Modelling the Formation of the Epidermis In Vitro to Investigate Barrier Function

Provision of a barrier against the outside environment and prevention of inside-out evaporation is a major function of epidermal differentiation. The key cellular and molecular components of the epidermal barrier are cell-cell junctions between keratinocytes, cornified envelopes (remnants of flattened cells in stratum corneum), and the lipid envelope consisting of lipids and free fatty acids. The fundamental importance of barrier function in human health is illustrated by examples where a compromised barrier leads to disease. There are several important reasons to develop in vitro models of the epidermal barrier: 1) A key factor in transdermal drug delivery systems is to be able to assess permeability and skin deposition of the drugs and carrier agents; 2) Both the pharmaceutical and cosmetic industries require reliable means for testing for skin irritation and toxicity caused by novel compounds and formulations especially given recent changes in legislation that prohibit certain animal tests; 3) The contribution of barrier function to diseases, such as eczema and psoriasis, that cause considerable health economic burden has highlighted the need for in vitro disease models to investigate both normal and pathological barrier physiology. In recent years several epidermal models have been developed. However, most organotypic cultures suffer from several impracticalities: (i) variability in the dermal substrates that reduces reproducibility of the model; (ii) long set up time required by each experiment; (iii) restricted life-span that makes most models unsuitable for long term experiments. Such limitations are not practical for the routine use of such technology. One major drawback of organotypic co-cultures based on collagen gel is their limited lifespan due to dramatic shrinkage and reduced stability of the reconstructed tissues, indicating the need for further optimisation. In addition, as the major applied use of these culture models is in skin irritant and toxicity testing, only some existing models have been evaluated for their barrier properties. The industrial and academic partners for this proposal currently collaborate and have together generated a prototype 3D culture model for human and mouse epidermal keratinocytes. This proof of concept model utilises Alvetex technology, a novel polystyrene scaffold that offers genuine routine 3D cell culture (see www.reinnervate.com). The model can be used to generate organotypic skin cultures at air-liquid interface either 1) as a stratified epidermis within the scaffold with flattened stratum corneum forming on the top surface of the scaffolds or 2) as a composite skin model where the scaffold harbours collagen gel and fibroblasts with the epidermis subsequently seeded on top. Initial characterisation shows that even without a dermal component the epidermis grown in Alvetex completes terminal differentiation with mature, lipid-coated cornified envelopes and abundant desmosomes in the underlying cell layers. The aim of the proposed CASE studentship is to take this model forward and: 1) Further develop and optimise the skin construct that has an established cornified layer that mimics highly keratinized plantar skin as closely as possible in terms of its cellular and extracellular structure and conformation; 2) Validate the skin model in terms of its structure and protein composition in comparison to known published structure of existing collagen gel systems and native skin; 3) Investigate the extent of epidermal terminal differentiation and barrier assembly. Optimized epidermal cultures will be mounted within Franz cell system to investigate the function and permeability of the epidermal barrier to test compounds; 4) Generate disease a model for barrier deficient epidermis through the culture of primary keratinocytes with selective knockdown (RNAi) of key cornified envelop scaffold proteins (involucrin, envoplakin, periplakin) to replicate changes seen in atopic eczema.