A culture medium and culture method for preparing umbilical cord mesenchymal stem cell exosomes

Abstract

The application discloses a culture medium and a culture method for preparing umbilical cord mesenchymal stem cell exosomes. The culture medium for preparing umbilical cord mesenchymal stem cell exosomes is composed of 99.79-99.99 wt% mesenchymal stem cell serum-free culture medium and 0.01-0.21 wt% additional agent; the additional agent is cyclodextrin modified astragalus extract; and the cyclodextrin modified astragalus extract is obtained by reaction of organic acid modified cyclodextrin and astragalus extract. The application also provides a preparation method thereof. Compared with the prior art, the culture medium for preparing umbilical cord mesenchymal stem cell exosomes has the advantages of good culture medium stability, can promote the proliferation of mesenchymal stem cells, and can effectively induce human umbilical cord mesenchymal stem cells to synthesize and secrete exosomes in large quantities.

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% additive;

[0006] The additive is cyclodextrin modified astragalus extract.

[0007] The cyclodextrin modified astragalus extract is obtained by reaction of organic acid modified cyclodextrin and astragalus plant extract.

[0008] Preferably, the preparation method of the cyclodextrin modified astragalus extract comprises the following steps:

[0009] 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.

[0010] Preferably, the preparation method of the astragalus extract comprises the following steps:

[0011] 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 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.

[0012] As a further description of the application, the astragalus extract contains various effective components such as astragalus polysaccharide, saponin, flavonoid compound and alkaloid, 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.

[0013] Preferably, the preparation method of the modified cyclodextrin comprises the following steps: in terms of mass parts,

[0014] 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 DEG C for 3-5 hours, then increasing the temperature to 120-160 DEG C for 4-30 minutes, 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 modified cyclodextrin.

[0015] Further preferably, the modifier is selected from at least one of malic acid, succinic acid and citric acid.

[0016] 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, be suitable for inclusion of medium-sized molecules, 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.

[0017] The present application also provides a culture method for preparing umbilical cord mesenchymal stem cell exosomes, characterized by comprising the following steps:

[0018] S1, isolating human umbilical cord mesenchymal stem cells;

[0019] S2, subculturing the human umbilical cord mesenchymal stem cells in the mesenchymal stem cell serum-free culture medium, and taking 3-5 generations of mesenchymal stem cells to culture to a cell fusion rate of 70-80%, and then digesting and recovering the cells;

[0020] S3, inoculating the digested cells in a culture medium containing mesenchymal stem cell serum-free culture medium and additional agents, at an inoculation density of 1x10 5 -5x10 5 ML, culturing at 36-38 DEG C under hypoxic conditions for 48-72 hours, recovering the supernatant, gradient centrifuging, collecting exosomes, and obtaining human umbilical cord mesenchymal stem cell exosomes.

[0021] Preferably, in step S3, the hypoxic culture conditions are a CO2 volume fraction of 3-8%, an O2 volume fraction of ≤1%, and the remaining gas being N2.

[0022] Preferably, in step S3, the method of gradient centrifugation comprises the following steps: 10000-12000g centrifugation for 10-15 minutes, taking the supernatant, filtering, 100000-110000g centrifugation for 90-100 minutes.

[0023] The present application has the following beneficial effects:

[0024] 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.

[0025] 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

[0026] The parameters and sources of some raw materials in the embodiments of the present application are as follows:

[0027] Mesenchymal stem cell serum-free culture medium, product number: NC0107, from Youkang Biotechnology (Beijing) Co., Ltd.

[0028] Preparation of ginseng extract: grind ginseng through a 60-mesh sieve, 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.

[0029] Example 1

[0030] A preparation method of umbilical cord mesenchymal stem cell exosome, comprising the following preparation steps:

[0031] S1, isolating human umbilical cord mesenchymal stem cells;

[0032] S2, subculturing the human umbilical cord mesenchymal stem cells in the mesenchymal stem cell serum-free culture medium, and recovering the cells after digestion when the 4th generation mesenchymal stem cells are cultured to a cell fusion rate of 75%;

[0033] S3, inoculating the digested cells into the mesenchymal stem cell serum-free culture medium containing 0.15wt% cyclodextrin modified astragalus extract, with an inoculation density of 3x10 5 6 / 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.

[0034] The hypoxic culture condition is that the volume fraction of CO2 is 4%, the volume fraction of O2 is 1%, and the remaining gas is N2.

[0035] The preparation method of the cyclodextrin modified astragalus extract includes the following steps: uniformly mixing 0.3 g of modified cyclodextrin and 3 g of water, adding 0.3 g of astragalus extract, stirring for 3 h under light-proof conditions, centrifuging, collecting the supernatant, and freeze-drying to obtain the cyclodextrin modified astragalus extract.

[0036] The preparation method of the modified cyclodextrin includes the following steps: uniformly mixing 3 g of β-cyclodextrin, 3 g of citric acid, 4 g of sodium hypophosphite, and 45 g of water, then reacting at 100℃ for 4 h, then increasing the temperature to 140℃ for 15 min, decreasing to room temperature, uniformly mixing with 20 g of water, then adding 30 g of anhydrous ethanol, standing for precipitation, filtering, washing the filter cake with anhydrous ethanol, and drying to obtain the modified cyclodextrin.

[0037] The preparation method of the astragalus extract includes the following steps:

[0038] An alcohol solution of 5 wt% is adjusted to pH 12 with NaOH to obtain an alkali alcohol solution; dried astragalus root powder is sieved through a 30-mesh screen, 12 times the weight of the alkali alcohol solution is added, and reflux extraction is performed at 90℃ for 2 h, and then filtered; the filter residue is added with 10 times the weight of the alkali alcohol solution, and the above operation is repeated, and then filtered, and the filtrates of the two times are combined; a 5 wt% hydrochloric acid aqueous solution is used to adjust the pH to neutral, the filtrate is concentrated to one tenth of the original volume, then 95% ethanol aqueous solution is added to the original volume, and then it is left to stand overnight at 4℃; it is filtered, the filter cake is washed with anhydrous ethanol, and then dried; and then 2 times the weight of a 25 wt% ethanol aqueous solution is used for dissolution, and an astragalus extract is obtained.

[0039] Example 2

[0040] 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: uniformly mixing 3 g of β-cyclodextrin, 3 g of succinic acid, 4 g of sodium hypophosphite, and 45 g of water, then reacting at 100℃ for 4 h, then increasing the temperature to 140℃ for 15 min, decreasing to room temperature; uniformly mixing with 20 g of water, then adding 30 g of anhydrous ethanol, standing for precipitation, filtering, washing the filter cake with anhydrous ethanol, and drying to obtain the modified cyclodextrin.

[0041] Example 3

[0042] A culture method for preparing umbilical cord mesenchymal stem cell exosomes, the difference from example 2 is only that the preparation method of the modified cyclodextrin comprises the following steps: 3g of β-cyclodextrin, 3g of malic acid, 4g of sodium hypophosphite and 45g of water are uniformly mixed, then reacted at 100℃ for 4h, then heated to 140℃ for 15min, and then cooled to room temperature; after uniformly mixing with 20g of water, 30g of anhydrous ethanol is added, precipitated, filtered, the filter cake is washed with anhydrous ethanol, and dried to obtain the modified cyclodextrin.

[0043] Comparative example 1

[0044] A culture method for preparing umbilical cord mesenchymal stem cell exosomes, comprising the following preparation steps:

[0045] S1, isolating human umbilical cord mesenchymal stem cells;

[0046] S2, subculturing the human umbilical cord mesenchymal stem cells in the mesenchymal stem cell serum-free culture medium, and taking the 4th generation of mesenchymal stem cells to culture until the cell fusion rate reaches 75%, and then digesting and recovering the cells;

[0047] S3, inoculating the digested cells into the mesenchymal stem cell serum-free culture medium containing 0.15wt% of ginseng extract, with an inoculation density of 3×10 5 / mL, culturing at 37℃ under hypoxia for 60h, recovering the supernatant, gradient centrifuging, centrifuging at 11000g for 10min, taking the supernatant, filtering, centrifuging at 100000g for 90min, collecting the exosomes, and obtaining human umbilical cord mesenchymal stem cell exosomes. The hypoxic culture condition is.

[0048] The hypoxic culture condition is that the CO2 volume fraction is 4%, the O2 volume fraction is 1%, and the remaining gas is N2.

[0049] Comparative example 2 A culture method for preparing umbilical cord mesenchymal stem cell exosomes, comprising the following preparation steps:

[0050] S1, isolating human umbilical cord mesenchymal stem cells;

[0051] S2, subculturing the human umbilical cord mesenchymal stem cells in the mesenchymal stem cell serum-free culture medium, and taking the 4th generation of mesenchymal stem cells to culture until the cell fusion rate reaches 75%, and then digesting and recovering the cells;

[0052] S3, inoculating the digested cells into the mesenchymal stem cell serum-free culture medium containing 0.15wt% of ginseng extract, with an inoculation density of 3×10 5 / mL, culturing at 37℃ under hypoxia for 60h, recovering the supernatant, gradient centrifuging, centrifuging at 11000g for 10min, taking the supernatant, filtering, centrifuging at 100000g for 90min, collecting the exosomes, and obtaining human umbilical cord mesenchymal stem cell exosomes.

[0053] The hypoxic culture condition is that the volume fraction of CO2 is 4%, the volume fraction of O2 is 1%, and the remaining gas is N2.

[0054] The preparation method of the astragalus extract includes the following steps:

[0055] The 5wt% ethanol aqueous solution is adjusted to pH 12 by NaOH powder to obtain an alkali alcohol solution, the 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℃ for 2h. Filtration is performed, 10 times the weight of the 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 by using a 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 at 4℃ overnight. Filtration is performed, the filter cake is washed with anhydrous ethanol, and drying is performed. The astragalus extract is obtained.

[0056] Comparative Example 3

[0057] A culture method for preparing umbilical cord mesenchymal stem cell exosomes, which is different from that of Example 2 only in that the step S3 is that the digested cells are inoculated into a mesenchymal stem cell serum-free culture medium containing 0.15wt% cyclodextrin astragalus extract, the inoculation density is 3×10 5 6 / mL, and the supernatant is recovered after hypoxic culture at 37℃ for 60h, gradient centrifugation is performed, 11000g centrifugation is performed for 10min, the supernatant is taken, filtration is performed, 100000g centrifugation is performed for 90min, and the exosomes are collected to obtain human umbilical cord mesenchymal stem cell exosomes.

[0058] The hypoxic culture condition is that the volume fraction of CO2 is 4%, the volume fraction of O2 is 1%, and the remaining gas is N2.

[0059] The preparation method of the cyclodextrin astragalus extract includes the following steps: 0.3g of cyclodextrin and 3g of water are uniformly mixed, 0.3g of astragalus extract is added, stirring is performed under light shielding conditions for 3h, centrifugation is performed, the supernatant is collected, and freeze-drying is performed. The cyclodextrin astragalus extract is obtained.

[0060] The preparation method of the astragalus extract is consistent with that of Example 1.

[0061] Test Example 1

[0062] Culture medium performance test

[0063] 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.

[0064] Table 1 Culture medium performance test

[0065]

[0066] By comparing examples 1-3 and comparative examples 1-3, it can be found that the properties of example 3 are the best. The possible reason for analysis is that example 3 adds apple acid modified cyclodextrin astragalus extract, the cavity structure formed by apple acid modified cyclodextrin is more flexible and the matching degree with astragalus extract is higher; example 1 adds lemon acid modified cyclodextrin astragalus extract, the molecule of lemon acid is larger, which may affect the space structure, and the inclusion effect is lower; example 2 adds succinic acid modified cyclodextrin astragalus extract, succinic acid has strong hydrophobicity, which may reduce the polarity of the cavity and is not conducive to the inclusion of water-soluble molecules.

[0067] Test example 2

[0068] Exosome protein concentration and purity

[0069] Exosome protein concentration determination: BCA protein quantitative kit was used, and ND-2000 ultramicro ultraviolet visible spectrophotometer was used to detect the total protein amount of exosomes. 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 nanoflow, and the purity of the exosomes was calculated. The protein concentration and exosome purity are shown in Table 2.

[0070] Table 2 Exosome protein concentration and purity

[0071]

[0072] 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 possible reason for analysis is that the astragalus extract in the medium of comparative example 2 contains polysaccharides, saponins, flavonoids and other components, which have 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 astragalus extract.

[0073] 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 possible reason for analysis is that example 3 adds apple acid modified cyclodextrin astragalus extract, 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 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 in that: The culture medium consists of 99.79-99.99 wt% serum-free culture medium for mesenchymal stem cells and 0.01-0.21 wt% additives; The additive is cyclodextrin-modified Astragalus membranaceus extract; The preparation method of the cyclodextrin-modified Astragalus extract includes the following steps: Mix 0.1-0.5 parts of modified cyclodextrin and 1-5 parts of water evenly, add 0.1-0.5 parts of Astragalus extract, stir for 2-4 hours under light-protected conditions, centrifuge, collect the supernatant and freeze-dry to obtain cyclodextrin-modified Astragalus extract. The preparation method of the Astragalus extract includes the following steps: To obtain an alkaline-alcoholic solution, adjust the pH of a 3-8 wt% ethanol aqueous solution to 11-13 using NaOH powder. Pass dried Astragalus root powder through a 20-40 mesh sieve, add 10-15 times its weight of the alkaline-alcoholic solution, and reflux at 80-100℃ for 1-3 hours. Filter. Repeat the above operation with 8-12 times its weight of the alkaline-alcoholic solution on the filter residue, filter, and combine the two filtrates. Adjust the pH to neutral using a 1-10 wt% hydrochloric acid aqueous solution, and concentrate the filtrate to one-tenth of its original volume. Then, add 95 wt% ethanol aqueous solution to bring the volume back to the original volume and let it stand overnight at 0-4℃. Filter, wash the filter cake with anhydrous ethanol, and dry. Then, dissolve the cake in a 1-3 times its weight of a 20-30 wt% ethanol aqueous solution to obtain the Astragalus extract. The preparation method of the modified cyclodextrin includes the following steps, in parts by mass: Mix 1-6 parts of β-cyclodextrin, 1-5 parts of modifier, 1-6 parts of sodium hypophosphite and 30-60 parts of water evenly, then react at 80-120℃ for 3-5 hours, then raise the temperature to 120-160℃ and react for 4-30 minutes, cool to room temperature, add 10-30 parts of water and mix evenly, then add 20-40 parts of anhydrous ethanol, let stand to precipitate, filter, wash the filter cake with anhydrous ethanol and dry to obtain modified cyclodextrin. The modifier is malic acid.

2. The method for preparing umbilical cord mesenchymal stem cell exosomes as described in claim 1, characterized in that, Includes the following steps: S1. Isolation of human umbilical cord mesenchymal stem cells; S2. Human umbilical cord mesenchymal stem cells are passaged in serum-free mesenchymal stem cell culture medium. After passages 3-5, mesenchymal stem cells are cultured until the cell fusion rate reaches 70-80%, and then the cells are digested and recovered. S3. Seed the digested cells into serum-free mesenchymal stem cell culture medium containing adjuvants at a density of 1×10⁶ cells / mL. 5 -5×10 5 Human umbilical cord mesenchymal stem cell exosomes were obtained by culturing at 36-38℃ under hypoxic conditions for 48-72 hours, collecting the supernatant, performing gradient centrifugation, and collecting the exosomes.

3. The method for preparing umbilical cord mesenchymal stem cell exosomes as described in claim 2, characterized in that: In step S3, the conditions for the low-oxygen culture are: CO2 volume fraction of 3-8%, O2 volume fraction of ≤1%, and the remaining gas is N2.

4. The method for preparing umbilical cord mesenchymal stem cell exosomes as described in claim 2, characterized in that: In step S3, the gradient centrifugation method includes the following steps: centrifuge at 10000-12000g for 10-15min, take the supernatant, filter, and centrifuge at 100000-110000g for 90-100min.

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