Method for promoting proliferation of neural stem cells

A neural stem cell technology, applied to nervous system cells, animal cells, vertebrate cells, etc., can solve the problems of single reagent and weak effect of cell proliferation

Inactive Publication Date: 2019-02-22
赛璟生物医药科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The reagents currently used to promote the proliferation of neural stem cells are relatively si...

Method used

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  • Method for promoting proliferation of neural stem cells
  • Method for promoting proliferation of neural stem cells
  • Method for promoting proliferation of neural stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Different concentrations of VX-702 interfere with the proliferation of neural stem cells

[0034] The culture medium of the neural stem cell control group used basic culture medium: DMEM / F12 culture medium + 2% B27 (50×) + epidermal growth factor EGF (20ng / mL) + basic fibroblast growth factor bFGF (20ng / mL) + green / streptomycin (penicillin 100U / mL, streptomycin 0.1mg / mL) + amphotericin B (250ng / mL); the culture solution of the experimental group was added 1μM, 3μM, 5μM, 7μM, 10μM respectively on the basis of the control group VX-702.

[0035] Proceed as follows:

[0036] (1) After counting the neural stem cells cultured to the logarithmic growth phase, each well was divided into 3×10 3 Neural stem cells were seeded into 96-well plates, and each group was set up with 5 replicate wells.

[0037] (2) Divide the cells into blank wells (containing medium only), control wells (containing cells, medium, and no VX-702), and experimental wells (adding 1 μM, 3 μM, ...

Embodiment 2

[0041] Example 2 Detection of 7μM VX-702 Interfering with the Proliferation of Neural Stem Cells

[0042] The neural stem cells were divided into the control group and the experimental group. The culture medium of the control group was DMEM / F12 medium + 2% B27 (50×) + EGF (20ng / mL) + bFGF (20ng / mL) + penicillin / streptomycin ( Penicillin 100U / mL, streptomycin 0.1mg / mL) + amphotericin B (250ng / mL), the culture medium of the experimental group was added 7μM VX-702 on the basis of the control group.

[0043] Proceed as follows:

[0044](1) After the neural stem cells were in the logarithmic growth phase and counted on a counting plate, 1000 cells / well were planted in a 96-well plate, and each group was repeated for 5 wells, and 100 μL of ordinary medium was added to each well. Keep the cell volume in all wells equal, if the gap is too large, re-see the plate.

[0045] (2) In order to prevent the culture medium in the culture plate from being dried by the incubator, at the end of...

Embodiment 3

[0050] Example 3 7μM VX-702 Intervenes in the Formation of Neural Stem Cell Neurospheres

[0051] According to the method of steps (1) to (3) of Example 2, a control group and an experimental group were set up. The experimental group cultured neural stem cells with a medium added with 7 μM VX-702, and replaced the medium every 2 days for 3 days.

[0052] Such as image 3 As shown, after culturing for 3 days, observed under a phase-contrast microscope, the neural stem cell spheres in the experimental group gradually increased ( image 3 B).

[0053] The size and number of neural stem cell spheroids are as follows Figure 4 shown. Regardless of the neural stem cell spheres with a diameter of 20-50 μm, 51-80 μm or 81-110 μm, the number of the experimental group (VX-702) was far more than that of the control group (Ctrl), indicating that VX-702 (7 μM) can promote Proliferation of neural stem cells.

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Abstract

The invention belongs to the field of biotechnology, relates to a method for stem cell culture and discloses application of VX-702 to preparation of a neural stem cell culture medium for promoting proliferation of the neural stem cells or maintaining dryness of the neural stem cells. The VX-702 can effectively increase the proliferation of the neural stem cells, promote formation of neural stem cell neurospheres, and significantly increase the expression of neural stem cell markers, such as NESTIN and SOX2. The invention also provides a method for promoting the proliferation of the neural stemcells. The method includes the steps of culturing the neural stem cells with the neural stem cell culture medium containing the VX-702 under the conditions of 35 -39 DEG C and 3%-8% of CO2 in an adherent culture mode. The VX-702 is used as an additive, the neural stem cell culture medium and the method for promoting the proliferation of the neural stem cells are provided, and building of a stableand reliable in-vitro culture model of the neural stem cells is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for culturing stem cells, in particular to a method for promoting the proliferation of neural stem cells. Background technique [0002] Neural stem cells are a cell population with self-renewal ability and multi-lineage differentiation potential existing in the mammalian central nervous system. During brain development, neural stem cells can differentiate into new neurons, astrocytes and oligodendrocytes, which can build brain structural and functional units; after the brain matures, neural stem cells still have limited regenerative capacity , to provide the possibility for the repair of brain damage. [0003] The proliferation and differentiation of neural stem cells has always been a difficult and hotspot in research, and it is of great significance to effectively increase the proliferation of neural stem cells. Due to the small number of neural stem cells in the body, the...

Claims

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Application Information

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IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2501/11C12N2501/115C12N2501/599
Inventor 张瑶郭芙蓉
Owner 赛璟生物医药科技(上海)有限公司
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