Polyvinylpyrrolidone (PVP) mitigates the damaging effects of intracellular ice formation in adult stem cells.

The objective of this work was to assess the effect of 10% (w/v) polyvinylpyrrolidone (PVP) on the pattern of intracellular ice formation (IIF) in human adipose tissue derived adult stem cells (ASCs) in the absence of serum and other cryoprotective agents (CPAs). The freezing experiments were carried out using a fluorescence microscope equipped with a Linkam cooling stage using two cooling protocols. Both the cooling protocols had a common cooling ramp: cells were cooled from 20 degrees C to -8 degrees C at 20 degrees C/min and then further cooled to -13 degrees C at 1 degrees C/min. At this point we employed either cooling protocol 1: the cells were cooled from -13 degrees C to -40 degrees C at a pre-determined cooling rate of 1, 5, 10, 20, or 40 degrees C/min and then thawed back to 20 degrees C at 20 degrees C/min; or cooling protocol 2: the cells were re-warmed from -13 degrees C to -5 degrees C at 20 degrees C/min and then re-cooled at a pre-determined rate of 1, 5, 10, 20, or 40 degrees C/min to -40 degrees C. Almost all (>95%) of the ASCs frozen in 1x PBS and protocol 1 exhibited IIF. However, almost none (<5%) of the ASCs frozen in 1x PBS and protocol 2 exhibited IIF. Similarly, almost all (>95%) of the ASCs frozen in 10% PVP in PBS and protocol 1 exhibited IIF. However, ~0, ~40, ~47, ~67, and ~100% of the ASCs exhibited IIF when frozen in 10% PVP in PBS and utilizing protocol 2 at a cooling rate of 1, 5, 10, 20, or 40 degrees C/min, respectively.