ULTRASTRUCTURE OF SIZE-REGULATED HETERO-SPHEROIDS COMPOSED OF HUMAN KERATINOCYTES AND FIBROBLASTS

In order to develop a new in vitro skin model multicellular floating h etero-spheroids were prepared by culturing keratinocytes and fibroblas ts on hydrophilic culture dishes coated with type I collagen and a the rmo-responsive polymer. Upon decreasing the substratum's temperature t o an ambient temperature, the spheroids detached from the substratum a nd were thereafter maintained in either: Medium I, a medium mixture of keratinocyte growth medium (KGM) and supplemented Dulbecco's modified Eagle's medium (DMEM) at a ratio of 1 to 2; or medium II, KGM for the initial 24 h followed by supplemented DMEM for the remainder of the c ulture periods. The spheroids displayed a typical pattern of an extern al rim of keratinocytes with an internal core of fibroblasts. A minute space separated the keratinocytes and fibroblasts, The stratification of cells cultured in medium II was more prominent than that of the ce lls cultured in medium I. Markers of the advanced stages of keratiniza tion such as keratohyalin granules, membrane coating granules and the cornified envelope were nor observed. Interestingly, keratinocytes und erwent the same differentiation pathway as non-keratinized stratified epithelia such as esophagus. With consideration to keratinocyte-fibrob last interactions, it may be of interest to incorporate the study of s uch morphological impairments when investigating the effects of growth factors and their ligands.