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Serum-free culture medium for maintaining in-vitro culture of neural stem cells

A serum-free medium, neural stem cell technology, applied in nervous system cells, cell culture active agents, culture process, etc., can solve the problems of not being able to support neural stem cell growth for a long time, lack of cell growth requirements, and poor cell adhesion, etc. Achieve the effect of maintaining self-renewal ability, low cost and simple formula

Inactive Publication Date: 2019-04-19
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

B27 has complex components, including bovine serum albumin, which can support the growth of neural stem cells; although N2 contains five components: insulin, transferrin, progesterone, putrescine, and sodium selenate, it lacks some cell growth requirements and cannot support for a long time growth of neural stem cells
[0005] The existing neural stem cell culture medium has problems such as poor cell adhesion, limited proliferation ability, and severe spontaneous differentiation, and cannot maintain the culture of neural stem cells well. Therefore, it is necessary to provide a new type of culture medium that can maintain neural stem cells in vitro. serum medium to address the above

Method used

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  • Serum-free culture medium for maintaining in-vitro culture of neural stem cells
  • Serum-free culture medium for maintaining in-vitro culture of neural stem cells
  • Serum-free culture medium for maintaining in-vitro culture of neural stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Detecting the Apoptosis of Neural Stem Cells

[0025] The neural stem cells were divided into two groups. The neural stem cells in the control group used a traditional medium (medium 1): DMEM / F12+N2+B27+10ng / ml EGF+10ng / ml bFGF; the neural stem cells in the experimental group used a new medium ( Medium 2): DMEM / F12+N2+B27+10ng / ml EGF+10ng / ml bFGF+5ng / ml LIF+10ng / ml IL6+10ug / ml Ascorbic acid.

[0026] The cell culture method is as follows:

[0027] (1) The neural stem cells were divided into two groups. The neural stem cells in the control group used traditional medium: DMEM / F12+N2+B27+10ng / ml EGF+10ng / ml bFGF, and the neural stem cells in the experimental group used a new medium: DMEM / F12+N2+B27+10ng / ml EGF+10ng / ml bFGF+5ng / ml LIF+10ng / ml IL6+10μg / ml ascorbic acid;

[0028] (2) After the two groups of cells were cultured for 24 hours (placed at 37° C., incubated in an incubator with 5% CO 2 ), the cells were counted, and 10 6 Place the cells in a 5ml flow ...

Embodiment 2

[0035] Example 2 Detection of proliferation status of neural stem cells

[0036] Adopt the control group identical with embodiment 1 and experimental group culture medium, method is as follows:

[0037] (1) After the neural stem cells are counted, each well is divided into 3×10 3 Inoculate the neural stem cells into a 96-well plate, set up 5 duplicate wells, add 100 μL of traditional medium to the control group, add 100 μL of new medium to the experimental group, and culture them in a cell culture box for 24 hours (37 ° C, 5% CO 2 ).

[0038] (2) Add 10 μl CCK8 to each well, place in an incubator and incubate for 30 min-1 h, then measure the OD value at a wavelength of 450 nm with a microplate reader.

[0039] (3) The proliferation of the neural stem cells cultured in the two kinds of media after 1, 2, 3, and 4 days was detected by the same method.

[0040] Such as figure 1As shown in B, there is no difference in the proliferation of neural stem cells between the control g...

Embodiment 3

[0041] Example 3 Detection of stem cell markers when maintaining neural stem cell proliferation in vitro

[0042] (1) For the neural stem cells in two different media (the control group was added with the traditional media, and the real group was added with the new media for the experimental type), they were cultured in vitro for 1 week, 2 weeks, 3 weeks and 4 weeks respectively (placed at 37°C , 5% CO 2 Incubate in an incubator, replace the medium every two days), clamp the sterilized cell slides into a 24-well plate with tweezers, make 2 replicate wells for each group, and take 20,000 cells to plant in 24 wells after cell counting The cells on the plate were placed on the slide and cultured in a cell culture incubator.

[0043] (3) After 24 hours, when the cells were attached to the wall, the medium was sucked out, PBS buffer was added, placed on a shaker at 200 rpm for 5 minutes, rinsed 3 times, and the cell debris and excess medium were washed away.

[0044] (4) Aspirate...

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Abstract

The invention belongs to the technical field of biology and discloses a serum-free culture medium for neural stem cells. The serum-free culture medium contains a basic culture medium, a nutritional additive, a growth factor, cytokines and an antioxidant. The cytokines are at least one of LIF and IL-6. The antioxidant is ascorbic acid. The culture medium is simple in formula and low in cost, can beused for in-vitro culture of neural stem cells, remarkably reduces the apoptosis rate of the neural stem cells in in-vitro culture, improves the proliferation rate of the neural stem cells, effectively reduces the apoptosis rate of the neural stem cells in in-vitro culture, can be used for maintaining the long-time in-vitro culture of the neural stem cells, and maintains the dryness of the neuralstem cells and the self-renewal capacity after long-time in-vitro culture.

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 serum-free culture medium for neural stem cells. Background technique [0002] Neural stem cells are a type of cells with self-renewal and multi-directional differentiation potential, which exist in the mammalian central nervous system, and can generate most cell types in the central nervous system through symmetrical and asymmetrical divisions: neural cells, astrocytes, and oligodendrocytes. [0003] 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. [0004] As the seed cells in biomedical engineering and in vitro model research, neural stem cells need to be expanded and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2500/38C12N2500/90C12N2501/11C12N2501/115C12N2501/2306C12N2501/235
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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