A team at the University of Alberta have been using the Linkam Freeze Drying system FDCS196 to study how intracellular ice formation during cryopreservation of cells is affected by the degree of supercooling and the cell volume, in the absence of cryoprotectants.
Representative image of SYTO®13 (green)/EB (red) fluorescence used for membrane integrity assay. Green cells have intact membranes and red cells have damaged membranes. Picture from Prickett et al., 2015.
EFFECT OF SUPERCOOLING AND CELL VOLUME ON INTRACELLULAR ICE FORMATION (PRICKETT ET AL., 2015)
A team at the University of Alberta have been studying how intracellular ice formation during cryopreservation of cells is affected by the degree of supercooling and the cell volume, in the absence of cryoprotectants.
Cryopreservation is the use of very low temperatures to preserve living and structurally intact cells and tissues.
Successful cryopreservation of mammalian cells is crucial for medical use and research purposes. However, ice formation at these temperatures — and the resultant increase of concentration of solutes in the remaining liquid fraction — can be damaging to cells; especially intracellular ice formation (IIF) which is the main cause of cell death for cells cryopreserved in solution.
While the exact mechanistic process for the damage caused by IIF is not known, the plasma membrane plays a key role in all popular theories: either allowing extracellular ice to enter the cell or catalysing the nucleation of intracellular ice.
Cells can generally tolerate supercooling of about 2-10°C before IIF occurs but this is affected by extracellular temperatures and different osmalities of solutions (which affects cell volume).
In this study the number of human umbilical vein endothelial cells (HUVECs) undergoing intracellular ice formation at different degrees of supercooling were examined on a cryostage. Intracellular freezing can be detected by the darkening of cells. Cell survival after thawing was determined using a membrane integrity assay. In the picture above, the cells with intact membranes can be seen in green and the cells with damaged membranes can be seen in red.
The cryomicroscope system consisted of the Linkam freeze drying stage (FDCS196), T94 temperature controller, liquid nitrogen pump and a Nikon Eclipse 80i microscope.
The team found that the smaller cells in a heterogeneous cell population (and those that were smaller as a result of osmotic dehydration) could withstand more supercooling before experiencing IIF. This knowledge could be used to develop novel cryopreservation techniques.
They told us: "cryomicroscopy allowed us to record the formation of intracellular ice (lethal for cells in suspension) in individual HUVECs, and link this to individual cell volume and the amount of solution supercooling. Our work demonstrates that larger cells are more likely to form intracellular ice. This type of detailed understanding will lead to improved ability to design superior cryopreservation protocols for cells for medical use or distribution for research."
You can read more on their work here.