Keratinocytes grown at the air-liquid interface

Summary A procedure is described which allows primary cultures of rat keratinocytes grown at the liquid-air interface to develop and maintain multilayered strata and to produce highly keratinized sheets morphologically similar to those seen in epidermis in situ. Various substrata were tested and compared as to their ability to support growth and stratification of keratinocytes. It was found that when cultured on plastic surfaces, keratinocytes adhered tightly to the substratum and produced a confluent monolayer that later stratified to two to three layers. Cells plated on Vitrogen 100 collagen failed to reach confluence and, in addition, exhibited the “clustering” phenomenon and deterioration of collagen after 3 to 4 d of growth. Significantly better attachment and spreading were observed for cells grown on rat-tail collagen as compared with plastic and Vitrogen 100 collagen. The best results, including maximal and uniform stratification, were seen in cells grown on a mixture, of rat-tail and Vitrogen 100 collagens. The system that was developed in the present study offers a model for use in the study of epidermal toxicity from topically applied environmental chemicals. This investigation was supported by grant 5 T32 AM 07236 and 5 R01 AM 15206 from the National Institutes of Health, contract DAMD17-82-C-2198 from the US Army Medical Research and Development Command, and contract N-2 from the Proctor and Gamble Company The contents of this paper do not represent the opinion of the sponsors nor should findings in this report be construed as their official position.