Method for promoting balling culture of mesenchymal stem cells

Abstract

The invention discloses a method for promoting mesenchymal stem cell balling culture, and belongs to the field of stem cell biology. The method comprises the following steps: inoculating mesenchymal stem cells into a culture medium to obtain a cell culture solution, adding a DMSO solution of a compound combination CT into the cell culture solution, uniformly mixing, and carrying out balling culture to obtain the three-dimensional spherical mesenchymal stem cells, wherein the compound combination CT is a combination of Chroman 1 and Trans-ISRIB, and the molar ratio of the Chroman 1 to the Trans-ISRIB is (1: 10) to (1: 20). The compound combination CT can significantly improve the efficiency and consistency of MSC sphere formation, and effectively alleviate cell apoptosis, thereby enhancing the biological activity of MSC spheres and the extracellular vesicle generation ability. The method is suitable for a three-dimensional culture system without a bracket, and can effectively promote spontaneous assembly of MSC into spheres in an in-vitro culture process.

Description

Technical Field

[0001] The invention relates to the field of stem cell biology, and in particular to a method for promoting spheroidization culture of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSC) are multipotent stem cells with the ability of self-renewal and differentiation. They can differentiate into a variety of tissue cells and have broad prospects for clinical application. In the process of clinical transformation and application of mesenchymal stem cells, cell culture is the most basic and important part. The traditional two-dimensional cell culture method refers to immersing stem cells in culture medium and extending and growing along a two-dimensional plane on the surface of ordinary glass or plastic culture dishes. The two-dimensional cell culture method has been developed for a long time and the technology is mature. It is easy to observe the cell state during culture. Although the two-dimensional stem cell culture method can effectively expand cells, it has the disadvantages of too large a footprint of cell culture containers and uneven cell quality in large-scale culture. In addition, studies have shown that the two-dimensional culture method cannot maintain the original cell characteristics of stem cells for a long time, and this culture method cannot accurately describe and simulate the rich environment and complex processes in the body, such as signal transduction and spatial structure changes. Therefore, the data collected by the two-dimensional cell culture method may be misleading for its in vivo application prediction.

[0003] Cell spheroid culture technology refers to the process of promoting cells to form cell clusters in three-dimensional space without scaffold materials, so that they can grow, proliferate and migrate in three-dimensional space. The three-dimensional conditions of spheroid culture better simulate the growth environment of cells in the body, and can more closely imitate the complex interactions between cells and tissues and the microenvironment in the body, providing cells with a microenvironment suitable for growth and differentiation, and having the ability to construct tissue-like structures in vitro. Studies have shown that stem cells cultured in spheroids exhibit a higher nuclear-cytoplasmic ratio and differentiation ability, and increased expression of chemokines.

[0004] Therefore, it is necessary to screen a combination of compounds that promote MSC sphere culture to promote MSC sphere culture in vitro and provide new methods and tools for the clinical application of MSC. Summary of the invention

[0005] The purpose of the present invention is to provide a method for promoting the spheroidization culture of mesenchymal stem cells, which effectively promotes the formation and maintenance of three-dimensional spheroids of MSCs without a scaffold by screening a specific small molecule compound combination CT.

[0006] To achieve the above objectives, the following technical solutions are adopted: In a first aspect, the present invention provides a method for promoting spheroidization of mesenchymal stem cells, comprising: Mesenchymal stem cells are inoculated into a culture medium to obtain a cell culture fluid, a DMSO solution of a compound combination CT is added to the cell culture fluid and mixed, and sphere culture is performed to obtain three-dimensional sphere mesenchymal stem cells; wherein the compound combination CT is a combination of Chroman 1 and Trans-ISRIB, and the molar ratio of Chroman 1 to Trans-ISRIB is 1:10-1:20.

[0007] In some embodiments, the culture medium is composed of: mesenchymal stem cell culture medium plus 5% V / V human platelet lysate.

[0008] In some embodiments, the molar ratio of Chroman 1 to Trans-ISRIB is 1:14.

[0009] In some embodiments, in the DMSO solution of the compound combination CT, the concentration of Chroman 1 is 40-60 μM, and the concentration of Trans-ISRIB is 0.6-0.8 mM.

[0010] In some embodiments, in the DMSO solution of the compound combination CT, the concentration of Chroman 1 is 50 μM, and the concentration of Trans-ISRIB is 0.7 mM.

[0011] In some embodiments, the volume ratio of the added amount of the DMSO solution of the compound combination CT to the cell culture medium is 1:1000.

[0012] In some embodiments, the method comprises the following steps: S1: After the mesenchymal stem cells are expanded in two-dimensional culture, they are digested into single cells using TrypleLE digestion enzyme, and then digestion is terminated with PBS buffer. After centrifugation, the supernatant is discarded and the cells are resuspended in culture medium into a single cell suspension; S2: Single cell suspension was prepared at 5×10 4 / mL~20×10 4 The cells were inoculated into a culture plate at a density of 1:1 / mL to obtain a cell culture solution, a DMSO solution of the compound combination CT was added to the cell culture solution and mixed evenly, and sphere culture was performed to obtain three-dimensional sphere mesenchymal stem cells.

[0013] In some embodiments, the sphere formation culture comprises: culturing in a culture plate at 36.5-37.5°C, 4.5-5.5% CO2, on a horizontal shaker at 60-80 rpm / min for 12-48 hours, preferably culturing at 37°C, 5% CO2, on a horizontal shaker at 70 rpm / min for 12-48 hours.

[0014] In some embodiments, the cell density in the cell culture medium is 15×104 / mL.

[0015] The present invention found that the compound combination (CT) composed of Chroman 1 and Trans-ISRIB can significantly improve the efficiency and consistency of MSC sphere formation, and effectively reduce cell apoptosis, thereby enhancing the biological activity of MSC spheres and the ability to generate extracellular vesicles. In addition, the compound combination CT can also promote the proliferation of MSC under two-dimensional culture conditions. The compound combination CT is suitable for a scaffold-free three-dimensional culture system, and can effectively promote the spontaneous assembly of MSC into spheres during in vitro culture, simulating the in vivo microenvironment, thereby providing new methods and tools for the clinical application of MSC.

[0016] The advantages and positive effects of the method for promoting spheroidization of mesenchymal stem cells described in the present invention are: 1. The present invention establishes a MSC sphere culture system and determines that the compound combination CT effectively accelerates the MSC sphere process and promotes the formation of uniform spheres by MSC; 2. The present invention establishes a CT intervention MSC sphere culture system, which effectively improves the stemness of MSC cells and increases their secretory activity; 3. The CT intervention MSC sphere culture system effectively promotes the secretion of MSC extracellular vesicles, which can be used for extracellular vesicle production in mesenchymal stem cell therapy, providing a new tool for stem cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The morphology of MSC spheres cultured after 12 hours of intervention with compound CT in the present invention, wherein A is the DMSO control group, and B is the MSC sphere morphology in the CT group, and the scale bar is 100 μm; Figure 2 Comparison of cell stemness gene mRNA expression between the DMSO control group and the CT group after spheroid culture for 12 hours in the embodiment of the present invention, wherein A is a statistical graph of stemness gene Nanog expression, B is a statistical graph of stemness gene Oct4 expression, and C is a statistical graph of stemness gene Sox2 expression, n=4, all data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 3Comparison of MSC paracrine-related gene expression between the DMSO control group and the CT group after spheroid culture for 12 hours in the embodiment of the present invention, wherein A is the IGFBP3 gene expression statistical graph, B is the SCF gene expression statistical graph, C is the Ang2 gene expression statistical graph, and D is the GDF15 gene expression statistical graph, n=4, all data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 4 The cell apoptosis was detected after 48 hours of spheroid culture in the embodiment of the present invention, wherein A is the CalceinAM / PI staining confocal three-dimensional scanning reconstruction image of the spheroids in the DMSO control group and the CT group, and the scale bar is 100 μm; B is the CalceinAM / PI staining confocal three-dimensional scanning statistical graph, all data are expressed as mean ± standard deviation, * p <0.05; C is the Western Blotting result of apoptosis protein Cas3 in spheroids of DMSO group and CT group; D is the statistical graph of Western Blotting result of apoptosis protein Cas3 in spheroids of MSO group and CT group, all data are expressed as mean ± standard deviation, ***p<0.001; Figure 5 The experimental results of increasing the secretion of extracellular vesicles of spheroidized MSCs by CT in the embodiments of the present invention, wherein A, B, and C are the comparison of the mRNA expression of extracellular vesicle generation genes between the DMSO control group and the CT group after spheroidization for 12 hours, wherein A is a statistical graph of CD63 gene expression, B is a statistical graph of CD9 gene expression, C is a statistical graph of CD81 expression, n=4, D is a statistical graph comparing the extracellular vesicle content in the supernatant of the DMSO control group and the CT group, n=3, and E is the result of Western Blotting to identify the extracellular vesicle markers of spheroidized MSCs in the DMSO control group and the CT group; all data are expressed as mean ± standard deviation, *p<0.05, ***p<0.001. DETAILED DESCRIPTION

[0018] The technical scheme of the present invention is further described below by means of the accompanying drawings and examples. Unless otherwise defined, the technical terms or scientific terms used in the present invention should be the common meanings understood by people with ordinary skills in the field to which the present invention belongs. Unless otherwise defined, the instruments, equipment and reagents used in the present invention are all conventionally commercially available.

[0019] Chroman 1 is a highly effective ROCK-II inhibitor purchased from MedChemExpress, catalog number HY-15392, with a specification of 10 mM*1 mL dissolved in DMSO.

[0020] Trans-ISRIB is an integrated stress response (ISR) inhibitor purchased from Tocris, catalog number 5284, with a specification of 2 mg.

[0021] In this embodiment, in the DMSO solution of the compound combination CT, the concentration of Chroman 1 is 50 μM, and the concentration of Trans-ISRIB is 0.7 mM. The preparation method is: weigh Trans-ISRIB according to the marked molecular weight, dissolve it in DMSO to prepare a 10 mM Trans-ISRIB DMSO solution. Take 5 μL of 10 mM Chroman 1 DMSO solution and 70 μL of 10 mM Trans-ISRIB DMSO solution, add 925 μL DMSO and mix well to prepare 1 mL of DMSO solution of the compound combination CT (wherein the concentration of Chroman 1 is 50 μM and the concentration of Trans-ISRIB is 0.7 mM).

[0022] The composition of the culture medium used in the present application is: mesenchymal stem cell culture medium (Sartorius, 05-200-1A) added with 5% V / V human platelet lysate (Sartorius, PLTGOLD1000R).

[0023] Example 1: Experimental method MSC cell 2D expansion culture After MSCs were revived, they were expanded and subcultured at a ratio of 1:3 until the cell number was sufficient.

[0024] MSC sphere culture Preparation of single cell suspension: After discarding the supernatant of 2D cultured MSCs, wash once with an appropriate amount of PBS buffer, add an appropriate amount of trypleLE digestion enzyme, digest at 37°C for 2 min, add 2 times the volume of PBS buffer to terminate digestion, blow thoroughly, centrifuge at 1000 rpm / min for 5 min, add an appropriate amount of culture medium to resuspend into a single cell suspension; Cell counting: Pipette 10 uL of single cell suspension and mix thoroughly with 10 uL of trypan blue dye solution, then count the cells; Cell seeding: 15×10 4 / mL cell density, 2.5 mL culture system per well, inoculate the corresponding volume of single cell suspension and culture base into a low-adhesion culture six-well plate to obtain cell culture medium, where the single cell suspension volume (mL) = 2.5 × 15 × 10 4 / single cell suspension cell density, inoculation medium volume (mL) = 2.5-single cell suspension volume (mL), 2.5 uL DMSO was added to each well of the control group, and 2.5 uL DMSO solution of compound combination CT was added to each well of the experimental group; Spheroid culture: The low-adhesion culture six-well plate was placed on a horizontal shaker with the speed adjusted to 70 rpm / min and placed in an incubator for spheroid culture; Observation of spheroid formation: After 12 hours and 24 hours of spheroid culture, the low-adhesion culture plate was removed and allowed to stand at room temperature for 5 minutes until the cells settled to the bottom. The spheroid morphology of the cells was observed under a microscope.

[0025] MSC spheroidization extracellular vesicle extraction The single cell suspension preparation and cell counting methods were the same as above; The principle of cell inoculation was the same as above. To increase the yield of cell supernatant, spheroid cells were cultured in 125 mL cell culture conical spinner flasks using serum-free mesenchymal stem cell medium (Youkang, NC0105.S). The volume of the spheroid culture system in the spinner flask was 30 mL, so the volume of the single cell suspension (mL) = 30 × 15 × 10 4 / single cell suspension cell density, inoculation medium volume (mL) = 30-single cell suspension volume (mL), 30 uL DMSO was added to each well of the control group, and 30 uL DMSO solution of compound combination CT was added to each well of the experimental group; The method of sphering culture is the same as above; Collection and pretreatment of sphere culture supernatant: After MSC sphere culture for 24 hours, the cell flask was removed and the cell supernatant was transferred to a 50 mL centrifuge tube. After centrifugation at 2000 g and 4°C for 20 min, the supernatant was transferred to a new centrifuge tube and centrifuged at 13500 g and 4°C for 30 min. Ultracentrifugation: The pretreated cell supernatant was then subjected to ultracentrifugation at 100,000 g for 120 min. After centrifugation, the supernatant was discarded, leaving only the precipitate. The extracellular vesicle precipitate was resuspended in PBS buffer and stored at -80°C for subsequent experiments.

[0026] Identification of EVs in spheroid culture 4.1 Nanoparticle Size Analysis (NTA) First, the extracellular vesicle sample was appropriately diluted and loaded into the NTA instrument. The data was analyzed and detected using Zetaview 8.04.02 software to obtain the size distribution and particle size concentration of the vesicles, providing accurate information on the physical properties of the sample extracellular vesicles; Western Blot Experiment The protein of the extracellular vesicle sample was extracted, and the protein concentration was determined by BCA experiment. Then, SDS-PAGE electrophoresis and membrane transfer were performed to calculate the relative expression level of the marker protein in the sample.

[0027] Example 2: Experimental results CT promotes MSC spheroidization. Figure 1As shown in the figure, after 12 hours of spheroid culture, MSCs in the CT group formed three-dimensional spheres of uniform size, with fewer free cells that did not form spheres, while a small amount of cell aggregation occurred in the DMSO control group, and no obvious spheres were formed. This directly confirms that CT effectively promotes MSC spheroid culture. (As shown in the figure Figure 1 shown) CT enhances the biological activity of MSC after spheroidization. Figure 2 As shown in the figure, the mRNA expressions of MSC stemness genes Nanog, Sox2, and Oct4 in the sphere-forming CT group were significantly higher than those in the DMSO group, indicating that CT can enhance the stemness of MSC cells in sphere-forming CT. Figure 3 As shown in the figure, the mRNA expressions of IGFBP, SCF, Ang2, and GDF15 paracrine factors of MSC in sphere cultured CT group were significantly higher than those in DMSO group, indicating that CT can enhance the secretory activity of MSC in sphere cultured.

[0028] CT reduces cell apoptosis after MSC spheroidization. Calcein-AM / PI staining is performed on MSC spheroids to distinguish cell states, where live cells show green fluorescence, dead cells show red fluorescence, and blue fluorescence is the cell nucleus. Confocal scanning three-dimensional reconstruction is performed on the stained MSC spheroids. The results are as follows Figure 4 As shown in A, the spheroid structure in the CT group is more three-dimensional and uniform. Confocal scans of different layers of the two groups of spheroids were selected to count the red fluorescence intensity of PI, as shown in Figure 4 As shown in B, the red fluorescence intensity of the CT group was significantly lower than that of the DMSO control group, indicating that CT effectively reduced the cell death of MSC spheres. Western Blotting was used to detect the expression level of apoptosis-related protein Caspase3 in the two groups. Figure 4 As shown in C and D, the level of Caspase3 in the CT group was lower than that in the DMSO control group, indicating that CT effectively reduced the apoptosis of MSC sphere-forming cells.

[0029] CT promotes the secretion of extracellular vesicles by MSC spheroidization, such as Figure 5 As shown in A, B, and C, the expression of genes CD63, CD9, and CD81 related to the generation of extracellular vesicles in spheroids in the CT group was upregulated. In addition, equal amounts of supernatants from spheroid culture cells in the DMSO control group and the CT group were collected to extract extracellular vesicles. The concentrations of extracellular vesicle particles in the two groups were detected by NTA. The results are shown in Figure 5. Figure 5 As shown in D, the extracellular vesicle production in the supernatant of the CT group was higher than that in the DMSO control group. The results of Western blotting to identify the extracellular vesicle protein markers of the two groups are shown in Figure 5 As shown in E, both groups of extracellular vesicles expressed extracellular vesicle positive marker proteins Alix, CD63, CD81, and TSG101, and did not express extracellular vesicle negative marker GM130. These results indicate that CT effectively enhances the secretion of extracellular vesicles in MSC spheres.

[0030] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for promoting spheroidization of mesenchymal stem cells, characterized in that: include: Mesenchymal stem cells are inoculated into a culture medium to obtain a cell culture fluid, a DMSO solution of a compound combination CT is added to the cell culture fluid and mixed, and sphere culture is performed to obtain three-dimensional sphere mesenchymal stem cells; wherein the compound combination CT is a combination of Chroman 1 and Trans-ISRIB, and the molar ratio of Chroman 1 to Trans-ISRIB is 1:10-1:

20.

2. The method according to claim 1, characterized in that The composition of the culture medium is as follows: 5% V / V human platelet lysate is added to the mesenchymal stem cell culture medium.

3. The method according to claim 1, characterized in that The molar ratio of Chroman 1 to Trans-ISRIB was 1:

14.

4. The method according to claim 1, characterized in that: In the DMSO solution of the compound combination CT, the concentration of Chroman1 is 40-60 μM, and the concentration of Trans-ISRIB is 0.6-0.8 mM.

5. The method according to claim 4, characterized in that In the DMSO solution of the compound combination CT, the concentration of Chroman1 is 50 μM, and the concentration of Trans-ISRIB is 0.7 mM.

6. The method according to claim 1, characterized in that The volume ratio of the added amount of the DMSO solution of the compound combination CT to the cell culture medium is 1:1000.

7. The method according to claim 1, characterized in that The following steps are involved: S1: After the mesenchymal stem cells are expanded in two-dimensional culture, they are digested into single cells using TrypleLE digestion enzyme, and then digestion is terminated with PBS buffer. After centrifugation, the supernatant is discarded and the cells are resuspended in culture medium into a single cell suspension; S2: Single cell suspension was prepared at 5×10 4 / mL~20×10 4 The cells were inoculated into a culture plate at a density of 1:1 / mL to obtain a cell culture solution, a DMSO solution of the compound combination CT was added to the cell culture solution and mixed evenly, and sphere culture was performed to obtain three-dimensional sphere mesenchymal stem cells.

8. The method according to claim 1 or 7, characterized in that: The sphere formation culture comprises: culturing in a culture plate at 36.5-37.5° C., 4.5-5.5% CO 2 , and on a horizontal shaker at 60-80 rpm / min for 12-48 hours.

9. The method according to claim 8, characterized in that Incubate the cells in a culture plate at 37°C, 5% CO2, and on a horizontal shaker at 70 rpm / min for 12 to 48 hours.

10. The method according to claim 7, characterized in that The cell density in the cell culture medium was 15×10 4 / mL.

Images