**4. Cryo-protecting agents**

The biophysical changes that take place during temperature depression, extracellular ice formation and the creation of a potentially lethal hyperosmotic environment leads to further dehydration of the cell. Termed "freeze dehydration" this effect is implicated in organelle disruption and loss, as well as fusion or changes in cell membranes [73]. At more depressed temperatures, the viscosity of the highly concentrated solution inside and outside of the cells remains as a glassy matrix, which is relatively stable for long-term preservation.

Additional cell damage may be caused by intracellular ice formation, which is more prominent during inappropriate rapid cooling as time might be insufficient for water to move down the chemical potential gradient established by the difference in solution concentrations between the two sides of the membrane. If a cell can be cooled to a 'glass region', under conditions inhibiting ice crystal formation, successful preservation can be achieved. This is termed vitrification as previously described.

While the methodologies of slow freeze and vitrification technologies may vary within clinics, the underlying principles are fundamentally the same. Combinations of reagents provide a delicate balance between the protective and toxic effects of CPAs aiming to maintain the functional capacity of organelles, while avoiding the two main causes of cell death associated with cryopreservation: solute toxicity [74] and ice formation [34].

CPAs are generally small molecular weight solutes with high aqueous solubility, bearing polar groups that interact weakly with water [75]. CPAs act to (i) moderate the effects of the rising solute (electrolyte) concentrations in the intra and extracellular environment, (ii) stabilize intracellular protein structure and (iii) provide increasing viscosity during temperature depression that may kinetically slow or inhibit ice crystal formation. There has been a myriad of solutes that exhibit some CPA activity: amino acids (e.g. alanine, glycine, proline), amides (e.g. acetamide, formamide), diols (e.g. 1,2-propanediol, ethanediol), sugars (glucose, lactose, ribose raffinose, dextrans, hydroxyethyl starch), large polymers (polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol) and alcohols (methanol) although some at low efficiencies [76, 77]. Modern cryopreservation protocols are largely based on few reported CPAs that are considered moderately or very effective in preserving nucleated cells [78].

CPAs can also be deleterious as osmotic effects resulting in too rapid excursion of water across a cell membrane can cause membrane rupture. Similarly, hydrogen bonding may disrupt the hydration shell around macromolecules. Chemical toxicity of high concentrations of CPAs is another cause for concern, largely in vitrification methods, the nature of which is not entirely understood [79, 80]. The suggested protein denaturation effect, even DNA conformational changes and fragmentation have been debated [80, 81]. Finally, CPAs have been shown to alter cytoskeletal components in mammalian oocytes, particularly filaments and the meiotic spindle [82]. The reversibility of this disruption is concentration and CPA dependent and varies amongst species [54, 83–85].CPA reagents are classified as permeating, non-permeating and stabilizing.

may implore the few labs resistant to convert to vitrification technologies to reconsider; albeit

The biophysical changes that take place during temperature depression, extracellular ice formation and the creation of a potentially lethal hyperosmotic environment leads to further dehydration of the cell. Termed "freeze dehydration" this effect is implicated in organelle disruption and loss, as well as fusion or changes in cell membranes [73]. At more depressed temperatures, the viscosity of the highly concentrated solution inside and outside of the cells

Additional cell damage may be caused by intracellular ice formation, which is more prominent during inappropriate rapid cooling as time might be insufficient for water to move down the chemical potential gradient established by the difference in solution concentrations between the two sides of the membrane. If a cell can be cooled to a 'glass region', under conditions inhibiting ice crystal formation, successful preservation can be achieved. This is termed

While the methodologies of slow freeze and vitrification technologies may vary within clinics, the underlying principles are fundamentally the same. Combinations of reagents provide a delicate balance between the protective and toxic effects of CPAs aiming to maintain the functional capacity of organelles, while avoiding the two main causes of cell death associated

CPAs are generally small molecular weight solutes with high aqueous solubility, bearing polar groups that interact weakly with water [75]. CPAs act to (i) moderate the effects of the rising solute (electrolyte) concentrations in the intra and extracellular environment, (ii) stabilize intracellular protein structure and (iii) provide increasing viscosity during temperature depression that may kinetically slow or inhibit ice crystal formation. There has been a myriad of solutes that exhibit some CPA activity: amino acids (e.g. alanine, glycine, proline), amides (e.g. acetamide, formamide), diols (e.g. 1,2-propanediol, ethanediol), sugars (glucose, lactose, ribose raffinose, dextrans, hydroxyethyl starch), large polymers (polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol) and alcohols (methanol) although some at low efficiencies [76, 77]. Modern cryopreservation protocols are largely based on few reported CPAs that are

CPAs can also be deleterious as osmotic effects resulting in too rapid excursion of water across a cell membrane can cause membrane rupture. Similarly, hydrogen bonding may disrupt the hydration shell around macromolecules. Chemical toxicity of high concentrations of CPAs is another cause for concern, largely in vitrification methods, the nature of which is not entirely understood [79, 80]. The suggested protein denaturation effect, even DNA conformational changes and fragmentation have been debated [80, 81]. Finally, CPAs have been shown to alter cytoskeletal components in mammalian oocytes, particularly filaments and the meiotic spindle

remains as a glassy matrix, which is relatively stable for long-term preservation.

with cryopreservation: solute toxicity [74] and ice formation [34].

considered moderately or very effective in preserving nucleated cells [78].

cost considerations excluded.

146 Cryopreservation in Eukaryotes

**4. Cryo-protecting agents**

vitrification as previously described.

Permeating cryoprotectants (e.g., glycerol, propane diol, dimethyl sulphoxide or ethylene glycol) cross the cell membrane through an osmotic gradient displacing water. These agents act to reduce ice crystallization and reduce cell dehydration but are toxic at higher concentrations. More specifically, in vitrification, the role is to completely inhibit ice formation. Permeating cryoprotectants also stabilize intracellular solutes which otherwise would be lethal in a hyperosmotic state. A similar dehydration effect is mimicked in the extracellular environment with temperature reduction promoting further dehydration. Dehydration is dependent upon the rate of temperature depression and limited by the cell permeability to water [35, 86].

Non-permeating cryoprotectants are generally higher molecular weight polymers (e.g., sucrose, polyethylene glycol, polyvinylpyrrolidone, ficoll, dextran). These agents mimic the dehydration mechanism of penetrating cryoprotectants but remain outside of the cell.

Generally, less toxic than penetrating cryoprotectants at the same concentration a successful vitrification strategy is to create a mixture of non-toxic level of permeating cryoprotectant(s) by the addition of non-permeating cryoprotectant. Interestingly as toxic effects of permeating cryoprotectants have been shown to be at least somewhat biochemical and unique in action, total molarity of a mixture may not be a reliable indicator of cryosolution embryotoxicity [53].

For application, CPAs are contained within a "carrier" solution that will help keep cells alive during cryopreservation. They act to provide osmotic and physiological support and avoid deviations from isotonicity, which could result in dehydration or swelling and burst of cells. It is important to note that the efficacies of carrier solutions are unpredictable and vary based on the individual or mixture of CPAs present [19].

Concern of long-term putative effects of these chemicals has paved the way for investigation into and application of extracted or modified natural biological agents, which are evolutionarily found in extreme environments. While conventional cryoprotectants interact with water, the application of uniquely acting, naturally based complementary agents, is an attractive proposition.

Biological anti-freeze molecules of sorts (e.g., cyclohexanediol and polyvinyl alcohol) [87, 88] selectively adsorb to the surface of ice crystals inhibiting ice crystal growth and ice recrystallization. Ice blockers, including polyvinyl alcohol and polyglycerol (i.e., X-1000 and Z-1000) [89], specific to vitrification solutions act to prevent ice crystal formation. Together, these agents may also play a role in potentially damaging re-crystallization of ice growth during warming [90].

Ice-nucleating agents act to achieve deliberate ice growth in defined sites. This phenomenon is largely important in intact tissues and organs where integrated cell cooperation is essential to normal function. Unlike small tissue sections, organs are unable to effectively absorb cryoprotectant solution by simply soaking in a solution. In the case of whole organs, introduction of cryoprotectants by perfusion (through existing vasculature) is necessary. As perfusion, due to capillary distribution and time requirement of CPA diffusion, may not be equivalent throughout a larger structure, random ice crystal growth can be lethal simply by mechanical disruption. By achieving deliberate ice growth in specific sites, the damaging effects of super-cooling and likewise intracellular ice formation can be mediated and potentially avoided.

Lastly as organisms synthesize solutes and metabolites in response to cold survival strategies (e.g., trehalose [91], glycerol [92], polyols [93, 94]), understanding how biological structures interact with these mixtures may offer added benefits to current freezing regimes.
