Nerve stem cell special-purpose culture medium and culture method thereof

A technology of neural stem cells and culture methods, applied in the direction of nervous system cells, animal cells, vertebrate cells, etc., can solve the problems of low survival rate of neural stem cells, complex serum components, unstable quality, etc., to promote adhesion and proliferation, Maintain high activity rate, reasonable and effective formula

Pending Publication Date: 2017-03-08
BEIJING YULONG SHENGSHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because serum has the disadvantages of complex components, unstable quality, and high price, although the expansion efficiency of serum is higher, serum-free culture is still a development trend. At present, the addition of basic fibroblast growth factor (bFGF) is generally used. epidermal growth factor (EGF), antibiotics, N 2 and / or B 24 DMEM or DMEM / F12 culture medium with additives and other ingredients is used as a serum-free culture medium for neural stem cells. Although this culture medium solves the problems of potential viruses and contamination, and has the advantages of clearer culture medium components and high repeatability, the survival of neural stem cells Problems such as low rate, slow proliferation, low subculture yield, and severe differentiation are still difficult to solve

Method used

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  • Nerve stem cell special-purpose culture medium and culture method thereof
  • Nerve stem cell special-purpose culture medium and culture method thereof
  • Nerve stem cell special-purpose culture medium and culture method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] A special medium for neural stem cells, the medium is composed of DMEM / F12 culture fluid and solute, the solute and its concentration in DMEM / F12 culture fluid are: 300 μg / ml 2′,3′-di Deoxyadenosine-5-triphosphate, 5 μg / ml N2 Additives, 15ng / ml stem cell growth factor, 5mg / ml thioglycerol, 50ng / ml nerve growth factor, 0.05mmol / ml acetylcholine, 10mg / ml carboxymethylcellulose sodium, 0.005mmol / ml retinoic acid and 0.110mmol / ml folic acid.

Embodiment 2

[0031] A special medium for neural stem cells, said medium is composed of DMEM / F12 culture fluid and solute, said solute and its concentration in DMEM / F12 culture fluid are: 350μg / ml 2′,3′-di Deoxyadenosine-5-triphosphate, 8 μg / ml N 2 Additives, 22ng / ml stem cell growth factor, 6mg / ml thioglycerol, 63ng / ml nerve growth factor, 0.06mmol / ml acetylcholine, 18mg / ml sodium carboxymethylcellulose and 0.15mmol / ml folic acid.

Embodiment 3

[0033] A method for culturing neural stem cells, comprising the steps of:

[0034] 1) The isolated brain nerve tissue is obtained under sterile conditions, and rinsed with PBS buffer containing 5% double antibody;

[0035] 2) The brain nerve tissue obtained in step 1) is cut into 1mmol 3 Fragments, add DMEM / F12 medium containing 0.5mg / ml type I collagenase, 0.5mg / ml type IV collagenase and 0.1mg / ml deoxyribonucleic acid I, at 8 ℃ CO 2 Digest and decompose in the incubator for 20 hours to obtain tissue suspension;

[0036] 3) Centrifuge the tissue suspension obtained in step 2), remove the supernatant, add DMEM / F12 medium to wash 3 times, add cell digestion solution, digest at 35°C for 57min, gently pipette and mix to stop the digestion, and place in DMEM Rinse in / F12, pass through a 200-400 mesh cell sieve, collect the filtrate, centrifuge and discard the supernatant to obtain a single cell suspension;

[0037] 4) Add the special medium for neural stem cells to the single ...

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Abstract

The invention relates to a special-purpose culture medium for culturing neural stem cells and a culture method thereof. The neural stem cell special-purpose culture medium is based on a DMEM/F12 culture medium and comprises the following concentration components: 300-450[mu]g/ml of 2',3'-dideoxyadenosine-5-triphosphate, 5-15[mu]g/ml of a N2 additive, 15-30ng/ml of stem cell growth factors, 5-7mg/ml of thioglycerol, 50-70ng/ml of nerve growth factors, 0.05-0.07mmol/ml of acetylcholine, 10-25mg/ml of sodium carboxymethylcellulose, 0.005-0.01mmol/ml of retinoic acid and 0.110-0.20mmol/ml of folic acid. The culture medium formula is reasonable and effective. The undifferentiated state of neural stem cells can be maintained and the cell expansion rate is effectively improved. The differentiation performance is not influenced and the passage number is prolonged.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a special medium for neural stem cells and a culture method thereof. Background technique [0002] Neural stem cells (Neural Stem Cells, NSCs) are a kind of stem cells that exist in nervous tissues such as embryonic and adult brains and spinal cords. Various types of cells that replicate and renew or differentiate into neurons, astrocytes, oligodendrocytes and other nervous tissues can also be transdifferentiated into blood cells and skeletal muscle cells. Therefore, the purpose of cultivating neural stem cells is to obtain a sufficient number of neural stem cells with proliferation and differentiation potential for transplantation, transgenic manipulation to attack tumor growth, autologous reinfusion, and treatment of central nervous system dysfunction. [0003] Because the nerve is the most complex and highly differentiated tissue in the body, the culture of neural stem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
Inventor 张晓南吴芳春冯传前邵来
Owner BEIJING YULONG SHENGSHI BIOTECH CO LTD
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