Establishment and characteristics of Gottingen minipig skin in organ culture and monolayer cell culture: relevance to drug safety testing.

Skin from Gottingen minipigs was used as a source of tissue for organ and cell culture and compared to human skin for growth conditions and sensitivity to irritants. Optimal organ culture conditions were determined, based on the preservation of the histological structure. These included serum-free, growth factor-free conditions with a calcium concentration of 1.5mM. Formulations in which the calcium concentration were low (0.075-0.15mM) failed to support tissue viability (even in the presence of dialyzed serum). Epidermal keratinocytes were grown from tissue explants and as single cells from enzyme-disrupted tissue. Optimal keratinocyte growth was achieved using a serum-free, growth factor-supplemented culture medium with a calcium concentration of 0.15mM. Fibroblasts were optimally grown from explant cultures using a medium with 1.5mM calcium and 10% fetal bovine serum. The conditions that were optimal for maintenance of intact pig skin, as well as for the isolated cells, are the same conditions that have been shown previously to be optimal for intact human skin and skin cells. In additional studies, pig skin keratinocytes and fibroblasts were exposed to a panel of contact irritants and contact sensitizers. Using growth inhibition as the response, the median effective dose values with each agent were very similar to the values previously determined for human epidermal keratinocytes and human dermal fibroblasts. Taken together, these data suggest that the skin from the Gottingen minipig can be used as a surrogate for human skin in ex vivo skin safety studies.

Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer culture for assessment of chemically induced skin damage.

Human skin cells (epidermal keratinocytes and dermal fibroblasts) in monolayer culture and human skin in organ culture were exposed to agents that are known to produce irritation (redness, dryness, edema and scaly crusts) when applied topically to skin. Among the agents used were three well accepted contact irritants (i.e., all-trans retinoic acid [RA], sodium lauryl sulfate [SLS] and benzalkonium chloride) as well as the corrosive organic mercury compound, aminophenyl mercuric acetate (APMA), and 5 contact sensitizers (oxazolone, nickel sulfate, eugenol, isoeugenol and ethylene glycol dimethacrylate [EGDM]). As a group, the contact irritants (including the corrosive mercuric compound) were cytotoxic for keratinocytes and fibroblasts and suppressed growth at lower concentrations than the contact sensitizers. The contact irritants also produced histological changes (hyperplasia, incomplete keratinization, loss of the granular layer, acantholysis and necrosis) in organ-cultured skin at dose levels at which the contact sensitizers appeared to be inert. Finally, the profile of secreted molecules from organ-cultured skin was different in the presence of contact irritants versus contact sensitizers. Taken together, these data suggest that the use of organ-cultured skin in conjunction with cells derived from the skin in monolayer culture may provide an initial approach to screening agents for deleterious changes in skin.