Cryopreservation is the technology used to stabilize cells for a variety of applications, including diagnosis and treatment of disease. Because we don’t completely understand the mechanisms of freezing damage, poor or inadequate methods of preservation have limited our ability to use cells for cell therapy. Also, observations of cell responses could not be correlated to viability on a cell-by-cell basis using conventional low-temperature microscopy techniques. This study establishes our ability to measure the viability of individual frozen cells based on the correlation of cytochrome c distribution, a signal that can be detected using Raman spectroscopy, with trypan blue staining. With Raman spectroscopy, we are able to observe cells during freezing, and identify specific chemical and morphological changes inside the cell that result in life or death.
The cover image for the June 20 issue of the Biophysical Journal is an artistic rendering of frozen cells surrounded by extracellular ice and unfrozen solution. The background image is Lake Michigan in cold winter. Floating ice is separated by unfrozen water. The distribution pattern of the floating ice and unfrozen water is just like a frozen sample: ice crystals are separated by unfrozen concentrated solution. Schematic diagrams of frozen cells were imbedded in the background image to mimic a real frozen cell sample. In the diagram, the blue area represents unfrozen solution, the white area represents extracellular ice, the red line represents a region of cell membrane in close proximity to extracellular ice, and the black line represents a region of cell membrane in unfrozen solution between adjacent extracellular ice crystals. The schematic diagrams were precisely positioned in the background image such that the unfrozen solution in the diagram was co-located with unfrozen water in the background image and the extracellular ice crystals in the diagram were co-located with the floating ice in the background image. Our studies found that interactions between the cell membrane and extracellular ice resulted in intracellular ice formation (IIF), and increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF.
Raman spectroscopy has enhanced our understanding of freezing damage. These studies can enable the development of new and improved cell preservation protocols and eventually improve the growth of cellular therapies and our ability to treat patients.
– Guanglin Yu, Yan Rou Yap, Katie Pollock, Allison Hubel