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Method for separately culturing hippocampus neural stem cells and neurons

A neural stem cell, isolation and culture technology, applied to nervous system cells, animal cells, vertebrate cells, etc., can solve problems such as waste, and achieve the effects of maintaining differentiation potential, stable passage, and good proliferation.

Inactive Publication Date: 2018-01-12
冯世庆
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide a method for separating and culturing hippocampal neural stem cells and neurons in view of the problems existing in the prior art, and to solve the problem that the existing neuron cell culturing method will cause great waste

Method used

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  • Method for separately culturing hippocampus neural stem cells and neurons
  • Method for separately culturing hippocampus neural stem cells and neurons
  • Method for separately culturing hippocampus neural stem cells and neurons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] This example provides a method for isolating and culturing hippocampal neural stem cells and neurons. This method uses an improved neonatal mouse nerve culture process and improved medium for primary culture of neonatal rat hippocampal tissue, and can directly make cell slides. 24 After 1 hour, extract the cell culture supernatant and transfer it to another culture system to directly culture neural stem cells, and replace the adherent cells in the original system with the improved neuron culture medium for neuron culture. Concrete steps of the present invention are as follows:

[0044] 1. Lay the board with poly-lysine first, rinse with double distilled water after finishing, and then dry naturally;

[0045] 2. Execute the mice on the day of birth or the first day, spray the mice with alcohol, collect the brains of the mice in a centrifuge tube filled with culture medium + penicillin / streptomycin solution, and place them on ice;

[0046] 3. Remove the meninges under a ...

Embodiment 2

[0056] This example provides a method for isolating and culturing hippocampal neural stem cells and neurons. This method uses an improved neonatal mouse nerve culture process and improved medium for primary culture of neonatal rat hippocampal tissue, and can directly make cell slides. 24 After 1 hour, extract the cell culture supernatant and transfer it to another culture system to directly culture neural stem cells, and replace the adherent cells in the original system with the improved neuron culture medium for neuron culture. Concrete steps of the present invention are as follows:

[0057] 1. Spread the plates with 50ug / ml poly-lysine 2 hours in advance, rinse with double distilled water 3 times after finishing, and dry naturally.

[0058] 2. Execute the mice on the day of birth or the first day, spray the mice with 70% alcohol, collect the brains of the mice in a 50ml centrifuge tube filled with culture medium + 2% penicillin / streptomycin solution, and place them on ice ....

Embodiment 3

[0069] Material preparation:

[0070] C57blmouse (C57BL / 6 mice) P0-1,; ice DMEM+P / S 30ml (DMEM culture medium (Gibco)+1% penicillin / streptomycin solution 30ml placed in ice); sterilized blades, scissors, Tweezers; microscope; neural stem cell medium (Neurobasal medium (Neurobasal medium (Gibco)) + 2% B27 (B-27 cell culture supplement) + 1% N2 (N2 cell culture supplement) + 20ng / ml EGF (epidermal growth factor )+20ng / ml bFGF (basic fibroblast growth factor)+1%P / S (penicillin / streptomycin solution)); neuron medium (Neurobasal medium+2%B27+1%L-G+1 %P / S); 0.05% trypsin (trypsin); FBS (fetal bovine serum); PDL (polylysine) 50ug / ml; 15mm coverslips (cover slip).

[0071] step:

[0072] 1. Spread the plates with 50ug / ml poly-lysine 2 hours in advance, rinse with double distilled water for 3 times, and dry naturally. Among them: 1ml per well of 6-well plate; 0.5ml per well of 24-well plate.

[0073] 2. Execute the P0-1 mice (the young mice on the day of birth and the first day), s...

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Abstract

The invention provides a method for separately culturing hippocampus neural stem cells and neurons. The method comprises the following steps: adopting an improved newborn rate nerve cell culturing process and an improved culture medium for performing primary culture on newborn rate hippocampus tissue; directly preparing hepatocytes growing on glass coverslips; extracting cell culture supernatant after 24 hours and transferring to another culture system for directly culturing neural stem cells; and replacing the improved neuron culture medium for the adherent cells in original system, thereby culturing the neurons. According to the invention, the stable culturing for neural stem cells and neurons is completed on the basis of killing an animal at one time, so that the experimental animals can be saved; the neurons with excellent culture density and form can be cultured and an in vitro experimental system related to the neurons can be constructed; the cultured neural stem cells have excellent proliferation, generation stability and excellent differentiation potential.

Description

technical field [0001] The invention relates to the field of nerve cell culture, in particular to a method for separating and culturing hippocampal neural stem cells and neurons. Background technique [0002] Nerve cell culture is a basic technique for studying neuroscience, especially primary cell culture, which is very strict with sterile technique / separation techniques, etc. Due to the non-proliferative nature of neurons (terminal differentiated cells), the in vitro culture of neurons requires Consume large numbers of laboratory animals. Neural stem cells are another hot spot in current neuroscience research. Neural stem cells are mostly derived from newborn mice or embryos. Due to the particularity of neural stem cell culture conditions (serum-free culture, otherwise it will differentiate), an experimental animal often can only It is simply used for the extraction of neural stem cells, causing great waste. Contents of the invention [0003] The purpose of the present...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/0793
Inventor 冯世庆魏志坚刘洋周新福姚雪周先虎宁广智史仲举樊保佑丁汉
Owner 冯世庆
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