Formation of multilayers in the caco-2 cell culture model: a confocal laser scanning microscopy study.
OBJECTIVE
To introduce confocal laser scanning microscopy (CLSM) combined with digital image restoration to characterise Caco-2 cells under different culture conditions, and thus to define additional valid criteria for the optimisation of culture models.


METHODS
Growth curves were established and transepithelial electrical resistance (TEER) measured for cells grown in EMEM or DMEM medium on Cyclopore membranes. Cytoskeleton, cell nuclei and tight junctions (TJ) were investigated by CLSM.


RESULTS
Cultures reached a plateau of approximately 4.5 x 10(5) cells/cm2 after approximately 10 days. At the same time TEER reached 750 omega cm2. An irregular, fairly complete network of TJ was present at confluence (approximately 2 d). Between 15 and 30 days a regular TJ network was established. Cells formed mixed mono- and multilayers under most conditions with two exceptions: flat monolayers were observed on polycarbonate filters with EMEM and with the Biocoat intestinal epithelium differentiation environment system. In multilayers TJ were found in the upper as well as in the lower cell layers although the regular vertical polarity was disturbed.


CONCLUSIONS
CLSM represents an important tool to investigate the cytoarchitecture of Caco-2 cells. 3D-analysis of confocal data gives important clues on the characteristics of cell layers and thus helps to validate optimisation strategies.
Rothen-Rutishauser
B
B
Biopharmacy, Department of Applied Biosciences, ETH Zurich, Switzerland. wunderli@pharma.ethz.ch
Braun
A
A
Günthert
M
M
Wunderli-Allenspach
H
H
D000230:Adenocarcinoma / Q000473:pathology*; D018938:Caco-2 Cells; D002455:Cell Division; D003110:Colonic Neoplasms / Q000473:pathology*; D003599:Cytoskeleton / Q000648:ultrastructure; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*