Damage to cells during freezing comes from a variety of mechanisms. We have established that ice formation inside of cells can be damaging when the fraction of the cell containing ice exceed a critical level. Larger ice crystals are more damaging than small ice crystals. Stabilization of cells during freezing results from both stabilizing critical structures in the cell (i.e. the cell membrane) as well as altering the freezing behavior of water.
Ongoing research in this area uses low temperature Raman spectroscopy to characterize with great chemical specificity and spatial resolution the response of single cells and tissue to the freezing environment. These techniques are being used to quantify the unique freezing characteristics of tissues and the manner by which those characteristics result in the greater sensitivity of tissues to freezing. Other techniques to characterize mechanisms of stabilization/damage include differential scanning calorimetry, multiphoton microscopy, and Fourier Transform Infrared (FTIR) Spectroscopy.
The addition of mechanistic preservation molecules, to target cell-specific mechanisms of damage, are being explored and compared to other cell types. This mechanism is distinct from biochemical/osmotic and freezing damage as it is dependent on the cell type and not easily predicted. Molecules, polymers, and other additives are being explored to improve preservation outcomes such as targeting surface protein stabilization, nuclei preservation, mitochondrial function, membrane stabilization, and more. Assays and high-throughput testing are being explored to identify the specific mechanism of all cryoprotective agents and additives.
Research into alternative methods for preservation including biostabilization and vitrification is also being explored in BioCoR and ATP-Bio. This includes methods for cryogenic, high-subzero, and room temperature storage.
A combination of Raman and differential scanning calorimetry (DSC) methodology
Cryopreservation of Human iPS Cell Aggregates in a DMSO-Free Solution—An Optimization and Comparative Study - Rui Li, Kathlyn Hornberger, James R. Dutton, and Allison Hubel