The effect of two different freezing methods on the immediate post-thaw membrane integrity of adipose tissue derived stem cells

Abstract

The effect of directional cooling on the immediate post-thaw membrane integrity of adipose tissue derived adult stem cells (ASCs) was investigated using a directional solidification stage (DSS). Several passages, including Passage-0 (P0), Passage-1 (P1), and Passage-2 (P2), obtained from the suspended culture of stromal vascular fraction (SVF) of the ASCs were used for this study. ASCs from P0, P1, and P2 were cooled at either 1, 5, 20, or 40 °C/min to an end temperature of −80 °C in the presence and absence of a cryoprotective agent (dimethylsulfoxide, DMSO). After freezing to −80 °C, the samples were thawed at 200 °C/min and the ability of the frozen/thawed ASCs to exclude fluorescent dyes was assessed. ASCs frozen using the DSS in the absence of DMSO were found to have a lower post-thaw cell membrane integrity (confidence level of 95%), when compared with the ASCs frozen in the presence of 10% volume/volume (v/v) ratio of DMSO. Intriguingly, a comparison with corresponding data for ASCs that were frozen using a commercially available controlled rate freezer (CRF) suggests that the directionally cooled ASCs (both in the absence and presence of DMSO) exhibit a significantly lower post-thaw cell membrane integrity (confidence level of 95%). This lowering of post-thaw cell membrane integrity for ASCs frozen using the DSS is postulated to be related to the differences in the nature and the associated damaging effects of ice crystals formed in the DSS vs. the commercial freezer.