GROWTH AND DIFFERENTIATION PROPERTIES OF NORMAL AND TRANSFORMED HUMANKERATINOCYTES IN ORGANOTYPIC CULTURE

The growth and differentiation of human normal keratinocytes and their transformed counterparts were examined in organotypic cultures in whi ch the keratinocytes were grown at the air-liquid interface on top of contracted collagen gel containing fibroblasts. We developed a modifie d culture procedure including the use of a mixed medium for keratinocy tes and fibroblasts. Normal keratinocytes formed a three-dimensional s tructure of epithelium that closely resembled the epidermis in vivo, c onsisting of basal, spinous, granular and cornified layers. Cells synt hesizing DNA were located in the lowest basal layer facing the collage n gel. Expressions of proteins involved in epidermal differentiation w ere examined by immunohistochemical staining and compared with those i n skin in vivo. In the organotypic culture, transglutaminase, involucr in and filaggrin were expressed, as in the epidermis in vitro, most pr ominently in the granular layer. Type IV collagen, a component of base ment membrane, was expressed at the interface between the keratinocyte sheet and the contracted collagen gel. Keratinocytes transformed by s imian virus 40 or human papilloma virus (HPV) exhibited a highly disor ganized pattern of squamous differentiation. In particular, HPV-transf ormed cells invaded the collagen gel. Organotypic culture is unique in that regulatory mechanisms of growth and differentiation of keratinoc ytes can be investigated under conditions mimicking those in vivo.