Towards optimization of an organotypic assay system that imitates human hair follicle-like epithelial-mesenchymal interactions
Abstract
Background Human hair growth can currently be studied in vitro by the use of organ-cultured scalp hair follicles (HFs). However, simplified organotypic systems are needed for dissecting the underlying epithelial-mesenchymal interactions and as screening tools for candidate hair growth-modulatory agents.
Objectives To optimize the design and culture conditions of previously published organotypic systems that imitate epithelial-mesenchymal interactions in the human HF 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 MatrigelTM basement membrane matrix (BD Biosciences, Bedford, MA, U.S.A.) and follicular dermal papilla fibroblasts (DPC), with outer root sheath keratinocytes (ORSK) layered on the top ('layered' system), or a mixture of MatrigelTM, 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). In the 'mixed' sandwich model the size of the epithelial cell aggregates was smaller, but the numbers of ORSK were significantly higher than in the 'layered' model at day 14 in the culture.
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, which retained their characteristic expression of versican, were similar in both systems.
However, proliferation and apoptosis of DPC were higher in the presence of serum and/or under high calcium conditions. Conclusions Our results underscore the importance of structural design and medium composition for epithelial-mesenchymal interactions as they occur in the human HF.
Specifically, we report 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. This system allows the standardized assessment of the effects of a test agent on the proliferation, apoptosis and key marker expression of human ORSK and DPC under substantially simplified in vitro conditions which approximate the in vivo situation.