MDCK cell cultures as an epithelial in vitro model: cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy.
OBJECTIVE
Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier.


METHODS
Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, alpha-tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration.


RESULTS
TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0-1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2-6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7-14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV (> or = 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer.


CONCLUSIONS
Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitro model to study drug transport and/or interactions with drugs and excipients.
Biopharmacy, Department of Pharmacy, ETH Zürich, Switzerland. brothen@pharma.ethz.ch
B
B
Rothen-Rutishauser
S D
SD
Krämer
A
A
Braun
M
M
Günthert
H
H
Wunderli-Allenspach
D000818:Animals; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D003599:Cytoskeleton / Q000502:physiology*; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D007668:Kidney / Q000166:cytology; D018613:Microscopy, Confocal; D019108:Tight Junctions / Q000502:physiology*