Abstract
Epithelial tissue guarantees proper performance of many organs, e.g., the kidneys, the gastrointestinal organs, and endocrine glands. Epithelial layers are responsible for the formation and maintenance of separate compartments with distinct solute composition. This is achieved by epithelial layers forming a barrier between the two compartments and concomitantly allowing site-directed transepithelial transport, uptake or secretion of electrolytes, energy substrates, proteins, and other solutes.
Research on epithelial tissue functions has highly profited from the establishment of tissue culture technologies allowing to cultivate primary epithelial cells or established epithelial cell lines. A property of transporting epithelia cultured in vitro that has long been noted is the formation of the so-called domes on solid growth supports, which represent fluid filled blisters between the solid growth surface and the cell layer. Formation of domes is regarded as a sign of active transport processes and an intact epithelial barrier function due to functional tight junctional cell–cell contacts. A novel methodology for long-term live-cell light microscopy is described in the present article, which allows the monitoring of the dynamic nature of structures, such as epithelial domes over days to weeks of tissue culture (“under the microscope”).
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References
Shin K, Fogg VC, and Margolis B (2006) Tight Junctions and Cell Polarity. Annu Rev Cell Dev Bi 22: 207–235.
Van Itallie CM, and Anderson JM (2004) The Molecular Physiology of Tight Junction Pores. Physiology 19: 331–338.
Schneeberger EE, and Lynch RD (2004) The tight junction: a multifunctional complex. Am J Physiol Cell Physiol 286: C1213-1228.
Tsukita S, Furuse M, and Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2: 285–93.
Misfeldt DS, Hamamoto ST, and Pitelka DR (1976) Transepithelial transport in cell culture. P Natl Acad Sci USA 73: 1212–1216.
Cereijido M, Robbins E, Dolan W, Rotunno C, and Sabatini D (1978) Polarized monolayers formed by epithelial cells on a permeable and translucent support. J. Cell Biol. 77: 853–880.
Leighton J, Brada Z, Estes LW, and Justh G (1969) Secretory Activity and Oncogenicity of a Cell Line (MDCK) Derived from Canine Kidney. Science 163: 472–473.
Lever JE (1979) Inducers of mammalian cell differentiation stimulate dome formation in a differentiated kidney epithelial cell line (MDCK). P Natl Acad Sci USA 76: 1323–1327.
Rizzoli R, and Bonjour J (1987) Effect of dexamethasone on parathyroid hormone stimulation of cyclic AMP in an opossum kidney cell line. J Cell Physiol 132: 517–23.
Grasset E, Pinto M, Dussaulx E, Zweibaum A, and Desjeux JF (1984) Epithelial properties of human colonic carcinoma cell line Caco-2: electrical parameters. Am J Physiol Cell Physiol 247: C260-267.
McKay DM, and Baird AW (1999) Cytokine regulation of epithelial permeability and ion transport. Gut 44: 283–289.
Nusrat A, Turner JR, and Madara JL (2000 Nov) Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol 279: G851-7.
Lechner J, Malloth N, Seppi T, Beer B, Jennings P, and Pfaller W (2008) IFN-{alpha} induces barrier destabilization and apoptosis in renal proximal tubular epithelium. Am J Physiol Cell Physiol 294: C153-C160 [Epub 2007, Nov 21].
Lechner J, Malloth NA, Jennings P, Hekl D, Pfaller W, and Seppi T (2007) Opposing roles of EGF in IFN-{alpha}-induced epithelial barrier destabilization and tissue repair. Am J Physiol Cell Physiol 293: C1843-1850 [Epub 2007, Oct 3].
Lechner J, Krall M, Netzer A, Radmayr C, Ryan M, and Pfaller W (1999) Effects of interferon alpha-2b on barrier function and junctional complexes of renal proximal tubular LLC-PK1 cells. Kidney Int 55: 2178–91.
Martin-Martin N, Ryan G, McMorrow T, and Ryan MP (2010) Sirolimus and cyclosporine A alter barrier function in renal proximal tubular cells through stimulation of ERK1/2 signaling and claudin-1 expression. Am J Physiol Renal Physiol 298: F672-682.
Forti E, Bulgheroni A, Cetin Y, Hartung T, Jennings P, Pfaller W, and Prieto P (2010) Characterisation of cadmium chloride induced molecular and functional alterations in airway epithelial cells. Cell Physiol Biochem 25: 159–68.
Acknowledgments
The presented work was part of scientific projects supported through grant No. P17583-B13 of the Austrian Science Fund (FWF, Dr. J. Lechner) and grant No. UNI-0404/229 of the Tyrolean Science Fund (Tiroler Wissenschaftsfond, Dr. J. Lechner). The live-cell imaging device was developed by Dr. T. Seppi, H. Gatt, and M. Voelp (patent AT 500473 B1 2006). Their work was supported through grants NanoBII-815828: PolyCell and NanoBII-0353: GEMID of the Austrian NANO Initiative (a thematic program of the FFG – Austrian Research Promotion Agency).
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1 Electronic Supplementary Material
Dynamics of dome formation monitored by cell culture under the microscope
Light microscopic images of LLC-PK1 cells in culture on borosilicate glass as growth support were obtained after the cells have reached confluence in cell culture. Phase contrast light microscopy was performed using a 20× objective (giving a total magnification of 200-fold). For the time-lapse motion picture series, single light microscopic images taken automatically every 2 min were combined.
The focal plane was set to sharply represent the cells that were well attached to the growth surface. Areas of dome formation comprise cell groups that detach from the surface forming fluid-filled blisters. Domes can be recognized by groups of cells moving out of the focal plane showing less sharp cellular structures and cell–cell interfaces.
At the beginning of the sequence, small regions with dome formation are already present, which merge into a very large dome in the course of the movie. It is noteworthy to see pulsating up and down movements of the whole dome area after the big dome has formed.
In the first few hours, sporadic apoptotic cell extrusion can be monitored at the margins of domes, which are highlighted in Fig. 2b. (Visit http://extras.springer.com/ to view the movie.)
Cell growth to confluence and apoptotic cell death induced by a toxin
A sequence of microscopic images of LLC-PK1 cells in culture on borosilicate glass as growth support is shown as a motion picture series. Single light microscopic images were taken every 2 min. Part 1 of the movie shows cells after attachment, which start to divide and fill up the growth support area using a 10× microscope objective, part 2 shows in more detail cells forming a confluent monolayer using a 20× objective, part 3 shows cellular movements and cell death after the addition of an apoptosis-inducing toxin (100 μM cisplatin) to the culture medium monitored by Nomarski differential interference contrast microscopy using a 40× objective. Part 1 and 2 were recorded using phase contrast microscopy. Copyright: inventive-ts, Innsbruck, Austria, published with permission. (Visit http://extras.springer.com/ to view the movie.)
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Lechner, J., Hekl, D., Gatt, H., Voelp, M., Seppi, T. (2011). Monitoring of the Dynamics of Epithelial Dome Formation Using a Novel Culture Chamber for Long-Term Continuous Live-Cell Imaging. In: Turksen, K. (eds) Permeability Barrier. Methods in Molecular Biology, vol 763. Humana Press. https://doi.org/10.1007/978-1-61779-191-8_11
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DOI: https://doi.org/10.1007/978-1-61779-191-8_11
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