CN110713982A - Culture medium for rapid amplification of neural stem cells - Google Patents
Culture medium for rapid amplification of neural stem cells Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 76
- 210000001178 neural stem cell Anatomy 0.000 title claims abstract description 50
- 230000003321 amplification Effects 0.000 title description 3
- 238000003199 nucleic acid amplification method Methods 0.000 title description 3
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 38
- 235000015097 nutrients Nutrition 0.000 claims abstract description 25
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 claims abstract description 24
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 claims abstract description 24
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims abstract description 24
- 102000004877 Insulin Human genes 0.000 claims abstract description 19
- 108090001061 Insulin Proteins 0.000 claims abstract description 19
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229940125396 insulin Drugs 0.000 claims abstract description 19
- 229910052711 selenium Inorganic materials 0.000 claims abstract description 19
- 239000011669 selenium Substances 0.000 claims abstract description 19
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 12
- 239000005700 Putrescine Substances 0.000 claims abstract description 12
- 102000004338 Transferrin Human genes 0.000 claims abstract description 12
- 108090000901 Transferrin Proteins 0.000 claims abstract description 12
- 239000008103 glucose Substances 0.000 claims abstract description 12
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229960003387 progesterone Drugs 0.000 claims abstract description 12
- 239000000186 progesterone Substances 0.000 claims abstract description 12
- 229960005322 streptomycin Drugs 0.000 claims abstract description 12
- 239000012581 transferrin Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 10
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims abstract description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 4
- 239000011259 mixed solution Substances 0.000 claims abstract description 4
- 238000012258 culturing Methods 0.000 claims description 10
- 229930182555 Penicillin Natural products 0.000 claims description 8
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 8
- 229940049954 penicillin Drugs 0.000 claims description 8
- 230000004927 fusion Effects 0.000 claims description 6
- 239000007640 basal medium Substances 0.000 claims description 4
- 210000004027 cell Anatomy 0.000 abstract description 17
- 230000004083 survival effect Effects 0.000 abstract description 13
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 abstract description 8
- 230000035755 proliferation Effects 0.000 abstract description 7
- 230000010261 cell growth Effects 0.000 abstract description 6
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 abstract description 5
- 230000002349 favourable effect Effects 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 5
- 108010024636 Glutathione Proteins 0.000 abstract description 4
- 229960003180 glutathione Drugs 0.000 abstract description 4
- 230000007062 hydrolysis Effects 0.000 abstract description 4
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 150000002978 peroxides Chemical class 0.000 abstract description 3
- 230000012010 growth Effects 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 5
- 239000006143 cell culture medium Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 3
- 210000004958 brain cell Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 210000001130 astrocyte Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 229940091258 selenium supplement Drugs 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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Abstract
The invention discloses a culture medium for rapid expansion of neural stem cells, which comprises: the culture medium comprises a basic culture medium and nutrient components, wherein the basic culture medium is a mixed solution of DMEM and F12, and the nutrient components comprise: glucose, glutamine, NaHCO3Penicillin, streptomycin, insulin, transferrin, progesterone, putrescine and selenium. The culture medium of the invention contains insulin and selenium, wherein the insulin plays an important role in the survival and growth of cells, provides sufficient nutrient substances for the survival and growth of the cells, and simultaneously, the selenium is a cooperative factor generated by glutathione, is beneficial to the hydrolysis of peroxide and superoxide, can prevent the damage of the cells, provides favorable conditions for the survival and rapid proliferation of the cells, ensures the integrity of the cells, and uses CO to the culture medium in the using method2The balance is carried out, the multiple culture medium is added, the sufficiency of nutrient substances in the culture medium is ensured, the content of the culture medium is gradually reduced by half, and resources are saved.
Description
Technical Field
The invention relates to the field of neural stem cells, in particular to a culture medium for rapid amplification of neural stem cells.
Background
Neural stem cells refer to a cell population that exists in the nervous system, has the potential to differentiate into neurons, astrocytes and oligodendrocytes, thereby being capable of producing a large amount of brain cells and performing self-renewal, and sufficiently providing a large amount of brain cells. Neural stem cells have a strong ability to divide, and need to be present in a culture medium for a long period of time to grow when cultured.
The existing culture medium has single nutrient component, brings unfavorable living environment for proliferation of the neural stem cells, has low proliferation amount in the same time, has poor proliferation dry cell quality, and is not favorable for later-period research or use. Meanwhile, the nutrient components in the culture medium do not protect the function of the cells, so that the proliferated stem cells are easy to die, and an unreasonable environment is brought to the survival of other stem cells.
Disclosure of Invention
In order to solve the above-mentioned deficiencies in the background art, the present invention aims to provide a culture medium for rapid expansion of neural stem cells, wherein the culture medium comprises insulin and selenium, wherein the insulin plays an important role in the survival and growth of cells and provides sufficient nutrients for the survival and growth of cells, and meanwhile, the selenium is a cofactor generated by glutathione, which facilitates the hydrolysis of superoxide and superoxide, can prevent the damage of cells, provides favorable conditions for the survival and rapid proliferation of cells and ensures the integrity of cells;
meanwhile, in the using method of the culture medium, CO is used for the culture medium2The balance is carried out, the balance of the culture medium is ensured, meanwhile, the multiple culture mediums are added, the sufficiency of nutrient substances in the culture medium is ensured, the content of the culture medium is gradually reduced by half, and resources are saved.
The purpose of the invention can be realized by the following technical scheme:
a culture medium for rapid expansion of neural stem cells, the culture medium comprising: basal medium and nutrient components;
the basic culture medium is a mixed solution of DMEM and F12, and the volume ratio is 1: 1;
the nutrient components comprise: glucose, glutamine, NaHCO3Penicillin, streptomycin, insulin, transferrin, progesterone, putrescine and selenium.
Further, the contents of the components of the nutrient components are as follows: glucose 6-15g/L, glutamine 3 x 10- 3mol/L-8*10-3mol/L，NaHCO35*10-3mol/L-12*10-3mol/L, penicillin 60-90mg/ml, streptomycin 65-85mg/ml, insulin 2.8-3.5 mu g/ml, transferrin 130-250ng/ml, progesterone 8 x 10-9mol/L-15*10-9mol/L, putrescine 30 x 10-6mol/L-50*10-6mol/L, selenium 50 x 10-9mol/L-60*10-9mol/L。
Further, the contents of the components of the nutrient components are as follows: glucose 10g/L, Glutamine 5 x 10- 3mol/L，10*10-3mol/L, penicillin 75mg/ml, streptomycin 75mg/ml, insulin 3.0 μ g/ml, transferrin 190ng/ml, progesterone 12 x 10-9mol/L, putrescine 40 x 10-6mol/L, selenium 55 x 10-9mol/L。
A method of using a culture medium for rapid expansion of neural stem cells, the method of using comprising the steps of:
firstly, placing the culture medium in a sterile box, and using CO2Balancing for 30-40 minutes;
secondly, placing the neural stem cells in a culture medium for culturing, and standing for 5-10 hours;
thirdly, adding the culture medium in the first step into the sterile box again, repeating the second step for a plurality of times, wherein the number of times of adding the culture medium is 3-5 times;
and fourthly, digesting the neural stem cell after the fusion degree of the neural stem cell reaches 85-92%, and carrying out subculture according to the proportion of 1:2 or 1:4 to obtain the neural stem cell.
Further, CO in the first step2The concentration is 5-10%, and the temperature is 32-34 ℃.
Further, in the second step, the temperature in the sterile box is increased by 0.5 ℃ every 2 hours until the temperature is increased to 36.5-37.5 ℃.
Further, the content of the culture medium added in the third step is gradually reduced by half.
The invention has the beneficial effects that:
1. the culture medium contains insulin and selenium, wherein the insulin plays an important role in the survival and growth of cells, provides sufficient nutrient substances for the survival and growth of the cells, and meanwhile, the selenium is a cooperative factor generated by glutathione, is beneficial to the hydrolysis of peroxide and superoxide, can prevent the damage of the cells, provides favorable conditions for the survival and rapid proliferation of the cells, and ensures the integrity of the cells;
2. in the method for using the culture medium, CO is used for the culture medium2The balance is carried out, the balance of the culture medium is ensured, meanwhile, the multiple culture mediums are added, the sufficiency of nutrient substances in the culture medium is ensured, the content of the culture medium is gradually reduced by half, and resources are saved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. 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.
A culture medium for rapid expansion of neural stem cells, the culture medium comprising: a basal medium and nutrient components, wherein the basal medium is a mixed solution of DMEM and F12, and the volume ratio is 1: 1; the nutrient components comprise: glucose, glutamine, NaHCO3Penicillin, streptomycin, insulin, transferrin, progesterone, putrescine and selenium.
Example 1:
taking 1L of a basic culture medium, wherein the contents of all components of the nutrient components are as follows: glucose 6g/L, Glutamine 3 x 10- 3mol/L，NaHCO35*10-3mol/L, penicillin 60mg/ml, streptomycin 65mg/ml, insulin 2.8 μ g/ml, transferrin 130ng/ml, progesterone 8 x 10-9mol/L, putrescine 30 x 10-6mol/L, selenium 50 x 10-9mol/L。
The preparation method of the neural stem cell culture medium comprises the following steps:
firstly, placing the culture medium in a sterile box, and using CO2Equilibrating for 30-40 minutes with CO2The concentration is 5-10%, and the temperature is 32-34 ℃;
secondly, placing the neural stem cells in a culture medium for culturing, raising the temperature in a sterile box to 0.5 ℃ every 2 hours until the temperature is raised to 36.5-37.5 ℃, and standing for culturing for 5-10 hours;
thirdly, adding the culture medium in the first step into the sterile box again, repeating the second step for a plurality of times, wherein the number of times of adding the culture medium is 3-5 times, and the content of the added culture medium is gradually reduced by half;
and fourthly, digesting the neural stem cell after the fusion degree of the neural stem cell reaches 85-92%, and carrying out subculture according to the proportion of 1:2 or 1:4 to obtain the neural stem cell.
Example 2:
taking 1L of a basic culture medium, wherein the contents of all components of the nutrient components are as follows: glucose 15g/L, Glutamine 8 x 10- 3mol/L，NaHCO312*10-3mol/L, penicillin 90mg/ml, streptomycin 85mg/ml, insulin 3.5 μ g/ml, transferrin 250ng/ml, progesterone 15 x 10-9mol/L, putrescine 50 x 10-6mol/L, selenium 60 x 10-9mol/L。
The preparation method of the neural stem cell culture medium comprises the following steps:
firstly, placing the culture medium in a sterile box, and using CO2Equilibrating for 30-40 minutes with CO2The concentration is 5-10%, and the temperature is 32-34 ℃;
secondly, placing the neural stem cells in a culture medium for culturing, raising the temperature in a sterile box to 0.5 ℃ every 2 hours until the temperature is raised to 36.5-37.5 ℃, and standing for culturing for 5-10 hours;
thirdly, adding the culture medium in the first step into the sterile box again, repeating the second step for a plurality of times, wherein the number of times of adding the culture medium is 3-5 times, and the content of the added culture medium is gradually reduced by half;
and fourthly, digesting the neural stem cell after the fusion degree of the neural stem cell reaches 85-92%, and carrying out subculture according to the proportion of 1:2 or 1:4 to obtain the neural stem cell.
Example 3:
taking 1L of a basic culture medium, wherein the contents of all components of the nutrient components are as follows: glucose 10g/L, Glutamine 5 x 10-3mol/L，10*10-3mol/L, penicillin 75mg/ml, streptomycin 75mg/ml, insulin 3.0 μ g/ml, transferrin 190ng/ml, progesterone 12 x 10-9mol/L, putrescine 40 x 10-6mol/L, selenium 55 x 10-9mol/L。
The preparation method of the neural stem cell culture medium comprises the following steps:
firstly, placing the culture medium in a sterile box, and using CO2Equilibrating for 30-40 minutes with CO2The concentration is 5-10%, and the temperature is 32-34 ℃;
secondly, placing the neural stem cells in a culture medium for culturing, raising the temperature in a sterile box to 0.5 ℃ every 2 hours until the temperature is raised to 36.5-37.5 ℃, and standing for culturing for 5-10 hours;
thirdly, adding the culture medium in the first step into the sterile box again, repeating the second step for a plurality of times, wherein the number of times of adding the culture medium is 3-5 times, and the content of the added culture medium is gradually reduced by half;
and fourthly, digesting the neural stem cell after the fusion degree of the neural stem cell reaches 85-92%, and carrying out subculture according to the proportion of 1:2 or 1:4 to obtain the neural stem cell.
Comparative example:
taking 1L of a basic culture medium, wherein the contents of all components of the nutrient components are as follows: glucose 10g/L, Glutamine 5 x 10-3mol/L，10*10-3mol/L, penicillin 75mg/ml, streptomycin 75mg/ml, transferrin 190ng/ml, progesterone 12 x 10-9mol/L, putrescine 40 x 10-6mol/L。
The preparation method of the neural stem cell culture medium comprises the following steps:
firstly, placing the culture medium in a sterile box, and using CO2Equilibrating for 30-40 minutes with CO2The concentration is 5-10%, and the temperature is 32-34 ℃;
secondly, placing the neural stem cells in a culture medium for culturing, raising the temperature in a sterile box to 0.5 ℃ every 2 hours until the temperature is raised to 36.5-37.5 ℃, and standing for culturing for 5-10 hours;
thirdly, adding the culture medium in the first step into the sterile box again, repeating the second step for a plurality of times, wherein the number of times of adding the culture medium is 3-5 times, and the content of the added culture medium is gradually reduced by half;
and fourthly, digesting the neural stem cell after the fusion degree of the neural stem cell reaches 85-92%, and carrying out subculture according to the proportion of 1:2 or 1:4 to obtain the neural stem cell.
The neural stem cells were obtained using the contents in examples 1 to 3 and comparative example, respectively, and then observed using a microscope, and the results of observation were as follows:
the number of neural stem cells obtained in examples 1 to 3 was normal, the number of neural stem cells obtained in comparative example was significantly lower than that obtained in examples 1 to 3, and a certain number of post-dead stem cells were present in comparative example.
The content of the comparative example is lacking insulin and selenium, and insulin plays an important role in the survival and growth of cells and provides sufficient nutrients for the survival and growth of cells. Meanwhile, selenium is a co-factor generated by glutathione, contributes to hydrolysis of peroxide and superoxide, can prevent damage of cells, provides favorable conditions for survival and rapid proliferation of the cells, and ensures the integrity of the cells.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (7)
1. A culture medium for rapid expansion of neural stem cells, comprising: basal medium and nutrient components;
the basic culture medium is a mixed solution of DMEM and F12, and the volume ratio is 1: 1;
the nutrient components comprise: glucose, glutamine, NaHCO3Penicillin, streptomycin, insulin, transferrin, progesterone, putrescine and selenium.
2. The culture medium for the rapid expansion of the neural stem cells as claimed in claim 1, wherein the contents of the components of the nutrient components are as follows: glucose 6-15g/L, glutamine 3 x 10-3mol/L-8*10-3mol/L，NaHCO35*10-3mol/L-12*10-3mol/L, penicillin 60-90mg/ml, streptomycin 65-85mg/ml, insulin 2.8-3.5 mu g/ml, transferrin 130-250ng/ml, progesterone 8 x 10-9mol/L-15*10-9mol/L, putrescine 30 x 10-6mol/L-50*10-6mol/L, selenium 50 x 10-9mol/L-60*10-9mol/L。
3. The culture medium for the rapid expansion of the neural stem cells as claimed in claim 1, wherein the contents of the components of the nutrient components are as follows: glucose 10g/L, Glutamine 5 x 10-3mol/L，10*10-3mol/L, penicillin 75mg/ml, streptomycin 75mg/ml, insulin 3.0 μ g/ml, transferrin 190ng/ml, progesterone 12 x 10-9mol/L, putrescine 40 x 10-6mol/L, selenium 55 x 10-9mol/L。
4. A use method of a culture medium for rapid expansion of neural stem cells, the use method comprises the following steps:
firstly, placing the culture medium in a sterile box, and using CO2Balancing for 30-40 minutes;
secondly, placing the neural stem cells in a culture medium for culturing, and standing for 5-10 hours;
thirdly, adding the culture medium in the first step into the sterile box again, repeating the second step for a plurality of times, wherein the number of times of adding the culture medium is 3-5 times;
and fourthly, digesting the neural stem cell after the fusion degree of the neural stem cell reaches 85-92%, and carrying out subculture according to the proportion of 1:2 or 1:4 to obtain the neural stem cell.
5. The method for using the culture medium for the rapid expansion of the neural stem cells as claimed in claim 4, wherein the CO in the first step2The concentration is 5-10%, and the temperature is 32-34 ℃.
6. The method for using the culture medium for the rapid expansion of the neural stem cells as claimed in claim 4, wherein the temperature in the sterile box is raised by 0.5 ℃ every 2 hours in the second step until the temperature is raised to 36.5-37.5 ℃.
7. The method for using the culture medium for the rapid expansion of the neural stem cells as claimed in claim 4, wherein the content of the culture medium added in the third step is gradually reduced by half.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999021966A1 (en) * | 1997-10-24 | 1999-05-06 | Neurospheres Holdings Ltd. | Erythropoietin-mediated neurogenesis |
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