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CN-118291382-B - Neural stem cell and preparation method and application thereof

CN118291382BCN 118291382 BCN118291382 BCN 118291382BCN-118291382-B

Abstract

The invention belongs to the technical field of biology, and particularly discloses a neural stem cell and a preparation method and application thereof, and the neural stem cell comprises the following steps of (1) rinsing hippocampal tissues with PBS buffer solution, putting the rinsed hippocampal tissues into digestive juice for digestion, adding digestive stop solution for stopping digestion, centrifuging to obtain cell sediment, washing the cell sediment with the PBS buffer solution, centrifuging, re-suspending cells with DMEM/F12 culture medium, adjusting the cell concentration to 1X 10 4 /mL, (2) primary culture, and (3) expansion culture. The amplification culture medium can provide a carrier with viscosity in the process of culturing the neural stem cells, promote division and proliferation of the neural stem cells, improve activity and stability of the neural stem cells, improve survival rate and cell number, and have wide application prospects.

Inventors

  • XU ZHIFENG
  • ZHANG XIN
  • LI ZHIYAO

Assignees

  • 广州沙艾生物科技有限公司

Dates

Publication Date
20260505
Application Date
20240117

Claims (8)

  1. 1. A method for preparing neural stem cells, comprising the steps of: (1) Rinsing the rat hippocampal tissue with PBS buffer solution, placing the rinsed hippocampal tissue into digestive juice for digestion, adding digestive stop solution to stop digestion, centrifuging to obtain cell precipitate, washing the cell precipitate with PBS buffer solution, centrifuging, re-suspending cells with DMEM/F12 culture medium, and adjusting the cell concentration to 1×10 4 cells/mL; (2) Adding the cells in the step (1) into a DMEM/F12 culture medium, and culturing until the cells are fused to 80-90% to obtain primary cells; (3) Inoculating 1.5X10 4 primary cells/mL into an amplification culture medium for amplification culture, transferring half of the liquid into the 5 th generation every day, and obtaining the neural stem cells; The amplification culture medium comprises a 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 luffa powder, 10-40 mg/mL MnFe-LDH, 10-40 mg/mL hydrolyzed chitin, 10-30 mg/mL fetal bovine serum and 10-50 mg/mL albumin; The neural stem cells are rat neural stem cells.
  2. 2. The preparation method of the neural stem cells according to claim 1, wherein the digestive juice comprises, by mass, 0.6-1% of sodium chloride injection, 1-4% of collagenase I, 2-5% of papain, 4-6% of glucose, 8-15% of calcium chloride, 15-25% of albumin and the balance of water.
  3. 3. The method for preparing neural stem cells according to claim 1, wherein the digestion terminator is a PBS solution of fetal bovine serum with a mass fraction of 10%.
  4. 4. The method of claim 1, wherein the step (2) is performed under conditions of a temperature of 37.0.+ -. 0.5 ℃ and a concentration of 5.0.+ -. 0.2% CO 2 .
  5. 5. The method of claim 1, wherein the step (3) is performed under conditions of a temperature of 37.0.+ -. 0.5 ℃ and a concentration of 5.0.+ -. 0.2% CO 2 .
  6. 6. The preparation method of the neural stem cells according to claim 1, wherein the amplification culture medium comprises a 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 luffa powder, 10-30 mg/mL MnFe-LDH, 20-40 mg/mL hydrolyzed chitin, 15-30 mg/mL fetal bovine serum and 30-50 mg/mL albumin.
  7. 7. The method according to claim 1, wherein the amplification medium comprises DMEM/F12 serum-free medium, 30 ng/mL tocopherol, 60ng/mL glutathione, 300 ng/mL EGF, 400 ng/mL bFGF, 1.8mg/mL eicosapentaenoic acid, 8mg/mL retinervus Luffae fructus powder, 20mg/mL MnFe-LDH, 30mg/mL hydrolyzed chitin, 20mg/mL fetal bovine serum, 45mg/mL albumin.
  8. 8. The method for preparing neural stem cells according to claim 1, wherein the MnFe-LDH has a sheet diameter of 50 to 400nm.

Description

Neural stem cell and preparation method and application thereof Technical Field The invention relates to the technical field of biology, in particular to a neural stem cell and a preparation method and application thereof. Background Neural Stem Cells (NSCs) are critical for the development, regeneration and repair of the nervous system. In the brain of adult mammals, most NSCs in a static state can be reactivated when being subjected to external stimulus and the coordination among a plurality of factors such as inherent signal molecules, transcription factors and the like, and bring prospect for cell therapy of nervous system diseases. The neural stem cells obtained by the existing method have low survival rate and cannot meet the requirement on quantity, and the application is provided in view of the above. Disclosure of Invention The invention provides a neural stem cell, a preparation method and application thereof, and the amplification culture medium can provide a carrier with viscosity in the process of culturing the neural stem cell, promote division and proliferation of the neural stem cell, improve activity and stability of the neural stem cell, improve survival rate and cell number, and has wide application prospect. A method for preparing neural stem cells, comprising the steps of: (1) Rinsing Hippocampus tissue with PBS buffer solution, placing the rinsed Hippocampus tissue into digestive juice for digestion, adding digestive stop solution to stop digestion, centrifuging to obtain cell precipitate, washing cell precipitate with PBS buffer solution, centrifuging, re-suspending cells with DMEM/F12 medium, and adjusting cell concentration to 1×10 4/mL; (2) Adding the cells in the step (1) into a DMEM/F12 culture medium, and culturing until the cells are fused to 80-90% to obtain primary cells; (3) The primary cells are inoculated into an amplification culture medium at the rate of 1.5 multiplied by 10 4/mL for amplification culture, half a day of liquid exchange is carried out, and the culture medium is transferred to the 5 th generation, so that the neural stem cells are obtained. The digestion liquid comprises, by mass, 0.6-1% of sodium chloride injection, 1-4% of collagenase I, 2-5% of papain, 4-6% of glucose, 8-15% of calcium chloride, 15-25% of albumin and the balance of water. As a preferred embodiment of the present invention, the digestion terminator is a PBS solution of fetal bovine serum with a mass fraction of 10%. As a preferred embodiment of the present invention, the step (2) is performed under the conditions of a temperature of 37.0.+ -. 0.5 ℃ and a concentration of CO 2 of 5.0.+ -. 0.2%. As a preferred embodiment of the present invention, the step (3) is performed under the conditions of a temperature of 37.0.+ -. 0.5 ℃ and a concentration of CO 2 of 5.0.+ -. 0.2%. As a preferred embodiment of the invention, the amplification medium consists of 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 retinervus Luffae fructus powder, 10-40 mg/mL MnFe-LDH, 10-40 mg/mL hydrolyzed chitin, 10-30 mg/mL fetal bovine serum and 10-50 mg/mL albumin. The MnFe-LDH and the hydrolyzed chitin are combined, the MnFe-LDH and the hydrolyzed chitin can be uniformly dispersed in a culture medium, a three-dimensional network is formed in the culture medium, a carrier can be provided for the culture of the neural stem cells, a larger space is provided for the adhesion and proliferation of the neural stem cells, the amplification effect is effectively improved, the cell viability is improved, and the two have remarkable synergistic effects. As a preferred embodiment of the invention, the amplification medium consists of 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 retinervus Luffae fructus powder, 10-30 mg/mL MnFe-LDH, 20-40 mg/mL hydrolyzed chitin, 15-30 mg/mL fetal bovine serum and 30-50 mg/mL albumin. As a preferred embodiment of the present invention, the amplification medium consists of DMEM/F12 serum-free medium, 30 ng/mL tocopherol, 60ng/mL glutathione, 300 ng/mL EGF, 400 ng/mL bFGF, 1.8mg/mL eicosapentaenoic acid, 8mg/mL retinervus Luffae fructus powder, 20mg/mL MnFe-LDH, 30mg/mL hydrolyzed chitin, 20mg/mL fetal bovine serum, 45mg/mL albumin. As a preferred embodiment of the present invention, the sheet diameter of the MnFe-LDH is 50-400 nm. The invention also provides an application of the neural stem cells prepared by the preparation method in preparing medicines for treating nervous system diseases. The invention has the beneficial effects that (1) the expansion culture medium can provide a carrier with viscosity in the culture process of the neural stem cells, promote the division and proliferation of the neural stem cells, improve the activity and stability of the neural st