As part of our attempt to develop a hybrid artificial liver support system using cultured hepatocytes, we investigated the long-term metabolic function of hepatocytes incubated in a packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Long-term (up to 1 week) perfusion culture experiments using the packed-bed reactor (20 mm i.d.) loaded with 500 PVF resin cubes (mean pore size 250 mum, 2 x 2 x 2 mm), together with conventional monolayer culture experiments as controls, were performed in serum-free or serum-containing medium. Ammonium metabolism and urea synthesis activities were evaluated quantitatively based on reaction kinetic analyses. Initial rates of ammonium metabolism and urea-N synthesis, as well as GPT enzyme activities, were adopted as indexes of the metabolic performance of the reactor and activities of the cultured hepatocytes.When serum-free medium was used in the perfusion cultures, ammonium metabolic and urea-N synthetic rates showed significant decay with elapse of the culture period, being less than 10% of those measured on day 1. This loss of activity was more prominent in the perfusion culture than in the monolayer cultures using this medium. In contrast, when serum-containing medium was used, approximately 50% of these activities obtained on day 1 were maintained even at the end of the cultures both in the perfusion and monolayer culture experiments.We concluded that the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes, and showed a satisfactory ability to maintain the metabolic function of immobilized hepatocytes for relatively long periods of up to 1 week. This type of reactor is thus considered to represent a breakthrough in overcoming the difficulties involved in the development of a hybridtype artificial liver support system. (c) 1994 John Wiley & Sons, Inc.

No abstract

To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2 x 2 x 2 mm, mean pore size: 100, 250 or 500 microns) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6-11.3 x 10(7) hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2 x 10(7) cells/cm3 PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes.

Our results suggest that local tissue cooling, similar to cryotherapy, improves edema and inflammatory reaction, and may be useful for reducing inflammatory response without inhibiting blood flow after contusion.

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.