Culture medium and culture method for preparing umbilical cord mesenchymal stem cell exosome

Abstract

The invention discloses a culture medium and a culture method for preparing umbilical cord mesenchymal stem cell exosomes. The culture medium for preparing the umbilical cord mesenchymal stem cell exosome is prepared from 99.79 to 99.99 weight percent of a mesenchymal stem cell serum-free culture medium and 0.01 to 0.21 weight percent of an additive, the additive is a cyclodextrin modified radix astragali seu hedysari extract; the cyclodextrin modified astragalus membranaceus extract is obtained by reacting organic acid modified cyclodextrin with an astragalus membranaceus extract. The invention also provides a preparation method of the composition. Compared with the prior art, the culture medium for preparing the umbilical cord mesenchymal stem cell exosome has the advantages that the culture medium stability is good, proliferation of mesenchymal stem cells can be promoted, and human umbilical cord mesenchymal stem cells are effectively induced to synthesize a large amount and secrete exosome.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of exosome culture, and particularly relates to a culture medium and a culture method for preparing umbilical cord mesenchymal stem cell exosomes. BACKGROUND

[0002] The action mode of mesenchymal stem cells mainly includes direct cell action and paracrine action, and currently, researchers believe that the most important mode of mesenchymal stem cells is paracrine action. Exosomes refer to small vesicles with a diameter of 30-150 nm, which contain RNA, microRNA, protein, DNA fragments and other structures. Almost all cells in the human body can secrete exosomes, which naturally exist in body fluids, including saliva, urine, cerebrospinal fluid, blood and milk. Umbilical cord mesenchymal stem cells are no exception, and umbilical cord mesenchymal stem cells can secrete exosomes under normal and pathological conditions. Exosomes are mainly derived from multivesicular bodies formed by endocytic lysosomal microvesicle invagination, and are released into the extracellular matrix after fusion of the multivesicular body outer membrane with the cell membrane. Compared with umbilical cord mesenchymal stem cells, exosomes contain proteins, lipids and nucleic acids derived from parent cells, and can act as signal molecules on other cells due to their small molecular weight and strong permeability.

[0003] CN119614492A discloses an induced culture medium for preparing human umbilical cord mesenchymal stem cell exosomes and its application. The induced culture medium includes DMEM / F12 basic culture medium and an additional agent, and the additional agent includes the following components: 0.8-1.5% ITS, 0.03-0.05% osmanthus extract, 0.04-0.06% dandelion extract, 0.02-0.04% ginseng extract, 0.03-0.05% resveratrol, 0.01-0.03% vitamin C, 0.03-0.05% sheep red pheasant extract, 0.01-0.03% yam extract and 0.01-0.02% sodium citrate, which improves the secretion performance of exosomes. However, the direct addition of plant extracts in the basic culture medium may form a precipitate and reduce the promotion effect. CN118879627A discloses a method for culturing umbilical cord mesenchymal stem cell exosomes and its application. The method includes the following steps: S1, resuscitating P3 generation of frozen umbilical cord mesenchymal stem cells, culturing the cells in a DMEM culture medium under the condition of 5% CO2 and 37℃ for 48 hours, and adding IL-1β and TNF-α; S2, high-oxygen culture for 12-16 hours after normal-oxygen culture; S3, low-oxygen culture for 48-72 hours after high-oxygen culture; and S4, after the culture is completed, the cell culture supernatant is harvested, and human umbilical cord mesenchymal stem cell exosomes are obtained by ultracentrifugation separation and purification, which improves the secretion performance of exosomes. However, the application has high requirements for equipment. SUMMARY

[0004] In view of the above defects of the prior art, the prepared umbilical cord mesenchymal stem cell exosome culture medium has good culture medium stability, can promote the proliferation of mesenchymal stem cells, and effectively induces human umbilical cord mesenchymal stem cells to synthesize and secrete a large amount of exosomes.

[0005] To achieve the above-mentioned purpose, the application provides a culture medium for preparing umbilical cord mesenchymal stem cell exosomes, which is composed of 99.79-99.99wt% mesenchymal stem cell serum-free culture medium and 0.01-0.21wt% additional agent. The additional agent is cyclodextrin modified astragalus extract. The cyclodextrin modified astragalus extract is obtained by reaction of organic acid modified cyclodextrin and astragalus plant extract.

[0006] Preferably, the preparation method of the cyclodextrin modified astragalus extract comprises the following steps: Mix 0.1-0.5 parts of modified cyclodextrin and 1-5 parts of water uniformly, add 0.1-0.5 parts of astragalus extract, stir in the dark for 2-4h, centrifuge, collect the supernatant and freeze-dry, to obtain the cyclodextrin modified astragalus extract.

[0007] Preferably, the preparation method of the astragalus extract comprises the following steps: Adjust the pH of 3-8wt% ethanol aqueous solution to 11-13 with NaOH to obtain alkali alcohol solution, pass the dried astragalus root powder through a 20-40 mesh sieve; add 10-15 times the weight of alkali alcohol solution, reflux extract at 80-100℃ for 1-3h, and filter; add 8-12 times the weight of alkali alcohol solution to the filter residue and repeat the above operation, filter, and combine the two filtrates; adjust the pH of the filtrate to neutral with 1-10wt% hydrochloric acid aqueous solution, concentrate the filtrate to one tenth of the original volume, then supplement to the original volume with 95% ethanol aqueous solution, and stand overnight at 0-4℃; filter, wash the filter cake with anhydrous ethanol, and dry; then dissolve with 1-3 times the weight of 20-30wt% ethanol aqueous solution, to obtain the astragalus extract.

[0008] As a further description of the application, the astragalus extract contains various effective components such as astragalus polysaccharide, saponin, flavonoid compound alkaloid, etc., can activate the antioxidant pathway to reduce the accumulation of intracellular ROS, thereby protecting cells from oxidative damage, and can promote exosome secretion by stabilizing HIF-1α, simulating a hypoxic environment, etc.

[0009] Preferably, the preparation method of the modified cyclodextrin comprises the following steps: in terms of mass parts, Mixing 1-6 parts of beta-cyclodextrin, 1-5 parts of modifier, 1-6 parts of sodium hypophosphite and 30-60 parts of water uniformly, then reacting at 80-120℃ for 3-5h, then increasing the temperature to 120-160℃ for 4-30min, decreasing to room temperature, adding 10-30 parts of water uniformly, then adding 20-40 parts of anhydrous ethanol, standing for precipitation, filtering, washing the filter cake with anhydrous ethanol, and drying to obtain the modified cyclodextrin.

[0010] Further preferably, the modifier is selected from at least one of malic acid, succinic acid, and citric acid.

[0011] As a further illustration of the present application, beta-cyclodextrin has an inner hydrophobic and outer hydrophilic cavity structure, but has low solubility in water. The carboxyl groups of the modifiers malic acid, succinic acid, and citric acid can react with the hydroxyl groups of cyclodextrin to introduce hydrophilic groups to modify beta-cyclodextrin, which can improve the cavity activity, maintain high flexibility, and is suitable for inclusion of medium-sized molecules. This can reduce the interference of astragalus extract on the culture medium, improve the utilization of astragalus extract in the culture medium, and further promote the secretion of exosomes.

[0012] The present application also provides a culture method for preparing umbilical cord mesenchymal stem cell exosomes, characterized by comprising the following steps: S1, isolating human umbilical cord mesenchymal stem cells; S2, subculturing the human umbilical cord mesenchymal stem cells in a mesenchymal stem cell serum-free culture medium, and collecting the cells after the 3-5th generation mesenchymal stem cells are cultured to a cell fusion rate of 70-80%; S3, inoculating the digested cells in a culture medium containing a mesenchymal stem cell serum-free culture medium and an additional agent, with an inoculation density of 1×10 5 -5×10 5 ML, culturing at 36-38℃ under hypoxia for 48-72h, collecting the supernatant, performing gradient centrifugation, collecting exosomes, and obtaining human umbilical cord mesenchymal stem cell exosomes.

[0013] Preferably, in step S3, the hypoxic culture condition is a CO2 volume fraction of 3-8%, an O2 volume fraction of ≤1%, and the remaining gas being N2.

[0014] Preferably, in step S3, the method of gradient centrifugation comprises the following steps: 10000-12000g centrifugation for 10-15min, taking the supernatant, filtering, 100000-110000g centrifugation for 90-100min.

[0015] The present application has the following beneficial effects: 1. Compared with the prior art, the umbilical cord mesenchymal stem cell exosome preparation culture medium is prepared by reasonably matching the mutual action of various substances and optimizing the preparation process, has good stability, can promote the proliferation of mesenchymal stem cells, effectively induce human umbilical cord mesenchymal stem cells to synthesize and secrete exosomes in a large amount, and has other advantages.

[0016] 2. Compared with the prior art, the induction culture medium provided by the present application forms a synergistic culture environment by accurately matching a plurality of functional additives. The astragalus extract used can protect the integrity of the exosome structure and promote cell growth by removing free radicals and enhancing antioxidant enzyme activity; can promote the proliferation of mesenchymal stem cells and inhibit their replicative senescence by activating the cell proliferation signaling pathway; and can enhance the solubility and availability of the effective components that can promote exosome secretion in the culture medium by modifying the cyclodextrin to modify the astragalus extract. DETAILED DESCRIPTION

[0017] The parameters and sources of some raw materials in the embodiments of the present application are as follows: Mesenchymal stem cell serum-free culture medium, product number: NC0107, from Youkang Biotechnology (Beijing) Co., Ltd.

[0018] Preparation of ginseng extract: grind ginseng to 60 mesh, add 6 times the weight of anhydrous ethanol, reflux extract at 60℃ for 2h, filter, repeat the above operation for the filter residue, filter, combine the two filtrates, and concentrate under reduced pressure to obtain the ginseng extract.

[0019] Example 1 A preparation method of umbilical cord mesenchymal stem cell exosomes, comprising the following preparation steps: S1, isolating human umbilical cord mesenchymal stem cells; S2, subculturing the human umbilical cord mesenchymal stem cells in the mesenchymal stem cell serum-free culture medium, and recovering the cells after digesting when the 4th generation mesenchymal stem cells are cultured to a cell fusion rate of 75%; S3, inoculating the digested cells in the mesenchymal stem cell serum-free culture medium containing 0.15wt% cyclodextrin modified astragalus extract, with an inoculation density of 3x10 5 individuals / mL, recovering the supernatant after low-oxygen culture at 37℃ for 60h, gradient centrifugation, 11000g centrifugation for 10min, taking the supernatant, filtering, 100000g centrifugation for 90min, and collecting the exosomes to obtain the human umbilical cord mesenchymal stem cell exosomes.

[0020] The low-oxygen culture condition is that the CO2 volume fraction is 4%, the O2 volume fraction is 1%, and the remaining gas is N2.

[0021] The preparation method of the cyclodextrin modified astragalus extract includes the following steps: 0.3 g of modified cyclodextrin and 3 g of water are uniformly mixed, 0.3 g of astragalus extract is added, stirring is performed under light shielding conditions for 3 h, centrifugation is performed, the supernatant is collected, and freeze-drying is performed, thereby obtaining the cyclodextrin modified astragalus extract.

[0022] The preparation method of the modified cyclodextrin includes the following steps: 3 g of β-cyclodextrin, 3 g of citric acid, 4 g of sodium hypophosphite, and 45 g of water are uniformly mixed, then reaction is performed at 100℃ for 4 h, then the temperature is increased to 140℃ and reaction is performed for 15 min, the temperature is decreased to room temperature, 20 g of water is added and uniformly mixed, then 30 g of anhydrous ethanol is added, precipitation is performed after standing, filtration is performed, the filter cake is washed with anhydrous ethanol, and drying is performed, thereby obtaining the modified cyclodextrin.

[0023] The preparation method of the astragalus extract includes the following steps: 5wt% ethanol aqueous solution is adjusted to pH 12 with NaOH to obtain alkali alcohol solution; dried astragalus root powder is passed through a 30 mesh sieve, 12 times the weight of the alkali alcohol solution is added, reflux extraction is performed at 90℃ for 2 h, and filtration is performed; the filter residue is added with 10 times the weight of the alkali alcohol solution, the above operation is repeated, filtration is performed, and the filtrates of the two times are combined; 5wt% hydrochloric acid aqueous solution is used to adjust the pH to neutral, the filtrate is concentrated to 1 / 10 of the original volume, then 95% ethanol aqueous solution is used to supplement to the original volume, and standing is performed at 4℃ overnight; filtration is performed, the filter cake is washed with anhydrous ethanol, and drying is performed; then 2 times the weight of 25wt% ethanol aqueous solution is used for dissolution, thereby obtaining the astragalus extract.

[0024] Example 2 A culture method for preparing umbilical cord mesenchymal stem cell exosomes, which is different from example 2 only in that the preparation method of the modified cyclodextrin includes the following steps: 3 g of β-cyclodextrin, 3 g of succinic acid, 4 g of sodium hypophosphite, and 45 g of water are uniformly mixed, then reaction is performed at 100℃ for 4 h, then the temperature is increased to 140℃ and reaction is performed for 15 min, the temperature is decreased to room temperature; 20 g of water is added and uniformly mixed, then 30 g of anhydrous ethanol is added, precipitation is performed after standing, filtration is performed, the filter cake is washed with anhydrous ethanol, and drying is performed, thereby obtaining the modified cyclodextrin.

[0025] Example 3 A culture method for preparing umbilical cord mesenchymal stem cell exosomes, which is different from example 2 only in that the preparation method of the modified cyclodextrin includes the following steps: 3 g of β-cyclodextrin, 3 g of malic acid, 4 g of sodium hypophosphite, and 45 g of water are uniformly mixed, then reaction is performed at 100℃ for 4 h, then the temperature is increased to 140℃ and reaction is performed for 15 min, the temperature is decreased to room temperature; 20 g of water is added and uniformly mixed, then 30 g of anhydrous ethanol is added, precipitation is performed after standing, filtration is performed, the filter cake is washed with anhydrous ethanol, and drying is performed, thereby obtaining the modified cyclodextrin.

[0026] Comparative example 1 A culture method for preparing umbilical cord mesenchymal stem cell exosomes, comprising the following preparation steps: S1, isolating human umbilical cord mesenchymal stem cells; S2, subculturing the human umbilical cord mesenchymal stem cells in a mesenchymal stem cell serum-free culture medium, and recovering the cells after the 4th generation of mesenchymal stem cells are cultured to a cell fusion rate of 75%; S3, inoculating the digested cells into a mesenchymal stem cell serum-free culture medium containing 0.15wt% ginseng extract at a seeding density of 3x10 5 / mL, recovering the supernatant after low-oxygen culture at 37°C for 60h, performing gradient centrifugation, centrifuging at 11000g for 10min, taking the supernatant, filtering, centrifuging at 100000g for 90min, and collecting the exosomes to obtain human umbilical cord mesenchymal stem cell exosomes. The low-oxygen culture condition is.

[0027] The low-oxygen culture condition is that the CO2 volume fraction is 4%, the O2 volume fraction is 1%, and the remaining gas is N2.

[0028] A culture method for preparing umbilical cord mesenchymal stem cell exosomes, comprising the following preparation steps: S1, isolating human umbilical cord mesenchymal stem cells; S2, subculturing the human umbilical cord mesenchymal stem cells in a mesenchymal stem cell serum-free culture medium, and recovering the cells after the 4th generation of mesenchymal stem cells are cultured to a cell fusion rate of 75%; S3, inoculating the digested cells into a mesenchymal stem cell serum-free culture medium containing 0.15wt% ginseng extract at a seeding density of 3x10 5 / mL, recovering the supernatant after low-oxygen culture at 37°C for 60h, performing gradient centrifugation, centrifuging at 11000g for 10min, taking the supernatant, filtering, centrifuging at 100000g for 90min, and collecting the exosomes to obtain human umbilical cord mesenchymal stem cell exosomes.

[0029] The low-oxygen culture condition is that the CO2 volume fraction is 4%, the O2 volume fraction is 1%, and the remaining gas is N2.

[0030] The preparation method of the astragalus extract comprises the following steps: An alkali alcohol solution is obtained by adjusting 5wt% ethanol aqueous solution to pH 12 with NaOH powder, dried astragalus root powder is sieved through a 30-mesh sieve, 12 times the weight of the alkali alcohol solution is added, and reflux extraction is performed at 90°C for 2h, filtration is performed, 10 times the weight of alkali alcohol solution is added to the filter residue, and the above operation is repeated, filtration is performed, and the filtrates of the two times are combined; the pH is adjusted to neutral with 5wt% hydrochloric acid aqueous solution, the filtrate is concentrated to one tenth of the original volume, 95% ethanol aqueous solution is added to the original volume, and the mixture is left to stand overnight at 4°C; filtration is performed, the filter cake is washed with anhydrous ethanol, and drying is performed; and the astragalus extract is obtained.

[0031] Comparative Example 3 A culture method for preparing umbilical cord mesenchymal stem cell exosomes, which is different from Example 2 only in that the step S3, the cells after digestion are inoculated in the mesenchymal stem cell serum-free culture medium containing 0.15wt% cyclodextrin astragalus extract, the inoculation density is 3x10 5 4 / mL, and the supernatant is recovered after 60h of hypoxic culture at 37℃, gradient centrifugation, 11000g centrifugation for 10min, taking the supernatant, filtering, 100000g centrifugation for 90min, collecting the exosomes, and obtaining human umbilical cord mesenchymal stem cell exosomes.

[0032] The hypoxic culture condition is that the CO2 volume fraction is 4%, the O2 volume fraction is 1%, and the remaining gas is N2.

[0033] The preparation method of the cyclodextrin astragalus extract comprises the following steps: uniformly mixing 0.3g of cyclodextrin and 3g of water, adding 0.3g of astragalus extract, stirring for 3h under light-proof conditions, centrifuging, collecting the supernatant, and freeze-drying to obtain the cyclodextrin astragalus extract.

[0034] The preparation method of the astragalus extract is consistent with that of Example 1.

[0035] Test Example 1 Culture medium performance test The culture for preparing umbilical cord mesenchymal stem cell exosomes obtained in Examples 1-3 and Comparative Examples 1-3 is stored at 25℃±2℃ for 60 days, and the properties are observed.

[0036] Table 1 Culture medium performance test

[0037] Through comparison of Examples 1-3 and Comparative Examples 1-3, it can be found that the properties of Example 3 are the best, and the reason for analysis may be that the cavity structure formed by the malic acid modified cyclodextrin astragalus extract in Example 3 is more flexible and has a higher matching degree with the astragalus extract; the citric acid modified cyclodextrin astragalus extract is added in Example 1, the molecule of citric acid is relatively large, which may affect the spatial structure, and the inclusion effect is lower; the succinic acid modified cyclodextrin astragalus extract is added in Example 2, the hydrophobicity of succinic acid is relatively strong, which may reduce the polarity of the cavity and is not conducive to the inclusion of water-soluble molecules.

[0038] Test Example 2 Exosome protein concentration and purity Exosome protein concentration determination: BCA protein quantitative kit, ND-2000 ultramicro UV-visible spectrophotometer was used to detect the total protein amount of exosome. Parameters: sample 0.1 mL + reagent 2.0 mL; reaction time 30 min; reaction temperature 37℃; wavelength 562 nm. The supernatant obtained by culturing examples 1-3 and comparative examples 1-3 was separated to obtain 50 mL of exosome suspension, and the protein concentration was tested; the number of exosome particles with a particle size range of 20-150 nm in the sample was statistically analyzed using nano-flow, and the purity of the exosome was calculated. The protein concentration and exosome purity are shown in Table 2.

[0039] Table 2 Exosome protein concentration and purity

[0040] By comparing comparative examples 1-3, it can be found that the exosome protein concentration and purity in the medium of comparative example 2 added with astragalus extract are better than those in the medium of comparative example 1 added with ginseng extract. The analysis reason may be that the astragalus extract in the medium of comparative example 2 contains polysaccharides, saponins, flavonoids and other components, has stronger antioxidant and cell protection effects, can stabilize the structure of exosomes and promote secretion; although the ginseng extract in the medium of comparative example 1 contains active substances such as ginsenosides, its effect on promoting exosome secretion may not be as significant as that of the astragalus extract.

[0041] By comparing examples 1-3 and comparative examples 1-3, it can be found that the protein concentration and purity of example 3 are the highest. The analysis reason may be that the apple acid modified cyclodextrin astragalus extract is added in example 3. Apple acid is a small molecule organic acid, and the cavity of the modified cyclodextrin may be more suitable for the active ingredients in the astragalus extract, forming a more stable inclusion compound. Apple acid itself has antioxidant properties, which may synergistically enhance the antioxidant effect with the astragalus extract, reduce the oxidative damage of exosomes during secretion, and thus improve the integrity and protein concentration of exosomes.

Claims

1. A culture medium for preparing umbilical cord mesenchymal stem cell exosomes, characterized by: The culture medium is composed of 99.79-99.99wt% mesenchymal stem cell serum-free culture medium and 0.01-0.21wt% additive; The additive is cyclodextrin modified astragalus extract; The cyclodextrin modified astragalus extract is obtained by reaction of organic acid modified cyclodextrin and astragalus plant extract.

2. The medium for preparing umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that, The preparation method of the cyclodextrin modified astragalus extract comprises the following steps: Mix 0.1-0.5 parts of modified cyclodextrin and 1-5 parts of water uniformly, add 0.1-0.5 parts of astragalus extract, stir for 2-4h under light-proof condition, centrifuge, collect supernatant and freeze-dry, to obtain the cyclodextrin modified astragalus extract.

3. The medium for preparing umbilical cord mesenchymal stem cell exosomes according to claim 2, characterized in that, The preparation method of the astragalus extract comprises the following steps: Adjust pH of 3-8wt% ethanol aqueous solution to 11-13 with NaOH powder to obtain alkali alcohol solution; sieve dried astragalus root powder to 20-40 mesh, add 10-15 times weight of alkali alcohol solution, reflux extract at 80-100℃ for 1-3h, and filter; add 8-12 times weight of alkali alcohol solution to filter residue and repeat the above operation, filter, and combine the two filtrates; adjust pH to neutral with 1-10wt% hydrochloric acid aqueous solution, concentrate the filtrate to one tenth of the original volume, then supplement to the original volume with 95wt% ethanol aqueous solution, and stand overnight at 0-4℃; Filter, wash filter cake with anhydrous ethanol, and dry; then dissolve with 1-3 times weight of 20-30wt% ethanol aqueous solution, to obtain the astragalus extract.

4. The medium for preparing umbilical cord mesenchymal stem cell exosomes according to claim 2, characterized in that, The preparation method of the modified cyclodextrin comprises the following steps, by mass parts: Mix 1-6 parts of β-cyclodextrin, 1-5 parts of modifier, 1-6 parts of sodium hypophosphite and 30-60 parts of water uniformly, then react at 80-120℃ for 3-5h, then heat to 120-160℃ for 4-30min, cool to room temperature, mix uniformly with 10-30 parts of water, then add 20-40 parts of anhydrous ethanol, stand for precipitation, filter, wash filter cake with anhydrous ethanol, and dry, to obtain the modified cyclodextrin.

5. The medium for preparing umbilical cord mesenchymal stem cell exosomes according to claim 4, characterized by: The modifier is selected from one of malic acid, succinic acid and citric acid.

6. The culture method for preparing exosomes of umbilical cord mesenchymal stem cells according to any one of claims 1 to 5, characterized in that, Comprise the following steps: S1, isolate human umbilical cord mesenchymal stem cells; S2, passage culture the human umbilical cord mesenchymal stem cells in mesenchymal stem cell serum-free culture medium, and take 3-5 generation mesenchymal stem cells to culture until cell fusion rate reaches 70-80%, then digest and recover cells; S3, the digested cells are inoculated into the mesenchymal stem cell serum-free medium containing the additional agent at a seeding density of 1 x 10 5 -5 x 10 5 cells / mL, and after hypoxic culture at 36-38°C for 48-72 h, the supernatant is recovered, gradient centrifugation is performed, and exosomes are collected to obtain human umbilical cord mesenchymal stem cell exosomes.

7. The culture method for preparing exosomes of umbilical cord mesenchymal stem cells according to claim 6, characterized in that: In step S3, the hypoxic culture condition is that CO2 volume fraction is 3-8%, O2 volume fraction is ≤1%, and the remaining gas is N2.

8. The culture method for preparing exosomes of umbilical cord mesenchymal stem cells according to claim 6, characterized in that: In step S3, the method of gradient centrifugation comprises the following steps: centrifuge at 10000-12000g for 10-15min, take supernatant, filter, and centrifuge at 100000-110000g for 90-100min.

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