UBC Theses and Dissertations
Organotypic keratinocyte cultures on de-epithelialized tongue mucosa Hildebrand, H. Christopher
Organotypic cultures have been used to study epithelial cell behavior for many years. The aim of this study was to develop an organotypic culture method that better mimics the three-dimensional morphology of interdigitating rete ridges and connective tissue (CT) papillae and conserves the basement membrane zone (BMZ). Bovine tongue mucosa was chosen for the raft donor and incubated with cold 1 M sodium chloride solutions for 4 days to separate the epithelial tissue from the BMZ and underlying CT matrix. Level of separation was studied by electron microscopy (EM) and immunohistochemical staining of several integrin subunits, laminin-1 and -5, type IV and Vll collagen, tenascin, fibronectin, vitronectin and heparan sulfate proteoglycan. After the separation, IHC staining and EM observations suggest a separation through the lamina lucida. Rafts were used for growth of organotypic cultures of human keratinocytes. Three different keratinocyte cell lines were cultured submerged for 6 days after which the cultures were raised to air-liquid interface and maintained for up to 40 days. Sections were prepared for routine histology, EM and IHC staining. Histomorphology varied significantly between the cell lines. Sometimes more than 15 cell layers resembling normal epithelial histology were present. Keratinocytes in these raft cultures were found to express P4 integrins against the preexisting BMZ. In addition, human keratinocytes deposited their own matrix molecules, particularly tenascin, laminin-1 and -5. Expression of integrin subunits and differentiation markers revealed some differences from normal tissue, which depended on the cell line. The ultrastructure of the BMZ including hemidesmosomes was similar to the normal dermal-epidermal junction. This culture model seems to mimic normal epithelium including the BMZ and is potentially useful for multiple applications for studies on epithelial cell behavior in vitro.
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