A neural stem cell and a preparation method and application thereof

Abstract

This invention belongs to the field of biotechnology. Specifically, this invention discloses neural stem cells, their preparation methods and applications, including the following steps: (1) washing hippocampal tissue with PBS buffer, placing the washed hippocampal tissue in a digestive solution for digestion, adding a digestion termination solution to terminate digestion, centrifuging to obtain cell pellet, washing the cell pellet with PBS buffer, centrifuging, resuspending the cells in DMEM / F12 medium, and adjusting the cell concentration to 1×10⁻⁶. 4 (2) Primary culture; (3) Amplification culture. The amplification culture medium described in this invention can provide a carrier with viscosity during the culture of neural stem cells, promote the division and proliferation of neural stem cells, improve the activity and stability of neural stem cells, and increase the survival rate and cell number, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a neural stem cell, its preparation method, and its application. Background Technology

[0002] Neural stem cells (NSCs) are crucial for the development, regeneration, and repair of the nervous system. In the brains of adult mammals, most NSCs are in a quiescent state. However, they can be reactivated by external stimuli and the coordinated action of various factors, including internal signaling molecules and transcription factors, offering promise for cell therapy of nervous system diseases.

[0003] Existing methods yield neural stem cells with low survival rates and insufficient quantities to meet requirements; therefore, this application is hereby filed. Summary of the Invention

[0004] This invention provides a neural stem cell, its preparation method, and its application. The expansion culture medium described in this invention can provide a carrier with viscosity during the culture of neural stem cells, promote the division and proliferation of neural stem cells, improve the activity and stability of neural stem cells, increase the survival rate and cell number, and has broad application prospects.

[0005] A method for preparing neural stem cells includes the following steps:

[0006] (1) Wash the hippocampal tissue with PBS buffer, place the washed hippocampal tissue in a digestion solution for digestion, add digestion termination solution to stop digestion, centrifuge to obtain cell pellet, wash the cell pellet with PBS buffer, centrifuge, resuspend the cells in DMEM / F12 medium, and adjust the cell concentration to 1×10⁻⁶. 4 cells / mL;

[0007] (2) Add the cells from step (1) to DMEM / F12 medium and culture until the cells reach 80-90% confluence to obtain primary cells;

[0008] (3) Primary cells were subjected to a concentration of 1.5 × 10⁻⁶ 4 Neural stem cells were seeded at a density of 1 / mL in amplification medium and cultured. The medium was changed halfway through each day. The cells were passaged to the 5th generation to obtain neural stem cells.

[0009] As a preferred embodiment of the present invention, the digestive fluid comprises the following components by mass percentage: 0.6-1% sodium chloride injection, 1-4% collagenase I, 2-5% papain, 4-6% glucose, 8-15% calcium chloride, 15-25% albumin, and the balance being water.

[0010] In a preferred embodiment of the present invention, the digestion termination solution is a PBS solution containing 10% fetal bovine serum by mass.

[0011] As a preferred embodiment of the present invention, step (2) is carried out under the conditions of a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

[0012] As a preferred embodiment of the present invention, step (3) is carried out under the conditions of a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

[0013] As a preferred embodiment of the present invention, the amplification culture medium comprises the following components: DMEM / F12 serum-free culture medium, 20-50 ng / mL tocopherol, 50-100 ng / mL glutathione, 200-500 ng / mL EGF, 200-500 ng / mL bFGF, 0.5-2 mg / mL eicosapentaenoic acid, 5-10 mg / mL loofah powder, 10-40 mg / mL MnFe-LDH, 10-40 mg / mL hydrolyzed chitosan, 10-30 mg / mL fetal bovine serum, and 10-50 mg / mL albumin.

[0014] The combination of MnFe-LDH and hydrolyzed chitosan described in this invention allows them to disperse uniformly in the culture medium, forming a three-dimensional network that provides a carrier for the culture of neural stem cells. This provides greater space for the adhesion and proliferation of neural stem cells, effectively improving the amplification effect and enhancing cell viability. The two have a significant synergistic effect.

[0015] As a preferred embodiment of the present invention, the amplification culture medium comprises the following components: DMEM / F12 serum-free culture medium, 20-40 ng / mL tocopherol, 50-80 ng / mL glutathione, 200-400 ng / mL EGF, 300-500 ng / mL bFGF, 1-2 mg / mL eicosapentaenoic acid, 6-10 mg / mL loofah powder, 10-30 mg / mL MnFe-LDH, 20-40 mg / mL hydrolyzed chitosan, 15-30 mg / mL fetal bovine serum, and 30-50 mg / mL albumin.

[0016] As a preferred embodiment of the present invention, the amplification culture medium consists of the following: DMEM / F12 serum-free culture medium, 30 ng / mL tocopherol, 60 ng / mL glutathione, 300 ng / mL EGF, 400 ng / mL bFGF, 1.8 mg / mL eicosapentaenoic acid, 8 mg / mL loofah powder, 20 mg / mL MnFe-LDH, 30 mg / mL hydrolyzed chitosan, 20 mg / mL fetal bovine serum, and 45 mg / mL albumin.

[0017] In a preferred embodiment of the present invention, the MnFe-LDH flake diameter is 50~400nm.

[0018] The present invention also provides an application of neural stem cells prepared by the above-described preparation method in the preparation of drugs for treating nervous system diseases.

[0019] The beneficial effects of the present invention are as follows: (1) The amplification culture medium described in the present invention can provide a carrier for the viscosity of neural stem cells during the culture process, promote the division and proliferation of neural stem cells, improve the activity and stability of neural stem cells, increase the survival rate and cell number, and have broad application prospects; (2) The combination of MnFe-LDH and hydrolyzed chitosan described in the present invention can evenly disperse MnFe-LDH and hydrolyzed chitosan in the culture medium and form a three-dimensional network in the culture medium, which can provide a carrier for the culture of neural stem cells, provide more space for the adhesion and proliferation of neural stem cells, effectively improve the amplification effect, and improve cell vitality. The two have a significant synergistic effect. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0022] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0023] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0024] Unless otherwise specified, all reagents or instruments used in this invention are commercially available products.

[0025] Unless otherwise specified, all components and raw materials used in the embodiments and comparative examples of this invention are commercially available, and the same type of components and raw materials are used in each parallel experiment.

[0026] Example 1

[0027] A method for preparing neural stem cells includes the following steps:

[0028] (1) Hippocampal tissue was isolated from SD rats on day 10 under aseptic conditions. The hippocampal tissue was washed with PBS buffer and then digested in a digestive solution. Digestion was terminated by adding a digestion termination solution (the mass ratio of digestive solution to digestion termination solution was 1:2). The cells were centrifuged at 2000 rpm for 10 min to obtain cell pellet. The cell pellet was washed with PBS buffer and centrifuged at 2000 rpm for 10 min. The cells were resuspended in DMEM / F12 medium and the cell concentration was adjusted to 1×10⁻⁶. 4 cells / mL;

[0029] The digestive fluid comprises the following components by weight percentage: 0.9% sodium chloride injection, 3% collagenase I, 4% papain, 5% glucose, 10% calcium chloride, 20% albumin, and the remainder water.

[0030] The digestion termination solution is a PBS solution containing 10% fetal bovine serum by mass.

[0031] (2) Add the cells from step (1) to DMEM / F12 medium and culture them at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2% until the cells reach 85% confluence to obtain primary cells;

[0032] (3) Primary cells were subjected to a concentration of 1.5 × 10⁻⁶ 4Neural stem cells were seeded at a density of 1 / mL in amplification medium and cultured. The medium was changed halfway through each day. The cells were passaged to the 5th generation to obtain neural stem cells.

[0033] The step (3) is carried out at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

[0034] The amplification medium consists of the following components: DMEM / F12 serum-free medium, 30 ng / mL tocopherol, 60 ng / mL glutathione, 300 ng / mL EGF, 400 ng / mL bFGF, 1.8 mg / mL eicosapentaenoic acid, 8 mg / mL loofah powder, 20 mg / mL MnFe-LDH, 30 mg / mL hydrolyzed chitosan, 20 mg / mL fetal bovine serum, and 45 mg / mL albumin.

[0035] The MnFe-LDH (derived from Xianfeng Nano, XFL08) has a flake diameter of 50~400nm.

[0036] Example 2

[0037] A method for preparing neural stem cells includes the following steps:

[0038] (1) Hippocampal tissue was isolated from SD rats on day 10 under aseptic conditions. The hippocampal tissue was washed with PBS buffer and then digested in a digestive solution. Digestion was terminated by adding a digestion termination solution (the mass ratio of digestive solution to digestion termination solution was 1:2). The cells were centrifuged at 2000 rpm for 10 min to obtain cell pellet. The cell pellet was washed with PBS buffer and centrifuged at 2000 rpm for 10 min. The cells were resuspended in DMEM / F12 medium and the cell concentration was adjusted to 1×10⁻⁶. 4 cells / mL;

[0039] The digestive fluid comprises the following components by weight percentage: 0.9% sodium chloride injection, 3% collagenase I, 4% papain, 5% glucose, 10% calcium chloride, 20% albumin, and the remainder water.

[0040] The digestion termination solution is a PBS solution containing 10% fetal bovine serum by mass.

[0041] (2) Add the cells from step (1) to DMEM / F12 medium and culture them at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2% until the cells reach 85% confluence to obtain primary cells;

[0042] (3) Primary cells were subjected to a concentration of 1.5 × 10⁻⁶ 4Neural stem cells were seeded at a density of 1 / mL in amplification medium and cultured. The medium was changed halfway through each day. The cells were passaged to the 5th generation to obtain neural stem cells.

[0043] The step (3) is carried out at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

[0044] The amplification medium consists of the following components: DMEM / F12 serum-free medium, 20 ng / mL tocopherol, 100 ng / mL glutathione, 200 ng / mL EGF, 500 ng / mL bFGF, 0.5 mg / mL eicosapentaenoic acid, 10 mg / mL loofah powder, 10 mg / mL MnFe-LDH, 40 mg / mL hydrolyzed chitosan, 10 mg / mL fetal bovine serum, and 10 mg / mL albumin.

[0045] The MnFe-LDH (derived from Xianfeng Nano, XFL08) has a flake diameter of 50~400nm.

[0046] Example 3

[0047] A method for preparing neural stem cells includes the following steps:

[0048] (1) Hippocampal tissue was isolated from SD rats on day 10 under aseptic conditions. The hippocampal tissue was washed with PBS buffer and then digested in a digestive solution. Digestion was terminated by adding a digestion termination solution (the mass ratio of digestive solution to digestion termination solution was 1:2). The cells were centrifuged at 2000 rpm for 10 min to obtain cell pellet. The cell pellet was washed with PBS buffer and centrifuged at 2000 rpm for 10 min. The cells were resuspended in DMEM / F12 medium and the cell concentration was adjusted to 1×10⁻⁶. 4 cells / mL;

[0049] The digestive fluid comprises the following components by weight percentage: 0.9% sodium chloride injection, 3% collagenase I, 4% papain, 5% glucose, 10% calcium chloride, 20% albumin, and the remainder water.

[0050] The digestion termination solution is a PBS solution containing 10% fetal bovine serum by mass.

[0051] (2) Add the cells from step (1) to DMEM / F12 medium and culture them at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2% until the cells reach 85% confluence to obtain primary cells;

[0052] (3) Primary cells were subjected to a concentration of 1.5 × 10⁻⁶ 4Neural stem cells were seeded at a density of 1 / mL in amplification medium and cultured. The medium was changed halfway through each day. The cells were passaged to the 5th generation to obtain neural stem cells.

[0053] The step (3) is carried out at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

[0054] The amplification medium consists of the following components: DMEM / F12 serum-free medium, 50 ng / mL tocopherol, 50 ng / mL glutathione, 500 ng / mL EGF, 200 ng / mL bFGF, 2 mg / mL eicosapentaenoic acid, 5 mg / mL loofah powder, 40 mg / mL MnFe-LDH, 10 mg / mL hydrolyzed chitosan, 30 mg / mL fetal bovine serum, and 50 mg / mL albumin.

[0055] The MnFe-LDH (derived from Xianfeng Nano, XFL08) has a flake diameter of 50~400nm.

[0056] Example 4

[0057] A method for preparing neural stem cells includes the following steps:

[0058] (1) Hippocampal tissue was isolated from SD rats on day 10 under aseptic conditions. The hippocampal tissue was washed with PBS buffer and then digested in a digestive solution. Digestion was terminated by adding a digestion termination solution (the mass ratio of digestive solution to digestion termination solution was 1:2). The cells were centrifuged at 2000 rpm for 10 min to obtain cell pellet. The cell pellet was washed with PBS buffer and centrifuged at 2000 rpm for 10 min. The cells were resuspended in DMEM / F12 medium and the cell concentration was adjusted to 1×10⁻⁶. 4 cells / mL;

[0059] The digestive fluid comprises the following components by weight percentage: 0.9% sodium chloride injection, 3% collagenase I, 4% papain, 5% glucose, 10% calcium chloride, 20% albumin, and the remainder water.

[0060] The digestion termination solution is a PBS solution containing 10% fetal bovine serum by mass.

[0061] (2) Add the cells from step (1) to DMEM / F12 medium and culture them at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2% until the cells reach 85% confluence to obtain primary cells;

[0062] (3) Primary cells were subjected to a concentration of 1.5 × 10⁻⁶ 4Neural stem cells were seeded at a density of 1 / mL in amplification medium and cultured. The medium was changed halfway through each day. The cells were passaged to the 5th generation to obtain neural stem cells.

[0063] The step (3) is carried out at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

[0064] The amplification medium consists of the following components: DMEM / F12 serum-free medium, 40 ng / mL tocopherol, 80 ng / mL glutathione, 400 ng / mL EGF, 300 ng / mL bFGF, 2 mg / mL eicosapentaenoic acid, 6 mg / mL loofah powder, 30 mg / mL MnFe-LDH, 20 mg / mL hydrolyzed chitosan, 30 mg / mL fetal bovine serum, and 30 mg / mL albumin.

[0065] The MnFe-LDH (derived from Xianfeng Nano, XFL08) has a flake diameter of 50~400nm.

[0066] Example 5

[0067] A method for preparing neural stem cells includes the following steps:

[0068] (1) Hippocampal tissue was isolated from SD rats on day 10 under aseptic conditions. The hippocampal tissue was washed with PBS buffer and then digested in a digestive solution. Digestion was terminated by adding a digestion termination solution (the mass ratio of digestive solution to digestion termination solution was 1:2). The cells were centrifuged at 2000 rpm for 10 min to obtain cell pellet. The cell pellet was washed with PBS buffer and centrifuged at 2000 rpm for 10 min. The cells were resuspended in DMEM / F12 medium and the cell concentration was adjusted to 1×10⁻⁶. 4 cells / mL;

[0069] The digestive fluid comprises the following components by weight percentage: 0.9% sodium chloride injection, 3% collagenase I, 4% papain, 5% glucose, 10% calcium chloride, 20% albumin, and the remainder water.

[0070] The digestion termination solution is a PBS solution containing 10% fetal bovine serum by mass.

[0071] (2) Add the cells from step (1) to DMEM / F12 medium and culture them at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2% until the cells reach 85% confluence to obtain primary cells;

[0072] (3) Primary cells were subjected to a concentration of 1.5 × 10⁻⁶ 4Neural stem cells were seeded at a density of 1 / mL in amplification medium and cultured. The medium was changed halfway through each day. The cells were passaged to the 5th generation to obtain neural stem cells.

[0073] The step (3) is carried out at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

[0074] The amplification medium consists of the following components: DMEM / F12 serum-free medium, 20 ng / mL tocopherol, 50 ng / mL glutathione, 200 ng / mL EGF, 500 ng / mL bFGF, 1 mg / mL eicosapentaenoic acid, 10 mg / mL loofah powder, 10 mg / mL MnFe-LDH, 40 mg / mL hydrolyzed chitosan, 15 mg / mL fetal bovine serum, and 50 mg / mL albumin.

[0075] The MnFe-LDH (derived from Xianfeng Nano, XFL08) has a flake diameter of 50~400nm.

[0076] Example 6

[0077] A method for preparing neural stem cells includes the following steps:

[0078] (1) Hippocampal tissue was isolated from SD rats on day 10 under aseptic conditions. The hippocampal tissue was washed with PBS buffer and then digested in a digestive solution. Digestion was terminated by adding a digestion termination solution (the mass ratio of digestive solution to digestion termination solution was 1:2). The cells were centrifuged at 2000 rpm for 10 min to obtain cell pellet. The cell pellet was washed with PBS buffer and centrifuged at 2000 rpm for 10 min. The cells were resuspended in DMEM / F12 medium and the cell concentration was adjusted to 1×10⁻⁶. 4 cells / mL;

[0079] The digestive fluid comprises the following components by weight percentage: 0.9% sodium chloride injection, 3% collagenase I, 4% papain, 5% glucose, 10% calcium chloride, 20% albumin, and the remainder water.

[0080] The digestion termination solution is a PBS solution containing 10% fetal bovine serum by mass.

[0081] (2) Add the cells from step (1) to DMEM / F12 medium and culture them at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2% until the cells reach 85% confluence to obtain primary cells;

[0082] (3) Primary cells were subjected to a concentration of 1.5 × 10⁻⁶ 4Neural stem cells were seeded at a density of 1 / mL in amplification medium and cultured. The medium was changed halfway through each day. The cells were passaged to the 5th generation to obtain neural stem cells.

[0083] The step (3) is carried out at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

[0084] The amplification medium consists of the following components: DMEM / F12 serum-free medium, 25 ng / mL tocopherol, 70 ng / mL glutathione, 250 ng / mL EGF, 450 ng / mL bFGF, 1.2 mg / mL eicosapentaenoic acid, 7 mg / mL loofah powder, 15 mg / mL MnFe-LDH, 25 mg / mL hydrolyzed chitosan, 25 mg / mL fetal bovine serum, and 45 mg / mL albumin.

[0085] The MnFe-LDH (derived from Xianfeng Nano, XFL08) has a flake diameter of 50~400nm.

[0086] Example 7

[0087] A method for preparing neural stem cells includes the following steps:

[0088] (1) Hippocampal tissue was isolated from SD rats on day 10 under aseptic conditions. The hippocampal tissue was washed with PBS buffer and then digested in a digestive solution. Digestion was terminated by adding a digestion termination solution (the mass ratio of digestive solution to digestion termination solution was 1:2). The cells were centrifuged at 2000 rpm for 10 min to obtain cell pellet. The cell pellet was washed with PBS buffer and centrifuged at 2000 rpm for 10 min. The cells were resuspended in DMEM / F12 medium and the cell concentration was adjusted to 1×10⁻⁶. 4 cells / mL;

[0089] The digestive fluid comprises the following components by weight percentage: 0.9% sodium chloride injection, 3% collagenase I, 4% papain, 5% glucose, 10% calcium chloride, 20% albumin, and the remainder water.

[0090] The digestion termination solution is a PBS solution containing 10% fetal bovine serum by mass.

[0091] (2) Add the cells from step (1) to DMEM / F12 medium and culture them at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2% until the cells reach 85% confluence to obtain primary cells;

[0092] (3) Primary cells were subjected to a concentration of 1.5 × 10⁻⁶ 4Neural stem cells were seeded at a density of 1 / mL in amplification medium and cultured. The medium was changed halfway through each day. The cells were passaged to the 5th generation to obtain neural stem cells.

[0093] The step (3) is carried out at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

[0094] The amplification medium consists of the following components: DMEM / F12 serum-free medium, 35 ng / mL tocopherol, 60 ng / mL glutathione, 350 ng / mL EGF, 350 ng / mL bFGF, 1.8 mg / mL eicosapentaenoic acid, 9 mg / mL loofah powder, 25 mg / mL MnFe-LDH, 35 mg / mL hydrolyzed chitosan, 28 mg / mL fetal bovine serum, and 35 mg / mL albumin.

[0095] The MnFe-LDH (derived from Xianfeng Nano, XFL08) has a flake diameter of 50~400nm.

[0096] Comparative Example 1

[0097] The difference between Comparative Example 1 and Example 1 is that the amplification culture medium of Comparative Example 1 does not contain hydrolyzed chitosan, but all other aspects are the same.

[0098] The amplification medium consists of the following components: DMEM / F12 serum-free medium, 30 ng / mL tocopherol, 60 ng / mL glutathione, 300 ng / mL EGF, 400 ng / mL bFGF, 1.8 mg / mL eicosapentaenoic acid, 8 mg / mL loofah powder, 20 mg / mL MnFe-LDH, 20 mg / mL fetal bovine serum, and 45 mg / mL albumin.

[0099] Comparative Example 2

[0100] The difference between Comparative Example 2 and Example 1 is that the amplification medium of Comparative Example 2 does not contain MnFe-LDH, but all other aspects are the same.

[0101] The amplification medium consists of the following components: DMEM / F12 serum-free medium, 30 ng / mL tocopherol, 60 ng / mL glutathione, 300 ng / mL EGF, 400 ng / mL bFGF, 1.8 mg / mL eicosapentaenoic acid, 8 mg / mL loofah powder, 20 mg / mL MnFe-LDH, 20 mg / mL fetal bovine serum, and 45 mg / mL albumin.

[0102] Test case

[0103] Neural stem cells were prepared according to the same procedure as in Example 1 and the control group, and the process was repeated three times. Five samples were extracted each time, and the number of cells and the survival rate were counted. The results are shown in Table 1.

[0104] Table 1

[0105]

[0106] As can be seen from Table 1, the neural stem cells prepared by the preparation method described in this invention significantly improve the number and survival rate of neural stem cells.

[0107] Comparative examples 1-7 show that the present invention, by controlling the components of each raw material as follows: DMEM / F12 serum-free culture medium, 20-40 ng / mL tocopherol, 50-80 ng / mL glutathione, 200-400 ng / mL EGF, 300-500 ng / mL bFGF, 1-2 mg / mL eicosapentaenoic acid, 6-10 mg / mL loofah powder, 10-30 mg / mL MnFe-LDH, 20-40 mg / mL hydrolyzed chitosan, 15-30 mg / mL fetal bovine serum, and 30-50 mg / mL albumin, effectively improves the survival rate and cell number.

[0108] Comparing Example 1 with Comparative Examples 1 and 2, it can be seen that the hydrolyzed chitosan and MnFe-LDH described in this invention have a synergistic effect in improving the survival rate and quantity of neural stem cells.

[0109] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing neural stem cells, characterized in that, Includes the following steps: (1) Rinse rat hippocampal tissue with PBS buffer, place the rinsed hippocampal tissue in digestion solution for digestion, add digestion termination solution to stop digestion, centrifuge to obtain cell pellet, wash the cell pellet with PBS buffer, centrifuge, resuspend the cells in DMEM / F12 medium, and adjust the cell concentration to 1×10⁻⁶. 4 cells / mL; (2) Add the cells from step (1) to DMEM / F12 medium and culture until the cells reach 80-90% confluence to obtain primary cells; (3) Primary cells were subjected to a concentration of 1.5 × 10⁻⁶ 4 Neural stem cells were seeded at a density of 1 / mL in amplification medium and amplified. The medium was changed halfway every day. The cells were passaged to the 5th generation to obtain neural stem cells. The amplification medium consists of the following components: DMEM / F12 serum-free medium, 20-50 ng / mL tocopherol, 50-100 ng / mL glutathione, 200-500 ng / mL EGF, 200-500 ng / mL bFGF, 0.5-2 mg / mL eicosapentaenoic acid, 5-10 mg / mL loofah powder, 10-40 mg / mL MnFe-LDH, 10-40 mg / mL hydrolyzed chitosan, 10-30 mg / mL fetal bovine serum, and 10-50 mg / mL albumin. The neural stem cells mentioned are rat neural stem cells.

2. The method for preparing neural stem cells according to claim 1, characterized in that, The digestive fluid comprises the following components by weight percentage: 0.6-1% sodium chloride injection, 1-4% collagenase I, 2-5% papain, 4-6% glucose, 8-15% calcium chloride, 15-25% albumin, and the balance being water.

3. The method for preparing neural stem cells according to claim 1, characterized in that, The digestion termination solution is a PBS solution containing 10% fetal bovine serum by mass.

4. The method for preparing neural stem cells according to claim 1, characterized in that, The step (2) is carried out at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

5. The method for preparing neural stem cells according to claim 1, characterized in that, The step (3) is carried out at a temperature of 37.0±0.5℃ and a CO2 concentration of 5.0±0.2%.

6. The method for preparing neural stem cells according to claim 1, characterized in that, The amplification medium consists of the following components: DMEM / F12 serum-free medium, 20-40 ng / mL tocopherol, 50-80 ng / mL glutathione, 200-400 ng / mL EGF, 300-500 ng / mL bFGF, 1-2 mg / mL eicosapentaenoic acid, 6-10 mg / mL loofah powder, 10-30 mg / mL LMNFe-LDH, 20-40 mg / mL hydrolyzed chitosan, 15-30 mg / mL fetal bovine serum, and 30-50 mg / mL albumin.

7. The method for preparing neural stem cells according to claim 1, characterized in that, The amplification medium consisted of the following: DMEM / F12 serum-free medium, 30 ng / mL tocopherol, 60 ng / mL glutathione, 300 ng / mL EGF, 400 ng / mL bFGF, 1.8 mg / mL eicosapentaenoic acid, 8 mg / mL loofah powder, 20 mg / mL MnFe-LDH, 30 mg / mL hydrolyzed chitosan, 20 mg / mL fetal bovine serum, and 45 mg / mL albumin.

8. The method for preparing neural stem cells according to claim 1, characterized in that, The MnFe-LDH flakes have a diameter of 50~400 nm.

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