A medium additive for mesenchymal stem cells and use thereof

Abstract

This invention relates to the field of cell culture technology, and more particularly to a culture medium additive for mesenchymal stem cells and its application. The culture medium additive of this invention, using water as a solvent, comprises the following components at the following concentrations: 0.5-1.5 mg / L rhTXNDC5 protein, 10-20 mg / L vascular endothelial cadherin 5, 10-20 mg / L vascular endothelial cadherin 11, 10-20 mg / L recombinant human long-acting insulin-like growth factor-1, 400-600 mg / L dextran, 200-400 mg / L riboflavin, 300-500 IU / L superoxide dismutase, 10-20 mg / L ethanolamine, and 1-3 mg / L progesterone. Adding this culture medium additive to serum-free culture medium can increase the yield of mesenchymal stem cells and accelerate the emergence time of umbilical cord mesenchymal stem cells.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a culture medium additive for mesenchymal stem cells and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are adult stem cells belonging to the mesoderm, possessing multipotent differentiation potential, excellent in vitro expansion capacity, and immunomodulatory functions. MSCs exhibit strong differentiation capabilities; under specific culturing conditions, they can proliferate and differentiate into various cell types, including adipocytes, chondrocytes and osteocytes, myocytes, and nerve cells.

[0003] Mesenchymal stem cells (MSCs) are widely available and easily extracted, existing in various human tissues and organs such as adult bone marrow, dental pulp, adipose tissue, lung, liver, and perinatal umbilical cord blood, umbilical cord, and placenta. Among them, human umbilical cord-derived mesenchymal stem cells (UC-MSCs) derived from umbilical cord tissue are particularly convenient to obtain, easily isolated, cultured, expanded, and purified. Even after multiple passages and expansions, they retain significant stem cell characteristics, exhibiting minimal surface antigens, low rejection rates in allogeneic transplantation, and less stringent matching requirements. UC-MSCs also possess potent immunomodulatory functions, inhibiting T cell proliferation and immune responses through cell-cell interactions and cytokine production, thereby contributing to immune reconstitution. These characteristics make MSCs an ideal biological platform for scientific research and clinical applications, such as cell therapy, regenerative medicine, drug screening, the development of gene therapy vectors, research on the molecular regulatory mechanisms of cell development and differentiation, and seed cells for tissue engineering.

[0004] Umbilical cord tissue is an extremely abundant source of stem cells. While umbilical cords are typically disposed of as medical waste after childbirth, the legal and compliant exploitation of umbilical cord resources to obtain UC-MSCs for the treatment of human diseases would greatly benefit the development of human health. Despite the abundance of umbilical cord tissue, obtaining high-yield, highly active, highly homogeneous, and safer umbilical cord mesenchymal stem cells remains a crucial issue in mesenchymal stem cell research and application.

[0005] Traditional mesenchymal stem cell culture media typically use basal media such as MEM-alpha or DMEM / F12 supplemented with fetal bovine serum. These media have drawbacks such as uncertain composition, poor batch-to-batch consistency, presence of heterologous proteins, and low amplification yield.

[0006] There are two types of commercially available serum-free culture media for mesenchymal stem cells: one containing platelet lysate (hPL) and the other with a defined composition. Serum-free media containing hPL promotes rapid cell growth in mesenchymal stem cells, but it also suffers from drawbacks such as unclear composition and poor batch-to-batch consistency. Furthermore, hPL requires testing for various infectious diseases, such as HIV, before use in cell culture. On the other hand, serum-free media with a defined composition still lag significantly behind those containing hPL in culturing mesenchymal stem cells, particularly in isolating primary seed cells.

[0007] To address the existing problems, there is an urgent need for a culture medium that can improve the proliferation rate of mesenchymal stem cells when culturing them in serum-free medium with well-defined components, especially to improve its efficiency in isolating primary seed cells using the implantation method, accelerate the migration rate of seed cells, and increase the yield of seed cells. Summary of the Invention

[0008] The purpose of this invention is to provide a culture medium additive that improves the proliferation rate of mesenchymal stem cells cultured in serum-free culture media with well-defined composition, particularly improving its efficiency in the isolation of primary seed cells using the implantation method, accelerating the migration rate of primary seed cells, and increasing the yield of primary seed cells; particularly, it relates to a culture medium additive for mesenchymal stem cells and its application.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a culture medium additive for mesenchymal stem cells, comprising the following components in water as a solvent: 0.5~1.5 mg / L rhTXNDC5 protein, 10~20 mg / L endothelial cadherin 5, 10~20 mg / L endothelial cadherin 11, 10~20 mg / L recombinant human long-acting insulin-like growth factor-1, 400~600 mg / L dextran, 200~400 mg / L riboflavin, 300~500 IU / L superoxide dismutase, 10~20 mg / L ethanolamine, 1~3 mg / L progesterone.

[0010] Preferably, the water-based solvent comprises components with the following concentrations: 0.7~1.3 mg / L rhTXNDC5 protein, 12~18 mg / L endothelial cadherin 5, 12~18 mg / L endothelial cadherin 11, 12~18 mg / L recombinant human long-acting insulin-like growth factor-1, 450~550 mg / L dextran, 250~350 mg / L riboflavin, 350~450 IU / L superoxide dismutase, 12~18 mg / L ethanolamine, 1.5~2.5 mg / L progesterone.

[0011] Preferably, the water-based solvent comprises components with the following concentrations: 1.0 mg / L rhTXNDC5 protein, 15 mg / L endothelial cadherin 5, 15 mg / L endothelial cadherin 11, 15 mg / L recombinant human long-acting insulin-like growth factor-1, 500 mg / L dextran, 300 mg / L riboflavin, 400 IU / L superoxide dismutase, 15 mg / L ethanolamine, 2.0 mg / L progesterone.

[0012] The present invention also provides the application of the aforementioned culture medium additive in the preparation of products that improve the proliferation rate of mesenchymal stem cells.

[0013] The present invention also provides the application of the aforementioned culture medium additive in the preparation of primary seed cells for improving the isolation efficiency of mesenchymal stem cells; The separation efficiency includes the crawling rate and the output.

[0014] The present invention also provides a method for preparing a culture medium additive, comprising the following steps: mixing the components of the culture medium additive according to their concentrations, filtering the mixture through a filter membrane, and obtaining the culture medium additive. The pore size of the filter membrane is 0.2~0.3μm.

[0015] The present invention also provides a culture medium for improving the isolation efficiency of primary seed cells of mesenchymal stem cells, comprising the aforementioned culture medium additive and serum-free culture medium for mesenchymal stem cells; The volume ratio of the culture medium additive to the serum-free culture medium for mesenchymal stem cells is 1~3:97~99.

[0016] This invention also provides a method for preparing a culture medium that improves the isolation efficiency of primary seed cells of mesenchymal stem cells, comprising the following steps: mixing culture medium additives with serum-free culture medium for mesenchymal stem cells at a volume ratio of 1~3:97~99 to obtain the culture medium. The culture medium additive is the culture medium additive described above.

[0017] The present invention also provides the application of the culture medium described herein or the culture medium prepared by the preparation method described herein in improving the proliferation rate of mesenchymal stem cells.

[0018] The present invention also provides the application of the culture medium described herein or the culture medium prepared according to the preparation method described herein in improving the isolation efficiency of primary seed cells of mesenchymal stem cells.

[0019] The present invention has the following advantages: The culture medium additive of this invention includes rhTXNDC5 protein, an important endoplasmic reticulum (ER) resident protein involved in regulating collagen assembly and maturation, cell migration, and promoting the emergence of primary mesenchymal stem cells (MSCs). The addition of vascular endothelial cadherin 5 (rhCDH5) and vascular endothelial cadherin 11 (rhCDH11) helps maintain the homeostasis of the MSC microenvironment, regulates interactions between MSCs, and maintains MSC characteristics. Recombinant human long-acting insulin-like growth factor-1 (rhIGF-1) helps promote protein synthesis and increase amino acid uptake, thus promoting MSC proliferation and the emergence of primary MSCs. The addition of dextran and riboflavin, when added simultaneously, has a significant synergistic effect, significantly promoting MSC proliferation and the emergence of primary MSCs. This invention adds superoxide dismutase, which catalyzes the conversion of superoxide free radicals into hydrogen peroxide and molecular oxygen, playing a crucial role in resisting cell damage caused by oxygen free radicals and inhibiting apoptosis. Ethanolamine, a precursor molecule of phosphatidylethanolamine, plays an important role in cell proliferation and division; therefore, adding ethanolamine can promote cell proliferation. Progesterone, as a steroid hormone, participates in the functional regulation of stem cells and increases the secretion of immunomodulatory proteins such as HLA-G and PIBG.

[0020] The recombinant proteins, cytokines, antioxidant enzymes, nutrients, and hormones in the culture medium additive of this invention work synergistically without the addition of heterologous components. At the same time, it can efficiently improve the yield and emergence efficiency of primary seed cells isolated by umbilical cord tissue implantation, greatly shorten the cycle of primary cell isolation in serum-free culture system, and has good cell uniformity, which can effectively maintain the biological characteristics and differentiation potential of mesenchymal stem cells.

[0021] The mesenchymal stem cell culture medium additive of this invention has a clearly defined composition, does not contain heterologous components, and ensures the consistency of cell culture across multiple batches in the culture system.

[0022] The mesenchymal stem cell culture medium additive of the present invention, when added to a serum-free mesenchymal stem cell culture medium with clearly defined components, can further improve cell harvest yield and accelerate the emergence time of primary umbilical cord mesenchymal stem cells. Attached Figure Description

[0023] Figure 1 Mesenchymal stem cells cultured in the M1 group culture medium for 13 days (A and B are both mesenchymal stem cells cultured in the M1 group for 13 days). Figure 2 For the detection of surface markers of umbilical cord mesenchymal stem cells; Figure 3 The growth of umbilical cord mesenchymal stem cells cultured in different culture media to the P5 generation (A is the growth of P5 generation cells in group M1-1, and B is the growth of P5 generation cells in group M2-1). Figure 4 Image showing the adipogenesis results of umbilical cord mesenchymal stem cells cultured to passage P5; Figure 5 An image showing the osteogenic results of umbilical cord mesenchymal stem cells cultured to passage P5; Figure 6 This image shows the chondrogenic results of umbilical cord mesenchymal stem cells cultured to passage P5. Detailed Implementation

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] The serum-free mesenchymal stem cell culture medium described in this embodiment of the invention is the serum-free mesenchymal stem cell culture medium prepared in Example 1 of Chinese Patent CN118406647A "A Mesenchymal Stem Cell Culture Medium with Definite Composition and Its Application", specifically DMEM / F12 basal culture medium + FM9002.

[0026] In the embodiments of this invention, rhTXNDC5 protein, vascular endothelial cadherin 5 (rhCDH5), vascular endothelial cadherin 11 (rhCDH11), recombinant human long-acting insulin-like growth factor-1 (rhIGF-1) were purchased from MCE, dextran was purchased from Adamas, riboflavin was purchased from Aladdin, superoxide dismutase was purchased from Aladdin, ethanolamine was purchased from Sigma, and progesterone was purchased from Sigma.

[0027] In this embodiment of the invention, the mesenchymal stem cells are obtained from the umbilical cord.

[0028] Example 1

[0029] Culture medium additive for mesenchymal stem cells (FM9003)

[0030] Take 1 mg of rhTXNDC5 protein, 15 mg of rhCDH5 protein, 15 mg of rhCDH11 protein, 15 mg of rhIGF-1, 500 mg of dextran, 300 mg of riboflavin, 400 IU of superoxide dismutase, 15 mg of ethanolamine, and 2 mg of progesterone, and make up to 1 L with water. Mix and dissolve, filter through a 0.22 μm filter membrane to sterilize, and obtain the culture medium additive, denoted as FM9003.

[0031] Culture medium to improve the isolation efficiency of primary seed cells of mesenchymal stem cells

[0032] The culture medium additive FM9003 was prepared by mixing it with serum-free culture medium for mesenchymal stem cells at a volume ratio of 1:99.

[0033] Example 2

[0034] The effectiveness of FM9003 obtained in Example 1 was verified.

[0035] Extraction of umbilical cord mesenchymal stem cells: Extraction was performed from the umbilical cord of a healthy fetus at full term, provided by Daping Hospital. Extraction method: The umbilical cord was removed and disinfected by immersion in 75% alcohol for 2 minutes, then rinsed twice with physiological saline before separation preparation. The umbilical cord bloodstains were cleaned with physiological saline, cut into 2cm segments, the vein lumen was cut open, and the vein wall, artery, and epidermis were removed. Wharton's jelly was then separated and cut into 2mm pieces. 3 Small pieces of Wharton's gel were then evenly seeded into T75 culture flasks (the entire separation process was carried out in a clean bench), 15 mL of complete culture medium was added, and the flasks were incubated at 37°C in a 5% CO2 incubator. Before primary cells emerged, the culture medium was changed every 7 days. After primary cells began to emerge, the culture medium was changed every 2 days. When the primary cells emerged and reached confluence of 80% or more, they were passaged.

[0036] 1. Experimental Methods

[0037] Wharton's jelly from umbilical cord separation was seeded at a rate of 0.6 g per T75 culture flask. Three culture flasks were seeded with the serum-free culture medium (prepared in Example 1) to improve the isolation efficiency of primary mesenchymal stem cells, denoted as M1. Simultaneously, the serum-free mesenchymal stem cell culture medium (DMEM / F12 basal medium + FM9002) disclosed in CN118406647A "A Mesenchymal Stem Cell Culture Medium with Definitive Composition and Its Application" was seeded as a control, denoted as M2.

[0038] After inoculation, the primary cells were observed to emerge daily. The cells were counted at passage P0 and then passaged to passage P1.

[0039] In group M1, the primary cell migration time after Wharton's jelly inoculation was 12±2 days, and the passage time for P0 generation cells was 14±2 days. In group M2, the primary cell migration time after Wharton's jelly inoculation was 15±1 days, and the passage time for P0 generation cells was 17±2 days. Cell counts for P0 and P1 generations are shown in Table 1. A photograph taken on day 13 of mesenchymal stem cell culture medium supplemented with FM9003 components is shown below. Figure 1 As shown.

[0040] Table 1. Characteristics of cells isolated from different culture media

[0041] Table 1 shows that, compared with group M2, group M1 showed a 100.45% and 41.71% increase in cell yield and a 2% increase in cell viability when isolating and culturing P0 and P1 generation mesenchymal stem cells, respectively. This indicates that the additive FM9003 component provided by this invention, when added to a serum-free culture medium with clearly defined components for mesenchymal stem cells, can shorten the cycle of isolating primary cells in a serum-free culture system and efficiently improve the yield and emergence efficiency of primary seed cells isolated by the umbilical cord tissue implantation method.

[0042] Figure 1 The image shows tissue spheres adhering to the wall, with dense growth of mesenchymal stem cells, indicating that they can be passaged.

[0043] 2. Cell identification

[0044] Cellular immunostaining and flow cytometry were used to detect surface markers of mesenchymal stem cells cultured in M1 group culture medium. Positive markers included CD73, CD90, and CD105; negative markers included CD19, CD34, CD45, CD11b, and HLA-DR.

[0045] Test results as follows Figure 2 As shown.

[0046] Figure 2 The results showed that the expression levels of positive markers CD73, CD90, and CD105 were all above 99%, while the expression levels of negative markers CD34, CD45, CD11b, and HLA-DR were all below 1%. This indicates that the additive FM9003 component provided by this invention, when added to a serum-free culture medium for mesenchymal stem cells with well-defined components, can efficiently maintain the biological characteristics of the stem cells and meet the relevant indicator requirements.

[0047] Example 3

[0048] P1 generation cells from group M1 of Example 2 were cultured continuously in T75 culture flasks using the culture medium from group M1 (the culture medium prepared in Example 1 to improve the isolation efficiency of primary seed cells of mesenchymal stem cells). The volume of culture medium added was 15 mL (denoted as M1-1). P1 generation cells from group M2 of Example 2 were also cultured continuously in T75 culture flasks using the culture medium from group M2. The volume of culture medium added was 15 mL (denoted as M2-1). When the mesenchymal stem cell confluence reached approximately 85%, the cells were passaged, and the culture medium was replaced with fresh medium. Cells were counted during passage, and then cultured at 7000 / cm². 2 Cells were seeded at a density of [insert density here] in T75 culture flasks and passaged four times consecutively, with three replicates per sample. Cell counts at each passage are shown in Table 2. Photographs of P5 passage cells are shown below. Figure 3 As shown.

[0049] Table 2. Cell viability of P2-P5 generation cells after 72 h of culture in different culture media.

[0050] Table 2 and Figure 3 The results showed that, compared with the M2-1 group, the M1-1 group also had a cell yield increase of more than 10% when culturing P2~P5 generation mesenchymal stem cells, and a slight increase in cell viability. This indicates that the additive FM9003 component provided by the present invention can continuously increase the cell yield of mesenchymal stem cells after being added to the serum-free culture medium for mesenchymal stem cells with clearly defined components.

[0051] Example 4

[0052] P5 generation cells obtained from group M1 in Example 3 were collected and cultured for 3 weeks using an adipogenic differentiation induction medium kit (Pronosai). Cells were fixed with 4% paraformaldehyde, stained with Oil Red O, and observed under a microscope. Results are as follows: Figure 4 As shown.

[0053] P5 generation cells obtained from group M1 in Example 3 were collected and cultured for 3 weeks using an osteogenic induction differentiation culture kit (ScienCell). Cells were fixed with 4% paraformaldehyde, stained with alizarin red, and observed under a microscope. The results are as follows: Figure 5 As shown.

[0054] P5 generation cells obtained from group M1 in Example 3 were collected and cultured for 3 weeks using a chondrogenic differentiation induction culture kit (Cyagen Biosciences). Cells were fixed with 4% paraformaldehyde, stained with alexandrite blue, and observed under a microscope. The results are as follows: Figure 6 As shown.

[0055] Figure 4 The results showed that mesenchymal stem cells could still be successfully induced into adipocytes after multiple generations of culture. Figure 5The results showed that mesenchymal stem cells could still be successfully induced into osteoblasts after multiple generations of culture. Figure 6 The results show that mesenchymal stem cells can still be successfully induced into chondrocytes after multiple generations of culture. This indicates that the mesenchymal stem cell culture medium additive provided by this invention, when added to serum-free mesenchymal stem cell culture medium, can still efficiently maintain the differentiation potential of mesenchymal stem cells and meet relevant indicator requirements after multiple generations of continuous culture.

[0056] As can be seen from the above embodiments, the mesenchymal stem cell culture medium additive of the present invention has a clearly defined composition, does not contain heterologous components, and can ensure the consistency of the culture system. Adding this culture medium additive to serum-free mesenchymal stem cell culture medium can increase the yield of mesenchymal stem cells and accelerate the emergence time of umbilical cord mesenchymal stem cells.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A culture medium additive for mesenchymal stem cells, characterized in that, The components using water as a solvent include the following concentrations: 0.5~1.5 mg / L rhTXNDC5 protein, 10~20 mg / L endothelial cadherin 5, 10~20 mg / L endothelial cadherin 11, 10~20 mg / L recombinant human long-acting insulin-like growth factor-1, 400~600 mg / L dextran, 200~400 mg / L riboflavin, 300~500 IU / L superoxide dismutase, 10~20 mg / L ethanolamine, 1~3 mg / L progesterone.

2. The culture medium additive according to claim 1, characterized in that, The components using water as a solvent include the following concentrations: 0.7~1.3 mg / L rhTXNDC5 protein, 12~18 mg / L endothelial cadherin 5, 12~18 mg / L endothelial cadherin 11, 12~18 mg / L recombinant human long-acting insulin-like growth factor-1, 450~550 mg / L dextran, 250~350 mg / L riboflavin, 350~450 IU / L superoxide dismutase, 12~18 mg / L ethanolamine, 1.5~2.5 mg / L progesterone.

3. The culture medium additive according to claim 1, characterized in that, The components using water as a solvent include the following concentrations: 1.0 mg / L rhTXNDC5 protein, 15 mg / L endothelial cadherin 5, 15 mg / L endothelial cadherin 11, 15 mg / L recombinant human long-acting insulin-like growth factor-1, 500 mg / L dextran, 300 mg / L riboflavin, 400 IU / L superoxide dismutase, 15 mg / L ethanolamine, 2.0 mg / L progesterone.

4. The use of the culture medium additive according to any one of claims 1 to 3 in the preparation of products that improve the proliferation rate of mesenchymal stem cells.

5. The use of the culture medium additive according to any one of claims 1 to 3 in preparing primary seed cells for improving the isolation efficiency of mesenchymal stem cells; The separation efficiency includes the crawling rate and the output.

6. A method for preparing a culture medium additive, characterized in that, The process includes the following steps: mixing the components of the culture medium additive according to the concentrations of each component as described in any one of claims 1 to 3, filtering the mixture through a filter membrane, and obtaining the culture medium additive. The pore size of the filter membrane is 0.2~0.3μm.

7. A culture medium for improving the isolation efficiency of primary seed cells of mesenchymal stem cells, characterized in that, Includes the culture medium additives described in any one of claims 1 to 3 and serum-free culture medium for mesenchymal stem cells; The volume ratio of the culture medium additive to the serum-free culture medium for mesenchymal stem cells is 1~3:97~99.

8. A method for preparing a culture medium to improve the isolation efficiency of primary seed cells of mesenchymal stem cells, characterized in that, The process includes the following steps: mixing the culture medium additive with the serum-free culture medium for mesenchymal stem cells at a volume ratio of 1-3:97-99 to obtain the final product; The culture medium additive is the culture medium additive according to any one of claims 1 to 3.

9. The use of the culture medium according to claim 7 or the culture medium prepared according to the preparation method of claim 8 in improving the proliferation rate of mesenchymal stem cells.

10. The application of the culture medium according to claim 7 or the culture medium prepared according to the preparation method of claim 8 in improving the isolation efficiency of primary seed cells of mesenchymal stem cells.

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