Mitochondria and membrane cryoinjury in micropatterned cells: effects of cell-cell interactions

The maintenance of cell membrane integrity is an absolute minimum criterion for the selection of a successful cryopreservation process; however, it is often used as the sole determinant of cell "viability". Membrane stresses and strains that develop with cell volume fluctuations are only one component of the overall cellular response to freezing. Damage to organelles resulting from excessive concentration of intracellular solutes and/or the alternation of molecular signalling events may affect post-thaw outcomes. As the low temperature response of cells is affected by the presence of cell-cell interactions, the cryopreservation of tissues and tissue model systems would benefit from a more detailed understanding of the sites and mechanisms of cryoinjury. The purpose of this study was to examine the relationship between mitochondria and plasma membrane damage in frozen micropatterned cells and to identify the role of cell-cell interactions. Madin Darby Canine Kidney cells (MDCK) were micropatterned using a polydimethylsiloxane (PDMS) elastomeric stamp to create non-adhesive regions of agarose on untreated glass substrates. Five different cell arrangements were used to examine the effect of cell-cell contact: single cells, cell doublets, linear arrangement of cells, randomly arranged cells and confluent monolayers. Cells were cooled in a programmable alcohol bath at 1 degrees C/min to -40 degrees C after extracellular ice nucleation at -5 degrees C. Post-thaw plasma membrane integrity and mitochondria depolarization were determined using trypan blue and the lipophilic, cyanine derivative JC-1, respectively. alamarBlue was used to assess the post-thaw metabolic activity of the cell arrangements. We found that the incidence of plasma membrane damage and mitochondria integrity increased with decreasing temperature and was dependent on the degree of cell-cell interaction. Mitochondria damage was evident in cells that displayed intact plasma membranes, however this injury could be reversed in the micropatterned cells that are exposed to suprazero temperatures. The results from this study suggest that the exclusive use of membrane integrity as a measure of cell "viability" does not consider subcellular injury that may contribute to delayed recovery and/or cell death following low temperature exposures.