Liver decellularized scaffold, preparation method thereof, hydrogel and application of hydrogel

Abstract

The invention provides a liver decellularized scaffold, a preparation method of the liver decellularized scaffold, hydrogel and application of the hydrogel, and the preparation method of the liver decellularized scaffold comprises the step that the liver decellularized scaffold is perfused with a first buffer solution, a nonionic detergent, an ionic detergent and a second buffer solution in sequence to obtain the decellularized scaffold. According to the preparation method of the liver decellularized scaffold provided by the embodiment of the invention, the first buffer solution and other substances are sequentially used for respectively pouring the liver decellularized scaffold, so that DNA in liver cells can be removed in order, the DNA removal rate can be improved, effective extracellular matrix components can be reserved to a great extent, and the liver decellularized scaffold is prepared. The residue of harmful chemical agents is reduced, and the application prospect is relatively good.

Description

Technical Field

[0001] The present application relates to the technical field of biomaterials, and in particular to a liver decellularized scaffold and a preparation method thereof, a hydrogel and an application thereof. Background Art

[0002] Decellularized scaffolds are tissue engineering scaffolds that are constructed by removing cells from foreign or xenogeneic tissues through chemical and physical methods to form non-immunogenic or low-immunogenic materials. Due to the advantages of decellularized scaffolds, they can be used in tissue repair, tissue engineering, organoid culture and other fields.

[0003] At present, the commonly used method for preparing decellularized scaffolds includes treating biological tissues with enzymes and other substances to obtain decellularized scaffolds. However, for liver decellularized scaffolds, if traditional enzymes and other substances are used to treat the liver, it is easy to cause the loss of a large number of important components of the extracellular matrix, and physical methods cannot completely remove tissue DNA components and other defects. Summary of the invention

[0004] The present application provides a liver decellularized scaffold and a preparation method thereof, a hydrogel and an application thereof, in order to solve the problems that the current preparation of liver decellularized scaffolds easily leads to the loss of a large amount of important components of the extracellular matrix and the DNA removal effect is poor.

[0005] The embodiment of the present application provides a method for preparing a liver decellularized scaffold, comprising sequentially using a first buffer, a non-ionic detergent, an ionic detergent, and a second buffer to perfuse the liver decellularized scaffold to obtain the decellularized scaffold.

[0006] According to the preparation method of the liver decellularized scaffold provided in the embodiment of the present application, by sequentially using the first buffer and other substances to perfuse the liver decellularized scaffold, the DNA in the liver cells can be removed in an orderly manner, so as to improve the DNA removal rate, and at the same time, the effective extracellular matrix components can be retained to a great extent, and the harmful chemical residues can be reduced. The obtained liver decellularized scaffold can be used in the fields of tissue repair, tissue engineering, organoid culture, etc., and has good application prospects.

[0007] In some embodiments of the present application, the preparation method of a decellularized liver scaffold includes the following steps: obtaining a biological liver; using a first buffer to perfuse the liver through the portal vein to remove residual blood; then using a non-ionic detergent to perfuse the liver to lyse the cells; then using an ionic detergent to perfuse the liver to remove DNA; then using the first buffer to perfuse the liver to wash away the residual ionic detergent; then using a second buffer to perfuse the liver for sterilization to obtain a decellularized liver scaffold.

[0008] In some embodiments of the present application, the ionic detergent comprises sodium dodecyl sulfate solution.

[0009] In some embodiments of the present application, the mass fraction of the sodium dodecyl sulfate solution is 0.1% to 0.5%.

[0010] In some embodiments of the present application, the mass fraction of the sodium dodecyl sulfate solution is 0.1% to 0.4%.

[0011] In some embodiments of the present application, the mass fraction of the sodium dodecyl sulfate solution is 0.2%.

[0012] In some embodiments of the present application, the perfusion time of the sodium dodecyl sulfate solution is 2 to 3 hours.

[0013] In some embodiments of the present application, the infusion volume of the sodium dodecyl sulfate solution is 800-2000 mL.

[0014] In some embodiments of the present application, the first buffer comprises a PBS buffer containing ethylenediaminetetraacetic acid.

[0015] In some embodiments of the present application, the mass fraction of the EDTA contained in the first buffer solution is 0.01% to 0.1%, preferably 0.05%.

[0016] In some embodiments of the present application, the perfusion time of using the first buffer to perfuse the liver via the portal vein to remove residual blood is 10 to 30 minutes.

[0017] In some embodiments of the present application, the perfusion time of perfusing the liver with the first buffer to wash away the residual ionic detergent is 2 to 3 hours.

[0018] In some embodiments of the present application, the non-ionic detergent comprises TritonX-100 solution.

[0019] In some embodiments of the present application, the mass fraction of the TritonX-100 solution is 1% to 4%.

[0020] In some embodiments of the present application, the perfusion time of the TritonX-100 solution is 2 to 3 hours.

[0021] In some embodiments of the present application, the second buffer comprises a PBS buffer containing antibiotics.

[0022] In some embodiments of the present application, the antibiotic includes at least one of penicillin-streptomycin and gentamicin.

[0023] In some embodiments of the present application, the perfusion time of the PBS buffer containing antibiotics is 5 to 10 min.

[0024] Another embodiment of the present application provides a liver decellularized scaffold, which is prepared by any of the above methods for preparing a liver decellularized scaffold.

[0025] Another embodiment of the present application provides a method for preparing a hydrogel based on a liver decellularized scaffold, comprising the following steps: pre-treating the liver decellularized scaffold to obtain a powder; digesting the powder with a digestive enzyme to obtain a pregel; and mixing the pregel with a photosensitive material to obtain a hydrogel.

[0026] In some embodiments of the present application, the pretreatment includes freeze-drying the liver decellularized scaffold and grinding it to obtain a powder.

[0027] In some embodiments of the present application, the digestive enzyme comprises pepsin.

[0028] In some embodiments of the present application, the digestion time is 2 to 3 days.

[0029] In some embodiments of the present application, the photosensitive material includes at least one of methacrylated gelatin, methacrylated silk fibroin, and methacrylated hyaluronic acid.

[0030] Another embodiment of the present application provides an application of a hydrogel based on a liver decellularized scaffold in tissue repair and organoid culture.

[0031] Additional technical solutions and advantages of the present application will be partially given in the following description, and partially become apparent from the following description, or be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 A flow chart of a method for preparing a liver decellularized scaffold provided in some embodiments of the present application;

[0034] Figure 2 A flow chart of a method for preparing a hydrogel based on a liver decellularized scaffold provided in some embodiments of the present application;

[0035] Figure 3This is a picture of the decellularized liver scaffold after perfusion provided in Example 1 of the present application;

[0036] Figure 4 A diagram of the perfusion process of the liver decellularized scaffold provided in the embodiments and comparative examples of the present application;

[0037] Figure 5 Histopathological staining (HE, immunofluorescence staining) of the decellularized scaffolds provided in the examples and comparative examples of the present application;

[0038] Figure 6 The scanning electron microscopy results of the decellularized scaffolds provided in the examples and comparative examples of the present application;

[0039] Figure 7 Detection of some components of the decellularized scaffolds provided in the examples and comparative examples of the present application;

[0040] Figure 8 This is a diagram showing the effect of the decellularized scaffold hydrogel of Example 1 of the present application coagulating and stopping bleeding in a liver wound;

[0041] Fig. 9 This is a diagram showing the effect of the decellularized scaffold hydrogel of Example 1 of the present application being well integrated with liver tissue and promoting wound repair. DETAILED DESCRIPTION

[0042] "Scope" disclosed in the present application is limited in the form of lower limit and upper limit, and a given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a special range. The scope limited in this way can be including end values ​​or not including end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a scope. For example, if the scope of 60-120 and 80-110 is listed for a specific parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following scope can be all expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present application, unless otherwise specified, the numerical range "ab" represents the abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0043] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0044] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0045] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0046] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0047] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0048] The embodiment of the present application provides a method for preparing a liver decellularized scaffold, comprising sequentially using a first buffer, a non-ionic detergent, an ionic detergent, and a second buffer to perfuse the liver decellularized scaffold to obtain the decellularized scaffold.

[0049] According to the preparation method of the liver decellularized scaffold provided in the embodiment of the present application, by sequentially using the first buffer and other substances to perfuse the liver decellularized scaffold, the DNA in the liver cells can be removed in an orderly manner, so as to improve the DNA removal rate, and at the same time, the effective extracellular matrix components can be retained to a great extent, and the harmful chemical residues can be reduced. The obtained liver decellularized scaffold can be used in the fields of tissue repair, tissue engineering, organoid culture, etc., and has good application prospects.

[0050] Reference Figure 1 As shown, in some embodiments, the method for preparing a liver decellularized scaffold comprises the following steps:

[0051] S100. Obtain a biological liver.

[0052] S200. Use the first buffer to perfuse the liver via the portal vein to remove residual blood.

[0053] S300, then switch to non-ionic detergent to perfuse the liver to lyse the cells.

[0054] S400, then switch to ionic detergent to perfuse the liver to remove DNA.

[0055] S500, then perfusing the liver with the first buffer to wash away the residual ionic detergent.

[0056] S600, then use the second buffer to perfuse the liver for sterilization to obtain a decellularized liver scaffold.

[0057] In the embodiment of the present application, the first buffer solution is first used to perfuse the liver, which can remove residual blood and prevent residual blood in the liver from affecting the subsequent perfusion effect. Then, a non-ionic detergent is used to perfuse the liver, which can cause cell lysis to facilitate the removal of DNA. Then, an ionic detergent is used to perfuse the liver to fully remove DNA. Then, the first buffer solution is used to perfuse the liver, which can wash away the ionic detergent remaining in the liver and prevent the harmful properties of the ionic detergent from affecting liver health. Then, the second buffer solution is used to perfuse the liver to achieve a sterilization effect. Therefore, the method of the embodiment of the present application can effectively remove DNA, retain effective extracellular matrix components, and reduce the residual harmful chemicals, so as to obtain high-quality liver decellularized scaffolds.

[0058] In some embodiments, the ionic detergent includes sodium dodecyl sulfate (SDS) solution. Sodium dodecyl sulfate treatment has the advantages of being fast and thorough, and perfusing the liver with sodium dodecyl sulfate solution can separate DNA from protein and effectively remove DNA, thereby facilitating the improvement of DNA removal rate, thereby obtaining the desired liver decellularized scaffold.

[0059] Furthermore, when the perfusion conditions of the sodium dodecyl sulfate (SDS) solution are controlled within an appropriate range, the DNA removal effect can be optimized.

[0060] Sodium dodecyl sulfate itself has certain cytotoxicity, so it is necessary to control the appropriate concentration range as much as possible when using it. In some embodiments, the mass fraction of the sodium dodecyl sulfate solution is 0.1% to 0.5%. Exemplarily, the mass fraction of the sodium dodecyl sulfate solution can be 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% or any value between 0.1% and 0.5%. Under the above-mentioned suitable mass fraction range, it is beneficial to give full play to the role of sodium dodecyl sulfate in removing DNA, and to reduce the amount of sodium dodecyl sulfate used as much as possible to reduce the toxic effects of sodium dodecyl sulfate.

[0061] In some embodiments, the mass fraction of sodium dodecyl sulfate solution is 0.1% to 0.4%, which has a better effect on DNA removal. In some embodiments, the mass fraction of sodium dodecyl sulfate solution is 0.2%, which is conducive to further improving the DNA removal rate.

[0062] In some embodiments, the perfusion time of sodium dodecyl sulfate solution is 2 to 3 hours. Exemplarily, the perfusion time of sodium dodecyl sulfate solution can be 2 hours, 2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3 hours or any value between 2 to 3 hours. In some embodiments, the perfusion volume of sodium dodecyl sulfate solution is 800 to 2000 mL. Exemplarily, the perfusion volume of sodium dodecyl sulfate solution can be 800 mL, 1000 mL, 1200 mL, 1400 mL, 1600 mL, 1800 mL, 2000 mL or any value between 800 and 2000 mL. By controlling the perfusion time and perfusion volume in the above-mentioned appropriate ranges, sodium dodecyl sulfate solution can be used to fully remove DNA from sodium dodecyl sulfate, so as to obtain a liver decellularized scaffold with a high DNA removal rate.

[0063] In some embodiments, the first buffer comprises PBS buffer containing ethylenediaminetetraacetic acid (EDTA).

[0064] In some embodiments, the mass fraction of EDTA contained in the first buffer is 0.01% to 0.1%. For example, the mass fraction of EDTA contained in the first buffer may be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% or any value between 0.01% and 0.1%. Under the above appropriate mass fraction, it is beneficial to improve the effect of the first buffer used in removing residual blood and residual ionic detergent during perfusion.

[0065] Preferably, in some embodiments, the mass fraction of EDTA contained in the first buffer solution is 0.05%, which is beneficial to further improve the effect of the first buffer solution.

[0066] In some embodiments, the perfusion time of using the first buffer to perfuse the liver through the portal vein to remove residual blood is 10 to 30 minutes. Exemplarily, the perfusion time of using the first buffer to perfuse the liver through the portal vein to remove residual blood can be 10 minutes, 12 minutes, 14 minutes, 15 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 25 minutes, 26 minutes, 28 minutes, 30 minutes or any value between 10 and 30 minutes. Under the above-mentioned appropriate perfusion time, perfusing the liver with the first buffer can fully remove residual blood, avoid affecting the effects of cell lysis and DNA removal in subsequent steps, thereby facilitating the preparation of the target liver decellularized scaffold.

[0067] In some embodiments, the perfusion time of using the first buffer to perfuse the liver to wash away the residual ionic detergent is 2 to 3 hours. Exemplarily, the perfusion time of using the first buffer to perfuse the liver to wash away the residual ionic detergent can be 2 hours, 2.0 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3 hours or any value between 2 and 3 hours. Under the above-mentioned appropriate perfusion time, using the first buffer to perfuse the liver can fully remove the residual ionic detergent in the above steps, avoid the ionic detergent's own characteristics such as cytotoxicity from causing contamination to the liver, and remove the negative effects of the ionic detergent on the liver decellularized scaffold to the greatest extent, thereby facilitating the preparation of the target liver decellularized scaffold.

[0068] In some embodiments, the non-ionic detergent includes TritonX-100 solution. The TritonX-100 solution can be used to lyse cells, thereby facilitating subsequent operations such as DNA removal.

[0069] In some embodiments, the mass fraction of TritonX-100 solution is 1% to 4%. Exemplarily, the mass fraction of TritonX-100 solution can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or any value between 1% and 4%. In some embodiments, the perfusion time of TritonX-100 solution is 2 to 3h. Exemplarily, the perfusion time of TritonX-100 solution can be 2h, 2.0h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h or any value between 2 and 3h. By controlling the mass fraction and perfusion time of TritonX-100 solution in the above-mentioned suitable ranges, the effect of TritonX-100 solution can be fully exerted, thereby effectively lysing cells, so as to obtain a liver decellularized scaffold with high DNA removal rate and high sterilization rate.

[0070] In some embodiments, the second buffer includes a PBS buffer containing antibiotics, which can be used to effectively sterilize the liver, thereby facilitating the preparation of high-quality decellularized liver scaffolds.

[0071] In some embodiments, the antibiotics include at least one of penicillin-streptomycin and gentamicin. The selection of appropriate antibiotics is helpful in improving the sterilization effect.

[0072] In some embodiments, the perfusion time of the PBS buffer containing antibiotics is 5 to 10 minutes. Exemplarily, the perfusion time of the PBS buffer containing antibiotics can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes or any value between 5 and 10 minutes. Under the above-mentioned appropriate perfusion time, perfusion using the PBS buffer containing antibiotics can further improve the sterilization effect.

[0073] Another embodiment of the present application provides a liver decellularized scaffold, which is prepared by the preparation method of the liver decellularized scaffold of any of the above embodiments. The obtained liver decellularized scaffold has a high DNA removal rate, greatly retains effective extracellular matrix components, and has little residual harmful chemical agents, and has good application prospects.

[0074] Reference Figure 2 As shown, another embodiment of the present application provides a method for preparing a hydrogel based on a liver decellularized scaffold, comprising the following steps:

[0075] S10, pre-treating the decellularized liver scaffold of any of the above embodiments to obtain a powder;

[0076] S20, digesting the powder with a digestive enzyme to obtain a pre-gel;

[0077] S30, mixing the pregel with the photosensitive material to obtain a hydrogel.

[0078] The liver decellularized scaffold hydrogel prepared through the above steps in the embodiment of the present application is a photosensitive material, which can support the survival of organoids in vitro and can quickly coagulate at the wound in vivo to exert hemostasis and tissue repair effects. It can be widely used in tissue repair, tissue engineering, organoid culture and other fields, and has good results.

[0079] In some embodiments, the pretreatment comprises freeze-drying the liver decellularized scaffold and grinding it to obtain a powder. In some embodiments, the digestive enzyme comprises pepsin. In some embodiments, the digestion time is 2 to 3 days. In some embodiments, the photosensitive material comprises at least one of methacrylated gelatin (GelMA), methacrylated silk fibroin, and methacrylated hyaluronic acid.

[0080] By selecting the above-mentioned appropriate preparation conditions respectively, for example, first freeze-drying the liver decellularized scaffold and grinding it into powder, then using pepsin to digest the powder for 2 to 3 days to obtain pre-gel, and then mixing GelMA with the pre-gel to obtain the liver decellularized scaffold hydrogel, this is conducive to improving the effect of the obtained hydrogel.

[0081] Another embodiment of the present application provides an application of a hydrogel based on a liver decellularized scaffold in tissue repair and organoid culture.

[0082] The following examples describe the disclosure of the present application in more detail, and these examples are only used for illustrative purposes, because it is obvious to those skilled in the art that various modifications and variations are made within the scope of the disclosure of the present application. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on mass, and all reagents and raw materials used in the examples are commercially available or synthesized according to conventional methods, and the instruments used in the examples are commercially available.

[0083] Example 1

[0084] This embodiment provides a method for preparing a liver decellularized scaffold and a hydrogel, comprising the following steps:

[0085] Liver decellularized scaffold: After anesthetizing the rat, the laparotomy was performed to expose the liver; the portal vein was cannulated; PBS buffer containing 0.05% EDTA was used for perfusion for 10 min to remove residual blood; 1% TritonX-100 solution was used for perfusion for 2 h; 0.2% SDS solution was used for perfusion for 2 h, and the status was recorded when the perfusion volume reached 400 mL, 800 mL, 1200 mL, 1600 mL, and 2000 mL, respectively. Figure 4; Use PBS buffer containing 0.05% EDTA for perfusion for 2-3 hours to wash away the residual SDS solution; Use PBS buffer containing penicillin-streptomycin for perfusion for 10 minutes to prepare the liver decellularized scaffold.

[0086] Hydrogel: The obtained liver decellularized scaffold was freeze-dried and ground into powder; the powder was digested with pepsin for 3 days to obtain pre-gel; and GelMA was mixed with the pre-gel to obtain the liver decellularized scaffold hydrogel.

[0087] Example 2

[0088] The difference between this embodiment and embodiment 1 is that the concentration of the SDS solution is 0.4%.

[0089] Example 3

[0090] The difference between this embodiment and embodiment 1 is that the concentration of the SDS solution is 0.3%.

[0091] Example 4

[0092] The difference between this embodiment and embodiment 1 is that the concentration of the SDS solution is 0.1%.

[0093] Comparative Example 1

[0094] The difference from Example 1 is that a 1% TritonX-100 solution, 0.2 mg / mL DNase and 0.2 mg / mL RNase are used to prepare the rat liver decellularized scaffold, which is denoted as DnaseI in the figure.

[0095] Test Section

[0096] 1. HE (hematoxylin-eosin staining) staining test:

[0097] 1. Dewax the paraffin sections to water: put the paraffin sections into xylene (Ⅰ) for 20 min → xylene (Ⅱ) for 20 min → anhydrous ethanol (Ⅰ) for 5 min → anhydrous ethanol (Ⅱ) for 5 min → 75% alcohol for 5 min for gradient dewaxing → wash with water.

[0098] 2. Hematoxylin staining: Stain the sections with hematoxylin for 3-5 minutes, wash off the excess stain with tap water, differentiate with 1% hydrochloric acid aqueous solution for a few seconds, rinse with tap water, then with 0.6%-0.7% ammonia aqueous solution to blue, and rinse with running water for a few seconds.

[0099] 3. Eosin staining: Dehydrate the paraffin sections with gradient alcohol (85% ethanol → 95% ethanol) and stain them in eosin staining solution for 5 minutes.

[0100] 4. Dehydration and sealing: Put the paraffin sections into anhydrous ethanol (Ⅰ) for 5 min → anhydrous ethanol (Ⅱ) for 5 min → anhydrous ethanol (Ⅲ) for 5 min → n-butanol for 5 min → xylene (Ⅰ) for 5 min → xylene (Ⅱ) for 5 min to make them transparent. Take the sections out of xylene, dry them slightly, and seal them with neutral gum.

[0101] 2. Immunofluorescence staining (collagen I, DAPI) test:

[0102] 1. Dewax the paraffin sections to water: put the sections into xylene (I) for 15 min → xylene (II) for 15 min → anhydrous ethanol (I) for 5 min → anhydrous ethanol (II) for 5 min → 85% alcohol for 5 min → 75% alcohol for 5 min → wash with distilled water.

[0103] 2. Antigen repair: Place the slides in a repair box filled with EDTA antigen repair buffer (pH 8.0) in a microwave oven for antigen repair. Heat on medium heat for 8 minutes, stop for 8 minutes, and switch to medium-low heat for 7 minutes. After cooling naturally, place the slides in PBS (pH 7.4) and wash them on a decolorizing shaker 3 times, 5 minutes each time.

[0104] 3. Quenching of autofluorescence by drawing circles: After the slices are slightly dried, use a histochemical pen to draw circles around the tissue (to prevent the antibody from flowing away), add autofluorescence quencher in the circle for 5 minutes, and rinse with running water for 10 minutes.

[0105] 4. Serum blocking: Add bovine serum albumin (BSA) into the circle and incubate for 30 minutes.

[0106] 5. Add primary antibody: Gently shake off the blocking solution, add collagen I primary antibody prepared in a certain proportion with PBS on the slices, and lay the slices flat in a humidified box and incubate at 4°C overnight.

[0107] 6. Add secondary antibody: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody of the same species as the primary antibody to cover the tissue in the circle and incubate at room temperature for 50 minutes away from light.

[0108] 7. DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add DAPI (4',6-diamidino-2-phenylindole) staining solution in the circle and incubate at room temperature for 10 minutes away from light.

[0109] 8. Sealing: Place the slides in PBS (pH 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time. After the sections are dried slightly, seal them with anti-fluorescence quenching sealing agent and store them in a light-proof section box at 4 degrees.

[0110] 3. Scanning electron microscope (SEM):

[0111] 1. Sampling: Cut the extracted decellularized scaffold so that the observation surface is as flat as possible, with a size of 2-3 mm in thickness and 5-6 mm in length and width.

[0112] 2. Fixation: Rinse the excised tissue several times, centrifuge to remove the supernatant, add 4°C pre-cooled 2.5-3% glutaraldehyde, fix at 4°C overnight, aspirate the fixative, and rinse with PBS 3-5 times, 15 minutes each time.

[0113] 3. Dehydration: Dehydrate with a series of gradient alcohols (30%, 50%, 70%, 80%, 90%, 95%, 100%, each concentration of alcohol is passed once, each time for 10-20 minutes, and then use 100% alcohol to thoroughly dehydrate 1-2 times, and then use banana water to treat twice, each time for 20 minutes.

[0114] 4. Drying: Use critical drying method to dry the sample.

[0115] 5. Conductive treatment of samples: Use vacuum spraying method to evenly treat the samples and observe them under a microscope after completion.

[0116] 4. Collagen detection method:

[0117] 1. Digest 10 mg of freeze-dried decellularized scaffolds with 6M hydrochloric acid at 95°C for 20 hours. Treat collagen standards with 12M hydrochloric acid under the same conditions.

[0118] 2. Centrifuge at 13,000 g for 10 minutes and take the supernatant. Use QuickZyme's collagen detection kit to detect the collagen content.

[0119] 5. DNA testing method:

[0120] 1. Use ABclonal's DNA extraction kit to extract DNA from 10 mg of freeze-dried decellularized scaffold according to the method provided.

[0121] 2. Use Nanodrop2000 to detect DNA concentration.

[0122] The liver blank control group Liver was tested by the following test method: Figures 3 to 9 .

[0123] Reference Figure 4 , Figure 4The perfusion process diagram of the liver decellularized scaffold provided for the embodiment and the comparative example shows the perfusion process of the decellularized scaffold under different conditions of the embodiment of the present application, the enzymatic hydrolysis treatment conditions of the comparative example, and the liver blank control group. It can be seen from the comparison that the decellularization efficiency of the embodiment of the present application is higher than that of the enzymatic hydrolysis method, and the decellularization efficiency is higher when the SDS concentration is 0.2% or more, and the decellularization effect is better when the SDS perfusion volume is 800-2000mL.

[0124] Reference Figure 5 , Figure 5 The histopathological staining of the decellularized scaffolds provided in the examples and comparative examples of the present application shows a comparison of the staining results of the decellularized scaffolds obtained under different conditions. Comparison of the HE staining results shows that the cell structure can be completely removed by treatment under the conditions of the examples of the present application, while the enzymatic method has more residues; comparison of the immunofluorescence staining results shows that the collagen distribution obtained by different treatments is similar, and no nuclear structure is observed by DAPI staining.

[0125] Reference Figure 6 , Figure 6 The scanning electron microscopy results of the decellularized scaffolds provided in the examples and comparative examples of the present application, wherein A shows the scanning electron microscopy results of the decellularized scaffold obtained in Example 1, B shows the scanning electron microscopy results of the decellularized scaffold obtained in Comparative Example 1, and C shows the scanning electron microscopy results of normal liver. It can be seen that the decellularized scaffold obtained under the conditions of the examples of the present application retains the microstructure compared with the normal liver, and the cell structure is completely removed, while more cell structure residues can be seen in the enzymatic method.

[0126] Reference Figure 7 , Figure 7 Partial component detection of the decellularized scaffolds provided in the examples and comparative examples of the present application, wherein the comparison of collagen and DNA detection results of the decellularized scaffolds obtained under different conditions is shown. Comparison of the collagen content detection results shows that the decellularized scaffolds obtained under the conditions of the examples of the present application contain a higher collagen component, and the collagen content can reach the highest content under the conditions of Example 1. Comparison of the DNA residue analysis results shows that decellularization using the method of the examples of the present application can remove more than 90% of DNA, while enzymatic hydrolysis is difficult to effectively remove DNA.

[0127] Reference Figure 8 , Figure 8 This is a diagram showing the coagulation and hemostasis effect of the decellularized scaffold hydrogel of Example 1 of the present application in a liver wound, which shows an application example of the obtained hydrogel for wound hemostasis and the integration of the implant with the liver, and it can be seen that the effect is good.

[0128] Reference Fig. 9 , Fig. 9This is the integration of the obtained hydrogel implant with the liver after it is used for wound hemostasis. It can be seen that the decellularized scaffold hydrogel of Example 1 of the present application is well integrated with the liver tissue and has a good effect in promoting wound repair.

[0129] Reference Figures 3 to 9 From the comparison of the effects of Examples 1 to 4 with the comparative example, it can be seen that the liver decellularized scaffold preparation method provided by the embodiment of the present application uses a specific concentration of SDS solution, which can ensure the DNA removal rate of the decellularized scaffold while retaining the effective extracellular matrix components to a great extent and reducing the residual harmful chemical agents. The obtained liver decellularized scaffold can be applied to the field of tissue engineering, and after freeze-drying and digestion into gel, it can be used to support 3D organoid culture in vitro, and can be used in vivo to quickly coagulate at the wound to exert hemostasis and tissue repair effects.

[0130] The various technical features described above can be combined arbitrarily. Although all possible combinations of these technical features are not described, any combination of these technical features should be considered to be covered by this specification as long as there is no contradiction in such combination.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a liver decellularized scaffold, characterized in that: The method comprises sequentially using a first buffer, a non-ionic detergent, an ionic detergent and a second buffer to perfuse a liver decellularized scaffold to obtain the decellularized scaffold.

2. The method for preparing a decellularized liver scaffold according to claim 1, characterized in that: The following steps are involved: Obtaining biological liver; perfusing the liver via the portal vein with a first buffer to remove residual blood; Then, the liver is perfused with a non-ionic detergent to lyse the cells; The liver was then perfused with an ionic detergent to remove DNA; The liver is then perfused with the first buffer to wash away residual ionic detergent; Then, the liver is perfused with a second buffer to sterilize the liver, thereby obtaining a decellularized liver scaffold.

3. The method for preparing a decellularized liver scaffold according to claim 1 or 2, characterized in that: The ionic detergent includes sodium dodecyl sulfate solution; Optionally, the mass fraction of the sodium dodecyl sulfate solution is 0.1% to 0.5%; Optionally, the mass fraction of the sodium dodecyl sulfate solution is 0.1% to 0.4%; Optionally, the mass fraction of the sodium dodecyl sulfate solution is 0.2%; Optionally, the perfusion time of the sodium dodecyl sulfate solution is 2 to 3 hours; Optionally, the infusion volume of the sodium dodecyl sulfate solution is 800-2000 mL.

4. The method for preparing a decellularized liver scaffold according to claim 1 or 2, characterized in that: The first buffer comprises a PBS buffer containing ethylenediaminetetraacetic acid; Optionally, the mass fraction of the ethylenediaminetetraacetic acid contained in the first buffer is 0.01% to 0.1%, preferably 0.05%; Optionally, the perfusion time of using the first buffer to perfuse the liver via the portal vein to remove residual blood is 10 to 30 minutes; Optionally, the perfusion time of perfusing the liver with the first buffer to wash away the residual ionic detergent is 2-3 hours.

5. The method for preparing a decellularized liver scaffold according to claim 1 or 2, characterized in that: The non-ionic detergent includes TritonX-100 solution; Optionally, the mass fraction of the TritonX-100 solution is 1% to 4%; Optionally, the perfusion time of the TritonX-100 solution is 2 to 3 hours.

6. The method for preparing a decellularized liver scaffold according to claim 1 or 2, characterized in that: The second buffer comprises a PBS buffer containing antibiotics; Optionally, the antibiotics include at least one of penicillin-streptomycin and gentamicin; Optionally, the perfusion time using the PBS buffer containing antibiotics is 5-10 min.

7. A liver decellularized scaffold, characterized in that: The decellularized liver scaffold is prepared by the preparation method of any one of claims 1 to 6.

8. A method for preparing a hydrogel based on a liver decellularized scaffold, characterized in that: The following steps are involved: Pre-treating the liver decellularized scaffold to obtain a powder; digesting the powder using digestive enzymes to obtain pre-gel; The pregel is mixed with a photosensitive material to obtain a hydrogel.

9. The method for preparing a hydrogel based on a decellularized liver scaffold according to claim 8, characterized in that: The pretreatment includes freeze-drying the liver decellularized scaffold and grinding it to obtain a powder; Optionally, the digestive enzyme comprises pepsin; Optionally, the digestion time is 2 to 3 days; Optionally, the photosensitive material includes at least one of methacrylated gelatin, methacrylated silk fibroin, and methacrylated hyaluronic acid.

10. Application of a hydrogel based on liver decellularized scaffold in tissue repair and organoid culture.

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