Preparation method and application of mesenchymal stem cell preparation

Abstract

The invention provides a preparation method and application of a mesenchymal stem cell preparation, and the preparation method comprises the following steps: under a sterile condition, extracting adipose tissues from a donor, carrying out enzymolysis by using a mild protease lysate, filtering, centrifuging and collecting to obtain a single cell group; transferring the single cell group into a culture medium for culture to obtain adipose-derived mesenchymal stem cells, and treating the adipose-derived mesenchymal stem cells in an induction solution to obtain pretreated adipose-derived mesenchymal stem cells; performing centrifugal operation in the donor plasma, and sequentially adding a self-crosslinking inducer and a crosslinking agent into supernate to obtain fibrin gel; and mixing the pretreated adipose-derived mesenchymal stem cells with fibrin gel, and transferring into a culture environment containing 5% of oxygen to obtain the mesenchymal stem cell preparation. Therefore, the survival rate and quality uniformity of cells in the preparation are improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a preparation method of a mesenchymal stem cell preparation and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells derived from adult or embryonic tissues, with common sources including bone marrow, fat, and umbilical cord. They can differentiate into osteoblasts, chondrocytes, adipocytes, etc., and can also secrete anti-inflammatory and pro-repair factors, which can regulate the inflammatory microenvironment. Mesenchymal stem cell preparations refer to products that are prepared by using MSCs in a certain carrier or suspension form and can be directly used for in vivo or in vitro research and treatment. They are used for tissue repair and regeneration, such as fracture healing, cartilage repair, and burn skin reconstruction; for immunomodulatory treatment, such as autoimmune diseases (such as systemic lupus erythematosus) and graft-versus-host disease; for cardiovascular and neurological diseases, such as repair after myocardial infarction and regeneration after cerebral ischemia; and for inflammatory diseases, such as the treatment of chronic ulcers such as Crohn's disease and diabetic foot ulcers.

[0003] However, in related technologies, different donors, different tissues, and different isolation methods lead to differences in MSC function, making it difficult to standardize efficacy. After injection or implantation, a large number of MSCs have low survival rates in inflammatory or ischemic environments and cannot continue to function. Furthermore, preparations lack unified quality standards, and characteristics such as activity, purity, differentiation capacity, and secretory function vary significantly between batches. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a preparation method of a mesenchymal stem cell preparation and its application, aiming to solve the problems of low cell survival rate and uneven quality in the preparation.

[0005] To solve the above technical problems, the present invention is implemented as follows: a method for preparing a mesenchymal stem cell preparation is provided, characterized in that the steps include: S1. Under sterile conditions, adipose tissue was extracted from the donor and enzymatically hydrolyzed using a mild protease lysis buffer at 35-37°C for 40 minutes. Single cell populations were collected by filtration and centrifugation. S2. Transferring the single cell population into a culture medium containing 10% serum supplement to obtain adipose-derived mesenchymal stem cells, and treating the adipose-derived mesenchymal stem cells in the induction solution for 48 hours to obtain pretreated adipose-derived mesenchymal stem cells; S3, performing a centrifugation operation on the donor plasma at a rotation rate of 1200 rpm for 10 minutes, extracting the supernatant obtained by centrifugation, and sequentially adding a self-crosslinking inducer and a crosslinking agent to the supernatant to obtain a fibrin gel; S4. The pretreated adipose-derived mesenchymal stem cells were mixed with fibrin gel and transferred to a culture environment containing 5% oxygen for 5 days. The hypoxia-adapted culture medium containing donor serum supplement was replaced every 48 hours to obtain a mesenchymal stem cell preparation.

[0006] In some embodiments, in step S1, the mild protease lysis solution includes at least one of hyaluronidase, neutral protease, and collagenase type I.

[0007] In some embodiments, step S1 includes: S1.1. In a sterile operating room, use surgical scissors and forceps to extract approximately 5-10 grams of adipose tissue from the donor's inguinal or abdominal area and mince it. After mincing, wash it with cold sterile phosphate buffered saline (pH 7.2-7.4). S1.2. Place the adipose tissue in a centrifuge tube containing a mild protease lysis buffer and perform enzymatic hydrolysis by shaking at a temperature of 35-37°C for 40 minutes to obtain a mixed solution. S1.3. Add donor serum supplement to the mixture and filter the mixture using a 100 μm cell sieve to obtain a single cell population.

[0008] In some embodiments, in step S2, the induction solution includes at least one of dimethyloxoglutarate, acetylcysteine, and resveratrol.

[0009] In some embodiments, step S2 includes: S2.1. Place the single cell population in a culture medium supplemented with 10% serum and inoculate it into a sterile culture flask. Then, place it in a constant temperature incubator containing 5% carbon dioxide and adjust the temperature to 37°C. Change the culture medium after 48 hours of initial culture. S2.2. When adherent cell coverage in the sterile culture flask reaches 80-90%, digest the flask with 0.25% trypsin and ethylenediaminetetraacetic acid for 3-5 minutes, then add serum-supplemented culture medium and collect the isolated stem cells. S2.3. Add induction solution to the separated stem cells for 48 hours of pretreatment, and wash with phosphate-buffered saline to obtain pretreated adipose-derived mesenchymal stem cells.

[0010] In some embodiments, in step S3, the self-crosslinking inducer includes at least one of a polyglutamic acid subunit crosslinker, dopamine-modified chitosan, and a gelatin-glutaraldehyde system, and the crosslinker includes at least one of calcium chloride, thrombin, and an oxidase adjuvant.

[0011] In some embodiments, step S3 includes: S3.1. Collect 10–20 mL of peripheral blood from the donor vein, add anticoagulant, and centrifuge at 1200 rpm for 10 minutes at 4°C using a low-speed benchtop centrifuge. Collect the upper, transparent, pale yellow portion to obtain the supernatant. S3.2. Add a selected self-crosslinking inducer to the plasma supernatant to promote the natural crosslinking pre-activation reaction between fibrinogen. Mix by shaking in a 37°C water bath for 10 minutes to obtain a pre-treated mixture. S3.3. Add a cross-linking agent to the pre-treated mixture, set the temperature at 25-37°C, maintain the pH between 7.2 and 7.4, and react for 5-10 minutes. Pour the mixture into a mold and let it stand for 5-10 minutes to obtain a fibrin gel.

[0012] In some embodiments, step S4 includes: S4.1. Resuspend the pretreated adipose-derived mesenchymal stem cells in phosphate-buffered saline and count the cells to obtain a cell concentration of 1×10 5 ~5×10 5 cells / mL of supplemental solution; S4.2. Precool the fibrin gel to room temperature, add the additive solution, transfer it into a sterile mold, and let it stand for 10 minutes to obtain a cell-gel composite system. The mixing ratio of the two is 1×10 5 ~5×10 5 Pre-treated adipose-derived mesenchymal stem cells; S4.3. Place the cell-gel complex system in a hypoxic incubator, set the oxygen concentration to 5%, carbon dioxide to 5%, and the rest to nitrogen, maintain the temperature at 37°C, and culture continuously for 5 days. Use a pipette to remove the supernatant of the cell-gel complex system every 48 hours and replace it with a hypoxic adaptation medium supplemented with donor serum. After the culture is completed, a mesenchymal stem cell preparation is obtained.

[0013] The present invention provides an application of a mesenchymal stem cell preparation. The mesenchymal stem cell preparation is prepared by the above-mentioned preparation method of a mesenchymal stem cell preparation. The mesenchymal stem cell preparation is used in immunomodulatory and tissue repair drugs.

[0014] Compared with the prior art, the preparation method and application of a mesenchymal stem cell preparation in the present invention have the following beneficial effects: Using a mild protease lysis buffer, the extracellular matrix is efficiently degraded without damaging the cell membrane and adhesion molecules. The released single cell population contains stem cells with higher vitality and more complete surface receptors. The enzymatic hydrolysis process reduces mechanical and chemical damage to the cells, ensuring that high-survival starting cells can be obtained during the subsequent culture start-up phase. No exogenous animal serum or allogeneic serum is used throughout the process, eliminating serum batch differences and immunogenicity. This improves the consistency of formulations between batches of preparations, reduces functional gaps caused by fluctuations in serum composition, and is closer to the human physiological environment, enhancing cell adaptability. Hypoxic pretreatment enables stem cells to exhibit stronger survival and immunoregulatory functions in damaging microenvironments such as hypoxia and inflammation, reducing the risk of large-scale cell death after transplantation. The cells in the preparation have adapted to the hypoxic state before leaving the factory and have a stronger tolerance to the ischemic and hypoxic environment of tissues after transplantation. They quickly integrate into the host tissue after transplantation, reducing hypoxia-induced apoptosis and inflammatory responses, and improving in vivo survival rates. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 4 is a flow chart of a method for preparing a mesenchymal stem cell preparation in one embodiment of the present invention. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0017] Please refer to Figure 1 The present invention provides a method for preparing a mesenchymal stem cell preparation, comprising the following steps: S1. Under sterile conditions, adipose tissue was extracted from the donor and enzymatically hydrolyzed using a mild protease lysis buffer at 35-37°C for 40 minutes. Single cell populations were collected by filtration and centrifugation.

[0018] In step S1, the mild protease lysis solution includes at least one of hyaluronidase, neutral protease, and collagenase type I.

[0019] Step S1 includes: S1.1. In a sterile operating table, use surgical scissors and forceps to extract approximately 5-10 grams of adipose tissue from the donor's inguinal or abdominal area and mince it. After mincing, wash it with cooled sterile phosphate buffered saline (pH 7.2-7.4).

[0020] Removes residual blood and free lipid droplets, reducing interference from impurities with subsequent enzymatic hydrolysis. Increases tissue surface area, allowing the enzyme solution to more fully and evenly reach the tissue, improving enzymatic hydrolysis efficiency. Low-temperature cleaning inhibits cellular metabolism, reducing the impact of the cleaning process on cell viability. Smaller tissue areas provide a larger contact surface, accelerating enzyme penetration into the tissue. Low temperatures also reduce cellular metabolic rates, preventing excessive cell damage during the cleaning process.

[0021] S1.2. Then place the adipose tissue in a centrifuge tube containing mild protease lysis solution for oscillation enzymatic hydrolysis. The temperature is adjusted to 35-37°C and the oscillation time is 40 minutes to obtain a mixed solution.

[0022] Effectively degrades the extracellular matrix, loosening tissue structure. Highly releases stem cells and other supporting cells within the stromal vascular fraction. Gentle conditions preserve the integrity of cell membranes and surface receptors, ensuring cell viability. Different enzymes target collagen fibers, protein polymers, or hyaluronic acid, synergistically loosening tissue without disrupting cellular structure. Oscillation and temperature combine to ensure uniform enzyme distribution and maintain enzyme activity.

[0023] S1.3. Add donor serum supplement to the mixture and filter the mixture using a 100 μm cell sieve to obtain a single cell population.

[0024] Serum rapidly inhibits enzyme activity, protecting cell surface structures. Filtration removes undigested tissue and debris, yielding a pure single-cell suspension. Centrifugation concentrates the cells, washing away enzymes and impurities, providing highly pure starting material for subsequent culture. Protease inhibitors in autologous serum bind to and inactivate residual enzymes. Cell sieving and centrifugation work together to remove large particles and suspended impurities, ensuring the purity and viability of the single-cell population.

[0025] S2. The single cell population is transferred into a culture medium containing 10% serum supplement to obtain adipose-derived mesenchymal stem cells, and the adipose-derived mesenchymal stem cells are treated in an induction solution for 48 hours to obtain pretreated adipose-derived mesenchymal stem cells.

[0026] In step S2, the induction solution includes at least one of dimethyloxoglutarate, acetylcysteine, and resveratrol.

[0027] Step S2 includes: S2.1. Place the single cell population in a culture medium containing 10% serum supplement and inoculate it into a sterile culture flask. Then place it in a constant temperature incubator containing 5% carbon dioxide and adjust the temperature to 37°C. Replace the culture medium after 48 hours of initial culture.

[0028] By utilizing the excellent adherence of mesenchymal stem cells to the surface of the culture flask, non-adherent cells (blood components, immune cells, etc.) are removed during medium exchange to achieve initial enrichment. Growth factors (such as platelet-derived growth factor and insulin-like growth factor) and adhesion molecules in autologous serum can promote stem cell proliferation and fixation. Constant temperature and constant carbon dioxide conditions simulate the in vivo environment, maintaining stable pH and osmotic pressure in the culture medium to ensure cell survival and metabolic activity. Mesenchymal stem cells inherently have the characteristic of adherent growth, and 48 hours is sufficient for them to firmly attach to the bottom of the flask. Autologous serum replaces exogenous serum, avoiding the risk of immunogenicity while providing an appropriate amount of protein and nutrients. The constant temperature incubator maintains 37°C and 5% carbon dioxide, which stabilizes the pH of the culture medium (7.2–7.4) and promotes normal cell metabolism.

[0029] S2.2. When the adherent cell coverage of the sterile culture flask reaches 80-90%, digest with 0.25% trypsin and ethylenediaminetetraacetic acid for 3-5 minutes, then add culture medium containing serum supplement to collect the isolated stem cells.

[0030] Low-concentration pancreatic enzymes combined with EDTA chelate calcium ions, destroying cell adhesion molecules and allowing cells to completely detach. After digestion, enzyme activity is quickly terminated and centrifuged to remove pancreatic enzyme residues and debris, resulting in a high-purity, low-damage stem cell population. Gentle digestion and rapid termination maximize the retention of cell membrane receptors and signaling pathways, ensuring the effectiveness of subsequent functionalization. Pancreatic enzymes can enzymatically hydrolyze extracellular matrix proteins, while EDTA chelates calcium ions, inactivating adhesion molecules such as integrins. Antitrypsin components in autologous serum rapidly neutralize pancreatic enzymes and protect cells. Centrifugation concentrates cells and washes away the digestive fluid to minimize secondary damage to the cells.

[0031] S2.3. Add induction solution to the separated stem cells for 48 hours of pretreatment, and wash with phosphate-buffered saline to obtain pretreated adipose-derived mesenchymal stem cells.

[0032] Dimethyloxoglutarate simulates a hypoxic environment, stabilizes hypoxia-inducible factors, and enhances cell survival and function under hypoxic conditions. Acetylcysteine is a glutathione precursor that replenishes intracellular antioxidants and reduces damage to cells caused by reactive oxygen species. Resveratrol activates the longevity protein pathway, enhances intracellular stress repair and secretion of immune regulatory factors, and improves anti-inflammatory and repair capabilities. Dimethyloxoglutarate inhibits hypoxia-inducible factor-degrading enzymes, initiating the hypoxia response pathway; acetylcysteine is converted into glutathione within the cell, scavenging excess free radicals through glutathione peroxidase; resveratrol regulates gene expression networks, enhancing cells' ability to resist stress and secrete repair factors.

[0033] S3. Perform a centrifugation operation at a rotation rate of 1200 rpm in the donor plasma for 10 minutes, extract the supernatant obtained by centrifugation, and sequentially add a self-crosslinking inducer and a crosslinking agent to the supernatant to obtain a fibrin gel.

[0034] In step S3, the self-crosslinking inducer includes at least one of a polyglutamic acid subunit crosslinker, dopamine-modified chitosan, and a gelatin-glutaraldehyde system, and the crosslinking agent includes at least one of calcium chloride, thrombin, and an oxidase adjuvant.

[0035] Step S3 includes: S3.1. Collect 10-20 mL of peripheral blood from the donor vein, add anticoagulant, and use a desktop low-speed centrifuge with the speed set to 1200 rpm, the time set to 10 minutes, and the temperature set to 4°C. Collect the upper transparent light yellow portion to obtain the supernatant.

[0036] Centrifugation causes red blood cells, white blood cells, and platelets to settle to the bottom of the tube, leaving the upper layer of plasma virtually free of cellular components. This preserves coagulation factors, such as inactivated fibrinogen, which are abundant in the plasma and serve as raw materials for subsequent gelation. Low-temperature centrifugation inhibits thermal inactivation and degradation of plasma proteins, preserving their native conformation and biological activity. Due to the principle of density difference, blood cells have a greater specific gravity than plasma, enabling layered separation through low-speed centrifugation. Low temperatures stabilize protein structures, preventing enzymatic degradation and thermal denaturation.

[0037] S3.2. Add a selected self-crosslinking inducer to the plasma supernatant to promote the natural crosslinking pre-activation reaction between fibrinogen. Mix by shaking in a 37°C water bath for 10 minutes to obtain a pre-treated mixture.

[0038] Self-crosslinking inducers bind to functional groups such as amino and carboxyl groups on fibrinogen, promoting weak chemical bonds or physical crosslinking between protein chains to form a primary suspended network. The pre-activation stage makes subsequent gelation more uniform, without local aggregation or voids. Embedding natural polymers such as polyglutamic acid or dopamine can enhance the binding force between the scaffold and cells, facilitating subsequent cell adhesion. The carboxyl group of polyglutamic acid binds to the amino group on the protein molecule through a salt bridge or ester bond; the phenolic hydroxyl group of dopamine covalently crosslinks with the amino group on the protein through a Schiff base reaction; gelatin forms an imine bond under the action of glutaraldehyde, improving the stability of the protein network.

[0039] S3.3. Add a cross-linking agent to the pre-treated mixture, set the temperature at 25-37°C, maintain the pH between 7.2 and 7.4, and react for 5-10 minutes. Pour the mixture into a mold and let it stand for 5-10 minutes to obtain a fibrin gel.

[0040] Calcium chloride activates coagulation factors, thrombin specifically cleaves fibrinogen, and oxidase adjuvants synergistically enhance crosslinking, completing gelation within 5–10 minutes. Multiple crosslinking significantly enhances the density and strength of the gel network, making it less prone to collapse. Controlled reaction conditions and time create uniform pores within the gel, facilitating cell migration and nutrient exchange. Calcium chloride provides the essential calcium ions for the coagulation cascade, initiating the conversion of prothrombin to thrombin. Thrombin cleaves specific peptides of fibrinogen, causing it to self-assemble into a fibrin network. Oxidase (such as horseradish peroxidase), in the presence of adjuvants such as hydroquinone, further promotes protein phenolic crosslinking, enhancing network stability.

[0041] S4. The pretreated adipose-derived mesenchymal stem cells were mixed with fibrin gel and transferred to a culture environment containing 5% oxygen for 5 days. The hypoxia-adapted culture medium containing donor serum supplement was replaced every 48 hours to obtain a mesenchymal stem cell preparation.

[0042] Step S4 includes: S4.1. Resuspend the pretreated adipose-derived mesenchymal stem cells in phosphate-buffered saline and count the cells to obtain a cell concentration of 1×10 5 ~5×10 5 cells / mL of supplementary solution.

[0043] Accurate cell counting ensures a consistent cell count within each complex, improving the comparability and stability of formulation batches. During the resuspension step, buffered saline is used to wash away any remaining enzymes and serum components, preventing them from affecting gel structure or cell adhesion during subsequent mixing with the gel. Trypan blue exclusion can be used during the counting process to assess cell viability and ensure the proportion of live cells in subsequent formulations. Using buffered saline (pH 7.2-7.4) maintains a stable cellular osmotic pressure and ionic environment for a short period of time. Trypan blue exclusion can be used during the counting process, taking advantage of the fact that dead cells are stained but live cells are not, allowing for rapid determination of cell viability.

[0044] S4.2. Precool the fibrin gel to room temperature, add the additive solution, transfer it into a sterile mold, and let it stand for 10 minutes to obtain a cell-gel composite system. The mixing ratio of the two is 1×10 5 ~5×10 5 Pre-treated adipose-derived mesenchymal stem cells.

[0045] The gel, pre-cooled to room temperature, has a moderate viscosity, enabling cells to disperse evenly within the three-dimensional network and preventing aggregation. Allowing the gel to rest for ten minutes allows binding sites within the fibrin network to fully contact cell surface adhesion molecules, enhancing the initial retention and activity of cells after embedding. The three-dimensional gel provides mechanical support and a porous structure similar to that of a tissue matrix, supporting subsequent cell growth, extension, and secretory functions. The fibrin network naturally contains cell adhesion peptides that bind to integrin receptors on the surface of mesenchymal stem cells, promoting attachment. The three-dimensional porous structure ensures nutrient and gas exchange, simulating the extracellular matrix environment in the body.

[0046] S4.3. Place the cell-gel complex system in a hypoxic incubator, set the oxygen concentration to 5%, carbon dioxide to 5%, and the rest to nitrogen, maintain the temperature at 37°C, and culture continuously for 5 days. Use a pipette to remove the supernatant of the cell-gel complex system every 48 hours and replace it with a hypoxic adaptation medium supplemented with donor serum. After the culture is completed, a mesenchymal stem cell preparation is obtained.

[0047] Hypoxic conditions induce cells to produce hypoxia-inducible factor (HIF), which activates the secretion of angiogenesis-promoting and repair factors, improving the cells' ability to adapt in vivo. The culture medium is replaced every 48 hours to replenish growth factors and nutrients, while removing metabolic waste products such as lactate to ensure stable cell growth. Five consecutive days of hypoxic adaptive culture allow the cells to fully proliferate within the three-dimensional scaffold, secrete repair factors, and form a stable cell-matrix composite structure, ultimately resulting in a preparation suitable for transplantation. The continuous supply of growth factors in autologous serum meets the needs of cell proliferation and functional expression, and regular culture medium replacement prevents acid accumulation and nutrient deficiencies, ensuring cell activity and the secretion of immunomodulatory factors.

[0048] The present invention provides an application of a mesenchymal stem cell preparation, which is prepared by a preparation method of the mesenchymal stem cell preparation. The mesenchymal stem cell preparation is applied in immune regulation and tissue repair medicines.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a mesenchymal stem cell preparation, characterized in that the steps include: S1. Under sterile conditions, adipose tissue was extracted from the donor and enzymatically hydrolyzed using a mild protease lysis buffer at 35-37°C for 40 minutes. Single cell populations were collected by filtration and centrifugation. S2. Transferring the single cell population into a culture medium containing 10% serum supplement to obtain adipose-derived mesenchymal stem cells, and treating the adipose-derived mesenchymal stem cells in the induction solution for 48 hours to obtain pretreated adipose-derived mesenchymal stem cells; S3, performing a centrifugation operation on the donor plasma at a rotation rate of 1200 rpm for 10 minutes, extracting the supernatant obtained by centrifugation, and sequentially adding a self-crosslinking inducer and a crosslinking agent to the supernatant to obtain a fibrin gel; S4. The pretreated adipose-derived mesenchymal stem cells were mixed with fibrin gel and transferred to a culture environment containing 5% oxygen for 5 days. The hypoxia-adapted culture medium containing donor serum supplement was replaced every 48 hours to obtain a mesenchymal stem cell preparation.

2. The method for preparing a mesenchymal stem cell preparation according to claim 1, wherein: In step S1, the mild protease lysis solution includes at least one of hyaluronidase, neutral protease, and collagenase type I.

3. The method for preparing a mesenchymal stem cell preparation according to claim 1 or 2, characterized in that: Step S1 includes: S1.

1. In a sterile operating room, use surgical scissors and forceps to extract approximately 5-10 grams of adipose tissue from the donor's inguinal or abdominal area and mince it. After mincing, wash it with cold sterile phosphate buffered saline (pH 7.2-7.4). S1.

2. Place the adipose tissue in a centrifuge tube containing a mild protease lysis buffer and perform enzymatic hydrolysis by shaking at a temperature of 35-37°C for 40 minutes to obtain a mixed solution. S1.

3. Add donor serum supplement to the mixture and filter the mixture using a 100 μm cell sieve to obtain a single cell population.

4. The method for preparing a mesenchymal stem cell preparation according to claim 1, wherein: In step S2, the induction solution includes at least one of dimethyloxoglutarate, acetylcysteine, and resveratrol.

5. The method for preparing a mesenchymal stem cell preparation according to claim 1 or 4, characterized in that: Step S2 includes: S2.

1. Place the single cell population in a culture medium supplemented with 10% serum and inoculate it into a sterile culture flask. Then, place it in a constant temperature incubator containing 5% carbon dioxide and adjust the temperature to 37°C. Change the culture medium after 48 hours of initial culture. S2.

2. When adherent cell coverage in the sterile culture flask reaches 80-90%, digest the flask with 0.25% trypsin and ethylenediaminetetraacetic acid for 3-5 minutes, then add serum-supplemented culture medium and collect the isolated stem cells. S2.

3. Add induction solution to the separated stem cells for 48 hours of pretreatment, and wash with phosphate-buffered saline to obtain pretreated adipose-derived mesenchymal stem cells.

6. The method for preparing a mesenchymal stem cell preparation according to claim 1, wherein: In step S3, the self-crosslinking inducer includes at least one of a polyglutamic acid subunit crosslinker, dopamine-modified chitosan, and a gelatin-glutaraldehyde system, and the crosslinking agent includes at least one of calcium chloride, thrombin, and an oxidase adjuvant.

7. The method for preparing a mesenchymal stem cell preparation according to claim 1 or 6, characterized in that: Step S3 includes: S3.

1. Collect 10–20 mL of peripheral blood from the donor vein, add anticoagulant, and centrifuge at 1200 rpm for 10 minutes at 4°C using a low-speed benchtop centrifuge. Collect the upper, transparent, pale yellow portion to obtain the supernatant. S3.

2. Add a selected self-crosslinking inducer to the plasma supernatant to promote the natural crosslinking pre-activation reaction between fibrinogen. Mix by shaking in a 37°C water bath for 10 minutes to obtain a pre-treated mixture. S3.

3. Add a cross-linking agent to the pre-treated mixture, set the temperature at 25-37°C, maintain the pH between 7.2 and 7.4, and react for 5-10 minutes. Pour the mixture into a mold and let it stand for 5-10 minutes to obtain a fibrin gel.

8. The method for preparing a mesenchymal stem cell preparation according to claim 1, wherein: Step S4 includes: S4.

1. Resuspend the pretreated adipose-derived mesenchymal stem cells in phosphate-buffered saline and count the cells to obtain a cell concentration of 1×10 5 ~5×10 5 cells / mL of supplemental solution; S4.

2. Precool the fibrin gel to room temperature, add the additive solution, transfer it into a sterile mold, and let it stand for 10 minutes to obtain a cell-gel composite system. The mixing ratio of the two is 1×10 5 ~5×10 5 Pre-treated adipose-derived mesenchymal stem cells; S4.

3. Place the cell-gel complex system in a hypoxic incubator, set the oxygen concentration to 5%, carbon dioxide to 5%, and the rest to nitrogen, maintain the temperature at 37°C, and culture continuously for 5 days. Use a pipette to remove the supernatant of the cell-gel complex system every 48 hours and replace it with a hypoxic adaptation medium supplemented with donor serum. After the culture is completed, a mesenchymal stem cell preparation is obtained.

9. An application of a mesenchymal stem cell preparation, characterized in that: The mesenchymal stem cell preparation is prepared by the preparation method of a mesenchymal stem cell preparation according to any one of claims 1 to 8, and the mesenchymal stem cell preparation is used in immunomodulatory and tissue repair drugs.

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