P2.22 A NEW SIMPLE ORGANOTYPIC ASSAY SYSTEM THAT IMITATES HUMAN HAIR FOLLICLE-LIKE  EPITHELIAL-MESENCHYMAL INTERACTIONS

¹Department of Dermatology, 3^rd Medical Faculty, Charles’ University Hospital, Prague, Czech  Republic; ²Department of Dermatology, University Hospital Hamburg-Eppendorf; University of Hamburg, Hamburg, Germany; ³Department of Physiology, Medical and Health Science Center, University of Debrecen,  Debrecen, Hungary; ⁴Cutech Srl, Padova, Italy

Objectives: To prepare a simple organotypic system for dissecting the underlying epithelial-mesenchymal interactions and as screening tools for candidate hair growth-modulatory agents. To optimize the design and culture conditions of previously published organotypic systems that imitate epithelial-mesenchymal interactions in the human hair follicle as closely as possible.

Materials and methods: Continuous submerged organotypic “sandwich” cultures were established. These consist of a pseudodermis (collagen I mixed with and contracted by human interfollicular dermal fibroblasts) on which one of two upper layers is placed: either a mixture of Matrigel™ and follicular dermal papilla fibroblasts (DPC), with outer root sheath keratinocytes (ORSK) layered on the top (“layered” system), or a mixture of Matrigel™, DPC and ORSK (“mixed” system). Morphological and functional characteristics of these “folliculoid sandwiches” were then assessed by routine histology, histomorphometry and immunohistochemistry.

Results: In both “layered” and “mixed” systems, the ORSK formed spheroid epithelial cell aggregates, which retained their characteristic keratin expression pattern (i.e. cytokeratin 6), while the DPC retained their characteristic expression of Versican. ORSK proliferated better in the „mixed” than in the „layered” sandwich system, regardless of the calcium or serum content of the media, whereas apoptosis of ORSK was lowest in the „mixed” system in serum-free, low calcium medium. The kinetics of proliferation and apoptosis of DPC were similar in both systems. However, proliferation and apoptosis of DPC was higher in the presence of serum and/or under high calcium conditions.

Conclusions: We propose a new organotypic submerged “folliculoid sandwich” system with serum-free, low calcium medium and a mixture of interacting human DPC and ORSK, which offers several advantages over previously available assays and reasonable approximation of epithelial-mesenchymal interactions in human scalp hair follicles.