In situ image analysis of interactions between normal human keratinocytes and fibroblasts cultured in three-dimensional fibrin gels

The non-invasive investigation of different cells to interact and become spatially organised in a three-dimensional (3D) environment or scaffold is an important challenge in tissue engineering and tissue physiology. The aim of the present study was to develop 3D cell culture systems using fibrin gels, which would allow for the single and co-culture of different cell types with in situ image analysis. Two chambers were constructed for mono-culture and co-culture of human dermal fibroblasts and keratinocytes. During cell culture, in situ imaging and morphological characterisation of cells was assessed using brightfield light and/or fluorescence microscopy, and later confirmed by staining of fixed cells using immunofluorescence microscopy. The results showed that it was possible to investigate fibroblast and keratinocyte interactions in a fibrin scaffold for at least 12 days. Using this model system it was found that when a co-culture of fibroblasts and keratinocytes were plated on top of the fibrin gels, fibroblasts were seen to migrate into the gels within 2-3 days in contrast to keratinocytes, which did not enter. However, keratinocytes were found to retard fibroblast migration into gels when compared to fibroblasts cultured on their own, illustrating the dependency of intracellular communication on cell position for reconstructive approaches.