Assembly of intercellular junctions in epithelial cell monolayers following exposure to cryoprotectants

We investigated the effects of cryoprotectants (glycerol, propane-1,2-diol, dimethyl sulfoxide) on the ability of epithelial cells to assemble interce llular junctions. Madin-Darby canine kidney cells (MDCK, type IT) were grow n in S-MEM containing only 5 mu mol/L Ca2+ to allow attachment of cells to the growth surface but not the development of the junctional complex. In a first set of experiments, cells were exposed to 10% v/v cryoprotectant at r oom temperature for 30 min, After removal of the cryoprotectant, [Ca2+] was increased to 1.8 mmol/L (Ca-switch) and the assembly of junctions was foll owed immunocytochemically and by monitoring transepithelial resistance (TER ). In a second set of experiments, the development of junctions was followe d in the presence of 1% cryoprotectant, Addition and removal of 10% cryopro tectant had little effect on the assembly of junctions following the Ca-swi tch, with TER peaking >300 ohm cm(2) after 24 h. Immunocytochemical stainin g showed recruitment to cell borders of components of tight junctions, adhe rens junctions, and desmosomes and the presence of a distinct circumferenti al bundle of actin filaments. In the presence of 1% cryoprotectant, there w as a lag of more than 20 h before TER began to rise, There was then a progr essive rise in TER in all three cryoprotectant groups, indicating junction assembly, albeit at a lower rate than that in the absence of cryoprotectant . These results suggest that exposure to cryoprotectants per se will not in hibit cellular repair mechanisms aimed at restoring the integrity of epithe lial coil layers, but incomplete removal of cryoprotectant may delay repair . (C) 2000 Academic Press.