We describe direct determination of the state of intracellular water, measurement of the intercellular concentration of a cryoprotectant agent (dimethylsulfoxide), and the distribution of organic material in frozen mammalian cells. Confocal Raman microspectroscopy was utilized at cryogenic temperatures with single live cells to conduct high spatial resolution measurements (350 × 350 × 700 nm), which yielded two, we believe, novel observations: 1), intracellular ice formation during fast cooling (50°C/min) causes more pronounced intracellular dehydration than slow cooling (1°C/min); and 2), intracellular dimethylsulfoxide concentration is lower (by as much as 50%) during fast cooling, decreasing the propensity for intracellular vitrification. These observations have a very significant impact for developing successful biopreservation protocols for cells used for therapeutic purposes and for cellular biofluids.
Bibliographical noteFunding Information:
Funding for this research was provided by University of Minnesota Biopreservation Core Resource (BioCoR), an entity funded by the Academic Health Center, College of Science and Engineering, Medical School, Medical Devices Center, and the Institute for Engineering in Medicine at the University of Minnesota. Partial funding for pilot experiments was supplied by the Biotransport Center at the University of Minnesota.