Culture medium and method for promoting mesenchymal stem cells to secrete exosomes

Abstract

The invention provides a culture medium and a method for promoting mesenchymal stem cells to secrete exosomes, and belongs to the technical field of cell biology. The culture medium provided by the invention comprises a basic culture medium and the trichostatin A with the final concentration of 10-100nM. The TSA in the culture medium can significantly enhance MSCs exosome secretion under low-concentration and short-time stimulation, does not damage cell activity, does not affect basic biological characteristics and functions of the exosome, significantly increases the MSCs exosome secretion amount by more than 2 times, greatly improves the production efficiency, and provides a new strategy for large-scale production of the exosome.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology and relates to a culture medium and method for promoting the secretion of exosomes by mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are adult stem cells with self-renewal and multi-lineage differentiation potential, and are widely found in tissues such as bone marrow, adipose tissue, and umbilical cord.

[0003] Recent studies have found that the therapeutic effects of MSCs in tissue repair, immune regulation, and anti-fibrosis do not primarily rely on their direct differentiation into target cells, but rather on paracrine mechanisms. Exosomes (Exos) are one of the key effector factors in their function. Exosomes are nanoscale vesicles secreted by mesenchymal stem cells, carrying bioactive substances such as proteins, nucleic acids, and lipids. They can mediate intercellular communication and show great potential for application in disease treatment.

[0004] Exosome culture methods mainly include optimizing culture conditions (such as three-dimensional culture and hypoxic culture), stimulation with cytokines (such as IFN-γ and TNF-α), physical stimulation (such as fluid shear force), and gene editing. However, these methods have some limitations, such as: three-dimensional culture and bioreactors are expensive and complex to operate; cytokine stimulation may introduce uncertain immunogenicity; physical stimulation requires sophisticated equipment and is difficult to scale up; gene editing, such as overexpression of exosome-related proteins, involves technical complexity and safety risks. These limitations result in limited exosome secretion from cultured MSCs, severely restricting their development in basic research and clinical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium and method for promoting the secretion of exosomes by mesenchymal stem cells, so as to solve the problem of low exosome yield of existing MSCs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this application provides a culture medium for promoting the secretion of exosomes by mesenchymal stem cells, the culture medium comprising a basal culture medium and trogostatin A at a final concentration of 10-100 nM, wherein the mesenchymal stem cells are cultured in the culture medium for 12-24 hours.

[0007] Secondly, this application provides a method for promoting the secretion of exosomes by mesenchymal stem cells, the method comprising: When frozen MSCs are cultured to passage 3-5 and the cell confluence reaches 60-80%, the original culture medium is discarded, the cells are washed, and then replaced with the culture medium described in the first aspect for 12-24 hours. After culture, exosomes were separated and collected using differential centrifugation.

[0008] The present invention has the following beneficial effects: (1) In this application, it was discovered for the first time that TSA can significantly enhance the secretion of MSCs exosomes under low concentration and short-term stimulation without damaging cell activity or affecting the basic biological characteristics and functions of exosomes. At the same time, it significantly increases the secretion of MSCs exosomes by more than 2 times, greatly improving production efficiency and providing a new strategy for the large-scale production of exosomes.

[0009] (2) The method provided in this application significantly increases the yield of exosomes without significantly negatively affecting the cell viability of MSCs, the biological characteristics and functions of exosomes, thus ensuring the biosafety and effectiveness of the obtained exosomes.

[0010] (3) The stimulation intervention provided in this application is based on conventional two-dimensional cell culture only. It does not require complex equipment or expensive cytokines, has low production cost and high efficiency, and is easy to promote and implement in ordinary laboratories and production workshops. It is suitable for mass production. Attached Figure Description

[0011] Figure 1 Comparative TEM (Transmission Electron Microscope) images of exosomes cultured in Example 5 and Comparative Example 1; Figure 2 The particle size distribution diagrams of exosomes obtained from the culture of Example 5 and Comparative Example 1 are shown. Figure 3 This is a comparison diagram of the number of vesicles in exosomes obtained from Example 5 and Comparative Example 1. Figure 4 This is a comparison chart of the exosome protein concentrations obtained from the cultures of Example 5 and Comparative Example 1. Figure 5 A comparison of exosomal protein concentrations obtained from cultures with different TSA concentrations; Figure 6 A comparison of exosomal protein concentrations obtained from cultures with different TSA incubation times; Figure 7 Figure showing the effect of the CCK-8 kit on the proliferation toxicity of TSA on MSCs; Figure 8 The results of the cell scratch healing experiment are shown in Figure A, which is a microscopic image of the cell scratch, and Figure B is a statistical graph of the cell scratch healing rate. Detailed Implementation

[0012] In a first aspect, this application provides a culture medium for promoting the secretion of exosomes by mesenchymal stem cells, the culture medium comprising a basal culture medium and a final concentration of 10-100 nM of trogostatin A, wherein the mesenchymal stem cells are cultured in the culture medium for 12-24 hours.

[0013] TSA (trachostatin A) is a histone deacetylase inhibitor that increases the acetylation of intracellular histones, thereby altering chromatin structure and regulating gene expression. TSA can induce cell differentiation, apoptosis, and cell cycle arrest, particularly affecting cage protein gene expression and cytoskeleton stability. Cage proteins are involved in the formation and transport of multivesicular bodies (MVBs), and cytoskeleton regulation of MVB directional movement and membrane fusion includes microtubule acetylation and actin remodeling. Therefore, adding TSA during MSC culture can stimulate MSCs to secrete large amounts of exosomes.

[0014] In this application, the basal culture medium includes DMEM medium, α-MEM medium, or DMEM / F12 medium. Preferably, the final concentration of trogostatin A in the culture medium is 50 nM.

[0015] Secondly, this application provides a method for promoting the secretion of exosomes by mesenchymal stem cells, the method comprising: S01: When frozen MSCs are cultured to passage 3-5 and the cell confluence reaches 60-80%, discard the original culture medium, wash them, and replace them with the culture medium from the first aspect that promotes the secretion of exosomes by mesenchymal stem cells, and culture for 12-24 hours.

[0016] After thawing frozen MSCs, they were cultured in α-MEM medium containing 10% FBS in a humidified incubator at 37°C and 5% CO2, with the medium changed every 2-3 days. When the MSCs reached 80-90% confluence, they were passaged using trypsin. At passages 3-5, healthy MSCs were harvested at a rate of 5 × 10⁶ cells / year. 3 -6×10 3 / cm 2 The cells were inoculated at the desired density in α-MEM medium containing 10% FBS and cultured. When the cell confluence reached 60-80%, the original medium was discarded, and the MSCs were gently washed twice with PBS. The medium was then replaced with the medium provided in this application and cultured for another 12-24 hours.

[0017] Preferably, MSCs are cultured in the culture medium for 12 hours. MSCs are mesenchymal stem cells derived from human umbilical cord, adipose tissue, bone marrow, or dental pulp.

[0018] S02: After culture, the supernatant in the cell culture medium was transferred to centrifuge tubes, and exosomes were collected by differential centrifugation. The specific centrifugation process was as follows: centrifugation at 300×g for 10 min at 4℃ to remove MSCs cells from the supernatant; centrifugation at 2000×g for 20 min at 4℃ to remove dead MSCs cells; centrifugation at 10000×g for 30 min at 4℃ to remove cell debris; the supernatant was transferred to an ultracentrifuge tube and centrifuged at 1000000×g for 70 min at 4℃. After removing the supernatant, the resulting precipitate was crude exosomes; the exosome precipitate was resuspended in sterile PBS, and washed and purified by centrifugation at 1000000×g for 70 min at 4℃. After resuspending in PBS, the exosomes were obtained.

[0019] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0020] Example 1 This application provides a culture medium for promoting the secretion of exosomes by mesenchymal stem cells. The culture medium includes DMEM medium and trogostatin A at a final concentration of 10 nM. The mesenchymal stem cells are cultured in this medium for 12 hours.

[0021] Example 2 This application provides a culture medium for promoting the secretion of exosomes by mesenchymal stem cells. The culture medium includes α-MEM medium and trogostatin A at a final concentration of 50 nM. The mesenchymal stem cells are cultured in this medium for 12 hours.

[0022] Example 3 This application provides a culture medium for promoting the secretion of exosomes by mesenchymal stem cells. The culture medium includes DMEM / F12 medium and trogostatin A at a final concentration of 100 nM. The mesenchymal stem cells are cultured in this medium for 12 hours.

[0023] Example 4 This application provides a method for promoting the secretion of exosomes by mesenchymal stem cells, the method comprising: S401: After thawing frozen human umbilical cord MSCs, they were cultured in α-MEM medium containing 10% FBS in a humidified incubator at 37°C and 5% CO2, with the medium changed every 3 days. When the MSCs reached 80-90% confluence, they were digested and passaged using trypsin. At passage 4, healthy MSCs were harvested at a rate of 5 × 10⁻⁶ cells / year. 3 / cm 2The cells were inoculated at the desired density in α-MEM medium containing 10% FBS and cultured. When the cell confluence reached 70%, the original medium was discarded, and the MSCs were gently washed twice with PBS. The medium was then replaced with that from Example 1 and cultured for another 12 hours.

[0024] S402: After culture, the supernatant in the cell culture medium was transferred to centrifuge tubes, and exosomes were collected by differential centrifugation. The specific centrifugation process was as follows: centrifugation at 4℃ and 300×g for 10 min to remove MSCs cells from the supernatant; centrifugation at 4℃ and 2000×g for 20 min to remove dead MSCs cells; centrifugation at 4℃ and 10000×g for 30 min to remove cell debris; the supernatant was transferred to an ultracentrifuge tube and centrifuged at 4℃ and 1000000×g for 70 min. After removing the supernatant, the resulting precipitate was crude exosomes; the exosome precipitate was resuspended in sterile PBS, and washed and purified by centrifugation at 4℃ and 1000000×g for 70 min. After resuspending in PBS, the exosomes were obtained.

[0025] Example 5 This application provides a method for promoting the secretion of exosomes by mesenchymal stem cells. The method is the same as in Example 4, except that the culture medium in Example 2 is used to continue culturing for 12 hours.

[0026] Example 6 This application provides a method for promoting the secretion of exosomes by mesenchymal stem cells. The method is the same as in Example 4, except that the culture medium in Example 3 is used to continue culturing for 12 hours.

[0027] Example 7 This application provides a method for promoting the secretion of exosomes by mesenchymal stem cells. The method is the same as in Example 4, except that the culture medium in Example 2 is used to continue culturing for 12 hours.

[0028] Comparative Example 1 This application provides a comparative example of a method for promoting the secretion of exosomes by mesenchymal stem cells. The method is the same as in Example 5, except that in S401, after discarding the original culture medium, an equal volume of culture medium containing DMSO solvent and without FBS is used to continue culturing for 12 hours.

[0029] Test 1 This application uses transmission electron microscopy (TEM), a nanoparticle tracking analyzer, and a BCA protein quantification kit to detect the exosomes obtained in Example 5 and Comparative Example 1, respectively, to determine particle concentration and size distribution, and exosome protein concentration. Figure 1-4 .

[0030] From the appendix Figure 1 , 2As can be seen, the exosomes obtained in Example 5 and Comparative Example 1 both exhibit a typical saucer-shaped biconcave disc structure, with particle sizes mainly distributed between 60-100 nm, a peak at around 75 nm, and no significant difference in particle size distribution. (From Appendix...) Figure 3 , 4 As can be seen, the particle concentrations of exosomes obtained from the cultures in Example 5 and Comparative Example 1 were 1.06 × 10⁻⁶ and 10⁻⁶, respectively. 9 / mL, 4.82×10 8 / mL, there was a significant difference ( P <0.01); Compared with Comparative Example 1, the exosome vesicle yield and exosome protein yield obtained in Example 5 increased by 2.19 times and 2.29 times, respectively, with the increase factors being basically the same.

[0031] Detection 2 In this application, the BCA protein concentration assay was used to determine the protein yield of exosomes obtained from Examples 4, 5, 6, and Comparative Example 1, respectively, to determine the effect of TSA concentration on exosome yield. Figure 5 In Examples 4, 5, and 6, the concentrations of TSA were 10 nM, 50 nM, and 100 nM, respectively, while Comparative Example 1 contained an equal volume of DMSO solvent.

[0032] From the appendix Figure 5 As can be seen, compared with Comparative Example 1, the exosome yields obtained from Examples 4, 5, and 6 increased by 1.27 times, 2.29 times, and 1.46 times, respectively. This indicates that TSA can stimulate MSCs to produce the most exosomes at a concentration of 50 nM. The reason why higher TSA concentrations lead to a decrease in exosome secretion may be due to cytotoxicity causing a decrease in yield increase.

[0033] Detection 3 Referring to the methods in Example 5 and Comparative Example 1, this application uses the BCA protein concentration detection method to determine the protein yield of exosomes under different stimulation culture times, in order to determine the effect of TSA treatment time on exosome yield, and obtain the attached... Figure 6 The culture times after adding the culture medium from Example 2 and Comparative Example 1 were 6h, 12h, 24h, and 48h, respectively, with 3 replicates for each culture time.

[0034] From the appendix Figure 6 As can be seen, the exosome content in Comparative Example 1 continuously increased with the extension of culture time, while the exosome content in Example 5 first increased and then decreased. Furthermore, under the same culture time, the exosome content in Example 5 was higher than that in Comparative Example 1. This indicates that TSA can stimulate MSCs to produce the most exosomes when the culture time is 12 hours.

[0035] Detection 4 In this application, the CCK-8 assay was used to detect the effect of TSA on MSCs viability. The specific experimental procedure is as follows: P3 generation umbilical cord MSCs in good growth condition were digested with trypsin and resuspended for counting. 5 × 10⁶ cells per well were used. 3 Cells were seeded at a density of 100 μL in α-MEM complete medium containing 10% FBS into 96-well plates. The plates were pre-cultured at 37°C in a 5% CO2 incubator for 24 h to allow for full cell adhesion and entry into the logarithmic growth phase. The cells were then divided into three groups: a blank control group, a solvent control group, and an experimental group. The blank control group contained only complete medium, no cells, and was set up in 3 replicates for absorbance calibration. The solvent control group contained complete medium containing an equal volume of DMSO and was set up in 6 replicates. The experimental group used the medium from Example 2 and was set up in 6 replicates. MSCs cultured to the logarithmic growth phase were added to the blank control group, solvent control group, and experimental group, respectively, for 12 h of stimulation. Four h earlier, 10 μL of CCK-8 solution was added to each well. After 4 h, the absorbance of each well was measured at 450 nm using a microplate reader to obtain the absorbance values. Figure 7 .

[0036] From the appendix Figure 7 It can be seen that at a wavelength of 450 nm, there was no significant difference in the absorbance values ​​of each well between the solvent control group and the experimental group. P The result >0.05 indicates that TSA at a concentration of 50 nM and a stimulation time of less than 12 hours has no significant cytotoxicity to human mesenchymal stem cells. This provides direct quantitative data support for the optimization of TSA concentration and short-term stimulation in this application, ensuring that it has no significant negative impact on cell activity and guaranteeing its feasibility and reliability in subsequent large-scale exosome preparation.

[0037] Detection 5 Exosomes were extracted using conventional methods to form conventional exosomes. The content of conventional exosomes and exosomes obtained from Example 5 was determined using the BCA protein concentration assay.

[0038] Resuscitate human cardiac microvascular endothelial cells and adjust the cell concentration to 2 × 10⁻⁶. 5 / mL, seeded at 2mL / well in 6-well plates and cultured and observed. When the cell density reached about 80%, a straight scratch was made on the monolayer of cells using a 200μL sterile pipette tip. The cells were gently rinsed with PBS to remove detached cells, and then replaced with basal medium containing 1% FBS. The cells were divided into three groups: Control group (no additives); Control+Exos group (added with 50μg / mL of conventional MSCs exosomes); Control+TSA-Exos group (added with 50μg / mL of exosomes prepared in Example 5). Images were taken at the same location under a microscope at 0h and 24h. The scratch area was measured using ImageJ software, and the cell migration and healing rate was calculated. Healing rate (%) = [(A0-A...] 24 ) / A0] ×100%, where A0 and A24 represent the scratch areas at 0h and 24h respectively, to obtain the attached Figure 8 .

[0039] From the appendix Figure 8 As can be seen from A, compared to the Control group, the scratch areas of the Control+Exos group and the Control+TSA-Exos group are significantly reduced; (The last part, "from the attached...", appears to be a fragment and doesn't translate directly.) Figure 8 As shown in Figure B, there was no statistically significant difference in cell migration and healing rates between the Control+Exos group and the Control+TSA-Exos group. P >0.05), but both were significantly higher than the Control group ( P <0.01). This indicates that exosomes secreted by TSA-stimulated MSCs have equivalent biological activity in promoting endothelial cell migration as exosomes obtained by conventional culture.

[0040] In summary, TSA stimulation of MSCs can significantly promote exosome secretion, and the operation is simple. It successfully achieves the core goal of greatly increasing exosome production without sacrificing exosome function and quality, providing a new and efficient solution to address the bottleneck of exosome production in MSCs, and has important application value.

[0041] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A culture medium for promoting the secretion of exosomes by mesenchymal stem cells, characterized in that, The culture medium includes a basal culture medium and a final concentration of 10-100 nM of trogostatin A, and the mesenchymal stem cells are cultured in the culture medium for 12-24 hours.

2. The culture medium for promoting exosome secretion by mesenchymal stem cells according to claim 1, characterized in that, The final concentration of the quabustatin A was 50 nM.

3. The culture medium for promoting exosome secretion by mesenchymal stem cells according to claim 1, characterized in that, The basal culture medium includes DMEM medium, α-MEM medium, or DMEM / F12 medium.

4. A method for promoting the secretion of exosomes by mesenchymal stem cells, characterized in that, include: When frozen MSCs are cultured to passage 3-5 and the cell confluence reaches 60-80%, the original culture medium is discarded, the cells are washed, and then replaced with the culture medium for promoting the secretion of exosomes by mesenchymal stem cells as described in any one of claims 1-3 for 12-24 hours. After culture, exosomes were separated and collected using differential centrifugation.

5. The method for promoting the secretion of exosomes by mesenchymal stem cells according to claim 4, characterized in that, The MSCs are cultured for 12 hours in the culture medium for promoting the secretion of exosomes by mesenchymal stem cells as described in any one of claims 1-3.

6. The method for promoting the secretion of exosomes by mesenchymal stem cells according to claim 4, characterized in that, The differential centrifugation method includes: centrifuging at 300×g for 10 min, 2000×g for 20 min, 10000×g for 30 min, 1000000×g for 70 min, resuspending, and centrifuging at 1000000×g for 70 min at 4°C to obtain exosomes.

7. The method for promoting the secretion of exosomes by mesenchymal stem cells according to claim 6, characterized in that, Resuspended in sterile PBS.

8. The method for promoting the secretion of exosomes by mesenchymal stem cells according to claim 4, characterized in that, The MSCs are mesenchymal stem cells derived from human umbilical cord, fat, bone marrow, or dental pulp.

9. The method for promoting the secretion of exosomes by mesenchymal stem cells according to claim 4, characterized in that, The culture of cryopreserved MSCs to the 3rd-5th generation includes: After thawing, the frozen MSCs were inoculated into α-MEM medium containing 10% FBS and cultured in a saturated humidity incubator at 37°C and 5% CO2. When the MSCs reach a cell confluence of 80-90%, they are passaged using trypsin digestion to form MSCs of the 3rd to 5th generation.

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