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Method for inducing human neural stem cells to differentiate into dopaminergic neurons in vitro

A dopaminergic and differentiation-inducing technology, which is applied in the field of biomedicine, can solve the problems of low induction efficiency of dopaminergic neurons and the influence of serum on clinical use, and achieve the effects of easy acquisition, reduced probability, and avoiding cross-contamination

Active Publication Date: 2017-03-01
SHANGHAI ANGECON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention provides a method for using serum-free medium to adhere to the wall to culture neural stem cells and make them directionally induce and differentiate into dopaminergic neurons. Clinical use and other issues

Method used

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  • Method for inducing human neural stem cells to differentiate into dopaminergic neurons in vitro
  • Method for inducing human neural stem cells to differentiate into dopaminergic neurons in vitro
  • Method for inducing human neural stem cells to differentiate into dopaminergic neurons in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Adhesive culture of human neural stem cells:

[0021] Dilute laminin (Laminin, LN, purchased from Lifetechnologies) with DPBS (Dulbecco's Phosphate Buffered Saline) to 10 μg / mL; take a six-well plate for cell culture, coat it with Laminin solution, and store at 37°C Act for 2 hours, then wash the six-well plate once with DPBS, and store it at room temperature for later use; take human neural stem cells freshly passaged to passage P5-P8, count the cells, and use serum-free medium according to 5×10 4 / mL to make a cell suspension, evenly inoculated into the culture plate prepared above, at 35°C, 5% CO 2 Incubate in an incubator for 24 hours.

[0022] The above-mentioned serum-free medium contains DMEM / F12, B27 (1:50), N2 (1:50), bFGF (basic Fibroblast Growth Factor, basic fibroblast growth factor, 20ng / mL, purchased from Lifetechnologies Company) and EGF ((Epidermal Growth Factor, epidermal growth factor, 20ng / mL, purchased from Lifetechnologies company).A...

Embodiment 2

[0023] Example 2: Induction of dopaminergic neuronal precursor cells:

[0024] Replace with dopaminergic neuron precursor cell induction medium, and remove unattached cells and dead cells; 35°C, 5% CO 2 The culture is statically cultured in the incubator for 7 days, and the medium is changed every 3 days; when the cells grow to 60-70%, the directional induction of dopaminergic neurons can be carried out.

[0025] The above-mentioned dopaminergic neuron precursor cell induction liquid formula is as follows:

[0026]

Embodiment 3

[0027] Example 3: Cryopreservation and recovery of dopaminergic neuron precursor cells:

[0028] Cell cryopreservation step: Wash the dopaminergic neuron precursor cells in the culture plate once with DPBS without Ca and Mg, then add 0.05% trypsin-EDTA and place in a 37°C incubator Digest in medium for 2 minutes, add trypsin inhibitor (trypsin inhibitor) to stop digestion after digestion, then gently blow and blow to make cell suspension into 50ml centrifuge tube, centrifuge at 400g for 5min, add 20ml DMEM / F12 to wash once, Centrifuge at 400g for 5min, then resuspend with freezing solution to a final concentration of 1×10 6 / ml. Use 2ml cryopreservation tubes for aliquots, 1ml per tube, place in a programmed freezer box, store in a -80°C refrigerator overnight, take it out and store it in a liquid nitrogen tank the next day.

[0029] Cell recovery steps: Take 5 tubes of cells frozen in liquid nitrogen, put them into a thermos bottle filled with liquid nitrogen and transfer t...

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Abstract

The invention discloses a method for differentiating human neural stem cells into dopaminergic neuron by in-vitro directional induction. The method comprises the following steps of adherent culture of human neural stem cells, induction of dopaminergic neuron precursor cells, directional induction of dopaminergic neuron and cryopreservation and anabiosis of the human neural stem cells and the dopaminergic neuron precursor cells. By virtue of the steps of the method disclosed by the invention, abundant dopaminergic neuron can be obtained in vitro; compared with the plurality of dopaminergic neuron production ways at present, the method disclosed by the invention has characteristics of low cost, high safety, high production efficiency and strong operability. The in-vitro dopaminergic neuron production way can provide a novel idea for applying dopaminergic neuron to clinical transplant treatment of the Parkinson disease.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for using a serum-free medium to adhere to culture neural stem cells and induce differentiation into dopaminergic neurons. Background technique [0002] It is well known that the main cause of Parkinson's disease is the decrease of dopamine neurotransmitters caused by the degeneration of dopaminergic neurons in the substantia nigra and striatum of the midbrain. The current treatment methods are mainly exogenous supplementation of dopamine precursors (such as L-dopa) and application of some dopamine receptor agonists or similar drugs. The treatment result of this kind of method is not stable, it is easy to cause serious sequelae, and can not fundamentally solve the problem. The most ideal way to treat Parkinson's disease should be to stimulate the generation of endogenous dopaminergic neurons in the substantia nigra and striatum of the midbrain, or to replace and integrate exo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0793
Inventor 赵雄飞金宜强郑佳威杨立敏周均云
Owner SHANGHAI ANGECON BIOTECH
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