Method for digesting extracellular matrix by immobilized enzyme and application

Abstract

The invention discloses a method for digesting an extracellular matrix by an immobilized enzyme and application, and relates to the technical field of biology. The method comprises the following steps: (1) performing decellularization treatment on tissues to obtain an extracellular matrix; (2) dispersing the surface functionalized microspheres in a buffer solution, adding a coupling agent, mixing, and collecting the activated microspheres; (3) dispersing the activated microspheres in a buffer solution containing pepsin, mixing, and collecting immobilized enzyme microspheres; and (4) crushing the extracellular matrix, dispersing the crushed extracellular matrix in a solution, adding the immobilized enzyme microspheres for digestion, and separating and removing the immobilized enzyme microspheres after digestion is completed. According to the method provided by the invention, the enzymolysis of the immobilized enzyme on the extracellular matrix is realized by adopting a proper microsphere particle size and a proper zeta potential. According to the method disclosed by the invention, the residual quantity of free enzyme is relatively small, so that the biological safety of the extracellular matrix material is improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for digesting extracellular matrix with immobilized enzymes and an application thereof. Background Art

[0002] The extracellular matrix is ​​a material composed of a variety of functional macromolecules formed after the cells are removed from the tissue. The extracellular matrix not only provides structural support for cells but also regulates the behavior of cells through signal molecules. It plays an important role in maintaining cell homeostasis, ensuring the normal function of tissue functions, promoting wound healing and tissue repair. At present, extracellular matrix materials represented by decellularized small intestinal submucosa have been widely used in clinical practice. On this basis, converting the extracellular matrix into an injectable hydrogel form is an important direction for the application and development of extracellular matrix materials. Compared with traditional extracellular matrix materials, extracellular matrix hydrogels are less invasive and more suitable for irregular injury sites, and have good application prospects.

[0003] At present, the main method for preparing extracellular matrix gel is to crush the extracellular matrix and then digest it with pepsin to obtain a viscous liquid with uniform appearance, and then obtain the extracellular matrix gel by adjusting pH and temperature. For example, Chinese patent CN116726254A discloses a high-strength extracellular matrix film and its preparation method and application. The invention provides a preparation method of a high-strength extracellular matrix film, comprising the following steps: decellularization and freeze-drying, grinding the freeze-dried decellularized pig organ into powder and adding it to a pepsin-hydrochloric acid solution for digestion, forming a pregel, forming an extracellular matrix hydrogel, and obtaining an extracellular matrix film. The core of this method is that pepsin can selectively cut the end peptides of the collagen fibers that are cross-linked in the extracellular matrix, so that various substances in the extracellular matrix become relatively free, and a solution state is formed in appearance. The biggest advantage of this method is that it hardly affects the properties of various components in the extracellular matrix. However, this method also has a disadvantage: the existing pepsin preparation is extracted from the pig gastric mucosa, containing pepsin, miscellaneous proteins and other large amounts of blood type substances.

[0004] Prior art uses pepsin to digest the extracellular matrix, and the general addition amount is all above 0.5mg / ml. And after digestion, due to the need to retain multiple protein components in the extracellular matrix, there is no good method to remove the enzyme separately, but only to deactivate the enzyme, so that pepsin together with foreign proteins and blood group substances are retained in the extracellular matrix gel as impurities, and the content is higher. The immunogenicity of these impurities cannot be guaranteed, thereby increasing the risk of safety aspect of the extracellular matrix gel after implantation in vivo.

[0005] Therefore, there is an urgent need to provide a method for digesting extracellular matrix with higher safety. Summary of the invention

[0006] The purpose of the present invention is to provide a method for digesting extracellular matrix with an immobilized enzyme and its application.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a method for digesting an extracellular matrix using an immobilized enzyme, comprising the following steps:

[0009] (1) decellularizing the tissue to obtain the extracellular matrix;

[0010] (2) dispersing the surface functionalized microspheres in a buffer, adding a coupling agent, mixing, and collecting the activated microspheres;

[0011] (3) dispersing the activated microspheres in a buffer solution containing pepsin, mixing, and collecting the immobilized enzyme microspheres;

[0012] (4) The extracellular matrix is ​​crushed and dispersed in a solution, and the immobilized enzyme microspheres are added for digestion. After the digestion is completed, the immobilized enzyme microspheres are separated and removed.

[0013] Specifically, the tissue described in step (1) is mammalian tissue.

[0014] Furthermore, the mammal is selected from any one of pigs, cattle, sheep, rabbits, cats, horses, mice and deer;

[0015] Furthermore, the mammal is a pig.

[0016] Furthermore, the tissue is selected from any one of the following: small intestinal submucosa, dermis, blood vessels, bladder basement membrane, and greater peritoneal omentum;

[0017] Furthermore, the tissue is selected from any one of the small intestinal submucosa and dermis.

[0018] Specifically, step (1) comprises: taking mammalian tissue, adding peracetic acid solution and shaking and soaking for 0.5-2h, adding trypsin and EDTA and shaking and treating for 1-3h, adding sodium carbonate solution and shaking and soaking for 0.5-2h, adding deoxyribonuclease and ribonuclease and shaking and treating for 1-3h, and then adding sodium carbonate solution and shaking and soaking for 0.5-2h to obtain extracellular matrix.

[0019] According to some embodiments of the present invention, step (1) includes: taking mammalian tissue, adding peracetic acid solution and shaking and soaking for 1 hour, adding trypsin and EDTA and shaking for 2 hours, adding sodium carbonate solution and shaking and soaking for 1 hour, adding deoxyribonuclease and ribonuclease and shaking for 2 hours, and then adding sodium carbonate solution and shaking and soaking for 1 hour to obtain extracellular matrix.

[0020] Furthermore, the concentration of the peracetic acid solution is 0.1-1%; further, the concentration of the peracetic acid solution is 0.1%.

[0021] Furthermore, the concentration of the trypsin is 0.1-0.5%; further, the concentration of the trypsin is 0.25%.

[0022] Furthermore, the concentration of EDTA is 0.1-0.5%; further, the concentration of EDTA is 0.1%.

[0023] Furthermore, the concentration of the sodium carbonate solution is 0.1-0.5 mol / L; further, the concentration of the sodium carbonate solution is 0.1 mol / L.

[0024] Furthermore, the concentration of the deoxyribonuclease is 50-200 U / ml; further, the concentration of the deoxyribonuclease is 50 U / ml.

[0025] Furthermore, the concentration of the ribonuclease is 10-50 U / ml; further, the concentration of the ribonuclease is 10 U / ml.

[0026] Specifically, peracetic acid solution is added for disinfection and virus inactivation.

[0027] Specifically, the surface functionalized microspheres in step (2) are surface carboxyl-modified microspheres.

[0028] Furthermore, the microspheres are selected from any one or more of silica microspheres, polystyrene microspheres, and polymethacrylic acid microspheres.

[0029] Specifically, the average particle size of the surface functionalized microspheres described in step (2) is 1-15 μm; further, the average particle size of the surface functionalized microspheres described in step (2) is 1-10 μm.

[0030] Specifically, the surface Zeta potential of the surface functionalized microspheres described in step (2) is -35 to -50 mV.

[0031] Specifically, the coupling agent described in step (2) is selected from one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and 2-chloro-1-methyliodopyridine (CMPI);

[0032] Furthermore, the coupling agent in step (2) is a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide or 2-chloro-1-methylpyridine iodide.

[0033] Specifically, in step (2), the mass ratio of microspheres to coupling agent is 5:(1-3);

[0034] Furthermore, in step (2), the mass ratio of microspheres to coupling agent is 5:1, 5:2.5 or 5:1.2.

[0035] Specifically, the buffer described in step (2) is selected from any one of MES buffer and Tris-HCl buffer.

[0036] Further, the concentration of the buffer in step (2) is 10-50 mM;

[0037] Furthermore, the pH of the buffer solution in step (2) is 5.5-8.0.

[0038] Specifically, the mixing time in step (2) is 30 minutes.

[0039] Specifically, collecting the activated microspheres in step (2) also includes a washing step with a buffer solution.

[0040] Furthermore, the concentration of pepsin in the pepsin-containing buffer in step (3) is 5-10 mg / ml.

[0041] Furthermore, the buffer is citric acid-sodium citrate buffer.

[0042] Specifically, the concentration of the citric acid-sodium citrate buffer is 10 mM.

[0043] Furthermore, the pH of the citric acid-sodium citrate buffer is 4.0.

[0044] Specifically, the mixing time in step (3) is 8-16 hours.

[0045] Specifically, after collecting the immobilized enzyme microspheres in step (3), the step of washing with citric acid-sodium citrate buffer is also included.

[0046] Specifically, in step (4), the extracellular matrix is ​​crushed into particles with a size of 1-3 mm.

[0047] Specifically, the pH of the solution in step (4) is 3.0-4.5.

[0048] Specifically, the solution in step (4) is selected from any one or more of acetic acid solution and hydrochloric acid solution.

[0049] Furthermore, when the solution in step (4) is acetic acid, the concentration of acetic acid is 0.6%; further, when the solution in step (4) is hydrochloric acid, the concentration of hydrochloric acid is 1 mM.

[0050] Furthermore, in step (4), the mass volume ratio of the extracellular matrix to the solution is 1:(4-10).

[0051] Specifically, in step (4), the mass ratio of the extracellular matrix to the immobilized enzyme microspheres is 5:(1-2).

[0052] Furthermore, in step (4), the mass ratio of the extracellular matrix to the immobilized enzyme microspheres is 5:2 or 5:1.

[0053] Specifically, the digestion time in step (4) is 48-120 hours.

[0054] Specifically, the method for separating and removing the immobilized enzyme microspheres in step (4) is centrifugation or filtration.

[0055] Furthermore, the centrifugal speed is 4000-6000 rpm, and the centrifugal time is 20 min.

[0056] Furthermore, the filtration is performed by using a 2500 mesh nylon filter cloth to remove the immobilized enzyme microspheres.

[0057] In another aspect, the present invention provides an extracellular matrix solution, which is prepared by the above method.

[0058] In yet another aspect, the present invention provides use of the extracellular matrix solution in preparing extracellular matrix gel.

[0059] In another aspect, the present invention provides an extracellular matrix gel, wherein the extracellular matrix gel is prepared by adding a disodium hydrogen phosphate solution to the extracellular matrix solution prepared by the above method.

[0060] Specifically, the concentration of the disodium hydrogen phosphate solution is 0.1-0.5M; further, the concentration of the disodium hydrogen phosphate solution is 0.2M.

[0061] Specifically, the volume ratio of the disodium hydrogen phosphate solution to the extracellular matrix solution is 1:(8-10);

[0062] Furthermore, the volume ratio of the disodium hydrogen phosphate solution to the extracellular matrix solution is 1:9.

[0063] The beneficial effects of the present invention are:

[0064] (1) In the method for digesting extracellular matrix with immobilized enzyme provided by the present invention, the appropriate microsphere particle size helps the immobilized enzyme to fully contact with the extracellular matrix; the appropriate zeta potential can make the immobilized enzyme reach an appropriate density, thereby avoiding steric hindrance; the appropriate density and particle size of the microsphere material can also make the immobilized enzyme suspended in the digestion sample under stirring without sedimentation. These factors enable the immobilized enzyme to hydrolyze the extracellular matrix.

[0065] (2) The method for digesting the extracellular matrix with immobilized enzymes provided by the present invention can easily and thoroughly separate the microspheres from the digested extracellular matrix by filtration or centrifugation due to the appropriate density and rigidity of the microspheres. As a result, the amount of enzyme residue remaining in the digested extracellular matrix is ​​reduced by 4-5 orders of magnitude compared to the use of free enzymes, and the residue is almost negligible, thereby improving the biosafety of the extracellular matrix material. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a microscope picture of the immobilized enzyme obtained in Example 3.

[0067] Figure 2 This is a picture of the extracellular matrix solution obtained after digestion in Example 3.

[0068] Figure 3 This is the extracellular matrix solution obtained after digestion in Comparative Example 1.

[0069] Figure 4 This is the extracellular matrix gel obtained in Example 4. DETAILED DESCRIPTION

[0070] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0071] The manufacturer and item number of the experimental materials:

[0072] Carboxyl-modified silica microspheres were purchased from Xi’an Qiyue Biotechnology Co., Ltd., with the catalog number Q-0344557;

[0073] Carboxyl-modified polystyrene microspheres were purchased from Xi’an Qiyue Biotechnology Co., Ltd., with the catalog number Q-0344567;

[0074] Carboxyl-modified polymethyl methacrylate microspheres were purchased from Xi’an Qiyue Biotechnology Co., Ltd. with catalog numbers Q-0288128, Q-0292703, and Q-0288127;

[0075] Deoxyribonuclease was purchased from Solebao Biotechnology Co., Ltd., catalog number D8072;

[0076] Ribonuclease was purchased from Solebao Biotechnology Co., Ltd., catalog number R8020;

[0077] EDC was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E106172;

[0078] NHS was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number H109330;

[0079] CMPI was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with the catalog number C154059.

[0080] Example 1

[0081] The submucosal layer of the small intestine of the pig treated with an intestine scraper was taken, the fat was removed with a scraper, and the submucosal layer was cut into small pieces, and washed with pure water; it was shaken and soaked in a 0.1% peracetic acid solution for 1 hour, and washed with pure water; it was shaken and treated with a mixed solution containing 0.25% trypsin and 0.1% EDTA for 2 hours, and washed with pure water; it was shaken and soaked in a 0.1 mol / L sodium carbonate solution for 1 hour, and washed with pure water; it was shaken and treated with a mixed solution containing 50 U / ml deoxyribonuclease and 10 U / ml ribonuclease for 2 hours, and washed with pure water; it was shaken and soaked in a 0.1 mol / L sodium carbonate solution for 1 hour, and washed with pure water to obtain the extracellular matrix of the submucosal layer of the small intestine of the pig.

[0082] Take 5 g of surface carboxylated silica microspheres with an average particle size of 1 μm and a surface zeta potential of -35 mV, disperse them in 50 ml of MES buffer (10 mM, pH 5.5), add 1.0 g of EDC and 0.2 g of NHS, stir and mix at room temperature for 30 min, collect the activated microspheres, and wash them with MES buffer; disperse the activated microspheres in 50 ml of citric acid-sodium citrate buffer (10 mM, pH 4.0) containing 5 mg / ml pepsin, shake and mix at room temperature for 8 h, collect the immobilized enzyme microspheres, wash them with citric acid-sodium citrate buffer, and store them at 4°C.

[0083] Take 5g of the extracellular matrix of the submucosal layer of the small intestine of pigs, crush it into particles of 1-3mm in size, disperse it in 50ml of 0.6% acetic acid solution, add 1g of immobilized enzyme microspheres, keep stirring and digest at room temperature for 120h; centrifuge at 6000rpm for 20min, and collect the upper liquid.

[0084] Example 2

[0085] Take the pig dermis obtained by cutting with a skin slicer, remove the fat with a scraper, cut it into small pieces, and wash it with pure water; shake and soak it in 0.2% peracetic acid solution for 1 hour, and wash it with pure water; shake and treat it with a mixed solution containing 0.5% trypsin and 0.2% EDTA for 2 hours, and wash it with pure water; shake and soak it with 0.1mol / L sodium carbonate solution for 1 hour, and wash it with pure water; shake and treat it with a mixed solution containing 200U / ml deoxyribonuclease and 50U / ml ribonuclease for 1 hour, and wash it with pure water; shake and soak it with 0.1mol / L sodium carbonate solution for 1 hour, and wash it with pure water to obtain the pig dermal extracellular matrix.

[0086] 5 g of surface carboxyl-modified polystyrene microspheres with an average particle size of 15 μm and a surface zeta potential of -45 mV were dispersed in 50 ml of Tris-HCl buffer (50 mM, pH 8.0), 1.0 g of CMPI was added, and the mixture was stirred at room temperature for 30 min. The activated microspheres were collected and washed with Tris-HCl buffer; the activated microspheres were dispersed in 100 ml of citric acid-sodium citrate buffer (10 mM, pH 4.0) containing 5 mg / ml pepsin, and the mixture was shaken at room temperature for 16 h. The immobilized enzyme microspheres were collected, washed with citric acid-sodium citrate buffer, and stored at 4°C.

[0087] Take 5g of porcine dermal extracellular matrix, crush it into 1-3mm particles, disperse it in 20ml of 1mM hydrochloric acid solution, add 2g of immobilized enzyme microspheres, keep stirring and digest at room temperature for 48h; use 2500 mesh nylon filter cloth to filter out the immobilized enzyme microspheres and collect the filtrate.

[0088] Example 3

[0089] The submucosal layer of the small intestine of the pig treated with an intestine scraper was taken, the fat was removed with a scraper, and the submucosal layer was cut into small pieces, and washed with pure water; it was shaken and soaked in a 0.1% peracetic acid solution for 1 hour, and washed with pure water; it was shaken and treated with a mixed solution containing 0.25% trypsin and 0.1% EDTA for 2 hours, and washed with pure water; it was shaken and soaked in a 0.1 mol / L sodium carbonate solution for 1 hour, and washed with pure water; it was shaken and treated with a mixed solution containing 50 U / ml deoxyribonuclease and 10 U / ml ribonuclease for 2 hours, and washed with pure water; it was shaken and soaked in a 0.1 mol / L sodium carbonate solution for 1 hour, and washed with pure water to obtain the extracellular matrix of the submucosal layer of the small intestine of the pig.

[0090] Take 5g of surface carboxylated polymethyl methacrylate microspheres with an average particle size of 5μm and a surface zeta potential of -50mV, disperse them in 50ml MES buffer (10mM, pH 5.5), add 2.0g EDC and 0.5g NHS, stir and mix at room temperature for 30min, collect the activated microspheres, and wash them with MES buffer; disperse the activated microspheres in 100ml citric acid-sodium citrate buffer (10mM, pH 4.0) containing pepsin at a concentration of 10mg / ml, shake and mix at room temperature for 8h, collect the immobilized enzyme microspheres, wash them with citric acid-sodium citrate buffer, and store them at 4°C. The microscopic picture of the immobilized enzyme obtained is shown in the figure below. Figure 1 shown.

[0091] Take 5g of the extracellular matrix of the submucosal layer of the small intestine of pigs, crush it into particles of 1-3mm in size, disperse it in 50ml of 0.6% acetic acid solution, add 1g of immobilized enzyme microspheres, keep stirring and digest at room temperature for 72h; centrifuge at 4000rpm for 20min, and collect the upper liquid. The extracellular matrix solution obtained after digestion is as follows Figure 2 shown.

[0092] Example 4

[0093] Take the liquid obtained in Example 3, add 0.2M disodium hydrogen phosphate solution with a volume of 1 / 9 of the liquid, mix well and let stand at room temperature for 30 minutes to obtain an extracellular matrix gel such as Figure 4 shown.

[0094] Comparative Example 1

[0095] The only difference from Example 3 is that the average particle size of the microspheres used is 20 μm. The extracellular matrix solution obtained after digestion is as follows Figure 3 shown.

[0096] Comparative Example 2

[0097] The only difference from Example 3 is that the surface zeta potential of the microspheres used is -52.5 mV.

[0098] Experimental Example 1 Determination of immobilized enzyme activity

[0099] The enzyme activity of the immobilized enzyme was determined using the method of enzymatic casein hydrolysis. First, a tyrosine standard curve was drawn, and the specific operation was as follows: weigh the tyrosine standard and prepare solutions with concentrations of 20, 40, 60, 80, and 100 μg / ml, and measure the absorbance at 580 nm after adding the forin reagent to react, and draw a standard curve based on the absorbance and concentration. Afterwards, 3.0 ml of a 2% casein solution was divided into 3 portions, and each portion was treated with 1 mol / L hydrochloric acid to pH 2.0, 3.0, and 4.0, respectively. After 10 minutes of constant temperature at 37 degrees Celsius, 0.1 g of the immobilized enzyme microspheres in Example 1 was added to each portion, and after 10 minutes of reaction, 2.0 ml of 5% trichloroacetic acid was added to terminate the reaction, and the supernatant was collected by centrifugation. After reacting with the forin reagent, the absorbance was measured at 580 nm, and the concentration of tyrosine in the supernatant was obtained, and the enzyme activity was calculated accordingly. The same method was used to measure the immobilized enzyme activity of Examples 2, 3 and Comparative Examples 1, 2. The results of the determination of the enzyme activity of the immobilized enzyme are shown in Table 1. It can be seen that the immobilized enzymes obtained in the examples and comparative examples all have relatively large enzyme activity at pH 3.0.

[0100] Table 1 Enzyme activities (U / g) of immobilized enzymes of Examples 1-3 and Comparative Examples 1-2 at different pH values

[0101]

[0102] Experimental Example 2 Extracellular matrix digestion effect test

[0103] Due to the presence of a large amount of non-collagen components and multiple cross-links in the extracellular matrix, it is impossible to digest it to a transparent solution state in which all substances are completely dissolved using only pepsin, but only to achieve a colloidal solution state with a uniform appearance and translucent appearance. Therefore, when the extracellular matrix is ​​digested to obtain a viscous liquid state with a uniform appearance and translucent appearance, and can be smoothly pushed out with a fine injection needle, it can be considered that a good digestion effect has been achieved.

[0104] The digestion effect of the extracellular matrix of the present invention is determined based on the appearance of the sample and whether it can pass through a 30G injection needle. The results are shown in Table 2. It can be seen that although comparative examples 1 and 2 show good enzyme activity when detecting enzyme activity with casein, they cannot achieve good digestion effect when used to digest the extracellular matrix. The possible reason is that digestion requires good contact between the enzyme and the substrate. The state of the substrate is not free relative to casein in the extracellular matrix. When the particle size of the immobilized enzyme microspheres is too large, it cannot penetrate into the pores of the extracellular matrix and cannot fully contact; and the zeta potential on the surface of the microspheres is too low, indicating that the carboxyl content is too high, making the fixed enzymes and other proteins more dense, forming a larger steric hindrance, affecting the action of the active site of the enzyme and the substrate, and the effect is greater on relatively non-free substrates such as the extracellular matrix.

[0105] Table 2 The state of the extracellular matrix obtained after digestion in Examples 1-3 and Comparative Examples 1-2

[0106] Appearance 30G injection needle passing situation Example 1 Homogeneous, translucent liquid Completely passed Example 2 Homogeneous, translucent liquid Completely passed Example 3 Homogeneous, translucent liquid Completely passed Comparative Example 1 Inhomogeneous liquid Cannot pass Comparative Example 2 Inhomogeneous liquid Cannot pass

[0107] Experimental Example 3 Determination of residual pepsin in extracellular matrix solution

[0108] A porcine pepsin ELISA detection kit (purchased from Shanghai Keaibo Biotechnology, catalog number CB10495-PG) was used to detect the residual amount of porcine pepsin in the digested samples. The samples obtained in the examples were taken and tested according to the operating steps in the kit instructions. The residual amounts of pepsin in the samples obtained in Examples 1, 2, and 3 were detected to be 12.25 ng / ml, undetectable, and 7.75 ng / ml, respectively, indicating that the use of immobilized enzymes to digest the extracellular matrix can reduce the enzyme residues to an extremely low level.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for digesting extracellular matrix using immobilized enzymes, characterized in that: The following steps are involved: (1) decellularizing the tissue to obtain the extracellular matrix; (2) dispersing the surface functionalized microspheres in a buffer, adding a coupling agent, mixing, and collecting the activated microspheres; (3) dispersing the activated microspheres in a buffer solution containing pepsin, mixing, and collecting the immobilized enzyme microspheres; (4) The extracellular matrix is ​​crushed and dispersed in a solution, and the immobilized enzyme microspheres are added for digestion. After the digestion is completed, the immobilized enzyme microspheres are separated and removed.

2. The method according to claim 1, characterized in that Step (1) comprises: taking mammalian tissue, adding 0.1-1% peracetic acid solution and shaking and soaking for 0.5-2h, adding 0.1-0.5% trypsin and 0.1-0.5% EDTA and shaking and treating for 1-3h, adding 0.1-0.5mol / L sodium carbonate solution and shaking and soaking for 0.5-2h, adding 50-200U / ml deoxyribonuclease and 10-50U / ml ribonuclease and shaking and treating for 1-3h, and then adding 0.1-0.5mol / L sodium carbonate solution and shaking and soaking for 0.5-2h to obtain extracellular matrix.

3. The method according to claim 1, characterized in that The surface functionalized microspheres in step (2) are surface carboxylated microspheres, and the microspheres are selected from any one or more of silica microspheres, polystyrene microspheres, and polymethacrylic acid microspheres.

4. The method according to claim 1, characterized in that: The average particle size of the surface functionalized microspheres described in step (2) is 1-15 μm; the surface Zeta potential of the surface functionalized microspheres is -35 to -50 mV.

5. The method according to claim 1, characterized in that The coupling agent described in step (2) is selected from one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and 2-chloro-1-methylpyridine iodide; the mass ratio of the microspheres to the coupling agent in step (2) is 5:(1-3).

6. The method according to claim 1, characterized in that The pH of the solution in step (4) is 3.0-4.5; the mass volume ratio of the extracellular matrix to the solution is 1:(4-10).

7. The method according to claim 1, characterized in that In step (4), the mass ratio of the extracellular matrix to the immobilized enzyme microspheres is 5:(1-2).

8. An extracellular matrix solution, characterized in that The extracellular matrix solution is prepared by the method described in any one of claims 1-7.

9. Use of the extracellular matrix solution according to claim 8 in preparing extracellular matrix gel.

10. An extracellular matrix gel, characterized in that: The extracellular matrix gel is prepared by adding disodium hydrogen phosphate solution to the extracellular matrix solution prepared by the method described in any one of claims 1 to 7.

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