CN117904034A - Autologous stem cell culture solution and application thereof in gel for repairing female reproductive organs - Google Patents
Autologous stem cell culture solution and application thereof in gel for repairing female reproductive organs Download PDFInfo
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- CN117904034A CN117904034A CN202410084793.3A CN202410084793A CN117904034A CN 117904034 A CN117904034 A CN 117904034A CN 202410084793 A CN202410084793 A CN 202410084793A CN 117904034 A CN117904034 A CN 117904034A
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Abstract
The invention provides an autologous stem cell culture solution and application thereof in repairing female genital organ gel. The invention selects to add modified matrimony vine polysaccharide, modified astragaloside and hemoglobin into complete culture medium, transferrin and fibroblast growth factor, hemoglobin, transferrin and fibroblast growth factor added into the culture medium, and the invention cooperates with modified matrimony vine polysaccharide and modified astragaloside to promote proliferation of stem cells and delay aging phenomenon of stem cells in the culture process. Finally, the methacrylic acid modified hyaluronic acid hydrogel can effectively establish an immune isolation barrier, remarkably enhance the stability of the transplanted exosomes, and provide an efficient and effective way to maintain the bioactivity of the exosomes, so that the prepared hydrogel has high elasticity, good air permeability, no toxicity and good effect on repairing female reproductive organs.
Description
Technical Field
The invention belongs to the biomedical field, and relates to an autologous stem cell culture solution, in particular to an autologous stem cell culture solution and application thereof in repairing female genital organ gel.
Background
The integrity and functional integrity of the female reproductive system organs and tissue structures is critical to female health. Female reproductive system diseases are caused by various factors to cause the structural damage and dysfunction of reproductive organs, and further cause the weakening and even the loss of reproductive capacity. In recent years, stem cells and biological materials have brought new promise for the treatment of female reproductive system diseases. Mesenchymal stem cells have high proliferation, self-renewal capacity and multidirectional differentiation potential, and can repair damaged reproductive organs through various mechanisms, improve reproductive function and restore fertility.
Mesenchymal Stem Cells (MSCs) were originally isolated from human bone marrow and cultured, and due to their low immunogenicity, MSCs became one of the seed cells for tissue engineering. The placenta-derived MSCs are isolated from the decidua side placenta of a fetus, belong to cells with higher passaging and differentiation capacities and higher expansion capacities, and are derived from early stages. Because of the advantages of wide sources, low immunogenicity, low virus contamination rate, no ethical disputes and the like, pMSCs have become an attractive alternative cell for basic research and clinical application at present. The placenta mesenchymal stem cells can secrete a plurality of cytokines, improve the local microenvironment of the ovary, and obviously up-regulate the expression of B lymphoma-2 gene (BCL-2) protein. Mesenchymal stem cells have high proliferation, self-renewal capacity and multidirectional differentiation potential, and can repair damaged reproductive organs through various mechanisms, improve reproductive function and restore fertility. The hydrogel loaded with exosomes can promote cell migration, remarkably accelerate wound healing and collagen fiber deposition, and on the other hand, increase local concentration and retention of exosomes at the injured part through the extracellular matrix hydrogel. The hydrogel is used as a biological material, can simulate the environment required by tissue repair and regeneration, enhances biological activity, repairs damaged tissues, and has wide application prospect in the aspect of repairing a reproductive system.
The hydrogel prepared by adopting the hyaluronic acid gel loaded placenta stem cells has high elasticity, good air permeability, no toxicity and good mechanical property, and can be applied to female reproductive system diseases.
Disclosure of Invention
Aiming at the problems, the invention selects to add modified matrimony vine polysaccharide, modified astragaloside IV, added hemoglobin, transferrin and fibroblast growth factor into a complete culture medium to cooperatively promote proliferation of stem cells and delay aging of the stem cells in the culture process. And the methacrylic acid modified hyaluronic acid hydrogel can effectively establish an immune isolation barrier, so that the stability of the transplanted exosomes is obviously enhanced, and the prepared hydrogel is applied to female reproductive system diseases. The invention adopts the following specific steps:
In a first aspect, the invention provides a safe culture medium, which comprises the following specific preparation steps:
S1, modified wolfberry polysaccharide: taking 0.2-0.8 g of medlar polysaccharide (purchased from Shandong Santa Classification Biotechnology Co., ltd.) and adding a proper amount of water for dissolution, adjusting the pH to 9-10 by using 0.5mol/L NaOH solution, adding acetic anhydride reagent in batches while stirring, always maintaining the pH within the range of 8-10, and reacting for a period of time at constant temperature. After the reaction is finished, the pH value is regulated to 7-8 by 0.5mol/L HCl, then the solution is transferred into a dialysis bag with 3000Da, dialyzed for 40-50 h, and the modified wolfberry polysaccharide is obtained through vacuum freeze drying. In the step, the wolfberry polysaccharide is subjected to acetylation modification, the acetyl replaces the hydroxyl on the wolfberry polysaccharide chain, and the modified wolfberry polysaccharide has a certain antibacterial capacity and is obviously improved in oxidation resistance compared with the wolfberry polysaccharide before modification.
S2, modified astragaloside IV: 10-20 g of astragaloside IV (purchased from Shaanxi North Biotechnology Co., ltd.) is weighed and mixed with 0.4-0.5 g/mL of konjak gum and 10-20 g of chitosan dissolved in glacial acetic acid solution with the volume fraction of 3%. And (3) placing the mixture in a blast drying oven at 50-60 ℃ for drying for 2-4 hours to obtain the modified astragaloside IV. The step takes astragaloside IV and chitosan as materials and konjak gum as flocculant to prepare modified astragaloside IV, so that the obtained astragaloside IV not only maintains the original effect of promoting physiological metabolism of cells, but also can adsorb hemoglobin to enable the hemoglobin to transport oxygen for stem cells.
S3, preparing a complete culture medium: ultracentrifugation of serum at 120000r/min at 4 ℃ using an ultracentrifuge for 12h to prepare exosome-free serum; adding 90-95 mL of serum without exosomes and 5-10 mL of antibiotics into 400mL of low-sugar modified eagle medium in a biosafety cabinet, adding the modified matrimony vine polysaccharide prepared in the step S1 and the modified astragaloside prepared in the step S2, uniformly stirring for 50-60 min, adding hemoglobin, transferrin and fibroblast growth factor, uniformly stirring for 20-30 min, and finally adding vitamin D, wherein the mass ratio of penicillin, streptomycin and amphotericin B in 5mL of antibiotics is 1:10:2, the mass ratio of the modified wolfberry polysaccharide to the modified astragaloside IV to the hemoglobin to the transferrin to the fibroblast growth factor to the vitamin D is 8:4:3:1:1:1, wherein the added mass of the vitamin D is 10g; mixing uniformly, and placing in a refrigerator at the temperature of 2-8 ℃ to obtain the autologous stem cell culture solution, wherein the autologous stem cell culture solution is used up within one month. In the step, the wolfberry polysaccharide has the effects of resisting oxidation, reducing the oxygen partial pressure of stem cells, promoting the life of cytokines, and further promoting the growth and proliferation of cells by matching with astragaloside IV. Hemoglobin transports oxygen for stem cells; transferrin transports iron elements by binding to cell surface receptors, regulating the metabolism of iron elements in cells; the fibroblast growth factor has the effects of promoting repair and regeneration, and proper amount of vitamin D can be added to regulate cell growth and differentiation. The modified astragaloside adsorbs the hemoglobin, and the hemoglobin, transferrin and fibroblast growth factor added in the culture medium cooperate with the lycium barbarum polysaccharide and astragaloside to promote the proliferation of stem cells and delay the aging phenomenon of the stem cells in the culture process.
In a second aspect, the invention provides a method for obtaining exosomes of placenta stem cells, comprising the following steps:
s4, obtaining placenta stem cell exosomes by adopting a differential ultracentrifugation scheme: the 2 nd to 4 th generation placenta stem cells (purchased in Siro biotechnology, product number: YX-10089) are used for culturing and separating exosomes, when the cell density reaches 80 to 85%, the whole culture medium prepared in the exosome-free step S3 is replaced, namely the autologous stem cell culture solution obtained in the previous step is replaced, and cell supernatant is collected after 48 to 50 hours. The supernatant was centrifuged at a low speed (2000 r/min,20 min) to remove dead cells and cell debris, and then filtered using a 0.22 to 0.3 μm filter head, followed by centrifugation at a high speed (100 000r/min,90 min). The centrifuged contents were washed with PBS, centrifuged again at high speed (100 000r/min,90 min) to give exosomes, and 200-400 μg of dried exosomes were resuspended in 100-200 μ LPBS to give exosome solutions for further use. In the step, the exosomes are microvesicles secreted by cells and having the diameter of 40-140 nm, and have the advantages of non-immunity, non-tumorigenicity and the like. The placenta stem cell exosome contains various bioactive substances such as mRNA, lipid and protein, and has antiinflammatory and cell proliferation promoting effects.
In a third aspect, the present invention provides a hydrogel loaded with stem cell exosomes, namely a female genital organ repair gel, specifically prepared by the following steps:
S5, preparing female genital organ repair gel: adding 20-40 ml of 20% methacrylic anhydride solution (aqueous solution) into 4-8 g of hyaluronic acid solution, stirring and reacting for 1-2 h to prepare a mixed solution, dialyzing by a 3000Da dialysis bag, and freeze-drying and preserving to obtain the methacrylic acid hyaluronic acid hydrogel solution. 200-250 mug of the exosome solution in the step S4 and 0.05% of phenyl-2, 4, 6-trimethylbenzoyl phosphinic acid lithium are added into the methacrylic acid hyaluronic acid hydrogel solution to be evenly stirred, and then the mixture is put into a refrigerator at 4-5 ℃ for 12-18 hours to fully swell the gel. The mixed solution is crosslinked under ultraviolet light (UV) to form the exosome-loaded methacrylic hyaluronic acid hydrogel. The methacrylic acid modified hyaluronic acid hydrogel in the step can effectively establish an immune isolation barrier, remarkably enhance the stability of the transplanted exosomes, provide a high-efficiency and effective way to maintain the bioactivity of the exosomes, enable the prepared hydrogel to have good effect on repairing female genital organs, and have the advantages of high elasticity, high air permeability, no toxicity and good mechanical properties, and can be used as an ideal cell transport carrier.
Preferably: in the step S1, 0.2g of lycium barbarum polysaccharide is taken, a proper amount of water is added for dissolution, and 0.5mol/L NaOH solution is used for regulating the pH value to 9;
Preferably: in the step S1, the pH value is regulated to 7 by 0.5mol/L HCl, and then the solution is transferred into a dialysis bag with 3000Da for dialysis for 40 hours;
Preferably: in the step S2, 10g of astragaloside IV is weighed;
preferably: in the step S3, 90mL of fetal bovine serum and 5mL of antibiotics are added into 400mL of low-sugar modified eagle medium in a biosafety cabinet;
Preferably: in the step S4, the 3 rd generation placenta stem cells are used for culturing and separating exosomes;
preferably: filtering in the step S4 by using a 0.24 mu m filter head;
preferably: in the step S5, 20ml of a 20% methacrylic anhydride solution (aqueous solution) is added to 4g of a hyaluronic acid solution;
Preferably: in the step S5, 200 mug of exosomes extracted in the step S4 and 0.05% of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate are added into the methacrylic acid hyaluronic acid hydrogel solution, and after being uniformly stirred, the mixture is put into 4 ℃ for refrigeration for 12 hours to enable the gel to fully swell;
by adopting the technical scheme, the invention has the technical advantages that:
1. The invention selects to add the medlar polysaccharide, the astragaloside IV, the hemoglobin, the transferrin and the fibroblast growth factor into the complete culture medium, the medlar polysaccharide has the antioxidation function, reduces the oxygen partial pressure of stem cells, promotes the living of the cytokines, and can further promote the growth and proliferation of the cells by matching with the astragaloside IV. Hemoglobin transports oxygen for stem cells; transferrin transports iron elements by binding to cell surface receptors, regulating the metabolism of iron elements in cells; the fibroblast growth factor has the effects of promoting repair and regeneration, and proper amount of vitamin D can be added to regulate cell growth and differentiation.
2. The invention discovers that the placenta stem cells cultured by the complete culture medium (autologous stem cell culture solution) secrete exosomes which can increase the survival rate of the neuron cells, and the exosomes possibly have the effect of protecting the neuron structure damage of the placenta stem cells after the culture of the placenta stem cells by acetyl modification and modified astragaloside which are possibly prepared by the polysaccharide of the Chinese wolfberry, and can provide theoretical basis for treating ischemic cerebral apoplexy by the stem cells in female pregnancy.
3. In the invention, exosomes obtained by culturing placenta stem cells by a culture solution containing modified wolfberry polysaccharide and modified astragaloside IV have a synergistic effect on inhibiting migration of ovarian cancer cells.
4. The methacrylic acid modified hyaluronic acid hydrogel can effectively establish an immune isolation barrier, remarkably enhance the stability of transplanted exosomes, and provide an efficient and effective way to maintain the bioactivity of exosomes, so that the prepared hydrogel has good effect on repairing female genital organs, and has the advantages of high elasticity, high air permeability, no toxicity and good mechanical properties.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments or the description of the prior art will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the change of the structure of the primary culture of neurons under a microscope (200X) day 5 in example 1 of the present invention.
FIG. 2 is a change in the structure of the inventive comparative example 1 on day 5 of primary culture of neurons under light (200X).
FIG. 3 is a change in the structure of the inventive comparative example 2 on day 5 of primary culture of neurons under light (200X).
FIG. 4 is a graph showing the effect of the absence of exosomes on ovarian cancer cell migration.
FIG. 5 is a graph showing the effect of exosomes of example 2 of the present invention on ovarian cancer cell migration.
FIG. 6 is a graph showing the effect of the exosomes of comparative example 3 of the present invention on ovarian cancer cell migration.
FIG. 7 is a graph showing the effect of the exosomes of comparative example 4 of the present invention on ovarian cancer cell migration.
FIG. 8 is a drawing showing the observation of exosome particles of a sample of an active particle suspension of human placental stem cells after multiple centrifugation under a transmission electron microscope in accordance with example 3 of the present invention.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved by the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present patent.
Example 1
The embodiment provides a stem cell exosome, which is prepared by the following steps:
S1, modified wolfberry polysaccharide: 0.2g of wolfberry polysaccharide (purchased from Shanxi North Biotechnology Co., ltd.) is taken and added with proper amount of water for dissolution, the pH is adjusted to 9 by 0.5mol/L NaOH solution, acetic anhydride reagent is added in batches while stirring, the pH is always maintained within 8, and the reaction is carried out for a period of time at constant temperature. After the reaction is finished, the pH value is regulated to 7 by 0.5mol/L HCl, then the solution is transferred into a dialysis bag of 3000Da, dialyzed for 40 hours, and the modified matrimony vine polysaccharide is obtained after vacuum freeze drying. In the step, the wolfberry polysaccharide is subjected to acetylation modification, the acetyl replaces the hydroxyl on the wolfberry polysaccharide chain, and the modified wolfberry polysaccharide has a certain antibacterial capacity and is obviously improved in oxidation resistance compared with the wolfberry polysaccharide before modification.
S2, modified astragaloside IV: 10g of astragaloside IV (purchased from Shaanxi North Biotechnology Co., ltd.) was weighed and mixed with 0.4g/m L of konjak gum and 10g of chitosan dissolved in a 3% by volume glacial acetic acid solution. And (5) placing the mixture in a blast drying oven at 50 ℃ for drying for 2 hours to obtain the modified astragaloside IV. The step takes astragaloside IV and chitosan as materials and konjak gum as flocculant to prepare modified astragaloside IV, so that the obtained astragaloside IV not only maintains the original effect of promoting physiological metabolism of cells, but also can adsorb hemoglobin to enable the hemoglobin to transport oxygen for stem cells.
S3, preparing a complete culture medium: ultracentrifugation of serum at 120000r/min at 4 ℃ using an ultracentrifuge for 12h to prepare exosome-free serum; adding 90mL of serum without exosomes and 5mL of antibiotics into 400mL of low-sugar modified eagle medium in a biosafety cabinet, adding the modified matrimony vine polysaccharide prepared in the step S1 and the modified astragaloside prepared in the step S2, uniformly stirring for 50min, adding hemoglobin, transferrin and fibroblast growth factor, uniformly stirring for 20min, and finally adding vitamin D, wherein the antibiotics comprise penicillin, streptomycin and amphotericin B in a mass ratio of 1:10:2, the mass ratio of the modified wolfberry polysaccharide to the modified astragaloside IV to the hemoglobin to the transferrin to the fibroblast growth factor to the vitamin D is 8:4:3:1:1:1, wherein the added mass of the vitamin D is 10g; mixing, and placing in a refrigerator at 2deg.C to obtain autologous stem cell culture solution, which is used up within one month. In the step, the wolfberry polysaccharide has the effects of resisting oxidation, reducing the oxygen partial pressure of stem cells, promoting the life of cytokines, and further promoting the growth and proliferation of cells by matching with astragaloside IV. Hemoglobin transports oxygen for stem cells; transferrin transports iron elements by binding to cell surface receptors, regulating the metabolism of iron elements in cells; the fibroblast growth factor has the effects of promoting repair and regeneration, and proper amount of vitamin D can be added to regulate cell growth and differentiation. The modified astragaloside adsorbs the hemoglobin, and the hemoglobin, transferrin and fibroblast growth factor added in the culture medium cooperate with the lycium barbarum polysaccharide and astragaloside to promote the proliferation of stem cells and delay the aging phenomenon of the stem cells in the culture process.
S4, obtaining placenta stem cell exosomes by adopting a differential ultracentrifugation scheme: the 2 nd generation placenta stem cells (purchased in Sai industry biotechnology, product number: YX-10089) are used for culturing and separating exosomes, when the cell density reaches 80%, the exosome-free complete culture medium prepared in the step S3 is replaced, namely the autologous stem cell culture solution obtained in the previous step is replaced, and cell supernatant is collected after 48 hours. After removing dead cells and cell debris by centrifuging the supernatant at a low speed (2000 r/min,20 min), the supernatant was filtered using a 0.22 μm filter head and subjected to further high-speed centrifugation (100000 r/min,90 min). The centrifuged contents were washed with PBS, centrifuged again at high speed (100 000r/min,90 min) to give exosomes, and 200 μg of dried exosomes were resuspended in 100 μg LPBS to give exosome solution for further use. In the step, the exosomes are microvesicles secreted by cells and have the diameter of 40nm, and the exosomes have the advantages of non-immunity, non-tumorigenicity and the like. The placenta stem cell exosome contains various bioactive substances such as mRNA, lipid and protein, and has antiinflammatory and cell proliferation promoting effects.
Comparative example 1: in step S3, the procedure of example 1 was followed except that commercial lycium barbarum polysaccharide was used instead of the modified lycium barbarum polysaccharide of the invention.
Comparative example 2: in step S3, the procedure of example 1 was repeated except that commercial astragaloside was used instead of the modified astragaloside according to the present invention.
Taking a pregnant 16-18 d SD pregnant mouse, culturing cortical neurons, taking out uterus under aseptic condition after the SD pregnant mouse is anesthetized well, separating cerebral cortex tissue on ice surface by using microdissection forceps, cutting into tissue blocks with the size of 1mm multiplied by 1mm, putting into 0.125% pancreatin for digestion for 15min, then respectively resuspending cells by using DMEM culture medium containing 20% FBS, collecting supernatant for filtering by a 200-mesh filter, centrifuging at 1000r/min for 3min, adding 20 mu l of exosome solution obtained in step S4 of example 1, comparative example 1 and comparative example 2, inoculating the resuspension cells on a 6-hole plate by using serum-free neuron culture medium, culturing for 24h, and then completely replacing liquid every 2-4 d half liquid. On day 5 of neuronal culture, immunofluorescent staining was performed with mouse anti-rat class III beta-Tubulin antibodies, and then identified by laser confocal microscopy.
FIGS. 1 to 3 show changes in the structure of the primary culture of the cultured neurons of the exosomes prepared in example 1, comparative example 1 and comparative example 2 of the present invention on day 5 under a light microscope (200X), respectively. The cells grow on the wall, the cell bodies are full, the refraction is strong, the outline is clear, part of the cells gather and grow, the protrusions are smooth and complete, the morphology is obvious, and the cells are interwoven into a network shape (figure 1). The commercial Lycium barbarum polysaccharide is used for replacing the modified Lycium barbarum polysaccharide, then the modified Lycium barbarum polysaccharide is cultured, part of cells are dead, the cells are swollen and vacuolated, and part of neuronal protrusion injury breaks and disappears when the modified Lycium barbarum polysaccharide is observed under a light microscope (figure 2). The commercial astragaloside IV is used for replacing the modified astragaloside IV of the invention, and then the culture is carried out, so that the neuronal structure is seriously damaged, the cells are clustered and the embracing group grows, the vacuole shape and the refractive property of the cell disappear, and part of the protruding structure disappears (figure 3). From the above, it was found that the placental stem cells cultured in the complete medium of example 1 secrete exosomes that increase the survival rate of the cultured neuronal cells, and that the modified and acetylated Astragaloside IV modified by Lycium barbarum polysaccharides acts on the placental stem cells together with other components of the complete medium. The placenta stem cells secrete certain cytokines or metabolites (exosomes) thereof to protect neurons, can participate in the change of the morphological structure of the neurons after the neuronal injury and the occurrence and the development of cell survival and apoptosis, and provide theoretical basis for treating ischemic cerebral apoplexy by female gestational stem cells.
Example 2
The embodiment provides a stem cell exosome, which is prepared by the following steps:
S1, modified wolfberry polysaccharide: 0.3g of wolfberry polysaccharide (purchased from Shanxi North Biotechnology Co., ltd.) is taken and added with proper amount of water for dissolution, the pH is adjusted to 9 by 0.5mol/L NaOH solution, acetic anhydride reagent is added in batches while stirring, the pH is always maintained within 8, and the reaction is carried out for a period of time at constant temperature. After the reaction is finished, the pH value is regulated to 7 by 0.5mol/L HCl, then the solution is transferred into a dialysis bag of 3000Da, dialyzed for 42 hours, and the modified wolfberry polysaccharide is obtained after vacuum freeze drying. In the step, the wolfberry polysaccharide is subjected to acetylation modification, the acetyl replaces the hydroxyl on the wolfberry polysaccharide chain, and the modified wolfberry polysaccharide has a certain antibacterial capacity and is obviously improved in oxidation resistance compared with the wolfberry polysaccharide before modification.
S2, modified astragaloside IV: 12g of astragaloside IV (purchased from Shaanxi North Biotechnology Co., ltd.) are weighed and mixed with 0.4g/mL of konjac gum and 12g of chitosan dissolved in a 3% by volume glacial acetic acid solution. And (5) placing the mixture in a 52 ℃ forced air drying oven for drying for 3 hours to obtain the modified astragaloside IV. The step takes astragaloside IV and chitosan as materials and konjak gum as flocculant to prepare modified astragaloside IV, so that the obtained astragaloside IV not only maintains the original effect of promoting physiological metabolism of cells, but also can adsorb hemoglobin to enable the hemoglobin to transport oxygen for stem cells.
S3, preparing a complete culture medium: ultracentrifugation of serum at 120000r/min at 4 ℃ using an ultracentrifuge for 12h to prepare exosome-free serum; adding 92mL of serum without exosomes and 6mL of antibiotics into 400mL of low-sugar modified eagle medium in a biosafety cabinet, adding the modified matrimony vine polysaccharide prepared in the step S1 and the modified astragaloside prepared in the step S2, uniformly stirring for 52min, adding hemoglobin, transferrin and fibroblast growth factor, uniformly stirring for 22min, and finally adding vitamin D, wherein the antibiotics comprise penicillin, streptomycin and amphotericin B in a mass ratio of 1:10:2, the mass ratio of the modified wolfberry polysaccharide to the modified astragaloside IV to the hemoglobin to the transferrin to the fibroblast growth factor to the vitamin D is 8:4:3:1:1:1, wherein the added mass of the vitamin D is 10g; mixing, and placing in a refrigerator at 3deg.C to obtain autologous stem cell culture solution, which is used up within one month. In the step, the wolfberry polysaccharide has the effects of resisting oxidation, reducing the oxygen partial pressure of stem cells, promoting the life of cytokines, and further promoting the growth and proliferation of cells by matching with astragaloside IV. Hemoglobin transports oxygen for stem cells; transferrin transports iron elements by binding to cell surface receptors, regulating the metabolism of iron elements in cells; the fibroblast growth factor has the effects of promoting repair and regeneration, and proper amount of vitamin D can be added to regulate cell growth and differentiation. The modified astragaloside adsorbs the hemoglobin, and the hemoglobin, transferrin and fibroblast growth factor added in the culture medium cooperate with the lycium barbarum polysaccharide and astragaloside to promote the proliferation of stem cells and delay the aging phenomenon of the stem cells in the culture process.
S4, obtaining placenta stem cell exosomes by adopting a differential ultracentrifugation scheme: the 3 rd generation placenta stem cells (purchased in Sai industry biotechnology, cat# YX-10089) were used for culturing and separating exosomes, when the cell density reached 82%, the exosome-free medium prepared in step S3 was replaced, namely the autologous stem cell culture solution obtained above was replaced, and after 49 hours, the cell supernatant was collected. After removing dead cells and cell debris by centrifuging the supernatant at a low speed (2000 r/min,20 min), the supernatant was filtered using a 0.24 μm filter head and subjected to further high-speed centrifugation (100000 r/min,90 min). The centrifuged contents were washed with PBS, centrifuged again at high speed (100 000r/min,90 min) and 250. Mu.g of dried exosomes were resuspended in 120. Mu LPBS to give exosome solution for further use. In the step, the exosomes are microvesicles secreted by cells and having the diameter of 60nm, and have the advantages of non-immunity, non-tumorigenicity and the like. The placenta stem cell exosome contains various bioactive substances such as mRNA, lipid and protein, and has antiinflammatory and cell proliferation promoting effects.
Comparative example 3: the procedure of example 2 was repeated except that the modified Lycium barbarum polysaccharide of the invention was not added in step S3.
Comparative example 4: the procedure of example 2 was repeated except that the modified astragaloside IV of the present invention was not added in the step S3.
Transwell chamber migration experiments examine the effect of exosomes on the ability of human ovarian cancer cells to migrate:
1. Ovarian cancer cell suspension configuration: human ovarian cancer cells (purchased from Beijing Yi Rui Biotech Co., ltd.) were removed from liquid nitrogen and rapidly placed in a 37℃water bath, and the tube was gently frozen to dissolve the liquid therein. 1% penicillin-Lan Meisu double antibody was added to DMEM medium containing 10% fetal bovine serum from which bovine-derived exosomes were removed as a complete cell culture medium. Transferring the dissolved human ovarian cancer cells into an EP tube containing 2 times of culture medium, shaking uniformly, centrifuging, collecting precipitate, adding a small amount of culture medium, and culturing at 37 ℃ for 2-3 days to change the liquid. When the cell density reaches 80%, the ratio of the cell density to the cell density is 1:3, rinsing with PBS, digesting with 0.25% pancreatin, and stopping digestion with cell complete culture solution to obtain human ovarian cancer cell suspension.
2. The experimental steps are as follows:
(1) Taking 10 mu L of the exosome solutions obtained in the examples 2, 3 and 4, respectively adding the exosome solutions into 20ml of human ovarian cancer cell suspension, incubating for a certain period of time, washing the grown and well-grown human ovarian cancer cells with PBS, and digesting the cells with 0.25% pancreatin and no serum culture medium to obtain an ovarian cancer cell suspension containing exosomes of the example 2, an ovarian cancer cell suspension containing exosomes of the comparative example 3 and an ovarian cancer cell suspension containing exosomes of the comparative example 4; the exosome solutions obtained in example 2, comparative example 3 and comparative example 4 were sterilized by filtration through a 0.22 μm filter.
(2) 800. Mu.L of DMEM medium was added to the selected position of the 24-well plate and the time was 1 hour.
(3) The corresponding positions of DMEM medium added to the well plate were placed in a Transwell chamber in this order, and 200ul of ovarian cancer cell suspension (blank control group) having a cell concentration of 2X 10 5 cells/ml, ovarian cancer cell suspension containing the exosomes of example 2, ovarian cancer cell suspension containing the exosomes of comparative example 3, and ovarian cancer cell suspension containing the exosomes of comparative example 4 were cultured in an incubator at 37℃and 5% CO 2 for 24 hours.
(4) After the cultivation is completed, the upper indoor culture medium is removed, PBS is washed for 2 times, formaldehyde is fixed for a certain time (30 min), and the culture medium is air-dried at normal temperature.
(5) Removing formaldehyde in the room, washing with PBS for 2 times, staining with 0.5% crystal violet dye solution for 20min, sucking the dye solution, washing with PBS for 3-4 times, and observing cells in a microscope.
FIGS. 4-7 are the effect of human ovarian cancer cells without exosomes (blank), and after co-culturing exosomes with ovarian cancer cells in example 2, comparative example 3, and comparative example 4, respectively. The extracted placental stem cell exosomes contain various bioactive substances such as various mRNA, lipid, protein and the like. The migration changes of ovarian cancer cells of comparative example 3 and comparative example 4 were smaller than the blank, however, the migration ability of ovarian cancer cells was significantly decreased after example 2 of the present invention. According to the invention, the influence of the exosomes on the migration ability of the human ovarian cancer cells is detected by using a Transwell cell migration experiment, and the exosomes are found to be capable of remarkably inhibiting the migration ability of the human ovarian cancer cells; the reason is probably that exosomes obtained by culturing placental stem cells in a culture solution containing modified Lycium barbarum polysaccharide and modified Astragaloside IV have a synergistic effect on the inhibition of ovarian cancer cell migration.
Example 3
The embodiment provides a stem cell exosome, which is prepared by the following steps:
S1, modified wolfberry polysaccharide: 0.6g of wolfberry polysaccharide (purchased from Shanxi North Biotechnology Co., ltd.) is taken and added with proper amount of water for dissolution, the pH is adjusted to 10 by 0.5mol/L NaOH solution, acetic anhydride reagent is added in batches while stirring, the pH is always maintained within the range of 9, and the reaction is carried out for a period of time at constant temperature. After the reaction is finished, the pH value is regulated to 8 by 0.5mol/L HCl, then the solution is transferred into a dialysis bag of 3000Da, dialyzed for 48 hours, and the modified matrimony vine polysaccharide is obtained after vacuum freeze drying. In the step, the wolfberry polysaccharide is subjected to acetylation modification, the acetyl replaces the hydroxyl on the wolfberry polysaccharide chain, and the modified wolfberry polysaccharide has a certain antibacterial capacity and is obviously improved in oxidation resistance compared with the wolfberry polysaccharide before modification.
S2, modified astragaloside IV: 18g of astragaloside IV (purchased from North Biotechnology Co., ltd. In Shaanxi) was weighed and mixed with 0.5g/m L of konjak gum and 16g of chitosan dissolved in a 3% by volume glacial acetic acid solution. And (5) placing the mixture in a blast drying oven at 56 ℃ for drying for 3 hours to obtain the modified astragaloside IV. The step takes astragaloside IV and chitosan as materials and konjak gum as flocculant to prepare modified astragaloside IV, so that the obtained astragaloside IV not only maintains the original effect of promoting physiological metabolism of cells, but also can adsorb hemoglobin to enable the hemoglobin to transport oxygen for stem cells.
S3, preparing a complete culture medium: ultracentrifugation of serum at 120000r/min at 4 ℃ using an ultracentrifuge for 12h to prepare exosome-free serum; adding 94mL of serum without exosomes and 8mL of antibiotics into 400mL of low-sugar modified eagle medium in a biosafety cabinet, adding the modified matrimony vine polysaccharide prepared in the step S1 and the modified astragaloside prepared in the step S2, uniformly stirring for 58min, adding hemoglobin, transferrin and fibroblast growth factor, uniformly stirring for 28min, and finally adding vitamin D, wherein the antibiotics comprise penicillin, streptomycin and amphotericin B in a mass ratio of 1:10:2, the mass ratio of the modified wolfberry polysaccharide to the modified astragaloside IV to the hemoglobin to the transferrin to the fibroblast growth factor to the vitamin D is 8:4:3:1:1:1, wherein the added mass of the vitamin D is 10g; mixing, and placing in a refrigerator at 6deg.C to obtain autologous stem cell culture solution, which is used up within one month. In the step, the wolfberry polysaccharide has the effects of resisting oxidation, reducing the oxygen partial pressure of stem cells, promoting the life of cytokines, and further promoting the growth and proliferation of cells by matching with astragaloside IV. Hemoglobin transports oxygen for stem cells; transferrin transports iron elements by binding to cell surface receptors, regulating the metabolism of iron elements in cells; the fibroblast growth factor has the effects of promoting repair and regeneration, and proper amount of vitamin D can be added to regulate cell growth and differentiation. The modified astragaloside adsorbs the hemoglobin, and the hemoglobin, transferrin and fibroblast growth factor added in the culture medium cooperate with the lycium barbarum polysaccharide and astragaloside to promote the proliferation of stem cells and delay the aging phenomenon of the stem cells in the culture process.
S4, obtaining placenta stem cell exosomes by adopting a differential ultracentrifugation scheme: the 3 rd generation placenta stem cells (purchased in Sai industry biotechnology, cat# YX-10089) were used for culturing and separating exosomes, when the cell density reached 84%, the whole culture medium prepared in the exosome-free step S3 was replaced, namely the autologous stem cell culture solution obtained above was replaced, and after 49 hours, the cell supernatant was collected. After removing dead cells and cell debris by centrifuging the supernatant at a low speed (200 r/min,20 min), the supernatant was filtered using a 0.28 μm filter head and subjected to high-speed centrifugation (100000 r/min,90 min). The centrifuged contents were washed with PBS, centrifuged again at high speed (100 000r/min,90 min) to give exosomes, and 300 μg of dried exosomes were resuspended in 160 μg LPBS to give exosome solution for further use. In the step, the exosomes are microvesicles secreted by cells and with the diameter of 100nm, and have the advantages of non-immunity, non-tumorigenicity and the like. The placenta stem cell exosome contains various bioactive substances such as mRNA, lipid and protein, and has antiinflammatory and cell proliferation promoting effects.
FIG. 8 shows that the sample of the suspension of the movable particles of example 3 after the human placental stem cells are subjected to multiple centrifugation is observed by using a transmission electron microscope, a typical tea tray sample is visible under an electron microscope, a model structure is visible, and some particles are slightly bigger and probably caused by adhesion and adhesion among the particles, thus the result shows that the particles extracted from the supernatant of the placental stem cells are exosomes.
Example 4
The embodiment provides a female genital organ repair gel, which is prepared by the following steps:
S1, modified wolfberry polysaccharide: 0.8g of wolfberry polysaccharide (purchased from Shanxi North Biotechnology Co., ltd.) is taken and added with proper amount of water for dissolution, the pH is adjusted to 10 by 0.5mol/L NaOH solution, acetic anhydride reagent is added in batches while stirring, the pH is always maintained within the range of 10, and the reaction is carried out for a period of time at constant temperature. After the reaction is finished, the pH value is regulated to 8 by 0.5mol/L HCl, then the solution is transferred into a dialysis bag of 3000Da, dialyzed for 50 hours, and the modified wolfberry polysaccharide is obtained after vacuum freeze drying. In the step, the wolfberry polysaccharide is subjected to acetylation modification, the acetyl replaces the hydroxyl on the wolfberry polysaccharide chain, and the modified wolfberry polysaccharide has a certain antibacterial capacity and is obviously improved in oxidation resistance compared with the wolfberry polysaccharide before modification.
S2, modified astragaloside IV: 20g of astragaloside IV (purchased from Shaanxi North Biotechnology Co., ltd.) was weighed and mixed with 0.5g/m L of konjak gum and 20g of chitosan dissolved in a 3% by volume glacial acetic acid solution. And (5) placing the mixture in a blast drying oven at 60 ℃ for drying for 4 hours to obtain the modified astragaloside IV. The step takes astragaloside IV and chitosan as materials and konjak gum as flocculant to prepare modified astragaloside IV, so that the obtained astragaloside IV not only maintains the original effect of promoting physiological metabolism of cells, but also can adsorb hemoglobin to enable the hemoglobin to transport oxygen for stem cells.
S3, preparing a complete culture medium: ultracentrifugation of serum at 120000r/min at 4 ℃ using an ultracentrifuge for 12h to prepare exosome-free serum; adding 95mL of serum without exosomes and 10mL of antibiotics into 400mL of low-sugar modified eagle medium in a biosafety cabinet, adding the modified matrimony vine polysaccharide prepared in the step S1 and the modified astragaloside prepared in the step S2, uniformly stirring for 60min, adding hemoglobin, transferrin and fibroblast growth factor, uniformly stirring for 30min, and finally adding vitamin D, wherein the antibiotics comprise penicillin, streptomycin and amphotericin B in a mass ratio of 1:10:2, the mass ratio of the modified wolfberry polysaccharide to the modified astragaloside IV to the hemoglobin to the transferrin to the fibroblast growth factor to the vitamin D is 8:4:3:1:1:1, wherein the added mass of the vitamin D is 10g; mixing, and placing in a refrigerator at 8deg.C to obtain autologous stem cell culture solution. In the step, the wolfberry polysaccharide has the effects of resisting oxidation, reducing the oxygen partial pressure of stem cells, promoting the life of cytokines, and further promoting the growth and proliferation of cells by matching with astragaloside IV. Hemoglobin transports oxygen for stem cells; transferrin transports iron elements by binding to cell surface receptors, regulating the metabolism of iron elements in cells; the fibroblast growth factor has the effects of promoting repair and regeneration, and proper amount of vitamin D can be added to regulate cell growth and differentiation. The modified astragaloside adsorbs the hemoglobin, and the hemoglobin, transferrin and fibroblast growth factor added in the culture medium cooperate with the lycium barbarum polysaccharide and astragaloside to promote the proliferation of stem cells and delay the aging phenomenon of the stem cells in the culture process.
S4, obtaining placenta stem cell exosomes by adopting a differential ultracentrifugation scheme: the 4 th generation placenta stem cells (purchased in Sai industry biotechnology, product number: YX-10089) were used for culturing and separating exosomes, when the cell density reached 85%, the exosome-free medium prepared in step S3 was replaced, namely the autologous stem cell culture solution obtained above was replaced, and after 50 hours, the cell supernatant was collected. After removing dead cells and cell debris by centrifuging the supernatant at a low speed (200 r/min,20 min), the supernatant was filtered using a 0.3 μm filter head and subjected to high-speed centrifugation (100000 r/min,90 min). The centrifuged contents were washed with PBS, centrifuged again at high speed (100000 r/min,90 min) to give exosomes, and 400 μg of dried exosomes were resuspended in 200 μg LPBS to give exosome solution for further use. In the step, the exosomes are microvesicles secreted by cells and have the diameter of 140nm, and the exosomes have the advantages of non-immunity, non-tumorigenicity and the like. The placenta stem cell exosome contains various bioactive substances such as mRNA, lipid and protein, and has antiinflammatory and cell proliferation promoting effects.
S5, preparing female genital organ repair gel: 40ml of methacrylic anhydride (20%) was added to 8g of hyaluronic acid solution, and the mixture was stirred and reacted for 2 hours to prepare a mixed solution, which was dialyzed, freeze-dried and stored to obtain a methacrylic acid hyaluronic acid hydrogel solution. 250 μl of the exosome solution of step S4 and 0.05% of phenyl-2, 4, 6-trimethylbenzoyl phosphinate lithium are added into the above methacrylic acid hyaluronic acid hydrogel solution, and after uniform stirring, the mixture is put into a refrigerator at 4-5 ℃ for 12-18 h to fully swell the gel. The mixed solution is crosslinked under ultraviolet light (UV) to form the exosome-loaded methacrylic hyaluronic acid hydrogel. The methacrylic acid modified hyaluronic acid hydrogel in the step can effectively establish an immune isolation barrier, remarkably enhance the stability of the transplanted exosomes, provide a high-efficiency and effective way to maintain the bioactivity of the exosomes, enable the prepared hydrogel to have good effect on repairing female genital organs, and have the advantages of high elasticity, high air permeability, no toxicity and good mechanical properties, and can be used as an ideal cell transport carrier.
Comparative example 5: the procedure of example 4 was repeated except that methacrylic anhydride was not added in step S5.
Comparative example 6: in step S3, the procedure of example 4 was followed except that commercial lycium barbarum polysaccharide was used instead of the modified lycium barbarum polysaccharide of the invention.
Comparative example 7: in step S3, the procedure of example 4 was repeated except that commercial astragaloside was used instead of the modified astragaloside according to the present invention.
Comparative example 8: the procedure of example 4 was repeated except that the exosome extracted in step S4 was not added in step S5.
The hydrogel is used as a scaffold material of tissue engineering, and provides good physical support for proliferation and differentiation of transplanted cells in vivo. Animals were designed for experimental observation, and after vaginal smear observation, 70 female mice with normal estrus cycle were selected and randomly divided into a sham operation group, a model group, a example 4 group, a comparative example 5 group, a comparative example 6 group, a comparative example 7 group and a comparative example 8 group, each group having 10 animals. The rats were anesthetized by intraperitoneal injection with 2% sodium pentobarbital, and the sham surgery group only performed incision and suturing of the lower abdomen; the other 6 groups fully expose uterine horns at two sides, an incision of about 3mm is made at 1/3 of the proximal uterine body end, the incision is scraped for 10 times along 4 directions of the anterior, posterior, left and right directions of the uterine cavity from the incision to the distal end by using a self-made spatula with similar width, and the uterine incision is sutured by using a No. 0 thread. After molding, the model group, example 4 group, comparative example 5 group, comparative example 6 group, comparative example 7 group and comparative example 8 group were each injected with 0.2mLPBS, 0.2mL of methacrylic acid modified hyaluronic acid hydrogel-placental stem cell suspension of example 4 of the invention, 0.2mL of comparative example 5 hyaluronic acid hydrogel-placental stem cell suspension, 0.2mL of comparative example 6 hyaluronic acid hydrogel-placental stem cell suspension, 0.2mL of comparative example 7 hyaluronic acid hydrogel-placental stem cell suspension and 0.2mL of comparative example 8 hyaluronic acid hydrogel-placental stem cell suspension. After completion of the myo-layer suture, penicillin sodium powder is sprinkled, the skin is sutured, and after 7d, uterine tissue is taken for detection.
The uterine tissues of each group of rats were paraffin-embedded, sectioned with a vertical longitudinal axis, deparaffinized and hydrated, and stained with hematoxylin-eosin, and the morphological changes of the endometrium were observed under a microscope, and 4 fields were randomly selected for each section, and the endometrium was measured by imagesp 3.0 software, and the average value was taken as the endometrium thickness of the section.
TABLE 1
Table 1 shows the effect of hydrogels of inventive example 4 and comparative examples 5-8 on endometrium of rats in each group. As can be seen from Table 1, the rest of the groups had thinner endometrium than the sham group, but the endometrium of both the example 4 group and the comparative examples 5 to 8 group had increased compared to the model group, and in particular, the endometrium of the female genital organ repair gel group prepared in example 4 of the present invention was much higher than that of the model group and the comparative examples 5 to 8 group and was close to the sham group, with a significant difference. The methacrylic anhydride modified hyaluronic acid hydrogel provided by the invention can effectively establish an immune isolation barrier, remarkably enhance the stability of transplanted exosomes, provide a high-efficiency and effective way to maintain the bioactivity of exosomes, promote endometrial angiogenesis by secretion, and improve the endometrial receptivity, so that the prepared hydrogel has a good effect on treating endometrial injury, and can be applied to diseases of female reproductive systems.
The above embodiments are merely illustrative of the preparation process of the present invention, and not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. Autologous stem cell culture solution, its characterized in that: the preparation method comprises the following specific steps:
S1, modified wolfberry polysaccharide: dissolving 0.2-0.8 g of lycium barbarum polysaccharide in a proper amount of water, regulating the pH to 9-10 by using 0.5mol/L NaOH solution, adding acetic anhydride reagent in batches while stirring, always maintaining the pH within 8-10, reacting for a period of time at constant temperature, regulating the pH to 7-8 by using 0.5mol/L HCl after the reaction is finished, transferring the solution into a 3000Da dialysis bag, dialyzing for 40-50 h, and performing vacuum freeze drying to obtain modified lycium barbarum polysaccharide;
S2, modified astragaloside IV: weighing 10-20 g of astragaloside IV, mixing the astragaloside IV with 0.4-0.5 g/mL of konjak gum and 10-20 g of chitosan dissolved in glacial acetic acid solution with the volume fraction of 3%, and drying in a blast drying oven at 50-60 ℃ for 2-4 h to obtain modified astragaloside IV;
S3, preparing a complete culture medium: ultracentrifugation of serum at 120 000r/min at 4 ℃ using an ultracentrifuge for 12h to prepare exosome-free serum; adding 90-95 mL of serum without exosomes and 5-10 mL of antibiotics into 400mL of low-sugar modified eagle medium in a biosafety cabinet, adding the modified matrimony vine polysaccharide prepared in the step S1 and the modified astragaloside prepared in the step S2, uniformly stirring for 50-60 min, adding hemoglobin, transferrin and fibroblast growth factor, uniformly stirring for 20-30 min, and finally adding vitamin D, wherein the antibiotics comprise penicillin, streptomycin and amphotericin B, and the corresponding mass ratio is 1:10:2, the mass ratio of the modified wolfberry polysaccharide to the modified astragaloside IV to the hemoglobin to the transferrin to the fibroblast growth factor to the vitamin D is 8:4:3:1:1:1, wherein the added mass of the vitamin D is 10g; and (3) uniformly mixing and then placing in a refrigerator at the temperature of 2-8 ℃ to obtain the autologous stem cell culture solution.
2. The autologous stem cell culture medium of claim 1, wherein: in the step S1, 0.2g of wolfberry polysaccharide is taken, a proper amount of water is added for dissolution, and 0.5mol/L NaOH solution is used for regulating the pH value to 9.
3. The autologous stem cell culture medium of claim 2, wherein: in the step S1, the pH is regulated to 7 by 0.5mol/L HCl, and then the solution is transferred into a 3000Da dialysis bag for dialysis for 40 hours.
4. The autologous stem cell culture medium of claim 1, wherein: in the step S2, 10g of astragaloside IV is weighed.
5. The autologous stem cell culture medium of claim 1, wherein: 90mL of fetal bovine serum and 5mL of antibiotics were added to 400mL of low sugar modified eagle' S medium in a biosafety cabinet in step S3.
6. Stem cell exosomes, its characterized in that: the stem cell exosome is prepared by culturing the 2 nd-4 th generation placenta stem cells in the autologous stem cell culture solution according to any one of claims 1-5.
7. The stem cell exosome of claim 6, wherein: the placenta stem cell exosome is obtained by adopting a differential ultracentrifugation scheme, and specifically comprises the following steps: taking the 2 nd to 4 th generation placenta stem cells for culturing and separating exosomes, when the cell density reaches 80 to 85%, replacing the placenta stem cells with the complete culture medium prepared in the step S3, namely replacing the placenta stem cells with autologous stem cell culture solution, and collecting cell supernatant after 48 to 50 hours;
Removing dead cells and cell fragments by centrifuging the supernatant at a low speed (2000 r/min,20 min), filtering with a 0.22-0.3 μm filter head, and centrifuging at a high speed (100 000r/min,90 min); the content after high speed centrifugation is washed with PBS, centrifuged again at high speed (100 000r/min,90 min) to obtain exosomes, dried at 60 ℃ for 10h, 200-400 μg of dried exosomes is resuspended in 100-200 μ LPBS to obtain exosome solution for further use.
8. Use of the stem cell exosome of any one of claims 6-7 in repairing a female reproductive organ gel.
9. The use according to claim 8, characterized in that: the stem cell exosome is used for preparing the female genital organ repair gel by the following specific steps:
Adding 20-40 ml of 20% methacrylic anhydride solution into 4-8 g of hyaluronic acid solution with mass fraction of 2%, stirring and reacting for 1-2 h to prepare a mixed solution, dialyzing with a 3000Da dialysis bag, and freeze-drying and preserving to obtain methacrylic acid hyaluronic acid hydrogel solution; 200-250 μg of the exosome solution of any one of claims 6-7 and 0.05% phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate are added into the methacrylic acid hyaluronic acid hydrogel solution, stirred evenly, put into 4-5 ℃ for refrigeration for 12-18 hours to fully swell the gel, and the mixed solution is crosslinked under Ultraviolet (UV) to form the methacrylic acid hyaluronic acid hydrogel carrying exosome.
10. Use of autologous stem cell culture fluid according to claim 9 for repairing female genital organ gels, wherein: in the step S5, 20ml of a 20% methacrylic anhydride solution (aqueous solution) was added to 4g of the hyaluronic acid solution.
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