CN113234662A - Stem cell serum-free culture medium and preparation method and application thereof - Google Patents
Stem cell serum-free culture medium and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a stem cell serum-free medium and a preparation method and application thereof, relating to the technical field of biology. The stem cell serum-free culture medium provided by the invention consists of a basic culture medium and additive components, wherein the additive components consist of platelet lysate, transferrin and human insulin.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a stem cell serum-free culture medium and a preparation method and application thereof.
Background
Stem cells are defined as a population of cells that remain undifferentiated and naive during development of the body. During in vitro culture, stem cells are constantly regenerating and, under appropriate conditions, are capable of committed differentiation to a variety of tissue cell types. The regenerative capacity of certain tissues in the body is directly related to the role of stem cells. In general, stem cells can be classified into embryonic stem cells, induced pluripotent stem cells and adult stem cells 3 according to their development.
Stem cells, which are commonly used in regenerative medicine, are low in content in normal adult tissues and require amplification and culture in vitro, and thus, the in vitro amplification of stem cells is a necessary condition for promoting clinical research and application thereof. Factors influencing the in vitro expansion of stem cells include a culture mode and a culture medium, wherein in the in vitro culture process, the culture mode meeting the requirements of dynamic drug production management protocol (cGMP) is adopted, and a serum-free culture medium with definite components is used.
Currently, in vitro expansion of stem cells mainly uses a medium containing a certain proportion of Fetal Bovine Serum (FBS). FBS is an indispensable component for in-vitro amplification of various cells, contains a large amount of proteins, hormones, growth factors, adhesion-promoting factors and the like, and provides support for the survival of the cells in vitro. Clinical interstage mesenchymal stem cells need to achieve sufficient purity and meet quality standards of sterility, no foreign substances, no endotoxin and no pathogens; on the premise of meeting the quantity requirement, the biological characteristics of the stem cells need to be maintained. However, the ingredients of FBS are still unclear, the quality is closely related to the age, sex, health degree and the like of donors, and the content difference of substances in different batches is large, so that the quality of cultured cells is unstable, heterologous proteins remain, and the clinical application is at a high risk of allogeneic immune rejection. For the reasons, the use of FBS may result in that mesenchymal stem cell-related products cannot meet clinical treatment requirements. Therefore, in order to promote the development of stem cell in vitro culture technology and adapt to the clinical application requirements of stem cells, the development of a culture medium without animal-derived serum for stem cells is extremely important.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the present invention is to provide a serum-free culture medium for stem cells, which solves at least one of the above problems.
The second purpose of the invention is to provide a preparation method of the stem cell serum-free medium, which is simple and convenient and has low cost.
The third purpose of the invention is to provide an application in stem cell culture.
The fourth object of the present invention is to provide a method for culturing stem cells.
In a first aspect, the invention provides a serum-free culture medium for stem cells, which consists of a basic culture medium and an additive component;
the additive components consist of platelet lysate, transferrin and human insulin.
As a further technical aspect, the transferrin comprises recombinant transferrin;
the human insulin includes recombinant human insulin.
As a further technical scheme, the volume percentage of the platelet lysate in the stem cell serum-free medium is 5-10%.
As a further technical scheme, the concentration of transferrin in the serum-free culture medium of the stem cells is 5 to 100 mu g/mL.
As a further technical scheme, the concentration of human insulin in the stem cell serum-free culture medium is 5 to 100 mu g/mL.
As a further technical scheme, the basic culture medium comprises an alpha-MEM culture medium.
In a second aspect, the present invention provides a method for preparing a serum-free culture medium for stem cells, comprising the following steps: adding the platelet lysate, the transferrin solution and the human insulin solution into a basic culture medium to prepare the stem cell serum-free culture medium.
As a further technical scheme, the method also comprises the step of filtering the transferrin solution and the human insulin solution before adding the transferrin solution and the human insulin solution into a basic culture medium;
the filtration comprises filtration by a filter membrane of 0.20-0.25 μm.
In a third aspect, the invention provides an application of a stem cell serum-free medium in stem cell culture.
In a fourth aspect, the present invention provides a method for culturing stem cells, comprising the steps of:
culturing the stem cells in a stem cell serum-free culture medium;
the culture conditions at least satisfy one of the following conditions: the culture medium is replaced every 1-3 days at the temperature of 36-38 ℃ and the carbon dioxide concentration of 5%.
Compared with the prior art, the invention has the following beneficial effects:
the serum-free culture medium for the stem cells, provided by the invention, consists of a basic culture medium and an additive component, wherein the additive component consists of platelet lysate, transferrin and human insulin, and the basic culture medium provides basic nutrients for the stem cells; the platelet lysate contains rich growth factors and can replace bovine serum, so that the risk of introducing heterologous serum to carry bacteria and viruses is avoided; transferrin can avoid the generation of free radicals, promote the storage and transportation of extracellular iron, and protect and maintain the in vitro growth and proliferation of cells; human insulin promotes cell growth and metabolism. The invention has definite components and low cost, all the components are matched with each other, the proliferation of stem cells can be promoted, the in vitro culture speed is improved, and the invention has no serum addition and high safety, avoids adverse factors brought by a serum-containing culture medium, and is suitable for clinical application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a diagram showing the growth state of stem cells after culture using the culture media of examples 1 to 5 and comparative example 1;
FIG. 2 is a graph showing the expression rate of surface markers of stem cells (exemplified by dental pulp stem cells SHED, the same applies below) after culturing in the medium of comparative example 1;
FIG. 3 is a graph showing the expression rate of a marker on the surface of stem cells after culturing in the medium of example 1;
FIG. 4 is a graph showing the expression rate of a marker on the surface of stem cells after culturing in the medium of example 2;
FIG. 5 is a graph showing the expression rate of a marker on the surface of stem cells after culturing in the medium of example 3;
FIG. 6 is a graph showing the expression rate of a marker on the surface of stem cells after culturing in the medium of example 4;
FIG. 7 is a graph showing the expression rate of a marker on the surface of stem cells after culturing in the medium of example 5;
FIG. 8 shows the expression of NANOG sternness gene of stem cells after culture in different media as provided in Experimental example 4;
FIG. 9 shows the osteogenic adipogenic effect of stem cells cultured in different media as provided in test example 5;
FIG. 10 shows the expression of osteogenic lipid-associated genes in stem cells cultured in different media as provided in test example 5.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the invention provides a serum-free culture medium for stem cells, which consists of a basic culture medium and an additive component;
the additive components consist of platelet lysate, transferrin and human insulin.
In the present invention, the basic culture medium contains basic nutrients necessary for maintaining the growth of cells; the additive component is a substance with the characteristics of promoting cell growth and proliferation and maintaining cell function, and consists of platelet lysate, transferrin and human insulin. Wherein the basal medium provides basic nutrients for the stem cells; the platelet lysate contains rich growth factors, can replace fetal calf serum, and avoids the risk of introducing heterologous serum to carry bacteria and viruses; transferrin can avoid the generation of free radicals, promote the storage and transportation of extracellular iron, and protect and maintain the in vitro growth and proliferation of cells; human insulin promotes cell growth and metabolism. The invention has definite components and low cost, all the components are matched with each other, the proliferation of stem cells can be promoted, the in vitro culture speed is improved, and the invention has no serum addition and high safety, avoids adverse factors brought by a serum-containing culture medium, and is suitable for clinical application.
As a further technical aspect, the transferrin comprises recombinant transferrin. The recombinant transferrin completely avoids the possibility of pathogen contamination and provides good nutritional effect for the in vitro proliferation of cells.
The human insulin includes recombinant human insulin.
The brand of the products of platelet lysate, recombinant transferrin and recombinant human insulin is not specifically limited by the present invention, for example, platelet lysate can be selected from: brand name: BI, cargo number: PLTGOLD 010R; recombinant transferrin can be selected as: brand name: sigma, cat number T8158; recombinant human insulin may be selected as: brand name: solibao, cat # I8830.
In a further embodiment, the volume percentage of the platelet lysate in the stem cell serum-free medium is 5% to 10%, for example, but not limited to, 5%, 6%, 7%, 8%, 9% or 10%, preferably 5%.
In a further embodiment, the transferrin concentration in the serum-free medium of the stem cells is 5 to 100. mu.g/mL, but is not limited to, for example, 5, 6, 7, 8, 9 or 100. mu.g/mL, preferably 5. mu.g/mL.
As a further embodiment, the concentration of human insulin in the stem cell serum-free medium is 5. mu.g/mL-100. mu.g/mL, and may be, for example, but not limited to, 5. mu.g/mL, 6. mu.g/mL, 7. mu.g/mL, 8. mu.g/mL, 9. mu.g/mL, or 100. mu.g/mL, preferably 5. mu.g/mL.
In the invention, the contents of platelet lysate, transferrin and human insulin in the serum-free culture medium of the stem cells are further optimized and adjusted, so that the stem cells can grow better, and the in vitro culture efficiency is improved.
As a further embodiment, the basal medium includes, but is not limited to, alpha-MEM medium, or other suitable basal medium for animal cell culture known to those skilled in the art, preferably alpha-MEM medium.
In a second aspect, the present invention provides a method for preparing a serum-free culture medium for stem cells, comprising the following steps: adding the platelet lysate, the transferrin solution and the human insulin solution into a basic culture medium to prepare the stem cell serum-free culture medium.
The preparation method of the stem cell serum-free medium provided by the invention is simple and convenient.
As a further technical scheme, before the transferrin solution and the human insulin solution are added into the basic culture medium, the method also comprises the step of filtering the transferrin solution and the human insulin solution so as to remove insoluble substances in the transferrin solution and the human insulin solution and obtain a solution with uniform dissolution.
The filtration comprises the filtration by adopting a filter membrane of 0.20-0.25 μm;
preferably, filtration is carried out using a 0.22 μm filter.
In a third aspect, the invention provides an application of a stem cell serum-free medium in stem cell culture.
The serum-free culture medium for the stem cells, provided by the invention, has the advantages of definite components, low cost, high safety, capability of being used for culturing the stem cells and good culture effect.
In a fourth aspect, the present invention provides a method for culturing stem cells, comprising the steps of:
culturing the stem cells in a stem cell serum-free culture medium;
the culture conditions at least satisfy one of the following conditions: the culture medium is replaced every 1-3 days at the temperature of 36-38 ℃ and the carbon dioxide concentration of 5%.
Preferably, the conditions of the culture are: the culture medium was changed every 2 days at 37 ℃ and 5% carbon dioxide concentration.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
It should be noted that, unless otherwise specified, the platelet lysate used in the following examples is: brand name: BI, cargo number: PLTGOLD 010R; the recombinant transferrin is: brand name: sigma, cat number T8158; recombinant human insulin is: brand name: solibao, cat # I8830.
Example 1
The serum-free culture medium for the stem cells comprises an alpha-MEM culture medium and additive components, wherein the additive components comprise platelet lysate, recombinant transferrin and recombinant human insulin, the volume percentage of the platelet lysate in the serum-free culture medium for the stem cells is 5%, the concentration of the recombinant transferrin is 5 mug/mL, and the concentration of the recombinant human insulin is 5 mug/mL.
The preparation method comprises the following steps:
dissolving the recombinant human insulin to the concentration of 1mg/mL by using 10mM hydrochloric acid, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 5 mu g/mL; dissolving the recombinant transferrin to the concentration of 1mg/mL by using sterile water, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 5 mu g/mL; adding platelet lysate to the culture medium, wherein the volume ratio of the platelet lysate to the culture medium is 5: 95.
Example 2
The serum-free culture medium for the stem cells comprises an alpha-MEM culture medium and additive components, wherein the additive components comprise platelet lysate, recombinant transferrin and recombinant human insulin, the volume percentage of the platelet lysate in the serum-free culture medium for the stem cells is 5%, the concentration of the recombinant transferrin is 10 mug/mL, and the concentration of the recombinant human insulin is 10 mug/mL.
The preparation method comprises the following steps:
dissolving the recombinant human insulin to the concentration of 1mg/mL by using 10mM hydrochloric acid, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 10 mu g/mL; dissolving the recombinant transferrin to the concentration of 1mg/mL by using sterile water, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 10 mu g/mL; adding platelet lysate to the culture medium, wherein the volume ratio of the platelet lysate to the culture medium is 5: 95.
Example 3
The serum-free culture medium for the stem cells comprises an alpha-MEM culture medium and additive components, wherein the additive components comprise platelet lysate, recombinant transferrin and recombinant human insulin, the volume percentage of the platelet lysate in the serum-free culture medium for the stem cells is 5%, the concentration of the recombinant transferrin is 15 mug/mL, and the concentration of the recombinant human insulin is 15 mug/mL.
The preparation method comprises the following steps:
dissolving the recombinant human insulin to the concentration of 1mg/mL by using 10mM hydrochloric acid, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 15 mu g/mL; dissolving the recombinant transferrin to the concentration of 1mg/mL by using sterile water, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 15 mu g/mL; adding platelet lysate to the culture medium, wherein the volume ratio of the platelet lysate to the culture medium is 5: 95.
Example 4
The serum-free culture medium for the stem cells comprises an alpha-MEM culture medium and additive components, wherein the additive components comprise platelet lysate, recombinant transferrin and recombinant human insulin, the volume percentage of the platelet lysate in the serum-free culture medium for the stem cells is 5%, the concentration of the recombinant transferrin is 20 mug/mL, and the concentration of the recombinant human insulin is 20 mug/mL.
The preparation method comprises the following steps:
dissolving the recombinant human insulin to the concentration of 1mg/mL by using 10mM hydrochloric acid, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 20 mu g/mL; dissolving the recombinant transferrin to the concentration of 1mg/mL by using sterile water, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 20 mu g/mL; adding platelet lysate to the culture medium, wherein the volume ratio of the platelet lysate to the culture medium is 5: 95.
Example 5
The serum-free culture medium for the stem cells comprises an alpha-MEM culture medium and additive components, wherein the additive components comprise platelet lysate, recombinant transferrin and recombinant human insulin, the volume percentage of the platelet lysate in the serum-free culture medium for the stem cells is 5%, the concentration of the recombinant transferrin is 100 mu g/mL, and the concentration of the recombinant human insulin is 100 mu g/mL.
The preparation method comprises the following steps:
dissolving the recombinant human insulin to the concentration of 1mg/mL by using 10mM hydrochloric acid, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 100 mu g/mL; dissolving the recombinant transferrin to the concentration of 1mg/mL by using sterile water, filtering the solution by using a 0.22 micron filter membrane, and adding the solution into a basic culture medium according to the final concentration of 100 mu g/mL; adding platelet lysate to the culture medium, wherein the volume ratio of the platelet lysate to the culture medium is 5: 95.
Comparative example 1
A conventional commercial serum-free culture medium for certain brand of stem cells.
Test example 1 morphological Observation of cells
Cell recovery and culture: a15 mL centrifuge tube was prepared and 10mL of media was added to the tube. Taking out the cells from the liquid nitrogen tank, and quickly putting the cells into a 37 ℃ water bath kettle for warm bath for 1 min. After the freezing tube is sprayed with alcohol, the freezing tube is placed on a workbench, and all liquid in the freezing tube is transferred into a 15mL centrifuge tube filled with a culture medium. Placing in a centrifuge, centrifuging at 4 ℃ and 1000rpm for 5min, aspirating the medium from the centrifuge tube, adding 1mL of the medium into the centrifuge tube to resuspend the cells, seeding the stem cells in a dish, and culturing in the serum-free medium provided in examples 1-5 of the invention and the commercially available serum-free medium provided in comparative example 1, respectively.
And (3) morphological observation: at 24h after cell passage, the culture dish was removed from the incubator, placed under an inverted microscope for observation and photographed for recording, and the results are shown in fig. 1.
As can be seen from FIG. 1, the stem cells in the serum-free medium of the present invention have no difference in cell growth from the commercially available serum-free medium after culture, and the stem cells are in adherent growth under a microscope and show elongated fibroblasts.
Test example 2 cell Activity assay
In order to observe the effect of cells on the proliferation potency of cells after they passed through the medium of the present invention, the proliferation status of cells was examined using CCK-8. The experimental procedure was as follows:
1) culturing the cells until the fusion rate reaches more than 80%, and after digestion, according to about 10%4cells/mL were inoculated in 100. mu.L to 96-well plates, 6 wells in duplicate, and after 48h, CCK8 was added and incubated for 1h, followed by OD detection at 450 nm.
2) Calculating the relative proliferation rate of the cells by the following method:
relative proliferation rate is cell OD value/comparative cell OD value.
The results are shown in the table below, where the medium of the invention promotes the proliferation of stem cells compared to commercially available serum-free medium, and the formulation of the invention of example 5 is the best proliferation-promoting combination.
| Experimental group | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 | Comparative example |
| Relative increment rate | 121.62％ | 128.99％ | 132.42％ | 130.04％ | 148.82％ | 100.00％ |
Test example 3 detection of cell surface markers by flow cytometry
To understand whether the culture medium of the present invention affects the characteristics of the immunophenotype of stem cells, changes in immunophenotype of stem cells cultured in different formulations were examined by flow cytometry. The experimental procedure was as follows:
1) the stem cells cultured in test example 2 and having a confluency of 80% or more were digested and then cultured at a ratio of 0.5 to 1.0X 106The cells were harvested for each cell mass.
2) Labeling the flow tube according to the sample type, and adding the cells to the tube according to the label, respectively.
3) Add 2mL of room temperature PBS to the flow tube.
4) 1200rpm at ambient temperature for 5 min.
5) The supernatant was discarded and 100. mu.L of room temperature PBS was added to resuspend the cell pellet.
6) Labeling was performed by adding CD34, CD45, CD73, CD90, CD105 and HLA-DR, respectively, with a fluorescent label. The labeling condition is incubation for 25-30min at 2-8 ℃.
7) After incubation was complete, PBS was added to each flow tube for washing, followed by centrifugation at 1200rpm for 5min at 4 ℃. Finally, resuspend with 300. mu.L PBS, and test on the machine.
The test results are shown in the following table. The stem cells showed negative expression of CD34, CD45 and HLA-DR, while CD73, CD90 and CD105 showed positive expression. In addition, as shown in FIGS. 2 to 7, in both the examples and the comparative examples, the expression of the negative marker was 1% or less, and the positive rate of the positive marker was 98% or more.
| Marker substance | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 | Comparative example |
| CD34 | 0.43％ | 0.22％ | 0.22％ | 0.00％ | 0.00％ | 0.04％ |
| CD45 | 0.02％ | 0.09％ | 0.02％ | 0.10％ | 0.18％ | 0.32％ |
| CD73 | 99.91％ | 99.96％ | 100.00％ | 99.84％ | 98.59％ | 99.86％ |
| CD90 | 99.79％ | 99.81％ | 99.92％ | 99.75％ | 98.23％ | 99.40％ |
| CD105 | 99.79％ | 99.89％ | 100.00％ | 99.80％ | 98.29％ | 100.00％ |
| HLA-DR | 0.00％ | 0.02％ | 0.02％ | 0.00％ | 0.04％ | 0.28％ |
Test example 4 detection of Stem cell Stem
In this test example, the stem cells were cultured in 2 different media (the medium provided in example 1 and the medium provided in comparative example 1), wherein the medium containing platelet lysate (the medium of example 1) was designated as the experimental group and the conventional medium (the medium of comparative example 1) was designated as the control group. After stem cell culture, total RNA was extracted by Trizol method and reverse-transcribed into cDNA at 37 ℃, and expression of NANOG gene was detected by real-time fluorescent quantitative PCR (qPCR).
The test results are shown in fig. 8, and the culture of the stem cells in the platelet lysate-containing medium can promote the high expression of the sternness gene NANOG compared with the culture of the stem cells in the conventional medium.
Test example 5 osteogenic and adipogenic differentiation of Stem cells and detection of related genes
The present example was grouped as in test example 4. Osteogenic and adipogenic differentiation of stem cells was performed in 12-well plates. The stem cells cultured in 2 different ways were inoculated, and cultured in osteogenic induction medium and adipogenic induction medium, respectively. Fluid changes are carried out every 3 days, and staining observation of alizarin red and oil red O is carried out at 21 days respectively to determine the osteogenic differentiation and adipogenic differentiation capacities of stem cells. In addition, in order to compare the osteogenic capacity and the adipogenic capacity of 2 different culture modes, total RNA was extracted and the expression of osteogenic related genes ALP, OSTEOPONTIN, Runx2 and adipogenic related genes PPAR γ 2, LPL was examined by qPCR.
As shown in fig. 9 and 10, in fig. 9, the first and third line photographs are macro photographs of the well plate, the second and fourth line photographs are micro photographs under an optical microscope corresponding to the well plate, the macro photographs show the overall situation, and the micro photographs observe the calcium deposition and the oil staining, and it can be seen from fig. 9 that the stem cells of both media can be stained with alizarin red and oil red O after osteogenic differentiation and adipogenic differentiation induction. As shown in FIG. 10, the gene expression was examined by qPCR, and the results showed that the stem cells cultured in the platelet lysate-containing medium could enhance the expression of the osteogenic gene OSTEOPONTIN and the adipogenic genes PPAR γ 2 and LPL.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A serum-free culture medium for stem cells is characterized by consisting of a basic culture medium and an additive component;
the additive components consist of platelet lysate, transferrin and human insulin.
2. The stem cell serum-free medium of claim 1, wherein the transferrin comprises recombinant transferrin;
the human insulin includes recombinant human insulin.
3. The stem cell serum-free medium according to claim 1, wherein the volume percentage of the platelet lysate in the stem cell serum-free medium is 5-10%.
4. The stem cell serum-free medium according to claim 1, wherein the concentration of transferrin in the stem cell serum-free medium is 5 μ g/mL to 100 μ g/mL.
5. The stem cell serum-free medium according to claim 1, wherein the concentration of human insulin in the stem cell serum-free medium is 5 μ g/mL to 100 μ g/mL.
6. The stem cell serum-free medium according to claim 1, wherein the basal medium comprises α -MEM medium.
7. The method for preparing the serum-free culture medium for the stem cells according to any one of claims 1 to 6, which comprises the following steps: adding the platelet lysate, the transferrin solution and the human insulin solution into a basic culture medium to prepare the stem cell serum-free culture medium.
8. The method of claim 7, further comprising the step of filtering the transferrin solution and the human insulin solution before adding the transferrin solution and the human insulin solution to the basal medium;
the filtration comprises filtration by a filter membrane of 0.20-0.25 μm.
9. Use of the serum-free culture medium for stem cells according to any one of claims 1 to 6 or prepared by the preparation method according to claim 7 or 8 in stem cell culture.
10. A method for culturing stem cells, comprising the steps of:
culturing stem cells in a serum-free culture medium for stem cells according to any one of claims 1 to 6;
the culture conditions at least satisfy one of the following conditions: the culture medium is replaced every 1-3 days at the temperature of 36-38 ℃ and the carbon dioxide concentration of 5%.
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