Quantitative analysis of F-actin alterations in adherent human mesenchymal stem cells: Influence of slow-freezing and vitrification-based cryopreservation.

Cryopreservation is an essential tool to meet the increasing demand for stem cells in medical applications. To ensure maintenance of cell function upon thawing, the preservation of the actin cytoskeleton is crucial, but so far there is little quantitative data on the influence of cryopreservation on cytoskeletal structures. For this reason, our study aims to quantitatively describe cryopreservation induced alterations to F-actin in adherent human mesenchymal stem cells, as a basic model for biomedical applications. Here we have characterised the actin cytoskeleton on single-cell level by calculating the circular standard deviation of filament orientation, F-actin content, and average filament length. Cryo-induced alterations of these parameters in identical cells pre and post cryopreservation provide the basis of our investigation. Differences between the impact of slow-freezing and vitrification are qualitatively analyzed and highlighted. Our analysis is supported by live cryo imaging of the actin cytoskeleton via two photon microscopy. We found similar actin alterations in slow-frozen and vitrified cells including buckling of actin filaments, reduction of F-actin content and filament shortening. These alterations indicate limited functionality of the respective cells. However, there are substantial differences in the frequency and time dependence of F-actin disruptions among the applied cryopreservation strategies; immediately after thawing, cytoskeletal structures show least disruption after slow freezing at a rate of 1°C/min. As post-thaw recovery progresses, the ratio of cells with actin disruptions increases, particularly in slow frozen cells. After 120 min of recovery the proportion of cells with an intact actin cytoskeleton is higher in vitrified than in slow frozen cells. Freezing at 10°C/min is associated with a high ratio of impaired cells throughout the post-thawing culture.