Four-dimensional imaging of filter-grown polarized epithelial cells

Abstract

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell® filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.

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Acknowledgments

We thank Vicrotia C. Cogger for assistance with SEM; Roberto Weigert (NHLBI, NIH, Bethesda, MD, USA), Frederick J. Suchy (Mount Sinai School of Medicine, New York, NY, USA) and Adam D. Linstedt (Carnegie Mellon University, Pittsburgh, PA, USA) for providing reagents used in this study. Janet L. Larkin received support from the Oak Ridge Institute for Science and Education.

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Correspondence to Yoshiyuki Wakabayashi.

Electronic supplementary material

Movie 1 Z sections of VAMP 8-GFP positive endosomes in live MDCKmonolayer. (mov 2.87 MB)

Movie 2 Time lapse imaging of VAMP 8- GFP positive endosome inpolarized MDCK cells. (mov 2.85 MB)

Movie 3 Z sections of VAMP 8-GFP positive endosome in fixed MDCKmonolayer. (mov 2.77 MB)

Movie 4 4D imaging of Golgi fragmentation caused by BFA in polarizedMDCK cells. (mov 2.14 MB)

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Wakabayashi, Y., Chua, J., Larkin, J.M. et al. Four-dimensional imaging of filter-grown polarized epithelial cells. Histochem Cell Biol 127, 463–472 (2007). https://doi.org/10.1007/s00418-007-0274-x

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Keywords

  • Four-dimensional imaging
  • Live cell imaging
  • Polarized epithelial cells
  • Permeable filter insert
  • MDCK cells