[Fast harvest of seed cells for composite tissue engineered skin at one time]

To find a feasible method that can fast isolate seed cells, keratinocyte stem cell and fibroblasts, for composite tissue engineered skin.

The foreskin could be attained from posthectomy, the subcutaneous tissue was removed completely, and the full-thick skin was cut into pieces, 2 mm x 2 mm in size, then the pieces were submerged into a centrifuge tube containing collagenase I in a oscillator. After 3-hour digestion at 37 degrees C, the dermis was dissolved completely with all the fibroblasts in the digestion solution and the epidermis could be separated easily. With more than 10-minute digestion in trypsin at 37 degrees C, the epidermal cells could be harvested. Then flowcytometry and FITC-immunofluorescence for cytokeratin 19 of epidermal cells and FITC-immunofluorescence of vimentin of fibroblast were conducted to identify keratinocyte stem cells in the epidermal cells and fibroblasts in the digestion solution. Moreover, epidermal cells and fibroblasts were cultured in vitro for 7 days to investigate their biological behavior.

Using collagenase I combined with trypsin, epidermal cells and fibroblasts could be isolated at one time within 3 hours. Up to 17% cells demonstrated cytokeratin 19 positive in the epidermal cells, with fibroblast vimentin positive. The amount of fibroblast could be enlarged to more than 100 times within 6 days, but the putative keratinocyte stem cells were difficult for subculture.

Seed cells for composite tissue engineered skin could be harvested fast at one time, that made it possible to reconstruct composite tissue-engineered skin in vitro.