Channelling frozen cells to survival after thawing: opening the door to cryo‐physiology

Cryo-preservation is used for the long term storage of cells, including sperm and oocytes, and organs (e.g. cornea, kidney, liver, lung and heart) for transplant at ultra low temperatures. While cell and tissue death are known to result mainly from intracellular and extracellular ice crystal formation, osmotic stress and electrolyte disturbance, the nature of the primary damage suffered by cells and tissues during thawing is unclear. Since it is well known that cells undergo shrinkage, that is, dehydration, during freezing (Dong et al. 2010), rehydration upon thawing is necessary to restore the original cell volume and thus ensure the vitality of the recovered cells and tissues. Recovery of cell volume after ‘osmotic volume decrease’ (OVD) induced by hypertonic stress is called the ‘regulatory volume increase’ (RVI) and is achieved by water inflow driven by Na+ influx through the ‘hypertonicity-induced cation channels’ (HICCs) (Wehner et al. 2003), as schematically depicted in Fig. 1 (right scheme). However, the physiological mechanism of rewarming-induced rehydration after the ‘freezing volume decrease’ (FVD) was previously unknown. In the current issue of The Journal of Physiology, Christmann et al. (2016) demonstrate that HICCs are involved in RVI after FVD through a mechanism sensitive to the peptide hormone arginine vasopressin (AVP). In the temperature range of −5 to −15°C, ice forms in the extracellular solution, while the intracellular solution remains unfrozen even below the freezing point, called ‘supercooled’, especially in the presence of cryoprotectants (such as DMSO and glycerol). The chemical potential for water (μw) or the activity of water (aw) is thus increased within cells and decreased in the external medium containing pieces of ice, so water flows out of the cells (Mazur, 1984) leading to a passive FVD, similar to the case of OVD (Fig. 1, left upper scheme). Slight reductions in the cell size during cooling from +25 to around −10°C were previously observed even before ice formation in Chinese hamster ovary (CHO) cells (Griffiths et al. 1979). Christmann et al. (2016) found that progressive cooling from +35 to +10 or +5°C already reduced the osmotically active water space in human HepG2 and HeLa cells. The ‘cooling volume decrease’ (CVD) at over 0°C temperatures may be passively induced by the water efflux down the gradient of absolute temperature (T) or μw (which is expressed by the equation μw = μw0 + RTlnaw, where μw0 and R represent the standard chemical potential of water and the gas constant, respectively) between the intra- and extracellular media, if the plasma membrane more or less behaves as a thermal insulator. However, merely ‘passive’ thermodynamic behaviour caused by a microscopic temperature difference may not fully explain such drastic dehydration. Then there remains a question as to what kind of ‘active’ dehydration mechanism is associated with the CVD process. In this context, it is noted that apoptotic cell shrinkage, called the ‘apoptotic volume decrease’ (AVD) (Maeno et al. 2000), is attained ‘actively’ by water efflux that is driven by the exit of K+ and Cl− ions through some available K+ channels and the volume-sensitive outwardly rectifying anion channel (VSOR) (Maeno et al. 2000; Shimizu et al. 2004), as shown in Fig. 1 (left lower scheme). What is the mechanism of ‘active’ rehydration upon CVD or are there any cold-activated K+ and Cl− channels are questions awaiting future study. The most important discovery reported by Christmann et al. (2016) is that the HICC activity, and hence the RVI process, is augmented after conditions of cryo-preservation without major changes in the expression of aquaporins (AQPs). Thus, HICC is a principal player not only in RVI after OVD (Wehner et al. 2003) or AVD (Numata et al. 2008) but also in RVI after CVD or FVD (Fig. 1, right scheme). Some type of anion channel, here tentatively called the ‘hypertonicity-induced anion channel’ (HIAC), must be activated to produce net KCl efflux which drives water extrusion, because electrogenic Na+ inflow through the HICCs should be coupled to electrogenic Cl− inflow (Fig. 1, right scheme). The verification of this concept remains for the future, and so also must studies addressing the question as to how the process of RVI after FVD, especially via HICC activation, is affected by cryo-protectants. Another very important discovery reported by Christmann et al. (2016) is that pretreatment with AVP further increases the HICC current density which was already enhanced by freezing, in a manner sensitive to knockdown of αENaC and TRPM2, molecules that are responsible for HICCs (Bondarava et al. 2009; Numata et al. 2012), thereby improving post-cryo cell survival. AVP is known to upregulate AQP2 in the kidney collecting duct (Knepper, 1997) and VSOR in AVP-secreting neurons (Sato et al. 2011) through the G protein-coupled AVP receptor (VR) and cAMP. Further investigation is needed to elucidate the signalling mechanism for AVP-induced facilitation of HICCs. The study performed by Christmann et al. (2016) provides an important and innovative contribution to physiological sciences. Our understanding of the physiology of cell volume regulation is now extended to cooling, freezing and thawing conditions. In particular, a door has now been opened to cryo-physiology and this paper represents the first report that vasopressin is acting as a cryo-protectant for mammalian cells. None declared. This work was supported by JSPS KAKENHI Grant, 15K15028.

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