CN112458042A - Pluripotent stem cell culture medium - Google Patents

Pluripotent stem cell culture medium Download PDF

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Publication number
CN112458042A
CN112458042A CN202011389233.7A CN202011389233A CN112458042A CN 112458042 A CN112458042 A CN 112458042A CN 202011389233 A CN202011389233 A CN 202011389233A CN 112458042 A CN112458042 A CN 112458042A
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culture medium
cell culture
pluripotent stem
growth factor
concentration
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陈晓栋
尚倩雯
贾善粉
张胜超
王旻贤
马慧
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Wuxi Huatai Innovative Pharmaceutical Technology Research Institute Co ltd
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Wuxi Huatai Innovative Pharmaceutical Technology Research Institute Co ltd
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/148—Transforming growth factor alpha [TGF-a]
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Abstract

The invention discloses a pluripotent stem cell culture medium, which belongs to the technical field of bioengineering and comprises a basic culture medium and the following additional components; 150-; the normal proliferation of cells can be ensured under the action of recombinant transferrin, insulin and the like, and the possibility of pathogen pollution is reduced; the combined use of the allicin and the streptomycin can obviously increase the inhibition and killing effects of the allicin on bacteria, so that the cell culture medium has good inhibition and killing effects on the bacteria, thereby being beneficial to the growth and proliferation of the cells and reducing the possibility of bacterial infection in the cell culture medium.

Description

Pluripotent stem cell culture medium
Technical Field
The invention relates to the technical field of bioengineering, in particular to a pluripotent stem cell culture medium.
Background
Pluripotent Stem Cells (PSC) are a class of pluripotent cells that have the ability to self-renew and self-replicate. Under certain conditions, it can differentiate into a variety of APSC pluripotent cells, which have the potential to differentiate into a variety of cellular tissues. The human iPSC culture process goes through three stages using feeder layer cell culture, feeder layer-free cell culture and animal-derived feeder layer-free cell culture. Feeder cells are usually derived from Mouse Embryonic Fibroblasts (MEFs), and the judgment and further deep application research of experimental research are influenced by batch differences, animal sources, infection sources of various types, component uncertainty and the like.
The use of iPS cells to treat disease is the ultimate goal of the population. iPS cells can be used not only for differentiation and transplantation, but also for providing in vitro disease models, facilitating the study of the mechanism of disease formation, the screening of new drugs, and the development of new therapeutic methods. The establishment of human iPS cells is acknowledged as one of the most important scientific and technological advances in 2007, and the technology not only can establish an individual specific pluripotent stem cell line from somatic cells, solve the problem of immune rejection in cell transplantation treatment, but also provides a powerful method for researching the reprogramming mechanism of human cells and the occurrence mechanism of individual specific diseases. In the stem cell research field, the emergence of the iPS cell technology is undoubtedly a breakthrough with milestone significance, various somatic cells can be converted into stem cells with multidirectional differentiation potential through in vitro culture and induction, and the fact that several known transcription factors can invert differentiated somatic cells into an undifferentiated state is proved, and the huge plasticity of the cells is shown.
Traditional pluripotent stem cell culture medium has certain defect, and the cell in the process of culturing the cell culture medium is easily infected by external bacteria, and the bacteria can influence the growth of the cell and are not beneficial to the culture of the cell.
Disclosure of Invention
The invention aims to provide a pluripotent stem cell culture medium which comprises a basic culture medium and the following additional components, wherein the additional components comprise 400-g/mL allicin and 40-60-g/mL streptomycin, and the combined use of the allicin and the streptomycin can inhibit the survival of bacteria, so that the culture medium has good bactericidal effect, is beneficial to the culture of cells in the cell culture medium, and solves the problems in the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a pluripotent stem cell culture medium, which comprises a basic culture medium and the following additional components; 150-.
Preferably, the additional component further comprises glucose.
Preferably, the concentration of the glucose is 10-20 mmol/L.
Preferably, the additional component further comprises vitamin C.
Preferably, the concentration of the vitamin C is 6-10 mug/mL.
Preferably, the additional components further comprise basic fibroblast growth factor and transforming growth factor beta 1.
Preferably, the concentration of the basic fibroblast growth factor is 10-30ng/mL, and the concentration of the transforming growth factor beta1 is 1-2 ng/mL.
Preferably, the basal medium is DMEM/F12 cell culture medium.
Preferably, the osmotic pressure of the DMEM/F12 cell culture medium is 270-330 mosmol/L.
Corning BD matrix glue and Matrigel are polymerized to form a three-dimensional matrix with biological activity, the structure, the composition, the physical characteristics and the functions of an in-vivo cell basement membrane are simulated, the culture and the differentiation of in-vitro cells are facilitated, and the method can be used for researching the cell morphology, the biochemical function, the migration, the infection, the gene expression and the like.
Sodium selenite, one of the many antioxidants that helps protect against damaging cells in the body, is an essential mineral and micronutrient. It, together with vitamin E, traps the free radical of the singlet electron cell and makes it a stable cell.
Recombinant transferrin, which binds iron ions, reduces its toxicity and utilization by cells, completely avoids the possibility of pathogen contamination.
Allicin has strong antibacterial and bactericidal effects, has strong killing capability on escherichia coli, germs, bacteria, viruses and the like, can be used for treating diseases such as dysentery, infantile gastrointestinal diseases, diarrhea and the like, has a protection effect on digestive system diseases, and has a good protection effect on chronic diseases and gastrointestinal diseases. Has effects in treating cardiovascular and cerebrovascular diseases and blood diseases, and allicin has effects in lowering blood plasma total cholesterol, inhibiting platelet activity, reducing hematocrit, and protecting health of cardiovascular and cerebrovascular diseases.
Streptomycin, which is an aminoglycoside basic compound, binds to the protein of the ribosomal of ribonucleic acid of tubercle bacillus and acts to interfere with the synthesis of protein of tubercle bacillus, thus killing or inhibiting the growth of tubercle bacillus.
Vitamin C is also known as ascorbic acid and belongs to water-soluble vitamins. Vitamin C is an antioxidant that plays an important role in biological oxidation and reduction processes and in cellular respiration.
In summary, due to the adoption of the technical scheme, the method has the following beneficial effects:
the invention provides a pluripotent stem cell culture medium, which comprises a basic culture medium and the following additional components; 150-; the combined use of the allicin and the streptomycin can obviously increase the inhibition and killing effects of the allicin on bacteria, so that the cell culture medium has good inhibition and killing effects on the bacteria, thereby being beneficial to the growth of cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The cell culture medium is not only a basic substance for supplying cell nutrition and promoting cell reproduction in the cultured cells, but also a living environment for the growth and reproduction of the cultured cells. The existing pluripotent stem cell culture can supply cell nutrition and basic substances for promoting cell reproduction and proliferation, but has no inhibiting and killing effect on bacteria in the cell culture process. According to the embodiment of the invention, the combined use of the allicin and the streptomycin can obviously increase the inhibition and killing effects of the allicin on bacteria, so that the cell culture medium has good inhibition and killing effects on the bacteria, and is favorable for the culture and use of the cells.
The DMEM/F12 cell culture medium is a basic culture medium, and the basic culture medium is added with components, 150-200 mu L/cm2 corning BD matrigel, 10-20ng/mL sodium selenite, 20-200 ng/mL recombinant transferrin, 20-30 mu g/mL insulin, 400-600 mu g/mL allicin, 40-60 mu g/mL streptomycin, the concentration of glucose is 10-20mmol/L, the concentration of vitamin C is 6-10 mu g/mL, the concentration of basic fibroblast growth factor is 10-30ng/mL, the concentration of transforming growth factor beta1 is 1-2ng/mL, and the osmotic pressure of the cell culture medium is 270-330 mosmol/L.
Example 1
Firstly, 150 mu L/cm2 corning BD substrate gel is put on the bottom side of a DMEM/F12 cell culture medium, then 10ng/mL sodium selenite, 20ng/mL recombinant transferrin, 20 mu g/mL insulin, 400 mu g/mL allicin, 40 mu g/mL streptomycin, 10mmol/L glucose concentration, 6 mu g/mL vitamin C concentration, 10ng/mL basic fibroblast growth factor concentration and 1ng/mL transforming growth factor beta1 concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 are mixed first and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 2
160 mu L/cm2 corning BD substrate gel is firstly taken to the bottom side of a DMEM/F12 cell culture medium, and then 12ng/mL sodium selenite, 50ng/mL recombinant transferrin, 22 mu g/mL insulin, 440 mu g/mL allicin, 44 mu g/mL streptomycin, 12mmol/L glucose concentration, 7 mu g/mL vitamin C concentration, 14ng/mL basic fibroblast growth factor concentration and 1.2ng/mL transforming growth factor beta1 concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 are mixed first and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 3
170 mu L/cm2 corning BD substrate gel is firstly taken to the bottom side of a DMEM/F12 cell culture medium, and then 14ng/mL sodium selenite, 80ng/mL recombinant transferrin, 24 mu g/mL insulin, 480 mu g/mL allicin, 48 mu g/mL streptomycin, the concentration of glucose is 14mmol/L, the concentration of vitamin C is 7.5 mu g/mL, the concentration of basic fibroblast growth factor is 18ng/mL, and the concentration of transforming growth factor beta1 is 1.4 ng/mL.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 are mixed first and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 4
Firstly, 180 mu.L/cm 2 corning BD substrate gel is put on the bottom side of a DMEM/F12 cell culture medium, and then 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mu g/mL insulin, 500 mu g/mL allicin, 50 mu g/mL streptomycin, 15mol/L glucose concentration, 8 mu g/mL vitamin C concentration, 20ng/mL basic fibroblast growth factor concentration and 1.5ng/mL transforming growth factor beta1 concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 are mixed first and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 5
190 mu L/cm2 corning BD substrate gel is firstly taken to the bottom side of a DMEM/F12 cell culture medium, then 16ng/mL sodium selenite, 140ng/mL recombinant transferrin, 26 mu g/mL insulin, 540 mu g/mL allicin, 54 mu g/mL streptomycin, the concentration of glucose is 16mol/L, the concentration of vitamin C is 8.5 mu g/mL, the concentration of basic fibroblast growth factor is 24ng/mL, and the concentration of transforming growth factor beta1 is 1.6 ng/mL.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 are mixed first and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 6
195 mu.L/cm 2 of corning BD substrate gel is firstly taken to the bottom side of a DMEM/F12 cell culture medium, 18ng/mL of sodium selenite, 180ng/mL of recombinant transferrin, 28 mu g/mL of insulin, 580 mu g/mL of allicin, 58 mu g/mL of streptomycin, 18mol/L of glucose, 9 mu g/mL of vitamin C, 28ng/mL of basic fibroblast growth factor and 1.8ng/mL of transforming growth factor beta1 are then taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 are mixed first and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 7
200 mu L/cm2 corning BD substrate gel is firstly taken to the bottom side of a DMEM/F12 cell culture medium, and then 20ng/mL sodium selenite, 200ng/mL recombinant transferrin, 30 mu g/mL insulin, 600 mu g/mL allicin, 60 mu g/mL streptomycin, the concentration of glucose is 20mmol/L, the concentration of vitamin C is 10 mu g/mL, the concentration of basic fibroblast growth factor is 30ng/mL, the concentration of transforming growth factor beta1 is 2ng/mL, and the osmotic pressure of the cell culture medium is 330 mosmol/L.
Sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L, and transforming growth factor beta1 were then added to DMEM/F12 cell culture medium and stirred.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 8
180 mu.L/cm 2 of corning BD substrate gel is firstly taken to the bottom side of a DMEM/F12 cell culture medium, and then 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mu g/mL insulin, 500 mu g/mL allicin, 50 mu g/mL streptomycin, 15mol/L glucose concentration and 8 mu g/mL vitamin C concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose and vitamin C are mixed and added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 9
180 mu.L/cm 2 of corning BD substrate gel is firstly taken to the bottom side of a DMEM/F12 cell culture medium, and then 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mu g/mL insulin, 500 mu g/mL allicin, 50 mu g/mL streptomycin, 15mol/L glucose concentration, 8 mu g/mL vitamin C concentration and 20ng/mL basic fibroblast growth factor concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C and basic fibroblast growth factor are mixed and added into a DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 10
180 mu.L/cm 2 of corning BD substrate gel is firstly taken to the bottom side of a DMEM/F12 cell culture medium, and then 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mu g/mL insulin, 500 mu g/mL allicin, 50 mu g/mL streptomycin, 15mol/L glucose concentration, 8 mu g/mL vitamin C concentration and 1.5ng/mL transforming growth factor beta1 concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, transforming growth factor beta1 are mixed and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 11
Firstly, 180 mu.L/cm 2 corning BD substrate gel is put on the bottom side of a DMEM/F12 cell culture medium, and then 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mu g/mL insulin, 500 mu g/mL allicin, 50 mu g/mL streptomycin, 15mol/L glucose concentration, 8 mu g/mL vitamin C concentration, 20ng/mL basic fibroblast growth factor concentration and 1.5ng/mL transforming growth factor beta1 concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 are mixed first and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of a DMEM/F12 cell culture medium to 270mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 12
Firstly, 180 mu.L/cm 2 corning BD substrate gel is put on the bottom side of a DMEM/F12 cell culture medium, and then 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mu g/mL insulin, 500 mu g/mL allicin, 50 mu g/mL streptomycin, 15mol/L glucose concentration, 8 mu g/mL vitamin C concentration, 20ng/mL basic fibroblast growth factor concentration and 1.5ng/mL transforming growth factor beta1 concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 are mixed first and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 330mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Example 13
Taking 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mug/mL insulin, 500 mug/mL allicin, 50 mug/mL streptomycin, wherein the concentration of glucose is 15mol/L, the concentration of vitamin C is 8 mug/mL, the concentration of basic fibroblast growth factor is 20ng/mL, and the concentration of transforming growth factor beta1 is 1.5 ng/mL.
Then sodium selenite, recombinant transferrin, insulin, allicin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 are mixed first and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Comparative example 1
Firstly, 180 mu.L/cm 2 corning BD substrate gel is put on the bottom side of a DMEM/F12 cell culture medium, then 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mu g/mL insulin, 15mol/L glucose concentration, 8 mu g/mL vitamin C concentration, 20ng/mL basic fibroblast growth factor concentration and 1.5ng/mL transforming growth factor beta1 concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 were mixed and added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Comparative example 2
Firstly, 180 mu.L/cm 2 corning BD substrate gel is put on the bottom side of a DMEM/F12 cell culture medium, then 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mu g/mL insulin, 500 mu g/mL allicin, 15mol/L glucose, 8 mu g/mL vitamin C, 20ng/mL basic fibroblast growth factor and 1.5ng/mL transforming growth factor beta1 are taken.
Then sodium selenite, recombinant transferrin, insulin, allicin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 were mixed and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Comparative example 3
Firstly, 180 mu.L/cm 2 corning BD substrate gel is placed on the bottom side of a DMEM/F12 cell culture medium, then 15ng/mL sodium selenite, 100ng/mL recombinant transferrin, 25 mu g/mL insulin, 50 mu g/mL streptomycin, 15mol/L glucose concentration, 8 mu g/mL vitamin C concentration, 20ng/mL basic fibroblast growth factor concentration and 1.5ng/mL transforming growth factor beta1 concentration are taken.
Then sodium selenite, recombinant transferrin, insulin, streptomycin, glucose, vitamin C, basic fibroblast growth factor L and transforming growth factor beta1 were mixed and then added into DMEM/F12 cell culture medium.
Adjusting the osmotic pressure of the DMEM/F12 cell culture medium to 300mosmol/L, and performing constant volume to obtain the pluripotent stem cell culture medium.
Experimental example 1
(1) Firstly, corning BD stroma glue is selected to coat the pluripotent stem cells, and then IV collagenase is selected to digest the pluripotent stem cells.
(2) The pluripotent stem cells obtained in step 1 were placed in the additive components obtained in examples 1 to 6 and comparative examples 1 to 3, respectively.
(3) The cells were scraped and then placed in a centrifuge tube for centrifugation and the supernatant removed.
Then, the cells were placed in the wells of the culture medium plates obtained in examples 1 to 6 and comparative examples 1 to 3 at 3X 106iPS cells, divided into 3 groups, each group of 3 wells, were planted in six-well plates.
(4) Cells were cultured under 5% CO2, 95% humidity, 37 ℃.
(5) The following day, the medium described in example 1 was changed to fresh (total 4ml per well)
After five passages, the total amount of cells was counted and 100 sets of the same experiments were performed.
After the medium is infected with bacteria, the culture solution in the medium becomes turbid.
TABLE 1
Average turbidity ratio (%) Average cell number (ten thousand)
Example 1 30 950
Example 2 20 1140
Example 3 10 1350
Example 4 5 1560
Example 5 10 1350
Example 6 15 1300
Example 7 20 1140
Example 8 7 1490
Example 9 9 1410
Example 10 8 1440
Example 11 8 1440
Example 12 9 1410
Example 13 10 1350
Comparative example 1 57 740
Comparative example 2 15 1300
Comparative example 3 30 940
Experimental example 2
(1) Firstly, corning BD stroma glue is selected to coat the pluripotent stem cells, and then IV collagenase is selected to digest the pluripotent stem cells.
(2) The pluripotent stem cells obtained in step 1 were placed in the additive components obtained in examples 1 to 6 and comparative examples 1 to 3, respectively.
(3) The cells were scraped and then placed in a centrifuge tube for centrifugation and the supernatant removed.
(4) The cells were then placed in the wells of the culture medium plates obtained in examples 1-6 and comparative examples 1-3 at 6X 106iPS cells, divided into 3 groups, each group of 3 wells, were planted in six-well plates.
(5) Cells were cultured under 5% CO2, 95% humidity, 37 ℃.
(6) The following day, the medium described in example 1 was changed to fresh (total 4ml per well)
After five passages, the total amount of cells was counted and 100 sets of the same experiments were performed.
TABLE 2
Average turbidity ratio (%) Average cell number (ten thousand)
Example 1 31 1810
Example 2 20 2280
Example 3 10 2700
Example 4 5 3080
Example 5 10 2700
Example 6 15 2600
Example 7 19 2330
Example 8 7 2950
Example 9 9 2990
Example 10 8 2880
Example 11 8 2880
Example 12 9 2820
Example 13 11 2600
Comparative example 1 57 1480
Comparative example 2 15 2440
Comparative example 3 30 1800
Experimental example 3
(1) Firstly, corning BD stroma glue is selected to coat the pluripotent stem cells, and then IV collagenase is selected to digest the pluripotent stem cells. (2) The pluripotent stem cells obtained in step 1 were placed in the additive components obtained in examples 1 to 6 and comparative examples 1 to 3, respectively.
(3) The cells were scraped and then placed in a centrifuge tube for centrifugation and the supernatant removed.
(4) The cells were then placed in the wells of the culture medium plates obtained in examples 1-6 and comparative examples 1-3 at 9X 106iPS cells, divided into 3 groups, each group of 3 wells, were planted in six-well plates.
(5) Cells were cultured under 5% CO2, 95% humidity, 37 ℃.
(6) The following day, the medium described in example 1 was changed to fresh (total 4ml per well)
After five passages, the total amount of cells was counted and 100 sets of the same experiments were performed.
TABLE 3
Average turbidity ratio (%) Average cell number (ten thousand)
Experimental example 1 29 3600
Example 2 20 2560
Example 3 10 5400
Experimental example 2 5 6150
Example 5 10 5400
Example 6 15 5200
Experimental example 3 21 4610
Experimental example 4 8 5950
Experimental example 5 10 5080
Experimental example 6 8 5760
Experimental example 7 8 5760
Experimental example 8 10 5500
Experimental example 9 12 5210
Comparative example 1 57 2900
Comparative example 2 15 5880
Comparative example 3 30 3600
As can be seen from tables 1-3, the growth and proliferation of cells can be influenced after the cells in the cell culture medium are infected by bacteria, the allicin and the streptomycin respectively have certain inhibiting and killing effects on the bacteria, and the inhibiting and killing effects of the allicin on the bacteria can be obviously increased by the combined use of the allicin and the streptomycin, so that the cell culture medium has good inhibiting and killing effects on the bacteria, the growth and proliferation of the cells are facilitated, and the possibility of bacterial infection in the cell culture medium is reduced; matrigel is polymerized to form a three-dimensional matrix with biological activity, and the structure, the composition, the physical characteristics and the function of a cell basement membrane in vivo are simulated, so that the culture and the differentiation of cells in vitro are facilitated; can ensure the normal proliferation of cells under the action of recombinant transferrin, insulin and the like.
In summary, a pluripotent stem cell culture medium is provided, which comprises a basal medium and the following additional components; 150-; the normal proliferation of cells can be ensured under the action of recombinant transferrin, insulin and the like, and the possibility of pathogen pollution is reduced; the combined use of the allicin and the streptomycin can obviously increase the inhibition and killing effects of the allicin on bacteria, so that the cell culture medium has good inhibition and killing effects on the bacteria, thereby being beneficial to the growth and proliferation of the cells and reducing the possibility of bacterial infection in the cell culture medium.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (9)

1. A pluripotent stem cell culture medium is characterized by comprising a basic culture medium and the following additional components; 150-.
2. The pluripotent stem cell culture medium of claim 1, wherein the additional components further comprise glucose.
3. The culture medium of pluripotent stem cells according to claim 1, wherein the concentration of glucose is 10 to 20 mmol/L.
4. The pluripotent stem cell culture medium of claim 1, wherein the adjunct ingredients further comprise vitamin C.
5. The culture medium for the pluripotent stem cells according to claim 4, wherein the concentration of the vitamin C is 6-10 μ g/mL.
6. The pluripotent stem cell culture medium of claim 1, wherein the additional components further comprise basic fibroblast growth factor and transforming growth factor beta 1.
7. The culture medium for pluripotent stem cells according to claim 6, wherein the concentration of basic fibroblast growth factor is 10-30ng/mL, and the concentration of transforming growth factor beta1 is 1-2 ng/mL.
8. The pluripotent stem cell culture medium according to claim 1, wherein the basal medium is DMEM/F12 cell culture medium.
9. The culture medium for the pluripotent stem cells according to claim 8, wherein the osmotic pressure of the culture medium for the DMEM/F12 cells is 270-330 mosmol/L.
CN202011389233.7A 2020-12-03 2020-12-03 Pluripotent stem cell culture medium Pending CN112458042A (en)

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