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Abstract
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.
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