A Human Epidermal Model can be Assayed Employing a Multiple Fluorescent Endpoint Assay and the Cytofluor 2300

Abstract

A human epidermal model (HEM) was developed that could be rapidly and automatically assayed in the CytoFluor 2300 (Millipore Corporation, Bedford, MA) spectrofluorometer using multiple site-and activity-specific fluorescent probes. The HEM was cultured on the optically translucent Millipore Millicell CM microporous membrane. Application of a variety of fluorescent dyes to this membrane without the HEM revealed negligible nonspecific dye association. The HEM was differentiated on a cross-linked collagen matrix and the latter was also found to retain less dye than the HEM. Feasibility experiments using the site/activity-specific dyes calcein-AM (plasma membrane integrity indicator), sodium fluorescein (epidermal permeability indicator), 5-chloromethylfluorescein diacetateacetoxymethyl ester (CMFDA-AM; intracellular glutathione level indicator), rhodamine 123 (mitochondrial activity indicator), neutral red (lysosomal integrity indicator), Fluo3-AM (intracellular calcium indicator), and a similar human epidermal model, the Organogenesis Testskin living skin equivalent (LSE), indicated that in vitro human epidermis might be amenable to automated analysis in the CytoFluor 2300. To determine if these fluorescent probes might reveal mechanisms underlying cytotoxicity, normal human epidermal keratinocyte (NHEK) monolayers were exposed to the single arm mustard, 2-chloroethylethylsulfide (CEES) and labeled with the aforementioned fluorescent probes. The data reveal that there is a dose- and time-dependent alteration in cellular activities due to mustard exposure, which, in turn, suggested a possible sequence of mustard toxicity. The fact that these HEMs can be analyzed automatically using the CytoFluor 2300 and that changes in specific physiological parameters can be assessed using multiple fluorescent dyes suggests that this process might be a high throughput manner in which to screen further skin irritants.