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
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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.
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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