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

Yannik Müllers,Iris Riemann,Ina Meiser,Franziska Lautenschläger,Heiko Zimmermann,Julia C Neubauer,Frank Stracke,Yang Yu

doi:10.1371/journal.pone.0211382

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.

Highlights

The application of human stem cells is a promising approach for various fields in regenerative medicine
For cells fixed after 120 min of recovery, the ratio classified in class I is below 25%, 33.3% reveal severe actin disruptions while 31.1% of the cells were detached
We introduced a quantitative analysis based on characterization of the actin cytoskeleton of single, identical cells, thereby providing unique data on F-actin disruption at molecular level that allows us to draw conclusions about potential damaging mechanisms

Summary

Introduction

The application of human stem cells is a promising approach for various fields in regenerative medicine. Crystallization of the extracellular medium occurs, while the water inside the cell is still liquid [5]. Depending on the cooling rate, two different damaging mechanisms arise; cells either lose too much water, which leads to harming solution effects, or intracellular ice formation occurs [6] which in turn leads to a harmful loss of liquid intracellular water too. The glass transition temperature must be passed before crystallization starts This can be achieved by using highly viscous media to increase the glass transition temperature and ultra-fast cooling rates [8]. In some cases vitrification shows better results than slow freezing in regard to post-thaw survival rate, morphology and successful implantation of cryopreserved human embryos [9]. Cryopreservation of adherent cell systems remains a challenge in current research [10,11,12,13] but there is evidence that vitrification is generally more successful [14,15,16]

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