CN113801837A - Stem cell culture medium and preparation method thereof - Google Patents

Stem cell culture medium and preparation method thereof Download PDF

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Publication number
CN113801837A
CN113801837A CN202111069332.1A CN202111069332A CN113801837A CN 113801837 A CN113801837 A CN 113801837A CN 202111069332 A CN202111069332 A CN 202111069332A CN 113801837 A CN113801837 A CN 113801837A
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culture medium
growth factor
stem cell
mixture
cell culture
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付康
杨远方
田爱鹏
亢星亮
吴佳蔓
任新华
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Henan Intercell Biology Technology Co ltd
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Henan Intercell Biology Technology Co ltd
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Abstract

The invention relates to the technical field of cell culture media, in particular to a stem cell culture medium, which comprises a basal medium and an additive, wherein the basal medium is a DMEM/F12 liquid medium, and the additive components and the concentration thereof added into the basal medium are as follows based on the basal medium: 9-12 mu g/mL of transferrin, 1-3mg/mL of insulin, 2-4 mu g/mL of growth factor, 8-10mg/mL of vitamin C, 2-5mg/mL of folic acid, 1-3mg/mL of glucose, 6-8 mu g/mL of metal ions, 3-5 mu g/mL of calcium ions, 0.1-0.2mg/mL of streptomycin, 1-3mg/mL of fatty acid, 1-3mg/mL of amino acid and 1-3mg/mL of ethyl maltol. The stem cell culture medium has determined components, is convenient for batch production and standardization, does not add animal serum, can avoid the heterozygote in the animal serum, can improve the stability of cell growth on acid and heat, can ensure that the cell growth is continuously and stably released in the culture medium, and ensures that the cell is stably amplified.

Description

Stem cell culture medium and preparation method thereof
Technical Field
The invention relates to the technical field of cell culture media, in particular to a stem cell culture medium and a preparation method thereof.
Background
Stem cells do not survive in simple basal media, and certain trace nutrients and growth factors must be provided in cell culture to allow the cells to grow and maintain their growth state. The basal medium used is therefore often supplemented with serum, such as horse serum or fetal bovine serum; because serum is an extremely complex mixture of many biomolecules of different sizes, it provides growth factors, hormones, binding proteins, and provides protection for cells cultured in vitro; moreover, the serum is rich in mitotic factors, and is also beneficial to cell lines and primary culture.
However, the serum culture medium has certain disadvantages, so that the xenobiotics in animal serum cannot be avoided, and human xenobiotics rejection and conflict can exist if cells cultured by using the animal serum culture medium. Especially in the process of stem cell culture, a large amount of complex proteins in serum bring difficulties to the standardization of cell culture, wherein the complex proteins and multiple cytokines can cause the stem cells which are easy to differentiate to generate a plurality of completely different cell phenotypes, thereby generating a plurality of completely different cell differentiation trends, and also bringing great difficulties to the separation and purification of cell culture expression products and the repeatability of results.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a stem cell culture medium and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a stem cell culture medium comprises a matrix culture medium and additives, wherein the base culture medium is a DMEM/F12 liquid culture medium, and the additives are added into the matrix culture medium according to the matrix culture medium, and the concentrations of the additives are as follows: 9-12 mu g/mL of transferrin, 1-3mg/mL of insulin, 2-4 mu g/mL of growth factor, 8-10mg/mL of vitamin C, 2-5mg/mL of folic acid, 1-3mg/mL of glucose, 6-8 mu g/mL of metal ions, 3-5 mu g/mL of calcium ions, 0.1-0.2mg/mL of streptomycin, 1-3mg/mL of fatty acid, 1-3mg/mL of amino acid and 1-3mg/mL of ethyl maltol.
Preferably, the growth factors comprise fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor to the epidermal growth factor to the stem cell growth factor is 4:3: 3.
Preferably, the metal ions include equal amounts of iron ions, sodium ions, potassium ions, zinc ions, and magnesium ions.
A preparation method of a stem cell culture medium comprises the following steps:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 20-40min, then adding growth factors, stirring for 10-15min again, finally adding streptomycin, stirring for 10-15min, and standing for 3-4h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 20-30min, and standing for 1-2h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 6-8% into the mixture C, and adjusting the pH of the mixture C to 7.0-7.4;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 110-120 ℃, the sterilization time is 10-15min, and the stem cell culture medium is obtained after sterilization.
Preferably, the metal ion solution in S3 includes an iron ion solution, a sodium ion solution, a potassium ion solution, a zinc ion solution, and a magnesium ion solution in equal amounts.
Preferably, the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
Preferably, a 7% sodium carbonate solution is added to the mixture C in S5, and the pH of the mixture C is adjusted to 7.2.
The invention has the beneficial effects that:
the stem cell culture medium has determined components, is convenient for batch production and standardization, does not add animal serum, can avoid the heterozygote in the animal serum, can improve the stability of cell growth on acid and heat, can ensure that the cell growth is continuously and stably released in the culture medium, and ensures that the cell is stably amplified.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
In example 1
A stem cell culture medium and a preparation method thereof comprise a matrix culture medium and additives, wherein the matrix culture medium is a DMEM/F12 liquid culture medium, and the additives and the concentrations thereof added into the matrix culture medium are as follows: 9. mu.g/mL transferrin, 1mg/mL insulin, 2. mu.g/mL growth factor, 8mg/mL vitamin C, 2mg/mL folic acid, 1mg/mL glucose, 6. mu.g/mL metal ions, 3. mu.g/mL calcium ions, 0.1mg/mL streptomycin, 1mg/mL fatty acids, 1mg/mL amino acids, and 1mg/mL ethyl maltol.
Furthermore, the growth factors comprise fibroblast growth factors, epidermal growth factors and stem cell growth factors, and the concentration ratio of the fibroblast growth factors to the epidermal growth factors to the stem cell growth factors is 4:3: 3.
Further, the metal ions include iron ions, sodium ions, potassium ions, zinc ions, and magnesium ions in equal amounts.
A preparation method of a stem cell culture medium comprises the following steps:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 20-40min, then adding growth factors, stirring again for 10min, finally adding streptomycin, stirring for 10min, and standing for 3h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 20min, and standing for 1h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 6-8% into the mixture C, and adjusting the pH of the mixture C to 7.0;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 110 ℃, the sterilization time is 10min, and the stem cell culture medium is obtained after sterilization.
Further, the metal ion solution in S3 includes an iron ion solution, a sodium ion solution, a potassium ion solution, a zinc ion solution, and a magnesium ion solution in equal amounts.
Further, the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
In example 2
A stem cell culture medium and a preparation method thereof comprise a matrix culture medium and additives, wherein the matrix culture medium is a DMEM/F12 liquid culture medium, and the additives and the concentrations thereof added into the matrix culture medium are as follows: 10. mu.g/mL transferrin, 2mg/mL insulin, 3. mu.g/mL growth factor, 9mg/mL vitamin C, 3mg/mL folic acid, 2mg/mL glucose, 7. mu.g/mL metal ions, 4. mu.g/mL calcium ions, 0.15mg/mL streptomycin, 2mg/mL fatty acids, 2mg/mL amino acids, and 2mg/mL ethyl maltol.
Furthermore, the growth factors comprise fibroblast growth factors, epidermal growth factors and stem cell growth factors, and the concentration ratio of the fibroblast growth factors to the epidermal growth factors to the stem cell growth factors is 4:3: 3.
Further, the metal ions include iron ions, sodium ions, potassium ions, zinc ions, and magnesium ions in equal amounts.
A preparation method of a stem cell culture medium comprises the following steps:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 30min, then adding growth factors, stirring again for 12min, finally adding streptomycin, stirring for 12min, and standing for 3.5h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 25min, and standing for 1.5h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 7% into the mixture C, and adjusting the pH of the mixture C to 7.2;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 115 ℃, the sterilization time is 12min, and the stem cell culture medium is obtained after sterilization.
Further, the metal ion solution in S3 includes an iron ion solution, a sodium ion solution, a potassium ion solution, a zinc ion solution, and a magnesium ion solution in equal amounts.
Further, the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
In example 3
A stem cell culture medium and a preparation method thereof comprise a matrix culture medium and additives, wherein the matrix culture medium is a DMEM/F12 liquid culture medium, and the additives and the concentrations thereof added into the matrix culture medium are as follows: transferrin at 12. mu.g/mL, insulin at 3mg/mL, growth factor at 4. mu.g/mL, vitamin C at 10mg/mL, folic acid at 5mg/mL, glucose at 3mg/mL, metal ions at 8. mu.g/mL, calcium ions at 5. mu.g/mL, streptomycin at 0.2mg/mL, fatty acid at 3mg/mL, amino acid at 3mg/mL, and ethyl maltol at 3 mg/mL.
Furthermore, the growth factors comprise fibroblast growth factors, epidermal growth factors and stem cell growth factors, and the concentration ratio of the fibroblast growth factors to the epidermal growth factors to the stem cell growth factors is 4:3: 3.
Further, the metal ions include iron ions, sodium ions, potassium ions, zinc ions, and magnesium ions in equal amounts.
A preparation method of a stem cell culture medium comprises the following steps:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 40min, then adding growth factors, stirring again for 15min, finally adding streptomycin, stirring for 15min, and standing for 4h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 30min, and standing for 2h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 6-8% into the mixture C, and adjusting the pH of the mixture C to 7.4;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 120 ℃, the sterilization time is 15min, and the stem cell culture medium is obtained after sterilization.
Further, the metal ion solution in S3 includes an iron ion solution, a sodium ion solution, a potassium ion solution, a zinc ion solution, and a magnesium ion solution in equal amounts.
Further, the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
Detection of bacteria, fungi, pH indicators according to GMP standards, as shown in table 1:
bacteria Fungi pH
Example 1 Negative of Negative of 7.0
Example 2 Negative of Negative of 7.2
Example 3 Negative of Negative of 7.4
The culture media prepared in examples 1-3 were tested to meet the criteria.
The cell expansion fold of the medium in examples 1-3 was monitored for three weeks as shown in table 2:
first week Second week The third week
Example 1 144.24 1356.92 6358.57
Example 2 143.85 1349.84 6299.75
Example 3 145.17 1360.77 6433.43
According to the monitoring of cell expansion in the culture media of examples 1-3, the culture media of the present invention allow the cells to grow and be released continuously and smoothly in the culture media, and allow the cells to be expanded smoothly.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (7)

1. A stem cell culture medium comprises a matrix culture medium and additives, and is characterized in that the matrix culture medium is a DMEM/F12 liquid culture medium, and the additives are added into the matrix culture medium according to the matrix culture medium, wherein the additives comprise the following components in concentration: 9-12 mu g/mL of transferrin, 1-3mg/mL of insulin, 2-4 mu g/mL of growth factor, 8-10mg/mL of vitamin C, 2-5mg/mL of folic acid, 1-3mg/mL of glucose, 6-8 mu g/mL of metal ions, 3-5 mu g/mL of calcium ions, 0.1-0.2mg/mL of streptomycin, 1-3mg/mL of fatty acid, 1-3mg/mL of amino acid and 1-3mg/mL of ethyl maltol.
2. The stem cell culture medium according to claim 1, wherein the growth factors comprise fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor to the epidermal growth factor to the stem cell growth factor is 4:3: 3.
3. The stem cell culture medium of claim 1, wherein the metal ions comprise equal amounts of iron, sodium, potassium, zinc and magnesium ions.
4. A method of preparing the stem cell culture medium of claim 1, comprising the steps of:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 20-40min, then adding growth factors, stirring for 10-15min again, finally adding streptomycin, stirring for 10-15min, and standing for 3-4h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 20-30min, and standing for 1-2h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 6-8% into the mixture C, and adjusting the pH of the mixture C to 7.0-7.4;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 110-120 ℃, the sterilization time is 10-15min, and the stem cell culture medium is obtained after sterilization.
5. The method according to claim 4, wherein the metal ion solution in S3 includes equal amounts of iron ion solution, sodium ion solution, potassium ion solution, zinc ion solution and magnesium ion solution.
6. The method according to claim 4, wherein the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
7. The method for preparing the stem cell culture medium according to claim 4, wherein a 7% sodium carbonate solution is added to the mixture C in the S5 by mass fraction, and the pH of the mixture C is adjusted to 7.2.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN106434549A (en) * 2016-12-20 2017-02-22 江西宜信堂医疗科技有限公司 Serum-free stem cell culture medium and preparation method thereof
CN107043699A (en) * 2017-04-25 2017-08-15 徐子雁 A kind of kit of low-energy laser inducing mesenchymal stem cell vascularization
CN112313329A (en) * 2018-06-27 2021-02-02 味之素株式会社 Additive for stem cell culture, culture medium for stem cell culture, and culture method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434549A (en) * 2016-12-20 2017-02-22 江西宜信堂医疗科技有限公司 Serum-free stem cell culture medium and preparation method thereof
CN107043699A (en) * 2017-04-25 2017-08-15 徐子雁 A kind of kit of low-energy laser inducing mesenchymal stem cell vascularization
CN112313329A (en) * 2018-06-27 2021-02-02 味之素株式会社 Additive for stem cell culture, culture medium for stem cell culture, and culture method

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