Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions

Vitrification of aqueous cryoprotectant mixtures is essential in cryopreservation of proteins and other biological samples. Systematic measurements of critical cryoprotective agent (CPA) concentrations required for vitrification during plunge-cooling from T = 295 K to T = 77 K in liquid nitrogen are reported. Measurements on fourteen common CPAs, including alcohols (glycerol, methanol, 2-propanol), sugars (sucrose, xylitol, dextrose, trehalose), polyethylene glycols (ethylene glycol, PEG 200, PEG 2000, PEG 20000), glycols [dimethyl sulfoxide (DMSO), 2-methyl-2,4-pentanediol (MPD)], and salt (NaCl), were performed for volumes ranging over four orders of magnitude from ∼1 nl to 20 μl, and covering the range of interest in protein crystallography. X-ray diffraction measurements on aqueous glycerol mixtures confirm that the polycrystalline-to-vitreous transition occurs within a span of less than 2% w/v in CPA concentration, and that the form of polycrystalline ice (hexagonal or cubic) depends on CPA concentration and cooling rate. For most of the studied cryoprotectants, the critical concentration decreases strongly with volume in the range from ∼5 μl to ∼0.1 μl, typically by a factor of two. By combining measurements of the critical concentration versus volume with cooling time versus volume, the function of greatest intrinsic physical interest is obtained: the critical CPA concentration versus cooling rate during flash-cooling. These results provide a basis for more rational design of cryoprotective protocols, and should yield insight into the physics of glass formation in aqueous mixtures.