3D models for alternative testing

Irritants activate proinflammatory cytokines in skin via a predictable signalling cascade.

Above: (left) Irritants activate proinflammatory cytokines in skin via a predictable signalling cascade. By exploiting this keratinocytes and fibroblasts can be used to detect inflammatory activation when exposed to model irritants such as SDS and Formi® in monolayers (middle) and in 3D scaffolds (right).

Tissue engineered models originally developed for clinical purposes are now underpinning the design of physiologically relevant in vitro models as alternatives for animal testing. This is especially important for tissue engineered skin as European Council Directives now enforce the development of alternative tests for irritants and prohibit the use of animals for toxicological testing (as from 2009).

This highlights that alternative models must replace animals traditionally used for irritation, corrosivity and phototoxicity tests. In addition new European Registration, Evaluation and Authorisation of Chemicals (REACH) legislation requires all chemicals to be registered and labelled to establish an appropriate risk to human health.
To address this we have developed a reconstructed human skin using non-degradable polystryrene or cellulose scaffolds, and adapted this to develop a real-time method using transfected human fibroblasts containing a fluorescent reporter gene for directly detecting inflammatory signals within 3D scaffolds.
When damaging chemicals penetrate the stratum corneum of skin viable keratinocytes release interleukin-1 alpha (IL-1α). This in turn triggers a pro-inflammatory cascade of cytokines thereafter including interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor-alpha (TNF-α) and phospholipase A2 (PLA2). Fibroblast cells are highly sensitive to IL-1α and have an important role in cytokine amplification, contributing to the chemotactic recruitment of inflammatory cells and the propagation of an inflammatory response in the skin.

We have exploited these paracrine-interactions in vitro using keratinocytes and fibroblasts and in this way can discriminate between irritant-induced inflammatory reactions from a cytotoxic response. The irritant-triggered release of IL-1α from human keratinocytes and activation of NF-κB in human dermal fibroblasts (as the reporter method) can be used to detect the inflammatory effect of chemicals such as SDS and Formi® when used as model irritants.

This work is also being extended in to the creation of an immunocompetant skin model which includes dendritic cells to detect the response of allergens or sensitising chemicals, in collaboration with a clinical immunologist Dr Amir Ghaem Maghami (University of Nottingham).

People

John Haycock
Sheila MacNeil

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