Improved primary culture method for hippocampal neurons

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...

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. Add DMEM or high sugar to supply the brain in Hank's solution. Metabolism, but the concentration of glucose is too high, it is easy to increase the chance of bacterial contamination; adding DMEM medium will make it alkaline, which is not conducive to the survival of neuron cells

Chinese patent CN102978162A "neuron separation and culture method and reagent", Chinese patent CN102994452A "neuron separation and culture method with high efficiency" and Chinese patent CN102994451A "neuron separation and culture improved method", taken Put the brain tissue into a petri dish filled with 1×PBS, the cleaning solution used is PBS solution, DMEM-high glucose or DMEM-F12 and horse serum to soak the brain during dissection, to supply the metabolism of the brain, taking into account Needed for energy metabolism of neurons, but no antibacterial substances have been added. If the concentration of glucose is too high, it is easy to increase the chance of cell contamination

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