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

Abstract

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.

Fig 1. Time and frequency of actin-cytoskeleton disruption depending on cryopreservation strategy.

Cryopreserved hMSCs by slow freezing at 1°C/min (A), 10°C/min (B) and vitrification (C) are classified depending on quantified cytoskeletal disruption in class I (no disruption), class II (slight disruption), class III (severe disruption), and detached cells. Bars show mean value with standard deviation from three experimental replicates (N = 3, n = 45), 0, 15, and 120 min after thawing. Representative live-cryo images from two photon microscopy (D) show the actin cytoskeleton qualitatively during slow freezing and after subsequent thawing (37°C). The presented images originate from one out of three measurement with each cooling rate. For better visualization, contrast and brightness of the presented images were adjusted. Scale bars are 20 μm.

Fig 2. Circular standard deviation is significantly increased in the actin cytoskeleton of cryopreserved hMSCs.

(A) Boxplots show the difference of circular standard deviation between identical cells before and after cryopreservation with varying recovery time. Non-cryopreserved cells have been incubated for the respective recovery time in the corresponding cryo medium as a control. Horizontal bars represent the median, box size is defined by the 25^th and 75^th percentiles. Significant differences based on Mann-Whitney-U test with p-values smaller than 0.05 are indicated by *. (B) Cells, being classified in class II or III, are separated, depending on whether circular standard deviation increased or decreased after cryopreservation. The top x-axis states the recovery time in min. (C) Representative F-actin-morphologies of hMSCs with increased circular standard deviation after cryopreservation are shown with their related Δν-value. Images were captured with CLSM. Scale bar indicates 20 μm. For better visualization, contrast and brightness of the presented images were adjusted.

Fig 3. Vitrification and slow freezing result in reduced F-actin-content.

(A) Boxplots show the difference of F-actin-content between identical cells before and after cryopreservation with varying recovery time. Non-cryopreserved cells have been incubated for the respective recovery time as a control. Horizontal bars represent the median, box size is defined by the 25^th and 75^th percentiles. Significant differences based on Mann-Whitney-U test with p-values smaller than 0.05 are indicated by *. (B) Cells, being classified in class II or III, are separated, depending on whether F-actin-content increased or decreased after cryopreservation. The top x-axis states the recovery time in min. (C) Representative actin cytoskeletons of hMSCs with reduced F-actin-content after cryopreservation are shown with their related Δν-value. Images were captured with CLSM. Scale bar indicates 20 μm. For better visualization, contrast and brightness of the presented images were adjusted.

Fig 4. Cryopreserved hMSCs exhibit a reduced F-actin-length.

(A) Boxplots show the difference of F-actin-length between identical cells before and after cryopreservation with varying recovery time. Non-cryopreserved cells have been incubated for the respective recovery time as a control. Horizontal bars represent the median, box size is defined by the 25^th and 75^th percentiles. Significant differences based on Mann-Whitney-U test with p-values smaller than 0.05 are indicated by *. (B) Cells, being classified in class II or III, are separated, depending on whether F-actin-length increased or decreased after cryopreservation. The top x-axis states the recovery time in min. (C) Representative actin cytoskeletons of hMSCs with reduced F-actin-length after cryopreservation are shown with their related Δν-value. Images were captured with CLSM. Scale bar indicates 20 μm. For better visualization, contrast and brightness of the presented images were adjusted.