MSCs culture solution as well as preparation method and application thereof

Abstract

The invention discloses an MSCs culture solution and a preparation method and application thereof.An earthworm protein solution is added into an MSCs complete culture medium, namely a DMEM / F12 culture medium containing fetal calf serum with the volume fraction being 10%, the MSCs culture solution is obtained after sufficient and uniform mixing, the concentration of earthworm protein in the MSCs culture solution ranges from 1 microgram / ml to 500 microgram / ml, and the concentration of the earthworm protein in the MSCs culture solution ranges from 1 microgram / ml to 500 microgram / ml. By adopting the culture solution to culture the MSCs, the multiplication capacity, paracrine capacity and immunosuppression efficacy of the MSCs can be remarkably improved on the premise of not changing the form and basic phenotype of the MSCs, the culture solution is simple in additive component, a remarkable effect can be generated by short-time stimulation (24-48 hours), and the culture solution has a wide application prospect and is beneficial to popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a MSCs culture solution and a preparation method and application thereof. Background Art

[0002] Earthworm protein is a protein complex extracted from earthworms (earthworms). Its main components are plasmin, collagen, amino acids, and trace elements. Plasmin is the core active substance, with the potential to dissolve blood clots and improve blood circulation. Earthworm protein can activate the fibrinolytic system, dissolving fibrin clots within blood vessels and helping to improve thrombosis-related conditions (such as cerebral thrombosis and lower limb venous thrombosis). It can also reduce blood viscosity to a certain extent. Earthworm protein also exhibits anti-inflammatory, antioxidant, and immunomodulatory properties, making it useful in treating inflammatory and autoimmune diseases. In cancer treatment, earthworm protein has also demonstrated anti-tumor activity, inhibiting tumor cell proliferation and metastasis. Earthworm protein can also be used to prepare biomaterials, such as tissue engineering scaffolds and sustained-release drug delivery vehicles, offering new research directions and application prospects in the biomedical field.

[0003] Mesenchymal stem / stromal cells (MSCs) are a group of multipotent stem cells derived from the mesoderm. They possess diverse functions, including self-renewal, multidirectional differentiation (e.g., into adipocytes, osteocytes, and chondrocytes), immunomodulation, paracrine function, homing, and tissue repair. Given their diverse biological properties, MSCs hold broad application prospects in tissue repair and regenerative medicine. Currently, five MSC-based therapeutics are marketed globally, with many more entering clinical development. In China, 78 MSC-based therapeutics have received approval for Investigational New Drugs (INDs). Despite this, the industrialization of MSC-based therapeutics is steadily advancing. However, due to the inherent heterogeneity of MSCs, resulting from differences in donor and tissue origin, isolation and expansion processes, batch-to-batch variability is significant, making quality control of cultured cells difficult. To improve the yield and immunological efficacy of MSCs, both the scientific and industrial communities are exploring new research directions. The present invention uses a culture medium containing earthworm protein to culture MSCs, which can significantly improve their proliferation, paracrine and immunosuppressive efficacy, and related research has not been reported worldwide.

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

[0005] The purpose of the present invention is to provide a MSCs culture medium and its preparation method and application. Using this culture medium to culture MSCs can significantly enhance the proliferation ability, paracrine ability and immunosuppressive efficacy of MSCs without changing the morphology and basic phenotype of MSCs. The culture medium has simple additive components and can produce significant effects after a short stimulation time (24-48 hours). It has broad application prospects and is conducive to promotion and application.

[0006] In order to achieve the above-mentioned object, the present invention provides a method for preparing an MSCs culture fluid, comprising adding an earthworm protein solution to a complete MSCs culture medium, i.e., a DMEM / F12 culture medium containing 10% fetal bovine serum by volume, and thoroughly mixing to obtain an MSCs culture fluid, wherein the concentration of the earthworm protein in the MSCs culture fluid is 1 μg / ml-500 μg / ml.

[0007] Preferably, the earthworm protein solution is prepared by weighing earthworm protein powder and dissolving it in sterilized pure water, and 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 differentiated from embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).

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

[0011] The present invention also provides a use of the above MSCs culture solution in improving the proliferation ability of MSCs.

[0012] The present invention also provides a use of the above MSCs culture solution in improving the paracrine ability of MSCs.

[0013] The present invention also provides a use of the above MSCs culture solution in improving the immunosuppressive efficacy of MSCs.

[0014] The present invention provides a MSCs culture solution and a preparation method and application thereof, which have the following beneficial effects.

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

[0016] 2. The proliferation ability of MSCs can be significantly enhanced after culturing MSCs using the present invention.

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

[0018] 4. The immunosuppressive ability of MSCs can be significantly enhanced after culturing them using the present invention. That is, as the concentration of earthworm protein increases, its inhibitory level on the secretion of inflammatory factors TNFα and IFNγ by activated PBMC can be significantly enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of CCK8 assay to detect the effect of different concentrations of earthworm protein on the proliferation of MSCs (n=4); Figure 2 The morphological photos of MSCs observed under a microscope after being treated with different concentrations of earthworm protein for 24 hours and 48 hours; Figure 3 The results of flow cytometry phenotypic analysis of MSCs treated with different concentrations of earthworm protein for 24 hours; Figure 4 The flow cytometry phenotypic analysis results of MSCs treated with different concentrations of earthworm protein for 48 hours: Figure 5 The effects of different concentrations of earthworm protein treatment on the secretion of immunomodulatory cytokines (PGE2, HGF, IDO) by MSCs for 24 hours (n=8); Figure 6 The effect of different concentrations of earthworm protein 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 DESCRIPTION

[0020] The present invention will be further described below with reference to specific embodiments and accompanying drawings to facilitate understanding of the present invention.

[0021] The present invention provides a method for preparing an MSCs culture fluid, comprising adding an earthworm protein solution to a complete MSCs culture fluid, namely, a DMEM / F12 culture fluid containing 10% fetal bovine serum by volume, and thoroughly mixing the mixture to obtain an MSCs culture fluid, wherein the concentration of the earthworm protein in the MSCs culture fluid is 1 μg / ml-500 μg / ml.

[0022] The earthworm protein solution is prepared by weighing earthworm protein powder and dissolving it in sterilized pure water, and filtering it through a filter membrane with a pore size of 0.22 μm to obtain the earthworm protein solution. The MSCs culture medium is suitable for culturing MSCs from different tissue sources. Preferably, the MSCs are MSCs derived from umbilical cord, placenta, bone marrow, fat, dental pulp, endometrium, and embryonic stem cells or induced stem cell differentiation.

[0023] The present invention also provides a MSCs culture solution prepared by the above method.

[0024] The present invention also provides a use of the above MSCs culture solution in improving the proliferation ability of MSCs.

[0025] The present invention also provides a use of the above MSCs culture solution in improving the paracrine ability of MSCs.

[0026] The present invention also provides a use of the above MSCs culture solution in improving the immunosuppressive efficacy of MSCs.

[0027] Example: Preparation of MSCs culture medium containing different concentrations of earthworm protein, the specific method is as follows: Earthworm protein powder was weighed and dissolved in sterilized pure water. The solution was filtered through a 0.22 μm filter membrane to obtain the earthworm protein solution. Different volumes of the earthworm protein solution were added to complete MSC culture medium (DMEM / F12 medium supplemented with 10% fetal bovine serum) and thoroughly mixed to obtain MSC culture fluids containing four different concentrations of earthworm protein: 0, 50, 250, and 500 μg / ml.

[0028] Cell culture was performed using the MSCs culture medium containing different concentrations of earthworm protein prepared in the above example, and the expression level of surface basic antigens, proliferation ability, paracrine ability and immunosuppressive efficacy of MSCs were measured: (1) Determination of MSCs proliferation ability Methods: Human umbilical cord MSCs from four different donors were seeded at 5,000 cells / well in 96-well plates and cultured in MSC culture medium containing different concentrations of earthworm protein (0, 50, 250, and 500 μg / ml) for 24, 48, and 72 hours. Proliferation was assessed using the CCK8 assay. Two hours before each assay, the culture medium was replaced with complete MSC culture medium containing 10% CCK8. The absorbance (OD450) of each well was measured using a microplate reader. CCK8 is a cell proliferation and cytotoxicity assay based on a water-soluble tetrazolium salt (WST-8). It utilizes mitochondrial dehydrogenases to reduce WST-8 to a highly water-soluble yellow formazan product. The amount of formazan generated is proportional to the number of viable cells, allowing the absorbance at 450 nm (OD) to assess cell proliferation.

[0029] Experimental results: Figure 1 As shown, different concentrations of earthworm protein cultured human umbilical cord MSCs for 24h, 48h and 72h can significantly improve their proliferation ability, and the increase in proliferation level was significantly dose-dependent with the concentration of earthworm protein at 48h and 72h.

[0030] (2) Determination of MSCs cell morphology and basic phenotype Specific methods: In order to analyze whether earthworm protein affects the cell morphology and basic phenotype of MSCs, a MSC was cultured in MSC culture medium containing different concentrations of earthworm protein for 48 hours. The cell morphology was observed under a microscope and photographed. The results are as follows: Figure 2 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 0, 50, and 500 μg / ml for 24 and 48 hours. The cells were harvested and incubated with anti-human antibodies, 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), for 30 minutes at room temperature, according to flow cytometry antibody labeling protocols. All antibodies were purchased from BD Pharmingen. After the antibody incubation, the cells were washed three times with DPBS and centrifuged at 1500 rpm for 5 min. The cell pellet was retained and resuspended with 400 μl flow cytometry buffer and filtered. The cell phenotype was detected using a flow cytometer, BD FACS Calibur.

[0031] Take the flow cytometry phenotypic analysis of a MSC line after being cultured in MSC culture medium containing different concentrations of earthworm protein for 24h and 48h 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 hours and 48 hours, the cells were collected and the expression levels of surface antigens were analyzed by flow cytometry. It was found that compared with conventionally cultured MSCs, the expression levels of surface antigens PE-CD105, PE-CD73, PE-CD90, PE-HLA-DR, PE-CD45, and PE-CD11b were not significantly different (n=4) (see Table 1). The above results indicate that after MSCs are treated with different concentrations of earthworm protein for 24 hours and 48 hours, their surface antigen expression levels still meet the International Society for Cellular Therapy (ISCT) standards.

[0032] Table 1 Flow cytometry analysis of surface antigen expression levels of human umbilical cord MSCs cultured in MSC culture medium (containing different concentrations of earthworm protein) for 24 h and 48 h (positive rate%)

[0033] (3) Determination of the level of immunoregulatory factors secreted by MSCs Specific methods: 8 human umbilical cord MSCs of P6-P8 were cultured at a rate of 5×10 5 Cells were seeded into 6-well plates. After adherence, the culture medium was replaced with MSC culture medium containing different concentrations of earthworm protein (0, 50, and 500 μg / ml). The cells were then cultured at 37°C for 24 hours. The supernatant was collected after 24 hours of culture, centrifuged at 4000 rpm for 10 minutes, aliquoted, and stored at -80°C until further 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 determined using ELISA.

[0034] Experimental results: As shown in Table 2 and Figure 5 As shown in the figure, culturing human umbilical cord MSCs with MSC culture medium containing different concentrations of earthworm protein for 24 hours can significantly increase the secretion levels of HGF (A) and PGE2 (B) in a dose-dependent manner, while no significant change was observed in the secretion level of IDO (C).

[0035] Table 2 Cytokine levels secreted by MSCs after culturing with different concentrations of earthworm protein for 24 h (Mean ± SEM)

[0036] (4) Determination of the immunosuppressive effect of MSCs Specific methods: 8 human umbilical cord MSCs of P6-P8 were cultured at a rate of 5×10 5 Cells were seeded into 6-well plates. After adherence, the culture medium was replaced with MSC culture medium containing different concentrations of earthworm protein (0, 50, and 500 μg / ml). The cells were then cultured in a 37°C incubator for 24 hours. The 24-hour conditioned medium (24h-CM) was collected and centrifuged at 2000 rpm for 10 minutes. The cells were discarded and the supernatant was retained. The aliquots were then stored in a -80°C refrigerator. Healthy human peripheral blood mononuclear cells (HuPBMCs) were isolated and counted, and the HuPBMC concentration was adjusted to 1×10 6 MSCs were cultured at a constant volume of 1:1 at 24 h CM per well of a 96-well plate. The cells were then cultured at a constant volume of 1:1 at 24 h CM per well. The 24 h CM was then added to the plate at a constant volume of 200 μl. After thorough mixing, the 96-well plate was incubated at 37°C for 48 h. A positive control group consisted of a HuPBMC suspension activated with PHA, while a negative control group consisted of a HuPBMC suspension without PHA stimulation. Three replicate wells were set up for each group. The 48 h culture supernatant was collected and centrifuged at 2000 rpm for 10 min. The supernatant was carefully aspirated and stored at -80°C until further analysis. TNFα (Cat#EHC103a) and IFNγ (Cat#EHC102g) levels in the culture supernatant were determined by ELISA, and the inhibitory rates of these two inhibitors were calculated. The above ELISA kits were purchased from Xinbosheng Biotechnology Co., Ltd. The calculation formulas for TNFα and IFNγ inhibition rates are as follows:

[0037] Experimental results: Figure 6As shown in the figure, 24h-CM of MSCs can significantly inhibit the secretion of inflammatory factors TNFα (A) and IFNγ (B) by PHA-activated PBMC, with inhibition rates of 52.63%±2.95% (A) and 44.31%±5.51% (B), respectively. After being cultured in MSCs culture medium containing earthworm protein, the paracrine inhibition level of MSCs was significantly improved. After being cultured in MSCs culture medium containing 50μg / ml earthworm protein, the inhibition rates of activated HuPBMC secreting TNFα and IFNγ were 61.70%, respectively. ±3.50% (*p<0.05) and 65.89%±4.04% (*p<0.05), while after culturing with MSCs culture medium containing 500μg / ml earthworm protein, the inhibition rates of the two were 73.97%±1.59% (****p<0.0001) and 93.24%±0.47% (****p<0.0001), respectively; and there were significant statistical differences in the inhibition rates of TNFα (**p<0.01) and IFNγ (***p<0.001) between different earthworm protein concentrations.

[0038] This document uses specific examples to illustrate the inventive concept in detail. The above embodiments are only intended to help understand the core concept of the present invention. It should be noted that any obvious modifications, equivalent substitutions, or other improvements made by a person skilled in the art without departing from the inventive concept should be included within the scope of protection of the present invention.

Claims

1. A method for preparing MSCs culture medium, characterized in that: The earthworm protein solution is added to the 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 the MSCs culture medium. The concentration of the earthworm protein in the MSCs culture medium is 1 μg / ml-500 μg / ml.

2. The method for preparing a MSCs culture solution according to claim 1, wherein: The preparation method of the earthworm protein solution is as follows: weighing earthworm protein powder and dissolving it in sterilized pure water, and filtering it through a filter membrane with a pore size of 0.22 μm to obtain the earthworm protein solution.

3. The method for preparing a MSCs culture solution according to claim 2, wherein: The MSCs culture solution is suitable for culturing MSCs from different tissue sources.

4. The method for preparing a MSCs culture solution according to claim 3, wherein: The MSCs are MSCs derived from umbilical cord, placenta, bone marrow, fat, dental pulp, endometrium, embryonic stem cells or induced stem cell differentiation.

5. A MSCs culture solution prepared by the method according to any one of claims 1 to 4.

6. Use of the MSCs culture medium according to claim 5 in enhancing the proliferation ability of MSCs.

7. Use of the MSCs culture medium according to claim 5 in enhancing the paracrine capacity of MSCs.

8. Use of the MSCs culture solution according to claim 5 in enhancing the immunosuppressive efficacy of MSCs.

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