Acellular amnion matrix hydrogel and preparation method thereof

Abstract

The invention provides a decellularized amnion matrix hydrogel and a preparation method thereof. The preparation method comprises the following steps: amnion tissue pretreatment, cleaning, decellularization, grinding, freeze-drying, digestion and acid-base neutralization. According to the decellularized amnion matrix hydrogel, active cells are removed while collagen, fibronectin, glycoprotein and other components in an amnion cell matrix are reserved, and the immunogenicity of the decellularized amnion matrix hydrogel is reduced from the source. The decellularized amniotic membrane matrix hydrogel provided by the invention can simulate the microenvironment of in-vivo cells, provides support and signals required by cell adhesion and growth, can be used for constructing a tissue engineering scaffold, and provides support for directional growth and tissue regeneration of cells. Besides, the decellularized amniotic membrane matrix hydrogel can also be used as a drug carrier for in-vivo drug release and treatment, and has very high application potential in medical research and clinical application.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a decellularized amniotic membrane matrix hydrogel and a preparation method thereof. Background Art

[0002] All-synthetic hydrogels have recently been widely used in three-dimensional cell culture, including organoids, because of their stability and structural uniformity. However, all-synthetic materials often lack the cytokines required for cell growth and have poor biocompatibility. Therefore, the cell culture effect of all-synthetic hydrogels is often far inferior to that of biological matrix hydrogels.

[0003] Matrix hydrogel is a comprehensive complex composed of soluble extracellular matrix, and its basic components include collagen, laminin and nestin. Matrix hydrogel plays an important role in biological experiments and has a wide range of applications. First, it can be used to treat cell culture plates, which helps cell attachment and growth. Second, it can be laid in Transwell chambers to study the invasion ability of tumor cells. In addition, it can also be used for three-dimensional culture of cell lines or patient-derived cells to form organoid structures and provide models for anti-tumor drug screening. At present, EHS (Engelbreth-Holm-Swarm) matrix hydrogel is one of the main types, and the matrix hydrogel is mainly extracted from EHS cells. However, EHS cells themselves cannot be cultured in vitro, which limits their expansion scale in production and poses great challenges to the mass production of matrix hydrogel. In addition, the preparation of EHS matrix hydrogel requires osteosarcoma to be inoculated in mice, and this tumor-derived characteristic seriously restricts the application prospects of its related cell therapy in clinical practice.

[0004] The amniotic membrane is a natural thin membrane surrounding the fetus and is one of the important tissues formed during embryonic development. It is rich in collagen, elastic fibers, hyaluronic acid and other components, has good biocompatibility and bioactivity, and can be used for tissue repair in fields such as corneal transplantation and skin transplantation. It is an ideal source of extracellular matrix. Summary of the invention

[0005] In view of this, the present invention provides a method for preparing a decellularized amniotic membrane matrix hydrogel, comprising the following steps:

[0006] (1) Pretreatment and decellularization of amniotic membrane tissue: fresh amniotic membrane is placed in liquid nitrogen for freezing, then taken out and thawed, and repeated 3-5 times to obtain decellularized amniotic membrane;

[0007] (2) placing the decellularized amniotic membrane in step (1) into liquid nitrogen for freezing again, taking it out, grinding it, and sieving it in sequence to obtain decellularized amniotic membrane powder;

[0008] (3) freeze-drying the decellularized amniotic membrane powder of step (2) to obtain freeze-dried decellularized amniotic membrane powder;

[0009] (4) Mix the freeze-dried decellularized amniotic membrane powder described in step (3) with pepsin, and then add it to a 0.01M hydrochloric acid solution for digestion. After the digestion is completed, lower the ambient temperature to 0-4°C, and then adjust the pH of the system to neutral to obtain a mixed solution. Finally, add PBS buffer solution to the mixed solution and let it stand to obtain a decellularized amniotic membrane matrix hydrogel.

[0010] Furthermore, the freezing time in step (1) is 10-15 minutes, and the melting temperature is 37-40°C.

[0011] The present invention freezes cells to form ice particles inside the cells, and then melts them at room temperature (or 40° C.), so that the salt concentration of the remaining cell fluid increases after freezing, causing the cells to swell and break, thereby reducing the amount of reagents used.

[0012] Furthermore, the amniotic tissue pretreatment in step (1) is specifically as follows: fresh amniotic tissue is collected from postpartum women aged 25 to 35 years old who have been healthy and have negative results in serological screening for infectious diseases, and the tissue is cleaned with sterile saline, the chorion is removed, and the tissue is cut into small pieces of 2*2 cm; the tissue is placed in sterile saline at 0-4°C and rinsed at a rotation speed of 150-200 rpm for 30-35 minutes, and then rinsed with deionized water at 0-4°C at a rotation speed of 150-200 rpm for 30-35 minutes.

[0013] Furthermore, the sieving in step (2) is through a 200-mesh sieve.

[0014] Furthermore, the freeze-drying temperature in step (3) is -70 to -80°C, and the freeze-drying time is 36 to 48 hours.

[0015] Furthermore, in step (4), the mass ratio of the freeze-dried decellularized amniotic membrane powder to pepsin is 10-14:3.

[0016] Furthermore, the amount of the PBS buffer solution in step (4) is 9-10 times that of the mixed solution.

[0017] Furthermore, the present invention also provides the decellularized amniotic membrane matrix hydrogel prepared by the method, and the application of the decellularized amniotic membrane matrix hydrogel in the fields of cell culture, tissue engineering, and drug delivery system.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention prepares decellularized amniotic matrix hydrogel by introducing decellularization technology and hydrogel preparation technology. The decellularized amniotic matrix hydrogel removes active cells while retaining the components such as collagen, fibronectin, glycoprotein in the amniotic cell matrix, thereby reducing its immunogenicity from the source. In cell culture, the microenvironment in which cells in the body are located can be simulated, and the support and signals required for cell adhesion and growth can be provided, which is helpful for the in vitro culture and expansion of cells. In tissue engineering, the decellularized amniotic matrix hydrogel can be used to construct a tissue engineering scaffold to provide support for the directional growth of cells and tissue regeneration. In addition, the decellularized amniotic matrix hydrogel can also be used as a drug carrier for in vivo drug release and treatment. Due to its good biocompatibility and biological activity, the decellularized amniotic matrix hydrogel has high potential in medical research and clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison chart of the effects of the decellularized amniotic membrane (dAM) prepared in Example 1 of the present invention and the fresh amniotic membrane (fAM) prepared in Comparative Example 1.

[0021] Figure 2 It is a comparison diagram of the effects of the decellularized amniotic matrix hydrogel prepared in Examples 1 and 2 of the present invention and the Matrigel hydrogel prepared in Comparative Example 2.

[0022] Figure 3 It is an electron microscopic comparison picture of the decellularized amniotic matrix hydrogel prepared in Example 1 of the present invention and the Matrigel hydrogel prepared in Comparative Example 2. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0024] The experimental methods or test methods described in the following examples are conventional methods unless otherwise specified; the raw materials and auxiliary agents are obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0025] The test method according to the present invention is as follows:

[0026] 1. DNA content determination

[0027] 1. Take 20 mg of sample, freeze it in liquid nitrogen, and grind or mash it;

[0028] 2. After sample processing, add 5 μl of proteinase K to every 1 ml of sample lysis buffer and mix well;

[0029] 3. For the above treated samples, add 500 μL of sample lysis solution with proteinase K to 20 mg of tissue, invert and mix several times to fully lyse the tissue or cells;

[0030] 4. Digest in 50℃ water bath overnight;

[0031] 5. Add 500 μl Tris-equilibrated phenol to extract the sample;

[0032] 6. Aspirate the phenol phase and the middle phase (a small amount of aqueous phase liquid close to the middle phase can be aspirated), and extract the remaining aqueous phase again with an equal volume of Tris-equilibrated phenol;

[0033] 7. Aspirate the phenol phase and the middle phase (a small amount of aqueous phase liquid close to the middle phase can be aspirated), and extract the remaining aqueous phase again with an equal volume of chloroform;

[0034] 8. Aspirate about 300 μl of supernatant, add 60 μl of 10 M ammonium acetate and 600 μl of anhydrous ethanol, invert several times to mix, and DNA precipitation can be seen;

[0035] 9. Centrifuge at 20,000 rpm for 1 minute and discard the supernatant; add 70% ethanol to wash the DNA precipitate twice;

[0036] 10. Remove as much residual ethanol as possible, and when no obvious liquid is visible, immediately add 50-100 μl of ultrapure water to dissolve the DNA;

[0037] 11. Use an ultra-micro spectrophotometer to measure the concentration of DNA at a wavelength of 280 nm.

[0038] 2. Determination of collagen content;

[0039] Hydroxyproline (HYP) is one of the main components of collagen in the body. Collagen is mostly distributed in the skin, tendons, cartilage and blood vessels, so the HYP content is an important indicator reflecting the metabolism of collagen tissue and the degree of fibrosis. The sample is hydrolyzed by acid to produce free HYP, which is further oxidized by chloramine T. The oxidation product reacts with p-dimethylaminobenzaldehyde to produce a red compound with a characteristic absorption peak at 560nm. The HYP content can be calculated by measuring the absorbance of the sample hydrolyzate at 560nm.

[0040] extract:

[0041] Weigh about 0.2g of sample into a glass tube, cut the tissue into small pieces as much as possible for digestion, and keep the lid loose and not airtight; add 2mL of extraction solution (6mol / L hydrochloric acid solution), boil or place in a 110℃ oven for 2 to 6 hours to digest until no large lumps are visible (wrap the sealing film to prevent the lid from bursting), and after cooling, adjust the pH value to the range of 6-8 with NaOH solution (about 1mL of 10mol / L), and then make up to 4mL with distilled water; finally, centrifuge at 16000rpm, 25℃ for 20min, and take the supernatant for testing.

[0042] Determination:

[0043] 1. Preheat the spectrophotometer for more than 30 minutes, adjust the wavelength to 560nm, and use distilled water to zero;

[0044] 2. Dilute 0.5 mg / mL hydroxyproline standard (0.5 mg / mL hydroxyproline standard) with distilled water to 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.234, 0.117 μg / mL standard.

[0045] 3. Divide into 3 groups, namely blank tube, assay tube and standard tube; first, add 200μl chloramine T to the blank tube, add 200μl test sample and 200μl chloramine T to the assay tube, add 200μl standard and 200μl chloramine T to the standard tube, mix well and let stand at 25℃ for 20 minutes; then add 200μl dimethylaminobenzaldehyde and 600μl distilled water to the blank tube, add 200μl dimethylaminobenzaldehyde and 400μl distilled water to the assay tube, and add 200μl dimethylaminobenzaldehyde and 400μl distilled water to the standard tube;

[0046] Mix the above, place in a 60℃ water bath for 20min, take out and let stand at room temperature for 15min, use a 1mL glass cuvette to measure the absorbance of the blank tube, the test tube, and the standard tube at 560nm, record it as A 空白管 , A 测定管 , A 标准管 , calculate ΔA determination = A 测定管 -A 空白管 , ΔA standard = A 标准管 -A 空白管 ;

[0047] 4. Plotting of standard curve: Plotting the standard curve with the concentration of the standard as the x-axis and the ΔA standard as the y-axis to obtain the equation y=kx+b; Substitute the ΔA determination into the equation to obtain x (μg / mL);

[0048] 5. Calculate using the following formula: Tissue hydroxyproline content (μg / g mass) = x × V 样本 ÷(W×V样本 ÷V 组提 )×F=4x÷W×F;

[0049] Among them, V 样本 : added sample volume, 0.2mL; V 组提 : Volume of tissue extract, 4mL; V 胞提 : Volume of cell extract, 2mL; V 液提 : Liquid extract volume, 2mL; V 液体 : Volume of liquid added in pretreatment, 0.3 mL; W: sample mass, g.

[0050] 3. Glycosaminoglycan (GAG) determination

[0051] Glycosaminoglycan (GAG), also known as mucopolysaccharide, is the most abundant heteropolysaccharide in the body. GAG is an unbranched, negatively charged long-chain polysaccharide composed of repeated disaccharide units, including hexose or hexuronic acid, linked to hexoamine. GAG is covalently linked to core protein to form proteoglycan (PG), which is the main component of cell membrane and extracellular matrix (ECM). GAG in proteoglycan undergoes sulfated group during synthesis, and its location is at O ​​or N position, which is conducive to various functions, including enzyme regulation, cell adhesion, growth, migration, differentiation, and tissue damage response. Based on the highly negatively charged characteristics of GAG, the metachromatic cationic blue dye 1,9-dimethylmethylene blue (DMB) was used to detect the presence of GAG in proteoglycan. blue; DMMB), under acidic conditions, specifically binds to produce an insoluble purple or pink glycosaminoglycan dye complex (Dye-GAG complex), and releases the dye in the dissociation solution, which is measured by an enzyme reader (530nm wavelength) to quantitatively analyze the total content of glycosaminoglycans.

[0052] 1. Preparation of DMMB reagent

[0053] 1. Add 16 mg DMMB and 5 mL ethanol into a beaker and stir. Wrap with tin foil to avoid light.

[0054] 2. Add 3.04 g glycine, 2.37 g NaCl and 95 mL 0.1 N HCl;

[0055] 3. Add 800 mL of distilled water and adjust the pH to 3.0 with 0.1 N HCl;

[0056] 4. Add distilled water to make up to 1000mL;

[0057] 5. Stir with a magnetic stirrer at room temperature for 2h to 16h, away from light;

[0058] 6. Use 20μm to 25μm filter paper to remove impurities.

[0059] 2. Determination of samples

[0060] 1. Add 10.0 mg of 6-sulfate chondroitin (purity> 95%) to 20 mL of extract to prepare a 0.5 mg / mL 6-sulfate chondroitin solution, and stir for several minutes to completely dissolve it;

[0061] 2. Use the extract to prepare a series of 6-sulfate chondroitin solutions, namely 0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL;

[0062] 3. Turn on the ELISA reader;

[0063] 4. Add 0.02 mL of the extracted sample or 6-sulfate chondroitin standard to 0.2 mL of DMMB solution, mix well, and measure the absorbance at 530 nm.

[0064] 3. Normalization of GAG content

[0065] 1) Convert the absorbance to GAG concentration (μg / mL); then, multiply it by the volume of digestion solution added to each sample and the dilution factor to obtain the sGAG content (μg) in the sample;

[0066] 2) Divide the sGAG content (μg) by the wet weight of the parallel test samples to normalize it to the unit mass sGAG content (μg / mg); sGAG (μg / mg) = A×B×C / D, where,

[0067] A——GAG concentration, μg / mL;

[0068] B——dilution multiple;

[0069] C——test sample volume (volume of extract + volume of parallel samples), mL;

[0070] D——wet weight of parallel test specimens, mg.

[0071] IV. LNC culture method and proliferation method determination

[0072] 1. Get

[0073] Select specimens with intact cornea and limbal tissues and cut them radially into equal pieces of limbal tissue;

[0074] 2. Cultivation

[0075] The above-mentioned corneal limbal fragments were digested with 1 ml collagenase A solution at a temperature of 37° C. and a volume fraction of 5% CO2 for 8.0 to 10.0 h, and then digested with 1 ml trypsin solution for 10 min, and then inoculated on a gel-coated six-well plate (decellularized amniotic membrane hydrogel / Matrigel and culture medium (modified embryonic SC medium MESCM) were mixed on ice at a ratio of 5% and placed in an incubator to complete gel coating) to obtain primary corneal limbal microenvironment cells, and the fourth-generation corneal limbal microenvironment cells were obtained by continuous passage; wherein the trypsin solution is a PBS solution containing 0.25% Trypsin and EDTA (pH value is 7.2-7.4), and the digestion time is 10 to 20 min; the mass fraction of trypsin Trypsin is 0.25%; the mass fraction of EDTA is: 0.02%;

[0076] The configuration method of modified embryonic SC medium MESCM is as follows:

[0077]

[0078]

[0079] 3. Vaccination

[0080] Finally, the fourth-generation LNCs were inoculated at 3000 cells / 100 μL per well, and 5 replicates were set for each concentration. At the same time, blank wells (100 μL MESCM) were set up, and the cell density was observed every 12 hours. Three 96-well plates were seeded at the same time, and PBS was added around the experimental area to prevent the culture medium in the experimental area from evaporating in the incubator.

[0081] 4. Detection

[0082] On the 1st, 3rd and 5th days, a mixed solution (10 μL CCK-8 solution + 90 μL MESCM) was added to each well by changing the solution, and the bubbles were punctured with a sterile needle after burning. The cells were incubated in a 37°C incubator in the dark for 3 hours, and the absorbance at 450 nm was measured with an enzyme-labeled instrument.

[0083] Example 1

[0084] A method for preparing a decellularized amniotic membrane matrix hydrogel, the specific steps are as follows:

[0085] (1) Pretreatment of amniotic membrane tissue:

[0086] Fresh amniotic membrane tissue was collected from postpartum women aged 25 to 35 years old who were healthy and had negative serological screening for infectious diseases. The tissue was washed with sterile saline, the chorionic villi were removed, and the tissue was cut into small pieces of 2*2 cm.

[0087] (2) Cleaning:

[0088] The fresh amniotic membrane was placed in 4°C physiological saline and rinsed at a rotation speed of 150 rpm for 30 minutes, and then rinsed with 4°C deionized water at a rotation speed of 150 rpm for 30 minutes;

[0089] (3) Decellularization:

[0090] The cleaned amniotic membrane was frozen in liquid nitrogen for 10 minutes, then thawed in a 37°C water bath for 10 minutes, repeated three times, and washed again with sterile saline to obtain the decellularized amniotic membrane;

[0091] (4) Grinding:

[0092] The decellularized amniotic membrane was placed in liquid nitrogen again for freezing for 10 minutes, taken out and ground in a grinding mortar, and the ground amniotic membrane was passed through a 200-mesh sieve to obtain decellularized amniotic membrane powder;

[0093] (5) Freeze-drying:

[0094] The decellularized amniotic membrane powder is placed in a freeze dryer and freeze-dried at -80°C for 48 hours to obtain freeze-dried decellularized amniotic membrane powder;

[0095] (6) Digestion:

[0096] In an environment of 25°C, freeze-dried decellularized amniotic membrane powder (14 mg) and pepsin (1:3000, 3 mg) were added to 0.01 M HCl and the volume was fixed to 1 ml, and the mixture was continuously shaken for 96 hours for digestion to obtain a digested decellularized amniotic membrane solution;

[0097] (7) Acid-base neutralization:

[0098] In an environment of 4° C., 0.1 M NaOH was added to the decellularized amniotic membrane solution until the pH of the solution reached 7, and then 10 times the concentration of PBS was added and the solution was allowed to stand to obtain a decellularized amniotic membrane matrix hydrogel.

[0099] Example 2

[0100] The difference from Example 1 is that the added amount of freeze-dried decellularized amniotic membrane powder is 10 mg, and the other steps are consistent with Example 1.

[0101] Comparative Example 1

[0102] Untreated fresh amniotic membrane.

[0103] Comparative Example 2

[0104] Matrigel hydrogel.

[0105] (1) Figure 1 This is a comparison chart of the effects of the decellularized amniotic membrane (dAM) prepared in Example 1 and the fresh amniotic membrane (fAM) prepared in Comparative Example 1. By detecting the DNA content, collagen content and GAG content of the amniotic membrane before and after treatment, Figure 1 It can be seen that compared with fresh amniotic membrane, the DNA content in the decellularized amniotic membrane is significantly reduced (7.73±0.86μg / mg vs 0.16±0.07μg / mg); most of the collagen (313.00±37.40μg / mg vs 287.10±17.88μg / mg) and glycosaminoglycan GAG (dry weight 8.11±1.52μg / mg vs 6.50±0.59μg / mg) in the decellularized amniotic membrane are retained. ****P<0.001), which shows that the treatment method provided by the present invention can achieve the decellularization effect while retaining most of the effective substances.

[0106] (2) Figure 2 The effect comparison diagram of the decellularized amniotic matrix hydrogel prepared in Example 1 and Example 2 and the Matrigel hydrogel prepared in Comparative Example 2. The biocompatibility of the decellularized amniotic matrix hydrogel was tested by culturing limbal niche cells (LNCs) in a 6-well plastic plate coated with decellularized amniotic matrix hydrogel. Figure 2 It can be seen that the decellularized amniotic membrane matrix hydrogel provided by the present invention has similar biocompatibility as Matrigel hydrogel, and there is no statistically significant difference in the OD values ​​between the three hydrogels within 7 days, indicating that the decellularized amniotic membrane matrix hydrogel provided by the present invention has equivalent ability to Matrigel hydrogel in promoting the proliferation of LNCs, that is, the decellularized amniotic membrane matrix hydrogel provided by the present invention has good biocompatibility and can promote the proliferation of cells in the limbal microenvironment.

[0107] (3) Figure 3 This is an electron microscopic comparison of the decellularized amniotic matrix hydrogel prepared in Example 1 and the Matrigel hydrogel prepared in Comparative Example 2. Figure 3 Scanning electron microscopy images show that the dried decellularized amniotic membrane matrix hydrogel and Matrigel matrix hydrogel have similar highly porous network structures. The pore networks are interconnected, allowing cells to crawl and proliferate, and helping to promote the diffusion of nutrients and oxygen and the removal of waste.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a decellularized amniotic membrane matrix hydrogel, characterized in that: The following steps are involved: (1) Pretreatment and decellularization of amniotic membrane tissue: fresh amniotic membrane is placed in liquid nitrogen for freezing, then taken out and thawed, and repeated 3-5 times to obtain decellularized amniotic membrane; (2) placing the decellularized amniotic membrane in step (1) into liquid nitrogen for freezing again, taking it out, grinding it, and sieving it in sequence to obtain decellularized amniotic membrane powder; (3) freeze-drying the decellularized amniotic membrane powder of step (2) to obtain freeze-dried decellularized amniotic membrane powder; (4) Mix the freeze-dried decellularized amniotic membrane powder described in step (3) with pepsin, and then add it to a 0.01M hydrochloric acid solution for digestion. After the digestion is completed, lower the ambient temperature to 0-4°C, and then adjust the pH of the system to neutral to obtain a mixed solution. Finally, add PBS buffer solution to the mixed solution and let it stand to obtain a decellularized amniotic membrane matrix hydrogel.

2. The method for preparing the decellularized amniotic membrane matrix hydrogel according to claim 1, characterized in that: The freezing time in step (1) is 10 minutes.

3. The method for preparing the decellularized amniotic membrane matrix hydrogel according to claim 1, characterized in that: The melting temperature in step (1) is 37°C.

4. The method for preparing the decellularized amniotic membrane matrix hydrogel according to claim 1, characterized in that: The amniotic tissue pretreatment in step (1) is specifically as follows: fresh amniotic tissue from a 25 to 35-year-old pregnant woman who was previously healthy and had a negative result in serological screening for infectious diseases is collected, cleaned with sterile saline, and the chorion is removed and cut into small pieces of 2*2 cm; the pieces are placed in sterile saline at 0-4°C and rinsed at a speed of 150 rpm for 30 minutes, and then rinsed with deionized water at 0-4°C at a speed of 150 rpm for 30 minutes.

5. The method for preparing the decellularized amniotic membrane matrix hydrogel according to claim 1, characterized in that: The sieving in step (2) is through a 200-mesh sieve.

6. The method for preparing the decellularized amniotic membrane matrix hydrogel according to claim 1, characterized in that: The freeze-drying temperature in step (3) is -70 to -80°C, and the freeze-drying time is 36 to 48 hours.

7. The method for preparing the decellularized amniotic membrane matrix hydrogel according to claim 1, characterized in that: The mass ratio of the freeze-dried decellularized amniotic membrane powder to pepsin in step (4) is 10-14:

3.

8. The method for preparing the decellularized amniotic membrane matrix hydrogel according to claim 1, characterized in that: The amount of the PBS buffer solution in step (4) is 10 times that of the mixed solution.

9. The decellularized amniotic membrane matrix hydrogel prepared according to the method according to any one of claims 1 to 8.

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