Methods for analysis of apical lumen trafficking using micropatterned 3D systems

Abstract

Epithelial organs are made of interconnected branched networks of tubules, with a central lumen lined by a monolayer of epithelial cells. Certain epithelial cell lines can be converted into organotypic cultures by the addition of extracellular matrix components. When cultured in these conditions, epithelial cells reorient the axis of polarity, reorganize the membrane surfaces, and transport apical proteins to form the lumen in a process that recapitulates essential aspects of de novo apical membrane formation during epithelial organ morphogenesis. Micropatterns are a simple technique that allows cell culture in a controlled adhesive environment with extremely high precision, close to the nanometer scale. We have recently developed a method to culture MDCK cysts on micropatterns of different sizes and composition. Using this method we found that changes in micropattern shape and size can be used to modify cell contractility to understand its contribution to apical membrane formation. When imaging cysts on micropatterns the main advantage is that apical-directed vesicle trafficking is visualized in the x- y plane, which presents higher resolution on confocal microscopes. Thus, the use of micropatterns is an efficient setup to analyze polarized secretion with unprecedented higher resolution in both time and space. © 2013 Elsevier Inc.