Primary isolation method of animal skin fibroblasts

Abstract

The invention discloses a primary isolation method of animal skin fibroblasts. Disinfected animal skin is selected, and a full-layer skin wound is formed; an injured skin area is taken down after the skin is injured within 3-5 days, subcutaneous tissue and extra blood vessels are removed completely, and the skin is cleaned with PBS (phosphate buffer saline) containing 2% of a penicillin-streptomycin solution for 2-3 times; the skin is cut into tissue pieces ranging from 0.1 mm<3> to 0.5 mm<3> in size; PBS containing 1% of the penicillin-streptomycin solution is poured, centrifugation is performed at the low speed of 1,000 rpm / min for 5 min, and residual floating tissue pieces and a residual supernatant are removed; a cell culture dish covered with 0.2% of gelatin is evenly inoculated with centrifuged tissue pieces, a cell culture fluid containing 2 ml of a DMEM (Dulbecco modified eagle medium), 10% of the FBS and 2% of the penicillin-streptomycin solution is added, and the culture dish is placed in a 5% CO2 incubator at the temperature of 37 DEG C for culture. A skin injury healing mechanism is used, a proper healing period is selected, a large number of proliferative fibroblasts are obtained, meanwhile, removal of the subcutaneous tissue facilitates climbing out of the fibroblasts of corium layers, the number of cells can be increased, the cells can climb out more easily, the cell climbing-out cycle is shortened, and the adherence efficiency is enhanced.

Description

technical field [0001] The invention relates to a method for primary separation of cells, in particular to a method for primary separation of animal skin fibroblasts, which belongs to the field of stem cell culture. Background technique [0002] At present, fibroblasts are mainly used to study the aging of cells, the damage of various external factors to cells, the malignant transformation of cells in vitro, and some congenital metabolic abnormalities and enzyme defects. Because skin fibroblasts are easy to obtain and grow in vitro, the culture of skin fibroblasts has been widely used in basic and clinical medical research. [0003] For the acquisition of fibroblasts, there are mainly two primary separation methods currently available: (1) Enzyme digestion. Use trypsin or collagenase to digest the skin tissue rinsed with double-antibody-containing PBS and shred until the cells are separated, pass through 200 sieves, centrifuge, resuspend, count the cell density, and inocula...

AI Technical Summary

Problems solved by technology

[0004] For the enzymatic digestion method, although the cells can be obtained in a short period of time, the enzyme itself has a certain influence on the adhesion of the cells, and repeated centrifugation also causes certain mechanical damage to the cells, resulting in low cell viability and poor adhesion rate. Low

Compared with the enzymatic hydrolysis method, the tissue block attachment method has simpler steps, but the cycle is long, the attachment stability is low, and the cell crawling rate is low.

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