CN117535237A - Human umbilical cord mesenchymal stem cell culture medium and preparation method thereof - Google Patents
Human umbilical cord mesenchymal stem cell culture medium and preparation method thereof Download PDFInfo
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- CN117535237A CN117535237A CN202311428605.6A CN202311428605A CN117535237A CN 117535237 A CN117535237 A CN 117535237A CN 202311428605 A CN202311428605 A CN 202311428605A CN 117535237 A CN117535237 A CN 117535237A
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Abstract
The present disclosure describes a human umbilical cord mesenchymal stem cell culture medium and a method of preparing the same, the human umbilical cord mesenchymal stem cell culture medium comprising a basal medium, reduced glutathione, an epidermal growth factor, an alkaline fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin, the human umbilical cord mesenchymal stem cell culture medium excluding animal serum, the reduced glutathione concentration being 1mg/L to 3mg/L, the epidermal growth factor concentration being 30 μg/L to 80 μg/L, the alkaline fibroblast growth factor concentration being 10 μg/L to 30 μg/L, the recombinant human insulin concentration being 1mg/L to 5mg/L, the hyaluronic acid mass fraction being 0.1% to 0.6%, the fibronectin concentration being 10mg/L to 50mg/L. According to the present disclosure, a human umbilical cord mesenchymal stem cell culture medium free of animal serum, capable of supporting primary cell culture, facilitating cell proliferation, adherence and maintenance of dryness in vitro, and a method for preparing the same can be provided.
Description
Technical Field
The present disclosure relates generally to the technical field of biological agriculture and related industries, and in particular to a human umbilical cord mesenchymal stem cell culture medium and a preparation method thereof.
Background
Human umbilical cord mesenchymal stem cells (Human Umbilical Cord Mesenchymal Stem Cells, HUC-MSCs) are a type of stem cells existing in human umbilical cords, are wide in source and easy to obtain, have low immunogenicity, multidirectional differentiation potential and immunoregulatory performance, can secrete growth factors and cytokines, and have great application potential in the fields of regenerative medicine, tissue engineering, immunotherapy and the like.
HUC-MSCs are obtained from neonatal umbilical cord, the original concentration of cells is low, and in order to meet clinical application, rapid expansion culture in vitro is required while dryness is maintained. The most widely used human umbilical cord mesenchymal stem cell culture medium at present contains fetal bovine serum with a certain concentration, but the fetal bovine serum has complex components and is easy to cause in vivo immune response, so that adverse reactions can be generated when HUC-MSCs are clinically used. In partial clinical studies, researchers use human serum or human serum derivatives to replace fetal bovine serum for culturing mesenchymal stem cells, but the chemical composition is still unknown, in vivo immune response can be possibly caused, and the current human serum source is less, so that the clinical requirement of large-scale amplification of HUC-MSCs is not satisfied. There are also some commercially available serum substitutes for HUC-MSCs culture on the market today, which can be added to the basal human umbilical cord mesenchymal stem cell medium for the culture of human umbilical cord mesenchymal stem cells, wherein the most widely used serum substitute is platelet lysate (Pooled Human Platelet Lysate, pHPL), which can replace most functions of serum.
However, the proposal of using platelet lysate as serum substitute still has the problems of small platelet sources, difficult mass production, no guarantee of pHPL uniformity, relatively complex components, lack of relevant preparation and product standards, etc., and the effects are still not very ideal, and the problems of poor cell adherence, no support of primary cell culture, etc. exist. Thus, the human umbilical cord mesenchymal stem cell culture medium in the prior art is difficult to simultaneously satisfy the in vitro rapid proliferation, cell attachment, maintenance of cell stem property and support of primary cell culture of HUC-MSCs.
Disclosure of Invention
The present disclosure has been made in view of the above-mentioned prior art, and an object thereof is to provide a human umbilical cord mesenchymal stem cell culture medium free of animal serum, capable of supporting primary cell culture of human umbilical cord mesenchymal stem cells, facilitating rapid proliferation of human umbilical cord mesenchymal stem cells in vitro, cell attachment, and maintenance of cell stem properties, and a method for preparing the same.
To this end, a first aspect of the present disclosure provides a human umbilical cord mesenchymal stem cell medium comprising: basic 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 medium does not include animal serum, the concentration of reduced glutathione in the human umbilical cord mesenchymal stem cell medium is 1mg/L to 3mg/L, the concentration of the epidermal growth factor is 30 μg/L to 80 μg/L, the concentration of the basic fibroblast growth factor is 10 μg/L to 30 μg/L, the concentration of the recombinant human insulin is 1mg/L to 5mg/L, the mass fraction of hyaluronic acid is 0.1% to 0.6%, and the concentration of fibronectin is 10mg/L to 50mg/L.
In the human umbilical cord mesenchymal stem cell culture medium disclosed by the invention, the culture medium does not comprise animal serum, so that adverse reactions such as in-vivo immune response and the like caused by the animal serum when the human umbilical cord mesenchymal stem cells are clinically used can be reduced; the basic culture medium can provide nutrition and a growth environment for human umbilical cord mesenchymal stem cells; reduced glutathione can provide antioxidant protection, and enhance the survival capability and proliferation capability of human umbilical cord mesenchymal stem cells; the epidermal growth factor can promote proliferation of human umbilical cord mesenchymal stem cells, accelerate cell growth, inhibit expression of senescence genes and is beneficial to maintaining cell activity; the 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 multi-directional differentiation potential of the mesenchymal stem cells; the recombinant human insulin can promote the cell to utilize glucose and amino acid, regulate the sugar metabolism, fat metabolism and protein metabolism in the human umbilical cord mesenchymal stem cell, and play a role in promoting the proliferation and survival of the human umbilical cord mesenchymal stem cell; hyaluronic acid has good lubricity and viscoelasticity, can influence the adhesion, proliferation, differentiation and movement of human umbilical cord mesenchymal stem cells by being combined with cell surface receptors, and increases the cell activity, thereby being beneficial to the culture of primary cells of human umbilical cord mesenchymal stem cells; the 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 the polarity, differentiation and growth of cells, thereby facilitating the enhancement of the multidirectional differentiation potential of human umbilical cord mesenchymal stem cells and the promotion of the proliferation of human umbilical cord mesenchymal stem cells. In the human umbilical cord mesenchymal stem cell culture medium, the reduced glutathione, the epidermal growth factor, the basic fibroblast growth factor, the recombinant human insulin, the hyaluronic acid and the fibronectin which have proper concentrations can promote the growth and the adherence of the human umbilical cord mesenchymal stem cells under the concentration condition, thereby being beneficial to the culture of primary cells of the human umbilical cord mesenchymal stem cells and maintaining the stem property of the human umbilical cord mesenchymal stem cells. In addition, the components can act synergistically to further promote the growth, proliferation and adherence of the human umbilical cord mesenchymal stem cells, thereby being beneficial to the culture of primary cells of the human umbilical cord mesenchymal stem cells and maintaining the stem property of the human umbilical cord mesenchymal stem cells.
In the human umbilical cord mesenchymal stem cell medium according to the first aspect of the present disclosure, optionally, in the human umbilical cord mesenchymal stem cell medium, the concentration of reduced glutathione is 2mg/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 3mg/L, and the concentration of fibronectin is 25mg/L. Therefore, the growth and adherence of the human umbilical cord mesenchymal stem cells can be further promoted, and the culture of primary cells and the maintenance of the cell stem property are facilitated.
In the human umbilical cord mesenchymal stem cell medium according to the first aspect of the present disclosure, optionally, the hyaluronic acid has a molecular weight of 10kDa to 100kDa. The different molecular weights have different effects on the human umbilical cord mesenchymal stem cells, and in the present disclosure, hyaluronic acid with the molecular weight of 10kDa to 100kDa is selected, so that the adhesion, proliferation and migration of the human umbilical cord mesenchymal stem cells can be promoted, and the culture of primary cells of the human umbilical cord mesenchymal stem cells is facilitated.
In the human umbilical cord mesenchymal stem cell medium according to the first aspect of the present disclosure, optionally, hyaluronic acid having a molecular weight of 10kDa is included in the human umbilical cord mesenchymal stem cell medium, and the mass fraction is 0.5%. Therefore, the method can be beneficial to promoting the adhesion, proliferation and migration of the human umbilical cord mesenchymal stem cells and is beneficial to the culture of primary cells of the human umbilical cord mesenchymal stem cells.
In the human umbilical cord mesenchymal stem cell medium according to the first aspect of the present disclosure, optionally, hyaluronic acid having a molecular weight of 100kDa is included in the human umbilical cord mesenchymal stem cell medium, and the mass fraction is 0.1%. Therefore, the method can be beneficial to promoting the adhesion, proliferation and migration of the human umbilical cord mesenchymal stem cells and is beneficial to the culture of primary cells of the human umbilical cord mesenchymal stem cells.
In the human umbilical cord mesenchymal stem cell medium according to the first aspect of the present disclosure, optionally, the basal medium is DMEM/F12 medium. Therefore, the method can provide nutrient substances and a growth environment for the human umbilical cord mesenchymal stem cells, and is beneficial to the growth and proliferation of the human umbilical cord mesenchymal stem cells.
In the human umbilical cord mesenchymal stem cell medium according to the first aspect of the present disclosure, optionally, the pH (pH value) of the human umbilical cord mesenchymal stem cell medium is 7.2 to 7.4, and the osmotic pressure is 260mOsm to 320mOsm. Thereby, the growth and proliferation of human umbilical cord mesenchymal stem cells can be facilitated.
A second aspect of the present disclosure provides a method for preparing a human umbilical cord mesenchymal stem cell medium, comprising: preparing a basal medium, reduced glutathione, an epidermal growth factor, an alkaline fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin; mixing the basal medium, reduced glutathione, an epidermal growth factor, an alkaline 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 1mg/L to 3mg/L, the concentration of epidermal growth factor is 30 mug/L to 80 mug/L, the concentration of basic fibroblast growth factor is 10 mug/L to 30 mug/L, the concentration of recombinant human insulin is 1mg/L to 5mg/L, the mass fraction of hyaluronic acid is 0.1% to 0.6%, and the concentration of fibronectin is 10mg/L to 50mg/L.
In the preparation method disclosed by the invention, the prepared human umbilical cord mesenchymal stem cell culture medium does not comprise animal serum, so that adverse reactions such as in-vivo immune response and the like brought by the animal serum when the human umbilical cord mesenchymal stem cells are clinically used can be reduced; the basic culture medium is used, so that nutrition and a growth environment can be provided for human umbilical cord mesenchymal stem cells; the reduced glutathione can provide antioxidant protection and enhance the survival capability and proliferation capability of human umbilical mesenchymal stem cells; the use of the epidermal growth factor can promote the proliferation of human umbilical cord mesenchymal stem cells, accelerate the growth of cells, inhibit the expression of senescence genes and be beneficial to maintaining the activity of the cells; the basic fibroblast growth factor can stimulate the growth of mesoderm-derived cells, promote the proliferation of mesenchymal stem cells and enhance the multi-directional differentiation potential of the mesenchymal stem cells; the recombinant human insulin can promote the cell to utilize glucose and amino acid, regulate the sugar metabolism, fat metabolism and protein metabolism in the mesenchymal stem cells, and play a role in promoting the proliferation and survival of the mesenchymal stem cells; the hyaluronic acid has good lubricity and viscoelasticity, can influence the adhesion, proliferation, differentiation and movement of the human umbilical cord mesenchymal stem cells by being combined with cell surface receptors, and increases the cell viability, thereby being beneficial to the culture of primary cells of the human umbilical cord mesenchymal stem cells; the fibronectin can be used for facilitating the mutual adhesion of cells and extracellular matrixes, so as to promote the adhesion of human umbilical mesenchymal stem cells, and can regulate the polarity, differentiation and growth of the cells, so as to facilitate the enhancement of the multidirectional differentiation potential of the mesenchymal stem cells and the promotion of the proliferation of the mesenchymal stem cells. The human umbilical cord mesenchymal stem cell culture medium prepared by the preparation method disclosed by the invention has proper concentration of reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin, and can promote the growth, proliferation and adherence of human umbilical cord mesenchymal stem cells under the concentration condition, thereby being beneficial to the culture of primary cells and maintaining the cell stem property. In addition, the components can act synergistically to further promote the growth and adherence of human umbilical cord mesenchymal stem cells, thereby being beneficial to the culture of primary cells and the maintenance of cell stem property.
In the production method according to the second aspect of the present disclosure, optionally, in the human umbilical cord mesenchymal stem cell medium, the concentration of reduced glutathione is 2mg/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 3mg/L, the mass fraction of hyaluronic acid is 0.6%, and the concentration of fibronectin is 25mg/L. Therefore, the growth, proliferation and adherence of the human umbilical cord mesenchymal stem cells can be further promoted, and the culture of primary cells and the maintenance of the cell stem property are facilitated.
In the preparation method according to the second aspect of the present disclosure, optionally, the method further comprises filtering the human umbilical cord mesenchymal stem cell medium using a filter having a pore size of 0.22 μm. Therefore, microorganisms such as bacteria in the human umbilical cord mesenchymal stem cell culture medium can be reduced through filtration, and the growth of the human umbilical cord mesenchymal stem cells is facilitated.
According to the present disclosure, a human umbilical cord mesenchymal stem cell culture medium free of animal serum, capable of supporting primary cell culture of human umbilical cord mesenchymal stem cells, facilitating rapid proliferation of human umbilical cord mesenchymal stem cells in vitro, cell attachment and maintenance of cell stem properties, and a method for preparing the same can be provided.
Drawings
The present disclosure will now be explained in further detail by way of example only with reference to the accompanying drawings, in which:
fig. 1 is a flowchart illustrating a method of preparing a human umbilical cord mesenchymal stem cell medium according to the present disclosure.
Fig. 2A is a graph showing cell proliferation curves of medium 1 to medium 4 and the comparative example according to the present disclosure.
Fig. 2B is a graph showing cell proliferation curves of medium 5 to medium 8 and the comparative example according to the present disclosure.
Fig. 2C is a graph showing cell proliferation curves of medium 9 to medium 12 and the comparative example according to the present disclosure.
Fig. 3 is a diagram showing a morphology of cells climbing out of a tissue when cultured using the medium 9 according to the present disclosure.
Fig. 4 is a morphological diagram showing third generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure.
Fig. 5 is a morphological diagram showing eighth generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure.
Fig. 6 is a graph showing the expression level of a third generation human umbilical cord mesenchymal stem cell surface antigen cultured using medium 9 according to the present disclosure.
Fig. 7 is a graph showing the results of adipogenic differentiation staining of third generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure.
Fig. 8 is a graph showing the results of osteoinductive differentiation staining of third generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure.
Detailed Description
Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same members are denoted by the same reference numerals, and overlapping description thereof is omitted. In addition, the drawings are schematic, and the ratio of the sizes of the components to each other, the shapes of the components, and the like may be different from actual ones.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, in this disclosure, such as a process, method, system, article, or apparatus that comprises or has a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or apparatus, but may include or have other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In addition, headings and the like referred to in the following description of the disclosure are not intended to limit the disclosure or scope thereof, but rather are merely indicative of reading. Such subtitles are not to be understood as being used for segmenting the content of the article, nor should the content under the subtitle be limited only to the scope of the subtitle.
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 also referred to as "culture medium" for short. The present disclosure relates to a method of preparing a human umbilical cord mesenchymal stem cell medium, also sometimes referred to below simply as "preparation method". The human umbilical cord mesenchymal stem cell culture medium disclosed by the disclosure does not contain serum, can support primary cell culture of the human umbilical cord mesenchymal stem cells, and can meet the requirements of in vitro rapid proliferation, cell attachment and maintenance of cell stem property of the human umbilical cord mesenchymal stem cells.
In some examples, the human umbilical cord mesenchymal stem cell medium may 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.
In some examples, the human umbilical cord mesenchymal stem cell medium may not include animal serum. Such as fetal bovine Serum (Fetal Bovine Serum, FBS), horse Serum (Horse Serum), calf Serum (Calf Serum), sheep Serum (Sheep Serum), human Serum. Thus, adverse reactions such as in vivo immune response caused by animal serum when human umbilical cord mesenchymal stem cells are clinically used can be reduced.
In some examples, the human umbilical cord mesenchymal stem cell medium may comprise a basal medium. In some examples, the basal medium may be DMEM/F12 medium. The DMEM/F12 Medium is a mixed Medium in which two basic media, namely DMEM (Dulbecco's Modified Eagle's Medium) and F12 (Ham's F Medium), are mixed in a specific ratio. Therefore, the method can provide abundant nutrient substances and factors and a proper growth environment for the human umbilical cord mesenchymal stem cells, and is beneficial to the growth and proliferation of the human umbilical cord mesenchymal stem cells.
In some examples, components in the DMEM/F12 medium may include inorganic salts, sugars, amino acids, vitamins, and buffers. Thereby, nutrition and a growing environment can be provided for the human umbilical cord mesenchymal stem cells.
In some examples, DMEM/F12 medium may not include animal serum. Thus, adverse reactions such as an in vivo immune response by animal serum when human umbilical cord mesenchymal stem cells are clinically used can be reduced.
In some examples, the DMEM/F12 medium may be self-formulated. In some examples, DMEM/F12 medium is also commercially available.
In some examples, the human umbilical cord mesenchymal stem cell medium may include reduced glutathione. Thereby, can provide antioxidant protection, strengthen the survival ability and the proliferation ability of human umbilical cord mesenchymal stem cells.
In some examples, the concentration of reduced glutathione in human umbilical cord mesenchymal stem cell medium may be 1mg/L to 3mg/L. For example, in human umbilical cord mesenchymal stem cell medium, the concentration of reduced glutathione may be 1mg/L, 1.5mg/L, 2mg/L, 2.5mg/L, or 3mg/L. Thus, the method is beneficial to providing proper oxidation protection and enhancing the survival capability and proliferation capability of human umbilical cord mesenchymal stem cells.
In some examples, preferably, the concentration of reduced glutathione in human umbilical cord mesenchymal stem cell medium may be 2mg/L.
In some examples, the human umbilical cord mesenchymal stem cell medium may include an epidermal growth factor. Therefore, the proliferation of human umbilical cord mesenchymal stem cells can be promoted, the cell growth can be accelerated, the expression of senescence genes can be inhibited, and the cell activity can be maintained.
In some examples, the concentration of epidermal growth factor in the human umbilical cord mesenchymal stem cell medium may be 30 μg/L to 80 μg/L. For example, in human umbilical cord mesenchymal stem cell medium, the concentration of the epidermal growth factor may be 30 μg/L, 40 μg/L, 50 μg/L, 60 μg/L, 70 μg/L, or 80 μg/L. An excessively high concentration of epidermal growth factor may cause excessive proliferation of cells, may affect the stem property of cells, promote differentiation of cells, and an excessively low concentration of epidermal growth factor may affect the growth and proliferation of cells. Thus, an appropriate cell proliferation rate can be provided.
In some examples, preferably, the concentration of the epidermal growth factor may be 50 μg/L in the human umbilical cord mesenchymal stem cell medium. Thereby, the growth and proliferation of human umbilical cord mesenchymal stem cells can be facilitated.
In some examples, the human umbilical cord mesenchymal stem cell medium may comprise basic fibroblast growth factor. Thereby, the growth of mesoderm-derived cells can be stimulated, the proliferation of mesenchymal stem cells can be promoted, and the multipotent differentiation potential of the mesenchymal stem cells can be enhanced.
In some examples, the concentration of basic fibroblast growth factor in human umbilical cord mesenchymal stem cell medium may be 10 μg/L to 30 μg/L. For example, in human umbilical cord mesenchymal stem cell medium, the concentration of basic fibroblast growth factor may be 10 μg/L, 15 μg/L, 20 μg/L, 25 μg/L or 30 μg/L. Thus, it is possible to provide an appropriate effect of stimulating the growth of mesoderm-derived cells and an effect of promoting the proliferation of mesenchymal stem cells and enhancing the multipotent differentiation potential of mesenchymal stem cells.
In some examples, preferably, the concentration of basic fibroblast growth factor in human umbilical cord mesenchymal stem cell medium may be 20 μg/L. Thus, the method can be beneficial to stimulating the growth of mesoderm-derived cells, promoting the proliferation of mesenchymal stem cells and enhancing the multi-directional differentiation potential of the mesenchymal stem cells.
In some examples, the human umbilical cord mesenchymal stem cell medium may comprise recombinant human insulin. Therefore, the method can promote the cells to utilize glucose and amino acid to regulate the sugar metabolism, fat metabolism and protein metabolism in the mesenchymal stem cells, and plays a role in promoting the proliferation and survival of the human umbilical cord mesenchymal stem cells.
In some examples, the concentration of recombinant human insulin in human umbilical cord mesenchymal stem cell medium may be 1mg/L to 5mg/L. For example, in human umbilical cord mesenchymal stem cell medium, the concentration of recombinant human insulin may be 1mg/L, 2mg/L, 3mg/L, 4mg/L or 5mg/L. Thus, it is possible to provide an appropriate metabolic regulation effect, and promote proliferation and survival of human umbilical cord mesenchymal stem cells.
In some examples, preferably, the concentration of recombinant human insulin in human umbilical cord mesenchymal stem cell medium may be 3mg/L. Thereby, proliferation and survival of human umbilical cord mesenchymal stem cells can be facilitated.
In some examples, the human umbilical cord mesenchymal stem cell medium may comprise hyaluronic acid. In this case, the hyaluronic acid has good lubricity and viscoelasticity, can influence the adhesion, proliferation, differentiation and movement of the human umbilical cord mesenchymal stem cells by binding to the cell surface receptor, and increases the cell viability, thereby facilitating the culture of the primary cells of the human umbilical cord mesenchymal stem cells.
In some examples, the mass fraction of hyaluronic acid in the human umbilical cord mesenchymal stem cell medium may be 0.1% to 0.6%. For example, in human umbilical cord mesenchymal stem cell medium, the concentration of recombinant human insulin may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 0.6%. Therefore, the method is beneficial to the adhesion, proliferation, differentiation and movement of the human umbilical cord mesenchymal stem cells, increases the cell activity and is beneficial to the culture of primary cells of the human umbilical cord mesenchymal stem cells.
In some examples, preferably, the mass fraction of hyaluronic acid in human umbilical cord mesenchymal stem cell medium may be 0.6%. Therefore, the method is beneficial to the adhesion, proliferation, differentiation and movement of the human umbilical cord mesenchymal stem cells, increases the cell activity and is beneficial to the culture of primary cells of the human umbilical cord mesenchymal stem cells.
In some examples, the hyaluronic acid may have a molecular weight of 10kDa to 100kDa. For example, the molecular weight of hyaluronic acid may be 10kDa, 20kDa, 50kDa, 70kDa, 80kDa and/or 100kDa. The different molecular weights have different effects on human umbilical cord mesenchymal stem cells, in this disclosure, hyaluronic acid having a molecular weight of 10kDa to 100kDa is selected. Therefore, the adhesion, proliferation and migration of the human umbilical cord mesenchymal stem cells can be further promoted, the cell activity is increased, and the culture of the primary cells of the human umbilical cord mesenchymal stem cells is facilitated.
In some examples, preferably, the hyaluronic acid may include hyaluronic acid having a molecular weight of 10kDa and hyaluronic acid having a molecular weight of 100 kDa.
In some examples, the mass fraction of hyaluronic acid having a molecular weight of 10kDa may be 0.5% in human umbilical cord mesenchymal stem cell medium. Therefore, the adhesion, proliferation and migration of the human umbilical cord mesenchymal stem cells can be promoted, and the culture of the primary cells of the human umbilical cord mesenchymal stem cells is facilitated.
In some examples, the mass fraction of hyaluronic acid having a molecular weight of 100kDa may be 0.1% in human umbilical cord mesenchymal stem cell medium. Therefore, the adhesion, proliferation and migration of the human umbilical cord mesenchymal stem cells can be promoted, and the culture of the primary cells of the human umbilical cord mesenchymal stem cells is facilitated.
In some examples, in human umbilical cord mesenchymal stem cell medium, hyaluronic acid having a molecular weight of 10kDa may be included at a mass fraction of 0.5% together with hyaluronic acid having a molecular weight of 100kDa at a mass fraction of 0.1%. Therefore, the method can be beneficial to promoting the adhesion, proliferation and migration of the human umbilical cord mesenchymal stem cells and is beneficial to the culture of primary cells of the human umbilical cord mesenchymal stem cells.
In some examples, the human umbilical cord mesenchymal stem cell medium may comprise fibronectin. Therefore, the method can be beneficial to the adhesion of cells and extracellular matrixes, so as to promote the adhesion of human umbilical cord mesenchymal stem cells, and can regulate the polarity, differentiation and growth of the cells, so as to be beneficial to the enhancement of the multidirectional differentiation potential of the human umbilical cord mesenchymal stem cells and the promotion of the proliferation of the mesenchymal stem cells.
In some examples, the concentration of fibronectin in human umbilical cord mesenchymal stem cell medium may be 10mg/L to 50mg/L. For example, in human umbilical cord mesenchymal stem cell medium, the concentration of fibronectin may be 10mg/L, 20mg/L, 25mg/L, 30mg/L, 35mg/L, 40mg/L, or 50mg/L. Therefore, the method can be beneficial to promoting the adhesion, growth and proliferation of the human umbilical cord mesenchymal stem cells and enhancing the multidirectional differentiation potential of the human umbilical cord mesenchymal stem cells.
In some examples, preferably, the concentration of fibronectin in human umbilical cord mesenchymal stem cell medium may be 25mg/L. Therefore, the method can be beneficial to promoting the adhesion, growth and proliferation of the human umbilical cord mesenchymal stem cells and enhancing the multidirectional differentiation potential of the human umbilical cord mesenchymal stem cells.
In some examples, reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin are capable of acting synergistically to further promote growth, proliferation and adherence of human umbilical cord mesenchymal stem cells, facilitate culture of primary cells of human umbilical cord mesenchymal stem cells and maintain the stem properties of human umbilical cord mesenchymal stem cells.
In some examples, the pH of the human umbilical cord mesenchymal stem cell medium may be 7.2 to 7.4. Thereby, the growth of human umbilical cord mesenchymal stem cells can be facilitated.
In some examples, the osmolality of the human umbilical cord mesenchymal stem cell medium may be 260 to 320mOsm. Thereby, the growth of human umbilical cord mesenchymal stem cells can be facilitated.
The disclosure also relates to a preparation method of the human umbilical cord mesenchymal stem cell culture medium. It should be noted that, the aforementioned "human umbilical cord mesenchymal stem cell culture medium" part may be communicated with the content of the following "preparation method", and the content of the part may refer to the aforementioned "human umbilical cord mesenchymal stem cell culture medium" part, which is not described herein again.
Fig. 1 is a flowchart illustrating a method of preparing a human umbilical cord mesenchymal stem cell medium according to the present disclosure.
In some examples, as shown in fig. 1, the preparation method may include: preparing a basal medium and a raw material reagent (step S10); mixing the basal medium and the raw material reagent (step S20); obtaining human umbilical cord mesenchymal stem cell medium (step S30). Thus, a human umbilical cord mesenchymal stem cell medium can be obtained.
In some examples, in step S10, the starting agent may include reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid, and fibronectin.
In some examples, in step S10, the basal medium may be DMEM/F12 medium.
In some examples, in step S20, a basal medium may be used to dissolve the feedstock reagents. Specifically, a specific mass of a raw material reagent may be weighed, and then a basal medium is used to dissolve the raw material for a specific time and to fix the volume, thereby obtaining a human umbilical cord mesenchymal stem cell medium.
In some examples, in step S20 and step S30, reduced glutathione concentration of 2mg/L, epidermal growth factor concentration of 50 μg/L, basic fibroblast growth factor concentration of 20 μg/L, recombinant human insulin concentration of 3mg/L, hyaluronic acid mass fraction of 0.6% and fibronectin concentration of 25mg/L may be used in human umbilical cord mesenchymal stem cell culture medium. Therefore, the growth, proliferation and adherence of the human umbilical cord mesenchymal stem cells can be promoted, and the culture of primary cells and the maintenance of the cell stem property are facilitated.
In some examples, in step S20 and step S30, reduced glutathione concentration of 2mg/L, epidermal growth factor concentration of 50 μg/L, basic fibroblast growth factor concentration of 20 μg/L, recombinant human insulin concentration of 3mg/L, hyaluronic acid mass fraction of 0.6% and fibronectin concentration of 25mg/L may be used in human umbilical cord mesenchymal stem cell culture medium. Therefore, the growth, proliferation and adherence of the human umbilical cord mesenchymal stem cells can be promoted, and the culture of primary cells and the maintenance of the cell stem property are facilitated.
In some examples, the step of adjusting the pH is further included after mixing the basal medium and the feed reagent. In some examples, the pH of the human umbilical cord mesenchymal stem cell medium may be adjusted to 7.2 to 7.4. In some examples, the pH can be adjusted by adjusting the buffer salt concentration in the basal medium.
In some examples, the step of adjusting osmotic pressure is further included after mixing the basal medium and the feed agent. In some examples, the osmolality of the human umbilical cord mesenchymal stem cell medium may be adjusted to 260 to 320mOsm. In some examples, osmotic pressure may be adjusted by adjusting the concentration of solutes in the basal medium.
In some examples, filtering the prepared human umbilical cord mesenchymal stem cell medium using a filter having a pore size of 0.22 μm may be further included. Thus, the human umbilical cord mesenchymal stem cell culture medium can be subjected to sterilization treatment by filtration, and thereby the growth and propagation of the human umbilical cord mesenchymal stem cells can be facilitated.
According to the human umbilical cord mesenchymal stem cell culture medium and the preparation method thereof, the culture medium does not contain serum, can support primary cell culture of the human umbilical cord mesenchymal stem cells, and is beneficial to in vitro rapid proliferation, cell attachment and maintenance of cell stem property of the human umbilical cord mesenchymal stem cells. The human umbilical cord mesenchymal stem cell culture medium disclosed by the disclosure has the advantages that the added components are clear and simple, and the cell culture bottle can be not required to be coated in the culture process.
Hereinafter, the present disclosure will be described in detail by way of examples and comparative examples. However, the following examples or comparative examples are provided only for the purpose of specifically illustrating the present disclosure, and do not limit or restrict the scope of the invention disclosed in the present application.
The reagents, materials, and instruments used in the examples of the present disclosure and comparative examples are all commercially available unless otherwise specified.
First, a human umbilical cord mesenchymal stem cell medium was prepared. Specifically, DMEM/F12 medium, reduced glutathione, EGF, bFGF, recombinant human insulin, HA and fibronectin were prepared. Wherein, the components and the proportion of the DMEM/F12 medium can be shown in the following table:
then, human umbilical cord mesenchymal stem cell culture mediums of serial numbers 1 to 4 (i.e., culture mediums 1 to 4) were prepared based on the DMEM/F12 medium and according to the concentrations of the respective components in the following table:
human umbilical cord mesenchymal stem cell culture mediums with the serial numbers of 5 to 8 (namely culture mediums 5 to 8) are prepared according to the concentrations of the components in the following table on the basis of a DMEM/F12 culture medium:
human umbilical cord mesenchymal stem cell culture medium with serial numbers 9 to 12 (namely culture medium 9 to culture medium 12) is prepared based on DMEM/F12 culture medium according to the concentration of each component in the following table:
the culture media of comparative examples 1 and 2 were prepared based on DMEM/F12 medium:
comparative example 1: adding 5% volume fraction of platelet lysate into DMEM/F12 medium;
comparative example 2: a volume fraction of 10% fetal bovine serum was added to DMEM/F12 medium.
Subsequently, bacteria and fungi were detected on the medium 1 to the medium 12, the medium of comparative example 1 and the medium of comparative example 2, and the detection results were negative; detecting chlamydia and mycoplasma, wherein the detection result is negative; endotoxin detection was performed, and the results were 0EU/mL to 0.5EU/mL. Subsequently, the media 1 to 12, the media of comparative example 1 and comparative example 2 were sterilized by filtration through a 0.22 μm filter and stored at-20℃in a dark place.
Then, human umbilical cord mesenchymal stem cells were prepared. Specifically, the umbilical cord of a patient with fresh caesarean section is taken and placed in Phosphate Buffer (PBS) containing 1% penicillin streptomycin, residual blood stasis is squeezed and discharged, and 2 umbilical arteries, 1 umbilical vein and umbilical adventitia are removed in sequence, and the remaining tissue is Wharton's jelly. Shearing Whatman gum into 1mm in 50ml centrifuge tube 3 Tissue blocks with the size of 175mm are uniformly spread on the bottom area of 1.5ml 2 Is provided in the culture flask.
Then, medium 1 to medium 12, the medium of comparative example 1 and the medium of comparative example 2 were added, respectively, and the mixture was placed at 37℃with 5% CO 2 Culturing under saturated humidity, and supplementing fresh human umbilical cord mesenchymal stem cell culture medium every 3 days; removing the tissue block when the fibroblast (i.e. the cell climbs out from the tissue) growing in a colony form around the tissue block is observed under the microscope; digestion with recombinant trypsin analog (TrypLE) for 3 min, centrifugation at 1000rpm for 5 min and discarding supernatant, re-suspending cell pellet with medium 1 to medium 12, medium of comparative example 1, medium of comparative example 2, 10000 cells/cm, respectively 2 Inoculating with density in culture flask at 37deg.C and 5% CO 2 Continuously culturing under the condition of saturated humidity, and when the cells are fused to 80% -90%, performing the steps of digestion, centrifugation and resuspension again to obtain the mesenchymal stem cells according to 10000 mesenchymal stem cells/cm 2 Is inoculated in culture medium 1 to culture medium 12, culture medium of comparative example 1 and culture medium of comparative example 2 respectively for passage amplification, passage is carried out every 4 days, and the inoculation density of each passage is 10000 pieces/cm 2 . Cell count analysis was performed for each passage.
Fig. 2A is a graph showing cell proliferation curves of medium 1 to medium 4 and the comparative example according to the present disclosure. As can be seen from FIG. 2A, the mass fraction of added hyaluronic acid is 0.1%, the molecular weight of hyaluronic acid is 10kDa in culture medium 2 and the molecular weight of hyaluronic acid is 100kDa in culture medium 3, and the cell proliferation effect is good; medium 1 with a molecular weight of 1kDa of hyaluronic acid and medium 4 with a molecular weight of 1000kDa of hyaluronic acid have poor cell proliferation effect; in addition, the cell proliferation effect of medium 2 and medium 3 was better than that of the culture media of comparative examples 1 and 2. Medium 2 and Medium 3 are preferred embodiments of the present disclosure.
Fig. 2B is a graph showing cell proliferation curves of medium 5 to medium 8 and the comparative example according to the present disclosure. As can be seen from fig. 2B, the mass fraction of hyaluronic acid added was 0.5%, medium 6 having a molecular weight of 10kDa and medium 7 having a molecular weight of 100kDa, and the cell proliferation effect was good; medium 5 with a molecular weight of 1kDa for hyaluronic acid and medium 8 with a molecular weight of 1000kDa for hyaluronic acid, the cell proliferation effect is poor; in addition, the cell proliferation effect of medium 6 was better than that of the medium of comparative example 2, and the cell proliferation effect of medium 7 was better than that of the medium of comparative example 1. Media 6 and 7 are preferred embodiments of the present disclosure.
Fig. 2C is a graph showing cell proliferation curves of medium 9 to medium 12 and the comparative example according to the present disclosure. As is clear from fig. 2C, the cell proliferation effect was better than that of the culture medium of comparative examples 1 and 2 by adding 0.5% by mass of hyaluronic acid having a molecular weight of 10kDa and 0.1% by mass of hyaluronic acid having a molecular weight of 100kDa to the culture medium 9, and by adding 0.1% by mass of hyaluronic acid having a molecular weight of 10kDa and 0.5% by mass of hyaluronic acid having a molecular weight of 100kDa to the culture medium 10. Wherein the cell proliferation effect of the culture medium 9 is optimal. Medium 9 and medium 10 are preferred embodiments of the present disclosure, and medium 9 is a preferred embodiment of the present disclosure.
In summary, the cell proliferation effect is better when the molecular weight of the added hyaluronic acid is 10kDa to 100kDa, and the cell proliferation effect is better when the mass fraction of the added hyaluronic acid is 0.1% and 0.5%, wherein the cell proliferation effect of the culture medium 9 added with 0.5% of 10kDa hyaluronic acid and 0.1% of 100kDa hyaluronic acid is best.
Fig. 3 is a morphology diagram showing the climbing of cells from tissues when cultured using the medium 9 according to the present disclosure, fig. 4 is a morphology diagram showing third generation human umbilical cord mesenchymal stem cells cultured using the medium 9 according to the present disclosure, and fig. 5 is a morphology diagram showing eighth generation human umbilical cord mesenchymal stem cells cultured using the medium 9 according to the present disclosure. As can be seen from fig. 4 and 5, the human umbilical cord mesenchymal stem cells obtained by culturing in the medium 9 were uniform in cell morphology after multiple passages and were in line with the cell morphology of the human umbilical cord mesenchymal stem cells.
Cell surface antigen expression assay: taking p3 generation HUC-MSCs of the culture medium 9, adopting TrypLE to digest, centrifuging, removing supernatant, washing by using sterile PBS, adding corresponding antibodies to incubate, re-suspending the culture medium after washing by using PBS again, and detecting the expression of cell surface antigens CD73, CD90, CD105, HLA-DR, CD34, CD11b, CD19 and CD45 by using a flow cytometer. Fig. 6 is a graph showing the expression level of a third generation human umbilical cord mesenchymal stem cell surface antigen cultured using medium 9 according to the present disclosure. As shown in fig. 6, the human umbilical cord mesenchymal stem cells cultured by the human umbilical cord mesenchymal stem cell culture medium of the culture medium 9 can well ensure that the biological characteristics of the human umbilical cord mesenchymal stem cells are not changed, the expression rate of the positive marker CD73 of the human umbilical cord mesenchymal stem cells is more than 97%, the expression rate of CD90 is more than 99%, and the expression rate of CD105 is more than 96%; the expression rate of human umbilical cord mesenchymal stem cell negative markers CD34, CD45, CD19, CD11b and HLA-DR is lower than 1%.
In vitro adipogenic induced differentiation: taking p3 generation HUC-MSCs of the culture medium 9 according to 10000 pieces/cm 2 Is inoculated in a 6-well plate, is cultured by using a human umbilical cord mesenchymal stem cell culture medium of a culture medium 9, and is changed into a lipid induction culture solution (containing 10% fetal bovine serum, 10mg/L insulin, 1 mu M dexamethasone, 0.5mM IBMX (phosphodiesterase inhibitor) and 1% penicillin streptomycin solution) when the cells are fused to 90%; the lipid-induced culture medium was changed every 2 days, and the oil red O staining experiment was performed after 14 days of induction, and the staining results were observed under a microscope. Fig. 7 is a graph showing the results of adipogenic differentiation staining of third generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure. As can be seen from figure 7 of the drawings, Under the culture condition of the human umbilical cord mesenchymal stem cell culture medium using the culture medium 9, the human umbilical cord mesenchymal stem cell can maintain the differentiation potential thereof and can be differentiated into an adipocyte.
In vitro osteogenic induced differentiation: taking p3 generation HUC-MSCs of the culture medium 9 according to 10000 pieces/cm 2 Is inoculated in a 6-well plate, cultured by using the human umbilical cord mesenchymal stem cell culture medium of example 9, and when the cells are fused to 90%, the human umbilical cord mesenchymal stem cell culture medium is changed into an osteogenic induction culture medium (containing 10% fetal bovine serum, 10 mu M dexamethasone, 10mM beta-glycerophosphate, 1mM glutamine and 1% penicillin streptomycin solution), the osteogenic induction culture medium is changed every 2 days, and after 21 days of induction, alizarin red staining experiments are performed, and the staining results are observed under a microscope. Fig. 8 is a graph showing the results of osteoinductive differentiation staining of third generation human umbilical cord mesenchymal stem cells cultured using medium 9 according to the present disclosure. As can be seen from fig. 8, the human umbilical cord mesenchymal stem cells can maintain their differentiation potential and can differentiate into bone cells under the culture conditions of the human umbilical cord mesenchymal stem cell medium using the medium 9.
In summary, the human umbilical cord mesenchymal stem cell culture medium disclosed by the disclosure comprises reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin, and the components are synergistic to play roles, so that primary cell culture can be supported, growth and adherence of human umbilical cord mesenchymal stem cells can be promoted, culture of primary cells of human umbilical cord mesenchymal stem cells is facilitated, and stem property of human umbilical cord mesenchymal stem cells is maintained. In addition, the human umbilical cord mesenchymal stem cell medium disclosed by the invention does not contain animal serum, so that adverse reactions such as in-vivo immune response and the like caused by animal serum when the human umbilical cord mesenchymal stem cells are clinically used can be reduced.
While the disclosure has been described in detail in connection with the drawings and embodiments, it should be understood that the foregoing description is not intended to limit the disclosure in any way. Modifications and variations of the present disclosure may be made as desired by those skilled in the art without departing from the true spirit and scope of the disclosure, and such modifications and variations fall within the scope of the disclosure.
Claims (10)
1. A human umbilical cord mesenchymal stem cell culture medium, comprising: basal medium, reduced glutathione, epidermal growth factor, basic fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin,
wherein the human umbilical cord mesenchymal stem cell medium does not comprise animal serum, the concentration of reduced glutathione in the human umbilical cord mesenchymal stem cell medium is 1 mg/liter to 3 mg/liter, the concentration of epidermal growth factor is 30 micrograms/liter to 80 micrograms/liter, the concentration of basic fibroblast growth factor is 10 micrograms/liter to 30 micrograms/liter, the concentration of recombinant human insulin is 1 milligram/liter to 5 milligram/liter, the mass fraction of hyaluronic acid is 0.1% to 0.6%, and the concentration of fibronectin is 10 milligram/liter to 50 milligram/liter.
2. The human umbilical cord mesenchymal stem cell medium of claim 1, wherein in the human umbilical cord mesenchymal stem cell medium, the concentration of reduced glutathione is 2 mg per liter, the concentration of epidermal growth factor is 50 micrograms per liter, the concentration of basic fibroblast growth factor is 20 micrograms per liter, the concentration of recombinant human insulin is 3 mg per liter, and the concentration of fibronectin is 25 mg per liter.
3. The human umbilical cord mesenchymal stem cell medium of claim 2, wherein the hyaluronic acid has a molecular weight of 10 kilodaltons to 100 kilodaltons.
4. A human umbilical cord mesenchymal stem cell medium according to claim 3, comprising hyaluronic acid having a molecular weight of 10 kilodaltons in the human umbilical cord mesenchymal stem cell medium, and having a mass fraction of 0.5%.
5. The human umbilical cord mesenchymal stem cell medium according to claim 3 or 4, wherein hyaluronic acid having a molecular weight of 100 kilodaltons is included in the human umbilical cord mesenchymal stem cell medium, and the mass fraction is 0.1%.
6. The human umbilical cord mesenchymal stem cell medium of claim 1, wherein the basal medium is DMEM/F12 medium.
7. The human umbilical cord mesenchymal stem cell medium of claim 6, wherein the pH of the human umbilical cord mesenchymal stem cell medium is 7.2 to 7.4 and the osmotic pressure is 260 milli-osmotic per liter to 320 milli-osmotic per liter.
8. A method for preparing a human umbilical cord mesenchymal stem cell culture medium, which is characterized by comprising the following steps: preparing a basal medium, reduced glutathione, an epidermal growth factor, an alkaline fibroblast growth factor, recombinant human insulin, hyaluronic acid and fibronectin; mixing the basal medium, reduced glutathione, an epidermal growth factor, an alkaline 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/liter to 3 mg/liter, the concentration of epidermal growth factor is 30 micrograms/liter to 80 micrograms/liter, the concentration of basic fibroblast growth factor is 10 micrograms/liter to 30 micrograms/liter, the concentration of recombinant human insulin is 1 mg/liter to 5 mg/liter, the mass fraction of hyaluronic acid is 0.1% to 0.6%, and the concentration of fibronectin is 10 mg/liter to 50 mg/liter.
9. The method according to claim 8, wherein the concentration of reduced glutathione in the human umbilical cord mesenchymal stem cell medium is 2 mg/liter, the concentration of epidermal growth factor is 50. Mu.g/liter, the concentration of basic fibroblast growth factor is 20. Mu.g/liter, the concentration of recombinant human insulin is 3 mg/liter, the mass fraction of hyaluronic acid is 0.6%, and the concentration of fibronectin is 25 mg/liter.
10. The method of claim 8 or 9, further comprising filtering the human umbilical cord mesenchymal stem cell medium using a filter having a pore size of 0.22 μm.
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