By Willem F. Wolkers, Harriëtte Oldenhof
This quantity presents numerous common protocols used to cryopreserve or freeze-dry kinds of specimens. moreover, it presents chapters all in favour of the basic ideas of cryopreservation, vitrification, and freeze-drying. numerous state-of-the-art microscopic, spectroscopic in addition to calorimetric tools are highlighted that may be used to check mobile and macromolecular alterations in line with freezing or drying. Written within the hugely winning Methods in Molecular Biology sequence structure, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and tips about troubleshooting and keeping off recognized pitfalls.
Authoritative and functional, Cryopreservation and Freeze-Drying Protocols, 3rd Edition serves as a pragmatic guide for reports on freezing and drying strategies in addition to maintenance recommendations for organic specimens.
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Extra info for Cryopreservation and Freeze-Drying Protocols
Ice nucleation events will essentially be confined to discrete locations where heterogeneous nucleators are present. A third form of vitrification is stable vitrification, or “equilibrium vitrification” , which uses solute concentrations so high that ice cannot exist in the solution [2, 83]. A practical approximation to the edge of the stable vitrification zone can be defined as the concentration that is sufficient to make devitrification vanish during slow warming [18, 83], but complete stability requires the higher concentrations that are typical of those that preclude ice growth during previous slow freezing (the “unfreezable concentration,” or CU [15, 72]).
In accordance with the Gibbs-Thomson equation , ice crystals of small size have low melting temperatures (high vapor pressure) caused by sharp curvature of the crystal surface. Consequently, newly formed ice nuclei tend to melt at any temperatures warmer than about −38 °C in pure water. This defines the homogeneous nucleation temperature (Th), the lowest temperature to which small [30, 170] samples can be cooled under normal conditions without ice formation and the highest temperature at which small samples are likely to form ice when preexisting ice crystals or contaminants that mimic ice crystals (heterogeneous nucleators, discussed below) are absent [30, 70, 170–173].
Heterogeneous nucleators are particles or surfaces that mimic the structure of ice on a molecular scale or otherwise induce water to assume a more icelike configuration with a larger radius of curvature. Ice crystals with a larger radius of curvature upon their initial formation have a lower surface energy, allowing them to avoid melting at warmer temperatures . The most potent heterogeneous nucleators can cause ice to form at temperatures only 1 °C below Tm [175, 176]. Heterogeneous nucleators are ubiquitous environmental contaminants [168, 174].