Optimal subculture methods for passaging and growing human epidermal keratinocytes

Background: Human epidermal keratinocytes (HEKs) cover the outer layer of the skin and play a key role in wound repair. Although the methods for isolation and cultivation of primary HEKs from epidermis have been used successfully in both laboratory and clinical settings, the ability to subculture (passage) these cells has yet to be established and is the primary factor hindering their usage. Objectives: We conducted this study to identify optimal subculture conditions for HEKs. Methods: We first harvested the primary HEKs from prepuce tissue specimens, and then compared three different reagent compositions (0.25% trypsin, 0.25% trypsin plus 0.01% EDTA, and 0.25% dispase digestion solution) for various periods of time at 4 C with the conventional 0.25% trypsin or 0.25% trypsin plus 0.01% EDTA digestion at room temperature. Results: Our data indicated that the cold digestion conditions yielded higher cell numbers and more viable cells than the conventional methods. Furthermore, the subcultured HEKs also adhered and grew better after four hours of a 0.25% trypsin cold digestion or after six hours of a 0.25% dispase cold digestion. These procedures produced higher numbers of HEK passages than that commonly seen experienced with conventional methods. Conclusion: The data from the current study demonstrated that the optimal subculture condition for passaging and growing HEKs in vitro is four hours digestion with 0.25% trypsin.