Ultrastructure of size-regulated hetero-spheroids composed of human keratinocytes and fibroblasts.
In order to develop a new in vitro skin model multicellular floating hetero-spheroids were prepared by culturing keratinocytes and fibroblasts on hydrophilic culture dishes coated with type I collagen and a thermo-responsive polymer. Upon decreasing the substratum's temperature to an ambient temperature, the spheroids detached from the substratum and 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 culture periods. The spheroids displayed a typical pattern of an external 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 cells cultured in medium I. Markers of the advanced stages of keratinization such as keratohyalin granules, membrane coating granules and the cornified envelope were not observed. Interestingly, keratinocytes underwent the same differentiation pathway as non-keratinized stratified epithelia such as esophagus. With consideration to keratinocyte-fibroblast interactions, it may be of interest to incorporate the study of such morphological impairments when investigating the effects of growth factors and their ligands.
Butt
K I
KI
Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan.
Yamazaki
M
M
Manabe
M
M
D002449:Cell Aggregation; D018920:Coculture Techniques; D005347:Fibroblasts / Q000502:physiology / Q000648:ultrastructure*; D006358:Hot Temperature; D006801:Humans; D015603:Keratinocytes / Q000502:physiology / Q000648:ultrastructure*; D011108:Polymers; D018874:Spheroids, Cellular / Q000502:physiology / Q000648:ultrastructure*
D011108:Polymers