Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics

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Abstract
Epithelial cells grown in three‐dimensional (3‐D) cultures of extracellular matrix differentiate into a multicellular structure of polarized cells. This process shares many characteristics with the physiological development of an epithelial tissue and the formation of polarity in epithelial cells. Imaging 3‐D cultures of polarized epithelial cells is therefore a powerful tool to study epithelial architecture and morphogenesis under close‐to‐physiological conditions. The new generation of confocal microscopes allows live‐cell imaging of fluorescently tagged molecules in these cultures. This opens up new opportunities for studying how molecules behave and are distinguished asymmetrically within a 3‐D setting. This unit discusses technical aspects for culturing and imaging MDCK 3‐D culture for both fixed 3‐D cultures and live‐cell imaging. Curr. Protoc. Cell Biol. 43:4.22.1‐4.22.18. © 2009 by John Wiley & Sons, Inc.