Practical considerations for long-term time-lapse imaging of epithelial morphogenesis in three-dimensional organotypic cultures

Epithelia are one of the fundamental tissues in the animal body, and there is broad interest in understanding the cellular and molecular basis of their formation, growth, remodeling, and pathologic degeneration. Unfortunately, from an imaging perspective, many epithelial tissues develop deep within the animal and are inaccessible to high-resolution optical imaging with visible wavelengths. To circumvent this problem, researchers have long sought to model epithelial morphogenesis in culture systems. Protocols for culturing whole epithelial organs have existed since the 1950s, but the use of three-dimensional (3D) organotypic cultures of epithelial fragments has advanced dramatically in recent years. There has been a considerable increase in interest in imaging cell behaviors and molecular activities within these cultures. This article discusses the common technical challenges associated with imaging epithelial morphogenesis in 3D cultures and presents a range of specific strategies to address these challenges. Solutions are presented, first conceptually and then at several levels of sophistication and expense. The goal is to help you adapt those specific methods most useful to your own research, in a manner compatible with your needs and budget.