Preparation method of decellularized omentum majus

Abstract

The invention provides a preparation method of decellularized omentum majus, and relates to the technical field of biomedical materials.The method comprises the steps that in a closed container, omentum majus subjected to freeze-drying treatment is placed in a mixed solvent to be subjected to degreasing treatment, and a first intermediate is obtained; performing vacuum pulse treatment on the closed container in the degreasing treatment process; cleaning the first intermediate to obtain a second intermediate; performing circulating oscillation treatment on the second intermediate for at least one time to obtain a third intermediate; the third intermediate is cleaned in a phosphate buffer solution and then placed in a compound enzyme solution containing pancreatin and deoxyribonuclease I to be subjected to oscillation treatment, and a fourth intermediate is obtained; the fourth intermediate is sequentially washed with deionized water and a phosphate buffer solution and then placed in a sucrose solution to be soaked, then freeze-drying and sterilization are sequentially conducted, and the decellularized omentum majus is obtained. By adopting the method disclosed by the invention, the decellularized omentum majus with high purity, low residue and complete structure can be obtained.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to a method for preparing a decellularized omentum membrane. Background Technology

[0002] The greater omentum is a type of peritoneum, a mucous membrane in the abdominal cavity of higher vertebrates. It covers most of the organs within the abdominal cavity, secreting mucus to moisten their surfaces and reduce friction between them. The peritoneum migrates from the parietal layer to the visceral layer, or from one organ to another, forming a double-layered structure. The greater omentum connects the greater curvature of the stomach to the transverse colon. The greater omentum possesses excellent elasticity and flexibility, is rich in collagen, glycosaminoglycans, and angiogenesis-related signals, and exhibits a highly vascularized structure. These characteristics make the greater omentum an excellent natural biomaterial in tissue engineering and regenerative medicine. However, the greater omentum has a high lipid content, and deep lipid droplets and lipoprotein embedding hinder reagent penetration, resulting in incomplete degreasing. Furthermore, existing decellularization methods for the greater omentum easily damage the collagen network and leave high levels of harmful residues. Therefore, obtaining high-purity, low-residue, and structurally intact decellularized greater omentum is a pressing problem to be solved. Summary of the Invention

[0003] The problem solved by this invention is: how to obtain a decellularized omentum membrane with high purity, low residue, and intact structure.

[0004] To address the above problems, this invention provides a method for preparing a decellularized omentum membrane, comprising: Step S1: In a sealed container, the freeze-dried macromembrane is placed in a mixed solvent for degreasing to obtain a first intermediate; wherein, the mixed solvent is composed of ethyl acetate and n-hexane mixed in a preset volume ratio; the sealed container is subjected to vacuum pulse treatment during the degreasing process. Step S2: The first intermediate is washed sequentially with anhydrous ethanol, ethanol solution, and phosphate buffer to obtain the second intermediate; Step S3: Perform at least one cyclic oscillation treatment on the second intermediate to obtain the third intermediate; the cyclic oscillation treatment includes: first performing a first oscillation treatment on the second intermediate in sodium dodecyl sulfate solution, and then performing a second oscillation treatment in sodium deoxycholate solution; Step S4: After washing the third intermediate in phosphate buffer, place it in a complex enzyme solution containing trypsin and deoxyribonuclease I and shake to obtain the fourth intermediate; Step S5: The fourth intermediate is washed sequentially with deionized water and phosphate buffer, then soaked in sucrose solution, and then freeze-dried and sterilized sequentially to obtain decellularized omentum membrane.

[0005] Optionally, in step S1, the preset volume ratio is (2 to 3):(2 to 3).

[0006] Optionally, in step S1, the vacuum pulse processing includes: cyclically switching the sealed container between a vacuum state and an atmospheric pressure state.

[0007] Optionally, in step S2, the mass fraction of the ethanol solution is 65% to 75%.

[0008] Optionally, in step S2, the cleaning process further includes: after washing the first intermediate with the phosphate buffer solution, it is then placed in a sodium chloride solution for further washing.

[0009] Optionally, in step S3, the mass fraction of the sodium dodecyl sulfate solution is 0.4% to 0.6%, the frequency of the first oscillation treatment is 5 rpm to 10 rpm, and the time is 10 h to 14 h.

[0010] Optionally, in step S3, the mass fraction of the sodium deoxycholate solution is 0.09% to 0.11%, the frequency of the second oscillation treatment is 5 rpm to 10 rpm, and the time is 4 h to 8 h.

[0011] Optionally, in step S4, the concentration of pancreatic enzyme in the complex enzyme solution is 4 mg / mL to 6 mg / mL, and the concentration of deoxyribonuclease I in the complex enzyme solution is not less than 50 U / mL.

[0012] Optionally, in step S5, the concentration of the sucrose solution is from 0.04 g / mL to 0.06 g / mL.

[0013] Optionally, in step S5, the soaking time is 50 to 70 minutes.

[0014] Compared with related technologies, this invention uses a mixed solvent prepared from ethyl acetate and n-hexane to degrease the greater omentum membrane. Ethyl acetate is used to dissolve moderately polar lipids, while n-hexane is used to dissolve strongly nonpolar lipids. The combination of the two promotes more thorough degreasing. In addition, vacuum pulse treatment during the degreasing process helps eliminate gas resistance within the tissue, promotes deep penetration of reagents, and also contributes to more thorough degreasing. Subsequently, the sample is washed sequentially with anhydrous ethanol, ethanol solution, and phosphate buffer to remove residual solvent and lipoprotein complexes stepwise, reducing the amount of sodium dodecyl sulfate solution and sodium deoxycholate solution used in the subsequent process, which helps to reduce residues. During the decellularization process, the sample is first treated alternately with sodium dodecyl sulfate solution and sodium deoxycholate solution to reduce the damage to the sample caused by a single reagent, thereby improving the structural integrity of the decellularized greater omentum membrane. Then, a complex enzyme solution containing trypsin and deoxyribonuclease I is used to synergistically remove proteins and nucleic acids. In summary, the method of this invention can obtain a high-purity, low-residue, and structurally intact decellularized greater omentum membrane. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the preparation method of decellularized omentum membrane in an embodiment of the present invention. Detailed Implementation

[0016] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Although some embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the accompanying drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0018] The term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments". Definitions of other terms will be given in the following description. It should be noted that the concepts of "first," "second," etc., mentioned in this invention are used to distinguish different objects, not to describe a specific order or hierarchy. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0019] In response to the problems existing in the aforementioned related technologies, such as Figure 1 As shown, this embodiment of the invention provides a method for preparing a decellularized omentum membrane, comprising: Step S1: In a sealed container, the freeze-dried macromembrane is placed in a mixed solvent for degreasing to obtain a first intermediate; wherein, the mixed solvent is composed of ethyl acetate and n-hexane mixed in a preset volume ratio; the sealed container is subjected to vacuum pulse treatment during the degreasing process. Step S2: The first intermediate is washed sequentially with anhydrous ethanol, ethanol solution, and phosphate buffer to obtain the second intermediate; Step S3: Perform at least one cyclic oscillation treatment on the second intermediate to obtain the third intermediate; the cyclic oscillation treatment includes: first performing a first oscillation treatment on the second intermediate in sodium dodecyl sulfate solution, and then performing a second oscillation treatment in sodium deoxycholate solution; Step S4: After washing the third intermediate in phosphate buffer, place it in a complex enzyme solution containing trypsin and deoxyribonuclease I and shake to obtain the fourth intermediate; Step S5: The fourth intermediate is washed sequentially with deionized water and phosphate buffer, then soaked in sucrose solution, and then freeze-dried and sterilized sequentially to obtain decellularized omentum membrane.

[0020] This invention employs a mixed solvent of ethyl acetate and n-hexane to degrease the greater omentum membrane. Ethyl acetate dissolves moderately polar lipids, while n-hexane dissolves strongly nonpolar lipids; the combination of these two solvents promotes more thorough degreasing. Furthermore, vacuum pulse treatment during the degreasing process helps eliminate gas resistance within the tissue, promotes deeper reagent penetration, and further facilitates thorough degreasing. Subsequently, the sample is washed sequentially with anhydrous ethanol, ethanol solution, and phosphate buffer to remove residual solvent and lipoprotein complexes stepwise, reducing the amount of sodium dodecyl sulfate and sodium deoxycholate solution used in subsequent steps, thus minimizing residue. During the decellularization process, the sample is first treated alternately with sodium dodecyl sulfate and sodium deoxycholate solutions to reduce damage from single reagents, thereby improving the structural integrity of the decellularized greater omentum membrane. Then, a complex enzyme solution containing trypsin and deoxyribonuclease I is used to synergistically remove proteins and nucleic acids. In summary, the method of this invention can obtain a high-purity, low-residue, and structurally intact decellularized greater omentum membrane.

[0021] In some embodiments of the present invention, in step S1, the preset volume ratio is (2 to 3):(2 to 3); the mass ratio of the macromembrane to the volume ratio of the mixed solvent is 1:(15 to 25) g / mL; the degreasing treatment time is 2 to 4 hours, the temperature is 4°C to 8°C, and during the degreasing treatment, the sealed container is shaken at a frequency of 5 rpm to 10 rpm.

[0022] In some embodiments of the present invention, step S1, the vacuum pulse processing includes: cyclically switching the sealed container between a vacuum state and an atmospheric pressure state. For example, the sealed container is evacuated to a vacuum level of 0.06 MPa to 0.08 MPa, held for 5 minutes, then nitrogen gas is introduced until the sealed container reaches atmospheric pressure, held for 10 minutes, and then the above operation is repeated.

[0023] In some embodiments of the present invention, in step S2, the mass fraction of the ethanol solution is 65% to 75%.

[0024] In some embodiments of the present invention, step S2, the washing process further includes: washing the first intermediate in the phosphate buffer solution, and then washing it in a sodium chloride solution; the concentration of the sodium chloride solution is 1.0 mol / L to 1.5 mol / L. In this embodiment, adding the sodium chloride solution washing step is beneficial for disrupting the lipoprotein complex, thereby further reducing the lipid residue rate of the decellularized greater omentum membrane.

[0025] In some embodiments of the present invention, in step S3, the mass fraction of the sodium dodecyl sulfate (SDS) solution is 0.4% to 0.6%, the frequency of the first oscillation treatment is 5 rpm to 10 rpm, the temperature is 3°C to 5°C, and the time is 10 h to 14 h.

[0026] In some embodiments of the present invention, in step S3, the mass fraction of the sodium deoxycholate solution is 0.09% to 0.11%, the frequency of the second oscillation treatment is 5 rpm to 10 rpm, the temperature is 20°C to 25°C, and the time is 4 h to 8 h.

[0027] In some embodiments of the present invention, in step S4, the concentration of pancreatic enzyme in the complex enzyme solution is 4 mg / mL to 6 mg / mL, and the concentration of deoxyribonuclease I in the complex enzyme solution is not less than 50 U / mL.

[0028] In some embodiments of the present invention, in step S5, the concentration of the sucrose solution is from 0.04 g / mL to 0.06 g / mL.

[0029] In some embodiments of the present invention, in step S5, the soaking time is 50 min to 70 min.

[0030] In some embodiments of the present invention, the freeze-drying temperature is below -40°C and the time is above 2 hours, and the sterilization is carried out by gamma irradiation at a dose of 25 kGy to 30 kGy. The present invention will be further described below with reference to specific embodiments.

[0031] Example 1 A1. In a sealed container, the freeze-dried omentum membrane is placed in a mixed solvent for degreasing to obtain a first intermediate; wherein the omentum membrane is a porcine omentum membrane with a thickness of 3 mm, and the mixed solvent is composed of ethyl acetate and n-hexane mixed in a preset volume ratio; the degreasing process is subject to vacuum pulse treatment, which includes: evacuating the sealed container to a vacuum degree of 0.07 MPa, maintaining it for 5 min, then introducing nitrogen gas to bring the sealed container to atmospheric pressure, maintaining it for 10 min, and then repeating the above operation; the preset volume ratio is 1:1; the mass ratio of the omentum membrane to the volume of the mixed solvent is 1:20 g / mL; the degreasing time is 3 h, the temperature is 6 °C, and during the degreasing process, the sealed container is shaken at a frequency of 10 rpm.

[0032] A2. Cleaning treatment: The first intermediate was first washed in anhydrous ethanol for 60 min, then in an ethanol solution at 4°C for 12 h, and then in phosphate buffer for 90 min to obtain the second intermediate; wherein the mass fraction of the ethanol solution was 70%; the cleaning treatment was performed by shaking at a frequency of 80 rpm.

[0033] A3. The second intermediate is subjected to two cycles of oscillation treatment to obtain the third intermediate. The oscillation treatment includes: first, the second intermediate is subjected to a first oscillation treatment in a sodium dodecyl sulfate (SDS) solution, and then a second oscillation treatment in a sodium deoxycholate (DOC) solution. The mass fraction of the sodium dodecyl sulfate solution is 0.5%. The frequency of the first oscillation treatment is 8 rpm, the temperature is 4°C, and the time is 12 h. The mass fraction of the sodium deoxycholate solution is 0.1%. The frequency of the second oscillation treatment is 8 rpm, the temperature is 20°C, and the time is 6 h. Vacuum pulse treatment is performed during the oscillation treatment. The vacuum pulse treatment includes: evacuating the sealed container to a vacuum degree of 0.07 MPa, maintaining it for 5 min, then introducing nitrogen gas to bring the sealed container to atmospheric pressure, maintaining it for 10 min, and then repeating the above operation.

[0034] A4. After washing the third intermediate in phosphate buffer for 75 min, place it in a complex enzyme solution containing trypsin and deoxyribonuclease I and shake to obtain the fourth intermediate. The concentration of trypsin in the complex enzyme solution is 5 mg / mL, and the concentration of deoxyribonuclease I in the complex enzyme solution is 50 U / mL. The shaking frequency is 8 rpm, the temperature is 37°C, and the time is 12 h. During the shaking process, a vacuum pulse treatment is performed. The vacuum pulse treatment includes: evacuating the sealed container to a vacuum degree of 0.07 MPa and holding it for 5 min, then introducing nitrogen gas to bring the sealed container to atmospheric pressure and holding it for 10 min. Then, the above operation is repeated.

[0035] A5. The fourth intermediate is washed sequentially with deionized water and phosphate buffer, then immersed in a sucrose solution, followed by freeze-drying and sterilization to obtain a decellularized omentum membrane; wherein the concentration of the sucrose solution is 0.05 g / mL, the immersion time is 60 min, the freeze-drying temperature is -50°C for 2 h, and the sterilization is performed by gamma irradiation at a dose of 25 kGy.

[0036] Example 2 The difference from Example 1 is that in step A1, the preset volume ratio is 3:2.

[0037] Example 3 The difference from Example 1 is that step A2 is as follows: Cleaning treatment: the first intermediate is first cleaned in anhydrous ethanol for 60 min, then in an ethanol solution at 4°C for 12 h, then in phosphate buffer for 90 min, and finally in sodium chloride solution for 1.5 h to obtain the second intermediate; wherein the mass fraction of the ethanol solution is 70%; the concentration of the sodium chloride solution is 1.2 mol / L, and the cleaning treatment is performed by shaking at a frequency of 80 rpm.

[0038] Comparative Example 1 The difference from Example 1 is that no vacuum pulse processing was performed in steps A1, A3 and A4.

[0039] Comparative Example 2 The difference from Example 1 is that in step A1, the mixed solvent is a mixture of isopropanol and methanol in a volume ratio of 1:1.

[0040] Comparative Example 3 The difference from Example 1 is that in step A2, the washing process involves washing the sample in phosphate buffer for 90 minutes to obtain the second intermediate; the washing process is accompanied by an oscillation process at a frequency of 80 rpm.

[0041] Experimental Example The decellularized omentum membranes prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were measured for residual DNA, collagen retention rate, lipid residue rate, total residual solvent, mechanical retention rate, cell compatibility, HE staining, and Masson staining. The results are shown in Tables 1 and 2. As can be seen from Tables 1 and 2, compared with Comparative Examples 1 to 3, the decellularized omentum membranes prepared in Examples 1 to 3 had a higher collagen retention rate, indicating higher purity. Compared with Comparative Examples 1 to 3, the decellularized omentum membranes prepared in Examples 1 to 3 had lower residual DNA, lipid residue rate, and total residual solvent, indicating lower residue. Compared with Comparative Examples 1 to 3, the decellularized omentum membranes prepared in Examples 1 to 3 had higher mechanical retention rate, higher cell compatibility, no residual cell nuclei, intact tissue, and uniform and continuous distribution of collagen fibers.

[0042] Table 1

[0043] Table 2

[0044] It should be noted that the mechanical retention rate is the ratio of the elastic modulus of the decellularized omentum membrane to the elastic modulus of the omentum membrane used to prepare the decellularized omentum membrane. Residual DNA was measured spectrophotometrically, and collagen retention was measured using the hydroxyproline method. While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A method for preparing a decellularized omentum membrane, characterized in that, include: Step S1: In a sealed container, the freeze-dried macromembrane is placed in a mixed solvent for degreasing to obtain a first intermediate; wherein the mixed solvent is composed of ethyl acetate and n-hexane mixed in a preset volume ratio; the sealed container is subjected to vacuum pulse treatment during the degreasing process; Step S2: The first intermediate is cleaned to obtain the second intermediate; the cleaning process includes: the first intermediate is sequentially cleaned with anhydrous ethanol, ethanol solution, and phosphate buffer solution. Step S3: Perform at least one cyclic oscillation treatment on the second intermediate to obtain the third intermediate; the cyclic oscillation treatment includes: first performing a first oscillation treatment on the second intermediate in sodium dodecyl sulfate solution, and then performing a second oscillation treatment in sodium deoxycholate solution; Step S4: After washing the third intermediate in phosphate buffer, place it in a complex enzyme solution containing trypsin and deoxyribonuclease I and shake to obtain the fourth intermediate; Step S5: The fourth intermediate is washed sequentially with deionized water and phosphate buffer, then soaked in sucrose solution, and then freeze-dried and sterilized sequentially to obtain decellularized omentum membrane.

2. The method for preparing the decellularized omentum membrane according to claim 1, characterized in that, In step S1, the preset volume ratio is (2 to 3): (2 to 3).

3. The method for preparing the decellularized omentum membrane according to claim 1, characterized in that, In step S1, the vacuum pulse processing includes: cyclically switching the sealed container between a vacuum state and an atmospheric pressure state.

4. The method for preparing the decellularized omentum membrane according to claim 1, characterized in that, In step S2, the ethanol solution has a mass fraction of 65% to 75%.

5. The method for preparing the decellularized omentum membrane according to claim 1, characterized in that, In step S2, the cleaning process further includes: after washing the first intermediate with the phosphate buffer solution, it is then placed in a sodium chloride solution for further cleaning.

6. The method for preparing the decellularized omentum membrane according to claim 1, characterized in that, In step S3, the mass fraction of the sodium dodecyl sulfate solution is 0.4% to 0.6%, the frequency of the first oscillation treatment is 5 rpm to 10 rpm, and the time is 10 h to 14 h.

7. The method for preparing the decellularized omentum membrane according to claim 1, characterized in that, In step S3, the mass fraction of the sodium deoxycholate solution is 0.09% to 0.11%, and the frequency of the second oscillation treatment is 5 rpm to 10 rpm, with a duration of 4 h to 8 h.

8. The method for preparing the decellularized omentum membrane according to claim 1, characterized in that, In step S4, the concentration of pancreatic enzyme in the complex enzyme solution is 4 mg / mL to 6 mg / mL, and the concentration of deoxyribonuclease I in the complex enzyme solution is not less than 50 U / mL.

9. The method for preparing the decellularized omentum membrane according to claim 1, characterized in that, In step S5, the concentration of the sucrose solution is from 0.04 g / mL to 0.06 g / mL.

10. The method for preparing the decellularized omentum membrane according to claim 1, characterized in that, In step S5, the soaking time is 50 to 70 minutes.

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