Really Cool Cells (and how they got that way!)

Abstract

The ability to preserve tissues and organs for later use is of tremendous clinical importance. Whereas many types of individual cells can be frozen and later functionally restored, this has proven to be more difficult with tissues. This is due to intracellular ice formation (IIF), which for some reason is more common when cells are connected, as they are in tissues. Using the fastest high-speed video cryomicroscopy yet developed, Adam Higgins and Jens Karlsson have shown for the first time that penetration of extracellular ice into paracellular spaces at the cell-cell interface is a precursor to IIF. They termed this precursor phenomenon “paracellular ice penetration” (PIP), which is the growth of extracellular ice crystal protrusions into paracellular spaces that contain supercooled liquid (Figure 1). Because PIP is associated with cell-cell and cell-substrate interactions, and does not occur during freezing of suspended isolated cells, this mechanism may contribute to the observed increase in IIF in tissues. It has been thought that IIF moves from cell to cell through intercellular channels known as gap junctions, which are formed by connexin proteins. To test this hypothesis, Higgins and Karlsson tested three genetic variants of a mouse insulinoma cell line (MIN6): the wild-type containing connexin-36, as well as two other types of junctional proteins, E-cadherin and occludin. One of the genetically transformed strains contained almost none of those three proteins; whereas, another had only E-cadherin, and the third expressed only connexin-36. Pairs of the various cells were placed in a cryomicroscope stage chamber and then cooled rapidly to −60oC at 130oC/min. Images were acquired with their high-speed video camera at a sampling rate of 3,000–4,000 Hz. This gave a temporal resolution on the order of 0.3 ms. The IIF starting locations were classified into three