Animal skin flbroblast primary isolation method
A fibroblast and animal skin technology, applied in the field of stem cell culture, can solve the problems of low cell crawling rate, low cell viability rate, low adherence stability, etc., to increase the number of cells, reduce the influence of cells, and improve the Separation efficiency and effect of adherence effect
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Embodiment 1
[0021] A kind of mouse skin fibroblast primary separation method of the present embodiment, it comprises the steps:
[0022] 1.1 Select the abdominal skin of the mouse, use curved scissors to slightly cut off the hair, disinfect the skin with iodine and alcohol, make a circular full-thickness skin wound with a diameter of 2.0 cm, expose the wound and continue to raise.
[0023] 1.2 Within days of skin injury (3-5), the mice were sacrificed, the skin area after injury was removed, the subcutaneous tissue and all blood vessels were removed, and the skin was washed 3 times with PBS containing 2% double antibody.
[0024] 1.3 Transfer the skin to a 50mL centrifuge tube, add 7ml (according to the skin area 1mL / cm 2 Select medium volume) (0.1%-0.25%) trypsin, leave for 1-2 hours.
[0025] 1.4 Pass the digestive juice through a 200-mesh cell mesh sieve to remove indigestible tissues and magazines, centrifuge at 1000rpm / min at low speed for 5min, remove the supernatant, pour an appro...
Embodiment 2
[0029] This embodiment is the primary isolation method of conventional conventional mouse skin fibroblasts, comprising the following steps:
[0030] 2.1 The mice were killed, the hair was slightly trimmed with curved scissors, the skin was disinfected with iodine and alcohol, the skin tissue with a diameter of 2 cm was removed, and the skin was washed 3 times with PBS containing 2% double antibody.
[0031] 2.2 Transfer the skin to a 50mL centrifuge tube, add 7mL / cm according to the skin area 2 (0.1%--0.25%) trypsin, leave for 4-6 hours.
[0032] 2.3 Pass the digestive juice through a 200-mesh cell mesh sieve to remove undigestible tissues and magazines, centrifuge at a low speed of 1000rpm / min for 5min, remove the supernatant, pour an appropriate amount of PBS containing 1% double antibody, and remove residual trypsin, 1000rpm / min Centrifuge at low speed for 5 min, and remove the supernatant.
[0033] 2.4 Cells after centrifugation, according to (1-10)×10 5 density, add cu...
Embodiment 3
[0036] This embodiment is a comparison of the number of cells after the separation of mouse skin fibroblasts
[0037] The methods of Example 1 (experimental group) and Example 2 (control group) were used to treat three different batches of mice, and the number of cells per square centimeter of skin after separation was calculated. Cell growth was observed every day after inoculation.
[0038] The number of cells separated by the two schemes is as follows:
[0039] Table 1: Comparison of the number of skin fibroblasts in the experimental group and the control group after isolation
[0040]
[0041] As can be seen from the table, the number of cells separated by the experimental group is more than 2 times that of the control group, which proves that the scheme of the present invention can greatly improve the number of cells separated.
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