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In the Spotlight

Cryopreservation of cells and tissues for an unlimited ‘‘shelf life’’ is on the forefront of biology and medicine, with countless applications from the maintenance of cell lines for research to clinical tissue transplantation and banking. Effective cryopreservation is, however, difficult to achieve due to cell injury mostly because of intracellular ice formation (IIF). Therefore, understanding IIF and its propagation is essential for the development of new strategies to improve the process of keeping cells and tissues indefinitely alive at very low temperatures. Several studies have indicated that tissues are more susceptible to IFF than isolated cells. It has been suggested that cell– cell interactions increase the likelihood of IFF by enabling propagation of ice between neighboring cells, a process that has been proposed to be mediated by gap junction channels. Nevertheless, the issue remains unclear. Appropriately, a recently published report by Higgins and Karlsson (Biophys J. 2013;105(9):2006-2015) sheds new light in the process of IIF. In this study, Higgins and Karlsson investigated the effects of cell–cell interactions on IFF using a wild-type and 3 genetically engineered strains of the mouse insulinoma cell line MIN6, each of which expressed key intercellular junction proteins representing gap, adherens, and tight junctions (connexin 36, E-cadherin, and occluding) at different levels. The authors used high-speed video cryomicroscopy to visualize the freezing process in pairs of adherent cells and observed that the initial IIF event is the penetration of extracellular ice into the paracellular spaces at the cell–cell interface, independent of the cell strains used. Interestingly, the authors observed that the genetically modified cells exhibited a significantly higher frequency of ice penetration and a higher mean IFF temperature than the wild-type cells. In addition, Higgins and Karlsson used a 4-state Markov chain model to quantify the rate constants of the paracellular ice penetration process, the penetration-associated IFF initiation process, and the intercellular ice propagation process and concluded that in the initial stages of freezing, expression of junction proteins had a very modest effect on all these processes. In fact, even the cell strains lacking the gap junction protein connexin 36 exhibited ice propagation rates, negating the theory that these junctions would be the only mediators of such a process. Thus, the study presented by Higgins and Karlsson not only dismisses the dogma of the essential involvement of gap junctions for IFF but also clearly shows where freezing is initiated and allows the elucidation of the role of paracellular ice penetration. These findings add to a better understanding of cryopreservation that may lead to new advances in the field with outstanding clinical implications.