CN113564111B - Method for culturing umbilical cord-derived mesenchymal stem cells in low-oxygen mode - Google Patents

Method for culturing umbilical cord-derived mesenchymal stem cells in low-oxygen mode Download PDF

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CN113564111B
CN113564111B CN202110965758.9A CN202110965758A CN113564111B CN 113564111 B CN113564111 B CN 113564111B CN 202110965758 A CN202110965758 A CN 202110965758A CN 113564111 B CN113564111 B CN 113564111B
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stem cells
mesenchymal stem
umbilical cord
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CN113564111A (en
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李冉
高翔
李纬国
何泳恒
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Shanghai Taiyao Biomedical Co ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Abstract

The application relates to the technical field of mesenchymal stem cell culture methods, and particularly discloses a method for culturing umbilical cord-derived mesenchymal stem cells in a low-oxygen manner. The method comprises the following steps: inoculating primary umbilical cord source mesenchymal stem cells on a culture medium, placing the culture medium at a culture temperature of 37 ℃, placing the culture medium in an environment with an oxygen volume fraction of 1-3%, a carbon dioxide volume fraction of 2-2.5% and the balance of nitrogen for carrying out passage expansion for 96-108 hours, and collecting the mesenchymal stem cells; the culture medium comprises a basal culture medium and a passage additive; the basal medium is one or more of MEM, F12, low-sugar DMEM and high-sugar DMEM; the passage additive comprises 11-15 mu g/ml of glutathione, 0.5-2.5 mu g/ml of alpha-tocopherol, 0.5-2.0 mu g/ml of beta-tocopherol, 2.5-3.0ng/ml of acetazolamide, 10-20 mu g/ml of insulin and 8-10 mu g/ml of vitamin C based on the final concentration of the culture medium. The method can be used for carrying out amplification culture on the umbilical cord-derived mesenchymal stem cells, and the stem cells obtained by culture have high activity and multiplication factor, thereby being beneficial to popularization and application of biological products of the umbilical cord mesenchymal stem cells.

Description

Method for culturing umbilical cord-derived mesenchymal stem cells in low-oxygen mode
Technical Field
The application relates to the technical field of mesenchymal stem cell culture methods, in particular to a method for culturing umbilical cord-derived mesenchymal stem cells in a low-oxygen manner.
Background
Mesenchymal stem cells (mesenchymal stem cells, MSCs) are pluripotent stem cells of mesodermal origin with high self-renewal capacity and multipotent differentiation potential. At present, mesenchymal stem cells are mainly extracted from umbilical cords, bone marrow, adipose tissues, dental pulp and placenta, and have multidirectional differentiation potential after continuous subculture and cryopreservation, so that a new approach is provided for clinically treating various diseases such as nervous system diseases, nephropathy, autoimmune diseases, malignant tumors and the like.
The umbilical cord-derived mesenchymal stem cells refer to multifunctional stem cells existing in neonatal umbilical cord tissues, and have the characteristics of convenience in material acquisition, abundant sources, stable biological characteristics and low immunogenicity, so that the umbilical cord-derived mesenchymal stem cells become ideal seed cells for medical application of future stem cells. In addition, umbilical cord-derived mesenchymal stem cells can secrete various stem cell factors during the culture process, including various growth factors including Fibroblast Growth Factor (FGF), vascular Endothelial Growth Factor (VEGF), epidermal Growth Factor (EGF), etc., which can promote cell growth, maintain cell stem property, and have wide application in the cosmetic skin care field.
At present, the culture of umbilical cord-derived mesenchymal stem cells is mainly divided into two types, namely a serum culture method, which mostly uses DMEM/F12+ serum or adds some nutrients on the basis of the serum to help the cell growth, the method is suitable for the large-scale culture of umbilical cord-derived mesenchymal stem cells, but the serum used in the method may contain some unknown allergens and pathogens, and affects the safety of cell culture; secondly, the serum-free culture method simply uses a culture medium and various nutrient additives to culture the mesenchymal stem cells from expected sources, however, the cells cultured by the method have slow growth and poor cell activity, and the content of cell growth factors in the culture medium is low, so that the popularization and application of the umbilical cord-source mesenchymal stem cell biological products are influenced.
Disclosure of Invention
In order to solve the above-mentioned problems, the present application provides a method for culturing umbilical cord-derived mesenchymal stem cells in a low-oxygen manner.
The application provides a method for culturing umbilical cord source mesenchymal stem cells in a low-oxygen mode, which adopts the following technical scheme:
a method of hypoxia culturing umbilical cord-derived mesenchymal stem cells, the method comprising the steps of: inoculating primary umbilical cord source mesenchymal stem cells on a culture medium, placing the culture medium in an environment with the culture temperature of 37 ℃ and the oxygen volume fraction of 1-3%, the carbon dioxide volume fraction of 2-2.5% and the balance of nitrogen for carrying out passage expansion for 96-108 hours, and collecting the mesenchymal stem cells; the culture medium consists of a basic culture medium and passage additives; the basal medium is a composition of F12 and one of MEM, low-sugar DMEM or high-sugar DMEM; the passage additive comprises 11-15 mug/ml of glutathione, 0.5-2.5 mug/ml of alpha-tocopherol, 0.5-2.0 mug/ml of beta-tocopherol, 2.5-3.0ng/ml of acetazolamide, 10-20 mug/ml of insulin and 8-10 mug/ml of vitamin C according to the final concentration of a culture medium.
Preferably, when primary umbilical cord-derived mesenchymal stem cells are cultured, the initial oxygen volume fraction is 1%, and after the oxygen volume fraction is increased to 3% at a rate of 0.1%/2h, the culture is maintained for 10h, the oxygen volume fraction is reduced to 2.3-2.4% at a rate of 0.1%/5h, and the culture is maintained until the subculture is completed.
Preferably, the medium is changed every 2 days during the culture of the mesenchymal stem cells.
Preferably, the primary umbilical cord-derived mesenchymal stem cells are prepared by the steps of: taking an isolated umbilical cord by adopting a tissue adherence method, peeling amniotic membrane and blood vessels, shearing the tissue, and placing the tissue into an alpha-MEM complete culture medium containing 10% FBS for culturing until the cells climb out of a tissue block to obtain the primary umbilical cord-derived mesenchymal stem cells.
Preferably, the passaging additive further comprises TGF-beta 0.8-1.0ng/ml based on the final concentration of the culture medium.
Preferably, the passage additive comprises 12 mug/ml of glutathione, 2.1 mug/ml of alpha-tocopherol, 1.6 mug/ml of beta-tocopherol, 2.8ng/ml of acetazolamide, 16 mug/ml of insulin, 9 mug/ml of vitamin C and 0.8ng/ml of TGF-beta based on the final concentration of the culture medium.
In summary, the application has the following beneficial effects: the mesenchymal stem cells are placed in a low-oxygen environment, and after a plurality of passage additives are added into a culture medium, the mesenchymal stem cells are subjected to passage expansion culture, so that the proliferation times of the mesenchymal stem cells and the activity of the stem cells obtained by culture can be improved.
Detailed Description
The present application will be described in further detail with reference to examples 1 to 4 and comparative examples 1 to 2.
Preparation example
Preparation example 1
Preparation of primary umbilical cord-derived mesenchymal stem cells for expansion: taking fresh and healthy human umbilical cord, washing with PBS buffer solution, removing amniotic membrane and blood vessel with forceps, shearing separated Wharton's jelly into pieces of 1-3mm 3 The tissue blocks of (2) are spread and re-cultured at the bottom of a culture flask, added into alpha-MEM culture medium containing 10% FBS,100U/ml penicillin and 100U/ml streptomycin, and placed in an incubator with 5% carbon dioxide at 37 ℃ for culture; at seventh day of culture, umbilical cord mesenchymal stem cells appear around the tissue block, culturing is continued, and when umbilical cord mesenchymal stem cells reach 80% fusion, discardingWashing umbilical cord tissue blocks and an original culture medium with normal saline, and then adding 0.25% of pancreatin for digestion to obtain the primary umbilical cord source mesenchymal stem cells for amplification.
Examples
Example 1
The method for culturing umbilical cord-derived mesenchymal stem cells in this example 1 comprises the steps of:
inoculating the primary umbilical cord source mesenchymal stem cells in preparation example 1 on a culture medium, placing the culture medium in an incubator for cell expansion culture at 37 ℃, keeping the oxygen volume fraction in the incubator to be 2.3-2.4%, the carbon dioxide volume fraction to be 2-2.5% and the balance of nitrogen; the primary umbilical cord source mesenchymal stem cells are cultured for 96-108 hours in an incubator, the culture medium is replaced every 48 hours (2 days) in the culture process, and after the culture is finished, the mesenchymal stem cells obtained through culture are collected.
Wherein, the culture medium comprises basal medium and passage additive, and the basal medium is 1 of MEM and F12: 1, in other embodiments, the basal medium is a combination of F12 and one of MEM, low-sugar DMEM or high-sugar DMEM, and the passaging additive is used in an amount of 13 μg/ml glutathione, 1.6 μg/ml alpha-tocopherol, 1.1 μg/ml beta-tocopherol, 2.6ng/ml acetazolamide, 12 μg/ml insulin, and 9 μg/ml vitamin C based on the final concentration of the medium.
Example 2
This example 2 differs from example 1 in the content of the components of the passaging supplement in the medium, and in example 2 the passaging supplement was used in amounts of 15. Mu.g/ml glutathione, 2.5. Mu.g/ml alpha-tocopherol, 2.0. Mu.g/ml beta-tocopherol, 3.0ng/ml acetazolamide, 20. Mu.g/ml insulin and 10. Mu.g/ml vitamin C based on the final concentration of the medium.
Example 3
This example 3 differs from example 1 in that the passaging supplement in the medium also includes TGF-beta, the passaging supplement being used in amounts of 12. Mu.g/ml of glutathione, 2.1. Mu.g/ml of alpha-tocopherol, 1.6. Mu.g/ml of beta-tocopherol, 2.8ng/ml of acetazolamide, 16. Mu.g/ml of insulin, 9. Mu.g/ml of vitamin C, and 0.8ng/ml of TGF-beta, based on the final concentration of the medium.
Example 4
Example 4 is different from example 3 in that, when the culture of primary umbilical cord-derived mesenchymal stem cells is performed, the initial oxygen volume fraction in the incubator is 1%, the culture is maintained for 10 hours after the oxygen volume fraction is increased to 3% at a rate of 0.1%/2 hours, the oxygen volume fraction is decreased to 2.3-2.4% at a rate of 0.1%/5 hours, and the culture is maintained until the subculture is completed.
Comparative example
Comparative example 1
This comparative example 1 differs from example 1 in that the passaging supplement in the medium does not include alpha-tocopherol, and the passaging supplement is used in amounts of 13. Mu.g/ml of glutathione, 2.7. Mu.g/ml of beta-tocopherol, 2.6ng/ml of acetazolamide, 12. Mu.g/ml of insulin, and 9. Mu.g/ml of vitamin C based on the final concentration of the medium.
Comparative example 2
This comparative example 2 differs from example 1 in that the passage supplement in the medium does not include beta-tocopherol, and the passage supplement is used in amounts of 13. Mu.g/ml of glutathione, 2.7. Mu.g/ml of alpha-tocopherol, 2.6ng/ml of acetazolamide, 12. Mu.g/ml of insulin, and 9. Mu.g/ml of vitamin C, based on the final concentration of the medium.
Performance test
The mesenchymal stem cells amplified and cultured in examples 1 to 4 and comparative examples 1 to 2 were stained with 0.4% trypan blue, and after cell counting, the proliferation factor and the cell viability of the mesenchymal stem cells in examples 1 to 4 and comparative examples 1 to 2 were counted, and the proliferation factor=total number of cells after culture/number of cells initially inoculated, and the cell viability= (number of living cells/total number of cells after culture) ×100%, and the results are shown in table 1.
TABLE 1 proliferation fold and cell viability of mesenchymal stem cells of examples 1-4 and comparative examples 1-2
As can be seen from the combination of examples 1-4 and comparative examples 1-2 and the combination of Table 1, the method for culturing umbilical cord-derived mesenchymal stem cells according to the present application has a better effect on the culture of mesenchymal stem cells, and the proliferation factor and the cell viability of the cultured mesenchymal stem cells are better than those of comparative examples 1-2.
In combination with examples 1-2 and 3, and with Table 1, it can be seen that the proliferation factor and the cell viability of the cell culture can be improved by adding TGF-. Beta.to the medium.
In combination with examples 3 and 4 and table 1, it can be seen that when mesenchymal stem cells are cultured, the volume of oxygen in the incubator is changed with the extension of the culture time in a low-oxygen environment, and finally, the culture is stable continuously, so that the proliferation multiple and the cell viability of the mesenchymal stem cells can be improved.
It can be seen from the combination of example 1 and comparative examples 1 to 2, and the combination of Table 1 that the effect of adding alpha-tocopherol or beta-tocopherol alone to the medium was not remarkable in the culture promoting effect on mesenchymal stem cells when alpha-tocopherol and beta-tocopherol were used in combination.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims (6)

1. A method of hypoxia culturing umbilical cord-derived mesenchymal stem cells, the method comprising the steps of:
inoculating primary umbilical cord source mesenchymal stem cells on a culture medium, placing the culture medium in an environment with the culture temperature of 37 ℃ and the oxygen volume fraction of 1-3%, the carbon dioxide volume fraction of 2-2.5% and the balance of nitrogen for carrying out passage expansion for 96-108 hours, and collecting the mesenchymal stem cells;
the culture medium consists of a basic culture medium and passage additives;
the basal medium is a composition of F12 and one of MEM, low-sugar DMEM or high-sugar DMEM;
the passage additive comprises 11-15 mug/ml of glutathione, 0.5-2.5 mug/ml of alpha-tocopherol, 0.5-2.0 mug/ml of beta-tocopherol, 2.5-3.0ng/ml of acetazolamide, 10-20 mug/ml of insulin and 8-10 mug/ml of vitamin C according to the final concentration of a culture medium.
2. The method for culturing umbilical cord-derived mesenchymal stem cells in a low-oxygen manner according to claim 1, wherein: the primary umbilical cord source mesenchymal stem cells are cultured, wherein the initial oxygen volume fraction is 1%, the oxygen volume fraction is increased to 3% at the rate of 0.1%/2h, then the primary umbilical cord source mesenchymal stem cells are maintained for 10h, the oxygen volume fraction is reduced to 2.3-2.4% at the rate of 0.1%/5h, and the primary umbilical cord source mesenchymal stem cells are maintained until the subculture is finished.
3. The method for culturing umbilical cord-derived mesenchymal stem cells in a low-oxygen manner according to claim 1, wherein: in the process of culturing mesenchymal stem cells, the culture medium is replaced every 2 days.
4. The method for culturing umbilical cord-derived mesenchymal stem cells in a low-oxygen manner according to claim 1, wherein the primary umbilical cord-derived mesenchymal stem cells are prepared by the steps of: taking an isolated umbilical cord by adopting a tissue adherence method, peeling amniotic membrane and blood vessels, shearing the tissue, and placing the tissue into an alpha-MEM complete culture medium containing 10% FBS for culturing until the cells climb out of a tissue block to obtain the primary umbilical cord-derived mesenchymal stem cells.
5. The method for culturing umbilical cord-derived mesenchymal stem cells in a low-oxygen manner according to claim 1, wherein: the passage additive also comprises TGF-beta 0.8-1.0ng/ml based on the final concentration of the culture medium.
6. The method for culturing umbilical cord-derived mesenchymal stem cells under low oxygen according to claim 5, wherein: the passage additive comprises 12 mug/ml of glutathione, 2.1 mug/ml of alpha-tocopherol, 1.6 mug/ml of beta-tocopherol, 2.8ng/ml of acetazolamide, 16 mug/ml of insulin, 9 mug/ml of vitamin C and 0.8ng/ml of TGF-beta based on the final concentration of the culture medium.
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