AUTHOR: John W. Haycock (presenting) &
ADDRESS: Department of Engineering Materials, Kroto Research Institute
The European Commission wants to see alternative methods for replacing animals traditionally used for irritation, corrosivity and phototoxicity testing by 2009. We therefore report on the development of a non-destructive reporter system for assessing the response of human skin cells within a 3D tissue engineered scaffold to exogenous stress. Biologically stable and chemically resistant scaffolds, comprising nano-fibres of polystyrene, were formed by electrospinning. The spinning conditions, such as the dielectric constant of the solvent, polymer concentration and fibre collection geometry were optimised to give nanofibres of appropriate diameter and separation for use as templates for 3-D cell culture. Transiently transfected human fibroblasts with an NF-κB-GFP reporter construct were initially tested using pro-inflammatory or oxidative compounds in electrospun tissue scaffolds (TNF-α, IL-1, γ-IFN, H2O2 ), confirming in situ NF-κB activation. A 3D skin model was then constructed using human keratinocytes and NF-κB-GFP fibroblasts. Model irritants, SDS and Formi ™ when introduced to keratinocytes elevated IL-1α, which in turn activated NF- k B in the NF-κB-GFP fibroblasts. This reporter gene model represents a key step in developing a 3D skin model for detecting agents capable of inducing irritation or contact sensitisation in skin, and importantly has potential for the screening pharmaceutical and cosmetic compounds, as an alternative to existing in vivo methods.