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As a preliminary investigation to establish a cryopreservation method suited for bioartificial livers (BALs), three-dimensional (3-D) cryopreservation experiments with fibroblasts were performed, in which the cells were firstly seeded into a porous scaffold, and the scaffold containing the cells was then cryopreserved. After thawing, 65% of the initially applied cells were still attached to the scaffold, and this efficiency was significantly higher than that in the control experiments (39%), in which fibroblasts cryopreserved in a suspension were seeded into the scaffold. This higher efficiency was mainly caused by higher immobilization efficiency at the time of cell seeding (83%) than in the controls (54%). Collagen coating of the scaffold in the 3-D cryopreservation enhanced immobilization efficiency at the time of cell seeding, and 1-day precultures before the 3-D cryopreservation considerably improved cell growth after thawing. From these favorable results, this 3-D cryopreservation method may become useful for developing BALs.

In an attempt to develop a bioartificial liver using cultured hepatocytes, we investigated the short-term and long-term viability and metabolic functions of hepatocytes cultured in a new type of packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Perfusion culture experiments using this reactor, as well as monolayer cultures using conventional collagen-coated Petri dishes as control experiments, were performed. It was found that the highest density of immobilized hepatocytes attained with PVF resin was on the order of 10(7) cells/cm3 PVF and that hepatocytes cultured in this type of module for up to a week showed a sufficient level of liver-specific metabolic functions, such as ammonium metabolism, urea-N synthesis, and albumin secretion, to be comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising means to develop a bioartificial organ using hepatocytes.

Bubble blood oxygenators equipped with baffles of various types in the oxygenating column were studied. The rate of hemolysis, the volumetric coefficient for oxygen absorption into blood, and the fractional gas holdup were found to be affected mainly by the superficial gas velocity. When compared with the conventional bubble blood oxygenator without baffles, the bubble oxygenators equipped with various types of baffles (i.e., horizontal perforated baffles, radial vertical baffles, and a concentric hollow cylinder with and without horizontal perforated baffles) showed less hemolysis, larger gas holdup and higher values of the coefficient for oxygen absorption. Values of the heat transfer coefficient on the surface of the cylindrical baffle, which is useful as a built-in heat exchanger, were several times greater than those for single-phase heat transfer in conventional blood heat exchangers.

As a preliminary investigation to establish a cryopreservation method suited for bioartificial livers (BALs), three-dimensional (3-D) cryopreservation experiments with fibroblasts were performed, in which the cells were firstly seeded into a porous scaffold, and the scaffold containing the cells was then cryopreserved. After thawing, 65% of the initially applied cells were still attached to the scaffold, and this efficiency was significantly higher than that in the control experiments (39%), in which fibroblasts cryopreserved in a suspension were seeded into the scaffold. This higher efficiency was mainly caused by higher immobilization efficiency at the time of cell seeding (83%) than in the controls (54%). Collagen coating of the scaffold in the 3-D cryopreservation enhanced immobilization efficiency at the time of cell seeding, and 1-day precultures before the 3-D cryopreservation considerably improved cell growth after thawing. From these favorable results, this 3-D cryopreservation method may become useful for developing BALs.

Maintaining metabolic functions of cultured hepatocytes at higher levels is an essential requirement for the development of a bioartificial liver. We investigated the effect of oxygen tension (10–40%) of the medium on immobilization efficiency and metabolic functions of cultured hepatocytes obtained from a rat for up to 4 days. Immobilization efficiencies of cultures in 10% oxygen showed a significantly lower value from those for the other conditions. The ammonium metabolic rate and the albumin secretion rate were significantly improved with an increase of dissolved oxygen tension for up to 2 days. These values remained similar in the later stage of the culture. The urea secretion rate showed similar values in all conditions. In conclusion, higher oxygen tension improved immobilization efficiency and metabolic functions of cultured rat hepatocytes in the earlier stage of culture for up to 2 days.

A packed-bed reactor utilizing the porous polymer, polyvinyl formal (PVF) resin, as a supporting material was applied for the long-term culture of rat hepatocytes. Perfusion cultures using this reactor, as well as monolayer cultures, were performed for up to 1 week under 3 different media conditions. When serum-free or epidermal growth factor-containing medium was used, albumin secretion rates showed constant decreases in both the perfusion and the monolayer cultures, and approximately 10% of the activity exhibited on Day 1 was preserved at the end of the cultures. In contrast, the hepatocytes supplemented with serum-containing medium exhibited stable ability of albumin secretion throughout the culture period under the perfusion culture condition using PVF resin. From the microscopic observation, the immobilized hepatocytes in this medium revealed round shapes, and a cluster of cell aggregates was scarcely seen.