A Long-Term Coculture Model for the Study of Mast Cell–Keratinocyte Interactions

Abstract

Physiologic and pathologic events associated with cutaneous differentiation and repair are the result of a concerted action of various types of resident tissue cells. In vitro models simulating this complex in vivo situation are therefore needed to clarify the specific contribution and relevant interaction of, for example, dermal mast cells with other major cutaneous cells. The aim of this study was to establish a long-term coculture model that includes dermal mast cells, dermal fibroblasts, and keratinocytes in a human skin equivalent organotypic setting. Normal dermal mast cells and fibroblasts (1:4) were enclosed in collagen gel and normal keratinocytes were grown on top with exposure to the air interface. Under these conditions, mast cell integrity and functionality was preserved even after 4 wk of culture, as shown by electron microscopy and immunohistochemistry using antibodies against the mast-cell-specific granule enzyme tryptase and the receptors for stem cell factor and IgE. Mast cells also released histamine on stimulation with anti-IgE, and on ultrastructure were found to degranulate, with decrease of granule matrix density and formation of cell-cell contacts with fibroblasts. After 2 wk of culture, keratinocytes had formed an epidermis-like multilayer and were able to proliferate and differentiate, as shown by bromodeoxyuridine incorporation of basal cells and immunohistochemical staining for transglutaminase and cytokeratins 1 and 10. The model presented here thus provides a potentially relevant tool to further clarify the interaction of dermal mast cells with major other skin cells and their contribution to cutaneous physiology, repair processes, and pathology.