Use of Hypothermically Stored Keratinocyte Cultures for in vitro Toxicology

Abstract

A human epidermal model (HEM) was developed in conjunction with a multiple-endpoint fluorescent assay for use in in vitro toxicology studies. This HEM is comprised of normal human epidermal keratinocytes maintained on an acellular gel overlaid on a micro-porous membrane. The HEM exhibits many features of a multi-layered epidermis, including stratified cellular layers, desmosomes, a basal lamina, and hemidesmosomes at the gel/basal cell layer interface. The HEM and multiple-endpoint fluorescent assay have been used to examine the toxicity of a sulfur mustard simulant, 2-chloroethyl ethyl sulfide (CEES). A drawback to the use of this HEM, however, for in vitro toxicology studies is the inability to maintain sample viability during transport at 25%deg;C. Two hypothermic storage solutions were tested for their ability to maintain the viability of the HEM for extended periods at 4°C. Fluorescence-based viability assays showed that both solutions were useful to maintain HEM for at least 1 week at 4°C:. Electron microscopy was used to examine ultrastructural changes due to hypothermic storage. Stored samples were used in CEES exposure studies to demonstrate the ability to utilize refrigerated HEM for toxicology studies with results comparable to control samples.