Freezing monolayers of cells without gap junctions

Cells in monolayers have been reported to be more susceptible to freezing injury than the same cell type frozen in dispersed suspensions. There appears to be an enhanced susceptibility to intracellular freezing in the monolayers, which is thought to be facilitated by the presence of gap junctions allowing the spread of ice between neighbouring cells. MDCK Type II cells do not form gap junctions in monolayer culture. When frozen at rates of 0.2 to 10 degrees C/min, monolayers in 10% (v/v) propane-1,2-diol or dimethyl sulphoxide showed little influence of cooling rate on survival. This suggested that, in the absence of gap junctions, cells in monolayers did not display enhanced susceptibility to intracellular freezing. In contrast, however, monolayers frozen in glycerol showed a marked increase in cell damage when cooled at rates higher than 0.5 degrees C/min. This does not necessarily counter the suggestion that lack of gap junctions mitigates intracellular freezing as there is evidence that glycerol may itself promote intracellular freezing.