Use of MSCs culture medium in enhancing paracrine and immunosuppressive potency

Abstract

The application discloses application of MSCs culture solution in enhancing paracrine and immunosuppression efficiency, wherein a diplotene protein solution is added into a complete MSCs culture medium, i.e., a DMEM / F12 culture medium containing 10% fetal bovine serum, and the MSCs culture solution is obtained after sufficient mixing, wherein the concentration of the diplotene protein in the MSCs culture solution is 1 mu g / ml-500 mu g / ml, and the MSCs culture solution can significantly improve the proliferation capacity, paracrine capacity and immunosuppression efficiency of MSCs without changing the morphology and basic phenotype of the MSCs, the culture solution additive has simple components, can produce significant effects in a short time (24-48h), has wide application prospects, and is favorable for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of an MSC culture medium in enhancing paracrine and immunosuppressive efficacy. Background Technology

[0002] Earthworm protein is a protein complex extracted from earthworms (earthworms). Its main components include plasmin, collagen, amino acids, and trace elements. Plasmin is the core active substance, possessing the potential to dissolve blood clots and improve blood circulation. Earthworm protein can help dissolve fibrin thrombi in blood vessels by activating the fibrinolytic system, thus assisting in improving thrombosis-related problems (such as cerebral thrombosis and lower extremity venous thrombosis) and reducing blood viscosity to some extent. Furthermore, earthworm protein also has anti-inflammatory, antioxidant, and immunomodulatory effects, and can be used to treat inflammatory diseases and autoimmune diseases. In tumor treatment, earthworm protein has also shown certain anti-tumor activity, inhibiting the proliferation and metastasis of tumor cells. Simultaneously, earthworm protein can also be used to prepare biomaterials, such as tissue engineering scaffolds and drug delivery carriers, providing new research directions and application prospects for the biomedical field.

[0003] Mesenchymal stem cells (MSCs) are a group of pluripotent stem cells derived from the mesoderm, possessing multiple functions including self-renewal, multi-lineage differentiation (such as differentiation into adipocytes, osteocytes, and chondrocytes), immune regulation, paracrine function, migration and homing, and tissue repair. Given these diverse biological characteristics, MSCs show broad application prospects in tissue repair and regenerative medicine. Currently, globally, five MSC-based therapeutics are on the market, with more pipelines entering clinical trials. In China, 78 MSC-based drug applications (INDs) have been approved. Despite this progress in the industrialization of MSC-based drugs, the heterogeneity of MSCs due to different donor and tissue sources, as well as isolation and expansion processes, leads to significant batch-to-batch variations, making it difficult to control the quality of cultured cells. To improve MSC yield and the immunological efficacy of cultured cells, the scientific and industrial communities are exploring new research directions. This invention demonstrates that culturing MSCs with a culture medium containing earthworm protein can significantly enhance their proliferation, paracrine and immunosuppressive effects, and there are no related studies reported worldwide.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an application of MSC culture medium in enhancing paracrine and immunosuppressive efficacy. Using this culture medium to culture MSCs can significantly improve their proliferation, paracrine capacity, and immunosuppressive efficacy without altering their morphology and basic phenotype. The culture medium has simple additives and can produce significant effects with short-term stimulation (24-48 hours), showing broad application prospects and facilitating its widespread application.

[0006] To achieve the above objectives, the present invention provides a method for preparing MSCs culture medium, wherein earthworm protein solution is added to MSCs complete culture medium, i.e., DMEM / F12 culture medium containing 10% fetal bovine serum by volume, and the mixture is thoroughly mixed to obtain MSCs culture medium, wherein the concentration of earthworm protein in the MSCs culture medium is 1μg / ml-500μg / ml.

[0007] Preferably, the earthworm protein solution is prepared by weighing earthworm protein powder and dissolving it in sterilized pure water, then filtering it through a filter membrane with a pore size of 0.22 μm to obtain the earthworm protein solution.

[0008] Preferably, the MSCs culture medium is suitable for culturing MSCs from different tissue sources.

[0009] Preferably, the MSCs are MSCs derived from umbilical cord, placenta, bone marrow, fat, dental pulp, endometrium, and MSCs derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0010] The present invention also provides a culture medium for MSCs prepared by the above method.

[0011] The present invention also provides an application of the above-mentioned MSCs culture medium in enhancing the proliferation capacity of MSCs.

[0012] The present invention also provides an application of the above-mentioned MSCs culture medium in enhancing the paracrine capacity of MSCs.

[0013] The present invention also provides an application of the above-mentioned MSCs culture medium in enhancing the immunosuppressive efficacy of MSCs.

[0014] The present invention provides an application of MSCs culture medium in enhancing paracrine and immunosuppressive efficacy, which has the following beneficial effects.

[0015] 1. The use of this invention to culture MSCs does not change the expression levels of basic antigens on the surface of MSCs, including CD105, CD73, CD90, CD45, CD11b, and HLA-DR.

[0016] 2. The use of this invention to culture MSCs can significantly enhance the proliferation capacity of MSCs.

[0017] 3. Using the present invention to culture MSCs can significantly enhance the ability of MSCs to secrete paracrine cytokines, such as increasing the secretion of immunomodulatory factors PGE2 and HGF.

[0018] 4. The immunosuppressive ability of MSCs cultured using the present invention can be significantly enhanced. Specifically, as the concentration of earthworm protein increases, its inhibitory effect on the secretion of inflammatory factors TNFα and IFNγ by activated PBMCs can be significantly enhanced. Attached Figure Description

[0019] Figure 1 The results of CCK8 assay for detecting the proliferation of MSCs stimulated by different concentrations of earthworm protein (n=4).

[0020] Figure 2 Morphological images of MSCs observed under a microscope after treatment with different concentrations of earthworm protein for 24 h and 48 h.

[0021] Figure 3 Flow cytometry phenotypic analysis results of MSCs treated with different concentrations of earthworm protein for 24 h;

[0022] Figure 4 Flow cytometry phenotypic analysis results of MSCs treated with different concentrations of earthworm protein for 48 h;

[0023] Figure 5 The effect of treating MSCs with different concentrations of earthworm protein for 24 h on their secretion of immunomodulatory cytokines (PGE2, HGF, IDO) (n=8);

[0024] Figure 6 The effect of different concentrations of earthworm protein treatment on the immunosuppressive ability of MSCs, namely the inhibitory level on the secretion of inflammatory factors TNFα and IFNγ by activated PBMCs (n=8). Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments and accompanying drawings to help understand the content of the present invention.

[0026] The present invention provides a method for preparing MSCs culture medium, wherein earthworm protein solution is added to MSCs complete culture medium, namely DMEM / F12 culture medium containing 10% fetal bovine serum by volume, and the mixture is thoroughly mixed to obtain MSCs culture medium, wherein the concentration of earthworm protein in the MSCs culture medium is 1μg / ml-500μg / ml.

[0027] The earthworm protein solution is prepared as follows: weigh earthworm protein powder and dissolve it in sterilized pure water, then filter it through a 0.22 μm pore size filter membrane to obtain the earthworm protein solution. The MSCs culture medium is suitable for culturing MSCs from different tissue sources. Preferably, the MSCs are derived from umbilical cord, placenta, bone marrow, adipose tissue, dental pulp, endometrium, and embryonic stem cells or induced cellular stem cell differentiation.

[0028] The present invention also provides a culture medium for MSCs prepared by the above method.

[0029] The present invention also provides an application of the above-mentioned MSCs culture medium in enhancing the proliferation capacity of MSCs.

[0030] The present invention also provides an application of the above-mentioned MSCs culture medium in enhancing the paracrine capacity of MSCs.

[0031] The present invention also provides an application of the above-mentioned MSCs culture medium in enhancing the immunosuppressive efficacy of MSCs.

[0032] Example: Preparation of MSCs culture medium containing different concentrations of earthworm protein, the specific method is as follows:

[0033] Weigh the earthworm protein powder and dissolve it in sterilized pure water. Filter the solution through a 0.22 μm pore membrane to obtain the earthworm protein solution. Add different volumes of the earthworm protein solution to the complete MSC culture medium (DMEM / F12 medium containing 10% fetal bovine serum). Mix thoroughly to obtain MSC culture media containing four different concentrations of earthworm protein: 0, 50, 250, and 500 μg / ml.

[0034] MSCs with different concentrations of earthworm protein prepared in the above examples were cultured, and the expression levels of basic surface antigens, proliferation capacity, paracrine capacity, and immunosuppressive efficacy of MSCs were measured.

[0035] (1) Determination of MSC proliferation capacity

[0036] Specific methods: Four human umbilical cord MSCs from different donors were seeded at a rate of 5000 cells / well in 96-well plates and cultured in MSC medium containing different concentrations (0, 50, 250, and 500 μg / ml) of earthworm protein for 24 h, 48 h, and 72 h, respectively, before their proliferation was assessed. The CCK8 assay was used. Two hours before each assay point, the medium was replaced with complete MSC medium containing 10% CCK8, and the absorbance (OD450) of each well was measured using a microplate reader. CCK8 is a cell proliferation and toxicity assay based on water-soluble tetrazolium salt (WST-8). Its principle is based on the reduction of WST-8 to a highly water-soluble yellow formazan product by dehydrogenases in the mitochondria. The amount of formazan product generated is directly proportional to the number of viable cells; therefore, cell proliferation can be assessed by measuring the absorbance (OD value) at 450 nm.

[0037] Experimental results: such as Figure 1 As shown, different concentrations of earthworm protein can significantly improve the proliferation capacity of human umbilical cord MSCs after culturing for 24h, 48h and 72h, and the increase in proliferation level is significantly dose-dependent with the concentration of earthworm protein at 48h and 72h.

[0038] (2) Determination of MSC cell morphology and basic phenotype

[0039] Specific methods: To analyze whether earthworm protein affects the morphology and basic phenotype of MSCs, an MSC strain was cultured in MSC culture medium containing different concentrations of earthworm protein for 48 hours as an example. Cell morphology was observed and photographed under a microscope, and the results are as follows. Figure 2 As shown. Furthermore, to analyze the effect of earthworm protein on the immunophenotype of MSCs, four different human umbilical cord-derived MSCs were stimulated with earthworm protein at concentrations of 0, 50, and 500 μg / ml for 24 h and 48 h, respectively. Cells were collected and labeled with anti-human antibodies according to flow cytometry, including: PE-CD105 (Cat:560839), PE-CD73 (Cat: 550257), PE-CD90 (Cat:555596), PE-HLA-DR (Cat:555812), PE-CD45 (Cat:555483), and PE-CD11b (Cat:555388) antibodies, and incubated at room temperature for 30 min. All antibodies were purchased from BD Pharmingen. After antibody incubation, the cells were washed three times with DPBS, centrifuged at 1500 rpm for 5 min, and the cell pellet was retained. The pellet was then resuspended in 400 μl of flow cytometry wash buffer, filtered, and the cell phenotype was detected using a BDFACS Calibur flow cytometer.

[0040] Taking the flow cytometry phenotypic analysis of a single MSC strain cultured in MSC medium containing different concentrations of earthworm protein for 24 h and 48 h as an example ( Figure 3 and Figure 4 Experimental Results: As shown in Table 1, after culturing MSCs with different concentrations of earthworm protein (0, 50, 500 μg / ml) for 24 h and 48 h, cells were collected, and the expression levels of surface antigens were analyzed by flow cytometry. It was found that compared with conventionally cultured MSCs, there were no significant differences in the expression levels of surface antigens PE-CD105, PE-CD73, PE-CD90, PE-HLA-DR, PE-CD45, and PE-CD11b (n=4) (see Table 1). These results suggest that after treatment of MSCs with different concentrations of earthworm protein for 24 h and 48 h, the expression levels of their surface antigens still meet the standards of the International Society for Cell Therapy (ISCT).

[0041] Table 1. Flow cytometry detection of surface antigen expression levels of human umbilical cord MSCs cultured in MSC culture medium (containing different concentrations of earthworm protein) after 24 h and 48 h (positive rate %).

[0042]

[0043] (3) Measurement of the level of immunomodulatory factors secreted by MSCs

[0044] Specific method: Eight P6-P8 human umbilical cord MSCs were processed at a ratio of 5 × 10⁻⁶. 5 MSCs were seeded into 6-well plates. After adhesion, the culture medium was replaced, and MSCs culture medium containing different concentrations of earthworm protein (0, 50, and 500 μg / ml) was added. The plates were then incubated at 37°C for 24 h. The 24-h culture supernatant was collected, centrifuged at 4000 rpm for 10 min, aliquoted, and stored at -80°C for later analysis. The expression levels of PGE2 (Cayman Chemical, Cat: 500141), HGF (Xinbosheng, Cat: EHC138), and IDO (Elabscience, Cat: EE-H2162) in the culture supernatant were detected by ELISA.

[0045] Experimental results: as shown in Table 2 and Figure 5 As shown, culturing human umbilical cord MSCs in MSC culture medium containing different concentrations of earthworm protein for 24 hours can significantly increase the levels of HGF (A) and PGE2 (B) secreted by MSCs in a dose-dependent manner, while no significant change was observed in the level of IDO (C) secreted by MSCs.

[0046] Table 2. Cytokine content (Mean ± SEM) of MSCs after culturing in different concentrations of earthworm protein medium for 24 h.

[0047] HGF (pg / ml) 6363±1455 9686±1882 12774±2585 <![CDATA[PGE2(pg / ml)]]> 15156±2020 42440±7304 53272±7921 IDO (ng / ml) 18.05±1.157 19.17±1.313 19.07±1.188

[0048] (4) Determination of immunosuppressive efficacy against MSCs

[0049] Specific method: Eight P6-P8 human umbilical cord MSCs were processed at a ratio of 5 × 10⁻⁶. 5 Cells were seeded into 6-well plates. After adhesion, the culture medium was changed, and MSCs culture medium containing different concentrations of earthworm protein (0, 50, and 500 μg / ml) was added. The cells were then incubated at 37°C for 24 h. The 24-h conditioned medium (24h-CM) was collected, centrifuged at 2000 rpm for 10 min, the cells were discarded, and the supernatant was aliquoted and stored at -80°C. Peripheral blood mononuclear cells (HuPBMCs) were isolated from healthy individuals, counted, and the HuPBMC concentration was adjusted to 1×10⁻⁶. 6 = / ml, add a certain concentration of phytohemagglutinin-M (PHA-M) (Roche, Cat#11082132001) to stimulate activation. After thawing the culture supernatant for 24 h, add MSCs suspension and 24 h-CM at a volume ratio of 1:1 per well to a 96-well plate to make the final culture volume 200 μl. After thorough mixing, place the 96-well plate in an incubator at 37 ℃ for another 48 h. The positive control group is a PHA-activated HubBMC suspension, and the negative control group is a HubBMC suspension without PHA stimulation. Each group has 3 replicate wells. Collect the 48 h culture supernatant, centrifuge at 2000 rpm for 10 min, carefully aspirate the supernatant, aliquot it and store it at -80 ℃ for later analysis. The contents of TNFα (Cat#EHC103a) and IFNγ (Cat#EHC102g) in the culture supernatant are detected by ELISA and the inhibition rate of the two is calculated. All the above ELISA kits were purchased from Xinbosheng Biotechnology Co., Ltd. The formulas for calculating the inhibition rates of TNFα and IFNγ are shown below:

[0050] TNFα inhibition% = ( ) × 100%

[0051] IFNγ inhibition% = ( ) × 100%

[0052] Experimental results: such as Figure 6As shown, MSCs cultured for 24 hours significantly inhibited the secretion of inflammatory factors TNFα (A) and IFNγ (B) by PHA-activated PBMCs, with inhibition rates of 52.63% ± 2.95% (A) and 44.31% ± 5.51% (B), respectively. After MSCs were cultured in a medium containing earthworm protein, the paracrine inhibition level of MSCs was significantly enhanced. Specifically, MSCs cultured in a medium containing 50 μg / ml earthworm protein showed an inhibition rate of 61.70% on the secretion of TNFα and IFNγ by activated Hub PMCs. The inhibition rates of TNFα (*p<0.05) and IFNγ (***p<0.001) were 65.89%±4.04% (*p<0.05) and 65.89%±4.04% (*p<0.05), respectively, after culturing MSCs with 500 μg / ml earthworm protein, the inhibition rates of TNFα and IFNγ were 73.97%±1.59% (****p<0.0001) and 93.24%±0.47% (****p<0.0001), respectively. Moreover, there were also statistically significant differences in the inhibition rates of TNFα (**p<0.01) and IFNγ (***p<0.001) among different earthworm protein concentrations.

[0053] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. The application of an MSCs culture medium in enhancing paracrine and immunosuppressive efficacy, characterized in that, The MSCs culture medium was prepared by adding earthworm protein solution to DMEM / F12 medium containing 10% fetal bovine serum. The concentration of earthworm protein in the MSCs culture medium was 1 μg / ml-500 μg / ml. Using this culture medium to culture MSCs can significantly improve the paracrine capacity and immunosuppressive efficacy of MSCs without changing the expression level of basic antigens on the surface of MSCs.

2. The application of the MSCs culture medium according to claim 1 in enhancing paracrine and immunosuppressive efficacy, characterized in that, The earthworm protein solution is prepared by dissolving earthworm protein powder in sterilized pure water and filtering it through a filter membrane with a pore size of 0.22 μm.

3. The application of the MSCs culture medium according to claim 2 in enhancing paracrine and immunosuppressive efficacy, characterized in that, The MSCs culture medium is suitable for culturing MSCs from different tissue sources.

4. The application of the MSCs culture medium according to claim 3 in enhancing paracrine and immunosuppressive efficacy, characterized in that, The MSCs mentioned are MSCs derived from umbilical cord, placenta, bone marrow, fat, dental pulp, endometrium, and MSCs derived from embryonic stem cells or induced stem cell differentiation.

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