CN118370871B - Preparation method of decellularized biological omentum and decellularized biological omentum prepared by same - Google Patents
Preparation method of decellularized biological omentum and decellularized biological omentum prepared by same Download PDFInfo
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Abstract
The invention discloses a preparation method of a decellularized biological omentum and the decellularized biological omentum prepared by the preparation method, which comprises the following steps: 1) Degreasing fresh large omentum tissue; 2) The fat-removed large omentum tissue is immersed in the decellularized liquid I in an oscillating way for treatment; 3) Treating the large omentum tissue treated by the decellularized liquid I in a decellularized liquid II; 4) Treating the large omentum tissue treated by the decellularized liquid II in the decellularized liquid III; 5) And (3) freeze-drying and sterilizing the large omentum tissue treated by the decellularized liquid III. The method has the advantages of simpler and more convenient operation and shorter period, and the DNA content in the decellularized omentum is lower by adopting the decellularized liquid I, II and III with specific formulas.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of a decellularized biological omentum and the decellularized biological omentum prepared by the same.
Background
The decellularized matrix biological material is a novel biological material which is prepared by performing decellularized treatment on tissues/organs by a proper method, removing cells and other antigen molecules possibly causing rejection reaction in the tissues, retaining a three-dimensional structure and functional proteins, has a biological induction function, can form specific functional tissues in vivo/in vitro, and can be used for repairing/reconstructing tissue injury. The existing cell-free biological materials with more applications are mainly heterogeneous cell-free matrixes, and raw materials of the natural biological materials mainly come from animal small intestine submucosa, bladder submucosa, stomach submucosa, pericardium, amniotic membrane, peritoneum and dermis layers.
The large omentum (greater omentum) is a peritoneal tissue connecting the greater curvature of the stomach to the transverse colon, bulging forward from between the stomach and the intestine, sagging in front of the intestine to form folds, in the form of aprons, covering the empty and ileum. The large omentum has wide sources, is highly vascularized and rich in various growth factors, and is widely used for in vivo implantation platforms of various implants at present. For example, it may be used in surgical reconstructive surgery for repair of inflammatory or defective tissue, promoting vascularization and regeneration of tissue. The acellular matrix biomaterial is widely used in the medical field, and has the ability to induce tissue regeneration compared with non-absorbable materials or conventional polymer materials, and thus is considered to be an ideal tissue repair material. However, animal-derived biological materials also present some risks, on the one hand, in that the foreign residual DNA, fat, etc. carried by them may cause an immune response after implantation into the human body, and on the other hand, in that the reagents used in the treatment process easily break the three-dimensional structure of the extracellular matrix (Extracellular matrix, ECM), so that they lose the ability to induce tissue regeneration in the body.
Aiming at the technical problems, the Chinese patent application CN104771788B discloses a tissue engineering skin based on a large omentum acellular matrix and a construction method thereof, and the construction method of the tissue engineering skin comprises the following steps: (1) Preparing a macroreticular lamina decellularized matrix scaffold material, which comprises the steps of immersing the macroreticular lamina in a buffer solution containing sodium dodecyl sulfate and DNase for decellularizing treatment; and (2) tissue engineering skin construction and culture. Tissue engineering skin obtained by the method is also provided. The large omentum decellularized matrix is rich in the blood vessel promoting growth factors, so that capillary blood vessels can be promoted to grow into the transplanted tissue engineering skin from the wound surface, the vascularization and wound surface healing of the tissue engineering skin are facilitated, and the method has important significance for clinical application of the tissue engineering skin. However, the content of exogenous DNA in the large omentum decellularized matrix prepared by the method still has room for further improvement.
In addition, chinese patent application CN116832217a also discloses an improved method for preparing decellularized biological omentum, comprising the following pretreatment: taking fresh pig omentum, and washing with PBS; decellularization and dehydration treatment: the washed large omentum is put into a freeze dryer for freeze drying, and the freeze drying procedure is as follows: -40 ℃ for 5 hours, -25 ℃ for 2 hours, -10 ℃ for 2 hours, 4 ℃ for 4 hours, 25 ℃ for 8 hours. The lyophilization process is effective in decellularizing and maximally preserving ECM components. The improved preparation method of the decellularized biological omentum has the advantages that the traditional repeated freeze thawing method is used for performing the decellularization treatment, the required period is too long, the damage to the ECM components is relatively large, the method can be replaced by a freeze-drying step, the effects of dehydrating and inactivating the cells are better, and the ECM components such as collagen and glycosaminoglycan are better reserved. However, the above-described method has room for further improvement in how to remove exogenous DNA.
Therefore, how to provide a preparation method of a decellularized biological omentum with simpler operation, shorter period and lower DNA content in the decellularized omentum is still a problem to be solved by the person skilled in the art.
Disclosure of Invention
In summary, the present invention is directed to a method for preparing a decellularized biological omentum to reduce the DNA content in the decellularized omentum. In order to achieve the above purpose, the following technical scheme is adopted:
The invention relates to a preparation method of a decellularized biological omentum, which comprises the following steps:
1) Taking fresh large omentum tissue, cleaning in phosphate buffer solution, and oscillating and soaking the cleaned tissue in degreasing solution for 12-36h (preferably 20-28 h), wherein the oscillating frequency is 150-250r/min (preferably 180-220 r/min);
2) Washing the fat-removed large omentum tissue in phosphate buffer solution, and oscillating and soaking in decellularized solution I for 6-10h (preferably 7-9 h), wherein the oscillating frequency is 100-200r/min (preferably 120-180 r/min);
3) Washing the large omentum tissue treated by the decellularized liquid I in phosphate buffer solution, and oscillating and soaking the large omentum tissue in the decellularized liquid II for 6-10h (preferably 7-9 h), wherein the oscillating frequency is 100-200r/min (preferably 120-180 r/min);
4) Washing the large omentum tissue treated by the decellularized liquid II in phosphate buffer solution, and oscillating and soaking the large omentum tissue in the decellularized liquid III for 6-10h (preferably 7-9 h), wherein the oscillating frequency is 100-200r/min (preferably 120-180 r/min);
5) Freeze-drying the large omentum tissue treated by the decellularized liquid III and then sterilizing;
Wherein, the preparation steps of the decellularized liquid are as follows:
1) Decellularized liquid I: weighing sodium dodecyl sulfate, RNase and lipase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 1.5-2.5% by weight (preferably 1.8-2.2% by weight), the concentration of RNase in the phosphate buffer is 2500-3500U/L (preferably 2800-3200U/L), the concentration of lipase in the phosphate buffer is 1000-2000U/L (preferably 1200-1800U/L), the pH is 7.2-7.4, filtering and sterilizing;
2) Decellularized liquid II: weighing sodium dodecyl sulfate, RNase and lipase, dissolving 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 1500-2000U/L (preferably 1200-1800U/L), the concentration of lipase in the phosphate buffer is 500-1000U/L (preferably 600-800U/L), the pH is 7.2-7.4, filtering and sterilizing;
3) Decellularized liquid III: weighing sodium dodecyl sulfate, RNase and lipase, dissolving 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 1000-1500U/L (preferably 1100-1300U/L), the concentration of lipase in the phosphate buffer is 300-500U/L (preferably 350-450U/L), the pH is 7.2-7.4, filtering and sterilizing. It should be noted that the above-mentioned preferable amount of each component is advantageous for further reducing the DNA content in the decellularized biological omentum.
In one embodiment of the invention, the degreasing solution is a mixed solvent of methanol and isopropanol.
In a more specific embodiment, the methanol and isopropanol are present in a volume ratio of 1 to 2: 1-2.
The invention also relates to the decellularized biological net film prepared by the preparation method.
In one embodiment of the invention, the DNA content of the decellularized biological omentum is less than 12 μg/g; preferably less than 9 μg/g.
The invention has the beneficial effects that: the method has the advantages of simpler and more convenient operation and shorter period, and the DNA content in the decellularized omentum is lower by adopting the decellularized liquid I, II and III with specific formulas.
Drawings
FIG. 1 Decellularized biological omentum type I collagen immunohistochemical staining X200.
FIG. 2 Decellularized biological omentum type IV collagen immunohistochemical staining X200.
FIG. 3 immunohistochemical staining of decellularized biological omentum fibronectin X200.
FIG. 4 Decellularized biological omentum elastin immunohistochemical staining X200.
FIG. 5 DNA content detection in natural large omentum tissue and DNase decellularized and RNase decellularized biological omentump<0.01）。
Detailed Description
Example 1: decellularized treatment of large omentum of small pig origin
(1) Preparation of degreasing solution
Methanol and isopropanol are mixed according to the volume ratio of 1:1, mixing evenly;
(2) Preparation of cell-free liquid
1) Decellularized liquid I: weighing sodium dodecyl sulfate, RNase and lipase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 2% by weight, the concentration of RNase in the phosphate buffer is 3000U/L, the concentration of lipase in the phosphate buffer is 1500U/L, the pH is 7.3, filtering and sterilizing;
2) Decellularized liquid II: weighing sodium dodecyl sulfate, RNase and lipase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 1% by weight, the concentration of RNase in the phosphate buffer is 1800U/L, the concentration of lipase in the phosphate buffer is 750U/L, the pH is 7.3, filtering and sterilizing;
3) Decellularized liquid III: weighing sodium dodecyl sulfate, RNase and lipase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 0.3% by weight, the concentration of RNase in the phosphate buffer is 1200U/L, the concentration of lipase in the phosphate buffer is 400U/L, the pH is 7.3, filtering and sterilizing;
(3) Large omentum decellularization treatment:
fresh minipig peritoneum macroreticular tissue (purchased from slaughterhouse) (1 kg) was taken and washed 3 times in 1L PBS (Solarbio). The cleaned tissue is immersed in 2L degreasing solution for 24h under shaking with the shaking frequency of 200r/min. Subsequently, the fat-removed large omentum tissue was washed 3 times in PBS and treated by shaking immersion in decellularized liquid I (1L) for 8h at a shaking frequency of 150r/min. Subsequently, the large omentum tissue treated by the decellularized liquid I is washed 3 times in PBS, and is immersed in the decellularized liquid II (1L) for 8 hours in an oscillating way, and the oscillating frequency is 150r/min. Subsequently, the large omentum tissue treated by the decellularized liquid II is washed 3 times in PBS and is immersed in the decellularized liquid III (1L) for 8 hours in an oscillating way, and the oscillating frequency is 150r/min. Finally, the large omentum tissue treated by the decellularized liquid III is subjected to freeze drying (GIPP-100 FDB, shanghai secondary spectrum) and then irradiation sterilization (delivered golden bright radiation company Co., ltd.) and is preserved at normal temperature.
EXAMPLE 2 immunohistochemical staining and identification of decellularized biological omentum
The decellularized biological omentum prepared in example 1 was fixed with 4% paraformaldehyde solution, concentration-graded alcohol dehydrated, paraffin embedded, and sections with a thickness of 4 mm were prepared. The slices were dried on a pathological tissue bleaching and drying instrument (PH 60 model, langyi) for 2h. Subsequently, the tissue sections were subjected to hydration with gradient alcohol (concentration from high to low, 95%, 85%, 70%), and the sections were rinsed with distilled water. Adding 3% hydrogen peroxide to act for 10 minutes, and removing endogenous peroxidase; three rinses of PBS for 5 minutes each; performing microwave repair by using antigen repair liquid, cooling to room temperature after repair, and rinsing with PBS for three times, each time for 5 minutes; blocking for 30 min at 37 ℃ with 1% bovine serum albumin; anti-type I collagen (1:200), anti-type IV collagen (1:200), anti-fibronectin antibody (1:200) and anti-elastin primary antibody were added dropwise, respectively, at 4℃overnight. Washing 3 times with 0.01M PBS, dripping goat anti-mouse secondary antibody, incubating for 30 minutes at 37 ℃, and washing 3 times with PBS for 5 minutes each time; DAB color development for 10 minutes under the condition of light shading, and washing the slices with distilled water; gradient alcohol dehydration (concentration from low to high, 80%, 95%, 100%), xylene transparency, neutral resin sealing. The staining results were observed under an optical microscope (ICX 41, short duration) and photographed, and the results are shown in FIGS. 1 to 4.
The results show that the decellularized biological net membrane prepared by the invention has a typical reticular structure and is expressed in a large amount of type I collagen, type IV collagen, fibronectin and elastin, which shows that the main protein component of the natural extracellular matrix is well reserved after the decellularized treatment and processing of the prepared decellularized biological net membrane. In addition, no obvious cellular components were seen in any of the histological stained sections, indicating that the present invention has a good decellularization effect (FIGS. 1-4).
Comparative example 1 DNAzyme method Decellularization treatment
Changing the decellularized liquid I into: weighing sodium dodecyl sulfate and DNase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 2% by weight, the concentration of DNase in the phosphate buffer is 3000U/L, the pH is 7.3, filtering and sterilizing; the decellularized liquid II becomes: weighing sodium dodecyl sulfate and DNase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in phosphate buffer is 1% by weight, the concentration of DNase in PBS is 1800U/L, the pH is 7.3, filtering and sterilizing; the decellularized fluid III became: weighing sodium dodecyl sulfate and DNase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in phosphate buffer is 0.3% by weight, the concentration of DNase in PBS is 1200U/L, pH is 7.3, filtering and sterilizing. Other procedure referring to example 1, the large omentum of minipig origin was decellularized.
Comparative example 2 RNase-based decellularization treatment
Changing the decellularized liquid I into: weighing sodium dodecyl sulfate and RNase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 2% by weight, the concentration of RNase in the phosphate buffer is 3000U/L, the pH is 7.3, filtering and sterilizing; the decellularized liquid II becomes: weighing sodium dodecyl sulfate and RNase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in phosphate buffer is 1% by weight, the concentration of RNase in PBS is 1800U/L, the pH is 7.3, filtering and sterilizing; the decellularized fluid III became: weighing sodium dodecyl sulfate and RNase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in phosphate buffer is 0.3% by weight, the concentration of DNase in PBS is 1200U/L, pH is 7.3, filtering and sterilizing. Other procedure referring to example 1, decellularized treatment was performed on the minipig-derived macroreticular membrane.
Example 3 DNA content test of decellularized omentum matrix
About 30mg of natural large omentum tissue is rapidly weighed, ground, and subjected to genome DNA extraction by using a blood/cell/tissue genome DNA extraction kit (TIANGEN, root biochemistry). About 30mg of DNA was weighed by the same method, and about 30mg of DNA was extracted from the prepared material by the DNase method, RNase method and decellularized biological omentum of the present invention (average value was taken by repeating 5 times each). DNA content was determined using a nucleic acid protein meter (SMARTSPEC PLUS, BI 0-RAD). As a result, FIG. 5 shows that the DNA content in the natural large omentum tissue was 335.78 to 26.10. Mu.g/g, that in the DNase method used in comparative example 1 was 25.38 to 2.45. Mu.g/g, that in the RNase method used in comparative example 2 was 21.94 to 2.78. Mu.g/g, and that in the decellularized omentum of example 1 of the present invention was 8.14 to 3.72. Mu.g/g.
As a result, the DNA content in the present invention was significantly reduced (25.38. Mu.g/g, 21.94. Mu.g/g and 8.14. Mu.g/g) in the decellularized biological omentum compared with the natural macroomentum tissue, and there was a substantial difference (p < 0.01), indicating that the cellular DNA components contained in the natural macroomentum tissue were substantially removed under the present invention's decellularization method treatment, and that the DNA removal effect of the present invention was significantly superior to that of the DNase method and the RNase method alone (the differences of each group had a statistical significance, p < 0.01).
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations to the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (5)
1. A method for preparing a decellularized biological omentum, comprising the following steps:
1) Taking fresh large omentum tissue, cleaning in phosphate buffer solution, and oscillating and soaking the cleaned tissue in degreasing solution for 12-36h with oscillation frequency of 150-250r/min;
2) Washing the fat-removed large omentum tissue in phosphate buffer solution, and oscillating and soaking in cell-free solution I for 6-10h at oscillation frequency of 100-200r/min;
3) Washing the large omentum tissue treated by the decellularized liquid I in phosphate buffer solution, and oscillating and soaking the large omentum tissue in the decellularized liquid II for 6-10h at an oscillation frequency of 100-200r/min;
4) Washing the large omentum tissue treated by the decellularized liquid II in phosphate buffer solution, and oscillating and soaking the large omentum tissue in the decellularized liquid III for 6-10h at an oscillation frequency of 100-200r/min;
5) Freeze-drying the large omentum tissue treated by the decellularized liquid III and then sterilizing;
wherein, the preparation steps of the decellularized liquid are as follows:
1) Decellularized liquid I: weighing sodium dodecyl sulfate, RNase and lipase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 1.5-2.5% by weight, the concentration of RNase in the phosphate buffer is 2500-3500U/L, the concentration of lipase in the phosphate buffer is 1000-2000U/L, the pH is 7.2-7.4, filtering and sterilizing;
2) Decellularized liquid II: weighing sodium dodecyl sulfate, RNase and lipase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 0.6-1.5% by weight, the concentration of RNase in the phosphate buffer is 1500-2000U/L, the concentration of lipase in the phosphate buffer is 500-1000U/L, the pH is 7.2-7.4, filtering and sterilizing;
3) Decellularized liquid III: weighing sodium dodecyl sulfate, RNase and lipase, dissolving in phosphate buffer, wherein the concentration of sodium dodecyl sulfate in the phosphate buffer is 0.1-0.5% by weight, the concentration of RNase in the phosphate buffer is 1000-1500U/L, the concentration of lipase in the phosphate buffer is 300-500U/L, the pH is 7.2-7.4, filtering and sterilizing.
2. The preparation method according to claim 1, wherein the degreasing solution is a mixed solvent of methanol and isopropanol.
3. The production method according to claim 2, wherein the volume ratio of the methanol to the isopropanol is 1-2: 1-2.
4. A decellularized biological omentum prepared by the method of any one of claims 1-3.
5. The decellularized biofilm of claim 4 having a DNA content of less than 12 μg/g.
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| CN111110919A (en) * | 2019-12-30 | 2020-05-08 | 广东泓志生物科技有限公司 | Preparation method of omentum majus acellular matrix material and construction method of cartilage tissue |
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