Subzero osmotic characteristics of intact and disaggregated hepatocyte spheroids

This study has been conducted to examine basic transport characteristics of pig hepatocytes cultured as spheroids for use in a bioartificial liver. Static osmotic experiments were conducted by subjecting hepatocyte spheroids in solutions of increasing sucrose concentrations. A Boyle-van't Hoff plot was used to extrapolate an osmotically inactive volume, V(b) of 0.60, which is unusually high and might not represent the inactive volume of the individual cells. The spheroids were disaggregated and low-temperature cryomicroscopy experiments performed to examine the transport and intracellular ice formation (IIF) characteristics. A hydraulic permeability, L(pg), of 7.6 x 10¹⁵ m³/Ns and an activation energy, E(lp), of 82 kJ/mol was determined for the individual cells. The kinetic (Ω(o)) and thermodynamic (K(o)) coefficients for IIF were determined to be 5.9 x 10⁸ m^-2 s^-1 and 3.0 x 10⁹ K⁵, respectively. These results infer a decrease in the temperature range over which IIF is observed compared to freshly isolated pig hepatocytes. The technique of freeze substitution was used to examine the structure inside the spheroid during freezing. At a low cooling rate of 1°C/min, increasing amounts of intercellular ice formed between the cells. At a higher cooling rate of 100°C/min small intracellular ice crystals formed. This study shows the location of ice in a freezing hepatocyte spheroid and confirms that the cells cultured as spheroids do not transport water in the same manner as isolated cells.