Neural stem cell culture medium and culture method

Abstract

The invention belongs to the technical field of cells, and particularly relates to a neural stem cell culture medium and a culture method. The neural stem cell culture medium comprises a basic culture medium and the following components added into the basic culture medium in terms of final concentration: 0.5-10 mg / L of human insulin, 20-30 [mu] g / L of epidermal growth factors, 2-3 [mu] g / L of folic acid, 2-5 [mu] g / L of N-glutamine, 10-40 [mu] g / L of N-acetylcysteine, 0.1-5 mg / L of ethanolamine, 0.5-2 mg / L of carnitine, 0.5-5 mg / L of vitexin glucoside and 15-25 [mu] g / L of plecanatide. The vitexin glucoside and the plecanatide play a synergistic effect, so that the rapid division and proliferation of the neural stem cells are remarkably promoted, the cell differentiation potential of the neural stem cells is maintained, and the clinical application of the neural stem cells can be fully guaranteed.

Description

Technical Field

[0001] The invention belongs to the field of cell technology, and particularly relates to a neural stem cell culture medium and a culture method. Background Art

[0002] Neural stem cells (NSCs) are a type of adult stem cell with self-renewal and multipotential differentiation potential. They can differentiate into neurons, astrocytes, and oligodendrocytes, and are a core cell type for central nervous system development and injury repair. With the rapid development of biomedicine, NSCs have shown great clinical potential in the treatment of neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), spinal cord injury, and stroke sequelae.

[0003] Neural stem cells can be divided into embryonic-derived neural stem cells, umbilical cord blood-derived neural stem cells, and bone marrow mesenchymal stem cells based on their source. The in vitro culture of neural stem cells is the cornerstone for studying their biological characteristics, screening drugs, and developing cell therapies. However, neural stem cells are extremely fragile in the in vitro environment, and their self-renewal ability is easily affected by culture conditions. Primary neural stem cells are usually derived from embryonic or adult brain tissue, which is difficult to obtain and limited in number. During in vitro expansion, they are prone to spontaneous differentiation or aging, resulting in the loss of stem cell characteristics. In addition, the differentiation direction of neural stem cells is precisely regulated by growth factors, extracellular matrix, and physicochemical signals in the microenvironment. Existing culture systems often have many problems, resulting in low differentiation efficiency or abnormal cell function. However, the efficient in vitro expansion of neural stem cells and the stable maintenance of their differentiation potential remain the key bottlenecks restricting their basic research and clinical translation. Existing culture media have significant deficiencies in improving cell proliferation efficiency, differentiation potential, and even cost control, and there is an urgent need to develop more optimized technical solutions.

[0004] Therefore, there is an urgent need to develop a culture medium and culture method with clear ingredients that can effectively support the proliferation and differentiation of neural stem cells, so as to promote the clinical transformation of neural stem cell research. Summary of the Invention

[0005] The first objective of the present invention is to provide a neural stem cell culture medium comprising epidermal growth factor, folic acid, alanine glutathione, vitexin glucoside, and plecanatide, added to a basal culture medium. Vitexin glucoside and plecanatide exhibit a synergistic effect, significantly promoting the rapid division and proliferation of neural stem cells while maintaining their differentiation potential, thereby fully ensuring the clinical application of neural stem cells.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: A neural stem cell culture medium comprises a basal culture medium and the following components added to the basal culture medium: calculated in final concentrations, 0.5-10 mg / L human insulin, 20-30 μg / L epidermal growth factor, 2-3 μg / L folic acid, 2-5 μg / L alanine, 10-40 μg / L N-acetylcysteine, 0.1-5 mg / L ethanolamine, 0.5-2 mg / L carnitine, 0.5-5 mg / L vitexin glucoside, and 15-25 μg / L plecanatide.

[0007] Furthermore, the neural stem cell culture medium includes a basal culture medium and the following components added to the basal culture medium: in terms of final concentration, 5 mg / L human insulin, 25 μg / L epidermal growth factor, 2.5 μg / L folic acid, 2.5 μg / L alanine, 30 μg / L N-acetylcysteine, 3 mg / L ethanolamine, 1.2 mg / L carnitine, 3.5 mg / L vitexin glucoside, and 20 μg / L plecanatide.

[0008] Furthermore, the basal culture medium is DMEM / F12.

[0009] The second object of the present invention is to provide a method for culturing neural stem cells. The method is simple, feasible, and easy to operate, and provides a new technical approach for preparing neural stem cells with good proliferation activity and differentiation potential.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is: A method for culturing neural stem cells comprises the following steps: (1) inoculating the separated neural stem cells into the neural stem cell culture medium for culture, then digesting the cultured neural stem cells, and then centrifuging to obtain a precipitate; (2) Add neural stem cell culture medium to the centrifuged pellet to resuspend it, and then perform subculture.

[0011] Furthermore, the culturing time in step (1) is 4-6 days.

[0012] Furthermore, the culture conditions in step (1) and step (2) are both 37° C. and 5% CO 2 .

[0013] Furthermore, the cell density of the neural stem cells inoculated in step (1) and the cell density of the neural stem cells after resuspension in step (2) are both 1×10 5 -5×10 5 pieces / mL.

[0014] Furthermore, the ratio of the subculture in step (2) is 1:2-3, and the culture medium is replaced every 2-3 days during the subculture process.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly: 1. The present invention provides a neural stem cell culture medium, in which vitexin glucoside added to the basal culture medium has a significant antioxidant effect, helps protect neural stem cells from damage by oxidative stress, effectively scavenges free radicals, and can also provide sufficient energy and substances for neural stem cells, effectively promoting the proliferation and differentiation of neural stem cells. Plecanatide helps maintain the normal structure and function of cells, reduces cell aging, and provides a good microenvironment for the differentiation and proliferation of neural stem cells. Vitexin glucoside and Plecanatide both play a synergistic role, by reducing oxidative stress, protecting neural stem cells from damage, significantly promoting the rapid division and proliferation of neural stem cells, and maintaining their cell differentiation potential, which can fully guarantee the clinical application of neural stem cells.

[0016] 2. The present invention provides a method for culturing neural stem cells. The method is simple, feasible, and easy to operate, and provides a new technical idea for preparing neural stem cells with good proliferation activity and differentiation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a morphological diagram of the neural stem cells cultured in the present invention; Figure 2 This is a diagram showing the proliferation results of neural stem cells cultured according to the present invention; Figure 3 This is a diagram showing the expression of MAP2 and GFAP detected by Western blot in the present invention. DETAILED DESCRIPTION

[0018] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it is not intended that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art of the present invention, without departing from the inventive concept, several simple deductions or replacements can also be made, all of which should be considered to belong to the scope of protection of the present invention. The specific conditions not indicated in the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0019] Example 1: A neural stem cell culture medium comprises a DMEM / F12 basal culture medium and the following components added to the basal culture medium: the components, calculated in final concentrations, are: 5 mg / L human insulin, 25 μg / L epidermal growth factor, 2.5 μg / L folic acid, 2.5 μg / L alanine, 30 μg / L N-acetylcysteine, 3 mg / L ethanolamine, 1.2 mg / L carnitine, 3.5 mg / L vitexin glucoside, and 20 μg / L plecanatide.

[0020] This embodiment also provides a method for culturing neural stem cells, which specifically includes the following steps: (1) Isolation and collection of primary neural stem cells: After the newborn SD rats (within 48 hours of birth) were killed, the brain tissue was isolated in a clean bench and rinsed thoroughly with physiological saline. The meninges were peeled off under a microscope, and the hippocampus tissue was isolated. It was washed three times with PBS to remove blood and impurities, and transferred to DMEM / F12 medium. Accutase enzyme was added and digested at 37°C for 10 minutes, and then DMEM / F12 medium was added to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in DMEM / F12 medium, and then filter through a 70 μm cell sieve to remove undigested tissue blocks to obtain a single cell suspension. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and obtain neural stem cells; Neural stem cells were cultured at a density of 3×10 5 cells / mL were inoculated into the above-mentioned neural stem cell culture medium and cultured in a 37°C, 5% CO2 incubator for 5 days to form neurospheres. The size and density of the neurospheres were observed under a microscope. At this time, the diameter of the neurospheres was 100-200 μm. Accutase was added and digested at 37°C for 10 min. The cells were centrifuged at 1200 rpm for 5 min and the supernatant was discarded. (2) Subculture: Add fresh neural stem cell culture medium to the pellet after centrifugation to resuspend the neural stem cells and adjust the cell density to 3×10 5 cells / mL, placed in a 37°C, 5% CO2 incubator, and subcultured at a ratio of 1:2. The neural stem cell culture medium was replaced every 2 days. When the cells were subcultured to the third generation, the morphology of the P3 generation neural stem cells was observed under a microscope and used for subsequent experiments.

[0021] Example 2: A neural stem cell culture medium comprises a DMEM / F12 basal medium and the following components added to the basal medium: 1.5 mg / L human insulin, 20 μg / L epidermal growth factor, 2 μg / L folic acid, 2 μg / L alanine, 10 μg / L N-acetylcysteine, 1 mg / L ethanolamine, 0.5 mg / L carnitine, 1.5 mg / L vitexin glucoside, and 15 μg / L plecanatide, calculated in final concentrations.

[0022] This embodiment also provides a method for culturing neural stem cells, which specifically includes the following steps: (1) Isolation and collection of primary neural stem cells: After the newborn SD rats (within 48 hours of birth) were killed, the brain tissue was isolated in a clean bench and rinsed thoroughly with physiological saline. The meninges were peeled off under a microscope, and the hippocampus tissue was isolated. It was washed three times with PBS to remove blood and impurities, transferred to DMEM / F12 medium, and Accutase enzyme was added. Digestion was performed at 37°C for 10 minutes, and then DMEM / F12 medium was added to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in DMEM / F12 medium, and then filter through a 70 μm cell sieve to remove undigested tissue blocks to obtain a single cell suspension. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and obtain neural stem cells; Neural stem cells were cultured at a density of 1×10 5 cells / mL were inoculated into the above-mentioned neural stem cell culture medium and cultured in a 37°C, 5% CO2 incubator for 6 days to form neurospheres. The size and density of the neurospheres were observed under a microscope. At this time, the diameter of the neurospheres was 100-200 μm. Accutase was added and digested at 37°C for 10 min. The cells were centrifuged at 1200 rpm for 5 min and the supernatant was discarded. (2) Subculture: Add fresh neural stem cell culture medium to the pellet after centrifugation to resuspend the neural stem cells and adjust the cell density to 3×10 5 cells / mL, placed in a 37°C, 5% CO2 incubator, and subcultured at a ratio of 1:2. The neural stem cell culture medium was replaced every 3 days. When the cells were subcultured to the third generation, the morphology of the P3 generation neural stem cells was observed under a microscope and used for subsequent experiments.

[0023] Example 3: A neural stem cell culture medium comprises a DMEM / F12 basal medium and the following components added to the basal medium: human insulin 10 mg / L, epidermal growth factor 30 μg / L, folic acid 3 μg / L, alanine 5 μg / L, N-acetylcysteine ​​40 μg / L, ethanolamine 5 mg / L, carnitine 2 mg / L, vitexin glucoside 5 mg / L, and plecanatide 25 μg / L, calculated in final concentrations.

[0024] This embodiment also provides a method for culturing neural stem cells, which specifically includes the following steps: (1) Isolation and collection of primary neural stem cells: After the newborn SD rats (within 48 hours of birth) were killed, the brain tissue was isolated in a clean bench and rinsed thoroughly with physiological saline. The meninges were peeled off under a microscope, and the hippocampus tissue was isolated. It was washed three times with PBS to remove blood and impurities, transferred to DMEM / F12 medium, and Accutase enzyme was added. Digestion was performed at 37°C for 10 minutes, and then DMEM / F12 medium was added to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in DMEM / F12 medium, and then filter through a 70 μm cell sieve to remove undigested tissue blocks to obtain a single cell suspension. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and obtain neural stem cells; Neural stem cells were cultured at a density of 5×10 5 cells / mL were inoculated into the above-mentioned neural stem cell culture medium and cultured in a 37°C, 5% CO2 incubator for 4 days to form neurospheres. The size and density of the neurospheres were observed under a microscope. At this time, the diameter of the neurospheres was 100-200 μm. Accutase was added and digested at 37°C for 10 min. The cells were centrifuged at 1200 rpm for 5 min and the supernatant was discarded. (2) Subculture: Add fresh neural stem cell culture medium to the pellet after centrifugation to resuspend the neural stem cells and adjust the cell density to 3×10 5 cells / mL, placed in a 37°C, 5% CO2 incubator, and subcultured at a ratio of 1:2. The neural stem cell culture medium was replaced every 2 days. When the cells were subcultured to the third generation, the morphology of the P3 generation neural stem cells was observed under a microscope and used for subsequent experiments.

[0025] Comparative Example 1: A neural stem cell culture medium comprises a DMEM culture medium and the following components added to a basal culture medium: the components, calculated in final concentrations, are: 5 mg / L human insulin, 25 μg / L epidermal growth factor, 2.5 μg / L folic acid, 2.5 μg / L alanine, 30 μg / L N-acetylcysteine, 3 mg / L ethanolamine, 1.2 mg / L carnitine, and 3.5 mg / L vitexin glucoside; that is, compared with Example 1, no plecanatide is added.

[0026] Comparative Example 2: A neural stem cell culture medium comprises DMEM culture medium and the following components added to a basal culture medium: the components, calculated in final concentrations, are 5 mg / L human insulin, 25 μg / L epidermal growth factor, 2.5 μg / L folic acid, 2.5 μg / L alanine, 30 μg / L N-acetylcysteine, 3 mg / L ethanolamine, 1.2 mg / L carnitine, and 20 μg / L plecanatide, that is, compared with Example 1, no vitexin glucoside is added.

[0027] Comparative Example 3: A neural stem cell culture medium comprises DMEM culture medium and the following components added to a basal culture medium: the components, calculated in final concentrations, are: 5 mg / L human insulin, 25 μg / L epidermal growth factor, 2.5 μg / L folic acid, 2.5 μg / L alanine, 30 μg / L N-acetylcysteine, 3 mg / L ethanolamine, 1.2 mg / L carnitine, 3.5 mg / L ascorbyl glucoside, and 20 μg / L plecanatide; that is, compared with Example 1, vitexin glucoside is replaced with ascorbyl glucoside.

[0028] Comparative Example 4: A neural stem cell culture medium comprises a DMEM culture medium and the following components added to a basal culture medium: the components, calculated in final concentrations, are: 5 mg / L human insulin, 25 μg / L epidermal growth factor, 2.5 μg / L folic acid, 2.5 μg / L alanine, 30 μg / L N-acetylcysteine, 3 mg / L ethanolamine, 1.2 mg / L carnitine, 3.5 mg / L vitexin glucoside, and 5 μg / L plecanatide; that is, compared with Example 1, the amount of plecanatide used is reduced to 5 μg / L.

[0029] Comparative Example 5: A neural stem cell culture medium comprises a DMEM culture medium and the following components added to a basal culture medium: the components, calculated in final concentrations, are: 5 mg / L human insulin, 25 μg / L epidermal growth factor, 2.5 μg / L folic acid, 2.5 μg / L alanine, 30 μg / L N-acetylcysteine, 3 mg / L ethanolamine, 1.2 mg / L carnitine, 3.5 mg / L vitexin glucoside, and 40 μg / L plecanatide; that is, compared with Example 1, the amount of plecanatide is increased to 40 μg / L.

[0030] The beneficial effects of the present invention are described below with reference to specific test examples.

[0031] Test Example 1: The morphology of the P3 generation neural stem cells obtained by culture in Example 1 and Comparative Examples 1-5 was observed under a microscope. The results were as follows: Figure 1 shown.

[0032] Observation of the morphology of the neural stem cells cultured in Example 1 and Comparative Examples 1-5 revealed that the cells in each group existed as neurospheres. The cells in Example 1 were significantly more aggregated than those in Comparative Examples 1-5, demonstrating healthy growth. Compared to Example 1, the neural stem cells in Comparative Examples 1-5 were more dispersed, mostly single cells, indicating significantly lower cell viability than in Example 1.

[0033] Test Example 2: The neural stem cells of the P3 generation with good growth status in Example 1 and Comparative Examples 1-5 were cultured in vitro. Specifically, the neural stem cells of Example 1 and Comparative Examples 1-5 were seeded into 96-well plates, respectively, and cultured using the corresponding neural stem cell culture medium (e.g., the neural stem cells of Example 1 were cultured using the neural stem cell culture medium of Example 1). The cell seeding density was 1×10 5 Each group was set up with 5 replicate wells and cultured at 37°C and 5% CO2 for 3 days. The culture medium was changed every 2 days during the culture period. Then CCK-8 solution was added at 5 mg / mL and incubated at 37°C and 5% CO2 in the dark for 4 hours. The absorbance value was measured at 450 nm using a microplate reader, and the average value was taken. The test results were recorded in Figure 2 middle.

[0034] The absorbance value is proportional to the number of living cells. Figure 2From the experimental data, it can be seen that the absorbance value (OD value) of Example 1 is significantly higher than that of Comparative Examples 1-5. This result shows that the culture medium of the present invention can effectively promote the proliferation of neural stem cells and improve their activity. Specifically, compared with Example 1, Comparative Example 3 replaces vitexin glucoside with vitamin C glucoside, and its effect on the proliferation of neural stem cells is inferior to that of Example 1. Comparative Examples 5 and 4 adjusted the dosage of plecanatide, and the absorbance value decreased. It can be seen that plecanatide can promote the proliferation of neural stem cells within a certain dosage range. Comparative Examples 1 and 2 omitted plecanatide and vitexin glucoside, respectively, and the absorbance value decreased significantly. The above results show that the two components of plecanatide and vitexin glucoside can work synergistically in the culture medium, work together, enhance the activity of neural stem cells, and are beneficial to cell proliferation and growth. This may be attributed to the significant antioxidant effect of vitexin glucoside in the culture medium, which significantly reduces the levels of reactive oxygen species (ROS) and nitrogen free radicals (RNS) by regulating the intracellular redox balance, thereby protecting neural stem cells from oxidative stress-induced apoptosis and aging. The synergistic effect of plecanatide and vitexin glucoside helps maintain the normal structure and function of cells, reduces cell aging, and provides a good microenvironment for the differentiation and proliferation of neural stem cells, thereby promoting the proliferation of neural stem cells.

[0035] Test Example 3: Western Blot was used to analyze the differentiation ability of neural stem cells. 5 The cells were inoculated at a density of 100 μg / mL into a 24-well plate pre-coated with 0.1 mg / mL polylysine, and then the culture medium corresponding to Example 1 and Comparative Examples 1-5 was added respectively. The cells were cultured in an incubator at 37°C and 5% CO2. After the cells adhered, they were replaced with a differentiation medium consisting of 93% DMEM low-glucose medium, 2% B27 additive, 2% fetal bovine serum, 1% double antibody, 1% L-alanyl-L-glutamine solution (GlutaMAX), and 1% non-essential amino acids. After culturing for 7 days, the proliferated and differentiated cells were collected, washed 3 times with PBS buffer for 5 minutes each time, centrifuged at 4°C, and the supernatant was discarded. The cells were lysed on ice with RIPA lysis buffer for 15 minutes. The lysate was placed in a 4°C centrifuge and centrifuged at 12,000 rpm for 5 minutes to obtain a total protein extract.

[0036] The concentration of the resulting protein extract was determined according to the instructions of the BCA protein quantification kit. After the determination, 5× SDS loading buffer was added. The concentrations of Example 1 and Comparative Examples 1-5 were adjusted to the same concentration based on the measured neural stem cell protein concentration, and the samples were boiled for 10 minutes. After cooling to room temperature, the denatured proteins from the different groups were loaded and electrophoresed on a 10% polyacrylamide gel. Electrophoresis was terminated when bromophenol blue reached the bottom of the electrophoresis tank. The protein samples were transferred to a PVDF membrane using a wet transfer method. After transfer, the PVDF membrane was removed and blocked with 5% skim milk blocking buffer at room temperature for 1.5 hours. The primary antibodies (MAP2, GFAP, and β-actin) were diluted in blocking buffer and incubated on a shaker at 4°C overnight. The membranes were then rinsed three times with 1× TBST buffer for 10 minutes each. The secondary antibodies were then diluted in blocking buffer and incubated on a shaker at room temperature for 1.5 hours. The membranes were then rinsed three times with 1× TBST buffer for 10 minutes each. The developer solution A and solution B were mixed in a volume ratio of 1:1 to obtain the ECL reagent. The prepared reagent was evenly covered on the surface of the PVDF membrane, reacted for 2 minutes, and then detected using a chemiluminescence imaging system. The results are shown in Figure 2. Figure 3 shown.

[0037] Microtubule-associated protein 2 (MAP2) is a neuron-specific marker, and its expression level is closely correlated with neuronal maturity and synaptic formation capacity. Glial fibrillary acidic protein (GFAP) is a marker for astrocytes. During neural stem cell differentiation, changes in MAP2 and GFAP expression levels are important indicators for assessing differentiation direction and maturity.

[0038] Depend on Figure 3 The results show that the expression levels of MAP2 and GFAP in the neural stem cells after proliferation and differentiation in Example 1 are higher than those in Comparative Examples 1-5, indicating that the differentiation ability of the neural stem cells obtained by culturing with the culture medium of the present invention is significantly improved, and plecanatide and vitexin glucoside may maintain the intracellular redox balance through synergistic action, thereby promoting the differentiation of neural stem cells into functional neurons and astrocytes.

[0039] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A neural stem cell culture medium, characterized in that The invention comprises a basal culture medium and the following components added to the basal culture medium: in terms of final concentration, human insulin 0.5-10 mg / L, epidermal growth factor 20-30 μg / L, folic acid 2-3 μg / L, alanine 2-5 μg / L, N-acetylcysteine ​​10-40 μg / L, ethanolamine 0.1-5 mg / L, carnitine 0.5-2 mg / L, vitexin glucoside 0.5-5 mg / L, and plecanatide 15-25 μg / L.

2. The neural stem cell culture medium according to claim 1, wherein The medium includes the following components added to the medium: human insulin 5 mg / L, epidermal growth factor 25 μg / L, folic acid 2.5 μg / L, alanine 2.5 μg / L, N-acetylcysteine ​​30 μg / L, ethanolamine 3 mg / L, carnitine 1.2 mg / L, vitexin glucoside 3.5 mg / L, and plecanatide 20 μg / L, calculated at the final concentration.

3. The neural stem cell culture medium according to claim 1, characterized in that The basic culture medium is DMEM / F12.

4. A method for culturing neural stem cells, characterized in that: The following steps are involved: (1) inoculating the separated neural stem cells into the neural stem cell culture medium according to any one of claims 1 to 3 for culture, then digesting the cultured neural stem cells, and then centrifuging to obtain a precipitate; (2) Add neural stem cell culture medium to the centrifuged pellet to resuspend it, and then perform subculture.

5. The method for culturing neural stem cells according to claim 4, characterized in that: The culture time in step (1) is 4-6 days.

6. The method for culturing neural stem cells according to claim 4, characterized in that: The culture conditions in step (1) and step (2) are both 37°C and 5% CO2.

7. The method for culturing neural stem cells according to claim 4, characterized in that: The cell density of the neural stem cells inoculated in step (1) and the cell density of the neural stem cells after resuspension in step (2) were both 1×10 5 -5×10 5 pieces / mL.

8. The method for culturing neural stem cells according to claim 4, characterized in that: During the subculture process in step (2), the culture medium is replaced every 2-3 days.

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