The effect of isochoric freezing on mammalian cells in an extracellular phosphate buffered solution

Isochoric (constant volume) freezing has been recently suggested as a new method for cell and organ preservation. As a first step in studying the effect of isochoric freezing on mammalian cells, Madin-Darby canine kidney epithelial cells (MDCK), were frozen in an isochoric system, in a simple extracellular phosphate buffered solution to -10 degrees C (96.5 MPa), - 15 degrees C (162 MPa) and -20 degrees C (205 MPa) for 60 and 120 mM. Cell membrane integrity and cell metabolism were studied with a Live/Dead cell vitality assay and flow cytometry. We found that cell survival decreases with an increase in pressure (lower temperatures) and time of exposure. For example, 60% of cells survived 60 mM at -10 degrees C and only 18% survived 120 min at this temperature. Negligible survival was measured at -20 degrees C. This study may serve as the baseline towards further research on techniques to optimize the effects of isochoric freezing on living biological matter.