Human keratinocyte models: Assessment of cell adhesion and dermotoxicity using fluorescent probes

Abstract

To assess the molecular and cellular events that occur in the skin during biological and pharmaco-toxicological processes, we developed different in vitro models. Two major systems are described: (1) relatively simple ones such as normal human keratinocytes (NHK) grown in monolayer or continuous culture of spontaneously immortalized keratinocyte cells, the HaCaT cell line. This cell line forms a monolayer and displays the same phenotypic morphology, pattern of differentiation markers as NHK. (2) More complex models such as NHK multilayers differentiated on a synthetic porous membrane. Indeed, NHK grown at the air-liquid interface of culture inserts may undergo epidermal differentiation in 21 days (Noël-Hudson et al., 1995a). Under the same culture conditions, no stratification of the HaCaT cell line was obtained. NHK and/or HaCaT monolayers were used to study the cell surface molecules involved in heterologous cell interactions, and to estimate the cytotoxic effects of different compounds through a sensitive fluorimetric microtitration assay. When cell adhesion was measured with calcein-AM labelled lymphocytes, it appeared that lymphocytes display the same behaviour towards NHK or HaCaT cells. The importance of the activation status of each cell and the involvement of α 2 and α 3 β 1 integrins in lymphocyte-keratinocyte interactions were demonstrated. Likewise cytotoxicity of SDS and DNP was easily and rapidly detected with calcein-AM and Alamar blue probes. Skin models in combination with fluorescent probes offer promising alternatives for assessing cell interactions as well as cytotoxic effects.