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

Abstract

Vitrification of aqueous cryoprotectant mixtures is essential in cryopreservation of proteins and other biological samples. We report 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. Measurements on fourteen common CPAs including alcohols (glycerol, methanol, isopropanol), sugars (sucrose, xylitol, dextrose, trehalose), PEGs (ethylene glycol, PEG 200, PEG 2 000, PEG 20 000), glycols (DMSO, MPD), and salt (NaCl) were performed for volumes ranging over four orders of magnitude from ∼ 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, we obtain the function of greatest intrinsic physical interest: 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.

Progress in rational methods of cryoprotection in macromolecular crystallography

“…Because the cryosolution properties may be modulated inside the solvent channels and the critical concentration for vitrification should be lower (Berejnov et al, 2006) than in the $1 ml samples used for the contraction measurements, solutions that did not vitrify during these measurements can still be effective penetrating cryoprotective agents. Their expected contractions can be estimated by calculating the polar surface area of the solution (e.g.…”

Critical Droplet Theory Explains the Glass Formability of Aqueous Solutions

“…Second, the ice nucleation rate peaks strongly near 200 K, but the growth rate there is small and decreases rapidly on further cooling [12][13][14][15][16]. Third, x-ray diffraction experiments suggest that the transition versus cooling rate or concentration between crystalline and amorphous samples is discontinuous [8]; if growth was limiting, one would expect a continuous transition as samples were trapped with various ensembles of growing ice clusters after cooling at different rates. Finally, it is known that the formation of a small ($ 20 # A) cubic ice cluster precedes conversion to and growth of a larger hexagonal ice cluster [4,17].…”

“…As the rate increases, a transition to transparent samples is observed. This optical transition corresponds to a transition in the x-ray diffraction patterns obtained from the cooled samples [8]. Clear samples show diffuse rings characteristic of a glassy state, whereas opaque samples show a sharp ring characteristic of a crystalline powder.…”

Preservation of Embryonic Stem Cells

“…3 should be able to enhance vitrification of living cells encapsulated in the microcapsules at high cooling rates (e.g., > 10,000 o C/min). This is because it can not only depress ice formation and growth in the microcapsule but also prevent ice (if any) propagation into cells from the bulk solution where ice is usually formed first (because of its much bigger volume) (Berejnov et al 2006;Fahy et al 1987;Franks et al 1983;He et al 2008b;Karlsson et al 1994;Mazur et al 2005a;Mazur et al 2005b;Toner 1993;Toner et al 1990;Yavin and Arav 2007). This hypothesis is confirmed by a recent study where the C3H10T1/2 mouse mesenchymal stem cells encapsulated in ~100 µm alginate microcapsules were vitrified using a 400 µm, thin-walled quartz microcapillary at a low-CPA concentration (1.4 M DMSO) (Zhang et al 2010).…”

“…Vitrification can be done without a specialized machine and the time required is much shorter than that for slow-freezing. (He et al 2008b)) A comparison of the conventional French-type straw (top) used today for cell vitrification at an unusually high CPA concentration and the 200 µm (outer diameter), thin-walled (10 µm) quartz microcapillary (QMC, bottom) used to achieve ultrafast cooling to minimize the CPA concentration required for vitrification Another way to improve cell vitrification is to confine cells in a small space such as submilimeter (in diameter) sized liquid droplets of aqueous cell suspension (Berejnov et al 2006;Edd et al 2008;Franks et al 1983). A major disadvantage of using small liquid droplets to confine cells is that the droplets will merge with each other unless they are dispersed in an oil phase, which makes it difficult to retrieve cells from the droplets.…”