Formation of multilayers in the Caco-2 cell culture model: A confocal laser scanning microscopy study

Purpose. To introduce confocal laser scanning microscopy (CLSM) combined wi th digital image restoration to characterise Caco-2 cells under different c ulture conditions, and thus to define additional valid criteria for the opt imisation of culture models. Methods. Growth curves were established and transepithelial electrical resi stance (TEER) measured for cells grown in EMEM or DMEM medium on Cyclopore( R) membranes. Cytoskeleton, cell nuclei and tight junctions (TJ) were inves tigated by CLSM. Results. Cultures reached a plateau of similar to 4.5 x 10(5) cells/cm(2) a fter similar to 10 days. At the same time TEER reached 750 Ohm cm(2). An ir regular, fairly complete network of TJ was present at confluence (similar t o 2 d). Between 15 and 30 days a regular TJ network was established. Cells formed mixed mono- and multilayers under most conditions with two exception s: flat monolayers were observed on polycarbonate filters with EMEM and wit h the Biocoat(R) intestinal epithelium differentiation environment system. In multilayers TJ were found in the upper as well as in the lower cell laye rs although the regular vertical polarity was disturbed. Conclusions. CLSM represents an important tool to investigate the cytoarchi tecture of Caco-2 cells. 3D-analysis of confocal data gives important clues on the characteristics of cell layers and thus helps to validate optimisat ion strategies.