Improved primary culture method for hippocampal neurons

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, increase physiological The effect of improving the efficiency of breathing and digestion

Inactive Publication Date: 2014-05-14
刘洛贤
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  • 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 separation

Method used

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  • Improved primary culture method for hippocampal neurons

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Experimental program
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Effect test

Embodiment 1

[0041] The Bifidobacterium adolescentis LJM-001 of the present invention has been in the General Microbiology Center of the China Microbial Culture Collection and Management Committee on August 17, 2010, and its abbreviation is CGMCC (Address: 1 Beichen West Road, Chaoyang District, Beijing) No. 3, Institute of Microbiology, Chinese Academy of Sciences, 100101) preserved, classified and named as Bifidobacterium adolescentis (Bifidobacterium adolescentis), preservation number is CGMCC No.4090.

[0042] The Bifidobacterium adolescentis LJM-001 of the present invention was isolated from the stool of a healthy young man in Zhejiang Province.

[0043] The Bifidobacterium adolescentis LJM-001 strain of the present invention has the following microbiological characteristics:

[0044] (1) Colony morphology: The colony of strain LJM-001 on the plate is off-white or milky white, opaque, shiny, smooth, convex, soft, with neat edges, with a diameter of 1-1.5mm.

[0045] (2) Individual form: G + B...

Embodiment 2

[0051] Reagent purchase:

[0052] DMEM / F12 Neural Basal Medium, Neurobasal Medium and B 27 Purchased from Gibco; Polylysine, fetal bovine serum (FBS), trehalose and L-glutamine were purchased from Sigma, USA; Penicillin mixture (double antibody) was purchased from HyClone, USA.

[0053] Reagent configuration:

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

[0055] (2) The rinsing solution is obtained through the following steps: 2g of trehalose, 3g of glucose and 10mL of double antibody are dissolved in 100mL of D-Hank's solution, mixed well, and D-Hank's solution is added to make the volume to 1000mL, 0.4 Dissolve hydrogen gas under MPa atmospheric pressure fo...

Embodiment 3

[0066] The isolation and primary culture method of SD rat hippocampal neurons includes the following steps:

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

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

[0069] (2) Digestion: Cut the hippocampal tissue rinsed in step 1 to 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 form a porridge shape. Use cell planting solution to terminate the digestion, gently pipetting to the tissue block 10 times to disperse the cells.

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

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Abstract

The invention belongs to the field of cell biology and relates to a method and reagent for the separation and culture of neurons. The method and the reagent are used for solving problems in the prior art, the current in-vitro hippocampal neuron separation and culture methods are improved, and the problem that the proliferation and activity of hippocampal neurons during primary culture can not be maintained is solved. According to the method and the reagent, a relatively mature in-vitro hippocampal neuron culture method is established, the number of obtained nerve cells is sufficient, the growth status is relatively good, and a large number of high-activity hippocampal nerve cells can be separated from mammalian hippocampal tissue, so that the requirements for the primary culture of the hippocampal cells are met, and the demands on experiments of cell biology during neuroscience research can be met.

Description

Technical field [0001] The invention belongs to the field of biotechnology and relates to a method and reagent for separating 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 neuronal cell culture model is an important experimental model for studying neuron development and differentiation, nerve regeneration, and the mechanism of neurological diseases. In vitro culturing hippocampal neurons has become an important part of 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 conditions. Primary neuron...

Claims

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Application Information

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IPC IPC(8): C12N5/0793C12R1/01
Inventor 刘洛贤
Owner 刘洛贤
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