WO2023116891A1 - 封装细胞的间质材料及其制备方法和应用 - Google Patents

封装细胞的间质材料及其制备方法和应用 Download PDF

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WO2023116891A1
WO2023116891A1 PCT/CN2022/141455 CN2022141455W WO2023116891A1 WO 2023116891 A1 WO2023116891 A1 WO 2023116891A1 CN 2022141455 W CN2022141455 W CN 2022141455W WO 2023116891 A1 WO2023116891 A1 WO 2023116891A1
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cartilage
precipitate
solution
cells
cell
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PCT/CN2022/141455
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French (fr)
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贾启鹏
孙文全
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北京瑞健高科生物科技有限公司
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Publication of WO2023116891A1 publication Critical patent/WO2023116891A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the application belongs to the technical field of bioengineering, and in particular relates to a cell-encapsulated interstitial material and its preparation method and application.
  • Cell therapy refers to a treatment method in which normal cells or biotechnologically modified cells are expanded in vitro and then transplanted or infused into the patient. Newly imported cells can replace damaged cells to rebuild tissue structure and function (stem cell therapy technology), or have stronger immune killing function (immune cell therapy technology) to achieve the purpose of treating diseases.
  • Stem cell therapy technology uses the characteristics of stem cells' self-renewal ability, multi-differentiation potential and high proliferation ability to transplant healthy stem cells into the human body to achieve the purpose of repairing lesions or rebuilding normal tissue functions.
  • Immune cell therapy technology is to collect immune cells from the human body, reinfuse them into the human body after in vitro transformation, culture and expansion, and use the enhanced targeted killing function to kill pathogens, cancer cells, mutant cells, activate and enhance the body's immunity. immunity.
  • the role of cell therapy in clinical practice is becoming more and more prominent.
  • There are many types of cells currently used in clinical treatment including bone marrow stem cells, hematopoietic stem cells, neural stem cells, skin stem cells, islet stem cells, adipose stem cells, and various immune cells such as DC, CIK, NK, CD3AK, and ⁇ T; they are widely used in various clinical applications.
  • Treatment of various diseases including blood diseases, organ transplantation, cardiovascular system diseases, liver diseases, nervous system diseases, cartilage and bone tissue diseases, tissue trauma and malignant tumor diseases, etc.
  • the cultivation, proliferation, preservation and transportation of cells are indispensable links in the realization of cell therapy.
  • One is in vitro 2D culture. When the cells attach and proliferate to a confluent state, the cells are subcultured and continued to be subcultured.
  • the disadvantage of this method is that the in vitro 2D culture efficiency is low, which is not conducive to maintaining the phenotype of the cells.
  • the second is in vitro 3D culture, where cells are immobilized on a suitable carrier and combined with a bioreactor to achieve high-density cell culture and production, such as the use of porous microcarrier cell immobilization technology.
  • porous microcarriers There are a variety of materials that can be used to prepare porous microcarriers, including polymer synthetic materials with good biocompatibility, purified or structurally modified natural materials (such as starch, cellulose, chitosan, sodium alginate, collagen, etc.) , gelatin, etc.).
  • Cell mass culture technology can also be used to achieve carrier-free immobilization of cells, that is, to monitor cell growth and achieve efficient cell culture by controlling the formation and depolymerization of cell mass during cell culture.
  • Extracellular matrix is a biomacromolecular substance that exists between cells, is secreted by cells, is mainly composed of collagen, and contains elastin, proteoglycans, glycosaminoglycans, 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 great influence on cell functions and behaviors such as adhesion, growth, proliferation, differentiation, migration, and intercellular signal transduction. Delivery and so on have a regulating effect.
  • extracellular matrix materials can be used to prepare microcarriers for cell immobilization, and can also be used as active materials to promote the formation of cell clusters, and can also be used as interstitial materials for encapsulating and protecting cells during cell preservation and transportation.
  • stroma, CES stroma, CES
  • Patent CN105288737A discloses a tissue-engineered cartilage composite scaffold based on cartilage extracellular matrix and its preparation method, which involves using cartilage extracellular matrix particles to rapidly expand cartilage seed cells and induce stem cells to differentiate into chondrocytes.
  • the specific preparation method of the invention includes: 1) performing low-temperature wet crushing on fresh articular cartilage and sieving to obtain cartilage particles with a diameter of 100-500 ⁇ m, and preparing cartilage extracellular matrix microcarriers after decellularization; 2) chondrocytes The extracellular matrix microcarriers and cartilage seed cells are co-cultured in a bioreactor to form cartilage microtissues through the rapid expansion of chondrocytes or the induction of stem cells to differentiate into chondrocytes; 3) filling the cartilage microtissues in the three-dimensional porous matrix of the hydrogel precursor liquid In the pores of the scaffold, the cartilage microtissue is combined with the three-dimensional porous scaffold through a coagulant, and the tissue engineered cartilage composite scaffold is obtained through co-cultivation.
  • extracellular matrix particles or hydrogels for cell co-culture and promotion of directed differentiation of stem cells.
  • the present application provides a method for preparing a cell-encapsulated interstitial material, the preparation method comprising the following steps:
  • the present application provides the interstitial material encapsulating cells obtained by the above preparation method.
  • the present application provides the application of the above-mentioned interstitial material for encapsulating cells, that is, the interstitial material that can be used to promote the formation of cell clusters during cell culture, and can be used to encapsulate and protect cells during cell preservation and transportation; or in Applications in 3D bioprinting; or applications in the field of medical aesthetics.
  • Fig. 1 is a scanning electron micrograph of cartilage matrix microparticles after being treated with alkaline enzyme and decellularized in Example 1 of the present application.
  • Fig. 2 is the suspension stability of cartilage matrix microparticles after (left) treatment with alkaline enzyme and decellularization and (right) suspension stability of cartilage matrix microparticles without alkaline enzyme treatment and only decellularized treatment in Example 1 of the present application sex.
  • Fig. 3 is a differential scanning calorimetry diagram of (top) untreated porcine ear cartilage raw material and (bottom) cartilage matrix particles treated with alkaline enzyme and decellularized in Example 1 of the present application.
  • Fig. 4 is a diagram of co-cultivation of L929 cells on cartilage matrix microparticles after alkaline enzyme treatment and decellularization in Example 1 of the present application.
  • FIG. 5 is a graph showing the growth of cells 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.
  • Fig. 6 is a temperature response characteristic diagram of the cartilage matrix particulate material after alkaline enzyme treatment and decellularization in Example 1 of the present application.
  • Fig. 7 is an effect diagram of the subcutaneous implantation of cartilage matrix microparticles into rats to induce adipose tissue formation after alkaline enzyme treatment and decellularization in Example 1 of the present application.
  • Fig. 8 is a diagram of tissue sections of pig ear cartilage in Example 3 of the present application after being treated with alkaline enzyme alcalase for 5 hours (left) and 18 hours (right).
  • Fig. 9 is the general morphology of cartilage matrix material particles with different particle sizes in Example 5 of the present application.
  • the present application provides a method for preparing a cell-encapsulated interstitial material, which includes the following steps:
  • the mammal is selected from at least one of pigs, cattle, sheep, horses and deer.
  • the cartilage material is selected from more than one of elastic cartilage and hyaline cartilage.
  • the elastic cartilage is ear cartilage.
  • the hyaline cartilage is selected from one or more of articular cartilage, costal cartilage, scapular cartilage, and menisci.
  • the disinfectant is more than 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.
  • the alcohol solution is selected from one or more of ethanol or isopropanol.
  • the average particle diameter of the particulate material based on the number of particles is 2-20 ⁇ m, and the average particle diameter of the particulate material based on the particle volume is 20-200 ⁇ m.
  • the content of isopropanol is 50-70% w/v; the soaking time is 30-60 minutes, the centrifugation speed is 500-1500 ⁇ g, and the time is 5-20 minutes.
  • the soaking time is 30-60 minutes
  • the centrifugation speed is 500-1500 ⁇ g
  • the time is 5-20 minutes.
  • the buffer of the deoxyribonuclease solution is selected from more than one of hydroxyethylpiperazine ethylsulfuric acid buffer and trishydroxymethylaminomethane hydrochloride buffer; hydroxyethyl
  • concentration of the piperazine ethylsulfuric acid buffer solution is 5-100mmol/L
  • activity of the deoxyribonuclease in the deoxyribonuclease solution is 50-250U/L.
  • the soaking time is 8-12 hours
  • the centrifugation speed is 500-1500 ⁇ g
  • the time is 5-20 minutes.
  • the concentration of the alkaline enzyme (alcalase) in the alkaline enzyme (alcalase) solution is 0.02-0.2% w/v.
  • the soaking time is 30-120 min
  • the centrifugation speed is 500-1500 ⁇ g
  • the time is 5-20 min.
  • the detergent is selected from one or more of 0.5-2% w/v sodium deoxycholate, Triton X-100 or sodium lauryl sulfate.
  • the soaking time is 8-16 hours
  • the centrifugation speed is 500-1500 ⁇ g
  • the time is 5-20 minutes.
  • the concentration of disodium edetate in the disodium edetate solution is 5-50mmol/L, and it is prepared in a 10mmol/L sodium phosphate buffer.
  • the washing time is 2-4 hours
  • the centrifugation speed is 500-1500 ⁇ g
  • the time is 5-20 minutes.
  • step (9) the time for re-washing is 12-24 h, the speed of re-centrifugation is 500-1500 ⁇ g, and the time is 5-20 min.
  • the soaking time is 30-120 minutes
  • the centrifugation speed is 500-1500 ⁇ g
  • the time is 5-20 minutes.
  • the time for virus inactivation treatment is 30-60 min
  • the centrifugation speed is 500-1500 ⁇ g
  • the time is 5-20 min.
  • the soaking time is 30-120 min
  • the centrifugation speed is 500-1500 ⁇ g
  • the time is 5-20 min.
  • the phosphate buffer is sodium phosphate buffer.
  • the preparation method may be a method for non-disease diagnosis and treatment purposes.
  • %w/v means "g/100mL”.
  • the present application provides a cell-encapsulated interstitial material obtained by the above-mentioned preparation method.
  • the main components of the interstitial material encapsulating the cells are type II collagen and glycosaminoglycans.
  • the main components of the interstitial material encapsulating the cells are type II collagen, proteoglycans and glycosaminoglycans.
  • the glycosaminoglycans include chondroitin sulfate and hyaluronic acid.
  • the cell-encapsulating interstitial material comprises collagen type II, chondroitin sulfate, and hyaluronic acid.
  • the interstitial material encapsulating cells contains type II collagen, chondroitin sulfate and hyaluronic acid, wherein the sum of type II collagen, chondroitin sulfate and hyaluronic acid It is more than 85% w/w dry weight of the cell-encapsulating interstitial material, preferably more than 90% w/w dry weight, more preferably 96.5 ⁇ 0.5% w/w dry weight.
  • the cell-encapsulating interstitial material contains greater than 75% w/w type II collagen, less than 10% w/w elastin, and 1% to 10% glycosaminoglycan content w/w;
  • Glycosaminoglycans described herein include chondroitin sulfate and hyaluronic acid.
  • the interstitial material encapsulating cells contains type II collagen, chondroitin sulfate and hyaluronic acid, wherein the contents of type II collagen, chondroitin sulfate and hyaluronic acid are respectively : 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, 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; or more than 92% w/w (dry weight), more than 2% w/w (dry weight) and more than 1% w/w (dry weight).
  • the residual DNA content in the cell-encapsulating interstitial material is less than 20 ng/mg, preferably no more than 5 ng/mg, more preferably no more than 2.7 ⁇ 0.6 ng/mg .
  • the number of ⁇ -Gal epitopes in the interstitial material encapsulating cells is as low as 2.2 ⁇ 10 12 ⁇ 3.0 ⁇ 10 11 per gram of dry weight, or less than 5.0 ⁇ 10 12 pcs/g dry weight.
  • the cell-encapsulating interstitial material comprises 83.5 ⁇ 1.5% w/w to 90.5 ⁇ 1.5% w/w type II collagen.
  • the present application provides the above-mentioned interstitial material for encapsulating cells that can be used to promote the formation of cell clusters during cell culture, and can be used to encapsulate and protect cells during cell preservation and transportation; or in 3D bioprinting It can also be used for filling and inducing autologous fat formation in the field of medical aesthetics.
  • the above-mentioned interstitial material encapsulating cells is co-cultured with the cells, and does not show obvious cytotoxicity, and a large number of cells are attached to the surface of the interstitial material, which shows good support for stem cell adhesion and proliferation performance
  • the interstitial material can be used as a microcarrier for cell culture, and can also promote the formation of cell clusters.
  • the above-mentioned cell-encapsulating interstitial material can encapsulate cells through self-assembly, and can protect cells from adverse environmental influences during cell preservation and transportation, and improve cell survival rate.
  • the above-mentioned cell-encapsulating interstitial material has a protective effect on the cells under stress.
  • the above-mentioned interstitial material encapsulating cells can be used as a tissue scaffold material, which can be injected into the body for tissue regeneration and repair applications; or can be used for filling and inducing autologous fat formation in the field of medical aesthetics .
  • Existing cell culture microcarrier materials are mainly prepared by chemical cross-linking of polymer synthetic materials, purified or structurally modified natural materials (such as starch, cellulose, chitosan, sodium alginate, collagen, gelatin, etc.) And the present application uses the cartilage of mammals (such as pigs, cattle, sheep, horses, deer, etc.) Chondroitin and hyaluronic acid), no cross-linking agent toxicity, low immunogenicity, good biocompatibility, and can be used for human implantation.
  • polymer synthetic materials purified or structurally modified natural materials (such as starch, cellulose, chitosan, sodium alginate, collagen, gelatin, etc.)
  • the present application uses the cartilage of mammals (such as pigs, cattle, sheep, horses, deer, etc.) Chondroitin and hyaluronic acid), no cross-linking agent toxicity, low immunogenicity, good biocompatibility, and can be used for human implantation.
  • the cell-encapsulating interstitial particulate material prepared based on the extracellular matrix of cartilage in this application has a good surface topology structure and can provide a tissue microenvironment similar to that in vivo for in vitro cell culture. It can not only be used as a microcarrier for cell culture, but also Promote the formation of cell clusters; it has reversible temperature response characteristics in function, showing a lyosol-like state above 35°C, and a gel state at lower temperatures, and encapsulates cells through self-assembly when the sol transforms into a gel; using its When the sol is transformed into a gel, it can self-assemble and encapsulate the characteristics of the cells. During the storage and transportation of the cells, the cells are encapsulated in the gel-state interstitial, which can protect the cells against the influence of the adverse environment, thereby improving the cell survival rate.
  • the thermal effect in the grinding process causes protein in the material Modification or denaturation reduces the stability of the material.
  • the thermal stability of the cell-encapsulating interstitial particulate material prepared based on cartilage extracellular matrix in this application is above 40°C, and can be used alone or together with encapsulated cells for 3D bioprinting to form stable tissue and organoid structures.
  • the method of the present application for preparing the cell-encapsulating interstitial material based on the cartilage extracellular matrix uses alkaline enzyme (alcalase), which has the characteristics of simplicity and high efficiency, and is conducive to large-scale preparation.
  • alkaline enzyme alkaline enzyme
  • Mammalian cartilage is extremely dense, and the decellularization process takes a long time, making it difficult to completely remove cell components; repeated decellularization treatments and prolonged decellularization cycles to achieve complete cell removal not only take a long time, but also cause cartilage extracellular matrix It is not stable enough and has the disadvantage of a large loss of components.
  • alkaline enzyme (alcalase) treatment can also reduce the ⁇ -Gal epitope in the heterogeneous tissue.
  • the present application provides a cell-encapsulating interstitial material and its preparation method and application, which can be used to promote the formation of cell clusters during cell culture, self-assemble and depolymerize cells through reversible temperature-responsive properties, and preserve and transport cells.
  • the interstitial material that can be used to encapsulate and protect cells during the process is a composite natural biological material prepared from the cartilage of mammals (such as pigs, cattle, sheep, horses, deer, etc.), and the main component is type II Collagen and glycosaminoglycans (chondroitin sulfate and hyaluronic acid).
  • the interstitial material encapsulating cells in this application has more than 75% w/w type II collagen, less than 10% w/w elastin, and 1% to 10% glycosaminoglycan (chondroitin sulfate and hyaluronic acid) content w/w.
  • the present application is a method for preparing a cell-encapsulating interstitial material based on an animal cartilage material, which is optionally a method for non-disease diagnosis and treatment purposes, comprising the following steps:
  • Collect cartilage materials from mammals such as pigs, cattle, sheep, horses, deer, etc.
  • mammals such as pigs, cattle, sheep, horses, deer, etc.
  • remove excess tissue on the cartilage surface and fascia cut the cartilage tissue into thin slices with a thickness of less than 2mm;
  • Cartilage tissue slices are surface-sterilized with a disinfectant, washed with purified water after disinfection, and freeze-dried to a water content of less than 5% w/w after cleaning; wherein, the disinfectant is selected from 0.1-0.5% w/v sodium hypochlorite, 0.5 - More than one of 2.0% w/v sodium carbonate and 50-70% w/v ethanol (or isopropanol).
  • the particulate material is degreased with 50-70% w/v isopropanol (IPA), at a ratio of 1:5-1:20, and after soaking the particulate for 30-60min, 500-1500 ⁇ Centrifuge at g for 5-20min, discard the waste liquid, and obtain the first precipitate;
  • IPA isopropanol
  • the phosphate buffer is sodium phosphate buffer.
  • alkaline enzyme and decellularization treatment may be performed first, and then the cartilage tissue is pulverized.
  • the interstitial material for encapsulating cells of the present application is obtained by the above-mentioned preparation method.
  • the main components of the interstitial material encapsulating cells are type II collagen and glycosaminoglycans (chondroitin sulfate and hyaluronic acid).
  • the cell-encapsulating interstitial material of the present application can be applied in 3D bioprinting, and can also be used in the field of medical aesthetics to fill and induce autologous fat formation.
  • the method for preparing the cell-encapsulated interstitial material of this embodiment is optionally a method for non-disease diagnosis and treatment, including the following steps:
  • the concentration of hydroxyethylpiperazine ethylsulfuric acid buffer in the buffer of deoxyribonuclease solution is 10mmol/L
  • the residual DNA content of the cartilage matrix particulate material prepared by the above method was 2.7 ⁇ 0.6 ng/mg dry weight; compared with the untreated freeze-dried cartilage raw material (679.8 ⁇ 7.3 ng/mg dry weight), it decreased by 99.6 %.
  • the collagen content in the treated cartilage matrix particulate material was 92.4 ⁇ 0.4%w/w dry weight, and the collagen content in the untreated freeze-dried cartilage raw material was 50.9 ⁇ 5.2%w/w dry weight, indicating that the alkaline enzyme alcalase Treatment and decellularization effectively removes other tissue proteins.
  • the content of chondroitin sulfate in the processed cartilage matrix particulate material is 25.5 ⁇ 0.9ug/mg dry weight (2.55 ⁇ 0.09%w/w dry weight), and the content of chondroitin sulfate in the untreated freeze-dried cartilage raw material is 80.5 ⁇ 4.1 ug/mg dry weight (8.05 ⁇ 0.41% w/w dry weight).
  • the content of hyaluronic acid in the treated cartilage matrix particulate material was 16.6 ⁇ 0.4ug/mg dry weight (1.66 ⁇ 0.04%w/w dry weight), and the hyaluronic acid content in the untreated freeze-dried cartilage raw material was 136.6 ⁇ 7.3 ug/mg dry weight (13.66 ⁇ 0.73% w/w dry weight).
  • Component analysis results show that the treated cartilage matrix particle 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.
  • the particle size of cartilage matrix granular material increased after alkaline enzyme treatment and decellularization.
  • the cartilage matrix particulate material prepared by the above method with reference to the method in the standard "Detection of ⁇ -Gal Antigen Residual in Animal-derived Scaffold Materials of Tissue Engineering Medical Device Products" (YY/T 1561-2017), the epitope number of ⁇ -Gal antigen Quantitative detection was carried out.
  • the number of ⁇ -Gal antigen epitopes in untreated pig ear cartilage raw material is 5.4 ⁇ 10 13 ⁇ 5.2 ⁇ 10 12 per gram of dry weight material, and the number of ⁇ -Gal antigen epitopes in the treated cartilage matrix granule material is as low as 2.2 ⁇ 10 12 ⁇ 3.0 ⁇ 10 11 /g dry weight material, alkaline enzyme treatment and decellularization removed 95.9% of ⁇ -Gal epitopes.
  • the surface topology of cartilage matrix particles has an important impact on response behaviors such as cell adhesion and proliferation, and has important physiological effects on cell morphology, phenotype, and cell movement.
  • the surface structure of the cartilage matrix particles prepared by the alkaline enzyme treatment and decellularization process in this example was observed by a super-resolution field emission scanning electron microscope (SU8000), and the results showed that the cartilage matrix particles had a three-dimensional porous uneven rough surface structure, Has a very large surface area ( Figure 1).
  • the cartilage matrix particulate material prepared by the above method is formulated into a 4% w/v suspension with 0.9% w/v sodium chloride solution (physiological saline). After standing for 5 days, it does not settle or separate, and has good suspension stability. sex ( Figure 2). If there is no alkaline enzyme alcalase treatment, but only decellularization treatment, the particulate material will appear to settle, indicating that its suspension stability is poor.
  • the improved suspension stability of the cartilage matrix particles after alkaline enzyme treatment and decellularization was related to the increase in the particle size of the cartilage matrix particles after treatment, which decreased the density of the matrix particles.
  • the thermal stability of cartilage raw material rehydrated in saline and the prepared cartilage matrix granular material was compared by differential scanning calorimetry (DSC) (Fig. 3), prepared by the enzyme treatment and decellularization method of the present application
  • DSC differential scanning calorimetry
  • the thermal stability of the cartilage matrix particles is good, and above 40 °C, it is basically similar to the untreated cartilage raw material, indicating that there is no damage during the preparation process. Because of the increased collagen content, the enthalpy value of the protein denaturation of the prepared cartilage matrix particles was significantly increased.
  • the differential scanning calorimetry chart did not change, and the results also indicated the thermal stability of the cartilage matrix particles.
  • cartilage matrix microparticles In order to further examine the ability of cartilage matrix microparticles to support cell adhesion and proliferation after alkaline enzyme treatment and decellularization, human umbilical cord mesenchymal stem cells were co-cultured with cartilage matrix microparticles to observe their cell adhesion and cytotoxicity. The results of this experiment It shows that the co-culture of the material does not show obvious cytotoxicity, and a large number of cells adhere to the surface of the cartilage matrix particles, showing good performance in supporting the adhesion and proliferation of stem cells (Figure 5).
  • Cartilage-based matrix materials can be used as microcarriers for cell culture and can also promote cell cluster formation.
  • the cartilage matrix particulate material prepared by the above method has reversible temperature response characteristics functionally ( FIG. 6 ). At 37°C, it exhibits a liquid-sol-like state with fluidity; when the temperature drops, it exhibits a gel state without fluidity; when the sol turns into a gel, it can encapsulate cells through self-assembly. Utilizing the characteristics of self-assembly and encapsulation of cells when the sol is transformed into a gel, it can protect the cells from adverse environmental influences and improve the cell survival rate during cell preservation and transportation. Because the temperature-responsive properties of cartilage matrix particles are reversible, cells can depolymerize when the temperature rises to 37°C.
  • the cell suspension was transferred to a constant temperature metal bath at 25°C and stored in a refrigerator at 4°C to test the protective effect of cartilage matrix glue on cells in adversity.
  • the activity of L929 cells was detected with CCK-8 kit on the 1st and 4th day after being transferred to low temperature environment.
  • the cell viability of the first day and the fourth day in the constant temperature metal bath at 25°C were 0.424 ⁇ 0.008 and 0.459 ⁇ 0.022 respectively, and the cell viability did not change; the cell viability of the first day and the fourth day in the 4°C refrigerator were respectively 0.410 ⁇ 0.012 and 0.344 ⁇ 0.020, the cell viability decreased slightly.
  • the cartilage matrix particulate material prepared by the above method can play a good role as a scaffold and induce the formation of a large amount of adipose tissue when implanted subcutaneously in the back of rats by injection ( FIG. 7 ).
  • adipose tissue When implanted subcutaneously in the back of rats by injection ( FIG. 7 ).
  • At 3 weeks after implantation there was an inflammatory reaction, but the degree of inflammation decreased with time, and there was no chronic inflammatory reaction; at 6 weeks, new blood vessels formed in the area of the cartilage matrix particles, and the cartilage matrix particles degraded to a certain extent over time ;Adipocytes entered growth at 10 weeks and completely filled with adipocytes at 16 weeks. Therefore, it can also be used in the field of medical aesthetics to induce autologous fat formation.
  • the preparation method of the cartilage matrix material in this example is a method for non-disease diagnosis and treatment purposes, except that the following step is different from Example 1, and other steps are completely the same.
  • step (4) after soaking in the deoxyribonuclease solution, the sodium deoxycholate solution was added directly without being treated with the alkaline enzyme (alcalase) solution.
  • the cartilage matrix microparticles prepared by the method of Example 2 are quite different from the cartilage matrix microparticles prepared by the method of Example 1 (Table 1). After being treated by the method of Example 1, the residual DNA content of the cartilage matrix particles is low, and the number of residual ⁇ -Gal antigen epitopes is small; after being treated by the method of Example 2, the residual DNA content of the cartilage matrix particles is relatively high, and the residual ⁇ -Gal More antigenic epitopes.
  • the content of the cartilage matrix particulate material collagen, chondroitin sulfate and hyaluronic acid is 96.5 ⁇ 0.53% w/w dry weight; and after the method of Example 2, the cartilage matrix particulate material collagen
  • the total content of protein, chondroitin sulfate and hyaluronic acid is only 75.1 ⁇ 5.2% w/w dry weight.
  • the detection result shows that the effect of alkaline enzyme (alcalase) treatment is remarkable.
  • the preparation method of the cell-encapsulated interstitial material in this embodiment is a method for non-disease diagnosis and treatment purposes, including the following steps:
  • the content of collagen in the cartilage matrix after alkaline enzyme treatment was 76.5 ⁇ 6.5% w/w dry weight, indicating that the treatment with alkaline enzyme (alcalase) can also effectively carry out cartilage decellularization; ) were significantly less efficient using intact cartilage.
  • the preparation method of the cell-encapsulated interstitial material in this embodiment is a method for non-disease diagnosis and treatment purposes, including the following steps:
  • the cartilage tissue slices treated with isopropanol are added to the alkaline enzyme alcalase solution at a ratio of 1:10 for solid-liquid treatment.
  • the particulate material is sterilized by gamma ray or electron beam to prepare interstitial material which can be used for encapsulating cells, and the sterilizing dose is 15 kGy.
  • the cartilage matrix particulate material prepared by the method of Example 4 above has similar characteristics to the cartilage matrix particulate material prepared by the method of Example 1.
  • the cartilage interstitial materials prepared by cartilage microparticles with different particle sizes have different degrees of aggregation; as the particle size of cartilage particles increases, the degree of aggregation of matrix materials increases (Figure 9). It has good dispersion, and when the particle size is >0.3mm, there will be obvious aggregation and interweaving.
  • the DNA content of the pre-decellularized cartilage microparticles is about 411ng/mg, and the DNA content after decellularization is less than 20ng/mg; the collagen content in the cartilage matrix is 83.5 ⁇ 1.2%w/w-90.5 ⁇ 1.5%w/w. The larger the diameter, the higher the collagen content; the elastin content in the cartilage matrix is 1.6-4.0% w/w.
  • the Sulfated Glycosaminoglycan (sGAG) assay kit was used to determine the content of chondroitin sulfate in the cartilage decellularized matrix microcarrier, and the content of chondroitin sulfate was 7.1 ⁇ 0.7%w/w.
  • the cartilage matrix particulate material prepared by the method of Example 5 above has similar characteristics to the cartilage matrix particulate material prepared by the method of Example 1.

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Abstract

一种封装细胞的间质材料及其制备方法和应用，该材料以哺乳动物猪、牛、羊、马、鹿等的软骨为原料，经过裁切、低温微粒化研磨、酶解处理、清除细胞组分成分、病毒灭活、冻干和辐照灭菌处理制备而成，该方法用于非诊断目的，间质材料可单独或同封装细胞用于3D生物打印；材料的主要成分为II型胶原蛋白、硫酸软骨素和透明质酸，无交联剂毒性，具有低免疫原性，生物相容性好，可用于人体植入的优点；在结构上有良好的表面拓扑结构，为体外细胞培养提供类似体内的组织微环境，不仅可以作为细胞培养的微载体，还可以促进细胞团形成，在细胞保存和运输过程中，将细胞封存在凝胶态间质中，可保护细胞抵御不良环境的影响，提高细胞存活率。

Description

封装细胞的间质材料及其制备方法和应用
交叉引用
本申请要求于2021年12月24日提交的中国发明专利申请号CN202111598820.1的权益，该中国专利申请的内容以其全文并入本文。
技术领域
本申请属于生物工程技术领域，具体涉及一种封装细胞的间质材料及其制备方法和应用。
背景技术
细胞治疗是指将正常细胞或者经过生物技术改造的细胞经体外扩增后移植或输入患者体内的治疗方法。新输入的细胞可以替代受损细胞重建组织结构和功能(干细胞治疗技术)、或者具有更强的免疫杀伤功能(免疫细胞治疗技术)达到治疗疾病的目的。干细胞治疗技术利用干细胞的自我更新能力、多分化潜能和高度增殖能力的特点，把健康的干细胞移植到人体内，以达到修复病变或重建组织正常功能的目的。免疫细胞治疗技术是从人体采集免疫细胞，经过体外改造和培养扩增后再回输到人体，利用增强了的靶向性杀伤功能来杀灭病原体、癌细胞、突变细胞，激活和增强机体的免疫能力。随着分子生物学、干细胞生物学、组织工程学和再生医学等领域的快速发展，在临床中细胞治疗的作用越来越突出。目前用于临床治疗的细胞种类很多，包括骨髓干细胞、造血干细胞、神经干细胞、皮肤干细胞、胰岛干细胞、脂肪干细胞，以及DC、CIK、NK、CD3AK和γδT等多种免疫细胞；广泛应用于临床各类疾病的治疗，包括血液类疾病、器官移植、心血管系统疾病、肝脏疾病、神经系统疾病、软骨与骨组织疾病、组织创伤和恶性肿瘤疾病等。
细胞的培养增殖和保存运输是实现细胞治疗不可缺少的环节。目前细胞扩增主要用两种方式。其一是体外2D培养，当细胞附着增殖达到融合状态时将细胞进行传代继续继代扩增培养，该方法不足之处在于体外2D培养效率低，不利于细胞维持表型。其二是体外3D培养，将细胞固定在合适的载体上，与生物反应器相结合可以实现细胞高密度培养和生产，比如使用多孔 微载体细胞固定化技术。可用于制备多孔微载体的材料有多种，包括具有良好生物兼容性的高分子合成材料、纯化的或者经过结构修饰的天然材料(如淀粉、纤维素、壳聚糖、海藻酸钠、胶原蛋白、明胶等)。也可以利用细胞团培养技术实现细胞的无载体固定化，也即是在细胞培养过程中通过控制细胞团的形成和解聚来监测细胞生长和实现高效的细胞培养。
细胞外基质(extracellular matrix，ECM)是存在于细胞之间的、由细胞分泌的、以胶原蛋白为主和含有弹性蛋白、蛋白聚糖、糖胺聚糖以及细胞因子的生物大分子物质。细胞外基质具有复杂的空间网络结构，是细胞生长和活动的微环境，细胞与细胞外基质之间的相互作用对细胞的功能和行为如黏附、生长、增殖、分化、迁移、细胞间信号转递等具有调节作用。因此，细胞外基质材料可以用于制备细胞固定化的微载体，也可以用做促进细胞团形成的活性材料，在细胞保存运输过程中还可以用做封装和保护细胞的间质材料(cell encapsulation stroma，CES)。专利CN105288737A公开了一种基于软骨细胞外基质的组织工程软骨复合支架及其制备方法，其中涉及到用软骨细胞外基质颗粒快速扩增软骨种子细胞和诱导干细胞向软骨细胞分化。该发明的具体制备方法包括：1)将新鲜关节软骨进行低温湿法粉碎并过筛，得到直径100-500μm的软骨颗粒，脱细胞处理后制备得到软骨细胞外基质微载体；2)将软骨细胞外基质微载体和软骨种子细胞置于生物反应器中共培养，通过软骨细胞快速扩增或诱导干细胞向软骨细胞分化形成软骨微组织；3)将软骨微组织填充于含水凝胶前体液的三维多孔支架的孔隙中，通过致凝剂使软骨微组织与三维多孔支架结合，通过共培养获得组织工程软骨复合支架。使用细胞外基质微粒或水凝胶用于细胞共培养和促进干细胞定向分化已有很多研究。
发明概述
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
第一方面，本申请提供了一种封装细胞的间质材料的制备方法，所述的制备方法包括如下步骤：
(1)将离体哺乳动物的软骨材料剔除表面的组织和筋膜，然后切成薄片；
(2)将软骨组织薄片的表面采用消毒剂进行消毒，之后进行清洗和冻干；
(3)将冻干的软骨组织薄片进行液氮低温微粒化研磨，制备成微粒材料；
(4)按照料液比1∶5-1∶20，将微粒材料用异丙醇进行脱脂处理，浸泡后离心，弃废液，得到第一沉淀物；
(5)按照料液比1∶5-1∶20，在第一沉淀物中加入生理盐水或磷酸盐缓冲液浸泡处理，离心，弃废液，得到第二沉淀物；
(6)按照料液比1∶5-1∶20，在第二沉淀物中加入脱氧核糖核酸酶溶液的缓冲液浸泡处理，离心，弃废液，得到第三沉淀物；
(7)按照料液比1∶5-1∶10，在第三沉淀物中加入碱性酶(alcalase)溶液浸泡处理，离心，弃废液，得到第四沉淀物；
(8)按照料液比1∶5-1∶20，在第四沉淀物中加入洗涤剂，配制在磷酸盐缓冲液中浸泡处理，离心，弃废液，得到第五沉淀物；
(9)按照料液比1∶5-1∶20，在第五沉淀物中加入乙二胺四乙酸二钠溶液浸泡，清洗，离心，弃废液；更换溶液再次清洗，再次离心，弃废液，得到第六沉淀物；
(10)按照料液比1∶5-1∶20，在第六沉淀物中加入生理盐水或磷酸盐缓冲液浸泡处理，离心，弃废液；更换溶液重复操作，得到第七沉淀物；
(11)按照料液比1∶5-1∶10，在第七沉淀物中加入磷酸钠和过氧乙酸的混合液进行病毒灭活处理，离心，弃废液，得到第八沉淀物；
(12)按照料液比1∶5-1∶20，在第八沉淀物中加入生理盐水或磷酸盐缓冲液浸泡处理，离心，弃废液，得到第九沉淀物；
(13)按照料液比1∶5-1∶20，在第九沉淀物中加入无菌水清洗，冻干，冻干后用伽马射线或电子束灭菌。
第二方面，本申请提供了上述制备方法得到封装细胞的间质材料。
第三方面，本申请提供了上述封装细胞的间质材料的应用，即在细胞培养过程中可用于促进细胞团形成、在细胞保存运输过程中可用于封装和保护 细胞的间质材料；或在3D生物打印中的应用；或医美领域中的应用。
附图概述
图1为本申请的实施例1中用碱性酶处理和脱细胞后软骨基质微粒的扫描电镜图。
图2为本申请的实施例1中(左)用碱性酶处理和脱细胞后软骨基质微粒的悬浮稳定性和(右)未用碱性酶处理只有脱细胞处理的软骨基质微粒的悬浮稳定性。
图3为本申请的实施例1中(上)未处理猪耳软骨原料和(下)用碱性酶处理和脱细胞后软骨基质颗粒的差示扫描量热分析图。
图4为本申请的实施例1中L929细胞在碱性酶处理和脱细胞后软骨基质微粒上共培养情况图。
图5为本申请的实施例1中脐带间充质干细胞与软骨基质微粒共培养8h和8天后微粒上细胞生长情况图。
图6为本申请的实施例1中碱性酶处理和脱细胞后软骨基质微粒材料的温度响应特性图。
图7为本申请的实施例1中碱性酶处理和脱细胞后软骨基质微粒植入到大鼠皮下诱导脂肪组织形成的效果图。
图8为本申请的实施例3中猪耳软骨经碱性酶alcalase处理5h(左)和18h(右)后的组织切片图。
图9为本申请的实施例5中不同粒径软骨基质材料微粒的大体形貌。
详述
在第一方面的实施方案中，本申请提供了一种封装细胞的间质材料的制备方法，其包括如下步骤：
(1)将离体哺乳动物的软骨材料剔除表面的组织和筋膜，然后切成薄片；
(2)将软骨组织薄片的表面采用消毒剂进行消毒，之后进行清洗和冻干；
(3)将冻干的软骨组织薄片进行液氮低温微粒化研磨，制备成微粒材料；
(4)按照料液比1∶5-1∶20，将微粒材料用异丙醇进行脱脂处理，浸泡后离心，弃废液，得到第一沉淀物；
(5)按照料液比1∶5-1∶20，在第一沉淀物中加入生理盐水或磷酸盐缓冲液浸泡处理，离心，弃废液，得到第二沉淀物；
(6)按照料液比1∶5-1∶20，在第二沉淀物中加入脱氧核糖核酸酶溶液的缓冲液浸泡处理，离心，弃废液，得到第三沉淀物；
(7)按照料液比1∶5-1∶10，在第三沉淀物中加入碱性酶(alcalase)溶液浸泡处理，离心，弃废液，得到第四沉淀物；
(8)按照料液比1∶5-1∶20，在第四沉淀物中加入洗涤剂，配制在磷酸盐缓冲液中浸泡处理，离心，弃废液，得到第五沉淀物；
(9)按照料液比1∶5-1∶20，在第五沉淀物中加入乙二胺四乙酸二钠溶液浸泡，清洗，离心，弃废液；更换溶液再次清洗，再次离心，弃废液，得到第六沉淀物；
(10)按照料液比1∶5-1∶20，在第六沉淀物中加入生理盐水或磷酸盐缓冲液浸泡处理，离心，弃废液；更换溶液重复操作，得到第七沉淀物；
(11)按照料液比1∶5-1∶10，在第七沉淀物中加入磷酸钠和过氧乙酸的混合液进行病毒灭活处理，离心，弃废液，得到第八沉淀物；
(12)按照料液比1∶5-1∶20，在第八沉淀物中加入生理盐水或磷酸盐缓冲液浸泡处理，离心，弃废液，得到第九沉淀物；
(13)按照料液比1∶5-1∶20，在第九沉淀物中加入无菌水清洗，冻干，冻干后用伽马射线或电子束灭菌。
优选地，步骤(1)中，哺乳动物选自猪、牛、羊、马和鹿中的一种以上。
优选地，步骤(1)中，软骨材料选自弹性软骨和透明软骨中的一种以上。
优选地，弹性软骨为耳软骨。
优选地，透明软骨选自关节软骨、肋软骨、肩胛软骨和半月板中的一种以上。
优选地，步骤(2)中，消毒剂选自0.1-0.5％w/v次氯酸钠、0.5-2.0％w/v碳酸钠和50-70％w/v醇溶液中的一种以上。
优选地，醇溶液选自乙醇或异丙醇中的一种以上。
优选地，步骤(3)中，微粒材料的基于颗粒数量的平均粒径为2-20μm，微粒材料的基于颗粒体积的平均粒径为20-200μm。
优选地，步骤(4)中，异丙醇的含量为50-70％w/v；浸泡的时间为30-60min，离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(5)中，浸泡的时间为30-60min，离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(6)中，脱氧核糖核酸酶溶液的缓冲液选自羟乙基哌嗪乙硫磺酸缓冲液和三羟甲基氨基甲烷盐酸盐缓冲液中的一种以上；羟乙基哌嗪乙硫磺酸缓冲液的浓度为5-100mmol/L，脱氧核糖核酸酶溶液中脱氧核糖核酸酶的活性为50-250U/L。
优选地，步骤(6)中，浸泡的时间为8-12h，离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(7)中，碱性酶(alcalase)溶液中碱性酶(alcalase)的浓度为0.02-0.2％w/v。
优选地，步骤(7)中，浸泡的时间为30-120min，离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(8)中，洗涤剂选自0.5-2％w/v脱氧胆酸钠、曲拉通X-100或十二烷基硫酸钠中的一种以上。
优选地，步骤(8)中，浸泡的时间为8-16h，离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(9)中，乙二胺四乙酸二钠溶液中乙二胺四乙酸二钠的浓度为5-50mmol/L，配制在10mmol/L的磷酸钠缓冲液中。
优选地，步骤(9)中，清洗的时间为2-4h，离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(9)中，再次清洗的时间为12-24h，再次离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(10)中，浸泡的时间为30-120min，离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(11)中，病毒灭活处理的时间为30-60min，离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(12)中，浸泡的时间为30-120min，离心的转速为500-1500×g，时间为5-20min。
优选地，步骤(5)、步骤(8)、步骤(10)和步骤(12)中，磷酸盐缓冲液为磷酸钠缓冲液。
在本申请实施方案中，所述的制备方法可以为非疾病的诊断和治疗目的的方法。
在本申请实施方案中，除特别注明外，“％w/v”为“g/100mL”。
在第二方面的实施方案中，本申请提供了一种封装细胞的间质材料，其由上述的制备方法得到。
优选地，封装细胞的间质材料的主要成分为II型胶原蛋白和糖胺聚糖。
优选地，封装细胞的间质材料的主要成分为II型胶原蛋白、蛋白聚糖和糖胺聚糖。
优选地，糖胺聚糖包括硫酸软骨素和透明质酸。
在第二方面的一些实施方案中，所述封装细胞的间质材料含有II型胶原蛋白、硫酸软骨素和透明质酸。
在第二方面的一些实施方案中，所述封装细胞的间质材料含有II型胶原蛋白、硫酸软骨素和透明质酸，其中，II型胶原蛋白、硫酸软骨素和透明质酸三者之和为所述封装细胞的间质材料的85％w/w以上干重，优选地，为90％w/w以上干重，更优选地，为96.5±0.5％w/w干重。
在第二方面的一些实施方案中，所述封装细胞的间质材料含有II型胶原蛋白75％w/w以上，弹性蛋白少于10％w/w，糖胺聚糖含量1％至10％w/w； 这里所述的糖胺聚糖包括硫酸软骨素和透明质酸。
在第二方面的一些实施方案中，所述封装细胞的间质材料含有II型胶原蛋白、硫酸软骨素和透明质酸，其中，II型胶原蛋白、硫酸软骨素和透明质酸的含量分别为：92.4±0.4％w/w干重、2.5±0.09％w/w干重和1.6±0.04％w/w干重，或92.4±0.4％w/w干重、2.5±0.1％w/w干重和1.6±0.0％w/w干重；或92％w/w以上(干重)、2％w/w以上(干重)和1％w/w以上(干重)。
在第二方面的一些实施方案中，所述封装细胞的间质材料中残留的DNA含量低于20ng/mg，优选地，不超过5ng/mg，更优选地，不超过2.7±0.6ng/mg。
在第二方面的一些实施方案中，所述封装细胞的间质材料中α-Gal抗原表位数低至2.2×10 12±3.0×10 11个/克干重，或低于5.0×10 12个/克干重。
在第二方面的一些实施方案中，所述封装细胞的间质材料含有83.5±1.5％w/w至90.5±1.5％w/w的II型胶原蛋白。
第三方面，本申请提供了上述的封装细胞的间质材料在细胞培养过程中可用于促进细胞团形成、在细胞保存运输过程中可用于封装和保护细胞的间质材料；或在3D生物打印中的应用，还可以用于医美领域中填充诱导自体脂肪形成。
在第三方面的一些实施方案中，上述封装细胞的间质材料与细胞共培养，未表现出明显的细胞毒性，该间质材料的表面附着大量细胞，表现出良好的支持干细胞粘附与增殖的性能，该间质材料可以用作为细胞培养的微载体，还可以促进细胞团形成。
在第三方面的一些实施方案中，上述封装细胞的间质材料可通过自组装封装细胞，在细胞保存运输过程中可保护细胞抵御不良环境的影响，提高细胞存活率。
在第三方面的一些实施方案中，上述封装细胞的间质材料在逆境中对细胞具有保护作用。
在第三方面的一些实施方案中，上述封装细胞的间质材料可以用作组织支架材料，经注射进入体内用于组织再生修复的应用；或者，可以用于医美领域中填充诱导自体脂肪形成。
现有的细胞培养微载体材料，主要由高分子合成材料、纯化的或者经过结构修饰的天然材料(如淀粉、纤维素、壳聚糖、海藻酸钠、胶原蛋白、明胶等)化学交联制备而成，而本申请以哺乳动物(如猪、牛、羊、马、鹿等)的软骨为原料，制备的细胞封装间质材料主要成分是天然态的II型胶原蛋白和蛋白聚糖(硫酸软骨素和透明质酸)，无交联剂毒性，具有低免疫原性，生物相容性好，可用于人体植入的优点。
本申请基于软骨细胞外基质制备的细胞封装间质微粒材料，在结构上有良好的表面拓扑结构，可为体外细胞培养提供类似体内的组织微环境，不仅可以作为细胞培养的微载体，还可以促进细胞团形成；在功能上具有可逆的温度响应特性，在35℃以上呈现出类似液溶胶状态，在较低温度呈现出凝胶状态，溶胶转变为凝胶时通过自组装封装细胞；利用其溶胶转变为凝胶时可自组装封装细胞的特性，在细胞保存和运输过程中，将细胞封存在凝胶态间质中，可保护细胞抵御不良环境的影响，从而提高细胞存活率。
相比现有技术中用软骨材料研磨制备的软骨细胞基质微粒或水凝胶，经过酸碱处理或采用常规粉碎机进行研磨(如专利CN106075584A和CN112316211A)，研磨过程中的热效应引起材料中的蛋白改性或变性，降低材料的稳定性。本申请基于软骨细胞外基质制备的细胞封装间质微粒材料的热稳定性在40℃以上，可单独或同封装细胞一起用于3D生物打印形成稳定的组织和类器官结构。
本申请用于制备基于软骨细胞外基质的细胞封装间质材料的方法，使用碱性酶(alcalase)具有简单和高效的特点，有利于规模化制备。哺乳动物软骨极为致密，脱细胞过程时间长，难以实现完全彻底的细胞成分去除；通过多次反复脱细胞处理和延长脱细胞周期来实现彻底的细胞去除，不仅时间长，还造成软骨细胞外基质不够稳定，组成成分大量损失的缺点。而使用碱性酶(alcalase)处理还可减少异种组织中的α-Gal抗原表位。
具体实施方式
本申请提供了一种封装细胞的间质材料及其制备方法和应用，一种在细胞培养过程中可用于促进细胞团形成、通过可逆的温度响应特性自组封装和 解聚细胞、在细胞保存运输过程中可用于封装和保护细胞的间质材料，该材料是以哺乳动物(如猪、牛、羊、马、鹿等)的软骨为原料制备而成的复合天然生物材料，主要成分是II型胶原蛋白和糖胺聚糖(硫酸软骨素和透明质酸)。本申请中封装细胞的间质材料的II型胶原蛋白75％w/w以上，弹性蛋白少于10％w/w，糖胺聚糖(硫酸软骨素和透明质酸)含量1％至10％w/w。
本申请基于动物软骨材料制备封装细胞的间质材料的方法，该方法任选地为非疾病的诊断和治疗目的的方法，包括以下步骤：
(1)收集哺乳动物(如猪、牛、羊、马、鹿等)的软骨材料，包括耳软骨、关节软骨、肋软骨、肩胛软骨、半月板等弹性软骨和透明软骨；剔除软骨表面多余组织和筋膜，将软骨组织切成厚度2mm以下薄片；
(2)将软骨组织薄片采用消毒剂进行表面消毒，消毒后纯化水清洗，清洗后冻干至含水量小于5％w/w；其中，消毒剂选自0.1-0.5％w/v次氯酸钠、0.5-2.0％w/v碳酸钠和50-70％w/v乙醇(或异丙醇)中的一种以上。
(3)将冻干的软骨组织薄片进行液氮低温微粒化研磨，制备成基于颗粒数量的平均粒径为2-20μm和基于颗粒体积的平均粒径为20-200μm的微粒材料；
(4)将微粒材料用50-70％w/v异丙醇(IPA)进行脱脂处理，以料液比1∶5-1∶20的比例进行，微粒浸泡30-60min后，500-1500×g离心5-20min，弃废液，得到第一沉淀物；
(5)以料液比1∶5-1∶20的比例，在第一沉淀物中加入0.9％w/v生理盐水或10mmol/L的磷酸盐缓冲液(pH＝7.4)浸泡处理30-60min，500-1500×g离心5-20min，弃废液；重复清洗1次，得到第二沉淀物；
(6)以料液比1∶5-1∶20的比例，在第二沉淀物中加入脱氧核糖核酸酶溶液的缓冲液浸泡，脱氧核糖核酸酶溶液的缓冲液选自羟乙基哌嗪乙硫磺酸缓冲液和三羟甲基氨基甲烷盐酸盐缓冲液中的一种以上，该羟乙基哌嗪乙硫磺酸缓冲液的浓度为5-100mmol/L，pH＝7.6；脱氧核糖核酸酶溶液中脱氧核糖核酸酶的活性为50-250U/L，浸泡处理8-12h后，500-1500×g离心5-20min，弃废液，得到第三沉淀物；
(7)以料液比1∶5-1∶10的比例，在第三沉淀物中加入碱性酶(alcalase)溶液浸泡，该溶液中碱性酶(alcalase)的浓度为0.02-0.2％w/v，浸泡处理30-120min后，500-1500×g离心5-20min，弃废液，得到第四沉淀物；
(8)以料液比1∶5-1∶20的比例，在第四沉淀物中加入洗涤剂，该溶液中洗涤剂选自0.5-2％w/v脱氧胆酸钠、曲拉通X-100或十二烷基硫酸钠中的一种以上，配制在10mmol/L的磷酸盐缓冲液中，pH＝7.4；浸泡处理8-16h后，500-1500×g离心5-20min，弃废液，得到第五沉淀物；
(9)以料液比1∶5-1∶20的比例，在第五沉淀物中加入乙二胺四乙酸二钠(EDTA)溶液浸泡，乙二胺四乙酸二钠的浓度为5-50mmol/L，配制在10mmol/L的磷酸盐缓冲液中，溶液pH＝7.4；清洗2-4h后，500-1500×g离心5-20min，弃废液；换溶液再次清洗12-24h，500-1500×g再离心5-20min，弃废液，得到第六沉淀物；
(10)以料液比1∶5-1∶20的比例，在第六沉淀物中加入0.9％w/v生理盐水或10mmol/L的磷酸盐缓冲液(pH＝7.4)中浸泡处理30-120min，500-1500×g离心5-20min，弃废液；换溶液再浸泡30-120min，500-1500×g离心5-20min，弃废液，得到第七沉淀物；
(11)以料液比1∶5-1∶10的比例，在第七沉淀物中加入含有2-10％w/v的磷酸钠和0.05-0.5％w/v的过氧乙酸的混合液进行病毒灭活处理30-60min，500-1500×g离心5-20min，弃废液，得到第八沉淀物；
(12)以料液比1∶5-1∶20的比例，在第八沉淀物中加入0.9％w/v生理盐水或10mmol/L的磷酸盐缓冲液(pH＝7.4)浸泡处理30-120min，500-1500×g离心5-20min，弃废液，得到第九沉淀物；
(13)以料液比1∶5-1∶20的比例，在第九沉淀物中加入无菌水清洗30-60min；将处理后的微粒样品浓度调至3-6％w/w后冻干至含水量低于5％w/w；
(14)冻干后用伽马射线或电子束灭菌，灭菌剂量为15-35kGy。
其中，在步骤(5)中、步骤(8)中、步骤(10)中和步骤(12)中，磷酸盐缓冲液为磷酸钠缓冲液。
本申请的封装细胞的间质材料的制备方法过程中，还可以先进行碱性酶和脱细胞处理，再将软骨组织粉碎。
由上述的制备方法得到本申请的封装细胞的间质材料。
其中，封装细胞的间质材料的主要成分为II型胶原蛋白和糖胺聚糖(硫酸软骨素和透明质酸)。
本申请的封装细胞的间质材料在3D生物打印中得以应用，还可以用于医美领域中填充诱导自体脂肪形成。
下面结合实施例对本申请的技术内容做进一步的说明。下述实施例是说明性的，不是限定性的，不能以下述实施例来限定本申请的保护范围。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
本实施例的封装细胞的间质材料的制备方法任选地为非疾病的诊断和治疗目的的方法，包括如下步骤：
(1)从新鲜屠宰的6月龄猪体上收集猪耳软骨，剔除皮肤、结缔组织和软骨表面的筋膜；将软骨组织切成厚度2mm以下小块，用0.2％w/v次氯酸钠进行表面消毒10min后，用纯化水冲洗干净，冰冻干燥。
(2)将冻干的软骨放入200mL的研磨瓶，将装有软骨样品的密闭容器浸入液氮中，在-196℃环境中用SPEX 6875冷冻研磨仪，采用电磁撞子研磨技术快速粉碎样品。复温后使用100μm网筛选小粒径的软骨微粒。用激光衍射粒度分析仪检测，基于颗粒数量分布的粒径为Dx(10)＝1.8μm，Dx(50)＝2.9μm，Dx(90)＝6.0μm，其平均粒径为2-10μm；基于颗粒体积分布的粒径为Dv(10)＝15μm，Dv(50)＝67μm，Dv(90)＝144μm，其平均粒径为30-100μm。
(3)将软骨微粒放入70％w/v异丙醇溶液中浸泡脱脂，料液比1∶5的比例(即每升软骨微粒加入5升70％w/v异丙醇溶液)，脱脂处理1h后，500×g离心5min，弃上清废液，得到第一沉淀物。以料液比1∶5的比例加入10mmol/L的磷酸钠缓冲液(pH＝7.4)，浸泡处理30min后，500×g离心5min，弃上清 废液，得到第二沉淀物；重复用磷酸钠缓冲液清洗1次。
(4)将脱脂后的软骨微粒放入脱氧核糖核酸酶溶液的缓冲液中浸泡，脱氧核糖核酸酶溶液的缓冲液中羟乙基哌嗪乙硫磺酸缓冲液的浓度为10mmol/L，脱氧核糖核酸酶溶液中脱氧核糖核酸酶的活性为50U/L，pH＝7.6，料液比1∶5的比例；浸泡处理8h后，500×g离心5min，弃废液，得到第三沉淀物。然后按料液比1∶5的比例，在第三沉淀物中加入碱性酶(alcalase)溶液，溶液中碱性酶(alcalase)的浓度为0.05％w/v，配制在10mmol/L的磷酸钠缓冲液中，pH＝7.4，碱性酶(alcalase)处理120min后，500×g离心5min，弃上清废液。碱性酶(alcalase)处理后，按料液比1∶5的比例，在第四沉淀物中加入脱氧胆酸钠溶液，脱氧胆酸钠的浓度为1.0％w/v，配制在10mmol/L的磷酸钠缓冲液中，pH＝7.4，处理12h后，500×g离心5min，弃上清废液，得到第五沉淀物。
(5)将经过脱脂、酶处理和洗涤剂清洗后的材料加入EDTA溶液中，EDTA的浓度为10mmol/L，用10mmol/L磷酸钠缓冲液配制，pH＝7.4，料液比1∶5，浸泡清洗2h，500×g离心5min，弃上清废液，得到第六沉淀物；重复用EDTA溶液清洗1次。然后用10mmol/L磷酸钠缓冲液(pH＝7.4)浸泡清洗2h，500×g离心5min，弃上清废液，得到第七沉淀物；重复用磷酸钠溶液清洗1次。
(6)将清洗后的材料加入过氧乙酸的混合液做病毒灭活处理，按料液比1∶5的比例加入含有2％w/v磷酸钠和0.2％w/v的过氧乙酸的混合液，病毒灭活处理60min后，500×g离心5min，弃上清废液，得到第八沉淀物。加入料液比1∶5比例的0.9％w/v生理盐水，浸泡处理60min，500×g离心5min，弃上清废液，得到第九沉淀物。
(7)将病毒灭活后的材料用无菌纯化水清洗2次，料液比1∶5的比例，在第九沉淀物中加入无菌水清洗，每次30min后，500×g离心5min，弃上清废液。将处理后的微粒样品干重浓度调至3％w/v后进行冰冻干燥。冻干后用伽马射线灭菌，辐照剂量为17.1kGy。制备获得基于软骨材料的封装细胞的间质材料。
经上述方法制备的软骨基质微粒材料，其残留的DNA含量为2.7±0.6 ng/mg干重；与未经处理的冻干软骨原料相比(679.8±7.3ng/mg干重)，下降了99.6％。处理后的软骨基质微粒材料中胶原蛋白含量为92.4±0.4％w/w干重，未经处理的冻干软骨原料中胶原蛋白含量为50.9±5.2％w/w干重，说明碱性酶alcalase处理和脱细胞处理有效地去除了其它组织蛋白质。处理后的软骨基质微粒材料中硫酸软骨素含量为25.5±0.9ug/mg干重(2.55±0.09％w/w干重)，未经处理的冻干软骨原料中硫酸软骨素含量为80.5±4.1ug/mg干重(8.05±0.41％w/w干重)。处理后的软骨基质微粒材料中透明质酸含量为16.6±0.4ug/mg干重(1.66±0.04％w/w干重)，未经处理的冻干软骨原料中透明质酸含量为136.6±7.3ug/mg干重(13.66±0.73％w/w干重)。成分分析结果表明，处理后软骨基质微粒材料是胶原蛋白、硫酸软骨素和透明质酸天然复合材料，上述三者之和为96.5±0.53％w/w干重。
经上述方法制备的软骨基质微粒用0.9％w/v氯化钠溶液复水后，用激光衍射粒度分析仪检测，制备的软骨基质材料以数量分布的粒径为Dx(10)＝2.8μm，Dx(50)＝4.0μm，Dx(90)＝8.7μm，其颗粒数量的平均粒径为3-8μm；基于颗粒体积分布的粒径为Dv(10)＝13μm，Dv(50)＝80μm，Dv(90)＝400μm，其颗粒体积的平均粒径为40-200μm。用碱性酶处理和脱细胞处理后软骨基质颗粒材料的粒径有所增加。
经上述方法制备的软骨基质微粒材料，参照标准《组织工程医疗器械产品动物源性支架材料残留α-Gal抗原检测》(YY/T 1561-2017)中的方法，对α-Gal抗原表位数进行了定量检测。未处理猪耳软骨原料中的α-Gal抗原表位数为5.4×10 13±5.2×10 12个/克干重材料，处理后软骨基质颗粒材料的α-Gal抗原表位数低至2.2×10 12±3.0×10 11个/克干重材料，碱性酶处理和脱细胞去除了95.9％的α-Gal抗原表位数。
软骨基质颗粒的表面拓扑结构对细胞黏附和增殖等响应行为会产生重要影响，同时对细胞形态、表型和细胞运动具有重要生理作用。经本实施例碱性酶处理和脱细胞工艺制备的软骨基质微粒，经超高分辨场发射扫描电镜(SU8000)对其表面结构进行观察，结果显示软骨基质微粒为立体多孔凹凸不平糙面结构，具有极大的表面积(图1)。
经上述方法制备的软骨基质微粒材料，用0.9％w/v氯化钠溶液(生理盐 水)配制成4％w/v的悬浮液，静置5天后不沉降、不分离，具有良好的悬浮稳定性(图2)。如果没有碱性酶alcalase处理，而只有脱细胞处理，微粒材料会出现沉降，表明其悬浮稳定性较差。碱性酶处理和脱细胞后软骨基质微粒的悬浮稳定性提高与处理后软骨基质颗粒的粒径增加有关，降低了基质颗粒的密度。
用差示扫描量热仪(DSC)对在生理盐水中复水的软骨原料和制备的软骨基质颗粒材料的热稳定性做了比较(图3)，用本申请的酶处理和脱细胞方法制备的软骨基质颗粒的热稳定性良好，在40℃以上，与未处理的软骨原料基本相似，表明制备过程中没有受到破坏。因为胶原蛋白含量增加，所以制备的软骨基质颗粒蛋白质变性时的热焓值获得了显著升高。此外，将软骨基质微粒在37℃放置24h后再做DSC检测，差示扫描量热分析图也没有发生变化，结果也表明软骨基质颗粒的热稳定性。
为检验碱性酶处理和脱细胞后软骨基质微粒是否具有良好的生物安全性，将L929细胞与软骨脱基质微粒共培养48h，结果表明细胞出现较为明显的聚集生长现象，表明该材料具有良好安全性，在一定程度上提示具有良好的生物相容性(图4)。
为了进一步检验碱性酶处理和脱细胞后软骨基质微粒支持细胞粘附与增殖的能力，将人脐带间充质干细胞与软骨基质微粒共培养，观察其细胞粘附、细胞毒性作用，该实验结果表明该材料共培养未表现出明显的细胞毒性，软骨基质微粒表面附着大量细胞，表现出良好的支持干细胞粘附与增殖的性能(图5)。基于软骨基质材料可以用作为细胞培养的微载体，还可以促进细胞团形成。
经上述方法制备的软骨基质微粒材料，在功能上具有可逆的温度响应特性(图6)。在37℃时呈现出类似液溶胶状态，具有流动性；在温度下降时呈现出凝胶状态，不具有流动性；溶胶转变为凝胶时可通过自组装封装细胞。利用其溶胶转变为凝胶时可自组装封装细胞的特性，在细胞保存运输过程中可保护细胞抵御不良环境的影响，提高细胞存活率。因为软骨基质微粒的温度响应特性具有可逆性，温度升至37℃时细胞可以解聚。
L929细胞与2.5％软骨脱基质微粒在37℃共培养48h后，将细胞悬浮液 转入25℃恒温金属浴和4℃冰箱中存放，用于检验软骨基质胶在逆境中对细胞的保护作用。转入低温环境后第1天和第4天用CCK-8试剂盒检测L929细胞的活性。在25℃恒温金属浴中第1天和第4天的细胞活性分别是0.424±0.008和0.459±0.022，细胞活性没有发生变化；在4℃冰箱中第1天和第4天的细胞活性分别是0.410±0.012和0.344±0.020，细胞活性略有下降。
经上述方法制备的软骨基质微粒材料，通过注射的方式植入到大鼠背部皮下能起到良好的支架作用，可诱导大量脂肪组织形成(图7)。植入后3周时，可见有炎性反应，但随时间延长炎性程度减少，无慢性炎症反应；6周时软骨基质微粒区域出现新血管形成，软骨基质微粒随时间而发生一定程度的降解；10周时出现脂肪细胞进入生长，16周时已完全填满脂肪细胞。故也可以用于医美领域中填充诱导自体脂肪形成。
实施例2
本实施例的软骨基质材料的制备方法为非疾病的诊断和治疗目的的方法，除以下一个步骤与实施例1不同以外，其它步骤完全相同。在步骤(4)中、在脱氧核糖核酸酶溶液浸泡后，没有经过碱性酶(alcalase)溶液处理，直接加入了脱氧胆酸钠溶液处理。
经实施例2方法制备的软骨基质微粒与经实施例1方法制备的软骨基质微粒有很大不同(表1)。经实施例1方法处理后软骨基质微粒的残留DNA含量较低，残留的α-Gal抗原表位数少；经实施例2方法处理后软骨基质微粒的残留DNA含量较高，残留的α-Gal抗原表位数较多。经实施例1方法处理后软骨基质微粒材料胶原蛋白、硫酸软骨素和透明质酸三者的含量累加为96.5±0.53％w/w干重；而经实施例2方法处理后软骨基质微粒材料胶原蛋白、硫酸软骨素和透明质酸三者的含量累加仅为75.1±5.2％w/w干重。检测结果说明碱性酶(alcalase)处理的效果显著。
表1实施例1方法与实施例2方法的比较
Figure PCTCN2022141455-appb-000001
Figure PCTCN2022141455-appb-000002
实施例3
本实施例的封装细胞的间质材料的制备方法为非疾病的诊断和治疗目的的方法，包括如下步骤：
(1)从新鲜屠宰的6月龄猪体上的耳软骨，去除其它组织，将厚度为约2mm软骨切成2cm×3cm小块，以料液比1∶5的比例进行加入0.5％w/v次氯酸钠溶液进行消毒处理10min后弃废液；加入70％w/v异丙醇脱脂60min后弃废液，再放入0.125％w/v的碱性酶(alcalase)溶液中浸泡18h。
(2)将碱性酶处理后的材料用过氧乙酸的混合液做病毒灭活处理，按料液比1∶5的比例加入含有2％w/v磷酸钠和0.15％w/v的过氧乙酸的混合液，病毒灭活处理120min后，弃废液；然后加入EDTA溶液，EDTA的浓度为10mmol/L，用10mmol/L磷酸钠缓冲液配制，pH＝7.4，料液比1∶5，浸泡清洗24h，弃废液后，重复用EDTA溶液清洗24h。
(3)使用生理盐水清洗24h。
按实施例3方法制备软骨脱细胞基质材料过程中，碱性酶(alcalase)处理5h后取样检测脱细胞进展，碱性酶(alcalase)处理5h后软骨组织中间还有与未处理软骨相似的含有细胞区域，但是碱性酶处理18h后的，整片软骨已完全变化，残留的DNA含量为6.79±0.65ng/mg干重。猪耳软骨经碱性酶alcalase处理5h(左)和18h(右)后的组织切片如图8。碱性酶处理后的软骨基质胶原蛋白含量为76.5±6.5％w/w干重，说明用碱性酶(alcalase)处理也能有效的进行软骨脱细胞；但是与微粒化的材料(实施例1)相比，使用完整软骨的效率明显较低。
实施例4
本实施例的封装细胞的间质材料的制备方法为非疾病的诊断和治疗目的的方法，包括如下步骤：
(1)从新鲜屠宰的22月龄牛体上的肋软骨，去除其它组织，将软骨组 织切成厚度2mm以下薄片；将软骨组织薄片以料液比1∶5的比例进行加入50％w/v异丙醇进行脱脂和消毒处理，浸泡30min后，1500×g离心20min，弃废液，得到第一沉淀物。
(2)将异丙醇处理后的软骨组织薄片以料液比1∶10的比例加入碱性酶alcalase溶液中处理，溶液中的碱性酶浓度为0.2％w/v，配制在10mmol/L的磷酸钠缓冲液中，pH＝7.4；处理30min后，1500×g离心20min，弃废液，得到第二沉淀物。
(3)将碱性酶alcalase处理后的软骨组织薄片以料液比1∶20的比例，加入曲拉通X-100，配制在10mmol/L的磷酸钠缓冲液中，pH＝7.4；处理16h后，1500×g离心20min，弃废液，得到第三沉淀物。
(4)将洗涤液浸泡后的软骨组织薄片以料液比1∶5的比例放入0.9％w/v生理盐水中浸泡处理30min，1500×g离心20min，弃废液；换溶液再浸泡30min，1500×g离心20min，弃废液，得到第四沉淀物。
(5)将清洗后的软骨组织薄片以料液比1∶20的比例，放入脱氧核糖核酸酶溶液的缓冲液中浸泡，该缓冲液中含5mmol/L羟乙基哌嗪乙硫磺酸缓冲液，脱氧核糖核酸酶溶液中脱氧核糖核酸酶的活性为250U/L，pH＝7.6；处理12h后，1500×g离心20min，弃废液，得到第五沉淀物。
(6)将脱氧核糖核酸酶处理后的软骨组织薄片以料液比1∶20的比例，加入10mmol/L的磷酸钠缓冲液(pH＝7.4)中浸泡处理120min，1500×g离心5min，弃废液；换溶液再浸泡120min，1500×g离心5min，弃废液，得到第六沉淀物。
(7)以料液比1∶10的比例放入含有10％w/v的氯化钠和0.5％w/v的过氧乙酸的混合液中病毒灭活处理30min，1500×g离心20min，弃废液，得到第七沉淀物。
(8)将病毒灭活后的软骨组织薄片以料液比1∶20的比例放入EDTA溶液浸泡，EDTA的浓度为50mmol/L，溶液pH＝7.4；清洗4h后，1500×g离心20min，弃废液重复用EDTA溶液清洗1次，1500×g离心20min，弃废液，得到第八沉淀物。
(9)将处理后的软骨组织薄片以料液比1∶20的比例放入无菌水中清洗60min，将清洗后的软骨组织薄片冻干至含水量低于5％w/w。
(10)将冻干的软骨组织薄片放入200mL的研磨瓶，将装有软骨样品的密闭容器浸入液氮中，在-196℃环境中用SPEX 6875冷冻研磨仪，采用电磁撞子研磨技术快速粉碎样品。复温后使用100μm网筛选小粒径的软骨微粒。
(11)将微粒材料用伽马射线或电子束灭菌制备成可用于封装细胞的间质材料，灭菌剂量为15kGy。
经上述实施例4方法制备的软骨基质微粒材料，与经实施例1方法制备的软骨基质微粒具有相似的特征。
实施例5：
本实施例的封装细胞的间质材料的制备方法包括如下步骤：
(1)从新鲜屠宰的6月龄猪体上的耳软骨，去除其它组织；加入70％w/v异丙醇脱脂60min后弃废液；用纯化水清洗干净后冻干；
(2)将冻干的软骨装入密闭容器浸入液氮中，在-196℃环境中冷冻研磨仪(参见实施例1)。低温研磨后使用筛网按粒径分筛，获得不同粒径的软骨微粒(＜0.1mm、0.1-0.2mm、0.2-0.3mm、0.3-0.4mm)；
(3)将不同粒径的软骨微粒分别用含有脱氧核糖核酸酶溶液的缓冲液中浸泡，每克软骨微粒干重加入12mL溶液；该缓冲液中含5mmol/L羟乙基哌嗪乙硫磺酸缓冲液，脱氧核糖核酸酶溶液中脱氧核糖核酸酶的活性为500U/L(pH＝7.6)，在37℃中处理16h后1500×g离心20min，弃废液；
(4)按每克软骨微粒干重加入6mL的0.05％w/v碱性酶Alcalase在37℃处理2h后1500×g离心20min，弃废液；
(5)按每克软骨微粒干重加入6mL的0.5％w/v脱氧胆酸钠(SDOC)室温处理6h，1500×g离心20min，弃废液；
(6)使用含青霉素/链霉素磷酸缓冲液溶液(pH＝7.0，含100U青霉素/链霉素/mL)室温清洗4次，每次8h；1500×g离心20min收集沉降的软骨基质微粒；
(7)用0.9％w/v氯化钠溶液稀释，使软骨基质微粒浓度为5％w/w；
(8)伽马射线辐照终端消毒(19.03kGy-20.08kGy)，取样分析。
不同粒径的软骨微粒制备的软骨间质材料出现了不同程度的聚集现象；随软骨微粒粒径增加，基质材料的发生聚集的程度增加(图9)，仅当粒径＜0.1mm时，才具有良好的分散性，当粒径＞0.3mm时出现明显的聚集、交织现象。
没有脱细胞前软骨微粒的DNA含量约为411ng/mg，脱细胞后DNA含量低于20ng/mg；软骨基质中胶原蛋白含量为83.5±1.2％w/w-90.5±1.5％w/w，粒径越大胶原蛋白含量越高；软骨基质中弹性蛋白含量1.6-4.0％w/w。使用Sulfated Glycosaminoglycan(sGAG)assay kit试剂盒测定软骨脱细胞基质微载体中硫酸软骨素含量，硫酸软骨素含量为7.1±0.7％w/w。
经上述实施例5方法制备的软骨基质微粒材料，与经实施例1方法制备的软骨基质微粒具有相似的特征。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本申请。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中，而不必经过创造性的劳动。因此，本申请不限于上述实施例。本领域技术人员根据本申请的原理，不脱离本申请的范畴所做出的改进和修改都应该在本申请的保护范围之内。

Claims (13)

  1. 一种封装细胞的间质材料的制备方法，其特征在于：其包括如下步骤：
    (1)将离体哺乳动物的软骨材料剔除表面的组织和筋膜，然后切成薄片；
    (2)将软骨组织薄片的表面采用消毒剂进行消毒，之后进行清洗和冻干；
    (3)将冻干的软骨组织薄片进行液氮低温微粒化研磨，制备微粒材料；
    (4)按照料液比1∶5-1∶20，将微粒材料用异丙醇进行脱脂处理，浸泡后离心，弃废液，得到第一沉淀物；
    (5)按照料液比1∶5-1∶20，在所述第一沉淀物中加入生理盐水或磷酸盐缓冲液浸泡处理，离心，弃废液，得到第二沉淀物；
    (6)按照料液比1∶5-1∶20，在所述第二沉淀物中加入脱氧核糖核酸酶溶液的缓冲液浸泡处理，离心，弃废液，得到第三沉淀物；
    (7)按照料液比1∶5-1∶10，在所述第三沉淀物中加入碱性酶(alcalase)溶液浸泡处理，离心，弃废液，得到第四沉淀物；
    (8)按照料液比1∶5-1∶20，在所述第四沉淀物中加入洗涤剂，配制在磷酸盐缓冲液中浸泡处理，离心，弃废液，得到第五沉淀物；
    (9)按照料液比1∶5-1∶20，在所述第五沉淀物中加入乙二胺四乙酸二钠溶液浸泡，清洗，离心，弃废液；更换溶液再次清洗，再次离心，弃废液，得到第六沉淀物；
    (10)按照料液比1∶5-1∶20，在所述第六沉淀物中加入生理盐水或磷酸盐缓冲液浸泡处理，离心，弃废液；更换溶液重复操作，得到第七沉淀物；
    (11)按照料液比1∶5-1∶10，在所述第七沉淀物中加入磷酸钠和过氧乙酸的混合液进行病毒灭活处理，离心，弃废液，得到第八沉淀物；
    (12)按照料液比1∶5-1∶20，在所述第八沉淀物中加入生理盐水或磷酸盐缓冲液浸泡处理，离心，弃废液，得到第九沉淀物；
    (13)按照料液比1∶5-1∶20，在所述第九沉淀物中加入无菌水清洗，冻干，冻干后用伽马射线或电子束灭菌。
  2. 根据权利要求1所述的封装细胞的间质材料的制备方法，其特征在于：步骤(1)中，所述哺乳动物选自猪、牛、羊、马和鹿中的一种以上；
    优选地，步骤(1)中，所述软骨材料选自弹性软骨和透明软骨中的一种以上；
    优选地，所述弹性软骨为耳软骨；
    优选地，所述透明软骨选自关节软骨、肋软骨、肩胛软骨和半月板中的一种以上。
  3. 根据权利要求1所述的封装细胞的间质材料的制备方法，其特征在于：步骤(2)中，所述消毒剂选自0.1-0.5％w/v次氯酸钠、0.5-2.0％w/v碳酸钠和50-70％w/v醇溶液中的一种以上；
    优选地，所述醇溶液选自乙醇或异丙醇中的一种以上；
    优选地，步骤(3)中，所述微粒材料的基于颗粒数量的平均粒径为2-20μm，所述微粒材料的基于颗粒体积的平均粒径为20-200μm。
  4. 根据权利要求1所述的封装细胞的间质材料的制备方法，其特征在于：步骤(4)中，所述异丙醇的含量为50-70％w/v；所述浸泡的时间为30-60min，所述离心的转速为500-1500×g，时间为5-20min；
    优选地，步骤(5)中，所述浸泡的时间为30-60min，所述离心的转速为500-1500×g，时间为5-20min；
    优选地，步骤(6)中，所述脱氧核糖核酸酶溶液的缓冲液选自羟乙基哌嗪乙硫磺酸缓冲液和三羟甲基氨基甲烷盐酸盐缓冲液中的一种以上；所述脱氧核糖核酸酶溶液中脱氧核糖核酸酶的活性为50-250U/L；
    优选地，所述羟乙基哌嗪乙硫磺酸缓冲液的浓度为5-100mmol/L；
    优选地，步骤(6)中，所述浸泡的时间为8-12h，所述离心的转速为500-1500×g，时间为5-20min。
  5. 根据权利要求1所述的封装细胞的间质材料的制备方法，其特征在于：步骤(7)中，所述碱性酶(alcalase)溶液中碱性酶(alcalase)的浓度为0.02-0.2％w/v；
    优选地，步骤(7)中，所述浸泡的时间为30-120min，所述离心的转速为500-1500×g，时间为5-20min；
    优选地，步骤(8)中，所述洗涤剂选自脱氧胆酸钠、曲拉通X-100或十二烷基硫酸钠中的一种以上；
    优选地，步骤(8)中，所述浸泡的时间为8-16h，所述离心的转速为500-1500×g，时间为5-20min。
  6. 根据权利要求1所述的封装细胞的间质材料的制备方法，其特征在于：步骤(9)中，所述乙二胺四乙酸二钠溶液中乙二胺四乙酸二钠的浓度为5-50mmol/L，配制在10mmol/L的磷酸钠缓冲液中；
    优选地，步骤(9)中，所述清洗的时间为2-4h，所述离心的转速为500-1500×g，时间为5-20min；
    优选地，步骤(9)中，所述再次清洗的时间为12-24h，所述再次离心的转速为500-1500×g，时间为5-20min；
    优选地，步骤(10)中，所述浸泡的时间为30-120min，所述离心的转速为500-1500×g，时间为5-20min。
  7. 根据权利要求1所述的封装细胞的间质材料的制备方法，其特征在于：步骤(11)中，所述病毒灭活处理的时间为30-60min，所述离心的转速为500-1500×g，时间为5-20min；
    优选地，步骤(12)中，所述浸泡的时间为30-120min，所述离心的转速为500-1500×g，时间为5-20min。
  8. 根据权利要求1至7中任一项所述的封装细胞的间质材料的制备方法，其特征在于：步骤(5)、步骤(8)、步骤(10)和步骤(12)中，所述磷酸盐缓冲液为磷酸钠缓冲液。
  9. 一种封装细胞的间质材料，其特征在于：其由权利要求1-8任一项所述的制备方法得到；
    优选地，所述封装细胞的间质材料的主要成分为II型胶原蛋白和糖胺聚糖；
    优选地，所述糖胺聚糖包括硫酸软骨素和透明质酸。
  10. 根据权利要求9所述的封装细胞的间质材料，含有II型胶原蛋白、硫酸软骨素和透明质酸，其中，II型胶原蛋白、硫酸软骨素和透明质酸三者之和为所述封装细胞的间质材料的85％w/w以上干重，优选地，为90％w/w以上干重；
    优选地，II型胶原蛋白75％w/w以上，弹性蛋白少于10％w/w，糖胺聚糖含量1％至10％w/w；这里所述的糖胺聚糖包括硫酸软骨素和透明质酸；
    优选地，II型胶原蛋白、硫酸软骨素和透明质酸的含量分别为：92％w/w以上(干重)、2％w/w以上(干重)和1％w/w以上(干重)；
    优选地，所述封装细胞的间质材料中残留的DNA含量低于20ng/mg，优选地，不超过5ng/mg；
    优选地，所述封装细胞的间质材料中α-Gal抗原表位数低于5.0×10 12个/克干重。
  11. 如权利要求9或10所述的封装细胞的间质材料在3D生物打印中的应用。
  12. 如权利要求9或10所述的封装细胞的间质材料在封装、或保护或封装和保护细胞中的应用。
  13. 如权利要求9或10所述的封装细胞的间质材料经注射进入体内用于组织再生修复的应用；或者，在医美领域中填充诱导自体脂肪形成的应用。
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