Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions

Toxicity of glycerol for the stallion spermatozoa: Effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential

“…Glycerol may induce cellular damage through two distinct mechanisms, either an osmotic effect and/or a biochemically [19,20]. The permeability of the sperm membrane to glycerol will control the osmotic damage, and glycerol is considered less permeable than other cryoprotectants [33].…”

“…An ideal cryoprotector must penetrate the membrane, otherwise it will itself dehydrate the cell osmotically and stimulate freezing injury and, at the same time must be nontoxic at the concentrations necessary to prevent excessive dehydration. It is well accepted that permeating cryoprotectants do have toxic effects (as distinghised from osmotic effects) [19,20], noteworthy has been pointed out that while cryoprotectant toxicity is well present in vitrification research, there is probably less awareness of the relevance of cryoprotectant toxicity on the part of those that use freezing as their method of cryopreservation [11] Recent evidence [21] indicates that the main factor explaining cryoinjury in the equine species, is an osmotic imbalance at thawing. This fact stresses the importance of a rapid penetration at freezing and exit at thawing of the cryoprotective agent to minimize cryoinjury.…”

Preservation of Embryonic Stem Cells

“…However, cryopreservation of cells encapsulated in the large (≥ ~ 250 µm) microcapsules by slow-freezing has been challenging because the inevitable ice formation always results in significant damage to the microcapsules, which in turn can damage the encapsulated cells (Heng et al 2004;Herrler et al 2006;Stensvaag et al 2004;Wu et al 2007). Although the conventional vitrification approach can overcome this problem, the unusually high concentration CPA needed is detrimental to stress-sensitive cells such as the ES cells (Fahy et al 1987;Fahy et al 1984;Fahy et al 2004b;Rall and Fahy 1985;Wu et al 2007). By careful cryomicroscopy and scanning calorimetry studies, it was identified in a recent publication that water enclosed in ~100 µm (in diameter) alginate microcapsules can be preferentially vitrified over the bulk water (where the microcapsules are suspended) with only 1.4 M DMSO at a cooling rate of 100 o C/min (Zhang et al 2010).…”

“…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).…”

“…Although a low, non-toxic CPA concentration (usually ≤ ~ 1.5 M) is used in slow-freezing, it is always associated with mechanical and physicochemical injury to cells due to ice formation and slow-freezing (usually ≤ 1 o C/min) induced cell dehydration (Bischof 2000;Gao and Critser 2000;Mazur 1984;Toner 1993). The conventional vitrification approach diminishes ice formation altogether to a harmless level (Fahy et al 1987;Fahy et al 1984;Fahy et al 2004b;Rall and Fahy 1985;Wu et al 2007). The unusually high (as high as 7 M) concentration of CPA required, however, can result in significant metabolic and osmotic injury to cells (Chen et al 2000;Chen et al 2001a;Fahy et al 2004a;Fowler and Toner 2005;Heng et al 2005;Hunt et al 2006).…”