Confocal Raman spectroscopy coupled with in vitro permeation testing to study the effects of formalin fixation on the skin barrier function of reconstructed human epidermis

Confocal Raman Spectroscopy is recognised as a potent tool for molecular characterisation of biological specimens. There is a growing demand for In Vitro Permeation Tests (IVPT) in the pharmaceutical and cosmetic areas, increasingly conducted using Reconstructed Human Epidermis (RHE) skin models. In this study, chemical fixation of RHE in 10 % Neutral Buffered Formalin for 24 h has been examined for storing RHE samples at 4 °C for up to 21 days. Confocal Raman Spectroscopy, combined with Principal Components Analysis, revealed the molecular-level effects of fixation, notably in protein and lipid conformation within the stratum corneum and viable epidermis. IVPT by means of high-performance liquid chromatography, using caffeine as a model compound, showed minimal impact of formalin fixation on the cumulative amount, flux, and permeability coefficient after 12 h. While the biochemical architecture is altered, the function of the model as a barrier to maintain rate-limiting diffusion of active molecules within skin layers remains intact. This study opens avenues for enhanced flexibility and utility in skin model research, promising insights into mitigating the limited shelf life of RHE models by preserving performance in fixed samples for up to 21 days.