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Culturing method of chick embryo forebrain neuron

A culture method, neuron technology, applied in the field of neuron culture, to achieve good responsiveness

Inactive Publication Date: 2016-03-16
PEKING UNIV SHENZHEN GRADUATE SCHOOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the electrophysiology of the neural network of CFBN based on MEA technology.

Method used

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  • Culturing method of chick embryo forebrain neuron
  • Culturing method of chick embryo forebrain neuron
  • Culturing method of chick embryo forebrain neuron

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Nerve tissue dissection of chicken embryonic forebrain

[0040] (1) Take the fertilized eggs of white Laihang chickens from the 7th to the 9th day, spray them with 70% alcohol for disinfection, and put all the dissection instruments into the dissection ultra-clean bench;

[0041] (2) Crack the egg head (big head) with blunt medium-sized tweezers, and carefully remove the eggshell, peel off the shell membrane, take out the chicken embryo and put it in a 35mm Petri dish filled with ice-cold HBSS (Gibco);

[0042] (3) Cut off the head of the chicken embryo with No. 5 tweezers, and put it into another new 35mm Petri dish with HBSS;

[0043] (4) Put the back of the head of the chicken embryo upwards, press the midbrain with the No. 5 tweezers with the left hand, and uncover the meninges of the forebrain with the No. 5 tweezers with the right hand, and then clamp out the two groups of white tissues;

[0044] (5) Transfer the two groups of white tissues to a new 35mm culture ...

Embodiment 2

[0051] Microelectrode array culture of chick embryo forebrain neurons

[0052] (1) Surface treatment of microelectrode array (60MEA200 / 30iR-Ti, MultichannelSystem, Germany):

[0053] Washing: If the MEA that has been cultured is reused, first soak it in distilled water for 10 minutes to swell and rupture the cells, and then rinse off the surface cells with distilled water. Add 0.5 mL of trypsin solution and place at 37°C for 30 min to wash off excess cell debris. Then add neutral detergent water with enzymes, soak at room temperature overnight, then rinse with distilled water. If there are still very stubborn fragments, ultrasonicate for 3 minutes, and then rinse with distilled water. Finally rinse the surface twice with deionized water and let the surface dry.

[0054] Sterilization: UV sterilization for 30min.

[0055] Plasma treatment: put it into a plasma surface treatment instrument (PDC-32G, Harrick), and then evacuate it to below 200mTORR, inject a small amount of o...

Embodiment 3

[0061] To confirm the cell type in the culture system, immunostaining was used to label neurons and glial cells for specific proteins. Among them, MAP2, microtubule-associated protein 2, is a neuron-specific skeletal protein, mainly distributed in the soma and dendrites. GFAP, glial fibrillary acidic protein, is a hallmark structural protein of glial cells. SynapsinI is a synapse-associated protein.

[0062] (1) Fixation: Wash the cell surface debris with PBS, and add 4% paraformaldehyde (containing 4% sucrose) for 15 minutes at room temperature.

[0063] (2) Membrane rupture: Aspirate the excess paraformaldehyde and wash it once with PBS. Add 1% Triton100 to break the membrane for 10min.

[0064] (3) Blocking: Aspirate the Triton100 permeabilization solution and wash it once with PBS. Add 1% albumin (BSA) to block non-specific groups.

[0065] (4) Primary antibody: Aspirate the blocking solution. Add the primary antibody combination for double staining according to the ...

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Abstract

The invention belongs to the field of neuron culturing, and more specifically relates to a culturing method of chick embryo forebrain neuron (CFBN). The culturing method comprises following steps: chick embryo forebrain nervous tissues are collected; a culture apparatus is coated with polyethyleneimine; and culturing of chick embryo forebrain neuron is carried out with Neurobasal culture medium containing fetal bovine serum. The culturing method is capable of realizing long time culturing of chick embryo forebrain neuron under high density conditions; it is confirmed by immunostaining results that neuron and gliocyte coexist, and synapse is formed on the 7th day; with gliocyte proliferation, compact cell layers are formed on the neural network of MEA (microelectrode array), and the cell layers are capable of surviving for as long as 6 months; and CFBN neural network possesses excellent response activity on neuroactive substances, and can be applied for neurotoxicity detection platforms.

Description

technical field [0001] The invention relates to neuron culture, in particular to a culture method for chicken embryo forebrain neurons. Background technique [0002] Chicken embryonic forebrain neurons (CFBN), similar to mammalian cerebral cortex neurons, have a wide range of sources, are simple to operate, and low in cost, and are often used as one of the models for in vitro neurotoxicity testing. As early as 1993, Weiss et al. used the CFBN model to do multi-center in vitro cytotoxicity detection and evaluation (MEIC), and found that it was highly correlated with the in vitro experimental data of rodents (murines), and was highly correlated with the in vivo experimental data of rodents. are also comparable. However, compared with the culture of rodent cerebral cortex cells, there are few reports on the culture of CFBN cells. The research method of Heidemann et al. is mainly aimed at low-density, short-term cell culture. But low-density neural networks are difficult to d...

Claims

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Application Information

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IPC IPC(8): C12N5/0793
CPCC12N5/0619C12N2500/70C12N2500/84
Inventor 盛立远赖琛甄珍都贝宁魏利娜王巧莉高志奚廷斐
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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