Human umbilical cord mesenchymal stem cell culture medium and preparation method thereof

Abstract

The present disclosure describes a human umbilical cord mesenchymal stem cell culture medium and a preparation method thereof, wherein the human umbilical cord mesenchymal stem cell culture medium includes a basal culture medium, reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin, and the human umbilical cord mesenchymal stem cell culture medium does not include animal serum, and the reduced glutathione concentration is 1 mg / L to 3 mg / L, the epidermal growth factor concentration is 30 μg / L to 80 μg / L, the basic fibroblast growth factor concentration is 10 μg / L to 30 μg / L, the recombinant human insulin concentration is 1 mg / L to 5 mg / L, the hyaluronic acid mass fraction is 0.1% to 0.6%, and the fibronectin concentration is 10 mg / L to 50 mg / L. According to the present disclosure, a human umbilical cord mesenchymal stem cell culture medium and a preparation method thereof that does not contain animal serum, can support primary cell culture, is conducive to cell proliferation in vitro, adheres to the wall and maintains stemness can be provided.

Description

Technical Field

[0001] The present disclosure generally relates to the field of bio-agriculture and related industrial technologies, and specifically relates to a human umbilical cord mesenchymal stem cell culture medium and a preparation method thereof. Background Art

[0002] Human umbilical cord mesenchymal stem cells (HUC-MSCs) are a type of stem cells found in the human umbilical cord. They are widely available and have low immunogenicity, multidirectional differentiation potential, and immunoregulatory properties. They can secrete growth factors and cytokines and have great application potential in the fields of regenerative medicine, tissue engineering, and immunotherapy.

[0003] HUC-MSCs are obtained from the umbilical cord of newborns, and the original concentration of cells is low. In order to meet clinical applications, they need to be rapidly expanded and cultured in vitro while maintaining their stemness. The most widely used human umbilical cord mesenchymal stem cell culture medium currently contains a certain concentration of fetal bovine serum, but the composition of fetal bovine serum is complex and can easily cause an immune response in the body, which may lead to adverse reactions when HUC-MSCs are used clinically. In some clinical studies, researchers use human serum or human serum derivatives instead of fetal bovine serum to culture mesenchymal stem cells, but the chemical composition is still unclear and may also cause an immune response in the body. In addition, there are currently few sources of human serum, which is insufficient to meet the clinical needs of large-scale expansion of HUC-MSCs. There are also some serum substitutes for HUC-MSCs culture that can be purchased on the market, which can be added to the basic human umbilical cord mesenchymal stem cell culture medium for the culture of human umbilical cord mesenchymal stem cells. Among them, the most widely used serum substitute is Pooled Human Platelet Lysate (pHPL), which can replace most of the functions of serum.

[0004] However, the scheme of using platelet lysate as a serum substitute still has problems such as a small source of platelets, difficulty in large-scale production, lack of guarantee of pHPL uniformity, relatively complex components, lack of relevant preparation and product standards, etc. In addition, its effect is still not ideal, and there are problems such as poor cell adhesion and failure to support primary cell culture. Therefore, the human umbilical cord mesenchymal stem cell culture medium in the prior art is difficult to simultaneously meet the requirements of rapid in vitro proliferation of HUC-MSCs, cell adhesion, maintenance of cell stemness, and support for primary cell culture. Summary of the invention

[0005] The present disclosure is proposed in view of the above-mentioned state of the prior art, and its purpose is to provide a human umbilical cord mesenchymal stem cell culture medium and a preparation method thereof that does not contain animal serum, can support primary cell culture of human umbilical cord mesenchymal stem cells, is conducive to rapid proliferation of human umbilical cord mesenchymal stem cells in vitro, cell adhesion and maintenance of cell stemness.

[0006] To this end, the first aspect of the present disclosure provides a human umbilical cord mesenchymal stem cell culture medium, comprising: a basal culture medium, reduced glutathione, epidermal growth factor (Epidermal Growth Factor, EGF), basic Fibroblast Growth Factor (basic Fibroblast Growth Factor, bFGF), recombinant human insulin, hyaluronic acid and fibronectin, wherein the human umbilical cord mesenchymal stem cell culture medium does not include animal serum, and in the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione is 1 mg / L to 3 mg / L, the concentration of epidermal growth factor is 30 μg / L to 80 μg / L, the concentration of basic fibroblast growth factor is 10 μg / L to 30 μg / L, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the mass fraction of hyaluronic acid is 0.1% to 0.6%, and the concentration of fibronectin is 10 mg / L to 50 mg / L.

[0007] In the human umbilical cord mesenchymal stem cell culture medium disclosed in the present invention, the culture medium does not include animal serum, thereby being able to reduce the occurrence of adverse reactions such as in vivo immune response caused by animal serum when human umbilical cord mesenchymal stem cells are used clinically; the basal culture medium can provide nutrition and growth environment for human umbilical cord mesenchymal stem cells; reduced glutathione can provide antioxidant protection, enhance the survival and proliferation ability of human umbilical cord mesenchymal stem cells; epidermal growth factor can promote the proliferation of human umbilical cord mesenchymal stem cells, accelerate cell growth, inhibit the expression of aging genes and help maintain cell activity; basic fibroblast growth factor can stimulate the growth of mesoderm-derived cells, promote the proliferation of human umbilical cord mesenchymal stem cells and enhance the multidirectional differentiation potential of mesenchymal stem cells; recombinant Human insulin can promote cells to utilize glucose and amino acids, regulate sugar metabolism, fat metabolism and protein metabolism in human umbilical cord mesenchymal stem cells, and promote the proliferation and survival of human umbilical cord mesenchymal stem cells; hyaluronic acid has good lubricity and viscoelasticity, and can affect the adhesion, proliferation, differentiation and migration of human umbilical cord mesenchymal stem cells by binding to cell surface receptors, and increase cell viability, which is beneficial to the culture of primary cells of human umbilical cord mesenchymal stem cells; fibronectin can facilitate the adhesion of cells and extracellular matrix to each other, thereby promoting the adhesion of human umbilical cord mesenchymal stem cells, and can regulate cell polarity, differentiation and growth, thereby enhancing the multidirectional differentiation potential of human umbilical cord mesenchymal stem cells and promoting the proliferation of human umbilical cord mesenchymal stem cells. The human umbilical cord mesenchymal stem cell culture medium disclosed herein has reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin at appropriate concentrations, which can promote the growth and adhesion of human umbilical cord mesenchymal stem cells under the concentration conditions, and is conducive to the culture of primary cells of human umbilical cord mesenchymal stem cells and maintain the stemness of human umbilical cord mesenchymal stem cells. In addition, the components can work synergistically to further promote the growth, proliferation and adhesion of human umbilical cord mesenchymal stem cells, which is conducive to the culture of primary cells of human umbilical cord mesenchymal stem cells and maintain the stemness of human umbilical cord mesenchymal stem cells.

[0008] In the human umbilical cord mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione is 2 mg / L, the concentration of epidermal growth factor is 50 μg / L, the concentration of basic fibroblast growth factor is 20 μg / L, the concentration of recombinant human insulin is 3 mg / L, and the concentration of fibronectin is 25 mg / L. Thus, the growth and adhesion of human umbilical cord mesenchymal stem cells can be further promoted, which is beneficial to the culture of primary cells and the maintenance of cell stemness.

[0009] In the human umbilical cord mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, the molecular weight of hyaluronic acid is 10kDa to 100kDa. Different molecular weights have different effects on human umbilical cord mesenchymal stem cells. In the present disclosure, selecting hyaluronic acid with a molecular weight of 10kDa to 100kDa can help promote the adhesion, proliferation and migration of human umbilical cord mesenchymal stem cells, and is beneficial to the culture of primary cells of human umbilical cord mesenchymal stem cells.

[0010] In the human umbilical cord mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, in the human umbilical cord mesenchymal stem cell culture medium, hyaluronic acid with a molecular weight of 10kDa and a mass fraction of 0.5% is included, thereby promoting the adhesion, proliferation and migration of human umbilical cord mesenchymal stem cells and facilitating the culture of primary cells of human umbilical cord mesenchymal stem cells.

[0011] In the human umbilical cord mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, hyaluronic acid with a molecular weight of 100 kDa and a mass fraction of 0.1% is included in the human umbilical cord mesenchymal stem cell culture medium, thereby promoting the adhesion, proliferation and migration of human umbilical cord mesenchymal stem cells and facilitating the culture of primary cells of human umbilical cord mesenchymal stem cells.

[0012] In the human umbilical cord mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, it is characterized in that the basal culture medium is DMEM / F12 culture medium. Thus, it can provide nutrients and a growth environment for human umbilical cord mesenchymal stem cells, which is conducive to the growth and proliferation of human umbilical cord mesenchymal stem cells.

[0013] In the human umbilical cord mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, the pH (acidity) of the human umbilical cord mesenchymal stem cell culture medium is 7.2 to 7.4, and the osmotic pressure is 260 mOsm to 320 mOsm, thereby facilitating the growth and proliferation of human umbilical cord mesenchymal stem cells.

[0014] A second aspect of the present disclosure provides a method for preparing a human umbilical cord mesenchymal stem cell culture medium, comprising: preparing a basal culture medium, reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin; mixing the basal culture medium, reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin to obtain the human umbilical cord mesenchymal stem cell culture medium; wherein, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione is 1 mg / L to 3 mg / L, the concentration of epidermal growth factor is 30 μg / L to 80 μg / L, the concentration of basic fibroblast growth factor is 10 μg / L to 30 μg / L, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the mass fraction of hyaluronic acid is 0.1% to 0.6%, and the concentration of fibronectin is 10 mg / L to 50 mg / L.

[0015] In the preparation method disclosed herein, the prepared human umbilical cord mesenchymal stem cell culture medium does not include animal serum, thereby being able to reduce the occurrence of adverse reactions such as in vivo immune response caused by animal serum when human umbilical cord mesenchymal stem cells are used clinically; the use of basal culture medium can provide nutrition and growth environment for human umbilical cord mesenchymal stem cells; the use of reduced glutathione can provide antioxidant protection and enhance the survival and proliferation ability of human umbilical cord mesenchymal stem cells; the use of epidermal growth factor can promote the proliferation of human umbilical cord mesenchymal stem cells, accelerate cell growth, inhibit the expression of aging genes, and help maintain cell activity; the use of basic fibroblast growth factor can stimulate the growth of mesoderm-derived cells, promote the proliferation of mesenchymal stem cells and enhance the multidirectionality of mesenchymal stem cells Differentiation potential; the use of recombinant human insulin can promote cells to utilize glucose and amino acids, regulate sugar metabolism, fat metabolism and protein metabolism in mesenchymal stem cells, and promote the proliferation and survival of mesenchymal stem cells; the use of hyaluronic acid, which has good lubricity and viscoelasticity, can affect the adhesion, proliferation, differentiation and migration of human umbilical cord mesenchymal stem cells by binding to cell surface receptors, and increase cell viability, which is beneficial to the culture of primary cells of human umbilical cord mesenchymal stem cells; the use of fibronectin can facilitate the adhesion of cells and extracellular matrix to each other, thereby promoting the adhesion of human umbilical cord mesenchymal stem cells, and can regulate cell polarity, differentiation and growth, thereby helping to enhance the multidirectional differentiation potential of mesenchymal stem cells and promote the proliferation of mesenchymal stem cells. The human umbilical cord mesenchymal stem cell culture medium prepared by the preparation method disclosed herein contains reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin at appropriate concentrations, which can promote the growth, proliferation and adhesion of human umbilical cord mesenchymal stem cells under the concentration conditions, and is conducive to the cultivation of primary cells and the maintenance of cell stemness. In addition, the components can work synergistically to further promote the growth and adhesion of human umbilical cord mesenchymal stem cells, which is conducive to the cultivation of primary cells and the maintenance of cell stemness.

[0016] In the preparation method involved in the second aspect of the present disclosure, optionally, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione is 2 mg / L, the concentration of epidermal growth factor is 50 μg / L, the concentration of basic fibroblast growth factor is 20 μg / L, the concentration of recombinant human insulin is 3 mg / L, the mass fraction of hyaluronic acid is 0.6%, and the concentration of fibronectin is 25 mg / L. Thus, the growth, proliferation and adhesion of human umbilical cord mesenchymal stem cells can be further promoted, which is beneficial to the culture of primary cells and the maintenance of cell stemness.

[0017] In the preparation method involved in the second aspect of the present disclosure, optionally, the human umbilical cord mesenchymal stem cell culture medium is further filtered using a filter with a pore size of 0.22 μm. Thus, bacteria and other microorganisms in the human umbilical cord mesenchymal stem cell culture medium can be reduced by filtering, thereby facilitating the growth of human umbilical cord mesenchymal stem cells.

[0018] According to the present disclosure, a human umbilical cord mesenchymal stem cell culture medium and a preparation method thereof can be provided which does not contain animal serum, can support primary cell culture of human umbilical cord mesenchymal stem cells, and is conducive to rapid proliferation of human umbilical cord mesenchymal stem cells in vitro, cell adhesion, and maintenance of cell stemness. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present disclosure will now be explained in further detail by way of example only with reference to the accompanying drawings, in which:

[0020] Figure 1 Detailed description of the invention is a flow chart showing a method for preparing a culture medium for human umbilical cord mesenchymal stem cells according to the present invention.

[0021] Figure 2A is a graph showing cell proliferation curves of medium 1 to medium 4 involved in the present disclosure and a comparative example.

[0022] Figure 2B is a graph showing cell proliferation curves of medium 5 to medium 8 involved in the present disclosure and a comparative example.

[0023] Figure 2C is a graph showing cell proliferation curves of medium 9 to medium 12 according to the present disclosure and a comparative example.

[0024] Figure 3 3 is a diagram showing the morphology of cells crawling out of a tissue when cultured using the culture medium 9 according to the present disclosure.

[0025] Figure 4 3 is a diagram showing the morphology of third-generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure.

[0026] Figure 5 3 is a diagram showing the morphology of human umbilical cord mesenchymal stem cells of the eighth passage cultured using medium 9 according to the present disclosure.

[0027] Figure 6 1 is a graph showing the expression level of surface antigens of third-generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure.

[0028] Figure 7 1 is a diagram showing the staining results of adipogenic differentiation of third-generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure.

[0029] Figure 8 1 is a graph showing the results of osteogenic differentiation staining of third-generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure. DETAILED DESCRIPTION

[0030] Hereinafter, the preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same symbols are assigned to the same components, and repeated descriptions are omitted. In addition, the accompanying drawings are only schematic diagrams, and the ratio of the dimensions of the components or the shapes of the components may be different from the actual ones.

[0031] It should be noted that the terms "including" and "having" and any variations thereof in the present disclosure, such as a process, method, system, product or device that includes or has a series of steps or units, are not necessarily limited to those steps or units clearly listed, but may include or have other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0032] In addition, the subheadings and the like involved in the following description of the present disclosure are not intended to limit the content or scope of the present disclosure, but are merely used as a reminder for reading. Such subheadings should neither be understood as being used to divide the content of the article, nor should the content under the subheading be limited to the scope of the subheading.

[0033] The present disclosure relates to a human umbilical cord mesenchymal stem cell culture medium for culturing human umbilical cord mesenchymal stem cells, which is sometimes referred to as "culture medium" below. The present disclosure relates to a method for preparing a human umbilical cord mesenchymal stem cell culture medium, which is sometimes referred to as "preparation method" below. The human umbilical cord mesenchymal stem cell culture medium disclosed herein does not contain serum, can support primary cell culture of human umbilical cord mesenchymal stem cells, and meet the requirements of rapid proliferation of human umbilical cord mesenchymal stem cells in vitro, cell adhesion, and maintenance of cell stemness.

[0034] In some examples, human umbilical cord mesenchymal stem cell culture medium can include basal medium, reduced glutathione, epidermal growth factor (Epidermal Growth Factor, EGF), basic Fibroblast Growth Factor (basic Fibroblast Growth Factor, bFGF), recombinant human insulin, hyaluronic acid (HA) and fibronectin.

[0035] In some examples, the culture medium for human umbilical cord mesenchymal stem cells may not include animal serum, such as fetal bovine serum (FBS), horse serum, calf serum, sheep serum, and human serum. This can reduce the occurrence of adverse reactions such as in vivo immune responses caused by animal serum when human umbilical cord mesenchymal stem cells are used clinically.

[0036] In some examples, the human umbilical cord mesenchymal stem cell culture medium may include a basal culture medium. In some examples, the basal culture medium may be a DMEM / F12 culture medium. DMEM / F12 culture medium is a mixed culture medium in which two basal culture media, DMEM (Dulbecco's Modified Eagle's Medium) and F12 (Ham's F12 Medium), are mixed in a specific ratio. Thus, it is possible to provide rich nutrients and factors and a suitable growth environment for human umbilical cord mesenchymal stem cells, which is conducive to the growth and proliferation of human umbilical cord mesenchymal stem cells.

[0037] In some examples, the components in the DMEM / F12 culture medium may include inorganic salts, sugars, amino acids, vitamins, and buffers, thereby providing nutrition and a growth environment for human umbilical cord mesenchymal stem cells.

[0038] In some examples, the DMEM / F12 medium may not include animal serum, thereby reducing the occurrence of adverse reactions such as in vivo immune response caused by animal serum when human umbilical cord mesenchymal stem cells are used clinically.

[0039] In some examples, DMEM / F12 culture medium can be prepared by oneself.In some examples, DMEM / F12 culture medium can also be purchased from commercial sources.

[0040] In some examples, the culture medium of human umbilical cord mesenchymal stem cells may include reduced glutathione, thereby providing antioxidant protection and enhancing the survival and proliferation ability of human umbilical cord mesenchymal stem cells.

[0041] In some examples, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione can be 1 mg / L to 3 mg / L. For example, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione can be 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, or 3 mg / L. Thus, it can be beneficial to provide suitable antioxidant protection and enhance the survival and proliferation ability of human umbilical cord mesenchymal stem cells.

[0042] In some examples, preferably, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione may be 2 mg / L.

[0043] In some examples, the culture medium of human umbilical cord mesenchymal stem cells may include epidermal growth factor, thereby promoting the proliferation of human umbilical cord mesenchymal stem cells, accelerating cell growth, inhibiting the expression of aging genes, and facilitating the maintenance of cell activity.

[0044] In some examples, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of epidermal growth factor can be 30 μg / L to 80 μg / L. For example, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of epidermal growth factor can be 30 μg / L, 40 μg / L, 50 μg / L, 60 μg / L, 70 μg / L or 80 μg / L. Too high a concentration of epidermal growth factor may cause excessive cell proliferation, may affect the stemness of the cells, and promote cell differentiation. Too low a concentration of epidermal growth factor may affect the growth and proliferation of cells. Thus, a suitable cell proliferation rate can be provided.

[0045] In some examples, preferably, in the culture medium of human umbilical cord mesenchymal stem cells, the concentration of epidermal growth factor can be 50 μg / L, thereby facilitating the growth and proliferation of human umbilical cord mesenchymal stem cells.

[0046] In some examples, the culture medium for human umbilical cord mesenchymal stem cells may include basic fibroblast growth factor, thereby stimulating the growth of mesoderm-derived cells, promoting the proliferation of mesenchymal stem cells, and enhancing the multidirectional differentiation potential of mesenchymal stem cells.

[0047] In some examples, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of basic fibroblast growth factor can be 10 μg / L to 30 μg / L. For example, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of basic fibroblast growth factor can be 10 μg / L, 15 μg / L, 20 μg / L, 25 μg / L or 30 μg / L. Thus, it is possible to provide a suitable effect of stimulating the growth of mesoderm-derived cells and promoting the proliferation of mesenchymal stem cells and enhancing the multidirectional differentiation potential of mesenchymal stem cells.

[0048] In some examples, preferably, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of basic fibroblast growth factor can be 20 μg / L, thereby stimulating the growth of mesoderm-derived cells, promoting the proliferation of mesenchymal stem cells, and enhancing the multidirectional differentiation potential of mesenchymal stem cells.

[0049] In some examples, the culture medium of human umbilical cord mesenchymal stem cells may include recombinant human insulin, thereby promoting the cells to utilize glucose and amino acids, regulating sugar metabolism, fat metabolism, and protein metabolism in mesenchymal stem cells, and promoting the proliferation and survival of human umbilical cord mesenchymal stem cells.

[0050] In some examples, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of recombinant human insulin can be 1 mg / L to 5 mg / L. For example, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of recombinant human insulin can be 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L or 5 mg / L. Thus, it is possible to provide a suitable metabolic regulation effect and promote the proliferation and survival of human umbilical cord mesenchymal stem cells.

[0051] In some examples, preferably, in the culture medium of human umbilical cord mesenchymal stem cells, the concentration of recombinant human insulin may be 3 mg / L, thereby facilitating the proliferation and survival of human umbilical cord mesenchymal stem cells.

[0052] In some examples, the culture medium of human umbilical cord mesenchymal stem cells may include hyaluronic acid. In this case, hyaluronic acid has good lubricity and viscoelasticity, and can affect the adhesion, proliferation, differentiation and migration of human umbilical cord mesenchymal stem cells by binding to cell surface receptors, and increase cell viability, which is beneficial to the culture of primary cells of human umbilical cord mesenchymal stem cells.

[0053] In some examples, the mass fraction of hyaluronic acid in the human umbilical cord mesenchymal stem cell culture medium can be 0.1% to 0.6%. For example, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of recombinant human insulin can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 0.6%. Thus, it can be beneficial to the adhesion, proliferation, differentiation and migration of human umbilical cord mesenchymal stem cells, increase cell viability, and be beneficial to the culture of primary cells of human umbilical cord mesenchymal stem cells.

[0054] In some examples, preferably, the mass fraction of hyaluronic acid in the human umbilical cord mesenchymal stem cell culture medium can be 0.6%, thereby facilitating the adhesion, proliferation, differentiation and migration of human umbilical cord mesenchymal stem cells, increasing cell viability, and facilitating the culture of primary cells of human umbilical cord mesenchymal stem cells.

[0055] In some examples, the molecular weight of hyaluronic acid can be 10kDa to 100kDa. For example, the molecular weight of hyaluronic acid can be 10kDa, 20kDa, 50kDa, 70kDa, 80kDa and / or 100kDa. Different molecular weights have different effects on human umbilical cord mesenchymal stem cells. In the present disclosure, hyaluronic acid with a molecular weight of 10kDa to 100kDa is selected. Thus, it is possible to further promote the adhesion, proliferation and migration of human umbilical cord mesenchymal stem cells, increase cell viability, and be beneficial to the cultivation of primary cells of human umbilical cord mesenchymal stem cells.

[0056] In some examples, preferably, the hyaluronic acid may include hyaluronic acid with a molecular weight of 10 kDa and hyaluronic acid with a molecular weight of 100 kDa.

[0057] In some examples, in the culture medium of human umbilical cord mesenchymal stem cells, the mass fraction of hyaluronic acid with a molecular weight of 10 kDa can be 0.5%, thereby promoting the adhesion, proliferation and migration of human umbilical cord mesenchymal stem cells and facilitating the culture of primary cells of human umbilical cord mesenchymal stem cells.

[0058] In some examples, in the culture medium of human umbilical cord mesenchymal stem cells, the mass fraction of hyaluronic acid with a molecular weight of 100 kDa can be 0.1%, thereby promoting the adhesion, proliferation and migration of human umbilical cord mesenchymal stem cells and facilitating the culture of primary cells of human umbilical cord mesenchymal stem cells.

[0059] In some examples, the culture medium for human umbilical cord mesenchymal stem cells may include 0.5% by mass of hyaluronic acid with a molecular weight of 10 kDa and 0.1% by mass of hyaluronic acid with a molecular weight of 100 kDa, thereby promoting the adhesion, proliferation and migration of human umbilical cord mesenchymal stem cells and facilitating the culture of primary cells of human umbilical cord mesenchymal stem cells.

[0060] In some examples, the culture medium for human umbilical cord mesenchymal stem cells may include fibronectin, which can facilitate the adhesion of cells and extracellular matrix to each other, thereby promoting the adhesion of human umbilical cord mesenchymal stem cells, and can regulate cell polarity, differentiation and growth, thereby facilitating the enhancement of the multidirectional differentiation potential of human umbilical cord mesenchymal stem cells and promoting the proliferation of mesenchymal stem cells.

[0061] In some examples, in the culture medium of human umbilical cord mesenchymal stem cells, the concentration of fibronectin can be 10 mg / L to 50 mg / L. For example, in the culture medium of human umbilical cord mesenchymal stem cells, the concentration of fibronectin can be 10 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L or 50 mg / L. Thus, it can be beneficial to promote the adhesion, growth and proliferation of human umbilical cord mesenchymal stem cells and enhance the multidirectional differentiation potential of human umbilical cord mesenchymal stem cells.

[0062] In some examples, preferably, in the culture medium of human umbilical cord mesenchymal stem cells, the concentration of fibronectin can be 25 mg / L, thereby promoting the adhesion, growth and proliferation of human umbilical cord mesenchymal stem cells and enhancing the multidirectional differentiation potential of human umbilical cord mesenchymal stem cells.

[0063] In some examples, reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin can work synergistically to further promote the growth, proliferation and adhesion of human umbilical cord mesenchymal stem cells, which is beneficial to the culture of primary cells of human umbilical cord mesenchymal stem cells and maintain the stemness of human umbilical cord mesenchymal stem cells.

[0064] In some examples, the pH of the human umbilical cord mesenchymal stem cell culture medium may be 7.2 to 7.4, which can be beneficial to the growth of the human umbilical cord mesenchymal stem cells.

[0065] In some examples, the osmotic pressure of the human umbilical cord mesenchymal stem cell culture medium may be 260 mOsm to 320 mOsm, which can be beneficial to the growth of the human umbilical cord mesenchymal stem cells.

[0066] The present disclosure also relates to a method for preparing a culture medium for human umbilical cord mesenchymal stem cells. It should be noted that the aforementioned "culture medium for human umbilical cord mesenchymal stem cells" section and the following "preparation method" may be interlinked, and some of the contents may refer to the aforementioned "culture medium for human umbilical cord mesenchymal stem cells", which will not be repeated here.

[0067] Figure 1 Detailed description of the invention is a flow chart showing a method for preparing a culture medium for human umbilical cord mesenchymal stem cells according to the present invention.

[0068] In some examples, such as Figure 1 As shown, the preparation method may include: preparing a basal culture medium and a raw material reagent (step S10); mixing the basal culture medium and the raw material reagent (step S20); and obtaining a human umbilical cord mesenchymal stem cell culture medium (step S30). Thus, a human umbilical cord mesenchymal stem cell culture medium can be obtained.

[0069] In some examples, in step S10 , the raw material reagents may include reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid, and fibronectin.

[0070] In some examples, in step S10, the basal culture medium may be DMEM / F12 culture medium.

[0071] In some examples, in step S20, a basal culture medium may be used to dissolve a raw material reagent. Specifically, a specific mass of a raw material reagent may be weighed, and then the basal culture medium may be used to dissolve the raw material reagent and dilute to a specific volume, thereby obtaining a human umbilical cord mesenchymal stem cell culture medium.

[0072] In some examples, in step S20 and step S30, the concentration of reduced glutathione in the culture medium of human umbilical cord mesenchymal stem cells can be 2 mg / L, the concentration of epidermal growth factor can be 50 μg / L, the concentration of basic fibroblast growth factor can be 20 μg / L, the concentration of recombinant human insulin can be 3 mg / L, the mass fraction of hyaluronic acid can be 0.6%, and the concentration of fibronectin can be 25 mg / L. Thus, the growth, proliferation and adhesion of human umbilical cord mesenchymal stem cells can be promoted, which is beneficial to the culture of primary cells and the maintenance of cell stemness.

[0073] In some examples, in step S20 and step S30, the concentration of reduced glutathione in the culture medium of human umbilical cord mesenchymal stem cells can be 2 mg / L, the concentration of epidermal growth factor can be 50 μg / L, the concentration of basic fibroblast growth factor can be 20 μg / L, the concentration of recombinant human insulin can be 3 mg / L, the mass fraction of hyaluronic acid can be 0.6%, and the concentration of fibronectin can be 25 mg / L. Thus, the growth, proliferation and adhesion of human umbilical cord mesenchymal stem cells can be promoted, which is beneficial to the culture of primary cells and the maintenance of cell stemness.

[0074] In some examples, the step of adjusting the pH value is further included after mixing the basal medium and the raw material reagent. In some examples, the pH value of the human umbilical cord mesenchymal stem cell culture medium can be adjusted to 7.2 to 7.4. In some examples, the pH value can be adjusted by adjusting the buffer salt concentration in the basal medium.

[0075] In some examples, the step of adjusting the osmotic pressure is further included after mixing the basal medium and the raw material reagent. In some examples, the osmotic pressure of the human umbilical cord mesenchymal stem cell culture medium can be adjusted to 260mOsm to 320mOsm. In some examples, the osmotic pressure can be adjusted by adjusting the solute concentration in the basal medium.

[0076] In some examples, the prepared human umbilical cord mesenchymal stem cell culture medium may be filtered using a filter with a pore size of 0.22 μm, thereby sterilizing the human umbilical cord mesenchymal stem cell culture medium through filtration, thereby facilitating the growth and reproduction of human umbilical cord mesenchymal stem cells.

[0077] According to the human umbilical cord mesenchymal stem cell culture medium and preparation method thereof disclosed herein, the culture medium does not contain serum and can support primary cell culture of human umbilical cord mesenchymal stem cells, which is beneficial to rapid proliferation of human umbilical cord mesenchymal stem cells in vitro, cell adhesion and maintenance of cell stemness. The human umbilical cord mesenchymal stem cell culture medium disclosed herein has clear and simple added components, and during the culture process, it is unnecessary to coat the cell culture flask.

[0078] The present disclosure is described in detail below through examples and comparative examples. However, the following examples or comparative examples are only provided as examples to specifically illustrate the present disclosure, and do not define or restrict the scope of the invention disclosed in this application.

[0079] Unless otherwise specified, the reagents, materials, and instruments used in the examples and comparative examples of the present disclosure are all commercially available.

[0080] First, prepare a human umbilical cord mesenchymal stem cell culture medium. Specifically, prepare DMEM / F12 culture medium, reduced glutathione, EGF, bFGF, recombinant human insulin, HA and fibronectin. The components and proportions in the DMEM / F12 culture medium can be shown in the following table:

[0081]

[0082]

[0083]

[0084] Then, based on DMEM / F12 medium, human umbilical cord mesenchymal stem cell medium numbered 1 to 4 (i.e., medium 1 to medium 4) were prepared according to the concentration of each component in the following table:

[0085]

[0086] Based on DMEM / F12 medium, human umbilical cord mesenchymal stem cell culture media No. 5 to 8 (i.e., culture media 5 to 8) were prepared according to the concentrations of the components in the following table:

[0087]

[0088] Based on DMEM / F12 medium, human umbilical cord mesenchymal stem cell culture media No. 9 to 12 (i.e., culture media 9 to 12) were prepared according to the concentrations of the components in the following table:

[0089]

[0090] Based on DMEM / F12 culture medium, the culture medium of Comparative Example 1 and Comparative Example 2 was prepared:

[0091] Comparative Example 1: Adding 5% platelet lysate into DMEM / F12 culture medium;

[0092] Comparative Example 2: 10% by volume of fetal bovine serum was added to the DMEM / F12 culture medium.

[0093] Subsequently, the culture media of medium 1 to medium 12, comparative example 1 and comparative example 2 were tested for bacteria and fungi, and the test results were negative; the chlamydia and mycoplasma tests were performed, and the test results were negative; the endotoxin test was performed, and the result was 0EU / mL to 0.5EU / mL. Subsequently, the culture media of medium 1 to medium 12, comparative example 1 and comparative example 2 were sterilized by filtering with a 0.22μm filter and stored at -20°C in the dark.

[0094] Then, human umbilical cord mesenchymal stem cells were prepared. Specifically, the umbilical cord of a fresh cesarean section patient was taken and placed in a phosphate buffered saline (PBS) containing 1% penicillin-streptomycin. The remaining blood was squeezed out, and the two umbilical arteries, one umbilical vein and the umbilical cord outer membrane were removed in turn. The remaining tissue was Wharton's jelly. The Wharton's jelly was placed in a 50ml centrifuge tube and cut into 1mm pieces. 3 Each 1.5 ml tissue block is evenly spread on a bottom area of ​​175 mm 2 in the culture bottle.

[0095] Then, medium 1 to medium 12, medium of comparative example 1 and medium of comparative example 2 were added respectively and placed at 37°C, 5% CO 2 , saturated humidity, and fresh human umbilical cord mesenchymal stem cell culture medium was added every 3 days; when fibroblasts growing in colonies around the tissue block were observed under the microscope (i.e., cells crawled out of the tissue), the tissue block was removed; digested with recombinant trypsin analog (TrypLE) for 3 minutes, centrifuged at 1000 rpm for 5 minutes, and then the supernatant was discarded, and the cell pellet was resuspended with culture medium 1 to culture medium 12, the culture medium of comparative example 1, and the culture medium of comparative example 2, respectively, at 10000 / cm 2 The cells were inoculated into culture flasks at a density of 1.5 × 10 and incubated at 37°C with 5% CO. 2The cells were cultured under saturated humidity and then digested, centrifuged and resuspended at 10,000 cells / cm2. 2 The cells were inoculated in medium 1 to medium 12, the medium of comparative example 1, and the medium of comparative example 2, respectively, and subcultured every 4 days, with the inoculation density of each generation being 10,000 cells / cm 2 Cell counting and analysis were performed at each passage.

[0096] Figure 2A is a graph showing the cell proliferation curves of medium 1 to medium 4 according to the present disclosure and the comparative example. Figure 2A It can be seen that the mass fraction of added hyaluronic acid is 0.1%, the culture medium 2 with a hyaluronic acid molecular weight of 10 kDa and the culture medium 3 with a hyaluronic acid molecular weight of 100 kDa have better cell proliferation effects; the culture medium 1 with a hyaluronic acid molecular weight of 1 kDa and the culture medium 4 with a hyaluronic acid molecular weight of 1000 kDa have poor cell proliferation effects; in addition, the cell proliferation effects of culture medium 2 and culture medium 3 are better than those of comparative examples 1 and 2. Culture medium 2 and culture medium 3 are preferred embodiments of the present disclosure.

[0097] Figure 2B is a graph showing the cell proliferation curves of medium 5 to medium 8 involved in the present disclosure and the comparative example. Figure 2B It can be seen that the mass fraction of added hyaluronic acid is 0.5%, the culture medium 6 with a hyaluronic acid molecular weight of 10 kDa and the culture medium 7 with a hyaluronic acid molecular weight of 100 kDa have better cell proliferation effects; the culture medium 5 with a hyaluronic acid molecular weight of 1 kDa and the culture medium 8 with a hyaluronic acid molecular weight of 1000 kDa have poor cell proliferation effects; in addition, the cell proliferation effect of culture medium 6 is better than that of the culture medium of comparative example 2, and the cell proliferation effect of culture medium 7 is better than that of the culture medium of comparative example 1. Culture medium 6 and culture medium 7 are preferred embodiments of the present disclosure.

[0098] Figure 2C is a graph showing the cell proliferation curves of the medium 9 to the medium 12 involved in the present disclosure and the comparative example. Figure 2CIt can be seen that the culture medium 9 added with 0.5% hyaluronic acid with a molecular weight of 10 kDa and 0.1% hyaluronic acid with a molecular weight of 100 kDa, and the culture medium 10 added with 0.1% hyaluronic acid with a molecular weight of 10 kDa and 0.5% hyaluronic acid with a molecular weight of 100 kDa, have better cell proliferation effects than the culture media of Comparative Examples 1 and 2. Among them, the cell proliferation effect of culture medium 9 is the best. Culture medium 9 and culture medium 10 are preferred embodiments of the present disclosure, and culture medium 9 is the best embodiment of the present disclosure.

[0099] In summary, when the molecular weight of the added hyaluronic acid is 10kDa to 100kDa, the cell proliferation effect is better, and when the mass fraction of the added hyaluronic acid is 0.1% and 0.5%, the cell proliferation effect is better, among which the cell proliferation effect of culture medium 9 with 0.5% 10kDa hyaluronic acid and 0.1% 100kDa hyaluronic acid added is the best.

[0100] Figure 3 is a morphological diagram showing cells crawling out of tissues when cultured using the culture medium 9 involved in the present disclosure, Figure 4 is a diagram showing the morphology of the third generation human umbilical cord mesenchymal stem cells cultured using the medium 9 involved in the present disclosure, Figure 5 is a diagram showing the morphology of the eighth generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure. Figure 4 and Figure 5 It can be seen that the human umbilical cord mesenchymal stem cells cultured with the medium of medium 9 have uniform cell morphology after multiple passages, which is consistent with the cell morphology of human umbilical cord mesenchymal stem cells.

[0101] Detection of cell surface antigen expression: HUC-MSCs of p3 generation in culture medium 9 were taken, digested with TrypLE, centrifuged and discarded the supernatant, washed with sterile PBS, incubated with corresponding antibodies, washed again with PBS and resuspended on the machine, and the expression of cell surface antigens CD73, CD90, CD105, HLA-DR, CD34, CD11b, CD19 and CD45 was detected by flow cytometry. Figure 6 is a graph showing the expression level of surface antigens of the third generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure. Figure 6It can be seen that human umbilical cord mesenchymal stem cells cultured with human umbilical cord mesenchymal stem cell culture medium 9 can well ensure that their biological characteristics are not changed. The expression rate of human umbilical cord mesenchymal stem cell positive marker CD73 is greater than 97%, the expression rate of CD90 is greater than 99%, and the expression rate of CD105 is greater than 96%; the expression rates of human umbilical cord mesenchymal stem cell negative markers CD34, CD45, CD19, CD11b, and HLA-DR are all lower than 1%.

[0102] In vitro adipogenic differentiation: HUC-MSCs at generation 3 in medium 9 were taken and plated at 10,000 cells / cm 2 The cells were seeded at a density of 1.50 μg / mL in a 6-well plate and cultured in a human umbilical cord mesenchymal stem cell culture medium of medium 9. When the cell fusion reached 90%, the human umbilical cord mesenchymal stem cell culture medium was replaced with an adipogenic induction culture medium (containing 10% fetal bovine serum, 10 mg / L insulin, 1 μM dexamethasone, 0.5 mM IBMX (phosphodiesterase inhibitor) and 1% penicillin-streptomycin solution); the adipogenic induction culture medium was replaced every 2 days, and an Oil Red O staining experiment was performed 14 days after induction, and the staining results were observed under a microscope. Figure 7 is a diagram showing the staining results of adipogenic differentiation of the third generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure. Figure 7 It can be seen that under the culture conditions of the human umbilical cord mesenchymal stem cell culture medium using medium 9, human umbilical cord mesenchymal stem cells can maintain their differentiation potential and differentiate into adipocytes.

[0103] In vitro osteogenic differentiation: HUC-MSCs at generation 3 in culture medium 9 were plated at 10,000 cells / cm 2 The cells were seeded at a density of 100 μg / cm2 in a 6-well plate and cultured using the human umbilical cord mesenchymal stem cell culture medium of Example 9. When the cell fusion reached 90%, the human umbilical cord mesenchymal stem cell culture medium was replaced with an osteogenic induction culture medium (containing 10% fetal bovine serum, 10 μM dexamethasone, 10 mM β-glycerophosphate disodium, 1 mM glutamine and 1% penicillin-streptomycin solution). The osteogenic induction culture medium was replaced every 2 days. After 21 days of induction, an Alizarin red staining experiment was performed and the staining results were observed under a microscope. Figure 8 is a graph showing the results of osteogenic differentiation staining of the third generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure. Figure 8 It can be seen that under the culture conditions of the human umbilical cord mesenchymal stem cell culture medium using medium 9, human umbilical cord mesenchymal stem cells can maintain their differentiation potential and differentiate into osteocytes.

[0104] In summary, the human umbilical cord mesenchymal stem cell culture medium disclosed in the present invention includes reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin, and the components work synergistically to support primary cell culture, promote the growth and adhesion of human umbilical cord mesenchymal stem cells, and are beneficial to the culture of primary cells of human umbilical cord mesenchymal stem cells and maintain the stemness of human umbilical cord mesenchymal stem cells. In addition, the human umbilical cord mesenchymal stem cell culture medium disclosed in the present invention does not contain animal serum, thereby reducing the occurrence of adverse reactions such as in vivo immune response caused by animal serum when human umbilical cord mesenchymal stem cells are used clinically.

[0105] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and embodiments, it is to be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. A human umbilical cord mesenchymal stem cell culture medium, characterized in that: include: basal culture medium, reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin, wherein the basal culture medium is DMEM / F12 culture medium, The human umbilical cord mesenchymal stem cell culture medium does not include animal serum. In the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione is 1 mg / L to 3 mg / L, the concentration of epidermal growth factor is 30 μg / L to 80 μg / L, the concentration of basic fibroblast growth factor is 10 μg / L to 30 μg / L, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the mass fraction of hyaluronic acid is 0.1% to 0.6%, the concentration of fibronectin is 10 mg / L to 50 mg / L, and the molecular weight of hyaluronic acid is 10 kilodaltons to 100 kilodaltons.

2. The human umbilical cord mesenchymal stem cell culture medium according to claim 1, characterized in that In the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione is 2 mg / L, the concentration of epidermal growth factor is 50 μg / L, the concentration of basic fibroblast growth factor is 20 μg / L, the concentration of recombinant human insulin is 3 mg / L, and the concentration of fibronectin is 25 mg / L.

3. The human umbilical cord mesenchymal stem cell culture medium according to claim 1, characterized in that The human umbilical cord mesenchymal stem cell culture medium includes hyaluronic acid with a molecular weight of 10 kilodaltons and a mass fraction of 0.5%.

4. The human umbilical cord mesenchymal stem cell culture medium according to claim 1 or 3, characterized in that: The human umbilical cord mesenchymal stem cell culture medium includes hyaluronic acid with a molecular weight of 100 kilodaltons and a mass fraction of 0.1%.

5. The human umbilical cord mesenchymal stem cell culture medium according to claim 1, characterized in that: The pH value of the human umbilical cord mesenchymal stem cell culture medium is 7.2 to 7.4, and the osmotic pressure is 260 to 320 milliosmolecular pressure.

6. A method for preparing a culture medium for human umbilical cord mesenchymal stem cells, characterized in that: include: Prepare basal medium, reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid, and fibronectin; The basal culture medium, reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin are mixed to obtain the human umbilical cord mesenchymal stem cell culture medium, wherein the basal culture medium is DMEM / F12 culture medium; wherein, in the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione is 1 mg / L to 3 mg / L, the concentration of epidermal growth factor is 30 μg / L to 80 μg / L, the concentration of basic fibroblast growth factor is 10 μg / L to 30 μg / L, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the mass fraction of hyaluronic acid is 0.1% to 0.6%, the concentration of fibronectin is 10 mg / L to 50 mg / L, and the molecular weight of hyaluronic acid is 10 kilodaltons to 100 kilodaltons.

7. The preparation method according to claim 6, characterized in that In the human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione is 2 mg / L, the concentration of epidermal growth factor is 50 μg / L, the concentration of basic fibroblast growth factor is 20 μg / L, the concentration of recombinant human insulin is 3 mg / L, the mass fraction of hyaluronic acid is 0.6%, and the concentration of fibronectin is 25 mg / L.

8. The preparation method according to claim 6 or 7, characterized in that The method also includes filtering the human umbilical cord mesenchymal stem cell culture medium using a filter with a pore size of 0.22 microns.

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