Buccal mucosa in vitro experiments I. Confocal imaging of vital staining and MTT assays for the determination of tissue viability

Delivery of drugs through the skin and the buccal mucosa has been considere d as an alternative to per oral dosing for those substances that are degrad ed in the gastro-intestinal tract, or are subject to first-pass metabolism in the liver. In the buccal mucosa, contrary to skin, the diffusion barrier s are located within living cell layers, hence the physiological state of t he tissue is likely to significantly affect in vitro diffusion profiles. In this study, we were interested in assessing the viability of excised bucca l mucosa and determining the limits of tissue usage under common in vitro e xperimental conditions. Using confocal laser scanning microscopy (CLSM), we have shown that optical sectioning of samples exposed to calcein AM and et hidium homodimer-1 (used as 'live' and 'dead' cell probes respectively) can be employed to accurately and reliably determine the viability of buccal m ucosa biopsies. The results of the CLSM assay were remarkably consistent wi th that of an MTT assay. In both studies, viability in PBS at 34 degrees C was lost after about 8 h post-mortem, whereas it could be sustained for up to 24 h in KBR. (C) 1999 Elsevier Science B.V. All rights reserved.