阅读说明：本技术 一种用于神经干细胞快速扩增的培养基 (Culture medium for rapid amplification of neural stem cells ) 是由 胡俊杰 于 2019-11-25 设计创作，主要内容包括：本发明公开一种用于神经干细胞快速扩增的培养基,所述培养基包括：基础培养基和营养成分,基础培养基为DMEM与F12的混合液,营养成分包括：葡萄糖、谷氨酰胺、NaHCO<Sub>3</Sub>、青霉素、链霉素、胰岛素、转铁蛋白、黄体酮、腐胺和硒。本发明中的培养基中含有胰岛素和硒,胰岛素对于细胞的存活和生长有重要作用,为细胞的存活和生长提供了充分的营养物质,同时,硒是谷胱甘肽产生的合作因子,有助于过氧化物和超氧化物的水解,可以防止细胞的损伤,为细胞的存活和快速增殖提供了有利的条件,保证了其完整性,在使用方法中对培养基使用CO<Sub>2</Sub>进行平衡,添加了多次培养基,保证了培养基中营养物质的充分性,且培养基的含量逐次减半,节省了资源。(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, NaHCO 3 Penicillin, 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 method 2 The 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.)

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.

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^{- 3}mol/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^{- 3}mol/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.