Figure 2

Three-dimensional in vitro epidermal cell cultures using COCA keratinocytes. A) H&E staining showing the formation of a 3D in vitro epidermal cell culture using passage 76 COCA keratinocytes. The micrographs show the progression of the culture for 1, 2 and 3 weeks after inducing the cells to differentiate by creating an air/liquid interface. B-F) Immunofluorescence staining of 3 week 3D cultures. B) K5 expression is expanded to suprabasal layers reflecting the hyperproliferative nature of the model (for details see inset). C) K6 is expressed mostly in suprabasal layers (for details see inset). B-E) K10 is expressed in suprabasal layers and some K10 positivity can be detected in the corneous stratum. Loricrin (D) and filaggrin (E) are also expressed in suprabasal layers. PCNA staining (F) showed that proliferation is mostly restricted to the basal layer cells. Dashed lines mark the contact of the basal layer of the 3D culture with the PCF membrane. Dotted lines mark the end of the corneous stratum. Scale bars: 100 μm.