Culture method for fast acquiring a lot of high-quality neural stem cells

A neural stem cell and stem cell culture technology, which is applied in the field of quickly obtaining a large number of high-quality neural stem cells, can solve the hidden dangers of limited number of cells, basic research of neural stem cells, limitations of clinical application industrial production, scientific research or clinical and industrial applications, etc. question

Active Publication Date: 2014-10-08
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, limited by factors such as cell culture conditions and tissue sources, the number of cells that can be obtained by conventional methods is very limited
In addition, most neural stem cells will degenerate after 20 passages in vitro, and neural stem cells have strict requirements on the culture environment, and are prone to problems such as changes in cell quality due to environmental changes. All these factors have led to basic research on neural stem cells. , clinical application and industrial production are limited[2,5-6]
At present, studies have found that some chemical molecules or biomolecules can promote the proliferation of neural stem cells, thereby obtaining a large number of cells in a short period of time. However, in addition to the effect on cell proliferation, chemical molecules or biomolecules often have additional effects on other aspects of stem cells. effects, that is, they may also cause changes in the quality of the cells themselves while affecting cell proliferation, so there are risks[7-10]
Another unavoidable problem is that the use of chemical molecules or biomolecules lays hidden dangers for subsequent scientific research or clinical and industrial applications, which are difficult to eliminate

Method used

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  • Culture method for fast acquiring a lot of high-quality neural stem cells
  • Culture method for fast acquiring a lot of high-quality neural stem cells
  • Culture method for fast acquiring a lot of high-quality neural stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, submagnetic cell culture environment control

[0025] On the support made of aluminum alloy, the magnetic shielding box is made of magnetic shielding metal Permalloy (thickness: 0.5mm, magnetic permeability: 20000, purchased from Beijing Capital Iron and Steel Co., Ltd.)), and the number of winding layers is 12 layers. The final size of the cabinet is 470mm*641mm*6511mm (length*width*height). Open several small holes (diameter 0.5mm) on the top of the box, respectively install the air intake fan and the air outlet fan, and open the door on the side of the box. In order to maintain an environment suitable for cell growth in the box, the hypomagnetic shielding box was placed in a cell culture box (HERA240, Thermo Fisher Scientific, USA), and the environment of the cell culture box was kept as a suitable condition for cell growth: temperature 37 ° C, humidity 95%, CO 2 Concentration 5%. By running the fan on the top of the magnetic shielding box, the envir...

Embodiment 2

[0026] Embodiment 2, primary culture of neural stem cells

[0027] Material: C57BL / 6 mice (purchased from Beijing Speifu Company)

[0028] Solution: HEM solution, a bag of MEM (GIBCO, 41500-018), 3.813g of HEPES (Sigma, H4034), add ddH 2 0 to 0.875L, add 2% double antibody (Penicillin / Streptomycin, Gibco, 14140-122)

[0029] NSA solution, (DMEM / F12 (Gibco, 12500-062) a bag, Glucose (Sigma, G7021) 3.75g, sodium bicarbonate (Sigma, S5761) 1.125g, HEPES1.192g, add ddH 2 O to 0.9L.

[0030] NSA- / - solution, (50 mL), NSA 44 mL, 10% BSA (Roche, 10711454001) 1 mL, Proliferation Supplement (Stem Cell Technologies, 05701) 5 mL, double antibody 1%.

[0031] NSA+ / + solution, (50mL), NSA- / -50mL, Heparin (Sigma, H3149, 3.78mg / mL) 50μL, EGF (BD Bioscience, 354010, 1μg / mL) 100μL, bFGF (Roche, 11104616001, 0.1μg / mL) mL) 50 μL.

[0032] 0.05% trypsin solution, trypsin (Sigma, T5266) 0.05g, EDTA (Sigma, E6158) 0.004g, add PBS 100mL.

[0033] Trypsin inhibitor solution, trypsin inhibitor (...

Embodiment 3

[0045] Embodiment 3, neural stem cell passage

[0046] Steps

[0047] 1. After the primary neural stem cells were cultured in a submagnetic environment for 7 days (as described in Example 2), all neurospheres were collected and centrifuged at 100 g for 5 minutes.

[0048] 2. Add 0.05% trypsin or accutase to digest at room temperature for 3 minutes, add the same amount of trypsin inhibitor, and mix gently. Centrifuge at 100g for 5 minutes.

[0049] 3. After aspirating the supernatant, add 1-2mL NSA+ / +, and pipette repeatedly to make a single cell suspension.

[0050] 4. Inoculate 100,000 cells into a 60mm cell culture dish (Corning), add 5mL NSA+ / +; 1000 cells per well, 200μL NSA+ / +, culture in geomagnetic and submagnetic environments respectively, and count the number of cells or statistics after 7 days Number and size of neurospheres.

[0051] Result analysis:

[0052] The results showed that the neurospheres grown in the geomagnetic and submagnetic environments could be...

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Abstract

The invention provides a method for improving stem cell culture efficiency. The method comprises culturing stem cells in a metamagnetism environment. The method can fast acquire a lot of high-quality stem cells without influence on cell properties, does not produce any additional biological or chemical pollution on cells, and has an important meaning for solving the problem of less cells in stem cell treatment and improving a yield of stem cell-related products.

Description

technical field [0001] The invention relates to a culture method capable of improving the culture efficiency of neural stem cells without affecting the quality of the stem cells. Background technique [0002] Currently, for the isolation and culture of neural stem cells, the growth factors EGF and bFGF serum-free medium are mainly used to maintain cell proliferation and form neural stem cell spheres, thereby separating stem cells from the cell mixture and maintaining the normal growth of cells[1-6] . However, limited by factors such as cell culture conditions and tissue sources, the number of cells that can be obtained by conventional methods is very limited. In addition, most neural stem cells will degenerate after 20 passages in vitro, and neural stem cells have strict requirements on the culture environment, and are prone to problems such as changes in cell quality due to environmental changes. All these factors have led to basic research on neural stem cells. , clinica...

Claims

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Application Information

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IPC IPC(8): C12N5/0797
Inventor 赫荣乔刘缨付晶鹏莫炜川
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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