Induced differentiation method of functional cerebral cortical cells

A technique for inducing differentiation of the cerebral cortex, applied in the direction of biochemical equipment and methods, animal cells, nervous system cells, etc., can solve problems such as limitations, long culture time, unsuitable for re-changing liquids, etc., to shorten the production cycle and reduce manufacturing costs. cost effect

Active Publication Date: 2018-08-03
ZHEJIANG HUODE BIOENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, in multi-well plates for high-throughput drug screening, neurons cannot be cultured stably and healthy for a long time, and it is not suitable to change the medium again, so the need for long culture time and immaturity greatly limit the ability of these cells in diseases. Large-scale application in research, drug screening and toxicity testing, also affects the representativeness and reliability of the obtained data

Method used

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  • Induced differentiation method of functional cerebral cortical cells
  • Induced differentiation method of functional cerebral cortical cells
  • Induced differentiation method of functional cerebral cortical cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Step 1. After digesting the neural precursor cells (hNPC) obtained by differentiation of the human induced pluripotent stem cell line DYR0100 with accutase, press 5×10 5 / cm 2 Inoculate onto poly-D-lysine and laminin-coated cell culture plates;

[0049] Step 2. Use Neural Differentiation Medium A from day 1 at 37°C, 5% CO 2 Cultured in the cell culture box for 7 days, half of the medium was changed every 3 days; the neural differentiation medium A contained final concentrations of 2 μM retinoic acid, 20ng / ml BDNF, 20ng / ml GDNF, 0.2mM ascorbic acid, 100nM SU5402, 200ng / ml The Neurobasal medium of ml BIBF1120, 10μM IBMX and 5mM glucose and the B27 supplement without vitamin A, wherein the dosage ratio of the Neurobasal medium and the B27 supplement is 50:1;

[0050] Step 3. Use Neural Differentiation Medium B from day 7 at 37°C, 5% CO 2 Culture in a cell culture incubator, half of the medium was changed every 3 days; neural differentiation medium B contained final conc...

Embodiment 2

[0065] Step 1. After digesting the neural precursor cells (hNPC) obtained by differentiation of the human induced pluripotent stem cell line DYR0100 with accutase, press 5×10 5 / cm 2 Inoculate onto poly-D-lysine and laminin-coated cell culture plates;

[0066] Step 2. Use Neural Differentiation Medium A from day 1 at 37°C, 5% CO 2 Cultivate in the cell culture box for 7 days, and change the medium in half every 3 days; the neural differentiation medium A contains Neurobasal culture with a final concentration of 2μM retinoic acid, 20ng / ml BDNF, 20ng / ml GDNF, 0.2mM ascorbic acid, and 10μM SU5402 Base and B27 supplement without vitaminA, wherein the dosage ratio of Neurobasal medium and B27 supplement is 50:1;

[0067] Step 3. Use Neural Differentiation Medium B from day 7 at 37°C, 5% CO 2 Culture in the cell culture incubator and change the medium every 3 days; neural differentiation medium B contains neurobasal medium with a final concentration of 20ng / ml BDNF, 20ng / ml GDNF,...

Embodiment 3

[0073] Step 1. After digesting the neural precursor cells (hNPC) obtained by differentiation of the human induced pluripotent stem cell line DYR0100 with accutase, press 5×10 5 / cm 2 Inoculate onto poly-D-lysine and laminin-coated cell culture plates;

[0074] Step 2. Use Neural Differentiation Medium A from day 1 at 37°C, 5% CO 2 Cultured in the cell culture box for 7 days, half of the medium was changed every 3 days; the neural differentiation medium A contained final concentrations of 2 μM retinoic acid, 20 ng / ml BDNF, 20 ng / ml GDNF, 0.2 mM ascorbic acid, 5 μM SU5402, 50 μM IBMX and the Neurobasal medium of 10mM glucose and the B27 supplement without vitamin A, wherein the dosage ratio of the Neurobasal medium and the B27 supplement is 50:1;

[0075] Step 3. Use Neural Differentiation Medium B from day 7 at 37°C, 5% CO 2 Culture in a cell culture incubator, and change the medium every 3 days; neural differentiation medium B contains neurobasal medium with a final concent...

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Abstract

The invention provides a culture medium for the induced differentiation of functional cerebral cortical cells. The culture medium is prepared from a nerve cell culture medium and a nutrient additive,wherein the nutrient additive is selected from one or more of SU5402, BIBF1120, IBMX and glucose. The invention provides an induced differentiation method of the functional cerebral cortical cells. Specific factors such as an inhibitor of FGF and VEGF signal channels and an activator of cAMP are added in a specific time of the induced differentiation of various nerve cells from the nerve stem cells or nerve precursor cells to accelerate the differentiation and maturation of the nerve cells, the stable and healthy nerve cells having a main function can be obtained in about 7 to 14 days of the induced differentiation from the human nerve precursor cells, and the nerve cells comprise excitable nerve cells and inhibitory nerve cells, so that the in-vivo cultured human nerve cells can be actually applied to the field of the drug screening and the like, the production cost can be reduced, and the production period can be shortened.

Description

technical field [0001] The invention belongs to the technical field of induction and differentiation of functional cerebral cortex cells, and in particular relates to a method for rapidly inducing differentiation of human neural stem cells into functional cerebral cortex cells. Background technique [0002] Since 2008 (Dimos et al.2008), human nerve cells have been induced and differentiated from human induced pluripotent stem cells (hiPSC) or embryonic stem cells (hESC) in vitro, and used for neurological disease research, drug screening, and neurotoxicity of drugs Tests, transplantation trials, etc. have always been hot spots in scientific research and drug development, and related articles have continued to grow. Among them, although the methods of inducing differentiation emerge in endlessly, it is still a big problem to obtain nerve cell populations with a composition and proportion close to that of the brain, as well as mature functions. [0003] It generally takes 30...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/0618C12N2501/165C12N2506/08C12N2501/13C12N2501/115C12N2500/38C12N2501/01C12N2533/52C12N2533/32C12N2500/34
Inventor 徐金翀范靖王安欣邹潭
Owner ZHEJIANG HUODE BIOENG CO LTD
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