Human umbilical cord mesenchymal stem cell expansion culture medium and culture method

Abstract

The application discloses a human umbilical cord mesenchymal stem cell expansion culture medium, which comprises a basic culture medium and other additive components. The culture medium of the application can significantly improve the proliferation efficiency of hUC-MSCs, maintain the cell stemness and the multi-directional differentiation capacity, and is high in efficiency, safety and practicability, and is suitable for large-scale expansion and application of hUC-MSCs.

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 suitable for the expansion of human umbilical cord mesenchymal stem cells (hUC-MSCs). Background Technology

[0002] In recent years, umbilical cord mesenchymal stem cells (hUC-MSCs), as a widely available, highly proliferative, and low-immunogenic pluripotent stem cell, have attracted widespread attention in the fields of regenerative medicine and cell therapy. Compared with mesenchymal stem cells from other sources, hUC-MSCs have advantages such as convenient sourcing, lack of ethical controversy, strong proliferative capacity, and outstanding immunomodulatory capabilities, thus being regarded as an ideal cell source for tissue repair, inflammation regulation, and the treatment of immune-related diseases. However, hUC-MSCs often face problems such as slow proliferation rate, easy differentiation, and difficulty in maintaining stemness during in vitro expansion, which limits their widespread application in clinical and research settings.

[0003] Currently, commonly used hUC-MSCs culture media typically contain fetal bovine serum (FBS), which has a complex composition and exhibits batch-to-batch variability, easily leading to unstable culture results. Furthermore, FBS carries potential immunogenicity and pathogen contamination risks, hindering its clinical application in cell therapy. Although some serum alternatives and serum-free culture media products are available on the market, their effectiveness in improving cell proliferation efficiency and maintaining cell stemness still needs improvement. Therefore, developing a serum-free or human serum alternative culture medium to ensure the stability and safety of cell quality during hUC-MSCs expansion is of great significance.

[0004] To meet the demands of hUC-MSCs in clinical treatment and large-scale production, numerous studies have focused on exploring the effects of various growth factors, cytokines, and small molecule compounds on hUC-MSC expansion. However, current culture medium formulations often fail to simultaneously maintain cell proliferation, stemness maintenance, and multi-lineage differentiation potential, leading to cell senescence or loss of pluripotency during long-term culture. Therefore, there is an urgent need for a highly efficient, stable, and safe human umbilical cord mesenchymal stem cell expansion medium capable of rapidly expanding hUC-MSCs in vitro while maintaining their stemness and multi-lineage differentiation potential to meet the needs of clinical research and applications. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium and a culture method for expanding human umbilical cord mesenchymal stem cells.

[0006] Therefore, on the one hand, the present invention discloses an anti-CD29 monoclonal antibody, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0007] Furthermore, this invention also discloses a culture medium for expanding human umbilical cord mesenchymal stem cells, the formulation of which is shown below:

[0008] Basic culture medium: DMEM / F12;

[0009] Human serum albumin: 5%;

[0010] Fibroblast growth factor: 5 ng / mL;

[0011] Epidermal growth factor: 10 ng / mL;

[0012] Vitamin C: 50 μg / mL;

[0013] Anti-CD29 monoclonal antibody: 10 μg / mL;

[0014] Stem cell factor: 10 ng / mL.

[0015] Furthermore, this invention also discloses a culture medium for expanding human umbilical cord mesenchymal stem cells, the formulation of which is shown below:

[0016] Basic culture medium: DMEM / F12;

[0017] Human serum albumin: 5%;

[0018] Fibroblast growth factor: 5 ng / mL;

[0019] Epidermal growth factor: 10 ng / mL;

[0020] Vitamin C: 50 μg / mL;

[0021] Anti-CD29 monoclonal antibody: 10 μg / mL;

[0022] Stem cell factor: 10 ng / mL;

[0023] Plerixafor: 1 μM (final concentration).

[0024] Preferably, the Plerixafor in the culture medium of the present invention is added before each use to ensure activity.

[0025] Furthermore, the present invention also discloses the application of the aforementioned anti-CD29 monoclonal antibody in the preparation of a culture medium for expanding human umbilical cord mesenchymal stem cells.

[0026] Furthermore, the present invention also discloses the application of the culture medium in the expansion of human umbilical cord mesenchymal stem cells.

[0027] The culture medium of this invention exhibits significant advantages in cell proliferation, stemness maintenance, multi-lineage differentiation potential, and immunomodulatory capabilities. CD29, an integrin molecule on the surface of hUC-MSCs, promotes cell-matrix adhesion and plays a crucial role in cell expansion. The addition of an anti-CD29 monoclonal antibody enhances the adhesion and proliferation of hUC-MSCs. Furthermore, Plerixafor, a CXCR4 receptor antagonist, activates proliferation-related signaling pathways in hUC-MSCs by blocking the interaction between CXCR4 and its ligand CXCL12, while simultaneously reducing apoptosis during culture. This small molecule improves the cell survival environment in vitro, thereby promoting cell proliferation. Moreover, the combined use of the anti-CD29 monoclonal antibody and Plerixafor results in even higher hUC-MSC proliferation efficiency. Attached Figure Description

[0028] Figure 1 Results of SDS-PAGE and Western blot analysis of anti-CD29 monoclonal antibody. Detailed Implementation

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0031] Example 1: Preparation of Culture Medium

[0032] Prepare the culture medium according to the following formula:

[0033] Basic culture medium: DMEM / F12;

[0034] Human serum albumin: 5%;

[0035] Fibroblast growth factor: 5 ng / mL;

[0036] Epidermal growth factor: 10 ng / mL;

[0037] Ascorbic acid (Vitamin C): 50 μg / mL;

[0038] Anti-CD29 monoclonal antibody (as shown in Example 5): 10 μg / mL;

[0039] Stem cell factor (SCF): 10 ng / mL.

[0040] Mix all the above ingredients according to the formula ratio, filter and sterilize before use.

[0041] Example 2: Preparation of culture medium

[0042] Prepare the culture medium according to the following formula:

[0043] Basic culture medium: DMEM / F12;

[0044] Human serum albumin: 5%;

[0045] Fibroblast growth factor: 5 ng / mL;

[0046] Epidermal growth factor: 10 ng / mL;

[0047] Ascorbic acid (Vitamin C): 50 μg / mL;

[0048] Anti-CD29 monoclonal antibody (as shown in Example 5): 10 μg / mL;

[0049] Stem cell factor (SCF): 10 ng / mL;

[0050] Plerixafor (AMD3100): 1 μM (final concentration).

[0051] Mix all the above ingredients according to the formula ratio, filter and sterilize before use. Add Plerixafor just before each use to ensure activity.

[0052] Example 3: Cell Expansion Culture

[0053] 1. Isolation of hUC-MSCs: Fresh umbilical cords obtained from cesarean sections were collected (in accordance with ethical requirements and with the consent of the recipient), and placed in sterile bottles containing PBS and 1% penicillin-dextrose antibodies; the umbilical cords were cut into 1-2 cm segments, the umbilical vessels were removed, and the tissue was minced to 1 mm. 3 Size; digest with 0.2% type I collagenase for 2 hours, filter and centrifuge (1000 rpm, 5 minutes) to obtain cell pellet; wash cell pellet twice with sterile PBS, and centrifuge again to collect cells.

[0054] 2. Cell seeding: The separated hUC-MSCs were resuspended in the culture medium prepared in Example 1 or Example 2, and the cell concentration was adjusted to 1×10⁻⁶. 6 cells / mL; after inoculation, the cells were placed in an incubator at 37°C and 5% CO2 for culture.

[0055] 3. Medium change: Use the same culture medium and change the medium every 2 days for 7 days.

[0056] 4. Cell passage: When the cell confluence reaches 80%-90%, digest the cells with 0.25% trypsin-EDTA solution and then passage, cryopreserve, or induce culture. The passage ratio is generally 1:3.

[0057] Example 4: Post-culture cell detection

[0058] 1. Effect of Culture Medium on hUC-MSC Proliferation: hUC-MSCs were seeded in the culture media of Example 1 and Example 2, as well as in conventional medium containing 10% FBSDMEM / F12. After 7 days of culture, cell proliferation was detected using the CCK-8 assay. The results (as shown in Table 1) showed that the cell proliferation rates in the culture media of Example 1 and Example 2 were significantly higher than those in the control group, with the culture medium of Example 2 showing better results than that of Example 1. Therefore, the culture medium of Example 2 was used to culture hUC-MSCs for further research.

[0059] Table 1 Results of proliferation detection

[0060]

[0061] 2. Multi-lineage differentiation capacity detection: 10th generation hUC-MSCs cultured in the culture medium of Example 2 and the control culture medium (containing 10% FBS DMEM / F12 medium) were induced to differentiate into adipocytes, osteoblasts and chondrocytes, and respectively stained.

[0062] (1) Adipocyte differentiation: After 14 days of induction, Oil Red O staining showed positive lipid droplets, and the adipocyte formation rate was 93.2%, which was 25% higher than that of the control group.

[0063] (2) Osteogenic differentiation: After 21 days of induction, alizarin red staining showed obvious calcified nodules, and the degree of calcification was 35% higher than that of the control group.

[0064] (3) Chondrogenesis: After 28 days of induction, safranin staining showed that a large number of chondrocyte clusters were formed, and the chondrocyte formation efficiency was 25% higher than that of the control group.

[0065] 3. Cell stemness detection: The 10th generation hUC-MSCs expanded in the culture medium of Example 2 were detected by flow cytometry. The positive expression rates of CD73, CD90 and CD105 in these cells were 97.2%, 98.5% and 96.8%, respectively, indicating that the culture medium of the present invention can effectively maintain cell stemness.

[0066] 4. Immunomodulatory capacity assay: 10th generation hUC-MSCs were co-cultured with human peripheral blood mononuclear cells (PBMCs) to detect their inhibitory capacity on T cell proliferation. Results showed that under co-culture conditions, hUC-MSCs inhibited T cell proliferation by 75.3%, significantly higher than hUC-MSCs expanded using the control medium (containing 10% FBSDMEM / F12 medium) (49.6%).

[0067] Example 5: Preparation of anti-CD29 monoclonal antibody

[0068] 1. A vaccine was prepared by emulsifying recombinant human CD29 protein (ab114157) with complete Freund's adjuvant, with a CD29 protein concentration of 100 μg / mL. Six-week-old female mice were then immunized with 0.2 mL of the vaccine subcutaneously. Subsequently, the recombinant human CD29 protein was emulsified with incomplete Freund's adjuvant, and secondary and tertiary immunizations were performed using the same method, with an interval of 2 weeks between immunizations. Seven days after the tertiary immunization, serum was collected, and the titer of specific antibodies was detected by ELISA. Mice with higher titers were selected for cell fusion. Spleens from mice with higher titers were selected, and spleen cell suspensions were prepared and mixed with myeloma cells at a ratio of 10:1. Cell fusion was performed using 40% PEG solution at 37°C. After fusion, the cell suspension was diluted and added to HAT-containing selective medium, and incubated at 37°C in a 5% CO2 incubator in the dark. After 7 days, hybridoma cells were selected, and cell lines with specific antibodies were screened using ELISA. The selected positive cell lines were then subjected to ELISA to confirm their binding activity to the CD29 protein. The positive cell lines were then cloned using a limiting dilution method until stable monoclonal cell lines were obtained. Finally, the obtained monoclonal antibody was purified using a protein G affinity column to obtain a specific monoclonal antibody against the CD29 protein. This antibody was sterilely filtered through a 0.22 μm filter membrane and stored at -80°C for later use. The concentration determined by BCA was 2.56 mg / mL.

[0069] 2. The purity and binding activity of the monoclonal antibody were analyzed using SDS-PAGE and Western blot methods. The results are shown in [Figure number missing]. Figure 1The monoclonal antibody exhibited a purity of over 95% on SDS-PAGE, and Western blot results showed that it specifically binds to CD29 protein (where 1 is CD29 protein with a molecular weight of approximately 115 kDa). The binding activity of this monoclonal antibody against commercially available monoclonal antibodies was detected by ELISA using a coating of recombinant human CD29 protein (both monoclonal antibodies were adjusted to a concentration of 1 mg / mL before dilution). The results are shown in Table 2, demonstrating that the binding activity of the monoclonal antibody of this invention is superior to that of commercially available monoclonal antibodies.

[0070] Table 2. Monoclonal antibody ELISA detection results (OD450nm)

[0071] 3. Next, this study analyzed the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody. The results showed that the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0072] Table 3. Results of monoclonal antibody sequence analysis

[0073]

[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An anti-CD29 monoclonal antibody, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

2. A human umbilical cord mesenchymal stem cell expansion medium, characterized by, The formulation of the medium is as follows: Base medium: DMEM / F12; Human serum albumin: 5%; Fibroblast growth factor: 5 ng / mL; Epidermal growth factor: 10 ng / mL; Vitamin C: 50 μg / mL; The anti-CD29 monoclonal antibody of claim 1: 10 μg / mL; Stem cell factor: 10 ng / mL.

3. A human umbilical cord mesenchymal stem cell expansion medium, characterized by, The formulation of the medium is as follows: Base medium: DMEM / F12; Human serum albumin: 5%; Fibroblast growth factor: 5 ng / mL; Epidermal growth factor: 10 ng / mL; Vitamin C: 50 μg / mL; The anti-CD29 monoclonal antibody of claim 1: 10 μg / mL; Stem cell factor: 10 ng / mL; Plerixafor: 1 μM.

4. The medium of claim 3, wherein, The Plerixafor in the medium is added before each use to ensure activity.

5. Use of the anti-CD29 monoclonal antibody of claim 1 in the preparation of a human umbilical cord mesenchymal stem cell expansion medium.

6. Use of the medium of claim 2 or 3 in the expansion of human umbilical cord mesenchymal stem cells.

Images