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Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof

A technique for primary culture of hippocampal neurons, which is applied in the field of primary culture medium and preparation of hippocampal neurons of newborn rats. and cell metabolism, ensuring purity and activity, increasing the effect of ATP synthesis

Inactive Publication Date: 2014-01-29
温州医学院附属第二医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Chinese patent CN1966674A discloses a kind of spider central nervous cell culture medium, and the culture medium that this invention adopts has added 200ml calf serum; The basal culture medium mixed at 1:1, add insulin, L-glutamine, butylenediamine hydrochloride, sodium selenide, human transferrin, hydrocortisone, progesterone, and add 5-20% fresh Serum; therefore, the formula and method of the above nerve culture medium can not effectively reduce the growth of glial cells and ensure the purity of neurons.
Although the purity of nerve cell culture has been improved and the growth of glial cells has been effectively reduced, it still cannot provide enough energy for the culture of hippocampal neurons, especially in the hypoxic environment, which is more likely to cause lethal damage and affect the hippocampus. Growth state of neurons, resulting in poor cell survival, slow cell growth, and poor synapse formation
By increasing the dose of glucose, the result will be even worse. Glucose as an energy source produces a large amount of lactic acid accumulation in anaerobic glycolysis, which will also reduce the pH value of the culture medium, increase the osmotic pressure, damage the nerve cells, and cause the growth of nerve cells in the medium. Inhibition, while also increasing the chance of neuronal contamination

Method used

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  • Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof
  • Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof
  • Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The formula and preparation method of embodiment 1 hippocampal neuron culture fluid

[0022] Prepare culture medium 1-9 according to the formula in the table below:

[0023]

[0024] The preparation method of above-mentioned nutrient solution 1-7 is as follows:

[0025] a. Add fructose 1,6 diphosphate and fructose into the basic culture medium Neurobasal Medium according to the ratio, and stir well;

[0026] b. Add the basic fibroblast growth factor, insulin, ferrous sulfate, human transferrin, amphotericin B, and vitamin mixture of the above-mentioned amount;

[0027] c. Adjust the pH to 7.4-8.0 with 1-10 normality of sodium hydroxide;

[0028] d. The laminar flow cell culture chamber is sterilized by suction and stored at 4°C for later use, and the above-mentioned amount of ATP and coenzyme A is added before use. Example 2 The method for primary culture of rat hippocampal neurons

Embodiment 2

[0028] d. The laminar flow cell culture chamber is sterilized by suction and stored at 4°C for later use, and the above-mentioned amount of ATP and coenzyme A is added before use. Example 2 The method for primary culture of rat hippocampal neurons

[0029] with the following steps:

[0030] ① Prepare the culture medium for the primary culture of hippocampal neurons as described in Example 1;

[0031] ②Material collection and digestion: Decapitate the brain of newborn rats, remove the brain tissue bluntly on ice, remove the vascular membrane, and take out the hippocampus tissue completely; cut it into small pieces of about 1mm3, and add a concentration of about the same volume as the tissue 2-4mg / mL papain, mix well, and digest at 37°C for 5-15min. The digestion was stopped with DMEM / F12 medium containing 10% fetal bovine serum, and 10 μl DNase I was added to open the flocculent adhesions, and the tissue block was gently blown with a pipette to disperse the cells.

[0032] ③...

Embodiment 3

[0034] Morphological observation and functional test of primary cultured rat hippocampal neurons of embodiment 3

[0035] Observed under an inverted microscope, rat hippocampal nerve cells began to adhere to the wall 6-8 hours after inoculation, scattered individually, and appeared round. After 24 hours, the cells were almost completely attached to the wall, and the cells began to flatten, and began to grow 1 to 2 protrusions. After 3 days, the number of neurons further increased, the processes further increased, and the glial cells also began to rapidly divide and proliferate, forming a supporting layer under the neurons. On the 10th day, the neurons were in good condition, and the synapses of the neurons were mature, forming an obvious neural network. After 14 days of culture, although the number of cells decreased, the cells reached the best state, and there were obvious halos around the cell body, and the cells were the most plump. After 17 days of culture, the cells beg...

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Abstract

Provided is a culture medium for the primary culture of hippocampus neurons of a neonatal rat. The culture medium is formed by adding 1,6-diphosphate fructose, fructose and nutritional supplements to a basic medium Neurobasal. Also provided is a method for the primary culture of hippocampus neurons of a rat using the abovementioned culture medium, comprising the steps of: (1) preparing the culture medium of hippocampus neurons; (2) sampling and digesting; (3) preparing a single cell suspension; (4) inoculating and culturing. Hippocampus neurons with good growing status and higher purity can be obtained using the abovementioned culture medium.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primary culture solution of neonatal rat hippocampal neurons, a preparation method and application thereof. Background technique [0002] Neuron is the basic unit of the structure and function of the nervous system, and it is also a good material for studying the physiological and pathological mechanisms of the nervous system and the functions of various neurotransmitters. The number and distribution of neurons in the hippocampus are relatively concentrated, and the culture of hippocampal neurons in vitro is one of the important means to study the structure, function, pathological and physiological changes of the nervous system. In the existing hippocampal neuron culture medium, serum is basically added to DMEM and F12 medium to culture hippocampal neurons. Serum increases the proliferation rate of non-neuronal cells such as glial cells, affects the purity of hippocamp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0793
CPCC12N2500/34C12N5/0619
Inventor 孙晶
Owner 温州医学院附属第二医院
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