Isolation and Differentiation of Human Keratinocytes

Human primary KCs were isolated as described previously [47 (link)] and cultured in serum-free KC growth medium (KGM-2, Lonza, Basel, Switzerland). Primary human KCs from three different adult donors were used for preparation of organotypic skin cultures as described previously [48 (link)] and, in parallel, cultured in two-dimensional monolayers. For analyzing proliferating KCs, 2nd passage cells at 50% confluency were used. Differentiation was induced by maintaining confluent KCs for 7 days at high calcium (1.1 mM Ca²⁺) conditions. The quality of keratinocyte differentiation was validated by analysis of well-known differentiation markers, including keratin 1, keratin 10, the S100 genes S100A7 and A9, desmoglein-1 and keratinocyte differentiation-associated protein. For in vitro assays, cytokines in the following final concentrations were used: IL-1β (10 ng/mL), TNF-α (10 ng/mL), IL-17 (10 ng/mL), INF-γ (100 ng/mL) (all R&D System, Minneapolis, MN, USA) and toll-like receptor (TLR) agonists (InvivoGen, Toulouse, France).