During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me₂SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B₅₀) was 70°C/min for cells frozen after 1 day in culture and decreased to 15°C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me₂SO + DMEM solution, the value of B₅₀ decreased from 70 to 50°C/min for cells in culture for 1 day and from 15 to 10°C/min for cells in culture for 7 days. The value of the average temperature for IIF (Tuf) for cultured cells was only slightly depressed by the addition of Me₂SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me₂SO on IIF characteristics implies that lower concentrations of Me₂SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B₅₀ measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.

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