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

Abstract

The invention discloses a human umbilical cord mesenchymal stem cell amplification culture medium. The culture medium comprises a basic culture medium and other additive components. According to the culture medium, the proliferation efficiency of the hUC-MSCs is remarkably improved, the cell dryness and the multidirectional differentiation capacity are maintained, and the culture medium has high efficiency, safety and practicability and is suitable for large-scale amplification and application of the hUC-MSCs.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell culture, and in particular to a culture medium suitable for expanding human umbilical cord mesenchymal stem cells (hUC-MSCs) and a culture method thereof. Background Art

[0002] In recent years, umbilical cord mesenchymal stem cells (hUC-MSCs), as a multipotent stem cell with a wide range of sources, strong proliferation ability and low immunogenicity, have attracted widespread attention in the field of regenerative medicine and cell therapy. Compared with mesenchymal stem cells from other sources, hUC-MSCs have the advantages of easy collection, no ethical controversy, strong proliferation ability, and outstanding immunoregulatory ability. Therefore, they are regarded as an ideal cell source for tissue repair, inflammation regulation, and treatment of immune-related diseases. However, hUC-MSCs often face problems such as slow proliferation, easy differentiation, and difficulty in maintaining stemness during in vitro expansion, which limits their wide application in clinical and scientific research.

[0003] At present, the commonly used hUC-MSCs culture medium usually contains fetal bovine serum (FBS), which has complex components and batch differences, which can easily lead to unstable culture results. In addition, FBS also has potential immunogenicity and pathogen contamination risks, which is not conducive to the clinical application of cell therapy. Although there are some serum substitutes and serum-free culture medium products on the market, their effects in improving cell proliferation efficiency and maintaining cell stemness still need to be improved. Therefore, it is of great significance to develop a serum-free or humanized serum substitute culture medium to ensure the stability and safety of cell quality during hUC-MSCs expansion.

[0004] In order to meet the needs of hUC-MSCs in clinical treatment and large-scale production, many studies have been devoted to exploring the effects of various growth factors, cytokines and small molecule compounds on the expansion of hUC-MSCs. However, current culture medium formulations usually fail to take into account cell proliferation, stemness maintenance and multi-directional differentiation potential at the same time, resulting in cell aging or loss of pluripotency in long-term culture. Therefore, there is an urgent need for an efficient, stable and safe human umbilical cord mesenchymal stem cell expansion medium that can rapidly expand hUC-MSCs in vitro while maintaining their stemness and multi-directional differentiation potential to meet the needs of clinical research and application. . Summary of the invention

[0005] The purpose of the present invention is to provide a human umbilical cord mesenchymal stem cell expansion culture medium and a culture method.

[0006] Therefore, in one aspect, the present invention discloses an anti-CD29 monoclonal antibody, 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.

[0007] In another aspect, the present invention further discloses a human umbilical cord mesenchymal stem cell expansion medium, the formula of the medium is as follows:

[0008] Basal 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] In another aspect, the present invention further discloses a human umbilical cord mesenchymal stem cell expansion medium, the formula of the medium is as follows:

[0016] Basal 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, Plerixafor in the culture medium of the present invention is added before each use to ensure activity.

[0025] In another aspect, the present invention also discloses the use of the anti-CD29 monoclonal antibody in preparing a culture medium for expansion of human umbilical cord mesenchymal stem cells.

[0026] In another aspect, the present invention also discloses the use of the culture medium in the expansion of human umbilical cord mesenchymal stem cells.

[0027] The culture medium of the present invention shows significant advantages in terms of cell proliferation, stemness maintenance, multidirectional differentiation potential and immunoregulatory ability. Among them, CD29 is an integrin molecule on the surface of hUC-MSCs, which can promote the adhesion of cells to the matrix and play a key role in the cell expansion process. By adding anti-CD29 monoclonal antibodies, the adhesion and proliferation ability of hUC-MSCs can be enhanced. In addition, Plerixafor is a CXCR4 receptor antagonist that can activate the proliferation-related signaling pathway of hUC-MSCs by blocking the interaction between CXCR4 and its ligand CXCL12, while reducing cell apoptosis during culture. This small molecule can improve the living environment of cells in in vitro culture, thereby promoting cell proliferation. And when anti-CD29 monoclonal antibodies and Plerixafor are used in combination, the proliferation efficiency of hUC-MSCs is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Anti-CD29 monoclonal antibody SDS-PAGE and western blot detection results. DETAILED DESCRIPTION

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0030] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] Example 1: Preparation of culture medium

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

[0033] Basal 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 (described 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 them for later use.

[0041] Example 2: Preparation of culture medium

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

[0043] Basal 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 (described 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 them for later use. Before each use, add Plerixafor temporarily to ensure activity.

[0052] Example 3: Cell expansion culture

[0053] 1. Isolation of hUC-MSCs: Fresh umbilical cords obtained by cesarean section (in accordance with ethical requirements and with the consent of the other party) were collected and placed in a sterile bottle containing PBS and 1% double antibody; the umbilical cords were cut into 1-2 cm segments, and after removing the umbilical blood vessels, the tissues were cut into 1 mm 3 Size; digested with 0.2% type I collagenase for 2 hours, filtered and centrifuged (1000 rpm, 5 minutes) to obtain cell pellets; washed the cell pellets twice with sterile PBS and centrifuged again to collect the cells.

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

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

[0056] 4. Cell passaging: When the cell confluence reaches 80%-90%, use 0.25% trypsin-EDTA solution to digest the cells and perform subculture, cryopreservation or induced culture. The subculture ratio is generally 1:3.

[0057] Example 4: Cell Detection after Culture

[0058] 1. Detection of the effect of culture medium on the proliferation ability of hUC-MSCs: hUC-MSCs were inoculated in the culture medium of Example 1, Example 2 and the traditional culture medium containing 10% FBSDMEM / F12, respectively. After 7 days of culture, the cell proliferation was detected by CCK-8 method. The results showed (as shown in Table 1) that the cell proliferation rate of the culture medium of Example 1 and Example 2 was much higher than that of the control group, and the culture medium of Example 2 was better than that of Example 1. Therefore, the culture medium of Example 2 was used to culture hUC-MSCs for the next step of research.

[0059] Table 1 Proliferation test results

[0060]

[0061] 2. Multidirectional differentiation ability detection: The 10th generation hUC-MSCs cultured in the culture medium of Example 2 and the culture medium of the control group (DMEM / F12 culture medium containing 10% FBS) were induced to differentiate into adipocytes, osteoblasts and chondrocytes, and the relevant staining was performed respectively.

[0062] (1) Adipose 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) Chondrogenic differentiation: After 28 days of induction, safranin staining results 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 amplified with the culture medium of Example 2 were detected by flow cytometry. The positive expression rates of CD73, CD90 and CD105 of the cells were 97.2%, 98.5% and 96.8%, respectively, indicating that the culture medium of the present invention can effectively maintain the stemness of the cells.

[0066] 4. Immunomodulatory capacity detection: The 10th generation hUC-MSCs were co-cultured with human peripheral blood mononuclear cells (PBMCs) to detect their ability to inhibit T cell proliferation. The results showed that under co-culture conditions, the inhibition rate of hUC-MSCs on T cell proliferation reached 75.3%, which was significantly higher than that of hUC-MSCs expanded using the control group culture medium (containing 10% FBSDMEM / F12 culture medium) (49.6%).

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

[0068] 1. The recombinant human CD29 protein (ab114157) was emulsified with complete Freund's adjuvant to prepare a vaccine, wherein the concentration of CD29 protein in the vaccine was 100 μg / mL, and then 6-week-old female mice were immunized, with each mouse subcutaneously injected with 0.2 mL of the vaccine. Then, after the recombinant human CD29 protein was emulsified with incomplete Freund's adjuvant, the second and third immunizations were performed in the same way, with an immunization interval of 2 weeks. Serum was collected 7 days after the third immunization, and the specific antibody titer was detected by ELISA, and mice with higher titers were screened for cell fusion. The spleen of mice with higher titers was selected, and after preparing spleen cell suspension, it was mixed with myeloma cells at a ratio of 10:1, and cell fusion was performed using a 40% PEG solution at 37°C. After the fusion was completed, the cell suspension was diluted and added to a HAT selection medium, and placed in a 37°C, 5% CO2 incubator for incubation in the dark. After 7 days, the grown hybridoma cells were selected, and the cell lines of specific antibodies were screened by ELISA. The screened positive cell lines were then further tested by ELISA to confirm their binding activity to the CD29 protein, and the positive cell lines were cloned by limiting dilution until a stable monoclonal cell line was obtained. Finally, the obtained monoclonal antibody was purified using a protein G affinity column to obtain a specific monoclonal antibody against the CD29 protein. The antibody was sterilized and filtered with a 0.22 μm filter membrane and stored at -80°C for later use. The concentration was 2.56 mg / mL by BCA detection.

[0069] 2. The purity and binding activity of the monoclonal antibody were analyzed by SDS-PAGE and western blot. Figure 1, wherein the purity of the monoclonal antibody by SDS-PAGE is above 95%; and the western blot results show that the monoclonal antibody can specifically bind to the CD29 protein (where 1 is the CD29 protein, and its molecular weight is about 115kD). At the same time, the binding activity of the monoclonal antibody and the commercially available monoclonal antibody was detected by ELISA method by coating the recombinant human CD29 protein (the concentration of the two monoclonal antibodies was adjusted to 1 mg / mL before dilution). The results are shown in Table 2. The binding activity of the monoclonal antibody of the present invention is better than that of the commercially available monoclonal antibody.

[0070] Table 2 Monoclonal antibody ELISA test 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 were as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0072] Table 3 Monoclonal antibody sequence analysis results

[0073]

[0074] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in 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 in that: The formula of the culture medium is as follows: Basal 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 in that: The formula of the culture medium is as follows: Basal 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 culture medium according to claim 1, characterized in that Plerixafor in the culture medium was added before each use to ensure activity.

5. Use of the anti-CD29 monoclonal antibody according to claim 1 in preparing a culture medium for expansion of human umbilical cord mesenchymal stem cells.

6. Use of the culture medium according to any one of claims 2 or 3 in the expansion of human umbilical cord mesenchymal stem cells.

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