Preparation method of universal culture medium for umbilical cord mesenchymal stem cells

Abstract

The invention relates to the field of stem cell culture, and discloses an umbilical cord mesenchymal stem cell universal culture medium preparation method, which comprises: mixing an RPMI culture medium and an IMDM culture medium to form a basic culture medium; adding growth factors and recombinant protein into the basic culture medium, and stirring to obtain a first culture medium; adding trace elements into the first culture medium added with the growth factors and the recombinant proteins, and lightly and uniformly stirring to obtain a second culture medium; and filtering and sterilizing the second culture medium at room temperature, and then subpackaging and storing to obtain the universal culture medium for the umbilical cord mesenchymal stem cells. According to the invention, pathogen pollution and immunoreaction risks are avoided, the safety and stability of cell culture are improved, the proliferation potential of cells can be effectively maintained, the proliferation efficiency is improved, and the cell culture medium has better stemness maintenance capability and stronger multidirectional differentiation potential.

Description

Technical Field

[0001] The present invention relates to the field of stem cell culture, and particularly to a method for preparing a universal medium for umbilical cord mesenchymal stem cells. Background Art

[0002] Currently, in the field of stem cell culture, there are many deficiencies in the media specifically for umbilical cord mesenchymal stem cells (MSCs). Traditional media usually add fetal bovine serum (FBS), growth factors and other components based on a basal medium. Common media formulations include the basal medium DMEM, and a certain amount of FBS, epidermal growth factor (EGF), fibroblast growth factor (FGF), etc. are added. These traditional media have some problems, big and small. For example: The uncertainty of the FBS source may lead to large differences between batches, affecting the stability and repeatability of experimental results, and affecting large-scale production. The presence of animal-derived components increases the risk of introducing pathogens during cell culture and may trigger immune responses in clinical applications. Slight differences in the components and proportions of media used by different laboratories or research institutions also make it difficult to measure the cell culture effects with a unified standard, which is not conducive to the large-scale industrial production of umbilical cord MSCs and the standardized development of clinical research. Summary of the Invention

[0003] The present invention aims to provide a method for preparing a universal medium for umbilical cord mesenchymal stem cells, which avoids the risks of pathogen contamination and immune responses, improves the safety and stability of cell culture, can effectively maintain the proliferation potential of cells, improves the proliferation efficiency, has better stemness maintenance ability, and stronger multi-directional differentiation potential.

[0004] To achieve the above object, the present invention provides the following methods and systems: A method for preparing a universal medium for umbilical cord mesenchymal stem cells provided by the present invention is as follows: S1: Cultivate a basal medium by mixing RPMI medium and IMDM medium; S2: Add growth factors and recombinant proteins to the basal medium and stir to obtain a first medium; S3: Add trace elements to the first medium after adding growth factors and recombinant proteins, and gently stir evenly to obtain a second medium; S4: Filter and sterilize the second medium at room temperature, then aliquot and store it to obtain a universal medium for umbilical cord mesenchymal stem cells.

[0005] Preferably, the volume ratio of the RPMI medium to the IMDM medium is 1:3.

[0006] Preferably, during the process of mixing the RPMI medium and the IMDM medium for culturing the basal medium, gently stir to fully mix the RPMI medium and the IMDM medium and avoid generating bubbles.

[0007] Preferably, the components of the growth factor include: basic fibroblast growth factor, insulin-like growth factor, platelet-derived growth factor, transforming growth factor-β, epidermal growth factor, hepatocyte growth factor, granulocyte-macrophage colony-stimulating factor, and stem cell factor.

[0008] Preferably, the dosage ratio of the components of the growth factor is: basic fibroblast growth factor: insulin-like growth factor: platelet-derived growth factor: transforming growth factor-β: epidermal growth factor: hepatocyte growth factor: granulocyte-macrophage colony-stimulating factor: stem cell factor = 1 - 10:5 - 20:5 - 15:1 - 5:5 - 10:5 - 15:1 - 5:5 - 10.

[0009] Preferably, the components of the recombinant protein include: recombinant human thrombopoietin, recombinant human insulin, recombinant human transferrin, and recombinant human serum albumin.

[0010] Preferably, the dosage ratio of the components of the recombinant protein is: recombinant human thrombopoietin: recombinant human insulin: recombinant human transferrin: recombinant human serum albumin = 5 - 10::1 - 10:1 - 10:5 - 10:1 - 5.

[0011] Preferably, the components of the trace element include: ethanolamine and sodium selenite; the dosage ratio of the ethanolamine and the sodium selenite is 1:10.

[0012] Preferably, the step of adding trace elements to the first medium after adding the growth factor and the recombinant protein and gently stirring evenly to obtain the second medium includes: adding trace elements to the first medium after adding the growth factor and the recombinant protein and gently stirring for 5 - 8 minutes to ensure uniform distribution, and performing the operation at room temperature of 20°C - 25°C to obtain the second medium.

[0013] Preferably, the step of filtering and sterilizing the second medium at room temperature, then aliquoting and storing it to obtain the umbilical cord mesenchymal stem cell universal medium includes: filtering and sterilizing the second medium through a 0.22 μm filter membrane at room temperature, then aliquoting and storing it to obtain the umbilical cord mesenchymal stem cell universal medium.

[0014] The beneficial effects of the present invention are as follows: In the serum-free medium adopted by the present invention, the replacement of serum components is mainly achieved by optimizing the medium formula. The optimized scheme can provide the nutrients and growth factors required for cell growth without relying on animal serum; the use of human recombinant growth factors to replace animal-derived components avoids the risks of pathogen contamination and immune reactions, and improves the safety and stability of cell culture; the optimized combination of growth factors and the concentration of extracellular matrix proteins promote the proliferation efficiency of umbilical cord MSCs; within the same culture time, the medium of the present invention can promote the proliferation of umbilical cord MSCs, accelerate the cell growth rate, and the number of cells is more than that proliferated using traditional media within the same culture time; at the same time, it can effectively maintain the proliferation potential of cells and improve the proliferation efficiency; by precisely controlling the sources and ratios of each component, the quality of different batches of media is highly consistent, reducing experimental errors and clinical application risks caused by media differences; the finally obtained cells have better ability to maintain stemness. By detecting the expression levels of cell surface markers such as CD73, CD90, CD105, etc., it is found that the positive rate of cells cultured in this medium is higher and the multi-directional differentiation potential is stronger. These advantages make the medium have broad application prospects in basic research on umbilical cord MSCs, regenerative medicine applications, and the development of cell therapy products, providing strong support for the realization of standardized and large-scale culture of umbilical cord MSCs. Brief Description of the Drawings

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual ratio.

[0016] Figure 1 It is a schematic flow chart of a method for preparing a universal medium for umbilical cord mesenchymal stem cells provided by an embodiment of the present invention; Figure 2 It is a schematic diagram of cell morphology under the culture of different media provided by an embodiment of the present invention; Figure 3 It is a schematic diagram of a series of flow cytometry detection results provided by an embodiment of the present invention; Figure 4 It is a schematic diagram of the comparison of cell growth curves provided by an embodiment of the present invention. Detailed Embodiments

[0017] In order to enable those skilled in the art to better understand the solution of the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0018] The terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish different objects, rather than to describe a specific order. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, device, product or terminal that includes a series of steps or units is not limited to the listed steps or units, but optionally further includes steps or units not listed, or optionally further includes other steps or units inherent to these processes, methods, products or terminals.

[0019] Referring to "embodiments" herein means that the specific features, structures or characteristics described in connection with the embodiments can be included in at least one embodiment of the present invention. The phrase appears in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0020] Currently, in the field of stem cell culture, there are many deficiencies in the culture media specifically for umbilical cord mesenchymal stem cells (MSCs). Traditional culture media usually add fetal bovine serum (FBS), growth factors and other components based on a basal medium. Common culture medium formulations include the basal medium DMEM and add a certain amount of FBS, epidermal growth factor (EGF), fibroblast growth factor (FGF), etc. These traditional culture media have some problems, big and small.

[0021] The present invention aims to provide a method for preparing a universal culture medium for umbilical cord mesenchymal stem cells, which avoids the risks of pathogen contamination and immune reactions, improves the safety and stability of cell culture, can effectively maintain the proliferation potential of cells, improves the proliferation efficiency, has better stemness maintenance ability, and stronger multi-directional differentiation potential.

[0022] As Figures 1-4 shown, the specific implementation manner of the present invention provides a method for preparing a universal culture medium for umbilical cord mesenchymal stem cells, including the following steps: S1: Cultivate the basal medium by mixing RPMI medium and IMDM medium.

[0023] In the embodiment of the present invention, the volume ratio of RPMI medium to IMDM medium is 1:3; during the process of mixing the RPMI medium and the IMDM medium to culture the basal medium, gently stir to fully mix the RPMI medium and the IMDM medium and avoid generating bubbles.

[0024] S2: Add growth factors and recombinant proteins to the basal medium and stir to obtain the first medium.

[0025] In the embodiment of the present invention, the components of the growth factors include: basic fibroblast growth factor, insulin-like growth factor, platelet-derived growth factor, transforming growth factor-β, epidermal growth factor, hepatocyte growth factor, granulocyte-macrophage colony-stimulating factor, and stem cell factor; the dosage ratio of the components of the growth factors is: basic fibroblast growth factor: insulin-like growth factor: platelet-derived growth factor: transforming growth factor-β: epidermal growth factor: hepatocyte growth factor: granulocyte-macrophage colony-stimulating factor: stem cell factor = 1 - 10:5 - 20:5 - 15:1 - 5:5 - 10:5 - 15:1 - 5:5 - 10; the components of the recombinant proteins include: recombinant human thrombopoietin, recombinant human insulin, recombinant human transferrin, recombinant human serum albumin; the dosage ratio of the components of the recombinant proteins is: recombinant human thrombopoietin: recombinant human insulin: recombinant human transferrin: recombinant human serum albumin = 5 - 10::1 - 10:1 - 10:5 - 10:1 - 5.

[0026] S3: Add trace elements to the first medium after adding growth factors and recombinant proteins and gently stir evenly to obtain the second medium.

[0027] In the embodiment of the present invention, the components of the trace elements include: ethanolamine and sodium selenite; the dosage ratio of the ethanolamine and the sodium selenite is 1:10; add trace elements to the first medium after adding growth factors and recombinant proteins and gently stir for 5 - 8 minutes to ensure uniform distribution, and carry out the operation at room temperature of 20 o C-25 o C to obtain the second medium.

[0028] S4: Filter and sterilize the second medium at room temperature, then carry out sub-packaging and storage to obtain the universal medium for umbilical cord mesenchymal stem cells.

[0029] In the embodiment of the present invention, filter and sterilize the second medium through a 0.22μm filter membrane at room temperature, then carry out sub-packaging and storage to obtain the universal medium for umbilical cord mesenchymal stem cells.

[0030] In practical applications, the umbilical cord MSC universal culture medium of the present invention is used for culturing umbilical cord MSCs. Fresh umbilical cord tissue is taken, and umbilical cord MSCs are obtained through isolation and purification. Then they are inoculated into this culture medium and cultured in an incubator at 37°C and 5% CO2. During the culture process, the cell morphology and growth status are observed regularly. It is found that the cells grow rapidly, the morphology is typically fibroblast-like, and the cell viability is high, and the passage is stable. After multiple passages of culture, the cells still maintain good proliferation ability and stemness characteristics and can be used for subsequent cell therapy research.

[0031] Figure 1 It is a schematic flow chart of a method for preparing a universal culture medium for umbilical cord mesenchymal stem cells provided by an embodiment of the present invention; Figure 2 It is a schematic diagram of cell morphology under different culture media provided by an embodiment of the present invention; cell morphology under different culture media; the cells cultured with the culture medium of the present invention have better cell morphology and growth status; (1) cells cultured with the culture medium of the present invention; (2) cells cultured with a commercially available culture medium.

[0032] Figure 3 It is a schematic diagram of a series of flow cytometry detection results provided by an embodiment of the present invention; a series of flow cytometry detection results show that the expression of cell surface markers of the cells cultured with the culture medium of the present invention shows high specificity. The positive markers CD73, CD90, and CD105 show high expression (>95%), while the negative markers CD34, CD45, CD11b, HLA-DR, and CD19 show low expression or no expression (<2%). These results indicate that the culture medium of the present invention can effectively induce cells to differentiate into specific cell types and has high purity and specificity. This specific expression pattern conforms to the typical characteristics of mesenchymal stem cells and can precisely regulate the differentiation direction of cells during cell culture, avoiding the mixing of other non-target cell types.

[0033] Figure 4 It is a schematic diagram of cell growth curve comparison provided by an embodiment of the present invention; Take the 4th-generation cells with good growth, inoculate them into a 24-well plate at a concentration of 2×10 4 / ml, 1 ml per well, change the liquid every 2 days, count the number of cells in 3 wells every 24 hours starting from the 2nd day, and take the average value to draw a growth curve for 6 days. The growth curve shows the change in the number of cells of two groups of cells (A and B) at the same culture days. The cells in group A show higher proliferation activity, and their proliferation rate rises rapidly in the initial stage of culture and reaches the peak on the 3rd day, and then enters the plateau phase, maintaining a relatively stable cell number. The proliferation rate of the cells in group B is relatively slow, reaching the peak on the 4th day, and then gradually decreasing. The proliferation rate of the cells in group A is significantly higher than that of the cells in group B. Example

[0034] Using aseptic operation technique, mix RPMI medium and IMDM medium in a ratio of 1:3. After mixing, gently stir to ensure full fusion of the two media and avoid generating bubbles; The dosages of growth factors and recombinant proteins are as follows: Basic fibroblast growth factor (bFGF): 1 - 10 ng / mL Insulin-like growth factor (IGF-1): 5 - 20 ng / mL Platelet-derived growth factor (PDGF): 5 - 15 ng / mL Transforming growth factor-β (TGF-β): 1 - 5 ng / mL Epidermal growth factor (EGF): 5 - 10 ng / mL Hepatocyte growth factor (HGF): 5 - 15 ng / mL Granulocyte-macrophage colony-stimulating factor (GM-CSF): 1 - 5 ng / mL Stem cell factor (SCF): 5 - 10 ng / mL Recombinant human thrombopoietin (TPO): 5 - 10 ng / mL Recombinant human insulin: 1 - 10 μg / mL Recombinant human apo-transferrin: 5 - 10 μg / mL Recombinant human serum albumin: 1 - 5 mg / mL Trace elements: Ethanolamine: 1 - 5 μM Sodium selenite: 10 - 50 nM The addition steps are as follows: thaw all the added components at room temperature until completely melted, and gently shake and mix them.

[0035] Sequentially add the above-mentioned growth factors, recombinant proteins and trace elements into the basal medium. After the addition is completed, gently stir for 5 - 8 minutes to ensure uniform distribution. The stirring speed should not be too fast to avoid generating bubbles or protein denaturation. The preparation and addition process of the medium should be carried out at room temperature (20 - 25 °C) to avoid the influence of too high or too low temperature on the components. Filter the mixed medium through a 0.22 μm filter membrane to sterilize and ensure sterility. The filtered medium can be used directly or stored after being aliquoted.

[0036] The beneficial effects of the present invention are reflected in that in the serum-free medium adopted by the present invention, the replacement of serum components is mainly achieved by optimizing the medium formula. The optimized scheme can provide the nutrients and growth factors required for cell growth without relying on animal serum; the use of human recombinant growth factors to replace animal-derived components avoids the risks of pathogen contamination and immune reactions, and improves the safety and stability of cell culture; the optimized combination of growth factors and the concentration of extracellular matrix proteins promote the proliferation efficiency of umbilical cord MSCs; within the same culture time, the medium of the present invention can promote the proliferation of umbilical cord MSCs, accelerate the cell growth rate, and within the same culture time, the cell number increases by about 10% compared with the use of traditional medium; at the same time, it can effectively maintain the proliferation potential of cells and improve the proliferation efficiency; by precisely controlling the sources and ratios of each component, the quality of different batches of medium is highly consistent, reducing the experimental errors and clinical application risks caused by medium differences; the finally obtained cells have better ability to maintain stemness. By detecting the expression levels of cell surface markers such as CD73, CD90, CD105, etc., it is found that the cells cultured in this medium have a higher positive rate and a higher proliferation efficiency. These advantages make the medium have broad application prospects in the basic research of umbilical cord MSCs, the application of regenerative medicine, and the development of cell therapy products, providing strong support for the realization of the standardized and large-scale culture of umbilical cord MSCs.

[0037] The above are only the embodiments of the present invention, and the common specific technical solutions or characteristics and the like in the solution are not described in detail here; it should be pointed out that for those skilled in the art, without departing from the solution of the present invention, several deformations and improvements can be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the effects of the present invention and the practicability of the patent. The protection scope required by this application should be subject to the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A method for preparing a universal culture medium for umbilical cord mesenchymal stem cells, characterized in that, The method includes the following steps: S1: Mix RPMI medium and IMDM medium to form a basal medium. S2: Add growth factors and recombinant proteins to the basal medium and stir to obtain a first medium. S3: Add trace elements to the first medium after adding growth factors and recombinant proteins, and gently stir evenly to obtain a second medium. S4: Filter and sterilize the second medium at room temperature, then aliquot and store it to obtain a universal medium for umbilical cord mesenchymal stem cells.

2. The method for preparing a universal medium for umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The volume ratio of the RPMI medium to the IMDM medium is 1:

3.

3. The method for preparing a universal medium for umbilical cord mesenchymal stem cells according to claim 2, characterized in that: During the process of mixing the RPMI medium and the IMDM medium to cultivate the basal medium, gently stir to fully mix the RPMI medium and the IMDM medium and avoid generating bubbles.

4. The method for preparing a universal medium for umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The components of the growth factors include basic fibroblast growth factor, insulin-like growth factor, platelet-derived growth factor, transforming growth factor-β, epidermal growth factor, hepatocyte growth factor, granulocyte-macrophage colony-stimulating factor, and stem cell factor.

5. The preparation method of a universal medium for umbilical cord mesenchymal stem cells according to claim 4, characterized in that, The dosage ratio of the components of the growth factors is: basic fibroblast growth factor: insulin-like growth factor: platelet-derived growth factor: transforming growth factor-β: epidermal growth factor: hepatocyte growth factor: granulocyte-macrophage colony-stimulating factor: stem cell factor = 1 - 10:5 - 20:5 - 15:1 - 5:5 - 10:5 - 15:1 - 5:5 - 10.

6. The method for preparing a universal medium for umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The components of the recombinant proteins include recombinant human thrombopoietin, recombinant human insulin, recombinant human apo-transferrin, and recombinant human serum albumin.

7. A method for preparing a universal culture medium for umbilical cord mesenchymal stem cells according to claim 6, characterized in that, The dosage ratio of the components of the recombinant proteins is: recombinant human thrombopoietin: recombinant human insulin: recombinant human apo-transferrin: recombinant human serum albumin = 5 - 10::1 - 10:1 - 10:5 - 10:1 - 5.

8. The method for preparing a universal medium for umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The components of the trace elements include ethanolamine and sodium selenite; The dosage ratio of ethanolamine to sodium selenite is 1:

10.

9. The preparation method of a universal medium for umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The step of adding trace elements to the first medium after adding growth factors and recombinant proteins and gently stirring evenly to obtain a second medium includes: Add trace elements to the first culture medium after adding growth factors and recombinant proteins, and gently stir for 5 - 8 minutes to ensure uniform distribution, and carry out the operation at room temperature of 20 o C - 25 o °C to obtain the second culture medium.

10. A method for preparing a universal medium for umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The step of filtering and sterilizing the second medium at room temperature, then aliquoting and storing it to obtain a universal medium for umbilical cord mesenchymal stem cells includes: Filter and sterilize the second culture medium through a 0.22 μm filter membrane at room temperature, then aliquot and store it to obtain a universal culture medium for umbilical cord mesenchymal stem cells.

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