A kind of culture method and application of human peripheral blood mesenchymal stem cells
A method for culturing and a technique for stromal stem cells, which is applied in the field of culturing human peripheral blood mesenchymal stem cells, and can solve the problems of not collecting and culturing PB-MSCs, and obvious side effects.
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Embodiment 1
[0039] Embodiment 1: Separation and culture of PB-MSC
[0040] Collect 20ml of peripheral blood, transfer to a 50mL centrifuge tube, and centrifuge at 2000rpm / min for 5min. Collect the upper layer of plasma and store it at 4°C for later use; the lower layer of blood cells was separated with Ficoll Lymph Separation Solution (Shenzhen Dakwei Biotechnology Co., Ltd.; article number: AS1114546) to obtain peripheral blood mononuclear cells (including PB-MSC, see figure 1 ).
[0041] Peripheral blood mononuclear cells were resuspended with M199 medium (Gibco, supplied by Guangzhou Suyan Biotechnology Co., Ltd.; product number: C11150500BT) containing 10% of the above-mentioned isolated plasma, and prepared according to 1×10 6The density of cells / ml was inoculated in the culture flask, and the final concentration of 20ng / ml EGF factor was added. After 24 hours, fibroblast-like cell attachment was seen (see figure 2 ), placed at 37°C, 5% CO 2 in a cell culture incubator for 3 day...
Embodiment 2
[0044] Embodiment 2: Separation and culture of PB-MSC
[0045] Collect 20ml of peripheral blood, transfer to a 50mL centrifuge tube, and centrifuge at 2000rpm / min for 5min. The upper layer of plasma was collected and stored at 4°C for later use; the lower layer of blood cells was separated with Ficoll lymphatic separation fluid (Shenzhen Dakwei Biotechnology Co., Ltd.; article number: AS1114546) to obtain peripheral blood mononuclear cells (including PB-MSC, refer to figure 1 ).
[0046] Peripheral blood mononuclear cells were resuspended with M199 medium (Gibco, supplied by Guangzhou Suyan Biotechnology Co., Ltd.; product number: C11150500BT) containing 10% of the above-mentioned isolated plasma, and prepared according to 1×10 6 The density of cells / ml was inoculated in the culture flask, and the final concentration of 20ng / ml EGF factor was added. After 24 hours, fibroblast-like cell adhesion was seen (refer to figure 2 ), placed at 37°C, 5% CO 2 in a cell culture incub...
Embodiment 3
[0049] Embodiment 3: Separation and culture of PB-MSC
[0050] Collect 20ml of peripheral blood, transfer to a 50mL centrifuge tube, and centrifuge at 2000rpm / min for 5min. The upper layer of plasma was collected and stored at 4°C for later use; the lower layer of blood cells was separated with Ficoll lymphatic separation fluid (Shenzhen Dakwei Biotechnology Co., Ltd.; article number: AS1114546) to obtain peripheral blood mononuclear cells (including PB-MSC, refer to figure 1 ).
[0051] Peripheral blood mononuclear cells were resuspended with M199 medium (Gibco, supplied by Guangzhou Suyan Biotechnology Co., Ltd.; product number: C11150500BT) containing 10% of the above-mentioned isolated plasma, and prepared according to 1×10 6 The density of cells / ml was inoculated in the culture flask, and the final concentration of 20ng / ml EGF factor was added. After 24 hours, fibroblast-like cell adhesion was seen (refer to figure 2 ), placed at 37°C, 5% CO 2 in a cell culture incub...
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