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Nerve cell serum-free medium and application thereof

A technology of serum-free medium and serum medium, which is applied to nervous system cells, cell culture active agents, and culture processes, and can solve problems such as poor repeatability, difficulty in culturing nerve cells, and affecting the reliability of experimental results.

Active Publication Date: 2017-09-29
辉源生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the relatively difficult culture of nerve cells, low survival rate, high proportion of non-neural cells, and poor reproducibility between different cell culture batches, this affects the reliability of the experimental results.

Method used

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  • Nerve cell serum-free medium and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] A serum-free medium for cultivating primary rat neuron cells and application thereof, comprising the following steps:

[0051] 1. Development of serum-free medium

[0052] 1. Basal medium D-MEM: a product from Thermo-Fisher, catalog number: 11995065;

[0053] 2. Serum substitutes:

[0054]2.1 Growth factors: basic fibroblast growth factor (bFGF), final concentration: 0.1-10 μg / ml; epidermal growth factor (EGF), final concentration: 0.1-10 μg / ml; vascular endothelial growth factor (VEGF), final concentration: 0.1-10μg / ml; brain-derived neurotrophic factor (BDNF), final concentration: 0.01-1μg / ml.

[0055] 2.2 Protein: bovine serum albumin, final concentration: 0.001-0.1%; transferrin, final concentration 10-500 μg / ml.

[0056] 2.3 Hormones: insulin, final concentration: 1-100 μg / ml; insulin-like growth factor 1 (IGF-1), final concentration: 0.1-10 μg / ml; corticosterone, final concentration: 1-100 μg / ml; progesterone, final Concentration: 0.1-10μg / ml.

[0057] 2.4 Ot...

Embodiment 2

[0075] Embodiment 2, rat hippocampal neuron cell adherent culture

[0076] 1. Take 15-20 day old SD rats, take out the bilateral hippocampus aseptically by conventional methods, cut them into pieces, put them in 0.125% trypsin / EDTA digestion solution, and store them at 37°C, 5% CO 2 , digest for 20-30 minutes;

[0077] 2. Aseptically transfer the above-mentioned digested tissue to a 15ml sterile centrifuge tube, add 3ml of serum-containing medium, and then gently pipette to disperse the tissue into single cells;

[0078] 3. Count the above cell suspension with a hemocytometer and adjust the cell concentration to ~5x10 5 cells / ml;

[0079] 4. Inoculate cells into 6-well culture plate, 1.5ml / well, at 37°C, 5% CO 2 Cultivate in the incubator for 24 hours;

[0080] 5. On the second day, replace the medium with serum or the medium without serum in different wells, and continue to culture for 7 days, during which the medium was changed twice;

[0081] 6. After the 7th day, add ...

Embodiment 3

[0084] Embodiment 3, rat cortical nerve cell adherent culture

[0085] 1. Take newborn SD rats, take out the cerebral cortex aseptically by conventional methods, peel off the meninges, cut them into pieces, put them in 0.125% trypsin / EDTA digestion solution, and store them at 37°C, 5% CO 2 , digest for 20-30 minutes;

[0086] 2. Aseptically transfer the above-mentioned digested tissue to a 15ml sterile centrifuge tube, add 5ml of serum-containing medium, and then gently pipette to disperse the tissue into single cells, and then filter the cell suspension with a 200-mesh sterile sieve to another in a centrifuge tube;

[0087] 3. Count the above cell suspension with a hemocytometer and adjust the cell concentration to ~3x10 5 cells / ml;

[0088] 4. Inoculate cells into 6-well culture plate, 1.5ml / well, at 37°C, 5% CO 2 Cultivate in the incubator for 24 hours;

[0089] 5. On the second day, replace the medium with serum or the medium without serum in different wells, and cont...

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Abstract

The invention relates to a nerve cell serum-free medium and application thereof. The nerve cell serum-free medium comprises the following components: a basic medium D-MEM (Dulbecco's Modified Eagle Medium); growth factors, namely a basic fibroblast growth factor of which the final concentration is 0.1-10mu g / ml, an epidermal growth factor of which the final concentration is 0.1-10mu g / ml, a vascular endothelial growth factor of which the final concentration is 0.1-10mu g / ml, and a brain-derived neurotrophic factor of which the final concentration is 0.01-1mu g / ml; proteins, namely a cattle serum protein of which the final concentration is 0.001-0.1mu g / ml, and transferrin of which the final concentration is 10-500mu g / ml; hormones, namely insulin of which the final concentration is 1-100mu g / ml, an insulin like growth hormone 1 of which the final concentration is 0.1-10mu g / ml, corticosterone of which the final concentration is 1-100mu g / ml, and progesterone of which the final concentration is 0.1-10mu g / ml; and other substances, namely a soybean trypsin inhibitor of which the final concentration is 0.1-20mu g / ml, butanediamine of which the final concentration is 1-100mu g / ml, and sodium selenite of which the final concentration is 1-1000mu g / ml. Compared with the prior art, the invention provides an efficient and reliable serum-free medium for rat nerve cell culture.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a cell culture medium, in particular to a serum-free culture medium for nerve cells and its application. Background technique [0002] Nerve cells are one of the research focuses in modern neuroscience. In a specific in vitro environment, the morphology, growth, differentiation, migration, synapse formation, physical and chemical changes of neurons or glial cells can be continuously and directly observed through culture. Reaction. In experiments, serum medium is often used to culture primary rat hippocampal neuron cells and cortical neuron cells in vitro for related research work. Due to the relative difficulty in culturing neural cells, low survival rate, high proportion of non-neural cells, and poor reproducibility between different cell culture batches, this affects the reliability of the experimental results. [0003] The main reasons for the above-mentioned nerve cell culture pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/0793
CPCC12N5/0618C12N5/0619C12N2500/25C12N2500/40C12N2500/46C12N2500/90C12N2501/105C12N2501/11C12N2501/115C12N2501/13C12N2501/165C12N2501/39C12N2501/392C12N2501/734
Inventor 陈景才严中华
Owner 辉源生物科技(上海)有限公司
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