Fetal Bovine Serum Limits Human Keratinocyte Differentiation and Tight Junction Function

Abstract

RATIONALE: Studying epidermal barrier function in vitro is challenging because the degree of keratinocyte differentiation and/or stratification in Ca+2-induced differentiation is limited and organotypic cultures are technically challenging. Therefore, we evaluated keratinocyte (KC) culture conditions to identify an approach, which would yield highly differentiated KC with well-developed tight junctions (TJ).METHODS: Primary human neonatal KC were grown to confluence in KC serum-free media, and then changed to DMEM (1.8mM CaCl2) with or without FBS. At several timepoints (1-7 d) KC were analyzed for differentiation markers and TJ proteins by qPCR or Western blot. These proteins were also visualized by immunofluorescence microscopy. TJ function was assessed by transepithelial electric resistance (TEER).RESULTS: KC in DMEM without FBS had greater expression of the differentiation markers, cytokeratin-10, desmoglein-1, loricrin, and filaggrin. In contrast, KC cultured with FBS had little to no expression of these proteins. Furthermore, TEER of KC cultured in DMEM without FBS was markedly higher (1746±208 ohms × cm2, n=10) than KC in DMEM with FBS (165±12 ohms × cm2, n=10; P<0.0001). Immunofluorescence staining for TJ proteins, ZO-1, occludin, and claudin-1, demonstrated that evidence for a TJ structure coincided with TEER increases in both culture conditions.CONCLUSIONS: DMEM media without FBS is a simple and useful alternative to organotypic cultures and more faithfully recapitulates human epidermis than high Ca+2 alone. Our results indicate that something in FBS limits KC differentiation. Additionally, we show that KC differentiation is a key determinant of TJ function, which is independent of TJ protein expression. RATIONALE: Studying epidermal barrier function in vitro is challenging because the degree of keratinocyte differentiation and/or stratification in Ca+2-induced differentiation is limited and organotypic cultures are technically challenging. Therefore, we evaluated keratinocyte (KC) culture conditions to identify an approach, which would yield highly differentiated KC with well-developed tight junctions (TJ). METHODS: Primary human neonatal KC were grown to confluence in KC serum-free media, and then changed to DMEM (1.8mM CaCl2) with or without FBS. At several timepoints (1-7 d) KC were analyzed for differentiation markers and TJ proteins by qPCR or Western blot. These proteins were also visualized by immunofluorescence microscopy. TJ function was assessed by transepithelial electric resistance (TEER). RESULTS: KC in DMEM without FBS had greater expression of the differentiation markers, cytokeratin-10, desmoglein-1, loricrin, and filaggrin. In contrast, KC cultured with FBS had little to no expression of these proteins. Furthermore, TEER of KC cultured in DMEM without FBS was markedly higher (1746±208 ohms × cm2, n=10) than KC in DMEM with FBS (165±12 ohms × cm2, n=10; P<0.0001). Immunofluorescence staining for TJ proteins, ZO-1, occludin, and claudin-1, demonstrated that evidence for a TJ structure coincided with TEER increases in both culture conditions. CONCLUSIONS: DMEM media without FBS is a simple and useful alternative to organotypic cultures and more faithfully recapitulates human epidermis than high Ca+2 alone. Our results indicate that something in FBS limits KC differentiation. Additionally, we show that KC differentiation is a key determinant of TJ function, which is independent of TJ protein expression.