The measurement and prediction of ice formation in cells and tissues is of importance in biomedical applications such as cryosurgery and cryopreservation which are used to selectively destroy or preserve biological materials by freezing. This presentation will survey some of the techniques used in our lab to study the effects of freezing (beneficial and detrimental) on cells and tissues. Experimental and theoretical techniques allow the measurement and prediction of biophysical changes due to ice formation in a variety of single cells and recently also in whole tissues during freezing after seeding of the extracellular space. Cellular cryomicroscopy methods allow direct dynamic visualization of single spherical cells during freezing. During the protocol, changes in the 2-D projected cell area can be extrapolated to cell volumetric changes (water transport) while cytoplasmic changes, such as twitching or darkening, indicate intracellular ice formation. In contrast, obtaining quantitative dynamic experimental data on water transport and/or intracellular ice formation in whole tissue (and non-spherical cells) during freezing has been much more difficult due to the opaque nature of tissue slices. Several new techniques based on freeze substitution cryomicroscopy and DSC (differential scanning calorimetry) have recently been developed in our lab to directly measure the water transport in a variety of tissues including rat liver and rat Dunning AT-1 prostate tumors. Studies are also currently underway to examine the use of the DSC to obtain intracellular ice formation data in whole tissue. The prediction of the experimentally determined water transport and intracellular ice formation in both single cells and whole tissues is possible with the use of theoretical models. These models are based on empirical parameters (for a given system) as well as general driving forces under idealized conditions, i.e. osmotic pressure for water transport and supercooling for intracellular ice nucleation. The experimental data is used to determine an empirical set of biophysical parameters in the model (by curve fitting) which then yields predictive ability for the given cell or tissue system. Once the models have been verified for a given biological system, the ability to optimize the destruction or preservation of that system (cryosurgery or cryopreservation) becomes possible.
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