Normal human epidermal keratinocytes were differentiated on Millipore Millicell CM microporous membranes overlaid with an acellular cross-linked collagen gel. The extracellular calcium concentration was elevated, and stratification of the monolayer to a 20-cell thick multilayer ensued within 5–7 days. Transmission electron microscopy of this stratified, human epidermal model (HEM) revealed typical ultrastructural markers of differentiation, including numerous desmosomes, keratinohyalin granules, keratin filaments, lamellar-type bodies, and squamous-shaped cells in the apical region, but not the basal region, of this HEM. Preparations lifted to the air–liquid interface had electron-dense apical cells suggestive of cornification. An electron-dense lamina appeared on the basal surface in both submerged and air–liquid interface cultures, suggesting that this structure can be produced without the help of underlying dermal cells and is independent of the air–liquid interface conditions. The uniform thickness of the epidermis, the absence of dermal fibroblasts, the lack of contraction of the epidermis, and the thinness of the underlying collagen gel, made this HEM a suitable candidate for use in an automated, fluorescence multiple endpoint assay. Fluorescent probes monitoring lysosomal integrity, epidermal permeability and plasma membrane integrity were monitored using the CytoFluor 2300 fluorescent plate reader. Our data suggest the feasibility of using multilayered human tissues for automated multiple endpoint analysis.
337 DermaCult™, a Serum- and BPE-free Medium Supporting Long-term Feeder-free Expansion of Primary Human Epidermal Keratinocytes that Retain Their Differentiation Potential at Air Liquid Interface
