Improved barrier function observed in cultured skin substitutes developed under anchored conditions

Abstract

The current method of producing cultured skin substitutes (CSS) is focused on providing treatments for severe skin wounds/burns. We have developed a modified growth method to make them more suitable for in vitro product-testing/toxicity-testing purposes. CSS grown in Petri dishes were either transferred to Franz diffusion cells on day 5 (modified method) or left in the Petri dish (standard method) and maintained in these environments for the remainder of the growth phase. Mitochondrial metabolism (MTT assay) was measured on days 5, 10 and 14 and histology was studied on days 5, 10 and 14. Barrier function for all tissues was evaluated by transferring them to Franz cells (standard method) and measuring transepidermal water loss (TEWL), 3H2O penetration and 14C-niacinamide permeability on days 7, 14 and 21. CSS grown by the standard and modified methods showed comparable cell viability and tissue morphology. Barrier function, however, was markedly improved in CSS grown by the modified method. The average improvement at days 7 and 14 was 1.3-fold for TEWL, 2.1-fold for 3H2O penetration and 6.4-fold for 14C-niacinamide permeability. The barrier function of CSS grown by the modified method was still significantly lower than that of human cadaver skin tested by the same methods. CSS developed using the anchored multi-cell system showed similar cell viability and morphology and improved barrier function compared with CSS produced by the standard Petri dish method, thereby improving its potential as an in vitro skin permeability and toxicity model.