at a solid-liquid ratio of 1:5, soaked for 30 minutes, and then centrifuged at 1500 x g for 20 minutes. 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 formulated in a 10 mmol / L sodium phosphate buffer solution at pH 7.4. After treatment for 30 minutes, the solution is centrifuged at 1500 x g for 20 minutes, the waste liquid is discarded, and a second precipitate is obtained.

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

[0092] In step (4), the washed and soaked cartilage tissue slices are processed by soaking in 0.9% w / v saline at a solid-liquid ratio of 1:5 for 30 minutes, centrifuged at 1500 x g for 20 minutes, and the waste liquid is discarded. Place again in 0.9% w / v saline. Soak again for 30 minutes, then centrifuge at 1500 x g for 20 minutes, discard the waste liquid, and obtain the fourth precipitate.

[0093] In step (5), the washed cartilage tissue slices are dissolved in a deoxyribonuclease solution at a solid-liquid ratio of 1:20. In liquid Put it in and soak it. solutionThe solution contained 5 mmol / L hydroxyethylpiperazine ethanesulfonic acid buffer, the deoxyribonuclease activity in the deoxyribonuclease solution was 250 U / L, and the pH was 7.6. After 12 hours of treatment, the solution was centrifuged at 1500 x g for 20 minutes, the waste liquid was discarded, and the fifth precipitate was obtained.

[0094] In step (6), the deoxyribonuclease-treated cartilage tissue slices are immersed in 10 mmol / L sodium phosphate buffer (pH = 7.4) at a solid-liquid ratio of 1:20 for 120 minutes, centrifuged at 1500 x g for 5 minutes, and the waste liquid is discarded. Put it again into 10mmol / L sodium phosphate buffer (pH=7.4) Soak again for 120 minutes, then centrifuge at 1500 x g for 5 minutes, discard the waste liquid, and obtain the sixth precipitate.

[0095] In step (7), the virus is inactivated in a mixture containing 10% w / v sodium chloride and 0.5% w / v peracetic acid at a solid-to-liquid ratio of 1:10 for 30 minutes, then centrifuged at 1500 x g for 20 minutes, the waste liquid 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 at a solid-to-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 more, 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 containing the cartilage sample is immersed in liquid nitrogen, and the sample is rapidly ground using an electromagnetic collider grinding technique using a SPEX 6875 freezer mill at -196°C. After temperature recovery, small cartilage particles are selected using a 100 μm mesh.

[0099] In step (11), the particulate material is sterilized by gamma rays or electron beams to prepare a stromal material for cell encapsulation, with a sterilization dose of 15 kGy.

[0100] The cartilage matrix microparticle material produced by the method of Example 4 above and the cartilage matrix microparticles 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 ear cartilage from a 6-month-old pig is removed from other tissues, degreased with 70% w / v isopropyl alcohol for 60 minutes, and then the waste liquid is discarded. The cartilage is then 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. It is then cryo-polished using a freezer mill (see Example 1) in an environment of -196°C, and then sieved 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 each dissolved in deoxyribonuclease. In liquid Add 12 mL of the solution per 1 gram of dry weight of the cartilage microparticles. solutionThe deoxyribonuclease solution contained 5 mmol / L hydroxyethylpiperazine ethanesulfonic acid buffer, and the deoxyribonuclease activity in the deoxyribonuclease solution was 500 U / L (pH = 7.6). After treatment at 37°C for 16 hours, the solution was centrifuged at 1500 x g for 20 minutes and the waste liquid was discarded.

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

[0107] In step (6), wash the sample with a penicillin / streptomycin-containing phosphate buffer solution (pH 7.0, containing 100 U penicillin / streptomycin / mL) four times at room temperature for 8 h each time. Then, centrifuge the sample 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 irradiation (19.03 kGy to 20.08 kGy), and the waste is sampled and analyzed.

[0110] Cartilage stromal materials produced from cartilage microparticles of different particle sizes exhibited different degrees of aggregation. As the particle size of the cartilage microparticles increased, the degree of aggregation of the stromal material increased (Figure 9). Only particles with a particle size of <0.1 mm exhibited good dispersibility, while particles with a particle size >0.3 mm exhibited significant aggregation and interweaving.

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

[0112] The cartilage matrix microparticle material produced by the method of Example 5 above and the cartilage matrix microparticles produced by the method of Example 1 have similar characteristics.

[0113] The above description of the embodiments is intended to enable 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 within the scope of protection 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 to obtain cartilage tissue slices; (2) a step of disinfecting the surface of the cartilage tissue slice with a disinfectant, washing it, and freeze-drying it; (3) performing liquid nitrogen low-temperature micro-grinding on the dry-frozen cartilage tissue slices to produce a micro-particle material; (4) degreasing the particulate material by soaking it in isopropyl alcohol at a solid-liquid ratio of 1:5 to 1:20, centrifuging, and discarding the waste liquid to obtain a first precipitate; (5) adding physiological saline or phosphate buffer to the first precipitate at a solid-liquid ratio of 1:5 to 1:20, treating the first precipitate by immersion, centrifuging, and discarding the waste liquid to obtain a second precipitate; (6) treating the second precipitate by adding and soaking in a deoxyribonuclease solution at a solid-liquid ratio of 1:5 to 1:20, centrifuging, and discarding the waste liquid to obtain a third precipitate; (7) treating the third precipitate by adding an alcalase solution at a solid-liquid ratio of 1:5 to 1:10, soaking the solution, centrifuging the solution, 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 it with a phosphate buffer solution, centrifuging it, and discarding the waste liquid to obtain a fifth precipitate; (9) adding a disodium ethylenediaminetetraacetic acid solution to the fifth precipitate at a solid-liquid ratio of 1:5 to 1:20, soaking the fifth precipitate, washing the fifth precipitate, centrifuging the fifth precipitate, discarding the waste liquid, washing the fifth precipitate again with a disodium ethylenediaminetetraacetic acid solution, centrifuging the fifth precipitate again, discarding the waste liquid, thereby obtaining a sixth precipitate; (10) adding physiological saline or phosphate buffer to the sixth precipitate at a solid-liquid ratio of 1:5 to 1:20, treating the sixth precipitate by immersion, centrifuging, discarding the waste liquid, and then adding physiological saline or phosphate buffer again to repeat the procedure to obtain a seventh precipitate; (11) adding a mixture of sodium phosphate and peracetic acid to the seventh precipitate at 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) adding physiological saline or phosphate buffer to the eighth precipitate at a solid-liquid ratio of 1:5 to 1:20, treating the eighth precipitate by immersion, centrifuging, and discarding the waste liquid to obtain a ninth precipitate; and (13) adding sterile water to the ninth precipitate at a solid-liquid ratio of 1:5 to 1:20 to wash the ninth precipitate, freeze-drying the ninth precipitate, and sterilizing the ninth precipitate with gamma rays or electron beams after freeze-drying.

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

3. In step (2), the disinfectant is at least one selected from 0.1 to 0.5% w / v sodium hypochlorite, 0.5 to 2.0% w / v sodium carbonate, and 50 to 70% w / v alcohol solution; The alcohol solution is at least one selected from ethanol and isopropyl alcohol, 2. The method for producing an interstitial material for cell encapsulation according to claim 1, wherein 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 minutes, the rotation speed of the centrifuge is 500 to 1500 x g, and the time is 5 to 20 minutes; In step (5), the immersion time is 30 to 60 minutes, the rotation speed of the centrifuge is 500 to 1500 xg, and the centrifuge time is 5 to 20 minutes; 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 deoxyribonuclease activity in the deoxyribonuclease solution is 50 to 250 U / L; the concentration of the hydroxyethylpiperazine ethanesulfonic acid buffer solution is 5 to 100 mmol / L; 2. The method for producing an interstitial material for cell encapsulation according to claim 1, wherein 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; In step (7), the immersion time is 30 to 120 minutes, the rotation speed of the centrifuge is 500 to 1500 x g, and the centrifuge time is 5 to 20 minutes; In step (8), the detergent is one or more selected from sodium deoxycholate, Triton X-100, and sodium dodecyl sulfate; 2. The method for producing an interstitial material for cell encapsulation according to claim 1, wherein 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 the solution is mixed with a 10 mmol / L sodium phosphate buffer solution; In step (9), the washing time is 2 to 4 hours, the rotation speed of the centrifuge is 500 to 1500 x g, and the washing time is 5 to 20 minutes; In step (9), the re-washing time is 12 to 24 hours, the re-centrifugation rotation speed is 500 to 1500 x g, and the time is 5 to 20 minutes; 2. The method for producing an interstitial material for cell encapsulation according to claim 1, wherein in step (10), the soaking time is 30 to 120 minutes, the rotation speed of the centrifuge is 500 to 1500 xg, and the time is 5 to 20 minutes.

7. In step (11), the virus inactivation treatment is carried out for 30 to 60 minutes, the centrifugation speed is 500 to 1500 x g, and the treatment time is 5 to 20 minutes; 2. The method for producing an interstitial material for cell encapsulation according to claim 1, wherein in step (12), the soaking time is 30 to 120 minutes, the rotation speed of the centrifuge is 500 to 1500 xg, and the time is 5 to 20 minutes.

8. The method for producing an interstitial material for cell encapsulation according to any one of claims 1 to 7, wherein in steps (5), (8), (10) and (12), the phosphate buffer is a sodium phosphate buffer.

9. The main components of the produced interstitial material for cell encapsulation are type II collagen and glycosaminoglycan, The method for producing an interstitial material for cell encapsulation according to any one of claims 1 to 7, wherein the glycosaminoglycan contains chondroitin sulfate and hyaluronic acid.

10. The produced interstitial material for cell encapsulation 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 of the dry weight of the interstitial material for cell encapsulation, Type II collagen is 75% w / w or more, elastin is less than 10% w / w, and the glycosaminoglycan content is 1% to 10% w / w, said glycosaminoglycans including chondroitin sulfate and hyaluronic acid; The contents of type II collagen, chondroitin sulfate, and hyaluronic acid are 92% w / w or more (dry weight), 2% w / w or more (dry weight), and 1% w / w or more (dry weight), respectively; the residual DNA content of the cell encapsulation stromal material is less than 20 ng / mg; The number of α-Gal antigen epitopes in the cell encapsulation stromal material is 5.0 × 10 12 The method for producing an interstitial material for cell encapsulation according to claim 9, characterized in that the cell encapsulation rate is lower than 1000 cells / gram dry weight.

Images