A method for preparing neural stem cells with high proliferative capacity

Abstract

The present application relates to a kind of preparation method for preparing neural stem cell with high proliferative capacity, belong to the field of biotechnology.The method is isolated from mammalian embryo brain tissue primary neural stem cell, with DMEM / F12 as base medium, add B27, N2, glutamine and double resistance, then join epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1, Y-27632 and heparin form optimized proliferation culture medium.Cell is seeded in the culture dish coated with polylysine and laminin, culture under suitable conditions, neural ball reaches certain diameter after digestion and passage.This method can improve the proliferation efficiency of neural stem cell, maintain stemness and differentiation potential, adopt serum-free system, simple operation, suitable for large-scale expansion and research application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for preparing neural stem cells with high proliferative capacity. Background Technology

[0002] Neural stem cells are a type of cell with self-renewal capacity and multi-lineage differentiation potential. They can differentiate into various neural cell types, such as neurons, astrocytes, and oligodendrocytes, and have broad application prospects in the treatment of nervous system diseases, neural regeneration research, and drug screening. However, the culture and expansion of neural stem cells currently face many challenges, limiting their development in basic research and clinical applications.

[0003] Traditional neural stem cell culture methods primarily rely on culture media containing fetal bovine serum or serum-free media supplemented with cytokines such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). While these methods can maintain neural stem cell proliferation to some extent, they suffer from low proliferation efficiency, short duration of stem cell maintenance, and susceptibility to differentiation or senescence. For example, under conventional culture conditions, neural stem cells have a long doubling time, typically requiring 48-72 hours, and after several generations of culture, their proliferative capacity significantly decreases. Simultaneously, the expression levels of stem cell markers (such as Nestin and Sox2) also decline, leading to impaired cell differentiation potential.

[0004] Furthermore, some measures taken in existing technologies to enhance the proliferation capacity of neural stem cells, such as increasing cytokine concentrations or adding serum, often trigger a series of side effects. High concentrations of cytokines may lead to abnormal cell proliferation or even carcinogenesis, while the addition of serum introduces foreign components, increasing the risk of immune rejection and batch-to-batch variability. It may also induce premature differentiation of neural stem cells, affecting their purity and function.

[0005] In recent years, researchers have attempted to improve the proliferative characteristics of neural stem cells through methods such as gene editing, small molecule compound regulation, or three-dimensional culture. However, these methods still suffer from problems such as technical complexity, high cost, or insufficient practicality. For example, gene editing technology may induce gene mutations or off-target effects, the long-term effects of small molecule compounds may be toxic to cells, and the large-scale scaling of three-dimensional culture systems is quite difficult.

[0006] Therefore, developing an efficient, stable, and controllable method for preparing neural stem cells, achieving high in vitro proliferation while maintaining good stemness and differentiation potential, has become a pressing technical problem in this field. This invention, through innovative culture system design, including specific combinations of culture medium components, signaling pathway regulators, and optimized culture conditions, significantly improves the proliferation efficiency of neural stem cells, providing a new technical means for basic research and clinical applications of neural stem cells. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing neural stem cells with high proliferative capacity. This method can significantly improve the in vitro proliferation efficiency of neural stem cells, prolong their self-renewal capacity, and maintain the multi-directional differentiation potential of neural stem cells, thus providing technical support for the large-scale expansion and application of neural stem cells.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] A method for preparing neural stem cells with high proliferative capacity includes the following steps:

[0010] W1. Isolation and acquisition of primary neural stem cells from mammalian embryonic brain tissue;

[0011] W2. A basal culture medium was prepared based on DMEM / F12 medium, with the addition of B27 additive, N2 additive, glutamine and penicillin-streptomycin antibiotics.

[0012] W3. Epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1, Y-27632 and heparin were added to the basic culture medium to form an optimized proliferation culture medium;

[0013] W4. The primary neural stem cells were subjected to a 2×10⁻⁶ 4 pcs / cm 2 The culture medium was seeded at a density of 100% in culture dishes coated with poly-L-lysine and laminin and cultured at 37°C and 5% CO2 using the optimized proliferation medium, with the medium being changed every 2 days.

[0014] 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 the specified density and cultured using the optimized proliferation medium.

[0015] As a preferred embodiment of the present invention, 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 embodiment of the present invention, 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 embodiment of the present invention, the amount of B27 additive added in step W2 is 1×, the amount of N2 additive added is 1×, the concentration of glutamine is 2mM, and the concentration of penicillin-streptomycin bispecific antibody is 100U / mL.

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

[0019] As a preferred embodiment of the present invention, the optimized proliferation medium in step W3 is further supplemented with LDN-193189 and SB-431542, 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 embodiment of the present invention, the concentration of LDN-193189 is 0.5 μM and the concentration of SB-431542 is 5 μM.

[0021] As a preferred embodiment of the present invention, the number of passages in step W5 shall not exceed 20, and cell stemness and differentiation potential shall be tested every 5 passages.

[0022] As a preferred embodiment of the present invention, the culture process in step W4 adopts low-oxygen culture conditions with an oxygen concentration of 2-5%.

[0023] In a preferred embodiment of the present invention, the mammal is a mouse, rat, or human.

[0024] The beneficial effects of this invention are:

[0025] (1) By optimizing the culture medium composition and adding a combination of various growth factors and signaling pathway inhibitors, the method of the present invention can shorten the doubling time of neural stem cells 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 invention can maintain high expression of stem markers Nestin, Sox2 and Oct4 in neural stem cells. After 20 generations of culture, the expression level of stem markers is still maintained at more than 80% of the initial level.

[0027] (3) The neural stem cells cultured by the method of the present invention 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 present invention adopts a serum-free culture system, which reduces the influence of heterologous components, ensures uniform culture conditions, and minimizes batch-to-batch differences, which is conducive to the large-scale expansion and quality control of neural stem cells.

[0029] (5) The preparation method of the present invention does not require complex gene editing or special equipment, but can be achieved by optimizing the culture medium composition and culture conditions, making it suitable for widespread application in laboratory and clinical research. Detailed Implementation

[0030] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0031] Experimental materials

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

[0033] Reagents: DMEM / F12 medium (Gibco, catalog number 11330032), B27 additive (Gibco, catalog number 17504044), N2 additive (Gibco, catalog number 17502048), glutamine (Gibco, catalog number 25030081), penicillin-streptomycin antibiotics (Gibco, catalog number 15140122), epidermal growth factor (EGF, PeproTech, catalog number 100-15), basic fibroblast growth factor (bFGF, PeproTech, catalog number 100-18B), islets IGF-1 (PeproTech, catalog number 100-11), Y-27632 (Selleck, catalog number S1049), Heparin (Sigma, catalog number H3149), LDN-193189 (Selleck, catalog number S7280), SB-431542 (Selleck, catalog number S1067), Trypsin-EDTA digestion solution (Gibco, catalog number 25200056), Poly-L-lysine (Sigma, catalog number P2636), Laminin (Sigma, catalog number L2020).

[0034] Main instruments: CO2 incubator (Thermo Fisher, model 3111), inverted microscope (Olympus, model CKX41), flow cytometer (BD, model FACSCalibur), real-time quantitative PCR instrument (Applied Biosystems, model 7500), microplate reader (Bio-Tek, model Synergy H1).

[0035] Isolation of primary neural stem cells

[0036] C57BL / 6 mice at 14 days of gestation were euthanized by cervical dislocation. Embryos were removed under aseptic conditions, and cerebral cortex tissue was isolated and placed in a culture dish containing ice-cold PBS. The tissue was minced using ophthalmic scissors, and 0.25% trypsin-EDTA digestion solution was added. Digestion was carried out at 37°C for 15 minutes with gentle agitation during digestion. After digestion, DMEM medium containing 10% fetal bovine serum was added to terminate digestion. The solution was filtered through a 100-mesh cell sieve, and the filtrate was collected. The filtrate was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended in basal medium to prepare a single-cell suspension.

[0037] Cell proliferation detection

[0038] Cell proliferation capacity was assessed using the MTT assay. Cells were cultured at 5 × 10⁶ cells / mL. 3 Cells were seeded at a density of 1 cell / well in 96-well plates, with 5 replicates per group. At 24h, 48h, 72h, 96h, and 120h post-seeding, 20μL of MTT solution (5mg / mL) was added to each well, and the plates were incubated at 37°C for 4 hours. The supernatant was discarded, and 150μL of DMSO was added to each well. The plates were shaken for 10 minutes to ensure complete dissolution of formazan. The absorbance (OD value) was measured at 570nm using a microplate reader, and the cell proliferation rate was calculated.

[0039] Dryness marker detection

[0040] The expression of stem cell markers was detected using real-time quantitative PCR (qPCR) and flow cytometry. The qPCR procedure was as follows: total RNA was extracted from cells, reverse transcribed into cDNA, and PCR amplification was performed using the cDNA as a 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℃ pre-denaturation for 5 minutes, 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, and 72℃ extension for 30 seconds, for a total of 40 cycles. Using 2... -ΔΔCt The relative expression level of the target gene is calculated using this method.

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

[0047] Differentiation potential testing

[0048] Neural stem cells were used at a rate of 1×10 4 pcs / cm 2 The culture medium was seeded at a density in poly-L-lysine-coated culture dishes, then replaced with differentiation medium (DMEM / F12 medium supplemented with 10% fetal bovine serum). After 7 days of culture, immunofluorescence staining was performed. The antibodies used are as follows:

[0049] Anti-β-III tubulin antibody (neuronal marker, Abcam, catalog number ab18207);

[0050] Anti-GFAP antibody (Astrocyte marker, Abcam, catalog number ab4674);

[0051] Anti-MBP antibody (oligodendrocyte marker, Abcam, catalog number ab40390).

[0052] Staining procedure: Fix with 4% paraformaldehyde for 15 minutes, permeabilize with 0.1% Triton X-100 for 10 minutes, block with 10% goat serum for 1 hour, add primary antibody and incubate overnight at 4°C, wash 3 times with PBS, add fluorescently labeled secondary antibody (Alexa Fluor 488), incubate at 37°C for 1 hour, stain nuclei with DAPI, mount the slide and observe and count the proportion of positive cells under a fluorescence microscope.

[0053] Example 1

[0054] Experimental steps

[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 × 10⁻⁶ 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 optimized proliferation medium was adjusted to 10 ng / mL, and the other steps were the same as in Example 1.

[0072] Comparative Example 5

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

[0074] Comparative Example 6

[0075] Same as Example 1, but without adding LDN-193189 and SB-431542.

[0076] Comparative Example 7

[0077] The basal culture medium was DMEM / F12 medium supplemented with 10% fetal bovine serum. Other steps were the same as in Example 1, but without the addition of cytokines such as EGF and bFGF.

[0078] Comparative Example 8

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

[0080] Comparative Example 9

[0081] No B27 additive was added to the basal culture medium, and the other steps were the same as in Example 1.

[0082] Comparative Example 10

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

[0084] Performance test results

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

[0086]

[0087] Note: The formula for calculating the proliferation rate is: (120h OD value - 24h OD value) / 24h OD value × 100%.

[0088] Table 2: Expression data of stem cell markers in passage 10 cells (relative expression levels, x±s, with primary cells as 1).

[0089]

[0090] Note: The relative expression levels of stem cell markers were set with GAPDH as an internal reference, and the expression level in primary cells was set to 1.

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

[0092] 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 mean ± standard deviation (x ± s) of three independent experiments.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing neural stem cells with high proliferative capacity, characterized in that, Includes the following steps: W1. Isolation and acquisition of primary neural stem cells from mammalian embryonic brain tissue; W2. Based on DMEM / F12 medium, 1×B27 additive, 1×N2 additive, 2mM glutamine and 100U / mL penicillin-streptomycin double antibiotic were added to prepare the basic medium; W3. Add epidermal growth factor (EGFR) at a final concentration of 20 ng / mL, basic fibroblast growth factor (BGF) at a final concentration of 20 ng / mL, insulin-like growth factor-1 (IGF-1) at a final concentration of 10 ng / mL, Y-27632 at a final concentration of 5 μM and heparin at a final concentration of 5 μg / mL to the basal culture medium. After thorough mixing, filter to remove bacteria to form an optimized proliferation culture medium. W4. The culture dish was coated with 10 μg / mL poly-L-lysine and 10 μg / mL laminin at 37°C for 2 hours; the primary neural stem cells were then introduced at a rate of 2 × 10⁻⁶. 4 pcs / cm 2 The culture medium was inoculated at the density of the above-coated culture dishes and cultured in the optimized proliferation medium at 37°C and 5% CO2, with the medium being changed every 2 days. 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 the specified density and cultured using the optimized proliferation medium.

2. The preparation method according to claim 1, characterized in that, The optimized proliferation medium in step W3 also contains LDN-193189 and SB-431542, wherein the concentration of LDN-193189 is 0.1-1 μM and the concentration of SB-431542 is 1-10 μM.

3. The preparation method according to claim 2, characterized in that, The concentration of LDN-193189 was 0.5 μM, and the concentration of SB-431542 was 5 μM.

4. The preparation method according to claim 1, characterized in that, The number of passages mentioned in step W5 shall not exceed 20, and cell stemness and differentiation potential shall be tested every 5 passages.

5. The preparation method according to claim 1, characterized in that, In step W4, a low-oxygen culture condition is used, with an oxygen concentration of 2-5%.

6. The preparation method according to claim 1, characterized in that, The mammal is a mouse, rat, or human.

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