Preparation method of neural stem cells with high multiplication capacity

Abstract

The invention relates to a method for preparing neural stem cells with high multiplication capacity, and belongs to the technical field of biology. The method comprises the following steps: separating primary neural stem cells from mammal embryo brain tissues, taking DMEM / F12 as a basic culture medium, adding B27, N2, glutamine and double antibodies, and then adding epidermal growth factors, basic fibroblast growth factors, insulin-like growth factor-1, Y-27632 and heparin to form an optimized proliferation culture medium. The method comprises the following steps: inoculating cells into a culture dish coated with polylysine and laminin, culturing under proper conditions, and digesting and subculturing after neurospheres reach a certain diameter; the method can improve the proliferation efficiency of neural stem cells and maintain stemness and differentiation potential, adopts a serum-free system, is simple and convenient to operate, and is suitable for large-scale amplification and research application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a preparation method of neural stem cells with high proliferation capacity. BACKGROUND

[0002] Neural stem cells are a type of cells with self-renewal ability and multi-directional differentiation potential, which can differentiate into neuron, astrocyte and oligodendrocyte, etc. neural cell types. They have broad application prospects in the fields of nervous system disease treatment, neural regeneration research and drug screening. However, the current culture and expansion of neural stem cells face many challenges, which limit their development in basic research and clinical application.

[0003] Traditional neural stem cell culture methods mainly rely on fetal bovine serum-containing medium or serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and other cytokines. Although these methods can maintain the proliferation of neural stem cells to some extent, they have low proliferation efficiency, short maintenance time of cell stemness, and are prone to differentiation or aging. For example, under conventional culture conditions, the doubling time of neural stem cells is long, usually 48-72 hours, and after several generations of culture, the proliferation capacity of the cells will decrease significantly, and the expression level of stemness markers such as Nestin and Sox2 will also decrease, resulting in impaired differentiation potential of the cells.

[0004] In addition, some measures taken in the prior art to improve the proliferation capacity of neural stem cells, such as increasing the concentration of cytokines or adding serum, often cause a series of side effects. High concentration of cytokines can cause abnormal cell proliferation and even carcinogenesis, while the addition of serum will introduce heterologous components, increase the risk of immune rejection and batch-to-batch differences, and also may induce premature differentiation of neural stem cells, affecting their purity and function.

[0005] In recent years, researchers have tried to improve the proliferation characteristics of neural stem cells through gene editing, small molecule compound regulation or three-dimensional culture, but these methods still have problems such as technical complexity, high cost or insufficient practicality. For example, gene editing technology may cause gene mutation or off-target effects, and long-term action of small molecule compounds may be toxic to cells, while scaling up of three-dimensional culture system is difficult.

[0006] Therefore, it is a technical problem to be solved in the field to develop an efficient, stable and controllable neural stem cell preparation method to achieve high proliferation capacity in vitro while maintaining good stemness and differentiation potential. The present application significantly improves the proliferation efficiency of neural stem cells through innovative culture system design, including specific combination of culture medium components, signal pathway regulation factors and optimization of culture conditions, providing a new technical means for basic research and clinical application of neural stem cells. SUMMARY

[0007] The present application aims to provide a preparation method of neural stem cells with high proliferation capacity, which can significantly improve the in vitro proliferation efficiency of neural stem cells, prolong the self-renewal capacity, and maintain the multi-directional differentiation potential of neural stem cells, thereby providing technical support for large-scale expansion and application of neural stem cells.

[0008] The object of the present application can be achieved by the following technical solutions:

[0009] A preparation method of neural stem cells with high proliferation capacity comprises the following steps:

[0010] W1. Primary neural stem cells are isolated from mammalian embryonic brain tissue;

[0011] W2. A basic culture medium is prepared by adding B27 additives, N2 additives, glutamine and penicillin-streptomycin double antibiotics to DMEM / F12 medium;

[0012] W3. An optimized proliferation culture medium is formed by adding epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1, Y-27632 and heparin to the basic culture medium;

[0013] W4. The primary neural stem cells are inoculated in a culture dish coated with polylysine and laminin at a density of 2×10 4 cells / cm 2 , and cultured in the optimized proliferation culture medium at 37℃ and 5% CO2, with the medium being replaced every 2 days;

[0014] W5. When the diameter of the neural spheres reaches 100-150 μm, the neural spheres are digested into a single cell suspension with trypsin-EDTA digestion solution, and subcultured at a density of 1×10 4 cells / cm 2 , and the optimized proliferation culture medium is continuously used for culture.

[0015] According to a preferred technical solution of the present application, the concentration of epidermal growth factor in step W3 is 10-30 ng / mL, the concentration of basic fibroblast growth factor is 10-30 ng / mL, the concentration of insulin-like growth factor-1 is 5-20 ng / mL, the concentration of Y-27632 is 1-10 μM, and the concentration of heparin is 1-10 μg / mL.

[0016] As a preferred technical solution of the present application, the concentration of epidermal growth factor in step W3 is 20 ng / mL, the concentration of basic fibroblast growth factor is 20 ng / mL, the concentration of insulin-like growth factor-1 is 10 ng / mL, the concentration of Y-27632 is 5 μM, and the concentration of heparin is 5 μg / mL.

[0017] As a preferred technical solution of the present application, the addition amount of B27 supplement in step W2 is 1x, the addition amount of N2 supplement is 1x, the concentration of glutamine is 2 mM, and the concentration of penicillin-streptomycin double antibody is 100 U / mL.

[0018] As a preferred technical solution of the present application, the concentration of polylysine in step W4 is 10 μg / mL, the concentration of laminin is 10 μg / mL, and the coating condition is 37°C incubation for 2 hours.

[0019] As a preferred technical solution of the present application, LDN-193189 and SB-431542 are further added to the optimized proliferation medium in step W3, wherein the concentration of LDN-193189 is 0.1-1 μM, and the concentration of SB-431542 is 1-10 μM.

[0020] As a preferred technical solution of the present application, the concentration of LDN-193189 is 0.5 μM, and the concentration of SB-431542 is 5 μM.

[0021] As a preferred technical solution of the present application, the number of passages in step W5 is not more than 20 generations, and the detection of cell stemness and differentiation potential is performed every 5 generations.

[0022] As a preferred technical solution of the present application, a low-oxygen culture condition is adopted during the culture process in step W4, and the oxygen concentration is 2-5%.

[0023] As a preferred technical solution of the present application, the mammal is a mouse, a rat, or a human.

[0024] The beneficial effects of the present application are:

[0025] (1) By optimizing the medium components and adding a combination of various growth factors and signal pathway inhibitors, the doubling time of neural stem cells in the method of the present application can be shortened to 24-36 hours, which improves the proliferation efficiency by more than 50% compared with the traditional method.

[0026] (2) The culture system of the present application can maintain high expression of stemness markers Nestin, Sox2 and Oct4 in neural stem cells, and after 20 generations of culture, the expression level of stemness markers remains more than 80% of the initial level.

[0027] (3) The neural stem cells cultured by the method of the present application can be efficiently differentiated into neurons, astrocytes and oligodendrocytes after induction differentiation, which is basically consistent with the differentiation ability of primary neural stem cells.

[0028] (4) The serum-free culture system used in the present application reduces the influence of heterologous components, and the culture conditions are uniform, which has small batch-to-batch difference and is beneficial to the large-scale expansion and quality control of neural stem cells.

[0029] (5) The preparation method of the present application does not need complex gene editing or special equipment, and can be realized only by optimization of culture medium composition and culture conditions, which is suitable for wide application in laboratory and clinical research. DETAILED DESCRIPTION

[0030] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object, the specific embodiments, structures, features and effects according to the present application are described in detail as follows in combination with examples.

[0031] Experimental materials

[0032] Experimental animals: C57BL / 6 mice at 14 days of pregnancy, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0033] Reagents: DMEM / F12 medium (Gibco, item number 11330032), B27 supplement (Gibco, item number 17504044), N2 supplement (Gibco, item number 17502048), glutamine (Gibco, item number 25030081), penicillin-streptomycin double antibody (Gibco, item number 15140122), epidermal growth factor (EGF, PeproTech, item number 100-15), basic fibroblast growth factor (bFGF, PeproTech, item number 100-18B), insulin-like growth factor-1 (IGF-1, PeproTech, item number 100-11), Y-27632 (Selleck, item number S1049), heparin (Sigma, item number H3149), LDN-193189 (Selleck, item number S7280), SB-431542 (Selleck, item number S1067), trypsin-EDTA digestion solution (Gibco, item number 25200056), polylysine (Sigma, item number P2636), laminin (Sigma, item number L2020).

[0034] Major instruments: CO2incubator (Thermo Fisher, model 3111), inverted microscope (Olympus, model CKX41), flow cytometer (BD, model FACSCalibur), real-time fluorescence quantitative PCR instrument (Applied Biosystems, model 7500), microplate reader (Bio-Tek, model Synergy H1).

[0035] Isolation of primary neural stem cells

[0036] Take 14-day pregnant C57BL / 6 mice, execute by cervical dislocation, take out the embryo under sterile conditions, separate the cerebral cortex tissue, and place it in a culture dish containing ice-cold PBS. The tissue is cut into small pieces with ophthalmic scissors, and 0.25% trypsin-EDTA digestion solution is added. Digestion is carried out at 37°C for 15 minutes, with gentle shaking during the process. After digestion, stop the digestion by adding DMEM medium containing 10% fetal bovine serum, filter with a 100-mesh cell sieve, collect the filtrate, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the precipitate in the base medium to prepare a single-cell suspension.

[0037] Cell proliferation detection

[0038] MTT method was used to detect cell proliferation. Cells were seeded in a 96-well plate at a density of 5×10 3 cells / well, with 5 replicate wells for each group. At 24h, 48h, 72h, 96h and 120h after seeding, 20μL of MTT solution (5mg / mL) was added to each well, and incubated at 37°C for 4 hours. The supernatant was discarded, and 150μL of DMSO was added to each well, and shaken for 10 minutes to fully dissolve the formazan. The absorbance value (OD value) was measured at 570nm wavelength using a microplate reader, and the cell proliferation rate was calculated.

[0039] Detection of stemness markers

[0040] Real-time fluorescence quantitative PCR (qPCR) and flow cytometry were used to detect the expression of stemness markers. The qPCR steps are as follows: total RNA is extracted, reverse transcribed into cDNA, and PCR amplification is performed using cDNA as the template. The primer sequences are as follows:

[0041] Nestin: upstream primer 5'-CAGCAGCTGAAGAGCAAGGA-3', downstream primer 5'-GCTGGTGGTGTTGGTGTTCT-3';

[0042] Sox2: upstream primer 5'-TCGCTGCTGCTGATGTTG-3', downstream primer 5'-GCTGCTGCTGCTGATGTT-3';

[0043] Oct4: Upstream primer 5'-CAGCAGCTGAAGAGCAAGGA-3', downstream primer 5'- GCTGGTGGTGTTGGTGTTCT-3';

[0044] GAPDH: Upstream primer 5'-GAAGGTGAAGGTCGGAGTC-3', downstream primer 5'- GAAGATGGTGATGGGATTTC-3'.

[0045] The reaction conditions were: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds, a total of 40 cycles. The relative expression amount of the target gene was calculated by 2 -ΔΔCt

[0046] When detecting by flow cytometry, the cells were collected, washed with PBS twice, and then fluorescently labeled anti-Nestin antibody (BD, item number 560513) was added and incubated at 4°C for 30 minutes. After washing with PBS, the proportion of Nestin positive cells was detected by flow cytometry.

[0047] Detection of differentiation potential

[0048] The neural stem cells were inoculated in culture dishes coated with polylysine at a density of 1 x 10 4 cells / cm 2 After 7 days of culture, immunofluorescence staining was performed. The antibodies used were as follows:

[0049] Anti-β-III tubulin antibody (neuron marker, Abeam, item number ab18207);

[0050] Anti-GFAP antibody (astrocyte marker, Abeam, item number ab4674);

[0051] Anti-MBP antibody (oligodendrocyte marker, Abeam, item number ab40390).

[0052] Staining steps: 4% paraformaldehyde fixation for 15 minutes, 0.1% Triton X-100 permeation for 10 minutes, 10% goat serum blocking for 1 hour, addition of primary antibody and incubation at 4°C overnight, PBS washing for 3 times, addition of fluorescently labeled secondary antibody (Alexa Fluor 488), incubation at 37°C for 1 hour, DAPI staining of the nucleus, and observation under a fluorescence microscope after mounting and counting of the proportion of positive cells.

[0053] Example 1

[0054] Experimental procedure

[0055] ​W1. Primary neural stem cells were isolated from the cerebral cortex of C57BL / 6 mouse embryos at 14 days of gestation, following the steps in the Materials and Methods section above.

[0056] W2. Take DMEM / F12 medium, add 1×B27 additive, 1×N2 additive, 2mM glutamine, and 100U / mL penicillin-streptomycin antibiotic, mix well, and store at 4℃.

[0057] W3. Add 20 ng / mL EGF, 20 ng / mL bFGF, 10 ng / mL IGF-1, 5 μM Y-27632 and 5 μg / mL heparin to the basal culture medium, mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, and store at 4°C.

[0058] W4. Cell Seeding and Culture: Primary neural stem cells were seeded at a rate of 2 × 10⁴ cells / year. 4 Inoculated at a density of cells / cm² into culture dishes coated with 10 μg / mL poly-L-lysine and 10 μg / mL laminin, and cultured in an optimized proliferation medium at 37°C and 5% CO2, with the medium being changed every 2 days.

[0059] W5. When the neurosphere diameter reaches 100-150 μm, digest it into a single-cell suspension using trypsin-EDTA digestion solution at a concentration of 1×10⁻⁶. 4 pcs / cm 2 The cells were passaged at high density and cultured using the optimized proliferation medium.

[0060] Example 2

[0061] Except for the addition of 0.5 μM LDN-193189 and 5 μM SB-431542 to the optimized proliferation medium, the other steps were the same as in Example 1.

[0062] Example 3

[0063] The culture conditions were set at 37°C, 5% CO2, and 3% O2, with other steps the same as in Example 1.

[0064] Comparative Example 1

[0065] The optimized proliferation medium does not contain IGF-1, and the other steps are the same as in Example 1.

[0066] Comparative Example 2

[0067] Y-27632 was not added to the optimized proliferation medium, and the other steps were the same as in Example 1.

[0068] Comparative Example 3

[0069] The optimized proliferation medium does not contain heparin, and the other steps are the same as in Example 1.

[0070] Comparative Example 4

[0071] The EGF concentration in the proliferation medium was optimized to 10 ng / mL, and other steps were the same as in Example 1.

[0072] Comparative Example 5

[0073] The bFGF concentration in the proliferation medium was optimized to 30 ng / mL, and other steps were the same as in Example 1.

[0074] Comparative Example 6

[0075] The same as in Example 1, without adding LDN-193189 and SB-431542.

[0076] Comparative Example 7

[0077] The base medium was DMEM / F12 medium added with 10% fetal bovine serum, and other steps were the same as in Example 1, without adding cell factors such as EGF and bFGF.

[0078] Comparative Example 8

[0079] The proliferation medium was optimized to only add 20 ng / mL EGF and 20 ng / mL bFGF, without adding IGF-1, Y-27632 and heparin, and other steps were the same as in Example 1.

[0080] Comparative Example 9

[0081] The B27 additive was not added in the base medium, and other steps were the same as in Example 1.

[0082] Comparative Example 10

[0083] The culture conditions were set to 37°C, 5% CO2, 20% O2, and other steps were the same as in Example 1.

[0084] Performance detection results

[0085] Table 1: Cell proliferation ability detection data (OD value, x ± s)

[0086]

[0087] Note: The proliferation rate calculation formula is: (120h OD value-24h OD value) / 24h OD value x 100%.

[0088] Table 2: Cell stemness marker expression data of the 10th generation (relative expression amount, x ± s, with the primary cells being 1)

[0089]

[0090] Note: The relative expression of dry markers was set to GAPDH as an internal control, and the expression of primary cells was set to 1.

[0091] Table 3: Proportion of positive cells after 7 days of induction (% x ± s)

[0092] Group neurons (β-III tubulin + )]]> Astrocytes (GFAP + )]]> Oligodendrocytes (MBP + )]]> Example 1 62.5±3.2 28.7±2.5 8.8±1.5 Example 2 65.2±3.5 27.3±2.3 9.5±1.2 Example 3 63.8±3.3 28.1±2.4 8.1±1.3 Comparative Example 1 55.3±3.0 32.1±2.7 7.6±1.1 Comparative Example 2 52.1±2.8 35.2±2.9 6.7±1.0 Comparative Example 3 57.6±3.1 30.5±2.6 7.9±1.2 Comparative Example 4 49.8±2.7 37.6±3.0 5.6±0.9 Comparative Example 5 58.9±3.2 29.8±2.7 7.3±1.1 Comparative Example 6 62.5±3.2 28.7±2.5 8.8±1.5 Comparative Example 7 40.2±2.5 45.6±3.5 4.2±0.8 Comparative Example 8 59.3±3.3 30.1±2.8 7.5±1.2 Comparative Example 9 53.7±2.9 33.4±2.8 6.9±1.0 Comparative Example 10 58.2±3.1 31.3±2.7 7.5±1.1

[0093] Note: All data are the average of 3 independent experiments ± standard deviation (x ± s).

[0094] The above description is only the preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content to make equivalent embodiments with equivalent changes, without departing from the technical solution of the present application. Any modification, equivalent change and modification of the above embodiments made in accordance with the technical essence of the present application, without departing from the technical solution of the present application, still belongs to the scope of the technical solution of the present application.

Claims

1. A method for preparing neural stem cells having high proliferative capacity, characterized by, The method comprises the following steps: W1. isolating primary neural stem cells from mammalian embryonic brain tissue; W2. preparing a basic culture medium based on DMEM / F12 medium, adding B27 supplement, N2 supplement, glutamine and penicillin-streptomycin double antibiotic; W3. adding epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1, Y-27632 and heparin to the basic culture medium to form an optimized proliferation culture medium; W4. seeding the primary neural stem cells at a density of 2 x 10 4 cells / cm 2 in culture dishes coated with poly-lysine and laminin, culturing at 37°C in 5% CO2 using the optimized proliferation medium, changing the medium every 2 days; W5. When the neurospheres reached a diameter of 100-150 pm, they were digested with a trypsin-EDTA solution into a single cell suspension, which was passaged at a density of 1 x 10 4 cells / cm 2 and continued to be cultured using the optimized proliferation medium.

2. The production method according to claim 1, characterized by, The concentration of the epidermal growth factor in step W3 is 10-30 ng / mL, the concentration of the basic fibroblast growth factor is 10-30 ng / mL, the concentration of the insulin-like growth factor-1 is 5-20 ng / mL, the concentration of Y-27632 is 1-10 μM, and the concentration of heparin is 1-10 μg / mL.

3. The production method according to claim 2, characterized by, The concentration of the epidermal growth factor in step W3 is 20 ng / mL, the concentration of the basic fibroblast growth factor is 20 ng / mL, the concentration of the insulin-like growth factor-1 is 10 ng / mL, the concentration of Y-27632 is 5 μM, and the concentration of heparin is 5 μg / mL.

4. The method of claim 1, wherein, The addition amount of the B27 supplement in step W2 is 1×, the addition amount of the N2 supplement is 1×, the concentration of glutamine is 2 mM, and the concentration of penicillin-streptomycin double antibiotic is 100 U / mL.

5. The preparation method according to claim 1, characterized in that, The concentration of the polylysine in step W4 is 10 μg / mL, the concentration of the laminin is 10 μg / mL, and the coating condition is 37℃ incubation for 2 hours.

6. The method of claim 1, wherein, The optimized proliferation culture medium in step W3 further comprises LDN-193189 and SB-431542, wherein the concentration of the LDN-193189 is 0.1-1 μM, and the concentration of the SB-431542 is 1-10 μM.

7. The preparation method according to claim 6, characterized in that, The concentration of the LDN-193189 is 0.5 μM, and the concentration of the SB-431542 is 5 μM.

8. The method of claim 1, wherein, The number of passages in step W5 is not more than 20 generations, and the detection of cell stemness and differentiation potential is performed once every 5 generations.

9. The method of claim 1, wherein, The low-oxygen culture condition is adopted in the culture process of step W4, and the oxygen concentration is 2-5%.

10. The method of claim 1, wherein, The mammal is a mouse, a rat or a human.

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