OBJECTIVETo establish an optimal method for serum-free and feeder layer-free culture of human keratinocytes and to investigate their biological characteristics.

METHODSThe keratinocytes were harvested from human foreskin of 5 children (aged 5-10 yr) and 5 adults (aged 20-30 yr). The samples were isolated by two-step digestion and the quantities of primary harvested HKCs were determined. The HKCs were then cultured in KCS serum-free culture medium. The morphology of HKCs were observed under light microscope. The HKCs and their growing speed were observed and identified under fluorescent microscope. The growth curve of HKCs was detected with MTT method, and the cell cycle was determined with flow cytometry.

RESULTSThe number of harvested HKCs from children [(1.780 +/- 0.010) x 10(6)/cm(2)] was obviously higher than that from adults [(1.490 +/- 0.120) x 10(6)/cm(2)], (P < 0.01). Freshly isolated primary HKCs were round and transparent, and 94% of them were trypan blue resistant. The adherent speed and rate and lucent degree of multiply passaged HKCs increased followed by each passage. Under the fluorescent microscope, the cells exhibited strong Kelly fluorescence in the cytoplasm and with no staining in the nucleolus, thus the cells were identified as HKCs. The HKCs from children for skin could be passaged for more times [(11.0 +/- 1.2) times] than that from adults [(9.2 +/- 0.8) times], (P < 0.05). There was no clear sign of incubation period in the growth curve of HKCs, and both cellular proliferating speed and rate of proliferation were high. The percentage of cells in G1, G2 and S phase and the proliferation index was 36.15%, 25.17%, 38.68% and 63.85%, respectively.

CONCLUSIONSerum-free and feeder layer-free culture seems to be an ideal method for the cultivation of HKCs.