Formation of Multilayers in the Caco-2 Cell Culture Model: A Confocal Laser Scanning Microscopy Study |
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Authors: | Rothen-Rutishauser Barbara Braun Annette Günthert Maja Wunderli-Allenspach Heidi |
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Institution: | (1) Biopharmacy, Department of Applied Biosciences, ETH Zurich, CH-8057 Zurich, Switzerland;(2) Biopharmacy, Department of Applied Biosciences, ETH Zurich, CH-8057 Zurich, Switzerland |
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Abstract: | Purpose. To introduce confocal laser scanning microscopy (CLSM)combined with digital image restoration to characterise Caco-2 cellsunder different culture conditions, and thus to define additional validcriteria for the optimisation of culture models.
Methods. Growth curves were established and transepithelial electricalresistance (TEER) measured for cells grown in EMEM or DMEMmedium on Cyclopore membranes. Cytoskeleton, cell nuclei and tightjunctions (TJ) were investigated by CLSM.
Results. Cultures reached a plateau of 4.5 × 105 cells/cm2 after 10 days. At the same time TEER reached 750 cm2. An irregular,fairly complete network of TJ was present at confluence (2 d).Between 15 and 30 days a regular TJ network was established. Cellsformed mixed mono- and multilayers under most conditions with twoexceptions: flat monolayers were observed on polycarbonate filterswith EMEM and with the Biocoat intestinal epithelium differentiationenvironment system. In multilayers TJ were found in the upper aswell as in the lower cell layers although the regular vertical polaritywas disturbed.
Conclusions. CLSM represents an important tool to investigate thecytoarchitecture of Caco-2 cells. 3D-analysis of confocal data givesimportant clues on the characteristics of cell layers and thus helps tovalidate optimisation strategies. |
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Keywords: | Caco-2 in vitro intestinal epithelial model confocal laser scanning microscopy tight junctions cytoarchitecture variability |
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