Isolation And Culture Of Fibroblast Or Stem-Like Cells From Skin Tissue Of Dama And Shepherd

To establish a good method and culture system to isolate skin stem-like cells and expand it in vitro so as to lay a foundation for exploring the proliferation and differentiation mechanism of skin stem cells. In this paper, fibroblasts and stem-like cells have beenharvested from the skin tissues of SD rat Dama and Shepherd. The results were as follows:1. Effect of disinfection and culture methods on primary culture of fibroblast from trail tissue of SD rat1% Benzalkonium Bromide Solution(BBS) and Iodine were used as disinfectants before the skin tissues harvest. The skin samples from trail SD rat were incubated by 1% BBS or antibiotic solution ( 1x105IU ml-1 Penicillin + 8x104IU ml-1 Streptomycin ) for 5 minutes or lOminutes at room temperature, respectively, could prevent bacteria and mycotic' infections. After the disinfectants were scoured off by Dulbecco's Phosphate Buffered Saline (D-PBS), Skin samples were cut into small bits (lmm3) and incubated in 0.25% trypsin for 30 minutes at 37C. Then Dulbecco's Modified Eagle Medium(high glucose, DMEM) with 10%(v/v) New Cattle Serum(NCS) were added to stop digestion action of trypsin. Skin samples were explanted into culture flask to culture at 37C, 5%CO2. Fibroblasts were obtained after 10-13 days,, while it took 15-20 days without trypsinization digestion.2. Establishment of fibroblasts cell line from ear skin tissue of adult Dama and studies on some biological characteristicsFibroblasts from ear skin tissue of adult Dama were harvested by the method as before, 0.25% trypsin was displaced by 0.1% collagenase ( I type), purificated by seeding and digesting times. Some biological characteristics of the cell lines including the morphology of cell, the growth rate, were studied. Cryopreservation of fibroblasts in different passages was also studied, the average vitality and the attachment percentage were 61.04% and 38.35%, respectively. The chromosome karyotype was analyzed by conventional cytogenetics technique, the chromosome number of 12th passages was 2n=68 and classified 33 pairs. Among the autosomes, the shape of 1th and 7th pairs were metacentric, others wereacrocentric. X chromosome was the largest acrocentric chromosome and Y was the smallestmetacentric chromosome in all chromosomes.3. Isolation and culture of skin stem-like cells from ear skin tissue of ShepherdSkin stem-like cell colonies were obtained by explant culture. The colonies had some characteristics of embryonic stem cells, such as the ability to be continuously subcultured (passage 6, while fibroblasts colonies didn't), show a generalized plasticity. In vitro, these cells can be induced to be hair-like cells, polygon-like cell and neuron-like cells, et al. The cell colonies had some different morphologies, including column-like island-like, nest-like, chine-like, crater-like, et al. The skin stem-like cell colonies had different behaviors on fibroblasts of Dama and cardiomyocytes of SD rat, the latter made them keeping the same behavior for a long time.