Serum-free medium for in-vitro amplification of neural stem cells and preparation method of serum-free medium

Abstract

The invention relates to a serum-free culture medium for in-vitro amplification of neural stem cells and a preparation method of the serum-free culture medium, and belongs to the technical field of biology. The culture medium comprises a DMEM / F12 and Neurobasic double-basal culture medium (the volume ratio is 1: 1), a bFGF / EGF / BDNF three-growth-factor combination (20 / 15 / 10ng / mL), a B27 / N2 supplement and a metabolism regulator (6g / L glucose + 0.1 mM beta-mercaptoethanol), and is prepared through the processes of stepwise dissolution of a buffer system, magnetic stirring and mixing, positive-pressure filtration and sterilization and the like. The innovation points are as follows: the double-basal culture medium synergistically balances amino acid and neurotransmitter nutrition, so that the glucose consumption rate is reduced by 25%; the three factors synergistically activate a proliferation pathway and inhibit differentiation, the amplification multiple reaches 14.8 times (7 days), and the Netin positive rate is gt; 95%; a metabolism regulation system controls lactic acid lt; the karyotype abnormality rate is 1t; and 0.3%. According to the scheme, the cost is reduced by 33%, and the method is completely free of animal-derived components and suitable for large-scale automatic production of clinical-grade neural stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a serum-free medium for in vitro expansion of neural stem cells and a preparation method thereof. Background Art

[0002] In vitro expansion of neural stem cells (NSCs) is a core technology in neural regenerative medicine, but the existing culture medium systems have significant limitations. Traditional serum-containing culture media have large batch differences and high risks of pathogen contamination due to animal-derived components (such as fetal bovine serum), and differentiation factors in the serum are prone to trigger spontaneous differentiation of NSCs (>25%).

[0003] Although serum-free media avoid the above problems, they generally have technical bottlenecks such as a single growth factor combination (such as only bFGF / EGF), low amplification efficiency (<8-fold / 7 days), and metabolic imbalance (lactic acid accumulation, oxidative stress). In addition, the existing preparation processes result in protein activity loss (>15%) due to unreasonable component addition sequences, and the storage conditions are harsh (requiring -80°C cold chain), severely restricting large-scale applications. For example, commercially available serum-free media (such as NSC SFM) still rely on high-cost imported components and cannot balance pluripotency maintenance and efficient amplification.

[0004] Therefore, developing a serum-free medium system with defined components, controllable costs, and the ability to synergistically optimize cell proliferation and stemness has become an urgent need for the clinical translation of neural stem cells. Summary of the Invention

[0005] The present invention provides a serum-free medium for in vitro expansion of neural stem cells and a preparation method thereof to solve the problems of the existing technology.

[0006] To solve the above technical problems, the present invention is achieved through the following technical solutions: In a first aspect, a serum-free medium for in vitro expansion of neural stem cells, calculated by 1 L, comprises the following components:

[0007] Basal medium: a mixed solution composed of DMEM / F12 and Neurobasal medium in a volume ratio of 1:1;

[0008] Growth factor combination: containing 20 ng / mL recombinant human basic fibroblast growth factor (bFGF), 15 ng / mL epidermal growth factor (EGF), and 10 ng / mL brain-derived neurotrophic factor (BDNF);

[0009] Nutritional components: 2% v / v B27 serum-free supplement, 1% v / v N2 supplement, 100 μg / mL human transferrin, and 5 μg / mL insulin;

[0010] Metabolic regulators: 2 mM L-glutamine, 6 g / L glucose, and 0.1 mM β-mercaptoethanol;

[0011] Buffering system: 1.2 g / L sodium bicarbonate and 15 mM HEPES.

[0012] In the first aspect, in the basal medium system, a 1:1 mixture (450 mL:450 mL) of DMEM / F12 (high-concentration amino acids) and Neurobasal (neuron-specific nutrition) is used. The complementarity between the two is as follows:

[0013] DMEM / F12 provides essential amino acids (such as 4 mM glutamine and 1.5 mM aspartic acid).

[0014] Neurobasal optimizes neurotransmitter precursors (such as 0.1 mM choline and 0.5 μM taurine).

[0015] Experimental verification: The glucose consumption rate in the mixed system is reduced by 25% compared to the single medium (p < 0.05).

[0016] Growth factor combination

[0017] Concentration ratio mechanism

[0018] bFGF (20 ng / mL): EGF (15 ng / mL): BDNF (10 ng / mL) = 4:3:2, and this ratio is optimized by the response surface method (RSM):

[0019] Synergistic effect: bFGF activates the MAPK pathway to promote proliferation, and BDNF inhibits differentiation genes (such as MASH1) through the TrkB receptor.

[0020] Verification data: The combination of the three factors increases the proportion of S-phase cells from 28% to 41% (detected by flow cytometry).

[0021] Stability control

[0022] The growth factors need to be pre-cooled to 4 °C before addition (final concentration deviation < 5%) to avoid the breakage of disulfide bonds caused by high temperature (> 25 °C).

[0023] Metabolic regulation system

[0024] Glucose / β-mercaptoethanol synergistic effect

[0025] 6 g / L glucose provides substrates for glycolysis, and 0.1 mM β-mercaptoethanol scavenges ROS (through the Nrf2 / ARE pathway). The combination maintains:

[0026] The lactate concentration < 4 mM (traditional protocol > 8 mM).

[0027] The GSH / GSSG ratio > 10:1 (traditional scheme 3:1).

[0028] In a specific embodiment of the first aspect, the mixing ratio of DMEM / F12 to Neurobasal medium in the basal medium is 450 mL:450 mL.

[0029] In a specific embodiment of the first aspect, the concentration ratio of bFGF, EGF to BDNF in the growth factor combination is 4:3:2.

[0030] In a specific embodiment of the first aspect, the pH value of the buffer system is 7.2 - 7.4, and the osmotic pressure is 280 - 320 mOsm / kg.

[0031] In the second aspect, a method for preparing a serum-free medium for in vitro expansion of neural stem cells includes the following steps:

[0032] S1: Sterilely mix DMEM / F12 and Neurobasal medium in a biosafety cabinet to obtain a basal solution;

[0033] S2: After dissolving the buffer system components in a 37°C water bath, sequentially add transferrin and insulin, and stir magnetically until completely dissolved;

[0034] S3: Stepwise add B27 and N2 supplements and stir to mix evenly;

[0035] S4: Add the growth factor combination pre-cooled to 4°C;

[0036] S5: Perform positive pressure filtration sterilization using a 0.22 μm pore size PES membrane;

[0037] S6: Aliquot into 50 mL tubes and store at -20°C in the dark.

[0038] In the second aspect, the buffer system is preferably dissolved

[0039] First dissolve sodium bicarbonate and HEPES (accelerate dissolution in a 37°C water bath) to avoid precipitation of subsequent protein components due to pH fluctuations (ΔpH > 0.5)

[0040] Stepwise addition strategy

[0041] Transferrin and insulin are dissolved in a buffer environment (pH 7.2 - 7.4) to ensure stable iron ion chelation (change in absorbance detected by UV280 < 0.05)

[0042] Sterilization process control

[0043] Positive pressure filtration parameters

[0044] Driven by a pressure of 5 - 15 psi, combined with a 5 - μm pre - filter membrane (such as Whatman GF / D) to remove protein aggregates (reducing the risk of membrane blockage by >70%)

[0045] Membrane material selection

[0046] Preferably select PES membrane (non - cellulose membrane) to avoid adsorbing growth factors (experimental results show that the adsorption loss is <2%, while that of nitrocellulose membrane reaches 15%)

[0047] Optimization of storage conditions

[0048] Aliquot volume and temperature

[0049] Aliquoting at 50 mL / tube can reduce the number of freeze - thaw cycles (each tube is for single use only), and the half - life of growth factors is extended to 6 months under dark storage at - 20°C (verified by HPLC)

[0050] Timing of adding β - mercaptoethanol

[0051] Fresh β - mercaptoethanol needs to be added before use (final concentration 0.1 mM), because its activity is lost by >50% after 30 days of storage at - 20°C.

[0052] In a specific embodiment of the second aspect, the rotation speed of magnetic stirring in step S2 is 200 - 300 rpm, the duration is 10 - 15 minutes, and a polypropylene container with silanization treatment is used.

[0053] In a specific embodiment of the second aspect, the pressure of positive pressure filtration in step S5 is controlled at 5 - 15 psi, and large particles are removed by a 5 - μm pre - filter membrane before filtration.

[0054] In the third aspect, after 7 days of amplification culture, the cell proliferation multiple reaches 12 - 15 times, and the Nestin positive rate >95%.

[0055] Quantitative verification of application effects in the third aspect

[0056] Mechanism of improved proliferation efficiency

[0057] Cell cycle analysis

[0058] Flow cytometry shows that the proportion of S - phase increases from 28% to 41% (PI staining method, n = 3), and the G0 / G1 - phase is shortened to 52±3% (traditional protocol 68±5%).

[0059] Colony - forming rate

[0060] Verified by the limiting dilution method: the colony - forming rate is 82.3±3.6% (Nestin+ clones with a diameter >50 μm).

[0061] Evidence for maintaining pluripotency

[0062] Marker detection

[0063] Immunofluorescence: Nestin positive rate > 95% (co-localized with DAPI).

[0064] qRT-PCR: The fluctuation of Oct-4 mRNA expression level < 15% (Ct value 27.3 ± 0.5).

[0065] Karyotype stability

[0066] G-banding analysis showed that normal diploid (46, XY) was still maintained after 3 consecutive passages, and no chromosomal breakage or translocation was detected.

[0067] Verification of differentiation potential

[0068] Differentiation induction after removing growth factors:

[0069] The proportion of neurons (β-IIITubulin+) was 78 ± 4%.

[0070] The proportion of astrocytes (GFAP+) was 12 ± 2%.

[0071] (The traditional protocols were 65 ± 6% and 25 ± 5% respectively).

[0072] In a specific embodiment of the third aspect, the neural stem cells are derived from a human cell line, including SH-SY5Y or ReNcellVM cell line.

[0073] Fourth aspect, an in vitro amplification system for neural stem cells, comprising:

[0074] Serum-free medium;

[0075] Cell culture device;

[0076] Temperature control module (37 ± 0.5 °C) and CO2 concentration control module (5 ± 0.2%);

[0077] Periodic feeding program: Fresh β-mercaptoethanol was supplemented to a final concentration of 0.1 mM every 48 hours.

[0078] In the fourth aspect, the modular design of the amplification system

[0079] Environmental control module

[0080] Precise temperature regulation

[0081] The heating plate was controlled by the PID algorithm (accuracy ± 0.1 °C), and the temperature of the culture medium was monitored in real time by an infrared sensor (37 ± 0.5 °C).

[0082] CO2 dynamic balance

[0083] An infrared CO2 sensor (such as ) feeds back to regulate the gas mixing ratio (5 ± 0.2% CO2) to maintain the pH of the culture medium at 7.2 - 7.4.

[0084] Optimization of the feeding program

[0085] β-mercaptoethanol supplementation strategy

[0086] Fresh mother liquor (100 mM stock solution) is supplemented every 48 hours to keep the concentration in the culture medium stable at 0.08 - 0.12 mM (monitored by HPLC-MS).

[0087] Automation implementation

[0088] An integrated peristaltic pump (such as Watson-Marlow323) is used to add a fixed amount at regular intervals, with an error < 2%.

[0089] Quality control module

[0090] Online monitoring indicators

[0091] Dissolved oxygen (DO) is maintained at 40 - 60% air saturation (through an optical fiber sensor).

[0092] The glucose concentration is dynamically monitored (sampled every 24 hours, by the glucose oxidase method).

[0093] Abnormal handling mechanism

[0094] When the lactic acid concentration > 5 mM or NH4 + > 2 mM, the automatic medium replacement program is triggered (replace 50% of the fresh culture medium by volume).

[0095] The beneficial effects of the present invention are as follows:

[0096] 1. Synergistic maintenance of efficient amplification and pluripotency: Through the synergistic effect of DMEM / F12 and Neurobasal dual basal media, combined with the precise ratio of bFGF / EGF / BDNF three factors (4:3:2), the amplification multiple of neural stem cells reaches 14.8 ± 1.3 times (in 7 days), while maintaining the Nestin positive rate > 95% and the stability of Oct-4 expression (ΔCt < 0.5), which is significantly better than the traditional serum-containing medium (amplification multiple 9.5 times, differentiation rate 28.6%).

[0097] 2. Metabolic regulation and improvement of genetic stability: The metabolic regulation system of 6 g / L glucose combined with 0.1 mM β-mercaptoethanol controls the lactic acid concentration at 3.8 ± 0.4 mM (8.9 mM in the traditional scheme), increases the GSH / GSSG ratio to 12.3 ± 1.1, and ensures that the karyotype abnormality rate < 0.3% after 3 consecutive passages, effectively solving the problems of oxidative damage and genetic aberration in high-density culture.

[0098] 3. Industrialization cost and clinical transformation advantages: Through component optimization (such as a 30% reduction in BDNF dosage) and preparation process innovation (positive pressure filtration reduces activity loss to <2%), the culture medium cost is reduced by 33% (280 / L vs. commercially available 420 / L), and it is completely free of animal-derived ingredients, meets GMP standards, and supports large-scale production of clinical-grade cell products. DETAILED DESCRIPTION

[0099] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0100] A serum-free culture medium for in vitro expansion of neural stem cells and a preparation method thereof.

[0101] Example 1: Preparation of basal culture medium and cell expansion.

[0102] 1. Materials and Equipment

[0103] Culture medium components

[0104] DMEM / F12(Gibco,Cat#11320033);

[0105] Neurobasal (Gibco, Cat#21103049);

[0106] Recombinant human bFGF / EGF / BDNF (PeproTech,

[0107] Cat#100-18B / 100-15 / 450-02).

[0108] B27 / N2 supplement (Gibco, Cat# 17504044 / 17502048)

[0109] equipment

[0110] Biological safety cabinet (ESCO, Class IIA2)

[0111] Magnetic stirrer (IKA, RHdigital)

[0112] Positive pressure filtration system (Millipore, 0.22μm PES)

[0113] CO2 incubator (Thermo, Heracell150i)

[0114] 2. Preparation Process

[0115] Step 1: Prepare the base solution

[0116] In a biosafety cabinet, measure 450 mL of DMEM / F12 and 450 mL of Neurobasal, mix them, and equilibrate them in a 37°C water bath for 30 minutes.

[0117] The osmotic pressure of the mixed solution was tested (using an Advanced Instruments 3250 osmometer) and adjusted to 300 ± 5 mOsm / kg.

[0118] Step 2: Buffer system dissolution

[0119] Weigh 1.2 g of sodium bicarbonate and 3.57 g of HEPES, add 50 mL of ultrapure water, and stir in a 37°C water bath until the solution becomes clear (250 rpm, 10 minutes).

[0120] Adjust the pH to 7.3 ± 0.1 with 0.1 M HCl.

[0121] Step 3: Add protein ingredients

[0122] The buffer solution was transferred to a magnetic stirring container, and 100 mg of human transferrin (Sigma, Cat#T8158) and 5 mg of insulin (Sigma, Cat#I9278) were added in sequence.

[0123] Stir (200 rpm, 15 minutes) until completely dissolved, and the absorbance change detected by UV280 is <0.05.

[0124] Step 4: Supplements and Growth Factors

[0125] 20 mL of B27 and 10 mL of N2 supplement were added stepwise and stirred for 5 minutes.

[0126] Pre-cool the growth factors to 4°C and add: bFGF 20 ng / mL, EGF 15 ng / mL, BDNF 10 ng / mL.

[0127] Step 5: Sterilization and packaging

[0128] The solution was pre-filtered using a 5 μm pre-filter membrane (Whatman GF / D) and then filtered through a 0.22 μm PES membrane under positive pressure (pressure 10 psi).

[0129] Aliquot into 50 mL / tube, store at -20℃ in the dark, and mark with batch number (e.g., NSCM-2023-001).

[0130] 3. Cell Culture Validation

[0131] Cell line: Human ReNcell VM cells (Millipore, Cat# SCC008).

[0132] Seeding conditions:

[0133] Density: 5×10 4 cells / cm 2 .

[0134] Culture dish: 6-well plate coated with Matrigel.

[0135] Culture conditions: 37°C, 5% CO2, 95% humidity.

[0136] Detection indicators:

[0137] Time point <![CDATA[Number of living cells (×10 6 )]]> Nestin positive rate Lactic acid (mM) Day0 0.5±0.1 98.2％ 1.2±0.3 Day7 6.8±0.5 96.5％ 3.8±0.4

[0138] Example 2: Integrated application of automated amplification system

[0139] 1. System hardware configuration.

[0140]

[0141]

[0142] 2. Dynamic amplification process

[0143] Step 1: System initialization

[0144] Load 500 mL of the culture medium prepared in Example 1 into the bioreactor.

[0145] Set parameters:

[0146] Temperature 37.0°C (PID control parameters: P = 2.0, I = 0.5, D = 0.1).

[0147] CO2 concentration 5.0% (feedback regulation rate 0.5 L / min).

[0148] Stirring rate 50 rpm (to avoid shear damage).

[0149] Step 2: Cell seeding and culture

[0150] Seeding density: 2×10 5 cells / mL (total cell number 1×10 8 cells).

[0151] Start the periodic feeding program:

[0152] Supplement 100 μL of β-mercaptoethanol (100 mM stock solution) every 48 hours.

[0153] When the glucose is < 3 g / L, 6 g / L glucose solution is automatically added (volume error < 2%).

[0154] Step 3: Real-time monitoring and regulation

[0155] Data collection:

[0156]

[0157] Abnormality handling:

[0158] When the lactic acid > 5 mM or NH4 + > 2 mM, the system automatically performs a 50% volume medium change (fresh medium preheated to 37 °C).

[0159] 3. Verification of amplification effect

[0160] Index Traditional static culture Dynamic amplification of this system Amplification multiple (7 days) 12 ± 1.5 times 35 ± 4.2 times Cell viability 92±3％ 98±1％ Oct-4 expression stability ΔCt = 2.1 ± 0.3 ΔCt = 0.5 ± 0.1 Yield per unit volume <![CDATA[1×10 6 cells / mL]]> <![CDATA[5×10 6 cells / mL]]>

[0161] 4. Quality control measures

[0162] Sterility test: Cultured by membrane filtration method according to USP <71> for 14 days, no microbial growth.

[0163] Mycoplasma test: Negative by PCR method ( Mycoplasma Kit).

[0164] Potency verification: The proportion of differentiated neurons is 78 ± 4% (β-IIITubulin immunostaining).

[0165] Experimental design

[0166] 1. Experimental grouping

[0167] Experimental group: The serum-free medium (NSCM) of the present invention

[0168] Control group 1: Traditional serum-containing medium (DMEM / F12 + 10% FBS + 20 ng / mL bFGF)

[0169] Control group 2: Commercially available serum-free medium (Gibco NSC SFM, Cat#A1647801).

[0170] 2. Cell line and culture conditions

[0171] Cell line: Human-derived ReNcell VM neural stem cells (passages 3 - 5);

[0172] Seeding density: 5×10 4 cells / cm 2(6-well plate coated with Corning Matrigel);

[0173] Cultivation time: 7 days (medium was changed by half every 48 hours);

[0174] Detection time points: 0h, 72h, 144h.

[0175] Comparison indicators and detection methods

[0176]

[0177] Experimental results

[0178] 1. Comparison of proliferation efficiency

[0179]

[0180] Mechanism analysis:

[0181] Three growth factors synergistically activate the MAPK / PI3K pathway, shortening the G1 phase (Cyclin D1 expression is up-regulated by 2.3 times); The B27 / N2 supplement provides lipid precursors, accelerating membrane synthesis.

[0182] 2. Comparison of pluripotency maintenance

[0183] Group Nestin positive rate (%) Oct-4 mRNA (ΔCt) Spontaneous differentiation rate (%) NSCM <![CDATA[96.5±1.2 ＊ > <![CDATA[0.5±0.1 ＊ > <![CDATA[7.3±0.9 ＊ > Control group 1 84.2±2.5 2.1±0.3 28.6±2.1 Control group 2 89.7±1.8 1.3±0.2 15.4±1.5

[0184] Key findings:

[0185] The HEPES buffer system maintains the pH at 7.2 - 7.4 (fluctuation <0.2), inhibiting the expression of differentiation-related genes (such as GFAP, MBP).

[0186] 3. Comparison of metabolic regulation

[0187] Group Lactic acid (mM) <![CDATA[NH4 + (μM)]]> GSH / GSSG ratio NSCM <![CDATA[3.8±0.4 ＊ > <![CDATA[182±15 ＊ > <![CDATA[12.3±1.1 ＊ > Control group 1 8.9±0.7 520±45 3.2±0.5 Control group 2 5.1±0.6 310±25 7.8±0.9

[0188] Technical advantages:

[0189] 6g / L glucose precisely matches the cell glycolysis rate, and β-mercaptoethanol scavenges ROS (MDA content in the NSCM group is reduced by 67%).

[0190] 4. Comparison of passage stability

[0191] Group Abnormal karyotype rate (%) after 3 passages Proportion of apoptotic cells (%) NSCM <![CDATA[0.3±0.1 ＊ > <![CDATA[4.2±0.5 ＊ > Control group 1 5.8±0.7 18.6±1.2 Control group 2 1.7±0.3 9.3±0.8

[0192] Innovations:

[0193] L-glutamine (2mM) maintains the stability of the NADPH pool, reducing DNA oxidative damage (8-OHdG content is reduced by 82%).

[0194] 5. Comparison of production costs (calculated based on 1L).

[0195] Component NSCM cost ($) Control group 2 cost ($) Cost reduction range B27 / N2 85 220 61％↓ Growth factor 65 150 57％↓ Basal medium 30 50 40％↓ Total 280 420 33％↓

[0196] Cost advantage:

[0197] By optimizing the component concentration (such as a 30% reduction in the dosage of BDNF) and domestic substitution (selecting Solarbio products for transferrin).

[0198] This technical solution is significantly superior to the existing technology in terms of core indicators such as amplification efficiency (a 55% increase), pluripotency maintenance (a 76% reduction in the spontaneous differentiation rate), metabolic regulation (a 58% reduction in lactate production), and production cost (a 33% reduction), providing a reliable solution for the clinical transformation of neural stem cell therapy.

[0199] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Serum-free medium for in vitro expansion of neural stem cells, characterized in that: Comprising the following components per 1 L: Basal medium: A mixed solution composed of DMEM / F12 and Neurobasal medium at a volume ratio of 1:1; Growth factor combination: Containing 20 ng / mL recombinant human basic fibroblast growth factor (bFGF), 15 ng / mL epidermal growth factor (EGF), and 10 ng / mL brain-derived neurotrophic factor (BDNF); Nutritional components: 2% v / v B27 serum-free supplement, 1% v / v N2 supplement, 100 μg / mL human transferrin, and 5 μg / mL insulin; Metabolic regulator: 2 mM L-glutamine, 6 g / L glucose, and 0.1 mM β-mercaptoethanol; Buffer system: 1.2 g / L sodium bicarbonate and 15 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

2. The serum-free medium for in vitro expansion of neural stem cells and its preparation method according to claim 1, characterized in that: The mixing ratio of DMEM / F12 and Neurobasal medium in the basal medium is 450 mL:450 mL.

3. The serum-free medium for in vitro expansion of neural stem cells and its preparation method according to claim 1, characterized in that: The concentration ratio of bFGF, EGF, and BDNF in the growth factor combination is 4:3:

2.

4. The serum-free medium for in vitro expansion of neural stem cells and its preparation method according to claim 1, characterized in that: The pH value of the buffer system is 7.2 - 7.4, and the osmotic pressure is 280 - 320 mOsm / kg.

5. Preparation method of serum-free medium for in vitro expansion of neural stem cells, characterized in that: Including the following steps: S1: Sterilely mix DMEM / F12 and Neurobasal medium in a biosafety cabinet to obtain a basal solution; S2: After dissolving the buffer system components in a 37°C water bath, sequentially add transferrin and insulin, and stir magnetically until completely dissolved; S3: Add B27 and N2 supplements step by step and stir to mix evenly; S4: Add the growth factor combination pre-cooled to 4°C; S5: Perform positive pressure filtration sterilization using a 0.22 μm pore size PES (polyethersulfone) membrane; S6: Aliquot into 50 mL / tube and store at -20°C in the dark.

6. The preparation method of the serum-free medium for in vitro expansion of neural stem cells according to claim 5, characterized in that: In step S2, the rotation speed of magnetic stirring is 200 - 300 rpm, the duration is 10 - 15 minutes, and a siliconized polypropylene container is used.

7. The method for preparing a serum-free medium for in vitro expansion of neural stem cells according to claim 5, characterized in that: In step S5, the pressure of positive pressure filtration is controlled at 5 - 15 psi, and a 5 μm pre-filter membrane is used to remove large particulate matter before filtration.

8. Use of a method for preparing a serum-free medium for in vitro expansion of neural stem cells, characterized in that: After 7 days of amplification culture, the cell proliferation multiple reaches 12 - 15 times, and the Nestin positive rate > 95%.

9. Use of the serum-free medium for in vitro expansion of neural stem cells according to claim 8, characterized in that: The neural stem cells are from a human cell line, including the SH-SY5Y or ReNcellVM cell line.

10. A neural stem cell in vitro expansion system, characterized in that, Containing: The serum-free medium according to any one of claims 1 - 4; A cell culture device; A temperature control module (37 ± 0.5°C) and a CO2 concentration control module (5 ± 0.2%); A periodic feeding program: Supplement fresh β-mercaptoethanol every 48 hours to a final concentration of 0.1 mM.

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