Intracellular Ice Formation during the Freezing of Hepatocytes Cultured in a Double Collagen Gel

Abstract

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 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees 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 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) 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.

Transport phenomena during freezing of isolated hepatocytes

“…On the other hand, for cooling rates faster than 100"C/min, hepatocytes do not have enough time to sufficiently dehydrate and the remaining intracellular water reaches thermodynamic equilibrium with the external partially frozen solution by a phase change from the liquid to the crystalline state. The existence of a cell-specific transition zone between slow and high cooling rates is consistent with observations from other cell types (Mazur, 1984;Hubel et al, 1991;Yarmush et al, 1991). For example, the transition zone for mouse oocytes has been determined to be between 0 and 1 "C/min both from the theory and experiments Toner et al, 1992).…”

“…This insensitivity of 50Tl,, to the cooling rate has been shown for many other cell types in the absence of cryoprotective agents (Morris and McGrath, 1981;Callow and McGrath, 1985;Scheiwe and Korber, 1987;Shabana and McGrath, 1988;Muldrew and McGann, 1990;Toner et al, 1992;Pitt et al, 1991;Hubel et al, 1991). Although the cooling rate dependence of 5oT[IF is negligible, the theoretical and experimental observations show a noticeable depression which occurs at -108"C/min (at the transition zone when the cumulative incidence of IIF increases from 0 to 100CVo).…”

Cryopreservation in micro‐volumes: Impact upon caco‐2 colon adenocarcinoma cell proliferation and differentiation

“…Cryopreservation must not reduce a cell's ability to differentiate. Several reports describe alternative cryopreservation technologies that have been tested for different cell-types intended for specific applications (Hornung et al, 1996;Hubel et al, 1991;Ji et al, 2004;Mahler et al, 2003).…”

Cryopreservation of Hepatocytes for Bioartificial Liver Devices

“…This suspension is then frozen at a freezing rate of the order of -0.3ºC/min to -20ºC/min. Faster cooling rates, -50ºC/min, invariably lead to IIF damage (Harris et al, 1991;Hubel et al, 1991), which causes reduced viability and increased morphological abnormalities such as blebbing (Griffiths et al, 1979). So, plunging suspensions directly in liquid nitrogen is not an option.…”

“…The pioneering work on hepatocytes cultured between two layers of collagen gel (sandwich system) was carried out by Dunn and co-workers (Dunn et al, 1989;. Their work showed that the collagen gel sandwich culture system supported the secretion of albumin, transferrin, fibrinogen, bile acids and urea for at least six weeks, and (Borel Rinkes et al, 1992a;Hubel et al, 1991;Koebe et al, 1990). The method was developed for freezing rat hepatocytes and showed a survival rate (in terms of morphology) on thawing of only 30%, signs of severe cytoplasmic damage in all cells, and a decrease in albumin secretion down to 20% of control cultures.…”