Modification of cellular membranes conveys cryoprotection to cells during rapid, non-equilibrium cryopreservation.

Jan Huebinger,Mária A Deli

doi:10.1371/journal.pone.0205520

Jan Huebinger, Mária A Deli

Open Access

https://doi.org/10.1371/journal.pone.0205520

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Journal: PloS one	Publication Date: Oct 10, 2018
Citations: 8	License type: CC BY 4.0

Affiliation: Max Planck Institute of Molecular Physiology

Abstract

Rapid cooling and re-warming has been shown promising to cryopreserve living cells, which cannot be preserved by conventional slow freezing methods. However, success is limited by the cytotoxicity of highly concentrated cryoprotective agents. Recent results have shown that cryoprotective agents do not need to suppress intracellular ice crystals completely to allow for survival after cryopreservation. Cryoprotective agents like DMSO or ethylene glycol can also lead to a tolerance of cells towards intracellular ice. It is however unclear by which mechanism this tolerance is achieved. These substances are also known to modulate properties of cellular membranes. It is shown here that cryoprotective DMSO and ethylene glycol have a clear influence on the mobility of lipids in the plasma membrane of HeLa cells. To isolate changes of the properties of plasma membranes from effects on ice formation, the membrane properties were modulated in absence of cryoprotective agents. This was achieved by changing their sterol content. In cells with elevated sterol content, an immobile lipid fraction was present, similar to cells treated with DMSO and ethylene glycol. These cells showed also significantly increased plasma membrane integrity after rapid freezing and thawing in the absence of classical cryoprotective agents. However, their intracellular lysosomes, which cannot be enriched with sterols, still got ruptured. These results clearly indicate that a modulation of membrane properties can convey cryoprotection. Upon slow cooling, elevated sterol content had actually an adverse effect on the plasma membranes, which shows that this effect is specific for rapid, non-equilibrium cooling processes. Unraveling this alternative mode of action of cryoprotection should help in the directed design of novel cryoprotective agents, which might be less cytotoxic than classical, empirically-found cryoprotective agents.

Highlights

Cryopreservation, i.e. the potentially infinite storage under very cold temperatures, of living cells is of fundamental interest for biomedical research, clinical application and the preservation of endangered species
To gain understanding about the effect that a medium of 15% DMSO and 15% ethylene glycol (DE-medium) has on membranes of living cells, living HeLa cells were labeled with the fluorescent lipid DiOC18 and treated with DE-medium
The plasma membrane of HeLa cells treated with 10 mM of cholesterol in medium without cryoprotective agents showed in fluorescence recovery after photobleaching (FRAP) experiments an overall decrease in diffusion speed (Fig 1B) and a similar increase in immobile lipids as cells with normal cholesterol content in DE-medium (Fig 1A)

Summary

Introduction

Cryopreservation, i.e. the potentially infinite storage under very cold temperatures, of living cells is of fundamental interest for biomedical research, clinical application and the preservation of endangered species. This approach suffers from toxicity of the relatively high concentrated cryoprotective agents that need to be applied to the cells at temperatures above 0 ̊C [1,5] These cryoprotecants were thought to be necessary to avoid ice-crystallization in cells, since ice-crystals were– in analogy to slow freezing approaches–considered to be absolutely lethal [1,5]. The term vitrification is not strictly correct for such applications, because it would imply the complete suppression of ice crystallization These approaches are called rapid-cooling and rewarming approaches here. Small polar molecules like DMSO, glycerol or ethylene glycol can modulate the hydration layer of membranes [11], which changes their properties at subzero temperatures [8,12] These substances generate a high tolerance against re-crystallization after rapid cooling [6]. The observed improved resistance to cryodamage by increasing sterol content was specific for rapid cooling processes

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