Abstract: Objective To improve the methods for primary culture and purification of type 2 diabetic human keratinocytes in serum-free medium in vitro. Methods Type 2 diabetic human keratinocytes was isolated by 2 step digestion of neutral protease and trypsin, cultured in serum-free medium and purified by two-step subculture, the keratinocytes were observed using inverted phase contrast microscope, the growth curve was obtained by the WST-8 assay and phenotype and purity were analyzed by flow cytometry. Results Type 2 diabetic human keratinocytes in serum-free medium showed typical epithelial morphology, the growth curve of the keratinocyte at passage 3 is S-shaped, the average doubling time is 36.9 hours during logarithmic growth phase and the keratin-positive rate was 98.9%. Conclusion The methods for culture of high-purity type 2 diabetic human keratinocytes in serum-free media have been established.

Key words: keratinocytes, type 2 diabetes, primary culture