A Fully Differentiating Epidermal Model with Extended Viability: Development and Partial Characterization

Abstract

A highly differentiated porcine skin organ culture model has been developed for future investigations of membrane-coating granules (MCG) and their role in epidermal differentiation. In contrast to many previous systems, cultures do not undergo necrosis of the upper epidermis or display dermo-epidermal separation, but survive for at least 3 weeks, at which time mitotic cells are still evident. Although rete projections are gradually smoothed out and the viable epidermis thins at a rate of approximately 0.35 cells per day, the stratum corneum gains approximately 1.5 corneocytes per day. Furthermore, at 3 weeks all the major differentiation markers are expressed, including keratohyalin granules, MCG, and an orthokeratotic stratum corneum. The system is inexpensive, simple to establish, and does not require elevated oxygen levels. The main requirements are 1) the use of Dulbecco's minimal essential medium supplemented with 2) hydrocortisone (100 micrograms/ml), 3) growth at an air/liquid interface, and 4) attached connective tissue. The further addition of vitamin C (300 micrograms/ml) and/or bovine serum albumin (2 mg/ml) offered no obvious advantage. Degeneration of organ cultures in standard cell culture media was discovered to be caused by fetal bovine serum (FBS). FBS-induced degeneration was not prevented by adding any of the supplements tested, or the inclusion of 3T3 fibroblasts, even when culturing at an air/liquid interface. Complete submersion rapidly killed specimens, presumably through oxygen starvation. The ability to maintain a fully keratinizing system for several weeks, in a totally chemically defined medium, will prove valuable for research not only into the role(s) of MCG in epidermal biology but also studies of desquamation and epidermal differentiation.