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To develop a tissue-engineered bioartificial liver (BAL), perfusion cultures of mouse and pig fetal liver cells (FLCs) immobilized within a three-dimensional (3D) porous scaffold were performed utilizing a packed-bed reactor system. These FLCs were cultured under different medium flow rate conditions, and the effects of the flow rates on cell growth and the hepatic functions of the FLCs were investigated. In the cultures of mouse FLCs, the medium flow suppressed cell growth and the albumin secretion activity of the FLCs, and considerably lower albumin secretion than that in the 3D stationary control cultures was obtained in the perfusion cultures. In the case of pig FLCs, cell growth was also inhibited by the medium flow, however, the cells exhibited higher tolerance to the medium flow compared with mouse FLCs. The albumin secretion activity of pig FLCs was well maintained under an extremely low flow rate condition (4.8 mm/min in the reactor), and activity higher than the 3D stationary cultures was detected at a later stage (after 20 days in the perfusion cultures). These results revealed that FLCs are quite sensitive to medium flow and an extremely low shear stress is required for the perfusion cultures of FLCs., (Copyright 2010 Elsevier B.V. All rights reserved.)

For the purpose of applying fetal liver cells (FLCs) as a cell source to tissue-engineered bioartificial livers, three-dimensional (3-D) cultures of FLCs using a porous polymer scaffold, as well as monolayer cultures as a control, were simultaneously performed. To achieve efficient growth and differentiation, the FLCs were cultured in the growth medium for the first 3 weeks and then cultured in the differentiation medium for 3 more weeks. In these cultures, stimulating factors (oncostatin M (OSM), epidermal growth factor (EGF), hepatocyte growth factor (HGF), or dimethyl sulfoxide (DMSO)) were added to the media, and their effects were examined. When the growth medium containing OSM and EGF was used, EGF stimulated the growth of FLCs synergistically with OSM. For the differentiation of FLCs into mature hepatocytes, DMSO added to the differentiation medium remarkably enhanced albumin secretion in the 3-D and monolayer cultures, although HGF was effective only in the monolayer culture. Microscopic observation proved that FLCs exhibited hepatocyte-like morphology only in the media containing DMSO. In conclusion, successive supply of the growth medium containing EGF and OSM and the differentiation medium containing DMSO efficiently induced the growth of the 3-D cultured FLCs and their differentiation into mature hepatocytes.

In order to develop a tissue engineered bioartificial liver (BAL), long-term three-dimensional (3-D) culture of fetal liver cells (FLCs) utilizing porous polymer as a scaffold was performed for up to 1 month. The effects of the basal medium and supplementation with oncostatin M (OSM) on the proliferation and differentiation of mouse FLCs were examined in both 3-D culture and conventional monolayer dish culture. Compared with monolayer culture, cell numbers and hepatic function of FLCs were better maintained by 3-D culture. When two kinds of basal media were tested in this study, Williams' medium E (WE) was superior to minimum essential medium alpha (alphaMEM) in expressing hepatic function of FLCs in both 3-D and monolayer cultures, although higher cell densities were obtained with alphaMEM. OSM potently stimulated both cell growth and metabolic activity, especially in 3-D culture. When WE supplemented with OSM was used for 3-D culture, albumin secretion by FLCs increased dramatically after day 5, and a high level of secretion was maintained until the end of culture. During a period of over 1 month, no decrease of albumin secretion was observed. In conclusion, this 3-D culture method was expected to be one of the realistic attempts to develop a tissue engineered BAL., (2004 Wiley-Liss, Inc.)

A packed-bed reactor using reticulated polyvinyl formal (PVF) resin as a support material is a useful configuration to achieve high density culture of hepatocytes for use as a bioartificial liver. The authors investigated the effects of oxygen concentrations of the culture medium on the metabolic performance of hepatocytes cultured in the reactor. A packed-bed reactor loaded with 250 PVF resin cubes (2 x 2 x 2 mm) was used. Hepatocytes obtained from male Wistar rats were inoculated into the reactor. Culture medium was perfused from the reservoir into the reactor through an oxygenator using a roller pump. Concentration of the dissolved oxygen in the medium was controlled by changing the gas mixture ratio supplied to the oxygenator. Hepatocytes cultured in the packed-bed reactor (cell density: 8.6 x 10(6) cells/cm3 PVF) under conditions of high dissolved oxygen concentrations ranging from 260 to 460 micromol/L showed 30% higher ammonium metabolic activity and 85% higher albumin secretion activity compared with those from the monolayer culture in the earlier culture stage (up to 2 days). However, low oxygen concentrations in the medium (<100 micromol/L) impaired activities of cultured hepatocytes.

To improve the culture conditions of hepatocytes for use as a bioartificial liver, the effects of shear flow on the co-culture system of hepatocytes/nonparenchymal cells (NPC) were investigated. A flow chamber with a collagen coated rectangular glass plate, where hepatocytes (5 x 10(4) cell/cm2) and NPC (2 x 10(5) cell/cm2) were seeded, was used to attain a shear stress of 4.7 dyne/cm2. Concentrations of ammonia and urea in the medium were measured daily during the 2 week experiment. The metabolic activity of hepatocytes in the homotypic culture were lower than those of the co-culture, especially when the cultivation time exceeded 1 week. In addition, the applied shear flow promoted activity of the co-culture system. An enhancement in the rates of ammonium removal and urea synthesis was obtained in the perfusion systems. Morphologic observation revealed that aggregates of hepatocytes formed abundantly in the perfusion system and hepatocytes developed a cuboid shape. This suggested that perfusion affected the function and morphology of hepatocytes in the co-culture system. Shear flow could induce cell-cell interactions and secretion of extracellular matrix through the activation of NPC.

To investigate the effects of culture conditions on the maintenance of metabolic functions of cultured hepatocytes, long-term hepatocyte culture lasting 20 days was performed under two different culture conditions, i.e. stationary cultures utilizing porous polymer (polyvinyl formal (PVF) resin) as a substratum and conventional monolayer dish cultures without PVF. Metabolic activities specific to hepatocytes were evaluated in terms of ammonia metabolism, urea synthesis, and albumin secretion. Concerning ammonia metabolic and urea synthetic activities, no significant differences in maintenance of these activities were found between the two culture conditions, and these activities rapidly decreased with the elapse of the culture period, especially during the early stage of the experiments. However, after day 10, these activities in the stationary cultures were maintained at a slightly more favorable level than in the monolayer cultures. On the other hand, compared with ammonia metabolism and urea synthesis, stable and well-maintained albumin secretion of hepatocytes (60% of the activity in day 1) was exhibited in the stationary culture experiments, despite that this particular activity under the monolayer culture condition gradually reduced to a very low level (5.7% of that on day 1) at the end of the culture. From the morphological observations, hepatocytes immobilized in the PVF resin revealed individual spherical shapes without forming multicellular aggregation, and it was suggested that this characteristic structure contributed to good albumin secretion of hepatocytes. In conclusion, the advantages of the hepatocyte culture technique utilizing PVF resin over the conventional dish culture in maintaining some representative metabolic function specific to hepatocytes were clarified.

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.

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.

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.

To enable high density culture of hepatocytes to be used as a hybrid artificial liver support or bioreactor system, collagen coated reticulated polyvinyl formal (PVF) resin (a filter material with a porosity of more than 80%) was used for the primary culture of hepatocytes. Stationary and perfusion culture experiments using PVF resin and monolayer culture were performed as control experiments. Due to the porous structure of the substrate material, hepatocytes were able to penetrate the reticulated pores of the PVF resin, and adhere to and spread on its surface. The densities of hepatocytes attained with PVF were about 10 times as high as those in the monolayer culture using conventional collagen coated Petri dishes. Hepatocytes immobilized in the PVF resin showed viability, as assessed by glutamic pyruvic transaminase (GPT) activity in the medium. Perfusion culture with PVF resin showed stable, high level metabolism, ammonium removal, and urea secretion comparable to the monolayer culture. It is concluded that perfusion culture with PVF resin is useful in attaining a high density culture of hepatocytes.

To develop a hybrid artificial liver support system (ALSS), the authors constructed a rotating-disk type ALSS using hepatocytes entrapped within a calcium alginate hydrogel. This module was designed in imitation of the gas-liquid contactor that uses the same principle. Forty disks with film-shaped hydrogel were mounted to a horizontal rotating axis and were kept in contact with blood. The concentration of hepatocytes in the gel varied from 1.9 X 10(9) to 1.7 X 10(10) cells/L gel. Entrapping hepatocytes within hydrogel kept the hepatocytes viable, and results of in vitro experiments showed ammonium metabolism and urea synthesis. The results of ex vivo perfusion experiments using cats with acute hepatic insufficiency indicated that this module had the ability to replace liver function in vivo.