Culture medium and culture method for delaying senescence of mesenchymal stem cells and improving immunosuppression function

Abstract

The invention discloses a culture medium for delaying senescence of mesenchymal stem cells and improving an immunosuppression function and a culture method, and belongs to the technical field of stem cell culture. The culture medium comprises the following components: a TLR3 agonist, a TGF beta agonist and VD. In order to solve the problems of function decline, immune regulation ability decline, insufficient dryness maintenance and the like in the existing mesenchymal stem cell (MSC) in-vitro amplification process, the mesenchymal stem cells are subjected to in-vitro culture in a culture medium containing a TLR3 agonist, a TGF beta agonist and VD, so that the aging of the mesenchymal stem cells can be delayed, and the immunosuppression function can be improved. An effective optimization strategy is provided for amplification and culture of the MSC before clinical application, the aging phenomenon of the MSC in the in-vitro culture process is delayed, meanwhile, the immunosuppression effect of the MSC is maintained, the multiplication capacity of the MSC is not affected, and the method has remarkable scientific research value and application prospects.

Description

Technical Field

[0001] This invention relates to the field of stem cell culture technology, and in particular to a culture medium and culture method for delaying the aging of mesenchymal stem cells and enhancing their immunosuppressive function. Background Technology

[0002] Mesenchymal stem cells (MSCs) possess self-renewal capacity, multi-lineage differentiation potential, and immunomodulatory functions, and belong to the category of adult stem cells. This cell type has shown great clinical application potential in various fields such as tissue engineering, regenerative medicine, treatment of autoimmune diseases, and anti-aging interventions. With in-depth research and ongoing clinical trials, the value of MSCs in the treatment of numerous diseases, including bone tissue repair, cartilage regeneration, cardiovascular diseases, and neurological disorders, is becoming increasingly significant, particularly in the treatment of graft-versus-host disease (GVHD).

[0003] Currently, the in vitro expansion and quality control of mesenchymal stem cells (MSCs) constitute a crucial step in stem cell applications, with the composition of the culture medium being a key factor in ensuring the maintenance of their stemness and functional stability. However, during in vitro expansion, MSCs often face problems such as weakened proliferation capacity, accelerated senescence characteristics, reduced gene stability, and functional degradation. These issues severely hinder their transformation efficiency and the predictability of therapeutic effects in clinical applications. Existing traditional MSC culture systems mainly rely on simple combinations of fetal bovine serum (FBS) or basal culture media, leading to significant senescence characteristics in cells after multiple in vitro passages, such as increased SA-β-gal activity and cell cycle arrest. This limits the large-scale expansion of MSCs and the maintenance of their function.

[0004] Therefore, developing a culture medium optimization strategy that can slow down the in vitro aging of MSCs, enhance their immune regulation and tissue repair functions, and has stable and controllable components is not only of great significance in the scientific research field, but also directly relates to the uniformity, safety and predictability of efficacy of stem cell drug products, and has significant clinical translation potential and industrial application value. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium and method for delaying the aging of mesenchymal stem cells (MSCs) and enhancing their immunosuppressive function, thereby addressing the problems existing in the prior art. This invention adds Vitamin D (VD), TGFβ agonist (TGFβ3), and poly(I:C) (PIPC) to the basal culture medium, which can significantly improve the aging state and immunomodulatory capacity of MSCs after in vitro culture. This provides a solid foundation and theoretical support for the quality control and industrial production of MSC-based cell therapy products, and has significant scientific research value and application prospects.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a culture medium for delaying the aging of mesenchymal stem cells and enhancing their immunosuppressive function, wherein the components of the culture medium include a TLR3 agonist, a TGFβ agonist, and vitamin D.

[0008] Furthermore, the TLR3 agonist is PIPC.

[0009] Furthermore, the TGFβ agonist is TGFβ3.

[0010] Furthermore, the total concentration of the TLR3 agonist, the TGFβ agonist, and the VD in the culture medium is 10 μg / mL.

[0011] The present invention also provides a culture method for delaying the aging of mesenchymal stem cells and enhancing their immunosuppressive function, comprising the step of culturing the mesenchymal stem cells in vitro in a culture medium containing a TLR3 agonist, a TGFβ agonist and vitamin D.

[0012] Furthermore, the TLR3 agonist is PIPC.

[0013] Furthermore, the TGFβ agonist is TGFβ3.

[0014] Furthermore, the total concentration of the TLR3 agonist, the TGFβ agonist, and the VD in the culture medium is 10 μg / mL.

[0015] Furthermore, the culture medium is based on DF12.

[0016] The present invention also provides mesenchymal stem cells obtained by the culture method described above.

[0017] The present invention discloses the following technical effects:

[0018] This invention addresses the problems of functional decline, decreased immunomodulatory capacity, and insufficient maintenance of stemness that occur during the in vitro expansion of mesenchymal stem cells (MSCs). By integrating single-cell transcriptome analysis and functional validation, it proposes a molecular mechanism-based culture medium optimization strategy. Adding Vitamin D (VD), TGFβ agonist (TGFβ3), and poly(I:C) (PIPC) to the basal culture medium significantly improves the senescence state and immunomodulatory capacity of MSCs after in vitro culture. This method provides an effective optimization strategy for the expansion and culture of MSCs before clinical application, helping to delay the senescence phenomenon during MSC in vitro culture while maintaining its immunosuppressive effect without affecting its proliferative capacity.

[0019] This invention overcomes the technical problems of existing culture systems, such as insufficient functional maintenance, unstable components, and poor clinical applicability. The optimized culture medium method provided by this invention has good reproducibility, safety, and clinical translatability, providing a solid foundation and theoretical support for the quality control and industrial production of MSC-based cell therapy products, and has significant scientific research value and application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Box plots showing the expression levels of IGF1R (A), MDM2 (B), and TGFβ3 (C) in P5 and P15 MSCs;

[0022] Figure 2 The effect of different culture medium treatments on the in vitro proliferation capacity of MSCs;

[0023] Figure 3 To investigate the effects of different culture medium treatments on the immunosuppressive capacity of MSCs;

[0024] Figure 4 The results of senescence staining of MSCs in different culture medium treatment groups. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] This invention addresses the problems of functional decline, decreased immunomodulatory capacity, and insufficient maintenance of stemness that occur during the in vitro expansion of mesenchymal stem cells (MSCs). By integrating single-cell transcriptome analysis and functional verification, it proposes a culture medium optimization strategy based on molecular mechanisms. Specifically, Vitamin D (VD), TGFβ agonist (TGFβ3), and poly(I:C) (PIPC) are added to the basal culture medium to form a targeted optimized culture system, which significantly improves the senescence state and immunomodulatory capacity of MSCs after in vitro culture.

[0031] In this invention, through in vitro expansion of MSCs, it was found that during the P5 to P15 generation expansion process, the immunomodulatory capacity of MSCs decreased significantly with cell senescence, and single-cell transcriptome sequencing showed that the expression of immune-related genes such as MDM2, IGF1R and TGFβ was reduced.

[0032] In this invention, starting from the key regulatory genes of MSC aging and immune regulation, three factors, namely Vitamin D (VD), TGFβ agonist (TGFβ3) and poly(I:C) (PIPC), are rationally screened and combined for in vitro culture intervention of MSCs.

[0033] Furthermore, MDM2 and IGF1R are common components of the VD signaling pathway and can be elevated by VD regulation; TGFβ3 is a widely used TGFβ agonist, and PIPC is a TLR3 agonist. Previous studies have shown that adding TLR3 agonists to the culture medium helps to improve the immunosuppressive capacity of MSCs.

[0034] In this invention, through combined intervention, it was found that when the three factors PIPC+VD+TGFβ3 were used in combination, although cell proliferation was not significantly affected, the immunomodulatory function of MSCs was significantly enhanced and cell senescence was delayed.

[0035] Example

[0036] 1. Acquisition and culture of MSC samples

[0037] First, umbilical cord MSCs were obtained from healthy clinical donors, and primary cells were obtained using collagenase digestion. Subsequently, the MSCs were expanded in vitro using DMEM / DF-12 medium containing 10% fetal bovine serum (FBS). Culture conditions were controlled at 37°C, pH 7.2-7.4, and 5% CO2 concentration. Cells were passaged after digestion when confluence reached 80%-90%. Cells were cultured to passage 5 (P5) and passage 15 (P15) for subsequent experiments.

[0038] 2. Single-cell transcriptome sequencing

[0039] P5 and P15 generation MSCs were isolated as single cells, and single-cell suspensions were prepared for high-throughput sequencing using single-cell transcriptomics technology. After data quality control, low-quality cells were removed, resulting in 52,153 high-quality cell samples. UMAP (Uniform Manifold Approximation and Projection) dimensionality reduction analysis was used to cluster the single cells, classifying them into three subpopulations: Biofunctional MSCs, Active proliferation MSCs, and Multipotent progenitor MSCs.

[0040] 3. Identifying differentially expressed genes

[0041] By comparing the differences between biofunctional MSC populations in P5 and P15 generations of MSCs, differential gene analysis revealed 453 upregulated genes and 763 downregulated genes in P15. Previous studies have shown that P5 MSCs possess superior immunosuppressive capabilities compared to P15 MSCs. Therefore, this invention uses GO and KEGG enrichment to specifically investigate the gene differences in immunosuppressive pathways between the two. Based on the mean logFoldChange > 1 and P < 0.0001, among the 763 downregulated genes, and considering literature reports related to MSC aging and reduced immunomodulatory capacity, three genes with significantly lower expression levels in P15 were selected: TGFβ3, MDM2, and IGF1R. Figure 1 TGFβ3 is a key molecule in the TGF signaling pathway, while MDM2 and IGF1R are key molecules in the vitamin D signaling pathway. Therefore, further experiments were designed to verify whether influencing these three genes could improve MSC culture conditions, thereby minimizing the aging phenotype and decreased immunosuppressive effects of MSCs during in vitro expansion.

[0042] 4. Effects of conditioned medium on MSC proliferation

[0043] MSCs in the logarithmic growth phase were harvested at a concentration of 7 × 10⁻⁶. 4 The well density was seeded in a 6-well plate, with five groups, each group containing 3 duplicate wells. The groupings are as follows:

[0044] (1) Basic culture (DF12) group;

[0045] (2) Basal culture medium + polyI:C (PIPC) group;

[0046] (3) Basic culture medium + polyI:C (PIPC) + TGF-β3 group;

[0047] (4) Basal culture medium + polyI:C (PIPC) + vitamin D (VD) group;

[0048] (5) Combined treatment group of basal culture medium + polyI:C (PIPC) + TGF-βa + VD.

[0049] The corresponding factors were added to the culture medium of the experimental groups. The standard concentration of PIPC was 10 μg / mL, the standard concentration of TGF-β3 was 10 ng / mL, and the standard concentration of VD was 83 ng / mL (i.e., 200 nM). To eliminate the influence of different total amounts of additives on the experimental results, the total amount of exogenous factors added in all experimental groups was kept consistent and adjusted to 10 μg / mL. In the PIPC+TGF-β3 group, TGF-β3 was used at the standard concentration, and PIPC was added to ensure that the total added mass of TGF-β3 and VD equaled the baseline total. In the PIPC+VD group, VD was used at the standard concentration, and PIPC was added to ensure that the total added mass of VD and VD equaled the baseline total. In the three-factor combination group, PIPC, TGF-β3, and VD were added separately according to the set ratio, and the total amount was still controlled at 10 μg / mL. By doing so, the total amount of additional additives in each group was ensured to be the same, thereby effectively eliminating experimental interference caused by differences in additive dosage. The control group and the experimental group were then cultured in 3 mL of the corresponding conditioned medium. Subculture continuously at 48-hour intervals, and change the corresponding culture medium.

[0050] 5. Assessment of MSC proliferation capacity

[0051] MSCs from both the control and experimental groups were sampled every 48 hours.

[0052] ① Discard the old culture medium and wash gently once with PBS.

[0053] ② Add trypLE Express enzyme to digest the cells, and after stopping the digestion, prepare a cell suspension.

[0054] The cells were stained using trypan blue staining, and live cells were counted using an automated cell counter. The number of cells in each group was recorded and a cell proliferation curve was plotted.

[0055] Cell proliferation curves showed no significant differences in MSC proliferation among the basal medium (DF12) control group, PIPC treatment group, PIPC+TGF-βa group, PIPC+VD group, and PIPC+TGF-βa+VD combined treatment group. This indicates that the optimized conditioned medium for delaying MSC senescence effectively inhibits MSC senescence without adversely affecting normal cell proliferation. Figure 2 ).

[0056] 6. Immunosuppressive capacity assessment

[0057] To evaluate the immunomodulatory capacity of MSCs, this invention co-cultured the human T-lymphoblastic leukemia cell line Jurkat with MSCs to verify changes in the immunosuppressive capacity of MSCs. Jurkat cells were co-cultured with MSCs in basal medium (DF12) control group, PIPC treatment group, PIPC+TGF-βa group, PIPC+VD group, and PIPC+TGF-βa+VD combined treatment group, respectively, at an effector-to-target ratio of 1:10 in 6-well plates, and then co-cultured in 3 mL of DMEM / DF12 medium for 24 hours. After 24 hours, Jurkat cells were collected, and the Jurkat apoptosis rate was analyzed by flow cytometry.

[0058] The experimental results showed that, compared with the control group, MSCs treated with PIPC, TGF-βa, and VD exhibited a certain degree of enhanced immunosuppressive ability when co-cultured with Jurkat cells. Figure 3 ).

[0059] 7. Cell senescence staining

[0060] To systematically evaluate the effects of different culture conditions on the senescence process of MSCs during long-term passage, MSCs were continuously passaged at 48-hour intervals in four different culture systems: a basal medium (DF12) control group, a PIPC treatment group, a PIPC+TGF-β3 group, a PIPC+VD group, and a PIPC+TGF-β3+VD combined treatment group, until the 15th passage (P15). The degree of senescence in each group was then quantitatively analyzed using β-galactosidase staining. The staining results showed that the MSCs in the basal medium (DF12) control group exhibited the highest degree of senescence, followed by the PIPC treatment group. The PIPC+TGF-β3 and PIPC+VD groups alleviated MSC senescence to some extent, while the MSCs in the PIPC+TGF-β3+VD combined treatment group showed the lowest degree of senescence. Figure 4 ).

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A culture medium for delaying the aging of mesenchymal stem cells and enhancing their immunosuppressive function, characterized in that, The components of the culture medium include TLR3 agonist, TGFβ agonist, and vitamin D.

2. The culture medium according to claim 1, characterized in that, The TLR3 agonist is PIPC.

3. The culture medium according to claim 1, characterized in that, The TGFβ agonist is TGFβ3.

4. The culture medium according to claim 1, characterized in that, The total concentration of the TLR3 agonist, the TGFβ agonist, and the VD in the culture medium was 10 μg / mL.

5. A method for culturing mesenchymal stem cells to delay aging and enhance immunosuppressive function, characterized in that, The procedure includes culturing the mesenchymal stem cells in vitro in a culture medium containing a TLR3 agonist, a TGFβ agonist, and vitamin D.

6. The cultivation method according to claim 5, characterized in that, The TLR3 agonist is PIPC.

7. The cultivation method according to claim 5, characterized in that, The TGFβ agonist is TGFβ3.

8. The cultivation method according to claim 5, characterized in that, The total concentration of the TLR3 agonist, the TGFβ agonist, and the VD in the culture medium was 10 μg / mL.

9. The cultivation method according to claim 5, characterized in that, The culture medium is based on DF12.

10. Mesenchymal stem cells obtained by the culture method according to any one of claims 5-9.

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