Solute effects during freezing

Cryoinjury to the specimen is caused directly by extra- or intracellular ice crystal formation as well as by ice-induced solution effects during cryopreservation. Ice crystals seriously deform cell components. Another disadvantage of the formation of ice crystals near the specimen surface is slowing the cooling rate in areas below the surface because their thermal conductivity is about half that of solid water in a noncrystalline state. Furthermore, ice crystal formation is accompanied by the generation of latent heat, which also slows down the freezing rate. 

Although one cannot always expect protein aggregation to be eliminated as effectively by addition of surfactant as demonstrated in the above examples, it is certainly prudent to test the effect of surfactants in situations where protein aggregation is a problem during freeze-thaw or during solution handling. 

As many excipients are capable of stabilizing proteins under different conditions, it is unlikely that the stabilization effect is excipient-specific. It is widely believed that the stabilizing mechanism of excipients involves the preferential exclusion of the added excipient from the protein in aqueous solution and during freeze-thawing [192]. Briefly, a protein in an aqueous environment is in dynamic equilibrium between the native and the denatured (unfolded) forms. As the exclusion of excipient from the unfolded form is greater than that from the native form, the equilibrium will shift toward increasing concentration of the native form. Thus, the unfolding of protein in the presence of excipient is a thermodynamically... 

De Antoni et al. [1.23] demonstrated, that the addition of trehalose during freezing and thawing of two strains of Lactobacillus bulgaricus improved the survival rate differentially, but in both cases considerably. The samples (1 mL) were frozen at 18 °C/min to -60 °C and thawed to 37 °C at 15 °C/min. The solution consisted of distilled water, culture medium and 10 % milk with or without trehalose. It was shown, that after three freezingthawing cycles, milk alone resulted in a survival rate of 24 % or 65 %, while with trehalose this was can be improved to 32 % and 100 % respectively. The efficacy in the case of both strains was clearly different. De Antoni et al. suggested, that the efficiency of milk was related to its Ca2+ content, while the trehalose could replace water molecules in the phospholipids of the membranes. However no mention was made wether other sugar molecules in milk showed any effect. 

If a firewater line needs to be temporarily isolated and an isolation means is not available on the immediate portion of the system needing work, a unique solution is to use a liquid nitrogen low temperature coil line freezing apparatus. This mechanism causes an ice plug to form in the line, effectively sealing the line from leakage, during the period the temporary isolation is needed. 

A stabilising effect in the presence of salt was also noted by Aronson and Petko [90]. Addition of various electrolytes was shown to lower the interfacial tension of the system. Thus, there was increased adsorption of emulsifier at oil/water interface and an increased resistance to coalescence. Salt addition also increased HIPE stability during freeze-thaw cycles. Film rupture, due to expansion of the water droplets on freezing, did not occur when aqueous solutions of various electrolytes were used. The salt reduced the rate of ice formation and caused a small amount of aqueous solution to remain unfrozen. The dispersed phase droplets could therefore deform gradually, allowing expansion of the oil films to avoid rupture [114]. 

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