Decellularized small intestinal submucosa as well as preparation method and application thereof
By using a combination of sodium dodecyl sulfate, ethanol solution, potassium chloride solution and NaOH/NaCl solution on the submucosa of the small intestine, the problems of incomplete cell decellularization and reagent residue in the prior art were solved, and cell completeness was achieved Improved removal and biocompatibility.
Patent Information
- Application Number
- CN202411267972.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-11
- Publication Date
- 2025-01-03
AI Technical Summary
In the prior art, when decellularizing the submucosal layer of the small intestine, physical methods are incomplete in decellularization and cell residues are high, while chemical reagents are prone to cause reagent residues, affecting biocompatibility.
Decellularization was performed using sodium dodecyl sulfate at a concentration of 10-30 mg/mL, followed by ethanol solution and potassium chloride solution, followed by 0.2-1 mol/L NaOH solution and 0.1-3 mol/L NaCl solution. Perform treatment to ensure complete removal of cells and reduce DNA residue.
Complete removal of cells in the submucosal layer of the small intestine is achieved, the DNA residue is low, and there is basically no reagent residue, which improves biocompatibility.
Smart Images
Figure CN119236180A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of biomaterial decellularization, and in particular to a decellularized small intestinal submucosa and a preparation method and application thereof. Background Art
[0002] The small intestinal submucosa is a natural extracellular matrix material with good mechanical properties. The SIS obtained by decellularization retains bioactive components such as fibronectin, glycosaminoglycans, and growth factors. Fibronectin and glycosaminoglycans play an important role in cell-cell and cell-extracellular matrix adhesion, and fibronectin plays an important role in regulating the information transmission between cells and extracellular matrix and the formation of vascular structure.
[0003] In the prior art, chemical reagents and physical methods are usually used for decellularization. However, the decellularization using physical methods is usually incomplete, with a high number of residual cells, while chemical reagents are prone to cause reagent residues, thereby affecting biocompatibility. Summary of the invention
[0004] The present application provides a decellularized small intestinal submucosa and a preparation method and application thereof, which can effectively remove all cells in the small intestinal submucosa, with low residual DNA, basically no reagent residue, and good biocompatibility.
[0005] This application is implemented as follows:
[0006] In a first aspect, the present application provides a method for preparing a decellularized small intestinal submucosa, comprising the following steps:
[0007] Pre-treatment of the small intestinal submucosa;
[0008] The pre-treated small intestinal submucosal layer is decellularized using sodium dodecyl sulfate at a concentration of 10 to 30 mg / mL, and then treated with an ethanol solution and a potassium chloride solution in sequence;
[0009] The surface is treated with a NaOH solution having a concentration of 0.2 to 1 mol / L, and then cleaned with a NaCl solution having a concentration of 0.1 to 3 mol / L.
[0010] In a possible embodiment, the treatment time of the sodium dodecyl sulfate is 1 to 4 hours.
[0011] In a possible implementation manner, the concentration of the NaOH solution is 0.5 to 1 mol / L, and the treatment time of the NaOH solution is 1 to 4 hours.
[0012] In a possible implementation manner, the concentration of the NaCl solution is 1 to 3 mol / L, and the treatment time of the NaCl solution is 1 to 4 h.
[0013] In a possible embodiment, the pre-treated small intestinal submucosal layer is decellularized with sodium dodecyl sulfate at a concentration of 10 to 30 mg / mL, and then treated with an ethanol solution and a potassium chloride solution in sequence; it also includes treating with trypsin, and then treating with a NaOH solution at a concentration of 0.2 to 1 mol / L;
[0014] Optionally, the concentration of trypsin is 1 to 3 mg / mL, and the treatment time is 9 to 24 hours.
[0015] In a possible implementation manner, the concentration of the ethanol solution is 70-80%, and the concentration of the potassium chloride solution is 0.3-0.8 mol / L.
[0016] In a possible implementation manner, the treatment time of the ethanol solution is 0.5 to 2 hours, and the treatment time of the potassium chloride solution is 0.5 to 3 hours.
[0017] In a possible embodiment, the pretreatment includes disinfecting and defatting the small intestinal submucosa.
[0018] In a possible implementation, the disinfection treatment comprises: treating the small intestinal submucosal layer with a peracetic acid solution and an ethanol solution in sequence; the concentration of the peracetic acid is 0.1-0.5%, and the concentration of the ethanol solution is 20-30%.
[0019] In a second aspect, an embodiment of the present application provides a decellularized small intestinal submucosa, which is prepared by the preparation method of the decellularized small intestinal submucosa according to the first aspect.
[0020] In a third aspect, an embodiment of the present application provides a use of the decellularized small intestinal submucosa according to the second aspect in preparing a patch or an oral membrane.
[0021] This application has at least the following beneficial effects:
[0022] The preparation method of the decellularized small intestinal submucosa of the embodiment of the present application is first to decellularize the small intestinal submucosa after pretreatment with sodium dodecyl sulfate at a concentration of 10 to 30 mg / mL, which can dissolve the cell membrane, thereby releasing substances such as proteins and nucleic acids in the cells. Then, ethanol solution and potassium chloride solution are used for treatment in turn, which can effectively remove the residual reagent of sodium dodecyl sulfate. Then, a NaOH solution with a concentration of 0.2 to 1 mol / L is used for treatment, so that the DNA can be unwound from a double strand to a single strand, and then the unwound DNA precipitate is easily cleaned by washing with a NaCl solution of 0.1 to 3 mol / L, so as to obtain a decellularized small intestinal submucosa with low DNA residue. If the concentration of sodium dodecyl sulfate, the concentration of NaOH solution, and the concentration of NaCl solution are not within the range specified in the present application, it will result in high DNA residue and incomplete cell removal. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying any creative work.
[0024] Figure 1 This is the HE staining image of the decellularized small intestinal submucosa of Example 1;
[0025] Figure 2 This is the HE staining image of the decellularized small intestinal submucosa of Example 2;
[0026] Figure 3 This is the HE staining image of the decellularized small intestinal submucosa of Example 3;
[0027] Figure 4 This is the HE staining image of the decellularized small intestinal submucosa of Example 4;
[0028] Figure 5 HE staining of the decellularized small intestinal submucosa of Comparative Example 1;
[0029] Figure 6 HE staining of the decellularized small intestinal submucosa of Comparative Example 2;
[0030] Figure 7 This is the HE staining image of the decellularized small intestinal submucosa of Comparative Example 3;
[0031] Figure 8 This is the HE staining image of the decellularized small intestinal submucosa of Comparative Example 4. DETAILED DESCRIPTION
[0032] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0033] The following is a detailed description of a decellularized small intestinal submucosa and its preparation method and application according to an embodiment of the present application:
[0034] In a first aspect, the present invention provides a method for preparing a decellularized small intestinal submucosa, comprising the following steps:
[0035] (1) Pretreatment of the small intestinal submucosa, which includes disinfection and degreasing of the small intestinal submucosa.
[0036] The disinfection treatment can clean away viral pathogens in the submucosa of the small intestine. In some embodiments, the disinfection treatment comprises sequentially treating the submucosa of the small intestine with a peracetic acid solution and an ethanol solution.
[0037] Optionally, the concentration of the peracetic acid is 0.1-0.5%, and the concentration of the ethanol solution is 20-30%. Exemplarily, the concentration of the peracetic acid is any one of 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, or a value between any two of them. Exemplarily, the concentration of the ethanol solution in the disinfection step is any one of 20%, 22%, 24%, 26%, 28% and 30%, or a value between any two of them.
[0038] In some embodiments, the degreasing treatment in step (1) is performed using a mixture of dichloromethane and anhydrous ethanol in a volume ratio of 2:1 for 6 to 18 hours, for example, the treatment time is 6 hours, 9 hours, 12 hours, 15 hours or 18 hours.
[0039] (2) The pre-treated small intestinal submucosal layer was decellularized using sodium dodecyl sulfate at a concentration of 10-30 mg / mL, and then treated with ethanol solution and potassium chloride solution in sequence.
[0040] Sodium dodecyl sulfate (SDS) can dissolve cell membranes, thereby releasing proteins, nucleic acids and other substances in cells. If the concentration of sodium dodecyl sulfate is low, the degree of cell membrane dissolution is low, resulting in cell residues, and DNA cannot be fully released, resulting in a high amount of DNA residue. After SDS is used to treat the submucosal layer of the small intestine, it is then treated with ethanol solution and potassium chloride solution in turn, which can effectively remove the residual sodium dodecyl sulfate and improve the biocompatibility of the decellularized submucosal layer of the small intestine.
[0041] Exemplarily, the concentration of sodium lauryl sulfate is any one of 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL and 30 mg / mL or a value between any two thereof. Optionally, the treatment time of sodium lauryl sulfate is 1 to 4 hours. Exemplarily, the treatment time of sodium lauryl sulfate is any one of 1 hour, 2 hours, 3 hours and 4 hours or a value between any two thereof.
[0042] Optionally, the concentration of the ethanol solution in step (2) is 70-80%, for example, any one of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% and 80%, or a value between any two of them. Optionally, the treatment time of the ethanol solution in step (2) is 0.5-2h, for example, 0.5h, 1.0h, 1.5h or 2h.
[0043] Optionally, the concentration of the potassium chloride solution is 0.3-0.8 mol / L, for example, any one of 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L and 0.8 mol / L, or a value between any two of them. Optionally, the treatment time of the potassium chloride solution is 0.5-3 h, for example, 0.5 h, 1 h, 2 h or 3 h.
[0044] Further, the pre-treated small intestinal submucosal layer is decellularized with sodium dodecyl sulfate at a concentration of 10 to 30 mg / mL, and then treated with ethanol solution and potassium chloride solution in sequence, and further includes: treating with trypsin, and then treating with NaOH solution at a concentration of 0.2 to 1 mol / L. Optionally, the concentration of trypsin is 1 to 3 mg / mL, and the treatment time is 9 to 24 hours.
[0045] (3) Treat with a NaOH solution with a concentration of 0.2 to 1 mol / L, and then clean with a NaCl solution with a concentration of 0.1 to 3 mol / L.
[0046] In the early process steps, sodium dodecyl sulfate is used to dissolve the cell membrane to release nucleic acids, proteins and other substances, and then treated with NaOH solution to unwind the DNA from double-stranded to single-stranded, which is beneficial for the subsequent reaction of NaCl solution with the unwound single-stranded DNA to precipitate the DNA and make it easy to clean, thereby reducing the residual DNA content in the decellularized small intestinal submucosal layer.
[0047] The concentration of the NaOH solution in step (3) is 0.2-1 mol / L, and the concentration of the NaCl solution is 0.1-3 mol / L. The inventors of the present application have found that if the concentration of the NaOH solution and / or the concentration of the NaCl solution are both low, the DNA content will be high.
[0048] Exemplarily, the concentration of the NaOH solution is any one of 0.2 mol / L, 0.5 mol / L, 0.8 mol / L and 1 mol / L or a value between any two of them. Optionally, the treatment time of the NaOH solution is 1 to 4 hours, for example, 1 hour, 2 hours, 3 hours or 4 hours.
[0049] Optionally, the concentration of the NaCl solution is any one of 0.1 mol / L, 0.5 mol / L, 1 mol / L, 2 mol / L and 3 mol / L or a value between any two thereof. Exemplarily, the treatment time of the NaCl solution is 1 to 4 h, for example, the treatment time of the NaCl solution is 1 h, 2 h, 3 h or 4 h.
[0050] In a second aspect, the present application provides a decellularized small intestinal submucosa, which is prepared by the preparation method of the decellularized small intestinal submucosa according to the first aspect.
[0051] In a third aspect, the present application provides a use of the decellularized small intestinal submucosa as described above in the preparation of a patch or an oral membrane.
[0052] The decellularized small intestinal submucosa of the present application can be applied to patches or oral membranes because the cells are completely removed and the DNA content is low.
[0053] The decellularized small intestinal submucosa of the present application and its preparation method and application are further described in detail below in conjunction with the examples.
[0054] Example 1
[0055] This embodiment provides a method for preparing acellular small intestinal submucosa, which comprises the following steps:
[0056] (1) Pretreatment of the small intestinal submucosa: The small intestinal submucosa was treated with a 0.2% peracetic acid solution and a 25% ethanol solution for 1 hour respectively.
[0057] (2) The pre-treated small intestinal submucosal layer was treated with 10 mg / mL sodium dodecyl sulfate for 3 h, and then treated with 75% ethanol solution and 0.5 mol / L potassium chloride for 1 h respectively.
[0058] (3) The small intestinal submucosa was treated with a 0.2 mol / L NaOH solution for 1.5 h and then cleaned with a 2 mol / L NaCl solution for 2 h.
[0059] Example 2
[0060] This embodiment provides a method for preparing acellular small intestinal submucosa, which is different from Embodiment 1 only in that the time for washing with NaCl solution in step (3) of Embodiment 2 is 4 hours.
[0061] Example 3
[0062] This embodiment provides a method for preparing decellularized small intestinal submucosa, which is different from Embodiment 1 only in that the concentration of the NaOH solution in step (3) of Embodiment 3 is 0.5 mol / L.
[0063] Example 4
[0064] This embodiment provides a method for preparing acellular small intestinal submucosa, which is different from Embodiment 3 only in that the time for washing with NaCl solution in step (3) of Embodiment 4 is 4 hours.
[0065] Example 5
[0066] This embodiment provides a method for preparing acellular small intestinal submucosa. Compared with Example 1, the only difference is that the concentration of sodium dodecyl sulfate in step (2) of Example 5 is 30 mg / mL.
[0067] Example 6
[0068] This embodiment provides a method for preparing acellular small intestinal submucosa. Compared with Example 1, the only difference is that the concentration of the NaOH solution in step (3) of Example 6 is 1.0 mol / L.
[0069] Example 7
[0070] This embodiment provides a method for preparing acellular small intestinal submucosa. Compared with Example 1, the only difference is that the concentration of the NaCl solution is 0.5 mol / L.
[0071] Example 8
[0072] The present embodiment provides a method for preparing a cell-free small intestinal submucosa. Compared with the embodiment 1, the only difference is that the step (2) of the embodiment 8 is different from the embodiment 1. The step (2) of the embodiment 8 is as follows: the pre-treated small intestinal submucosa is treated with 10 mg / mL sodium dodecyl sulfate for 3 h, and then treated with 75% ethanol solution and 0.5 mol / L potassium chloride for 1 h respectively; and then treated with 2.5 mg / mL trypsin for 18 h.
[0073] Comparative Example 1
[0074] This comparative example provides a method for preparing acellular small intestinal submucosa. Compared with Example 1, the only difference is that the concentration of the NaCl solution in step (3) of Comparative Example 1 is 0.015 mol / L.
[0075] Comparative Example 2
[0076] This comparative example provides a method for preparing acellular small intestinal submucosa. Compared with Example 1, the only difference is that the concentration of sodium dodecyl sulfate in step (2) of comparative example 2 is 5 mg / mL.
[0077] Comparative Example 3
[0078] This comparative example provides a method for preparing a decellularized small intestinal submucosa. Compared with Example 1, the only difference is that the concentration of sodium dodecyl sulfate in step (2) of comparative example 3 is 5 mg / mL, and the concentration of the NaOH solution in step (3) is 0.05 mol / L.
[0079] Comparative Example 4
[0080] This comparative example provides a method for preparing acellular small intestinal submucosa. Compared with Example 1, the only difference is that comparative example 4 omits the step of treating with a NaOH solution with a concentration of 0.2 mol / L in step (3).
[0081] Comparative Example 5
[0082] This comparative example provides a method for preparing a decellularized small intestinal submucosa, which comprises the following steps:
[0083] (1) Pretreatment of the small intestinal submucosa: The small intestinal submucosa was treated with a 0.2% peracetic acid solution and a 25% ethanol solution for 1 hour respectively.
[0084] (2) The pre-treated small intestinal submucosal layer was first treated with 2.5 mg / mL trypsin for 24 h, and then treated with 5 mg / mL Triton X-100 solution for 24 h.
[0085] (3) Use 2 mol / L NaCl solution for cleaning for 2 h.
[0086] Comparative Example 6
[0087] This comparative example provides a method for preparing a cell-free small intestinal submucosa. Compared with Example 1, the only difference is that in step (2) of comparative example 6, before the sodium dodecyl sulfate treatment step, a step of treating with trypsin at a concentration of 2.5 mg / mL for 18 hours is added.
[0088] Test Example 1
[0089] The decellularized small intestinal submucosal layer prepared in Examples 1 to 4 and Comparative Examples 1 to 4 was sectioned and HE stained. The HE staining images are shown in FIG. Figures 1 to 8 The circled part in the figure is the remaining cell nucleus component.
[0090] The experimental results show that the decellularized small intestinal submucosa of Examples 1 to 4 has no residual cell nuclei, while Comparative Examples 1 to 4 still have residual cell nuclei.
[0091] Test Example 2
[0092] The DNA content of the decellularized small intestinal submucosal layer of Examples 1 to 8 and Comparative Examples 1 to 6 was detected according to YYT 1876-2023 (Determination of DNA Residue in Animal-Derived Biomaterials for Tissue Engineering Medical Products: Fluorescence Staining Method), and the results are recorded in Table 1.
[0093]
[0094]
[0095] From the results in Table 1, it can be seen that the residual DNA content of the decellularized small intestinal submucosal layer obtained by the preparation methods of Examples 1 to 8 is less than 5 ng / mg, while the residual DNA content of the decellularized small intestinal submucosal layer obtained by the preparation methods of Comparative Examples 1 to 4 is greater than 20 ng / mg, and the residual DNA content of the decellularized small intestinal submucosal layer obtained by the preparation methods of Comparative Examples 5 and 6 is greater than 14 ng / mg. This indicates that the preparation methods of Examples 1 to 8 of the present application can effectively reduce the residual DNA content of the decellularized small intestinal submucosal layer.
[0096] Among them, by comparing Example 1, Example 5 and Comparative Example 2, it was found that the concentration of sodium dodecyl sulfate in Comparative Example 2 was too low, resulting in a DNA residue in the decellularized small intestinal submucosal layer greater than 39 ng / mg. This indicates that the concentration of sodium dodecyl sulfate affects the DNA residue, and when the concentration of sodium dodecyl sulfate is less than 10 mg / mL (for example, 5 mg / mL), the DNA residue in the decellularized small intestinal submucosal layer is high.
[0097] By comparing Example 1 with Comparative Example 4, it was found that the residual DNA in the decellularized small intestinal submucosal layer was higher than 45 ng / mg in Comparative Example 4 without the NaOH solution treatment, while the residual DNA in the decellularized small intestinal submucosal layer of Example 1 was less than 5 ng / mg. This indicates that the use of NaOH solution treatment before the NaCl solution cleaning step can effectively reduce the residual DNA in the decellularized small intestinal submucosal layer.
[0098] By comparing the experimental results of Example 1 and Comparative Example 3, it was found that the concentration of sodium dodecyl sulfate and the concentration of NaOH solution will jointly affect the result of DNA residue. When the step of treating with NaOH solution before the NaCl solution cleaning step is retained, if the concentration of sodium dodecyl sulfate is low and the concentration of NaOH solution is also low, the DNA residue in the decellularized small intestinal mucosa will be higher.
[0099] By comparing Example 1 with Comparative Example 1, it is found that the concentration of the NaCl solution in Comparative Example 1 is 0.015 mol / L, and the concentration of the NaCl solution in Example 1 is 2 mol / L, while the residual DNA amount in the decellularized small intestinal submucosal layer of Comparative Example 1 is greater than 20 ng / mg, and the residual DNA amount in the decellularized small intestinal submucosal layer of Example 1 is less than 5 ng / mg. This indicates that the concentration of the NaCl solution has an effect on the residual DNA amount, and when the concentration of the NaCl solution is low, the residual DNA amount will be large.
[0100] By comparing Example 1, Example 8, Comparative Example 5 and Comparative Example 6, it is found that, compared with Example 1, after the sodium dodecyl sulfate treatment, the ethanol solution and potassium chloride treatment, the step of trypsin treatment is added in Example 8, and the DNA residues in Example 1 and Example 8 are both less than 5 ng / mg, and the difference between the two is not large. Compared with Example 1, Comparative Example 6 further adds a trypsin treatment step before the sodium dodecyl sulfate treatment step, and the DNA residue in Comparative Example 6 is 14.25 ng / mg, while the DNA residue in Example 1 is 4.36 ng / mg, indicating that the trypsin treatment step before the sodium dodecyl sulfate treatment step is not conducive to the subsequent complete removal of cells.
[0101] In addition, by comparing Example 1 and Comparative Example 5, in Comparative Example 5, trypsin combined with Triton X-100 was used to treat the small intestinal submucosa, and the DNA residue of the decellularized small intestinal submucosa was 17.58 ng / mg, while the DNA residue of Example 1 was 4.36 ng / mg, indicating that the process of treating the small intestinal submucosa with trypsin combined with Triton X-100 is not conducive to the complete removal of cells in the small intestinal submucosa.
[0102] Test Example 3
[0103] The residual SDS content of the decellularized small intestinal submucosal layer of Example 1 was tested by methylene blue spectrophotometry: the sample was extracted with methanol-sodium carbonate solution, reacted with methylene blue to generate salt substances; the salt substances were extracted with chloroform, and then the absorbance value was measured at 651nm with a spectrophotometer. The results showed that the residual SDS content of the decellularized small intestinal submucosal layer of Example 1 was 0.77μg / mg, which met the requirements.
[0104] Test Example 4
[0105] The hemolysis test was performed on the decellularized small intestinal submucosa of Example 1 with reference to GB / T 16886.4 (Biological evaluation of medical devices Part 4: Selection of tests for interaction with blood). The test result was HI <5%, and the hemolysis test result was qualified.
[0106] Test Example 5
[0107] Referring to GB / T 16886.5-2017 (Biological evaluation of medical devices Part 5: In vitro cytotoxicity test), the extract of the decellularized small intestinal submucosa of Example 1 was tested for in vitro cytotoxicity. The cytotoxicity test method was based on the CCK-8 method. The cell survival rate obtained from the test was greater than 70%, and the cytotoxicity test result was qualified.
[0108] Among them, the results of the hemolysis test and the cytotoxicity test are both qualified, indicating that the residual SDS does not cause hemolysis and cytotoxicity, and the decellularized small intestinal submucosa of Example 1 of the present application has good biocompatibility.
[0109] The above description is only a specific embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A method for preparing decellularized small intestinal submucosa, characterized in that: The following steps are involved: Pre-treatment of the small intestinal submucosa; The pre-treated small intestinal submucosal layer is decellularized using sodium dodecyl sulfate at a concentration of 10 to 30 mg / mL, and then treated with an ethanol solution and a potassium chloride solution in sequence; The surface is treated with a NaOH solution having a concentration of 0.2 to 1 mol / L, and then cleaned with a NaCl solution having a concentration of 0.1 to 3 mol / L.
2. The method for preparing decellularized small intestinal submucosa according to claim 1, characterized in that: The treatment time of the sodium dodecyl sulfate is 1 to 4 hours.
3. The method for preparing decellularized small intestinal submucosa according to claim 1, characterized in that: The concentration of the NaOH solution is 0.5-1 mol / L, and the treatment time of the NaOH solution is 1-4 h; optionally, the concentration of the NaCl solution is 1-3 mol / L, and the treatment time of the NaCl solution is 1-4 h.
4. The method for preparing decellularized small intestinal submucosa according to claim 1, characterized in that: The pre-treated small intestinal submucosal layer is subjected to decellularization treatment with sodium dodecyl sulfate at a concentration of 10 to 30 mg / mL, and then treated with an ethanol solution and a potassium chloride solution in sequence; it also includes treating with trypsin, and then treating with a NaOH solution at a concentration of 0.2 to 1 mol / L; Optionally, the concentration of trypsin is 1 to 3 mg / mL, and the treatment time is 9 to 24 hours.
5. The method for preparing the decellularized small intestinal submucosa according to any one of claims 1 to 4, characterized in that: The concentration of the ethanol solution is 70-80%, and the concentration of the potassium chloride solution is 0.3-0.8 mol / L.
6. The method for preparing decellularized small intestinal submucosa according to claim 5, characterized in that: The treatment time of the ethanol solution is 0.5 to 2 hours, and the treatment time of the potassium chloride solution is 0.5 to 3 hours.
7. The method for preparing the decellularized small intestinal submucosa according to any one of claims 1 to 4, characterized in that: The pretreatment includes disinfection and degreasing of the small intestinal submucosal layer.
8. The method for preparing decellularized small intestinal submucosa according to claim 7, characterized in that: The disinfection treatment comprises: using peracetic acid solution and ethanol solution to treat the small intestinal submucosal layer in sequence; the concentration of the peracetic acid is 0.1-0.5%, and the concentration of the ethanol solution is 20-30%.
9. A decellularized small intestinal submucosa, characterized in that: The decellularized small intestinal submucosa is prepared by the preparation method of any one of claims 1 to 8.
10. Use of the decellularized small intestinal submucosa as claimed in claim 9 in preparing a patch or an oral membrane.