Development of a Three-dimensional Cell Culture Model for Epidermal Barrier Permeability Testing

Abstract

In this thesis, a novel 3D polystyrene scaffold was investigated for use as a 3D cell culture material to support keratinocyte culture. Methods of collagen-coating the scaffold were investigated and a keratinocyte model was developed at the air-liquid interface to form an in vitro epidermal equivalent. The procedures for culturing keratinocytes were analysed experimentally to promote optimal proliferation and differentiation of the cells.

Keratinocytes cultured in the scaffold showed signs of differentiation, visualised by immunofluorescence staining and ultrastructural analysis by scanning and transmission electron microscopy. The 3D model established is different to other epidermal models as keratinocytes differentiate inside the 3D substrate, rather than forming multiple layers on the air-exposed surface.

Mature cornified envelopes with covalently attached lipids were isolated from the cultures after fourteen days at the air-liquid interface, showing that keratinocytes reached terminal differentiation in the 3D scaffold. Lipid extractions, identification and quantitation showed that the cultures produced a lipid profile similar to native human epidermis, and optimisations to the culture media further improved this similarity.

In the final chapter, the barrier permeability of the 3D model to corticosterone was measured and the model was used to investigate the effect of inhibiting the JNK stress signalling pathway on keratinocyte differentiation and barrier function. This has previously not been investigated using a 3D model, and findings indicated that targeting this pathway may be useful for the treatment of psoriasis, a common skin disorder.