The Experts below are selected from a list of 219 Experts worldwide ranked by ideXlab platform
Norio Ohshima - One of the best experts on this subject based on the ideXlab platform.
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Perfusion of medium with supplemented growth factors changes metabolic activities and cell morphology of hepatocyte-nonparenchymal cell coculture.
Tissue Engineering, 2004Co-Authors: Pei Kan, Hirotoshi Miyoshi, Norio OhshimaAbstract:To develop a feasible perfusion-type Bioartificial Liver Device, perfusion of hepatocyte–nonparenchymal cell (NPC) cocultures with medium supplemented with hepatocyte growth factor (HGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) was carried out. On day 1 of culture, perfusion at a constant shear stress of 1.3 dyn/cm2 enhanced ammonia metabolic and urea synthetic activities of hepatocytes. These enhanced activities were sustained up to day 7 only when growth factors were present. In contrast, no beneficial effects of growth factors on these activities were observed in static cultures. In perfusion cultures, three-dimensional cell aggregates were formed. On the surface of these aggregates, flattened cell layers composed mainly of NPCs were found, and the central cluster of cell aggregates was composed of round-shaped hepatocytes and reticulin fibrils. These observations strongly suggested that the reconstruction of different types of Liver cells and connective tissues formed ti...
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Perfusion of medium with supplemented growth factors changes metabolic activities and cell morphology of hepatocyte-nonparenchymal cell coculture.
Tissue engineering, 2004Co-Authors: Pei Kan, Hirotoshi Miyoshi, Norio OhshimaAbstract:To develop a feasible perfusion-type Bioartificial Liver Device, perfusion of hepatocyte-nonparenchymal cell (NPC) cocultures with medium supplemented with hepatocyte growth factor (HGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) was carried out. On day 1 of culture, perfusion at a constant shear stress of 1.3 dyn/cm2 enhanced ammonia metabolic and urea synthetic activities of hepatocytes. These enhanced activities were sustained up to day 7 only when growth factors were present. In contrast, no beneficial effects of growth factors on these activities were observed in static cultures. In perfusion cultures, three-dimensional cell aggregates were formed. On the surface of these aggregates, flattened cell layers composed mainly of NPCs were found, and the central cluster of cell aggregates was composed of round-shaped hepatocytes and reticulin fibrils. These observations strongly suggested that the reconstruction of different types of Liver cells and connective tissues formed tissue-mimicking cell aggregates in the perfusion culture that was able to modulate the Liver-specific functions of hepatocytes. Thus, perfusion culture conditions of the hepatocyte--NPC coculture system should be appropriately designed to induce suitable reconstruction of the cultured cells for use as a Bioartificial Liver Device.
Frank B. Cerra - One of the best experts on this subject based on the ideXlab platform.
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Development of a Bioartificial Liver Device.
Methods in molecular medicine, 1999Co-Authors: Linda K. Hansen, Rory P Remmel, Julie R. Friend, Frank B. CerraAbstract:Liver disease continues to be a challenge clinically, with 30,000 patients dying each year from Liver failure (1). Although Liver transplantation can successfully treat many patients undergoing Liver failure, the scarcity of donor organs severely limits this treatment's application. For this reason, many investigators are pursuing alternatives to total organ transplantation, from living donors to cell transplantation. One additional approach is the development of a hybrid, Bioartificial Liver as an extracorporeal Device for the temporary treatment of acute Liver failure. This approach has demonstrated early success, and may provide an important clinical treatment in the near future. In addition, a Bioartificial Liver reactor is useful for prolonged in vitro studies of hepatocyte function. This chapter will provide information on the design and use of such a reactor for in vitro applications.
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Characterization of the three-compartment gel-entrapment porcine hepatocyte Bioartificial Liver.
Cell biology and toxicology, 1997Co-Authors: Timothy D. Sielaff, S L Nyberg, M D Rollins, B Amiot, A Lee, Frank B. CerraAbstract:A hybrid Bioartificial Liver Device supporting a large mass of cells expressing differentiated hepatocyte metabolic capabilities is necessary for the successful treatment of fulminant hepatic failure. The three-compartment gel-entrapment porcine hepatocyte Bioartificial Liver was designed to provide "bridge" support to transplantation or until native Liver recovery is achieved for patients with acute Liver failure. The Device is an automated mammalian cell culture system supporting 6-7 x 10(9) porcine hepatocytes entrapped in a collagen matrix and inoculated into the capillary lumen spaces of two 100 kDa molecular mass cut-off hollow fiber bioreactors. Gel contraction recreates a small lumen space within the hollow fiber which allows for the deLivery of a nutrient medium. This configuration supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis, oxygen consumption, and vital dye staining during both cell culture and ex vivo application. The hollow fiber membrane was also shown to isolate the cells from xenogenic immunoglobulin attack. The gel-entrapment Bioartificial Liver maintained a large mass of functional hepatocytes by providing a three-dimensional cell culture matrix, by deLivering basal nutrients through lumen media perfusion, and by preventing rejection of the xenocytes. These features make this Device a favorable candidate for the treatment of clinical fulminant hepatic failure.
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Extended Liver-specific functions of porcine hepatocyte spheroids entrapped in collagen gel
In Vitro Cellular & Developmental Biology - Animal, 1995Co-Authors: Arye Lazar, Henry J Mann, Rory P Remmel, Russell A. Shatford, Frank B. CerraAbstract:The potential use of porcine hepatocytes in a Bioartificial Liver Device requires large quantities of viable and highly active cells. To facilitate the scaling up of the system, Liver specific activities of hepatocytes should be maximized. One way of enhancing the specific activities is to cultivate hepatocytes as multicellular spheroids. Freshly isolated porcine hepatocytes form spheroids when cultivated in suspended cultures. These spheroids exhibit higher activities for a number of Liver specific functions compared to hepatocytes cultivated as monolayers. However, these activities decreased in a few days in culture. Entrappment of spheroids in collagen gel sustained their metabolic activities at a stable level over 21 days. Production of albumin and urea by spheroid hepatocytes entrapped in collagen gels were 2 to 3 times higher than those by freshly isolated single cells. P-450 activity was demonstrated by metabolism of lidocaine to its main metabolite, monoethylglycinexylidide. Phase II drug metabolism was demonstrated by glucuronidation of 4-methylumbelliferone. This work shows that porcine hepatocyte spheroids entrapped in collagen maintain differentiated functions for an extended time period. Such hepatocyte spheroid entrappment system may facilitate the development of a Bioartificial Liver support Device.
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Formation of porcine hepatocyte spheroids for use in a Bioartificial Liver.
Cell transplantation, 1995Co-Authors: Arye Lazar, Madhusudan V Peshwa, Chung Ming Chi, Frank B. CerraAbstract:Abstract Xenogeneic hepatocytes have recently been used in a Bioartificial Liver Device as a potential short-term extracorporeal support of acute Liver failure. Scaling up the system requires large quantities of viable and highly active cells. Hepatocytes grown as spheroids manifest higher metabolic activities for longer time periods as compared to those in monolayer cultures. Use of hepatocyte spheroids for application in a Bioartificial Liver can possibly alleviate the need of scaling up. Porcine hepatocytes when cultured under stirred conditions, form multicellular spheroids in a defined culture medium. Spheroids were formed 24 h after cell inoculation with an efficiency of 80–90°7o and a mean diameter of about 135 μm. Scanning electron microscopy revealed numerous microvilli projecting from the entire surface of the spheroids. Transmission electron microscopy revealed differentiated hepatocytes which displayed well-developed cytoplasmic structures separated by bile canaliculus-like structures. The morphological studies show a resemblance between cells in the spheroids and in the Liver in vivo. Ureagenesis by spheroids was twice as active and was sustained for a longer culture period than that by hepatocytes cultured as monolayers. Preparation of porcine hepatocyte spheroids in an agitated vessel is simple efficient and reproducible. It will allow for preparation of large quantities of spheroids to be employed in a Bioartificial Liver Device as well as in Liver metabolism studies.
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Effects of hepatocyte growth factor on viability and biotransformation functions of hepatocytes in gel entrapped and monolayer culture.
Critical care medicine, 1995Co-Authors: Mark D. Cipolle, Henry J Mann, Rory P Remmel, Timothy D. Sielaff, Michael J. Lovdahl, Frank B. CerraAbstract:Objectives : An extracorporeal Bioartificial Liver Device must maintain viability and differentiated function of hepatocytes cultivated at high cell density. Growth factors, such as hepatocyte growth factor, found in high concentrations in the plasma of patients with fulminant hepatic failure, have the potential to promote hepatocyte dedifferentiation and thus, decrease function. We tested the hypothesis that hepatocyte growth factor would improve viable cell density and decrease biotransformation functions of Liver cells in monolayer culture and in hepatocytes entrapped in collagen cylindrical gel noodles as found in the extracorporeal Bioartificial Liver. Design : In vitro, controlled study. Setting : University research laboratory. Subjects : Adult Sprague Dawley Rats. Interventions : Hepatocytes were harvested by a two-step collagenase technique. Harvested hepatocytes were plated onto type 1 collagen coated plates or entrapped in type 1 collagen cylindrical gels and cultured in different concentrations of hepatocyte growth factor. Interval measurements of 3 H-thymidine incorporation, albumin synthesis, biotransformation functions, and viability were made. Measurements and Main Results : In monolayer culture, the addition of hepatocyte growth factor caused a dramatic increase in 3 H-thymidine incorporation. This increase was accompanied by a decrease in the appearance of the lidocaine metabolite, monoethyglycin-exylidide. Albumin production was unchanged. In cylindrical gel entrapment cultures, hepatocyte growth factor caused a significant increase in 2-day viability but had no effect on the metabolite appearance of lidocaine or 4-methyl umbelliferone or albumin production. Conclusions : Hepatocyte growth factor induces dedifferentiation of hepatocytes in monolayer culture. Collagen matrix entrapment appears to abrogate this effect and improve Liver cell viability. There may be reciprocal regulation of hepatocyte reproductive and differentiated functions, such as biotransformation, which can be influenced by the entrapment of hepatocytes in an extracellular type 1 collagen matrix. (Crit Care Med 1995 ; 23 :1237-1242)
Chao-chun Hsu - One of the best experts on this subject based on the ideXlab platform.
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development of a 3d porous chitosan gelatin Liver scaffold for a Bioartificial Liver Device
Journal of Bioscience and Bioengineering, 2020Co-Authors: Yung-te Hou, Chao-chun HsuAbstract:Functional artificial Livers (FALs), with embedded hepatocytes that perform the functions of a normal Liver, have been developed during the past decades. It is important to note that the Liver scaffold, which is a biologically functional core of Bioartificial Livers, plays a vital role in the bio-cartridge within a Bioartificial Liver. In this study, a three-dimensional (3D) Liver scaffold for in vitro cultures was fabricated by freeze-drying a chitosan/gelatin (CG) solution. A CG scaffold has advantages such as (i) inexpensive and easy-to-make; (ii) easy to fabricate with varying compressive modulus by changing the concentration of glutaraldehyde; (iii) non-cytotoxicity; and (iv) porous structure is similar to extracellular matrix (ECM), thus facilitating hepatocyte adhesion and proliferation. The results revealed that the compressive modulus and maintainability of a CG scaffold was correlated to the increase in glutaraldehyde. Furthermore, hepatocyte viability and hepatic functions showed the best performances with a 0.61% glutaraldehyde-CG scaffold. This CG scaffold not only had higher hepatocyte biocompatibility and mechanical strength, but also maintained hepatic functions and viability in vitro cultures; especially, the mechanical properties of 0.61% glutaraldehyde-CG scaffold were very similar to those in normal Liver. The CG scaffold as a Liver scaffold may have high potential for further Bioartificial Liver design in the near future.
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Development of a 3D porous chitosan/gelatin Liver scaffold for a Bioartificial Liver Device
Journal of bioscience and bioengineering, 2020Co-Authors: Yung-te Hou, Chao-chun HsuAbstract:Functional artificial Livers (FALs), with embedded hepatocytes that perform the functions of a normal Liver, have been developed during the past decades. It is important to note that the Liver scaffold, which is a biologically functional core of Bioartificial Livers, plays a vital role in the bio-cartridge within a Bioartificial Liver. In this study, a three-dimensional (3D) Liver scaffold for in vitro cultures was fabricated by freeze-drying a chitosan/gelatin (CG) solution. A CG scaffold has advantages such as (i) inexpensive and easy-to-make; (ii) easy to fabricate with varying compressive modulus by changing the concentration of glutaraldehyde; (iii) non-cytotoxicity; and (iv) porous structure is similar to extracellular matrix (ECM), thus facilitating hepatocyte adhesion and proliferation. The results revealed that the compressive modulus and maintainability of a CG scaffold was correlated to the increase in glutaraldehyde. Furthermore, hepatocyte viability and hepatic functions showed the best performances with a 0.61% glutaraldehyde-CG scaffold. This CG scaffold not only had higher hepatocyte biocompatibility and mechanical strength, but also maintained hepatic functions and viability in vitro cultures; especially, the mechanical properties of 0.61% glutaraldehyde-CG scaffold were very similar to those in normal Liver. The CG scaffold as a Liver scaffold may have high potential for further Bioartificial Liver design in the near future.
Pei Kan - One of the best experts on this subject based on the ideXlab platform.
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Perfusion of medium with supplemented growth factors changes metabolic activities and cell morphology of hepatocyte-nonparenchymal cell coculture.
Tissue Engineering, 2004Co-Authors: Pei Kan, Hirotoshi Miyoshi, Norio OhshimaAbstract:To develop a feasible perfusion-type Bioartificial Liver Device, perfusion of hepatocyte–nonparenchymal cell (NPC) cocultures with medium supplemented with hepatocyte growth factor (HGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) was carried out. On day 1 of culture, perfusion at a constant shear stress of 1.3 dyn/cm2 enhanced ammonia metabolic and urea synthetic activities of hepatocytes. These enhanced activities were sustained up to day 7 only when growth factors were present. In contrast, no beneficial effects of growth factors on these activities were observed in static cultures. In perfusion cultures, three-dimensional cell aggregates were formed. On the surface of these aggregates, flattened cell layers composed mainly of NPCs were found, and the central cluster of cell aggregates was composed of round-shaped hepatocytes and reticulin fibrils. These observations strongly suggested that the reconstruction of different types of Liver cells and connective tissues formed ti...
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Perfusion of medium with supplemented growth factors changes metabolic activities and cell morphology of hepatocyte-nonparenchymal cell coculture.
Tissue engineering, 2004Co-Authors: Pei Kan, Hirotoshi Miyoshi, Norio OhshimaAbstract:To develop a feasible perfusion-type Bioartificial Liver Device, perfusion of hepatocyte-nonparenchymal cell (NPC) cocultures with medium supplemented with hepatocyte growth factor (HGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) was carried out. On day 1 of culture, perfusion at a constant shear stress of 1.3 dyn/cm2 enhanced ammonia metabolic and urea synthetic activities of hepatocytes. These enhanced activities were sustained up to day 7 only when growth factors were present. In contrast, no beneficial effects of growth factors on these activities were observed in static cultures. In perfusion cultures, three-dimensional cell aggregates were formed. On the surface of these aggregates, flattened cell layers composed mainly of NPCs were found, and the central cluster of cell aggregates was composed of round-shaped hepatocytes and reticulin fibrils. These observations strongly suggested that the reconstruction of different types of Liver cells and connective tissues formed tissue-mimicking cell aggregates in the perfusion culture that was able to modulate the Liver-specific functions of hepatocytes. Thus, perfusion culture conditions of the hepatocyte--NPC coculture system should be appropriately designed to induce suitable reconstruction of the cultured cells for use as a Bioartificial Liver Device.
F Berthiaume - One of the best experts on this subject based on the ideXlab platform.
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Homogeneous differentiation of hepatocyte-like cells from embryonic stem cells: applications for the treatment of Liver failure
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2007Co-Authors: Cheul H. Cho, A W Tilles, F Berthiaume, Jaesung Park, Natesh Parashurama, Eric Y. H. Park, Kazuhiro Suganuma, Yaakov Nahmias, M L YarmushAbstract:One of the major hurdles of cellular therapies for the treatment of Liver failure is the low availability of functional human hepatocytes. While embryonic stem (ES) cells represent a potential cell source for therapy, current methods for differentiation result in mixed cell populations or low yields of the cells of interest. Here we describe a rapid, direct differentiation method that yields a homogeneous population of endoderm-like cells with 95% purity. Mouse ES cells cultured on top of collagen-sandwiched hepatocytes differentiated and proliferated into a uniform and homogeneous cell population of endoderm-like cells. The endoderm-like cell population was positive for Foxa2, Sox17, and AFP and could be further differentiated into hepatocyte-like cells, demonstrating hepatic morphology, functionality, and gene and protein expression. Incorporating the hepatocyte-like cells into a Bioartificial Liver Device to treat fulminant hepatic failure improved animal survival, thereby underscoring the therapeutic ...
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a Bioartificial Liver Device secreting interleukin 1 receptor antagonist for the treatment of hepatic failure in rats
Journal of Surgical Research, 2007Co-Authors: Masahiro Shinoda, A W Tilles, Naoya Kobayashi, Kazuhiro Suganuma, Go Wakabayashi, Atsushi Takayanagi, Toshinori Totsugawa, Hirohisa Harada, Hideaki Obara, F BerthiaumeAbstract:Background Liver transplantation is the treatment of choice for many patients with fulminant hepatic failure (FHF). A major limitation of this treatment is the lack of available donors. An optimally functioning bio-artificial Liver (BAL) Device has the potential to provide critical hepatic support to patients with FHF. In this study, we examined the efficacy of combining interleukin-1 (IL-1) receptor blockade with the synthetic function of hepatocytes in a BAL Device for the treatment of FHF. Materials and methods We injected an adenoviral vector encoding human IL-1 receptor antagonist (AdIL-1Ra) into the Liver of D-galactosamine (GalN) intoxicated rats via the portal vein. We also transfected primary rat hepatocytes and reversibly immortalized human hepatocytes (TTNT cells) with AdIL-1Ra, and incorporated these transfected hepatocytes into our flat-plate BAL Device and evaluated their efficacy in our GalN-induced FHF rat model after 10 h of extracorporeal perfusion. Results Rats injected with AdIL-1Ra showed significant reductions in the plasma levels of hepatic enzymes. Primary rat hepatocytes transfected with AdIL-1Ra secreted IL-1Ra without losing their original synthetic function. Incorporating these cells into the BAL Device and testing in a GalN-induced FHF rat model resulted in significant reductions in plasma IL-6 levels and significantly improved animal survival. Incorporating the AdIL-1Ra transfected TTNT cells in the BAL Device and testing in the GalN-induced FHF rat model resulted in significantly reduced plasma IL-6 levels, and a trend toward improved survival was seen. Conclusion Hepatocytes producing IL-1Ra are a promising cell source for BAL Devices in the treatment of GalN-induced FHF.
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treatment of fulminant hepatic failure in rats using a Bioartificial Liver Device containing porcine hepatocytes producing interleukin 1 receptor antagonist
Tissue Engineering, 2006Co-Authors: Masahiro Shinoda, A W Tilles, F Berthiaume, Kazuhiro Suganuma, Go Wakabayashi, Atsushi Takayanagi, Hideaki Obara, Hirohisas Harada, Motohide Shimazu, Nobuyoshi ShimizuAbstract:Fulminant hepatic failure (FHF) is a serious clinical condition that is associated with high mortality. There is evidence that FHF is an inflammatory disease, which is supported clinically by elevated serum levels of cytokines. In an effort to develop hepatocytes with additional functions for use in our Bioartificial Liver (BAL) Device, we focused on interleukin-1 (IL-1) blockade as a therapeutic modality. Primary porcine hepatocytes were isolated from the Livers of miniature swine and then transfected with an adenoviral vector encoding human interleukin-1 receptor antagonist (AdIL- 1Ra). The transfected hepatocytes secreted human IL-1Ra. These transfected hepatocytes were incorporated into a flat-plate BAL Device to evaluate their efficacy in treating D-galactosamine (GalN)- induced FHF in a rat model. After extracorporeal perfusion with the BAL Device containing the transfected hepatocytes, there were significant reductions in the plasma levels of hepatic enzymes (aspartate aminotransferase and alanine ...
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microfabricated grooved substrates as platforms for Bioartificial Liver reactors
Biotechnology and Bioengineering, 2005Co-Authors: Jaesung Park, M L Yarmush, F Berthiaume, M Toner, A W TillesAbstract:An extracorporeal Bioartificial Liver Device has the potential to provide temporary hepatic support for patients with Liver failure. Our goal was to optimize the flow environment for the cultured hepatocytes in a flat- plate bioreactor, specifically focusing on oxygen deLivery using high medium flow rates while reducing the detri- mental effects of the resulting shear stresses. We used photolithographic techniques to fabricate microgrooves onto the underlying glass substrate. The microgrooves, perpendicular to the axial flow direction, protected the hepatocytes from the shear stress induced by the flowing medium. Using finite element analysis, we found that the velocity gradient change near the cell surface (i.e., bottom of the grooves) was smaller than that near the top surface of the flow channel, indicating that the grooves would provide protection to the attached cells from the mechan- ical effects of the flowing medium. We also determined that the shear stress at the cell surface could be reduced by as much as 30 times (channel height of 100 Am) in the grooved-substrate (0.5 dyn/cm 2 ) bioreactor compared to the flat-substrate (15 dyn/cm 2 ) bioreactor for a medi- um flow rate of 4.0 mL/min. Albumin and urea synthesis rates of hepatocytes cocultured with 3T3-J2 fibroblasts remained stable over 5 days of perfusion in the grooved- substrate bioreactor, whereas in the flat-substrate bio- reactor they decreased over the same time period. These studies indicate that under ''high'' flow conditions the microgrooved-substrate in the bioreactor can decrease the detrimental effects of shear stress on the hepatocytes while providing adequate oxygenation, thereby resulting in stable Liver-specific function. B 2005 Wiley Periodicals, Inc.
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Bioengineering of Liver assist Devices
Journal of Hepato-Biliary-Pancreatic Surgery, 2002Co-Authors: A W Tilles, F Berthiaume, M L Yarmush, Ronald G. Tompkins, M TonerAbstract:Over 30 000 patients die annually in the United States from Liver failure. In fulminant hepatic failure, a clinical syndrome associated with high mortality, orthotopic Liver transplantation is the primary therapeutic option for patients not responding to supportive therapy. However, the persistent scarcity of donor organs has limited this therapeutic modality, resulting in a continued increase in the number of patients who die waiting for a donor Liver. An extracorporeal Bioartificial Liver Device could provide vital support to a Liver failure patient until a donor Liver was available or until the patient's own Liver regenerated. Although it is unclear which Liver-specific functions must be provided by such a Device to be effective, a constant challenge has been to obtain stable, well-differentiated, and normally functioning hepatocytes that can be cultured at high cell densities. Many of the Devices currently undergoing clinical trials are limited by designs which are prone to substrate limitations, resulting in compromised hepatocyte function. In Devices that avoid substrate limitations, hepatocyte functions can be optimized, thereby leading to increased Device efficiency. In this overview, the authors describe the critical issues involved in Bioartificial Liver development and discuss their experiences in hepatocyte culture optimization within the context of a microchannel, flat-plate Bioartificial Liver Device with an internal membrane oxygenator.