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. St atic 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 indiv idual cells. The spheroids were disaggregated and low-temperature cryomicro scopy experiments performed to examine the transport and intracellular ice formation (IIF) characteristics. A hydraulic permeability, L-pg, of 7.6 x 1 0(15) m(3)/Ns and an activation energy, E-tp, of 82 kJ/mol was determined f or the individual cells. The kinetic (Omega(o)) and thermodynamic (kappa(o) ) coefficients for IIF were determined to be 5.9 x 10(8) m(-2) s(-1) and 3. 0 x 10(9) K-5, respectively. These results infer a decrease in the temperat ure range over which IIF is observed compared to freshly isolated pig hepat ocytes. The technique of freeze substitution was used to examine the struct ure inside the spheroid during freezing. At a low cooling rate of 1 degrees C/min, increasing amounts of intercellular ice formed between the cells. A t a higher cooling rate of 100 degrees C/min small intracellular ice crysta ls formed. This study shows the location of ice in a freezing hepatocyte sp heroid and confirms that the cells cultured as spheroids do not transport w ater in the same manner as isolated cells, (C) 1999 Academic Press.