MEASUREMENT of WATER TRANSPORT DURING FREEZING USING DIFFERENTIAL SCANNING CALORIMETRY

A new technique using Differential Scanning Calorimeter (DSC) was developed to investigate water transport in whole tissue slices (1-5 mm^3,) and suspended cells during freezing. The tissue and cellular DSC data were correlated to water transport data by freeze substitution tissue microscopy and standard cellular cryomicroscopy techniques respectively. Sprague Dawley liver tissue and a (non-attached) lymphocyte (Epstein Bar Virus Transformed, EBVT) human cell system, were chosen as our tissue and cell model systems. The DSC was used to quantitatively monitor the heat released by water transported from the cell to the frozen vascular/ extracellular space in both systems at 5°C/min. Cryomicroscopy experiments verified that at a slow cooling rate of 5°C/min no intracellular ice formation (HF) occurred in either system. The sub-zero volumes of the tissue and cells were obtained as a function of temperature by both DSC and cryomicroscopy. By fitting a model of water transport for cells and tissues, dV/dt = f (Vj), B, T (t), Lp {Lpg, Ej^p )), to the DSC data for both systems, the following biophysical parameters were obtained. for rat liver tissue: Lpg =2.25 jlm/min-atm. Eip =75.76 kcal/mole. and for EBVT lymphocytes; Lpg =0.15 fim/mina!m, Ei^p=2^.7S kcal/mole. These results compare favorably to a recent study which found water transport parameters in whole liver tissue (Pazhayannur and Bischof , 1996) and to the single cell cryomicroscopy data we obtained in this study. The DSC technique is shown to be a fast and powerful method to obtain dynamic water transport information during cell and tissue freezing.