WO2014008844A1 - 一种制备细胞外基质支架材料的脱细胞方法 - Google Patents

一种制备细胞外基质支架材料的脱细胞方法 Download PDF

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WO2014008844A1
WO2014008844A1 PCT/CN2013/079006 CN2013079006W WO2014008844A1 WO 2014008844 A1 WO2014008844 A1 WO 2014008844A1 CN 2013079006 W CN2013079006 W CN 2013079006W WO 2014008844 A1 WO2014008844 A1 WO 2014008844A1
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solution
tissue
glucopyranoside
hours
pericardium
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PCT/CN2013/079006
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English (en)
French (fr)
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董教明
李�雨
陈大凯
陈叶萌
王静
穆元姗
房圆
陈国明
张晓怡
乐承筠
罗七一
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上海微创医疗器械（集团）有限公司
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Priority to AU2013289661A priority Critical patent/AU2013289661B2/en
Priority to EP13817500.5A priority patent/EP2873429B1/en
Publication of WO2014008844A1 publication Critical patent/WO2014008844A1/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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/362—Skin, e.g. dermal papillae
    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3625—Vascular tissue, e.g. heart valves
    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the invention relates to the field of tissue engineering materials.
  • the present invention relates to a method of decellularizing a material for preparing an extracellular matrix scaffold and an extracellular matrix prepared by the method. Background technique
  • the tissue is composed of a cell and an extracellular matrix (ECM), in which the cellular component is recognized as an antigen by the host to cause inflammation or immune rejection.
  • ECM extracellular matrix
  • Bioscaffold materials composed of ECM are widely used in surgical reconstruction and regenerative medicine of tissues and organs, such as heart valves, blood vessels, skin, nerves, tendons, and small intestinal submucosa.
  • ECM is a secretory product of cells in tissues and organs. It has a dynamic interaction with these cells, which can reflect changes in the microenvironment in a timely manner, and plays an important role in cell migration, differentiation and proliferation.
  • the cells of heterologous or xenogenic connective tissues are removed by known conventional methods to form an extracellular matrix called decellularized, in decellularized cells. During the process, cells that cause tissue rejection are removed while retaining the complex structure and key biochemical components of the original tissue.
  • Different decellularization methods directly affect the composition and ultrastructure of the resulting acellular matrix, and ultimately affect the host's response to the acellular matrix after transplantation.
  • methods for preparing acellular matrix mainly including physical methods, chemical methods, and biological treatment methods.
  • Physical methods are mainly to remove cells by physical effects such as freezing and thawing, liquid high pressure, ultrasonic waves and electric shock. Their basic principle is to destroy the cell membrane of cells in tissues. Structure, changes in cell membrane structure lead to undesirable biochemical reactions in cells, and continued treatment will cause cell death, followed by removal of tissue by washing of the solution, removal of nucleic acids and lipids.
  • Chemical method is to use chemical reagents to break cells to achieve the purpose of removing cells.
  • Some specific chemical reagents such as acids, bases and detergents can penetrate various layers of the tissue, the bilayer phospholipids that dissolve the cell membrane and even destroy the cell membrane proteins to cause cell death and fragmentation, and then wash away the cell debris and antigen by shaking and washing steps.
  • substance. Biological treatment mainly uses enzyme reagents to lyse cells.
  • trypsin is commonly used in the decellularization method. It is a type of serine protease that selectively hydrolyzes a peptide chain composed of a carboxyl group of lysine or arginine in a protein. The action of trypsin makes intercellular The protein is hydrolyzed to discretize the cells.
  • 201110134511.9 relates to a preparation method of the same kind of acellular dermal matrix, which is decellularized by using sodium lauryl sulfate. Although the time is short, the sodium dodecyl sulfate has a great damage to the matrix structure. And sodium lauryl sulfate has obvious toxicity, which is not conducive to in vivo implantation.
  • Chinese Patent Application No. 200510002464.7 relates to a method for decellularizing a heterogeneous cardiovascular graft
  • Chinese Patent Application No. 200910076674.9 relates to a vascular matrix for removing cells in vascular tissue and a method for preparing the same. Both of these patent applications use trypsin.
  • n-Octyl beta-D-Glucopyranoside is a new type of green surfactant with excellent performance, both nonionic and anionic The characteristics of the surfactant. It not only has low surface tension, high activity, strong detergency, rich and delicate foam, but also has the advantages of no irritation to the skin, good biodegradation, no toxicity, no pollution to the environment.
  • Non-Patent Document 1 OGP has high surface activity, can destroy the cell membrane structure of bacteria, and has broad-spectrum antibacterial properties, such as antibacterial action against Gram-negative, positive bacteria, etc., and is highly antibacterial, and is ideal. A mild nonionic surfactant. Summary of the invention
  • the technical problem solved by the present invention is to provide a method for decellularizing a material for preparing an extracellular matrix scaffold and an extracellular matrix (ECM) prepared by the method.
  • the method can completely remove the cells and cell debris existing on the surface and inside of the tissue, and prepare an extracellular matrix material having better mechanical properties and biological properties, thereby obtaining a scaffold material for tissue engineering.
  • the present invention relates to a method of decellularizing a material for preparing an extracellular matrix scaffold comprising the following steps:
  • step b adding the tissue and organs treated in step b to the nuclease-containing solution to degrade various DNA and/or RNA components in the cells;
  • the tissue to be used is a heterologous or allogeneic or autologous tissue organ, including any one of a bovine pericardium, a pig pericardium, a heart valve, a skin, a blood vessel, a small intestinal submucosa, a nerve, and the like.
  • tissues containing more collagen fibers or spandex are suitable for removing cellular components by this method.
  • the pretreatment is to use a physiological saline containing each antibiotic, or a phosphate buffer having a pH of 6.8-8.6, or a D-Hanks solution having a pH of 6.8-8.6 (Hanks).
  • the solution is one of the common balanced salt solutions (BSS) and is also the tissue culture medium.
  • the D-Hanks solution is Hanks liquid (GIBCO) without calcium and magnesium ions, and the tissues and organs to be treated are routinely cleaned, disinfected and separated under the condition of 0-37 Torr.
  • the antibiotic comprises one of two types of antibiotics, each of which is usually used in combination, one of which includes amikacin, flucytosine, vancomycin, chloramphenicol; the other class includes penicillin, Streptomycin sulfate, amphotericin 8.
  • the antibiotic is preferably Pen Strep (Gibco).
  • the glucopyranoside is selected from the group consisting of n-octyl- ⁇ -D-glucopyranoside (OGP), octyl- ⁇ -D-thioglucopyranoside, decyl glucopyranoside Any one or a combination of decyl glucopyranoside, dodecyl glucopyranoside (this combination may also be collectively referred to as alkyl glucoside), preferably OGP.
  • OGP n-octyl- ⁇ -D-glucopyranoside
  • octyl- ⁇ -D-thioglucopyranoside decyl glucopyranoside Any one or a combination of decyl glucopyranoside, dodecyl glucopyranoside (this combination may also be collectively referred to as alkyl glucoside), preferably OGP.
  • the concentration of the glucopyranoside in the solution is less than 70% (w/v;), ie the weight: 70 g: 100 ml, preferably 0.1-10% (w/v), ie the weight
  • the volume is 0.1-10 g: 100 ml, more preferably 0.5-5% (w/v).
  • the temperature of the solution containing glucopyranoside ranges from rC to 40 °C, more preferably from 3 to 8 °C.
  • the pH of the solution containing glucopyranoside ranges from 5 to 12, more preferably from 6.8 to 8.6, and the treatment time is from 30 minutes to 96 hours, more preferably from 6 to 48 hours.
  • the hypotonic solution is oscillated by shaking in a 0.01 M sterile Tris-HCl buffer (pH 6.8-8.6) at 1 ° C to 40 ° C (more preferably 3 ° C to 8 ° C). From 30 minutes to 96 hours (more preferably from 6 to 48 hours), the oscillation rate is from 50 to 360 rpm, more preferably from 100 to 250 rpm.
  • the nuclease solution is prepared by adding 100-10000 u/ml in a sterile 0.02-0.05 M Tris-HCl buffer (pH 6.8-8.6) containing 0.15 M sodium chloride and 1-5 mM magnesium chloride ( Deoxyribonuclease (DNase) and ribonuclease (RNase) are preferred from 1000 to 8000 u/ml. Thereafter, the tissue organ is added to the nuclease solution and shaken for 12-72 hours at a rotational speed of 60-360 rpm (preferably 100-250 rpm) in an environment of 37 °C. The washing is performed using each physiological saline containing antibiotics, or a pH of 6.8-8.6. Phosphate buffer, or D-Hanks solution with a pH of 6.8-8.6, the conventional acellular matrix is routinely cleaned at C-25 °C for 6-48 hours. Evaluation criteria and methods for the acellular matrix of the present invention:
  • Residual cells The acellular matrix was fixed with 10% neutral formalin, embedded in paraffin, cut into 0.4 ⁇ m thick slices, dewaxed by xylene, dehydrated with series of alcohol, stained with hematoxylin-eosin, and observed for cell residue. happening.
  • Matrix fiber structure The acellular matrix was fixed with 10% neutral formalin, embedded in paraffin, cut into 0.4 ⁇ m thick slices, dewaxed by xylene, dehydrated with series of alcohol, stained with Movat Pentachrome, and observed for collagen in the matrix. Distribution and structural changes of fibers, elastic fibers and glycoproteins.
  • Cell surface residual The acellular matrix was fixed with 2.5% glutaraldehyde solution, ethanol was dehydrated step by step, dried, vacuum sprayed with gold, and the surface cell residue and fiber arrangement of the scanning electron microscope were observed.
  • elastin content The acellular matrix was boiled at 0-2 mmol/L oxalic acid at 100 ° C for 60 minutes, centrifuged, and the supernatant was taken, so that the extract was repeatedly extracted 3 times, and the supernatant was combined with elastin content (/dry weight). The measurement of the tissue was measured using the Fastin elastin kit (Biocolor).
  • the acellular matrix is cut into a sheet, in 10,000 The material testing machine will pull it off, and the maximum tensile force measured is divided by the cross-sectional area of the test material, which is the tensile strength of the matrix material, indicating the tensile strength of the matrix.
  • the acellular matrix is cut into a sheet shape, which is pulled off on a universal material testing machine to obtain the tensile stress and strain of the matrix material, and the stress-strain curve is linear. Fit, calculate the slope of the line, and derive the modulus of elasticity, reflecting the ability of the material to resist elastic deformation.
  • the glucopyranoside used in the present invention is a renewable fatty acid from a renewable fatty acid (a fatty alcohol is a fatty alcohol having a chain of 8 to 22 carbon atoms, and can be classified into a natural fatty alcohol and a synthetic fatty alcohol. Natural fatty alcohol is a natural animal and vegetable oil.
  • the fatty acid obtained by hydrolysis is reduced to alcohol, collectively referred to as natural fatty alcohol) and glucose synthesis
  • alcohol collectively referred to as natural fatty alcohol
  • glucose synthesis it is a new type of nonionic surfactant with comprehensive performance.
  • the characteristics of common nonionic and anionic surfactants, with high surface activity, good ecological safety and compatibility, are internationally recognized as the preferred "green" functional surfactants. It has good solubility, mildness and degreasing ability, is less irritating to the skin, non-toxic and easy to rinse. In addition, it also has the characteristics of sterilization and disinfection.
  • the glucopyranoside is stable in strong alkalis, strong acids and high-concentration electrolytes, has low corrosivity, and is easily biodegradable without causing environmental pollution. Mild performance with little damage to the ultrastructure and functional proteins of the extracellular matrix. It has the characteristics of non-toxic and easy degradation, and is harmless to the human body.
  • the tissue and organs to be used can be jointly treated by a physical method.
  • the hypotonic solution in the physical method is mainly used for immersing, oscillating and adjusting the temperature to increase the decellularization effect of glucopyranoside.
  • the combination of glucopyranoside, hypotonic solution and nuclease in the invention can effectively remove cellular components in tissues and organs, and overcome the disadvantages of traditional surfactants being toxic, decellularized or damaged extracellular matrix components.
  • the prepared acellular matrix has a greatly reduced immunogenicity, low cytotoxicity and good biocompatibility, and is a good tissue engineering scaffold material.
  • Figure 1 is a photomicrograph (HEx200) of fresh pig pericardial tissue containing cells.
  • Figure 2 is a photomicrograph (HEx200) of pig pericardium tissue after decellularization. As can be seen from the pictures, after treatment by the decellularization method of the present invention, the cell structure in the pericardial tissue of the pig disappears and the cells can be completely removed.
  • HEx200 photomicrograph
  • Figure 3 is a micrograph of a fresh pig pericardium tissue (Mavot pentachrome x400).
  • Figure 4 is a micrograph of the pig pericardium tissue after decellularization (Mavot pentachrome x400) o
  • the collagen fibers in the pericardium tissue of the pig are arranged neatly without significant breakage. , still arranged in a wave shape parallel, compact structure, clear structure of elastic fibers, no obvious edema in the tissue.
  • Figure 5 is an electron micrograph (800 x) of fresh pig pericardium tissue.
  • Figure 6 is a photomicrograph (800 x) of pig pericardium tissue after decellularization. It can be seen from the picture that after treatment by the decellularization method of the present invention, the collagen fibers in the pericardium tissue of the pig are arranged neatly, without obvious breakage, and are still arranged in a wavy parallel shape, and the structure is compact and continuous. detailed description
  • nuclease digestion oscillation 37 a C 24h was: sterile aliquot of 2.5 KU/ml DNase I (Sigma), 7.5 KU/ml RNase (Sigma), 0.15 M NaCl, 2 mM MgCI 2 (H 2 0) 6 and 1% double antibody. mM Tris-HCl buffer (pH 7.60).
  • Residual cells The decellularized pig pericardium was stained with conventional hematoxylin-eosin to confirm the absence of cell structure (see Figure 2).
  • Matrix fiber structure The decellularized pig pericardium was stained by Movat Pentachrome. It can be observed that after treatment by the decellularization method of the present invention, the collagen fibers in the pericardium tissue of the pig are arranged neatly, without obvious breakage, and are still arranged in a wavy parallel structure. Compact, elastic fiber structure is clear (see Figures 4 and 6).
  • the DNA content of the non-decellularized pig pericardium was 522.063 ⁇ 46.44 ng/mg dry weight tissue, which was subjected to the present invention.
  • the DNA content after decellularization was 5.642 ⁇ 1.75 ng / mg dry weight, and statistical analysis showed significant differences.
  • Residual cell surface on the surface of the stromal Scanning electron microscopy showed that there were a large number of cells on the surface of the non-decellularized porcine pericardium. After decellularization, the pericardial tissue of the pig showed the ordered fibers and no fiber breakage.
  • the elastin content of the undecellularized pig pericardium tissue determined by the Fastin elastin kit is 5.185 ⁇ 0.005 g / mg dry weight tissue, and the elastin content of the decellularized pig pericardium tissue is 4.316 ⁇ 0.001 g / mg dry weight tissue, statistically significant no significant difference.
  • the elastic modulus of the acellular matrix material was measured on a universal material testing machine.
  • the elastic modulus of the fresh pig pericard was 81.335 ⁇ 4.23 MPa.
  • the elastic modulus of the decellularized pig pericard was 66.302 ⁇ 6.13 MPa, there was no significant difference in statistical processing, and the tissue became slightly soft after treatment.
  • pericardium tissue After preparing the pericardium tissue according to the method of Example 1, 6 pieces of 3 cm x 4 cm pericardium were separately placed in 6 bottles of 2% OGP 10 mM Tris-HCl buffer solution for shaking for 16 h, 4 ° C, 150 rpm / min, each The bottle solution contained a solution of 1% double-antibiotics of streptomycin sulfate and penicillin sodium. After that, it was washed in sterile PBS (pH 7.30), 15 min/time, and washed 5 times. After washing, the pericardium was sequentially placed in a nuclease solution and shaken at 37 ° C for 24 h.
  • the nuclease solution was: 2.5 KU/ml DNase I, 7.5 KU/ml RNase, 0.15 M NaCl, 2 mM MgCl 2 (H 2 O) 6 and 1% double-antibody in sterile 50 mM Tris-HCl buffer (pH 7 .60).
  • the mixture was sterilized by shaking in a sterile double-antibody-containing PBS buffer (pH 7.30) for 24 hours at a rotation speed of 150 rpm. After washing, the pericardium was sequentially stored at 4 ° C in a sterile PBS solution containing a double antibody.
  • the pericardium was sequentially placed in a nuclease solution and shaken at 37 ° C for 24 hours.
  • the nuclease solution was: 2.5 KU/ml DNase I, 7.5 KU/ml RNase, 0.15 M NaCl, 2 mM MgCl 2 (H 2 O) 6 and 1% double-antibody in sterile 20 mM Tris-HCl buffer (pH 7). .60).
  • the mixture was sterilized by shaking in a sterile double-antibody-containing PBS buffer (pH 7.30) for 24 hours at a rotation speed of 150 rpm.
  • the pericardium was sequentially stored at 4 ° C in a sterile PBS solution containing a double antibody.
  • the nuclease solution was: 2.0 KU/ml DNase I, 8.0 KU/ml RNase, 0.15 M NaCl, 5 mM MgCl 2 (H 2 O) 6 and 1% double-antibody in sterile 50 mM Tris-HCl buffer (pH 7. 60).
  • the mixture was sterilized by shaking in a sterile double-antibody-containing PBS buffer (pH 7.30) for 24 hours at a rotation speed of 150 rpm. After washing, the pericardium was sequentially stored at 4 ° C in a sterile PBS solution containing a double antibody.
  • Non-patent Document 1 J Wo Detergent decellularization of heart valves for tissue engineering:. Toxicological effects of residual detergents on human endothelial cells Artif Organs, 2010, 34 (3): 206-210.
  • Non-patent literature 3 Does Sodium Dodecyl Sulfate Wash Out of Detergent-Treated Bovine Pericardium at Cytotoxic Concentrations? The Journal of Heart Valve Disease, 2009, 18: 101-.

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Abstract

一种制备细胞外基质支架材料的脱细胞方法及由该方法制备的细胞外基质（ECM）。所述方法包括使用吡喃葡萄糖苷的溶液和核酸酶溶液对待处理组织器官进行脱细胞处理。

Description

一种制备细胞外基质支架材料的脱细胞方法 技术领域
本发明涉及组织工程材料领域。 具体而言， 本发明涉及一种制备 细胞外基质支架材料的脱细胞方法及由该方法制备的细胞外基质。 背景技术
组织是由细胞和细胞外基质 (extracellular matrix, ECM)组成的， 其中的细胞成分作为抗原被宿主识别后会引发炎症或免疫排斥反应。 而 ECM是结构蛋白和功能蛋白的复合物， 该组分在不同物种间通常是 受保护的而且耐受性良好。 由 ECM组成的生物支架材料被广泛应用在 外科手术重建和组织与器官的再生医学领域， 如心脏瓣膜、 血管、 皮 肤、 神经、 肌腱和小肠粘膜下层等。 ECM是组织和器官中的细胞的分 泌产物， 与这些细胞之间有着动态的相互作用， 能够及时反映微环境 的改变， 同时在细胞迁移、 分化和增殖方面有着重要的作用。 其三维 结构与体内细胞生长的天然环境接近， 不仅可以起着支架材料的作用， 而且包含的多种生长因子在组织修复和重建中有重要促进作用， 即使 经过脱细胞处理仍有活性， 是细胞生长的理想环境。 利用已知的常规方法将异源或异种结締组织 (例如心包膜、 瓣膜、 皮肤、 血管和小肠粘膜下层)的细胞脱除， 形成一种称为脱细胞的细胞 外基质， 在脱细胞过程中， 去除导致组织排斥的细胞， 同时保留原始 组织的复杂结构和关键生化组分。 不同的脱细胞方法直接影响所得脱 细胞基质的成分和超微结构， 最终影响移植后宿主对脱细胞基质的反 应。 目前脱细胞基质的制备方法有很多， 主要包括物理法、 化学法和 生物处理法。 物理法主要是通过物理的作用如冻融、 液体高压、 超声 波和电击等脱除细胞， 它们的基本原理是破坏组织中细胞的细胞膜结 构， 细胞膜结构的变化导致细胞产生不良生物化学反应， 持续的处理 将使细胞死亡， 随后通过溶液的清洗、 核酸及脂质的去除而脱去组织 的细胞。 化学法就是使用化学试剂破碎细胞达到去除细胞的目的。 一 些特定的化学试剂如酸、 碱和去污剂等可以渗透组织的各层， 溶解细 胞膜的双分子层磷脂甚至破坏细胞膜蛋白导致细胞死亡和破碎， 之后 通过振荡清洗等步骤洗去细胞残渣和抗原物质。 生物处理法主要是用 酶试剂来裂解细胞。 例如脱细胞方法中通常会用到胰蛋白酶， 它是一 类丝氨酸蛋白酶， 能选择性地水解蛋白质中由赖氨酸或精氨酸的羧基 所构成的肽链， 胰蛋白酶的作用使细胞间的蛋白质水解从而使细胞离 散。 中国专利申请号 No.201110134511.9 涉及一种同种脱细胞真皮基 质制备方法， 使用十二烷基硫酸钠进行脱细胞处理， 虽然用时短， 但 十二烷基硫酸钠对基质结构有较大损伤， 且十二烷基硫酸钠有明显的 毒性， 不利于体内植入。 中国专利申请号 No.200510002464.7涉及异种 心血管移植物的脱细胞方法，而中国专利申请号 No.200910076674.9涉 及一种脱除血管组织内细胞的血管基质及其制备方法。 这两个专利申 请都使用了胰蛋白酶， 虽然处理时间和浓度各异， 但胰蛋白酶必然会 对支架不同程度的降解， 破坏支架材料的结构和组成成分， 且经胰蛋 白酶处理后的组织会有大约 30%的肿胀。 传统去污剂在脱细胞处理过程中， 虽能去除细胞， 但其残留会引 起细胞毒性及潜在的免疫性 [非专利文献 2和 3]。 避免这一毒性反应的 发生必须去除去污剂或替换新型无毒的试剂， 去除去污剂的方法很多 且过程繁琐、 不彻底， 因此需要寻找一种新型无毒可降解且能完全脱 除组织器官的细胞的替代试剂。 使用环保型生物降解完全无毒的去污 剂在脱细胞基质的制备上有着重要的意义。 n-辛基 -β-D-吡喃葡萄糖苷 (n-Octyl beta-D-Glucopyranoside , 简称 OGP)是一种性能优良的新型绿色表面活性剂， 兼具非离子与阴离子表 面活性剂的特性。 它不仅表面张力低、 活性高、 去污力强、 泡沫丰富 细腻而稳定， 而且具有对皮肤无刺激、 生物降解好、 无毒、 对环境无 污染等优点。 研究表明 [非专利文献 1]， OGP表面活性高， 能够破坏细 菌的细胞膜结构， 具有广谱抗菌性， 如对革兰氏阴、 阳性菌等均有抗 菌作用， 且抗菌性强， 是理想的温和非离子表面活性剂。 发明内容
本发明所解决的技术问题是提供一种制备细胞外基质支架材料的 脱细胞方法及由该方法制备的细胞外基质 （ECM) 。 该方法可彻底脱 除存在于组织表面和内部的细胞及细胞碎片， 制备出具有较好的力学 性能和生物学特性的细胞外基质材料， 从而得到组织工程所用的支架 材料。 本发明涉及一种制备细胞外基质支架材料的脱细胞方法， 包括以 下步骤：
a. 对待用组织器官进行预处理；
b. 将经过预处理的待用组织器官加入到含有吡喃葡萄糖苷的溶液 中， 通过低渗溶液振荡处理， 以破坏细胞膜结构和抽提细胞膜的脂质 和膜蛋白；
c 将步骤 b处理后的组织器官加入到含有核酸酶的溶液中， 以降 解细胞中各类 DNA和 /或 RNA成分； 以及
d. 洗涤制备的细胞外基质支架材料。 根据本发明， 所述的待用组织器官为异种或同种异体或自体组织 器官， 包括牛心包、 猪心包、 心脏瓣膜、 皮肤、 血管、 小肠粘膜下层、 神经等中任意一种。 一般来说， 含有较多胶原纤维或弹力纤维的组织 器官适宜用此方法去除细胞成分。 根据本发明， 所述的预处理是使用各含抗生素的生理盐水， 或 pH 值为 6.8-8.6的磷酸盐缓冲液，或 pH值为 6.8-8.6的 D-Hanks溶液 (Hanks 溶液是常见的平衡盐溶液 (BSS)之一，也是组织培养基本用液。 D-Hanks 溶液则是无钙镁离子的 Hanks液（GIBCO公司）， 在 0-37Ό条件下对待 用的组织器官进行常规清洗、 消毒、 分离。 所述的抗生素包括两类抗 生素中的一类， 每一类通常都会联合使用， 其中一类包括丁胺卡那霉 素、 氟胞嘧啶、 万古霉素、 氯霉素； 另一类包括青霉素、 硫酸链霉素、 两性霉素8。 例如， 所述的抗生素优选为 Pen Strep ( Gibco公司） 。 根据本发明， 所述的吡喃葡萄糖苷选自 n-辛基 -β-D-吡喃葡萄糖苷 (OGP)、 辛基 -β-D-硫代吡喃葡萄糖苷、 壬基吡喃葡萄糖苷、 癸基吡喃葡 萄糖苷、 十二垸基吡喃葡萄糖苷的任一种或其组合 (该组合也可统称为 烷基葡萄糖苷)， 优选 OGP。 根据本发明，所述的吡喃葡萄糖苷在溶液中的浓度小于 70%(w/v；)， 即重量:体积为 70克: 100毫升，优选 0.1-10%(w/v)，即重量:体积为 0.1-10 克: 100毫升， 更优选 0.5-5%(w/v)。 含有吡喃葡萄糖苷的溶液的温度范 围为 rC-40°C， 更优选 3 °C-8°C。 含有吡喃葡萄糖苷的溶液的 pH值范 围为 5-12， 更优选 6.8-8.6， 处理时间为 30分钟 -96小时， 更优选 6-48 小时。 所述的低渗溶液振荡处理是采用 0.01M 无菌的 Tris-HCl 缓冲液 (pH6.8-8.6)，在 1 °C-40°C (更优选 3 °C -8°C)中振荡处理 30分钟 -96小时 (更 优选 6-48小时）， 振荡速率为 50-360rpm， 更优选 100-250rpm。 所述的核酸酶溶液的配制过程是在含有 0.15M 氯化钠和 l-5mM 氯化镁的无菌的 0.02-0.05M Tris-HCl 缓冲液（pH6.8-8.6)中加入 100- 10000u/ml (优选 1000- 8000u/ml)的脱氧核糖核酸酶 (DNase)和核糖 核酸酶 (RNase)。 其后， 将组织器官加入到该核酸酶溶液中， 并在 37°C 环境中 60-360rpm (优选 100-250rpm)转速下振荡处理 12-72小时。 所述的洗涤是使用各含抗生素的生理盐水， 或 pH值为 6.8-8.6的 磷酸盐缓冲液， 或 pH值为 6.8-8.6的 D-Hanks溶液， 在 C-25 °C条件 下对所得的脱细胞基质进行常规清洗， 清洗时间为 6-48小时。 本发明脱细胞基质的评价标准及其方法：
1. 残留细胞： 脱细胞基质用 10%中性福尔马林固定， 石蜡包埋， 切成 0.4微米厚的薄片， 经二甲苯脱蜡、 系列酒精脱水， 苏木素一伊红 染色， 观察细胞残留情况。
2. 基质纤维结构： 脱细胞基质用 10%中性福尔马林固定， 石蜡包 埋， 切成 0.4 微米厚的薄片， 经二甲苯脱蜡、 系列酒精脱水， Movat Pentachrome染色， 观察基质中胶原纤维、 弹力纤维和糖蛋白的分布情 况和结构变化情况。
3. 脱细胞基质 DNA含量的测定： 将组织磨碎， 裂解， 将裂解后 的样品按 PureLink Genomic DNA Mini Kit 提纯 DNA， 用 PicoGreen® dsDNA Assay Kit(Invitrogen公司）测定， 在 545nm波长处测吸光度， 计 算 DNA含量。
4. 脱细胞基质含水量的测定： 将湿态样品夹在两层干滤纸中间， 滤纸上压一重 50 g的物体 30s， 测得样品湿重； 将样品真空冷冻干燥 24 h测得干重。 按含水量= (湿重 -干重 )/干重 χ 100%计算样品的含水量。
5. 基质表面细胞残留： 脱细胞基质用 2.5%戊二醛溶液固定， 乙醇 逐级脱水， 干燥， 真空喷金， 扫描电镜组织表面细胞残留和纤维排列 情况。
6. 胶原蛋白含量测定： 脱细胞基质在 6M HC1中 106°C溶解 24h。 水解产物用氯胺 T溶解 20°C处理 20min， 之后加入含高氯的醛酸中， 60°C水浴 15min， 550nm处测吸收值， 测定羟脯氨酸含量， 并按胶原蛋 白中羟脯氨酸约占 12.7%的比例， 推算出胶原蛋白含量。
7. 弹性蛋白含量测定： 脱细胞基质以 0-2mmol/L草酸 100°C煮沸 60分钟， 离心， 取上清液， 如此反复抽提 3次， 合并上清液， 弹性蛋 白含量 (/干重组织）的测定用 Fastin 弹性蛋白试剂盒测定 (Biocolor 公 司)。
8. 脱细胞基质拉伸强度的测定： 将脱细胞基质裁成板材状， 在万 能材料试验机上将将其拉断， 所测的最大拉力除以试材的截面积， 为 基质材料的拉伸强度， 表示基质的抗拉能力。
9. 脱细胞基质断裂伸长率的测定： 将脱细胞基质裁成板材状， 在 万能材料试验机上将其拉断， 记录试样从被拉断的长度， 除以试样的 长度所得的百分比， 即为基质材料的断裂伸长率， 表示材料的变形能 力。
10. 脱细胞基质弹性模量的测定： 将脱细胞基质裁成板材状， 在 万能材料试验机上将将其拉断， 得出基质材料的拉伸应力和应变， 作 应力-应变曲线图， 线性拟合， 计算直线的斜率， 得出弹性模量， 反映 材料抵抗弹性变形能力。
1 1. 脱细胞基质体外细胞毒性实验：根据 GB 16886.5-2003和 GB/T 14233.2-2005— 8的方法， 采用浸提液和 MTT的方法， 对材料的细胞毒 性进行测试。 本发明采用的吡喃葡萄糖苷由可再生资源天然脂肪醇 (脂肪醇为 具有 8至 22个碳原子链的脂肪醇， 可分为天然脂肪醇和合成脂肪醇。 天然脂肪醇以天然的动植物油脂为原料， 如来源比较丰富的椰子油、 棕榈油和牛油， 水解所得脂肪酸再还原为醇， 统称为天然脂肪醇)和葡 萄糖合成， 是一种性能较全面的新型非离子表面活性剂， 兼具普通非 离子和阴离子表面活性剂的特性，具有高表面活性、 良好的生态安全性 和相溶性， 是国际公认的首选"绿色"功能性表面活性剂。具有良好的溶 解性、 温和性和脱脂能力， 对皮肤刺激小， 无毒、 而且易漂洗。 此外， 还具有杀菌消毒、 降低刺激等特点。 吡喃葡萄糖苷在强碱、 强酸和高 浓度电解质中性能稳定， 腐蚀性小， 且易于生物降解不会造成对环境 的污染。 性能温和， 对细胞外基质的超微结构和功能蛋白几乎没有损 伤。 具有无毒易降解的特点， 对人体无害。 为进一步增强吡喃葡萄糖 苷脱细胞效果， 可以联合物理方法共同处理待用的组织器官。 本发明 中主要采用物理法中的低渗溶液浸泡， 振荡和调节温度的方式， 增加 吡喃葡萄糖苷的脱细胞效果。 本发明中的吡喃葡萄糖苷、 低渗溶液及核酸酶的联合应用可以高 效地去除组织器官中的细胞成分， 克服了传统表面活性剂有毒性、 去 细胞不充分或损伤细胞外基质成分等缺点， 制备的脱细胞基质的免疫 原性大幅降低且细胞毒性低， 生物相容性好， 是一种较好的组织工程 支架材料。 附图说明
为了更清楚地描述本发明的技术方案， 下面将结合附图作简要介 绍。 显而易见， 这些附图仅是本申请记载的一些具体实施方式。 本发 明的技术方案包括但不限于这些附图。 图 1为含有细胞的新鲜猪心包组织的显微镜照片 (HEx200)。
图 2为经脱细胞处理后的猪心包组织的显微镜照片 (HEx200)。 从 图片可以看出， 经本发明的脱细胞方法处理后， 猪心包组织中的细胞 结构消失， 能完全去除细胞。
图 3为新鲜猪心包组织的显微镜照片 (Mavot pentachrome x400)。 图 4 为经脱细胞处理后的猪心包组织的显微镜照片 (Mavot pentachrome x400)o 从图片可以看出， 经本发明的脱细胞方法处理后， 猪心包组织中的胶原纤维排列整齐， 无明显断裂， 仍呈波浪状平行排 列， 结构紧凑， 弹性纤维结构清晰， 组织无明显水肿。
图 5为新鲜猪心包组织的电子显微镜照片 (800 x)。
图 6为经脱细胞处理后的猪心包组织的显微镜照片 (800 x)。 从图 片可以看出， 经本发明的脱细胞方法处理后， 猪心包组织中的胶原纤 维排列整齐， 无明显断裂， 仍呈波浪状平行排列， 结构紧凑连续。 具体实施方式
为了进一步理解本发明， 下面将结合实施例对本发明的优选方案 进行描述。 这些描述只是举例说明本发明方法的特征和优点， 而非限 制本发明的保护范围。 实施例 1
1)材料制取： 猪心包取自当地屠宰厂， 健康成年猪宰杀后取出心 脏， 割取心包膜， 热缺血时间小于 2小时。 磷酸缓冲液 (PBS)反复冲洗 去除血凝块后置于保存液中， 带回实验室， 无菌条件下剔除外周脂肪， 取无损伤、 厚度均勾的前壁部分修剪为 3 cmx4 cm的小片， PBS溶液 充分漂洗， 4Ό保存含抗生素的无菌的 PBS溶液中。
2)猪心包的去细胞处理： 将 6片 3 cmx4 cm的心包分别放入 6瓶 1% OGP (阿拉丁试剂 (上海)有限公司）的 10mM Tris-HCl缓冲溶液中进 行振荡消化 24h， 4°C， 150rpm/min, 每瓶溶液含有 1%硫酸链霉素 (阿 拉丁试剂 (上海)有限公司， 批号： C1208010)和青霉素钠 (阿拉丁试剂 (上 海)有限公司， 批号： 16458)的双抗溶液。 之后在无菌的 PBS(pH7.30) 进行清洗， 15min/次， 清洗 5次； 清洗完后依次将心包放在核酸酶溶液 中 37aC振荡消化 24h。核酸酶溶液为： 2.5KU/ml DNase I (Sigma公司）、 7.5KU/ml RNase(Sigma公司） 、 0.15M NaCl 、 2 mM MgCI2(H20)6和 1%双抗的无菌的 50 mM Tris-HCl缓冲液 (pH7.60)。
3)洗涤：在无菌的含双抗的 PBS缓冲液 (pH7.30)进行振荡清洗 24h， 转速为 150rpm， 清洗完后依次将心包在 4°C保存含双抗的无菌的 PBS 溶液中。 结果评价：
1. 残留细胞： 脱细胞猪心包经常规苏木素一伊红染色， 证实无 细胞结构存在 (参见图 2)。
2. 基质纤维结构： 脱细胞猪心包经 Movat Pentachrome染色， 可 观察到经本发明的脱细胞方法处理后， 猪心包组织中的胶原纤维排列 整齐， 无明显断裂， 仍呈波浪状平行排列， 结构紧凑， 弹性纤维结构 清晰 (参见图 4和 6)。
3. 脱细胞基质 DNA含量的测定：经 DNA检测试剂盒测得未脱细 胞的猪心包 DNA含量是 522.063±46.44ng/mg干重组织，经过本发明的 脱细胞处理后的 DNA含量为 5.642±1.75 ng/mg干重组织， 统计学分析 有显著差异。
4. 脱细胞基质含水量的测定： 新鲜猪心包组织的含水量是 79.427±0.29%， 经脱细胞处理后的猪心包组织的含水量是 81.432±0.74%, 统计学处理无显著差异。
5. 基质表面细胞残留： 扫描电镜观察未脱细胞的猪心包组织表面 有大量的细胞存在， 经脱细胞处理后的猪心包组织可看到排列有序的 纤维， 且无纤维断裂。
6. 胶原蛋白含量测定： 由测得羟脯氨酸换算得未脱细胞的猪心包 组织的胶原蛋白含量是 46.218±1.27%， 经脱细胞处理的猪心包组织的 胶原蛋白含量是 46.785±0.42%， 统计学处理无显著差异。
7. 弹性蛋白含量测定： 由 Fastin 弹性蛋白试剂盒测定的未脱细胞 的猪心包组织的弹性蛋白含量是 5.185±0.005 g/mg干重组织， 经脱细 胞处理的猪心包组织的弹性蛋白含量是 4.316±0.001 g/mg干重组织， 统计学处理无显著差异。
8. 脱细胞基质拉伸强度的测定： 在万能材料试验机上测得基质材 料的拉伸强度， 新鲜猪心包的最大拉伸强度为 14.362±0.82MPa ， 经脱 细胞处理的猪心包的最大拉伸强度为 13.461±0.55MPa， 统计学处理无 显著差异。
9. 脱细胞基质断裂伸长率的测定： 在万能材料试验机上测得基质 材料的断裂伸长率， 新鲜猪心包的断裂伸长率为 70.621±5.09% ， 经脱 细胞处理的猪心包的断裂伸长率为 79.235±2.81%， 统计学处理有显著 差异。
10. 脱细胞基质弹性模量的测定： 在万能材料试验机上测得基质 材料的弹性模量， 新鲜猪心包的弹性模量为 81.335±4.23MPa ， 经脱细 胞处理的猪心包的弹性模量为 66.302±6.13MPa， 统计学处理无显著差 异， 组织经过处理后略微变得柔软。
1 1. 脱细胞基质体外细胞毒性实验： 根据浸提液和 MTT的方法测 得未脱细胞的猪心包组织的相对增值率是 102.325±1.77%， 细胞毒性级 别为 0级； 经脱细胞处理的猪心包组织的相对增值率是 92· 167±1.35%， 细胞毒性级别为 1级， 统计学处理无显著差异。 实施例 2
按实施例 1方法制取猪心包组织后， 将 6片 3 cmx4 cm的心包分 别放入 6瓶 2% OGP的 10mM Tris-HCl缓冲溶液中进行振荡消化 16h， 4°C， 150rpm/min, 每瓶溶液含有 1%硫酸链霉素和青霉素钠的双抗溶 液。 之后在无菌的 PBS(pH7.30)进行清洗， 15min/次， 清洗 5次； 清洗 完后依次将心包放在核酸酶溶液中 37°C振荡消化 24h。 核酸酶溶液为： 2.5KU/ml DNase I 、 7.5KU/ml RNase 、 0.15M NaCl 、 2 mM MgCl2(H2O)6和 1%双抗的无菌的 50 mM Tris-HCl缓冲液 (pH7.60)。 在 无菌的含双抗的 PBS 缓冲液 (pH7.30)进行振荡清洗 24h， 转速为 150rpm, 清洗完后依次将心包在 4°C保存含双抗的无菌的 PBS溶液中。 实施例 3
按实施例 1方法制取猪心包组织后， 将 6片 3 cmx4 cm的心包分 别放入 6瓶 1%十二垸基吡喃葡萄糖苷 (阿拉丁试剂 (上海)有限公司)的 10mM Tris-HCl缓冲溶液中进行振荡消化 24h， 4°C， 150rpm/min， 每 瓶溶液含有 1%硫酸链霉素和青霉素钠的双抗溶液。 之后在无菌的 PBS pH7.30)进行清洗， 15min/次， 清洗 5次； 清洗完后依次将心包放 在核酸酶溶液中 37°C振荡消化 24h。核酸酶溶液为：2.5KU/ml DNase I、 7.5KU/ml RNase 、 0.15M NaCl 、 2 mM MgCl2(H2O)6和 1%双抗的无菌 的 20 mM Tris-HCl缓冲液 (pH7.60)。 在无菌的含双抗的 PBS 缓冲液 (pH7.30)进行振荡清洗 24h，转速为 150rpm，清洗完后依次将心包在 4°C 保存含双抗的无菌的 PBS溶液中。 实施例 4
按实施例 1方法制取猪心包组织后， 将 6片 3 cmx4 cm的心包分 别放入 6 瓶 1%烷基葡萄糖苷 (河北石家庄金莫尔化学品有限公司）的 10mM Tris-HCl缓冲溶液中进行振荡消化 24h， 4°C， 150rpm/min, 每瓶 溶液含有 1%硫酸链霉素和青霉素钠的双抗溶液。之后在无菌的 PBS(pH 7.30)进行清洗， 15min/次， 清洗 5次； 清洗完后依次将心包放在核酸酶 溶液中 37°C振荡消化 24h。核酸酶溶液为：2.0KU/ml DNase I、8.0KU/ml RNase 、 0.15M NaCl 、 5 mM MgCl2(H2O)6和 1%双抗的无菌的 50mM Tris-HCl缓冲液 (pH7.60)。 在无菌的含双抗的 PBS缓冲液 (pH7.30)进行 振荡清洗 24h， 转速为 150rpm， 清洗完后依次将心包在 4°C保存含双抗 的无菌的 PBS溶液中。 使用可生物降解、 无毒无刺激、 对环境无污染的辛基 -β-D-吡喃葡 萄糖苷进行脱细胞处理， 不仅能完全去除细胞而且保持了支架的超微 结构和组成成分。 以上实施例的说明只是用于帮助理解本发明的核心思想。 应当指 出， 对于本领域的普通技术人员而言， 在不脱离本发明原理的前提下， 还可以对本发明方法进行若干改进和修饰， 但这些改进和修饰也落入 本发明权利要求请求保护的范围内。
参考文献 非专 禾 ll 文 献 1. Ecological properties of alkyl glucosides, Chemosphere, 1997, 35(3):545-556.
非专禾1 J文献 2. Detergent decellularization of heart valves for tissue engineering: toxicological effects of residual detergents on human endothelial cells. Artif Organs, 2010, 34(3):206-210.
非专利文献 3. Does Sodium Dodecyl Sulfate Wash Out of Detergent-Treated Bovine Pericardium at Cytotoxic Concentrations? The Journal of Heart Valve Disease, 2009, 18: 101-.

Claims

权 利 要 求
1. 一种制备细胞外基质支架材料的脱细胞方法， 包括以下步骤： a. 对待用组织器官进行预处理；
b. 将经过预处理的待用组织器官加入到含有吡喃葡萄糖苷的溶液 中， 通过低渗溶液振荡处理；
c 将步骤 b处理后的组织器官加入到含有核酸酶的溶液中； 以及 d. 洗涤制备的细胞外基质支架材料。
2. 权利要求 1的方法， 其中所述的待用组织器官为异种或同种异 体或自体组织器官， 选自牛心包、 猪心包、 心脏瓣膜、 皮肤、 血管、 小肠粘膜下层、 神经中的任意一种。
3. 权利要求 1或 2的方法， 其中所述的预处理是使用各含抗生素 的生理盐水， 或 pH值为 6.8-8.6的磷酸盐缓冲液， 或 pH值为 6.8-8.6 的 D-Hanks溶液， 在 0-37Ό条件下对待用的组织器官进行常规清洗、 消毒、 分离。
4. 前述权利要求任一项的方法， 其中所述的吡喃葡萄糖苷选自 n- 辛基 _β_Ε>_吡喃葡萄糖苷 (0GP)、 辛基 -β-D-硫代吡喃葡萄糖苷、 壬基吡 喃葡萄糖苷、 癸基吡喃葡萄糖苷、 十二垸基吡喃葡萄糖苷的任一种或 其组合， 优选 OGP。
5. 前述权利要求任一项的方法， 其中所述的吡喃葡萄糖苷在溶液 中的浓度小于 70%(w/v)， 优选 0.1-10%(w/v)， 更优选 0.5-5%(w/v)， 含 有吡喃葡萄糖苷的溶液的温度范围为 rC-40°C， 优选 3 °C-8°C， 含有吡 喃葡萄糖苷的溶液的 pH值范围为 5-12， 优选 6.8-8.6， 处理时间为 30 分钟 -96小时， 优选 6-48小时。
6. 前述权利要求任一项的方法， 其中所述的低渗溶液振荡处理是 采用无菌的 0.01M Tris-HCl 缓冲液 (pH6.8-8.6)， 在 Γ〇-40Ό (优选 3 °C-8°C)中振荡处理 30 分钟 -96 小时， 优选 6-48 小时， 振荡速率为 50-360rpm, 优选为 100-250rpm。
7. 前述权利要求任一项的方法， 其中步骤 c包括配制所述的核酸 酶溶液： 在含有 0.15M 氯化钠和 l-5mM 氯化镁的无菌的 0.02-0.05M Tris-HCl缓冲液 (pH6.8-8.6)中加入 100-lOOOOu/ml (优选 1000-8000u/ml) 的脱氧核糖核酸酶 (DNase)和核糖核酸酶 (RNase), 以及将处理后的组织 器官加入到所述的核酸酶溶液中， 并在 37°C环境中 60-360rpm (优选 100-250rpm)转速下振荡处理 12-72小时。
8. 前述权利要求任一项的方法， 其中所述的洗涤步骤 d是使用各 含抗生素的生理盐水， 或 pH值为 6.8-8.6的磷酸盐缓冲液， 或 pH值为 6.8-8.6的 D-Hanks溶液， 在 C-25O条件下对所得的脱细胞基质进行 常规清洗， 清洗时间为 6-48小时。
9. 通过前述权利要求任一项的方法制备得到的细胞外基质。
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