A kind of decellularized extracellular matrix and its preparation method and application

Abstract

The present invention provides a decellularized extracellular matrix and a preparation method and application thereof, which belong to the field of biomedical engineering technology. The present invention places soft tissue in a first organic solvent for immersion treatment to obtain soaked soft tissue; the soaked soft tissue is mixed with a second organic solvent, and decellularization is performed in the presence of supercritical carbon dioxide to obtain a decellularized extracellular matrix. The present invention first places the soft tissue in a first organic solvent for immersion treatment, which can dehydrate and defat the soft tissue. In addition, after the soft tissue is immersed, supercritical carbon dioxide can better enter the soft tissue, which is beneficial to improve the supercritical treatment effect; the present invention is based on supercritical carbon dioxide fluid technology for decellularization treatment. During the treatment process, the organic solvent will be taken away by carbon dioxide, and almost no organic solvent remains. The treatment efficiency is high and the damage to the active substances in the decellularized extracellular matrix is ​​small, and a whole large piece of membranous decellularized extracellular matrix can be obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to a decellularized extracellular matrix and a preparation method and application thereof. Background Art

[0002] Decellularized extracellular matrix refers to a mixture of biomacromolecules obtained by removing cells from tissues / organs using a specific method. It not only avoids adverse rejection reactions such as inflammation that may occur in the middle and late stages of tissues / organs, but also retains the three-dimensional structure (such as collagen and elastin) and functional biomacromolecules (such as aminoglycans, proteoglycans and growth factors) within the tissues / organs. It has good biocompatibility, induction of tissue regeneration and degradability, and has received widespread attention in the field of tissue repair and regenerative medicine in recent years.

[0003] Generally speaking, when preparing the decellularized extracellular matrix, the soft tissue is first cut into small pieces or minced, and then the cell membrane is destroyed by physical methods, chemical methods or biological methods, and finally treated with DNA enzyme to remove immunogenic DNA. Among them, physical methods such as ultrasound, repeated freezing and thawing, and mechanical stirring cannot completely remove cells. The chemical method is to use chemical solvents to change the permeability of the cell membrane, but due to the interaction between chemical solvents and proteins in the extracellular matrix, the components in the decellularized extracellular matrix are lost to a certain extent. The biological method mainly uses enzymes to hydrolyze cells, but when the enzyme concentration is too high or the action time is too long, the proteins in the extracellular matrix will also be damaged to a certain extent. In addition, it is usually necessary to chop the soft tissue into small pieces to achieve the removal of cells, and it is impossible to obtain a large piece of membrane-like decellularized extracellular matrix. Summary of the invention

[0004] The purpose of the present invention is to provide a decellularized extracellular matrix and a preparation method and application thereof. The method provided by the present invention has high processing efficiency, causes less damage to active substances in the decellularized extracellular matrix, and can obtain a whole large piece of membranous decellularized extracellular matrix.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a decellularized extracellular matrix, comprising the following steps:

[0007] placing the soft tissue in a first organic solvent for soaking treatment to obtain soaked soft tissue;

[0008] The soaked soft tissue is mixed with a second organic solvent, and decellularized in the presence of supercritical carbon dioxide to obtain a decellularized extracellular matrix.

[0009] Preferably, the soft tissue is animal membranous tissue.

[0010] Preferably, the soft tissue is pig skin, cow skin or fish skin.

[0011] Preferably, the first organic solvent and the second organic solvent are alcohol solvents.

[0012] Preferably, the immersion treatment is carried out at a temperature of 25 to 45° C. and for a time of 0.5 to 12 hours.

[0013] Preferably, the ratio of the soaked soft tissue to the second organic solvent is (0.5-2) g: 1 mL.

[0014] Preferably, the number of decellularization treatments is 1 to 5 times, and the conditions for each decellularization treatment include: the temperature is independently 32 to 65°C, the pressure is independently 7.38 to 40 MPa, and the time is independently 0.5 to 12 hours; the pressure release rate after each decellularization treatment is independently 5 to 20 MPa / s.

[0015] Preferably, the decellularization treatment further includes washing and drying in sequence.

[0016] The present invention provides a decellularized extracellular matrix prepared by the preparation method of the above technical solution, which is in the form of a membrane with an area of ​​80 to 120 cm 2 , DNA content is less than 50ng / mg.

[0017] The present invention provides the use of the decellularized extracellular matrix described in the above technical solution in the preparation of tissue repair materials or tissue regeneration materials.

[0018] The present invention provides a method for preparing a decellularized extracellular matrix, comprising the following steps: placing a soft tissue in a first organic solvent for soaking treatment to obtain soaked soft tissue; mixing the soaked soft tissue with a second organic solvent, and performing a decellularization treatment in the presence of supercritical carbon dioxide to obtain a decellularized extracellular matrix. The present invention first places the soft tissue in a first organic solvent for soaking treatment, which can dehydrate and defat the soft tissue. In addition, after the soaking treatment, the soft tissue can better allow supercritical carbon dioxide to enter the soft tissue, which is beneficial to improve the supercritical treatment effect; the present invention is based on supercritical carbon dioxide fluid technology for decellularization treatment, and the organic solvent will be taken away by carbon dioxide during the treatment process, and there is almost no organic solvent residue, the treatment efficiency is high, the operation time is short, and DNA enzyme is not used, and the active substances in the decellularized extracellular matrix are less damaged, and a whole large piece of membranous decellularized extracellular matrix can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the process of efficiently preparing a membrane-like decellularized matrix based on supercritical carbon dioxide fluid technology in the present invention;

[0020] Figure 2This is a comparison chart of DNA content in the original pig skin tissue, the pig skin decellularized extracellular matrix prepared in Example 1, and Comparative Examples 1-2;

[0021] Figure 3 The comparison photo of the pig skin decellularized extracellular matrix prepared in Example 1 and the original pig skin;

[0022] Figure 4 The scanning electron microscope photos of the pig skin decellularized extracellular matrix and the original pig skin prepared in Example 1;

[0023] Figure 5 The figure is a comparison of DNA content between the pig skin decellularized extracellular matrix prepared under different temperature conditions and the original pig skin;

[0024] Figure 6 The comparison of DNA content between the pig skin decellularized extracellular matrix prepared under different pressure conditions and the original pig skin;

[0025] Figure 7 The comparison of DNA content between the pig skin decellularized extracellular matrix prepared under different treatment time conditions and the original pig skin;

[0026] Figure 8 This is a comparison of the DNA content of the pig skin decellularized extracellular matrix prepared under different pressure release rates and the original pig skin.

[0027] Fig. 9 H&E staining and Masson staining images of the pig skin decellularized extracellular matrix prepared in Example 1 and the original pig skin tissue. DETAILED DESCRIPTION

[0028] The present invention provides a method for preparing a decellularized extracellular matrix, comprising the following steps:

[0029] placing the soft tissue in a first organic solvent for soaking treatment to obtain soaked soft tissue;

[0030] The soaked soft tissue is mixed with a second organic solvent, and decellularized in the presence of supercritical carbon dioxide to obtain a decellularized extracellular matrix.

[0031] The present invention places the soft tissue in a first organic solvent for soaking to obtain soaked soft tissue. In the present invention, the soft tissue is preferably an animal membranous tissue, more preferably pig skin, cow skin or fish skin; the area of ​​the soft tissue is preferably 80 to 120 cm 2 In the embodiment of the present invention, the specific area is 100cm 2A whole piece of fresh pig skin tissue. In the present invention, the first organic solvent is preferably an alcohol solvent, more preferably methanol and / or ethanol, and further preferably anhydrous ethanol. The present invention does not specifically limit the amount of the first organic solvent, as long as it can immerse the soft tissue. In the present invention, the temperature of the immersion treatment is preferably 25 to 45°C, more preferably 25 to 30°C; the time is preferably 0.5 to 12h, more preferably 1 to 5h. The present invention can dehydrate and defat the soft tissue through immersion treatment. In addition, after the immersion treatment, the soft tissue can better allow supercritical carbon dioxide to enter the soft tissue, which is beneficial to improve the supercritical treatment effect.

[0032] After obtaining the soaked soft tissue, the present invention mixes the soaked soft tissue with a second organic solvent, performs a decellularization treatment in the presence of supercritical carbon dioxide, and obtains a decellularized extracellular matrix. In the present invention, the second organic solvent is preferably an alcohol solvent, more preferably methanol and / or ethanol, and further preferably anhydrous ethanol. In the present invention, the amount ratio of the soaked soft tissue to the second organic solvent is preferably (0.5 to 2) g: 1 mL, more preferably 1 g: 1 mL. In the present invention, the number of decellularization treatments is preferably 1 to 5 times, more preferably 1 to 2 times. In the present invention, the conditions for each decellularization treatment preferably include: the temperature is independently preferably 32-65°C, specifically 32°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or 65°C; the pressure is independently preferably 7.38-40MPa, specifically 7.38MPa, 10MPa, 15MPa, 20MPa, 25MPa, 30MPa, 35MPa or 40MPa; the time is independently preferably 0.5-12h, specifically 0.5h, 1h, 2h, 4h, 6h, 8h, 10h or 12h; the pressure release rate after each decellularization treatment is completed is independently preferably 5-20MPa / s, specifically 5MPa / s, 10MPa / s, 15MPa / s or 20MPa / s. In the present invention, the decellularization treatment is preferably carried out in a supercritical autoclave, specifically, the soaked soft tissue is put into a supercritical autoclave, and a second organic solvent is added, carbon dioxide is introduced to increase the pressure, and the treatment is carried out under the above pressure and temperature conditions, and then the outlet valve is opened, and the pressure is released and rapidly expanded at the above pressure release rate to obtain a decellularized extracellular matrix.

[0033] Supercritical fluid has the characteristics of both gas and liquid, and has the characteristics of high density, low viscosity, good diffusivity, etc. Among them, carbon dioxide is stable in nature, and has good physical and chemical properties such as non-toxic, odorless, harmless, non-flammable and non-explosive, and its supercritical temperature and pressure are 31.26°C and 7.38MPa. The present invention is based on supercritical carbon dioxide fluid technology and can efficiently prepare a membranous decellularized matrix. The decellularized extracellular matrix uses supercritical carbon dioxide as a solvent and a second organic solvent as a co-solvent. By utilizing the rapid expansion of supercritical carbon dioxide, a large area of ​​soft tissue can be processed, and finally a large-area membranous decellularized extracellular matrix with a complete structure and few cell residues can be obtained. The present invention is based on supercritical carbon dioxide for decellularization, the method is a pure physical method, the processing efficiency is high, the obtained decellularized extracellular matrix DNA residue is low, and the DNA content is less than 50ng / mg; it avoids the use of chemical methods to cause organic solvent residues, does not add DNA enzymes during the treatment process, and avoids the destructive effect of DNA enzymes added to biological macromolecules in biological methods; the method is simple and easy, the preparation conditions are controllable, and it is not necessary to cut or chop the soft tissue into small pieces, and a large area of ​​membranous decellularized extracellular matrix can be obtained, which has strong practicality and great application prospects, and lays the foundation for the expanded application of decellularized extracellular matrix. The present invention preferably carries out decellularization under the above conditions, which is conducive to ensuring that there is a good cell removal effect; for example, the faster the pressure release rate, the faster the gas expands, and the better the decellularization effect will be.

[0034] In the present invention, the decellularization treatment preferably further includes washing and drying in sequence. In the present invention, the reagent used for washing is preferably pure water, and the washing is preferably performed under ultrasonic conditions; the number of washings is preferably 1 to 5 times, more preferably 2 to 3 times; and the drying is preferably freeze drying.

[0035] The present invention provides a decellularized extracellular matrix prepared by the preparation method of the above technical solution, which is in the form of a membrane with an area of ​​80 to 120 cm 2 , DNA content is less than 50ng / mg.

[0036] The present invention provides the use of the decellularized extracellular matrix described in the above technical solution in the preparation of tissue repair materials or tissue regeneration materials. In the present invention, the decellularized extracellular matrix can be specifically used to prepare hemostatic materials or artificial skin.

[0037] Figure 1 The schematic diagram of the process of efficiently preparing a membrane-like decellularized matrix based on supercritical carbon dioxide fluid technology in the present invention (the upper part is a schematic diagram of the process based on a macroscopic perspective, and the lower part is a schematic diagram of the process based on a microscopic perspective). Figure 1The embodiments of the present invention clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1

[0039] Take a whole piece of pig skin tissue (100cm 2 ) is placed in anhydrous ethanol and soaked at 25°C for 1h. The soaked pig skin tissue (100g) is put into a supercritical autoclave, and 25mL of anhydrous ethanol is added. Carbon dioxide is introduced to increase the pressure, and the decellularization treatment is carried out at a pressure of 15MPa and a temperature of 45°C for 4h; then the pressure is released at a rate of 10MPa / s for rapid expansion. After the pressure release is completed, the pig skin tissue is taken out, placed in pure water for ultrasonic cleaning twice, and then freeze-dried to obtain a membranous pig skin decellularized extracellular matrix.

[0040] Comparative Example 1

[0041] The pig skin decellularized extracellular matrix was prepared according to the method of Example 1, except that the pig skin tissue was not soaked in ethanol before the decellularization treatment.

[0042] Comparative Example 2

[0043] The porcine skin acellular extracellular matrix was prepared by chemical method, and the steps were as follows:

[0044] The whole piece of pig skin tissue (100cm 2 ) was added to a PBS solution containing 1% (w / v) Triton-100 and 25 mM EDTA, and treated for 14 h on a shaker at a speed of 120 rpm; after the treatment, the pig skin tissue was taken out and washed with deionized water for 3 times, and then freeze-dried to obtain the pig skin decellularized extracellular matrix.

[0045] Test Example 1

[0046] The original pig skin tissue, the pig skin decellularized extracellular matrix prepared in Example 1 and Comparative Examples 1-2 were used as samples to test their DNA content. The test method was as follows:

[0047] (1) Take 10 mg of freeze-dried porcine decellularized matrix and normal porcine skin tissue that has not been decellularized, cut them into small pieces, transfer them to a 1.5 mL centrifuge tube, add 220 μL of Buffer ATL and 20 μL of proteinase K, mix well, and digest in a 55°C warm water bath for 3 h, inverting and mixing several times during the process;

[0048] (2) Add 10 μL RNase A to the digestion solution, invert to mix, and leave at room temperature for 10 min;

[0049] (3) Add 250 μL of Buffer DL to the digestion solution, vortex at high speed for 10 seconds, and treat in a 70°C water bath for 10 minutes;

[0050] (4) Add 250 μL of anhydrous ethanol to the digestion solution and vortex for 15 seconds;

[0051] (5) Place the DNA binding column in a 2 mL collection tube, transfer the mixed solution (including the precipitate) obtained in step (4) into the DNA binding column, and centrifuge at 12,000 × g for 1 min;

[0052] (6) Discard the filtrate, put the DNA binding column back into the collection tube, add 500 μL of Buffer GW1 to the DNA binding column, and centrifuge at 12,000 × g for 1 min;

[0053] (7) Discard the filtrate, put the DNA binding column back into the collection tube, add 650 μL of Buffer GW2 to the DNA binding column, and centrifuge at 12,000 × g for 1 min;

[0054] (8) Discard the filtrate, put the DNA binding column back into the collection tube, and centrifuge at 12,000 × g for 1 min.

[0055] (9) Place the DNA binding column in a new 1.5 mL centrifuge tube, add 100 μL of Buffer AE preheated to 70°C to the center of the membrane of the DNA binding column, let stand for 3 min, and centrifuge at 12,000 × g for 1 min;

[0056] (10) Add 100 μL of Buffer AE preheated to 70°C to the center of the membrane of the DNA binding column, let it stand for 3 min, and centrifuge at 12,000 × g for 1 min.

[0057] (11) The DNA binding column is discarded, and the liquid to be tested is obtained in the centrifuge tube. The DNA concentration is detected using Nanodrap 2000.

[0058] The DNA content of the original pig skin tissue, the pig skin decellularized extracellular matrix prepared in Example 1 and Comparative Examples 1 to 2 is as follows: Figure 2 As shown. Figure 2It can be seen that the DNA content of the pig skin decellularized matrix prepared by supercritical carbon dioxide fluid technology in Example 1 (14.21 ng / mg) is less than the DNA content of the pig skin decellularized matrix prepared by the supercritical method but without ethanol immersion in Comparative Example 1 (66.25 ng / mg), and the DNA content of the pig skin decellularized extracellular matrix prepared by a chemical method (77.23 ng / mg), which meets the use standards of the decellularized extracellular matrix.

[0059] The pig skin decellularized extracellular matrix and the original pig skin tissue prepared in Example 1 were photographed. Figure 3 As shown. Figure 3 It can be seen that the pig skin tissue after decellularization in Example 1 changed from red to white, indicating that the main cellular substances were effectively removed.

[0060] The morphology of the pig skin decellularized extracellular matrix prepared in Example 1 was observed. Specifically, the pig skin decellularized extracellular matrix was cut into small pieces and pasted onto the conductive adhesive, and then gold was sprayed for 30 seconds. The morphology of different samples was observed using a scanning electron microscope and compared with the original pig skin tissue. The scanning electron microscope image is shown in FIG. Figure 4 As shown. Figure 4 It can be seen that the original pig skin tissue without decellularization treatment is in a paste state, and the pig skin tissue structure after decellularization treatment in Example 1 is in a fibrous filamentous structure with certain pores, and the main component is collagen fibers in the extracellular matrix, indicating that the decellularization process does not cause significant damage to the tissue structure.

[0061] Example 2

[0062] The porcine skin decellularized extracellular matrix was prepared according to the method of Example 1, except that the temperature of the decellularization treatment was 40°C.

[0063] Example 3

[0064] The porcine skin decellularized extracellular matrix was prepared according to the method of Example 1, except that the temperature of the decellularization treatment was 50°C.

[0065] The DNA content of the pig skin decellularized extracellular matrix prepared in Examples 2 to 3 was tested, and the original pig skin tissue was used as a control. The DNA content of the pig skin decellularized extracellular matrix prepared in Example 1 was compared. The results are as follows: Figure 5 As shown. Figure 5 It can be seen that different temperature conditions have a certain effect on the DNA content of pig skin decellularized extracellular matrix.

[0066] Example 4

[0067] The porcine skin decellularized extracellular matrix was prepared according to the method of Example 1, except that the pressure of the decellularization treatment was 10 MPa.

[0068] Example 5

[0069] The porcine skin decellularized extracellular matrix was prepared according to the method of Example 1, except that the pressure of the decellularization treatment was 20 MPa.

[0070] The DNA content of the pig skin decellularized extracellular matrix prepared in Examples 4 to 5 was tested, and the original pig skin tissue was used as a control. The DNA content of the pig skin decellularized extracellular matrix prepared in Example 1 was compared. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that different pressure conditions have a certain effect on the DNA content of pig skin decellularized extracellular matrix.

[0071] Example 6

[0072] The porcine skin decellularized extracellular matrix was prepared according to the method of Example 1, except that the decellularization treatment time was 8 h.

[0073] Example 7

[0074] The porcine skin decellularized extracellular matrix was prepared according to the method of Example 1, except that the decellularization treatment time was 12 h.

[0075] The DNA content of the pig skin decellularized extracellular matrix prepared in Examples 6 to 7 was tested, and the original pig skin tissue was used as a control. The DNA content of the pig skin decellularized extracellular matrix prepared in Example 1 was compared. The results are as follows: Figure 7 As shown. Figure 7 It can be seen that different treatment times have a certain effect on the DNA content of pig skin decellularized extracellular matrix.

[0076] Example 8

[0077] The porcine skin decellularized extracellular matrix was prepared according to the method of Example 1, except that the pressure relief rate after the decellularization treatment was 5 MPa / s.

[0078] Example 9

[0079] The porcine skin decellularized extracellular matrix was prepared according to the method of Example 1, except that the pressure relief rate after the decellularization treatment was 15 MPa / s.

[0080] The DNA content of the pig skin decellularized extracellular matrix prepared in Examples 8 to 9 was tested, and the original pig skin tissue was used as a control. The DNA content of the pig skin decellularized extracellular matrix prepared in Example 1 was compared. The results are as follows: Figure 8 As shown. Figure 8 It can be seen that different pressure release rates have a certain effect on the DNA content of pig skin decellularized extracellular matrix.

[0081] Test Example 2

[0082] The pig skin decellularized extracellular matrix and the original pig skin tissue prepared in Example 1 were subjected to H&E staining and Masson staining. The results are as follows: Fig. 9 As shown. Fig. 9 It can be seen that the results of H&E staining show that in normal pig skin tissue, the cell nuclei are clearly visible and have a good tissue structure; in the pig skin decellularized extracellular matrix prepared in Example 1, the cell nuclei are almost invisible, indicating that the cells have been completely removed. The results of Masson staining show that the pig skin decellularized extracellular matrix prepared in Example 1 contains a large amount of collagen fibers.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a decellularized extracellular matrix, comprising the following steps: The soft tissue is placed in a first organic solvent for soaking treatment to obtain soaked soft tissue; the soft tissue is an animal membranous tissue; the soaking treatment temperature is 25 to 45° C., and the time is 0.5 to 1 hour; The soaked soft tissue is mixed with a second organic solvent, and decellularized in the presence of supercritical carbon dioxide, followed by washing and drying to obtain a decellularized extracellular matrix; the decellularization is performed once, and the conditions of the decellularization include: The temperature is 32-65°C, the pressure is 7.38-20MPa, and the time is 0.5-12h; the pressure release rate after the decellularization treatment is completed is 5-20MPa / s; the reagent used for washing is pure water, and the washing is carried out under ultrasonic conditions; the first organic solvent and the second organic solvent are alcohol solvents.

2. The preparation method according to claim 1, characterized in that: The soft tissue is pig skin, cow skin or fish skin.

3. The preparation method according to claim 1, characterized in that: The usage ratio of the soaked soft tissue to the second organic solvent is (0.5-2) g:1 mL.

4. The decellularized extracellular matrix prepared by the preparation method according to any one of claims 1 to 3 is in the form of a membrane with an area of ​​80 to 120 cm 2 , DNA content is less than 50ng / mg.

5. Use of the decellularized extracellular matrix according to claim 4 in preparing tissue repair materials or tissue regeneration materials.

Images