Hippocampal neuron separation and primary culture method and reagent

A hippocampal neuron, primary culture technology, applied in the field of hippocampal neuron isolation and primary culture methods and reagents, can solve the problems of easy to increase bacterial contamination, lack of suitable, difficult to obtain, etc., to improve in vitro survival rate and digestion efficiency. Improve and increase the effect of physiological breathing

Inactive Publication Date: 2014-05-21
THE 2ND AFFILIATED HOSPITAL & YUYING CHILDRENS HOSPITAL OF WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the factors that make it difficult to obtain a sufficient number and vitality of primary hippocampal neurons are as follows: (1) During the isolation of hippocampal nerve tissue, most people use D-hank's or hank's as a rinse solution, and in vitro To remove impurities such as red blood cells, capsules, and connective tissue from mammalian hippocampal tissue, the separation process takes a long time, sometimes more than 2 hours, and the time in the rinsing solution will be longer, and the hippocampal neurons have partially died, resulting in false negative results. Results
The routine culture of hippocampal neurons cannot be carried out experimentally, mainly because there is no suitable rinse/dissecting solution for neuron culture in hippocampal tissue specimens
In the process of tissue separation, neurons still metabolize to a large extent even in the ice bath. The sugar-free environment of Hank's solution is not good for neuron separat...

Method used

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  • Hippocampal neuron separation and primary culture method and reagent

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Effect test

Embodiment 1

[0041] The Bifidobacterium sp. WZ002 of the present invention has been stored in the General Microbiology Center of the China Microbiological Culture Collection Management Committee on January 22, 2014, which is referred to as CGMCC (Address: Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing No. 3 Institute of Microbiology, Chinese Academy of Sciences, zip code 100101) is preserved, the classification is named Bifidobacterium sp, and the preservation number is CGMCC No.8809.

[0042] The bifidobacterium WZ002 of the present invention was isolated from the feces of a healthy young man in Zhejiang Province.

[0043] Bifidobacterium WZ002 bacterial strain of the present invention has following microbiological characteristics:

[0044] (1) Colony morphology: The colonies of the WZ002 strain on the plate are off-white or milky white, opaque, shiny, smooth, raised, soft in texture, with neat edges, and 1-1.5 mm in diameter.

[0045] (2) Individual form: G + Non-bacill...

Embodiment 2

[0051] Reagent purchase:

[0052] DMEM / F12 neural basal medium, Neurobasal medium and B 27 Purchased from Gibco Company; polylysine, fetal bovine serum (FBS), trehalose and L-glutamine were purchased from Sigma Company of the United States; penicillin and streptomycin mixed solution (double antibody) was purchased from HyClone Company of the United States.

[0053] Reagent configuration:

[0054] (1) The D-Hank's solution is obtained through the following steps: NaCl8.0g, KCl0.4g, NaCl 2 HPO 4 12H 2 O0.12g, KH 2 PO 4 0.06g, NaHCO 3 0.35g; Dissolve each component in approximately 500mL triple-distilled water and mix well, add triple-distilled water to make up to 1000mL, adjust the pH value to 7.2-7.4, subpackage, autoclave, subpackage, and store at 4°C for later use.

[0055] (2) The rinse liquid is obtained through the following steps: dissolve 2g trehalose, 3g glucose and 10mL double antibody in 100mL D-Hank's solution, mix well, add D-Hank's solution to 1000mL, 0.4 D...

Embodiment 3

[0066] The method for separating and primary culture of SD rat hippocampal neurons comprises the following steps:

[0067] Use the reagents prepared in Example 1 and Example 2 and configure the reagents.

[0068] (1) Rinsing: Take the hippocampus tissue of the isolated mammal, put it in the rinsing solution in an ice bath, remove red blood cells, capsules and connective tissue, and rinse it 2 to 5 times with the rinsing solution;

[0069](2) Digestion: cut the hippocampal tissue rinsed in step 1 into a diameter of 1mm 3 For small pieces, use 5 times the tissue volume of digestion solution at 37°C for 5-10 minutes to organize into a porridge-like shape, stop digestion with cell seeding solution, and gently pipette until the tissue piece is 10 times to disperse the cells.

[0070] (3) Preparation of cell suspension: Collect the initial cell suspension after digestion in step 2, filter through a 200-mesh cell sieve, centrifuge at 800-1000 rpm at 4°C for 5-10 minutes, discard the...

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Abstract

The invention belongs to the field of cell biology and relates to a hippocampal neuron separation and primary culture method and reagent. The existing hippocampal neuron separation and primary culture method is improved aiming at the problems of the prior art, and the problem that the proliferation property and the activity of primary culture of the hippocampal neuron cannot be kept is solved. A relatively mature method for culturing hippocampal neurons in vitro is established, the obtained nerve cells are sufficient, the growth condition is good, a number of hippocampal neurons with high activity can be separated from hippocampus of mammals, the requirement of primary hippocampus cell culture is met, and the requirement of cell biology experiments in neuroscience research is met.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method and a reagent for the separation and primary culture of hippocampal nerve cells. Background technique [0002] The hippocampus (hippocampal) is an important part of the central nervous system. As a highly concentrated area of ​​neurons, it has typical characteristics of the central nervous system and plays an important role in learning, memory, emotional response and autonomic nervous function. The neuron cell culture model is an important experimental model for studying neuron development and differentiation, nerve regeneration, and the mechanism of neurological diseases. In vitro culture of hippocampal neurons has become an important tool for studying neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. technical means. Primary culture refers to the first culture of cells, tissues or organs obtained from living cells under in vitro conditi...

Claims

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Application Information

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IPC IPC(8): C12N5/0793C12R1/01
Inventor 孙晶刘佳明
Owner THE 2ND AFFILIATED HOSPITAL & YUYING CHILDRENS HOSPITAL OF WENZHOU MEDICAL UNIV
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