Decellularized biological omentum preparation method and decellularized biological omentum prepared using same

Abstract

Disclosed are a decellularized biological omentum preparation method and a decellularized biological omentum prepared using same. The method comprises the following steps: 1) defatting a fresh greater omentum tissue; 2) shaking and soaking the defatted greater omentum tissue in a decellularization solution I; 3) treating the greater omentum tissue treated with the decellularization solution I with a decellularization solution II; 4) treating the greater omentum tissue treated with the decellularization solution II with a decellularization solution III; and 5) freeze-drying and then sterilizing the greater omentum tissue treated with the decellularization solution III. The method of the present invention is simpler to implement, and the period is shorter. Simultaneously using the decellularization solutions I, II, and III, which are of particular formulas, causes the DNA content in the decellularized omentum to be lower.

Description

Decellularized biomesh preparation method and decellularized biomesh prepared therefrom

[0001] The present application claims priority to the Chinese patent application No. 202410815430.2 filed on June 24, 2024, and entitled "Decellularized biomesh preparation method and decellularized biomesh prepared therefrom", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology, and specifically relates to a decellularized biomesh preparation method and a decellularized biomesh prepared therefrom. BACKGROUND

[0003] Decellularized matrix biomaterials are a new type of biomaterials that can be used for tissue damage repair / reconstruction. The decellularized matrix biomaterials are obtained by decellularizing tissues / organs using appropriate methods, removing cells and other antigen molecules that can cause rejection reactions in the tissues, and retaining three-dimensional structures and functional proteins. The decellularized matrix biomaterials have biological induction function and can form specific functional tissues in vivo / in vitro. Currently, the most commonly used decellularized biomaterials are xenogeneic decellularized matrices. The raw materials of such natural biomaterials are mainly derived from animal small intestinal submucosa, bladder submucosa, gastric submucosa, pericardium, amniotic membrane, peritoneum, and dermis.

[0004] The greater omentum is a peritoneal tissue connecting the greater curvature of the stomach to the transverse colon, which bulges forward between the stomach and the intestine, and forms a plica in front of the intestine, resembling a petticoat covering the jejunum and ileum. The greater omentum is widely available, highly vascularized, and rich in various growth factors, and has been widely used as an in vivo transplantation platform for various types of grafts. For example, it can be used to repair inflammatory or damaged tissues in surgical reconstruction surgery, and to promote tissue vascularization and regeneration. Decellularized matrix biomaterials are widely used in the medical field. Compared with non-absorbable materials or traditional polymer materials, they have the ability to induce tissue regeneration, and are therefore considered to be ideal tissue repair materials. However, animal-derived biomaterials also have some risks. On the one hand, they carry exogenous residual DNA, fat, and other immunogenic substances, which may cause an immune response when implanted in the human body. On the other hand, the reagents used in the processing process can easily destroy the three-dimensional structure of the extracellular matrix (ECM), which loses its ability to induce tissue regeneration in the body.

[0005] In view of the above technical problems, Chinese authorized invention patent CN104771788B discloses a tissue engineered skin based on large omental decellularized matrix and a construction method thereof. The construction method of the tissue engineered skin comprises the following steps: (1) preparing a large omental decellularized matrix scaffold material, which comprises the step of decellularizing the large omental by soaking it in a buffer solution containing sodium dodecyl sulfate and DNAase; and (2) tissue engineered skin construction and culture. The tissue engineered skin obtained by the method is also provided. Since the large omental decellularized matrix is rich in pro-angiogenic factors, it can promote the growth of capillaries into the transplanted tissue engineered skin from the wound surface, thereby facilitating the vascularization of the tissue engineered skin and wound healing, which is of great significance for the clinical application of the tissue engineered skin. However, the content of exogenous DNA in the large omental decellularized matrix prepared by the above method still has room for further improvement.

[0006] In addition, Chinese invention patent application CN116832217A also discloses an improved preparation method of decellularized biological omental membrane, which comprises the following steps: pretreatment: taking fresh pig large omental membrane and washing it with PBS; decellularization and dehydration treatment: placing the washed large omental membrane into a freeze dryer for freeze-drying, and the freeze-drying program is: -40°C for 5 hours, -25°C for 2 hours, -10°C for 2 hours, 4°C for 4 hours, and 25°C for 8 hours. The freeze-drying process can effectively decellularize and maximize the preservation of ECM components. The improved preparation method of decellularized biological omental membrane uses the traditional repeated freeze-thaw method for decellularization, which requires a long period and causes great damage to ECM components. The freeze-drying step can be used to replace the traditional repeated freeze-thaw method, which can better dehydrate and inactivate cells and better preserve ECM components such as collagen and glycosaminoglycans. However, the above method still has room for further improvement in terms of how to remove exogenous DNA.

[0007] Therefore, how to provide a decellularized biological omental membrane preparation method that is more convenient to operate, has a shorter period, and has lower DNA content in the decellularized omental membrane is still a problem that needs to be solved by those skilled in the art. SUMMARY

[0008] In summary, the purpose of the present application is at least to provide a decellularized biological omental membrane preparation method to reduce the DNA content in the decellularized omental membrane. In order to achieve the above-mentioned purpose of the application, the following technical solutions are adopted:

[0009] In one aspect, the present application relates to a decellularized biological omental membrane preparation method, which comprises the following steps:

[0010] 1) Take fresh omentum majus tissue, wash in phosphate buffer solution, and oscillatingly immerse in defatting solution for treatment, defatting solution immersion time is 12-36h (preferably 20-28h), oscillating frequency is 150-250r / min (preferably 180-220r / min);

[0011] 2) Wash the fat-removed omentum majus tissue in phosphate buffer solution, and oscillatingly immerse in decellularization solution I for treatment, immersion time is 6-10h (preferably 7-9h), oscillating frequency is 100-200r / min (preferably 120-180r / min);

[0012] 3) Wash the omentum majus tissue treated by decellularization solution I in phosphate buffer solution, and oscillatingly immerse in decellularization solution II for treatment, immersion time is 6-10h (preferably 7-9h), oscillating frequency is 100-200r / min (preferably 120-180r / min);

[0013] 4) Wash the omentum majus tissue treated by decellularization solution II in phosphate buffer solution, and oscillatingly immerse in decellularization solution III for treatment, immersion time is 6-10h (preferably 7-9h), oscillating frequency is 100-200r / min (preferably 120-180r / min);

[0014] 5) Freeze-dry the omentum majus tissue treated by decellularization solution III, and sterilize;

[0015] Wherein, the configuration steps of the decellularization solution are as follows:

[0016] 1) Decellularization solution I: weigh sodium dodecyl sulfate, RNAase and lipase, and dissolve in phosphate buffer solution, wherein, the concentration of sodium dodecyl sulfate in the phosphate buffer solution is 1.5-2.5% by weight (preferably 1.8-2.2% by weight), the concentration of RNAase in the phosphate buffer solution is 2500-3500U / L (preferably 2800-3200U / L), the concentration of lipase in the phosphate buffer solution is 1000-2000U / L (preferably 1200-1800U / L), pH is 7.2-7.4, and filter sterilization;

[0017] 2) Decellularization solution II: sodium dodecyl sulfate, RNase and lipase are weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 0.6-1.5% by weight (preferably 0.8-1.2% by weight), the concentration of RNase in the phosphate buffer is 1500-2000 U / L (preferably 1200-1800 U / L), the concentration of lipase in the phosphate buffer is 500-1000 U / L (preferably 600-800 U / L), the pH is 7.2-7.4, and sterilization is performed by filtration;

[0018] 3) Decellularization solution III: sodium dodecyl sulfate, RNase and lipase are weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 0.1-0.5% by weight (preferably 0.2-0.4% by weight), the concentration of RNase in the phosphate buffer is 1000-1500 U / L (preferably 1100-1300 U / L), the concentration of lipase in the phosphate buffer is 300-500 U / L (preferably 350-450 U / L), the pH is 7.2-7.4, and sterilization is performed by filtration. It should be noted that the use of the above-mentioned preferred amount of each component is conducive to further reducing the DNA content in the decellularized biological mesh.

[0019] In an embodiment of the present application, the defatting solution is a mixed solvent of methanol and isopropyl alcohol.

[0020] In a more specific embodiment, methanol and isopropyl alcohol are mixed in a volume ratio of 1-2:1-2.

[0021] Another aspect of the present application also relates to the decellularized biological mesh prepared by the above-mentioned preparation method.

[0022] In an embodiment of the present application, the DNA content in the decellularized biological mesh is less than 12 μg / g, preferably less than 9 μg / g.

[0023] The present application has at least the following advantages: the method of the present application is more convenient to operate and has a shorter cycle, and by simultaneously using the decellularization solutions I, II and III with specific formulations, the DNA content in the decellularized mesh is lower. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a photograph of immunohistochemical staining of type I collagen in the decellularized biological mesh (X200).

[0025] Figure 2 is a photograph of immunohistochemical staining of type IV collagen in the decellularized biological mesh (X200).

[0026] Figure 3 is a photograph of immunohistochemical staining of fibronectin in the decellularized biological mesh (X200).

[0027] Figure 4 is a photograph of an elasticin immunohistochemical staining of the decellularized biomesh (X200).

[0028] Figure 5 shows the results of DNA content detection in native greater omentum tissue and DNAase and RNAase decellularized biomeshes (*p<0.01). DETAILED DESCRIPTION

[0029] Example 1: Decellularization of the greater omentum from a minipig

[0030] (1) Preparation of the delipidation solution

[0031] Methanol and isopropyl alcohol were mixed in a volume ratio of 1:1;

[0032] (2) Preparation of the decellularization solution

[0033] 1) Decellularization solution I: Sodium dodecyl sulfate, RNAase and lipase were weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer was 2% by weight, the concentration of RNAase in the phosphate buffer was 3000 U / L, the concentration of lipase in the phosphate buffer was 1500 U / L, the pH was 7.3, and the solution was filtered to remove bacteria;

[0034] 2) Decellularization solution II: Sodium dodecyl sulfate, RNAase and lipase were weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer was 1% by weight, the concentration of RNAase in the phosphate buffer was 1800 U / L, the concentration of lipase in the phosphate buffer was 750 U / L, the pH was 7.3, and the solution was filtered to remove bacteria;

[0035] 3) Decellularization solution III: Sodium dodecyl sulfate, RNAase and lipase were weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer was 0.3% by weight, the concentration of RNAase in the phosphate buffer was 1200 U / L, the concentration of lipase in the phosphate buffer was 400 U / L, the pH was 7.3, and the solution was filtered to remove bacteria;

[0036] (3) Decellularization of the greater omentum:

[0037] Fresh omental tissue (1 kg) was obtained from a slaughterhouse and washed three times in 1 L PBS (Solarbio). The washed tissue was then treated with 2 L of defatting solution for 24 h with a shaking frequency of 200 r / min. Subsequently, the defatted omental tissue was washed three times in PBS and treated with 1 L of decellularization solution I for 8 h with a shaking frequency of 150 r / min. Then, the omental tissue treated with decellularization solution I was washed three times in PBS and treated with 1 L of decellularization solution II for 8 h with a shaking frequency of 150 r / min. Subsequently, the omental tissue treated with decellularization solution II was washed three times in PBS and treated with 1 L of decellularization solution III for 8 h with a shaking frequency of 150 r / min. Finally, the omental tissue treated with decellularization solution III was freeze-dried (GIPP-100FDB, Shanghai Jinpu) and sterilized by irradiation (Beijing Atomic High-tech Golden Ray Radiation Technology Application Co., Ltd.) and stored at room temperature.

[0038] Example 2: Immunohistochemical staining and identification of the decellularized biological omental membrane

[0039] The decellularized biological omental membrane prepared in Example 1 was fixed with a 4% paraformaldehyde solution, dehydrated with a concentration gradient of alcohol, embedded in paraffin, and prepared into sections with a thickness of 4 mm. The sections were dried on a pathological tissue drying instrument (PH60, Langyi) for 2 h. Subsequently, the tissue sections were hydrated with a gradient of alcohol (concentration from high to low, 95%, 85%, 70%), and then washed with distilled water. Endogenous peroxidase was removed by adding 3% hydrogen peroxide for 10 min, and then the sections were washed with PBS three times for 5 min each time. Microwave repair was performed using an antigen repair solution, and after cooling to room temperature, the sections were washed with PBS three times for 5 min each time. The sections were blocked with 1% bovine serum albumin at 37°C for 30 min. Anti-type I collagen (1:200), anti-type IV collagen (1:200), anti-fibronectin antibody (1:200), and anti-elastin primary antibodies were added dropwise, and the sections were incubated at 4°C overnight. The sections were washed with 0.01 M PBS three times, and goat anti-mouse secondary antibody was added dropwise and incubated at 37°C for 30 min. The sections were washed with PBS three times for 5 min each time. Color development was performed with DAB under light protection for 10 min, and the sections were washed with distilled water. The sections were dehydrated with a gradient of alcohol (concentration from low to high, 80%, 95%, 100%), transparentized with xylene, and mounted with neutral resin. The staining results were observed and photographed under an optical microscope (ICX41, Shunyu), and the results are shown in Figures 1-4.

[0040] The results show that the decellularized biological mesh prepared by the application has a typical mesh structure, and a large amount of collagen I, collagen IV, fibronectin and elastin is expressed, indicating that the decellularized biological mesh prepared by the application retains the main protein components of the natural extracellular matrix after the decellularization treatment and processing of the application. In addition, no obvious cell components are found in the histological staining sections of various types, indicating that the application has good decellularization effect (Figures 1-4).

[0041] Comparative Example 1: DNA enzyme decellularization treatment

[0042] The decellularization solution I is changed to: sodium dodecyl sulfate and DNA enzyme are weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 2% by weight, the concentration of DNA enzyme in the phosphate buffer is 3000 U / L, the pH is 7.3, and the bacteria are removed by filtration; the decellularization solution II is changed to: sodium dodecyl sulfate and DNA enzyme are weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 1% by weight, the concentration of DNA enzyme in PBS is 1800 U / L, the pH is 7.3, and the bacteria are removed by filtration; the decellularization solution III is changed to: sodium dodecyl sulfate and DNA enzyme are weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 0.3% by weight, the concentration of DNA enzyme in PBS is 1200 U / L, the pH is 7.3, and the bacteria are removed by filtration. Other steps refer to Example 1 for decellularization treatment of large intestinal from small pigs.

[0043] Comparative Example 2: RNA enzyme decellularization treatment

[0044] The decellularization solution I is changed to: sodium dodecyl sulfate and RNA enzyme are weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 2% by weight, the concentration of RNA enzyme in the phosphate buffer is 3000 U / L, the pH is 7.3, and the bacteria are removed by filtration; the decellularization solution II is changed to: sodium dodecyl sulfate and RNA enzyme are weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 1% by weight, the concentration of RNA enzyme in PBS is 1800 U / L, the pH is 7.3, and the bacteria are removed by filtration; the decellularization solution III is changed to: sodium dodecyl sulfate and RNA enzyme are weighed and dissolved in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 0.3% by weight, the concentration of RNA enzyme in PBS is 1200 U / L, the pH is 7.3, and the bacteria are removed by filtration. Other steps refer to Example 1 for decellularization treatment of large intestinal from small pigs.

[0045] Example 3: DNA content test of decellularized mesh matrix

[0046] About 30 mg of natural peritoneal tissue was weighed and ground, and genomic DNA was extracted using a "Blood / Cell / Tissue Genomic DNA Extraction Kit" (TIANGEN, Tian Gen Biochemical). About 30 mg of the decellularized biological mesh prepared by the DNA enzyme method (Comparative Example 1), the RNA enzyme method (Comparative Example 2), and the present application (Example 1) was weighed, and DNA in the prepared material was extracted (5 times for each, and the average value was taken). The DNA content was measured using a nucleic acid protein measuring instrument (SmartSpec Plus, BIO-RAD). The results are shown in FIG. 5, in which the DNA content in the natural peritoneal tissue was 335.78 ± 26.10 μg / g, the DNA content in the decellularized biological mesh prepared by the DNA enzyme method of Comparative Example 1 was 25.38 ± 2.45 μg / g, the DNA content in the decellularized biological mesh prepared by the RNA enzyme method of Comparative Example 2 was 21.94 ± 2.78 μg / g, and the DNA content in the decellularized biological mesh of Example 1 of the present application was 8.14 ± 3.72 μg / g.

[0047] As can be seen from the results, compared with the natural peritoneal tissue, the DNA content in the decellularized biological mesh prepared by the DNA enzyme method, the RNA enzyme method, and Example 1 of the present application was greatly reduced (25.38 ± 2.45 μg / g, 21.94 ± 2.78 μg / g, and 8.14 ± 3.72 μg / g), and there was a substantial difference (p < 0.01), indicating that the cellular DNA component contained in the natural peritoneal tissue was substantially removed by the decellularization method of the present application, and the DNA removal effect of the method of the present application was significantly better than that of the DNA enzyme method and the RNA enzyme method alone (the difference between each group was statistically significant, p < 0.01).

[0048] The preferred embodiments of the present application are described above, but are not intended to limit the present application. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present application.

Claims

1. A method for preparing a decellularized biological membrane, comprising the following steps: 1) Take fresh greater omentum tissue, wash it in phosphate buffer, and then immerse the washed tissue in a defatting solution with shaking for 12-36 hours and shaking at a frequency of 150-250 r / min. 2) The greater omentum tissue after fat removal was washed in phosphate buffer and then immersed in decellularized solution I with shaking for 6-10 hours at a shaking frequency of 100-200 r / min. 3) The greater omentum tissue treated with the decellularization solution I was washed in phosphate buffer and then immersed in decellularization solution II with shaking for 6-10 hours at a shaking frequency of 100-200 r / min. 4) The greater omentum tissue treated with the decellularization solution II was washed in phosphate buffer and then immersed in decellularization solution III with shaking for 6-10 hours at a shaking frequency of 100-200 r / min. 5) The greater omentum tissue treated with the decellularized solution III was sterilized by freeze-drying; in, The steps for preparing the decellularization solution are as follows: 1) Decellularization solution I: Weigh sodium dodecyl sulfate, RNase, and lipase, and dissolve them in phosphate buffer. The concentration of sodium dodecyl sulfate in phosphate buffer is 1.5-2.5% by weight, the concentration of RNase in phosphate buffer is 2500-3500 U / L, the concentration of lipase in phosphate buffer is 1000-2000 U / L, and the pH is 7.2-7.

4. Filter to remove bacteria. 2) Decellularization solution II: Weigh sodium dodecyl sulfate, RNase, and lipase, and dissolve them in phosphate buffer. The concentration of sodium dodecyl sulfate in phosphate buffer is 0.6-1.5% by weight, the concentration of RNase in phosphate buffer is 1500-2000 U / L, the concentration of lipase in phosphate buffer is 500-1000 U / L, and the pH is 7.2-7.

4. Filter to remove bacteria. 3) Decellularization solution III: Weigh sodium dodecyl sulfate, RNase, and lipase, and dissolve them in phosphate buffer. The concentration of sodium dodecyl sulfate in phosphate buffer is 0.1-0.5% by weight, the concentration of RNase in phosphate buffer is 1000-1500 U / L, the concentration of lipase in phosphate buffer is 300-500 U / L, and the pH is 7.2-7.

4. Filter to remove bacteria.

2. The preparation method according to claim 1, wherein the degreasing solution is a mixed solvent of methanol and isopropanol.

3. The preparation method according to claim 2, wherein the methanol and isopropanol are mixed in a volume ratio of 1-2:1-2.

4. The decellularized biomembrane prepared by the preparation method according to any one of claims 1-3.

5. The decellularized biomembrane according to claim 4, wherein the DNA content in the decellularized biomembrane is less than 12 μg / g.

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