Applications of a human epidermal model: Storage and cytotoxicity studies.

摘要

The goal of this dissertation project was to cryopreserve a human epidermal model (HEM) for storage and transportation. Human epidermal keratinocytes were seeded and stratified on an aldehyde cross-linked collagen gel overlaid on microporous membranes. The resulting HEM demonstrated many ultrastructural characteristics of a normal human epidermis, including keratin filaments, desmosomes, hemidesmosomes, and a basement membrane.;A fluorescent multiple endpoint assay was developed to examine changes in cellular function during transportation and cryopreservation. The fluorescent multiple endpoint assay incorporates the utilization of markers which can assess changes in plasma membrane integrity, lysosomal integrity, intracellular calcium, intracellular glutathione, free radical accumulation, mitochondrial activity and barrier properties of a monolayer or an epidermis. This assay system revealed that barrier function was compromised prior to a loss in plasma membrane integrity in HEM transported at ambient temperatures.;Cryopreservation-induced changes in single cells or an HEM were examined with the multiple endpoint assay and optimal concentrations of cryoprotectants were determined for single cells. The HEM, however, could not be preserved in frozen storage using standard cryopreservation protocols so an alternative procedure was examined.;Hypothermic solutions were tested for their abilities to preserve both monolayers and the HEM during refrigerated storage. The multiple endpoint assay indicated that successful preservation could be achieved with these hypothermic solutions. Ultrastructural analysis using transmission electron microscopy provided further evidence regarding cellular viability.;Stored and non-stored monolayers were assessed in a study of sulfur mustard-induced toxicity using the multiple endpoint assay. The stored and non-stored monolayers demonstrated similar toxicity profiles. Additionally, stored and non-stored HEM were compared for their abilities to demonstrate mustard-induced injury to the plasma membrane. The profiles for plasma membrane disruption were similar for both stored and non-stored HEM.;In conclusion, the data indicate that an HEM can be stored and subsequently utilized for in vitro toxicology. The ability to successfully store and transport an HEM facilitates the use of synthetic tissues as an alternative to animal testing. Furthermore, the data indicate that the fluorescent multiple endpoint assay could be used to reformulate future hypothermic maintenance solutions so that tissue- and organ-specific solutions can be defined.