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Chamuleau RAFM, Hoekstra R. End-stage liver failure: filling the treatment gap at the intensive care unit. J Artif Organs 2019; 23:113-123. [PMID: 31535298 PMCID: PMC7228976 DOI: 10.1007/s10047-019-01133-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2019] [Accepted: 09/09/2019] [Indexed: 12/14/2022]
Abstract
End-stage liver failure is a condition of collapsing liver function with mortality rates up to 80. Liver transplantation is the only lifesaving therapy. There is an unmet need for therapy to extend the waiting time for liver transplantation or regeneration of the native liver. Here we review the state-of-the-art of non-cell based and cell-based artificial liver support systems, cell transplantation and plasma exchange, with the first therapy relying on detoxification, while the others aim to correct also other failing liver functions and/or modulate the immune response. Meta-analyses on the effect of non-cell based systems show contradictory outcomes for different types of albumin purification devices. For bioartificial livers proof of concept has been shown in animals with liver failure. However, large clinical trials with two different systems did not show a survival benefit. Two clinical trials with plasma exchange and one with transplantation of mesenchymal stem cells showed positive outcomes on survival. Detoxification therapies lack adequacy for most patients. Correction of additional liver functions, and also modulation of the immune system hold promise for future therapy of liver failure.
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Affiliation(s)
- Robert A F M Chamuleau
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Academic Medical Center, Meibergdreef 69-71, S1-176, 1105 BK, Amsterdam, The Netherlands.
| | - Ruurdtje Hoekstra
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Academic Medical Center, Meibergdreef 69-71, S1-176, 1105 BK, Amsterdam, The Netherlands
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2
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Kojima H, Yasuchika K, Fukumitsu K, Ishii T, Ogiso S, Miyauchi Y, Yamaoka R, Kawai T, Katayama H, Yoshitoshi-Uebayashi EY, Kita S, Yasuda K, Sasaki N, Komori J, Uemoto S. Establishment of practical recellularized liver graft for blood perfusion using primary rat hepatocytes and liver sinusoidal endothelial cells. Am J Transplant 2018; 18:1351-1359. [PMID: 29338127 DOI: 10.1111/ajt.14666] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2017] [Revised: 12/29/2017] [Accepted: 01/03/2018] [Indexed: 01/25/2023]
Abstract
Tissue decellularization produces a three-dimensional scaffold that can be used to fabricate functional liver grafts following recellularization. Inappropriate cell distribution and clotting during blood perfusion hinder the practical use of recellularized livers. Here we aimed to establish a seeding method for the optimal distribution of parenchymal and endothelial cells, and to evaluate the effect of liver sinusoidal endothelial cells (LSECs) in the decellularized liver. Primary rat hepatocytes and LSECs were seeded into decellularized whole-liver scaffolds via the biliary duct and portal vein, respectively. Biliary duct seeding provided appropriate hepatocyte distribution into the parenchymal space, and portal vein-seeded LSECs simultaneously lined the portal lumen, thereby maintaining function and morphology. Hepatocytes co-seeded with LSECs retained their function compared with those seeded alone. Platelet deposition was significantly decreased and hepatocyte viability was maintained in the co-seeded group after extracorporeal blood perfusion. In conclusion, our seeding method provided optimal cell distribution into the parenchyma and vasculature according to the three-dimensional structure of the decellularized liver. LSECs maintained hepatic function, and supported hepatocyte viability under blood perfusion in the engineered liver graft owing to their antithrombogenicity. This recellularization procedure could help produce practical liver grafts with blood perfusion.
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Affiliation(s)
- Hidenobu Kojima
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Kentaro Yasuchika
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - KenIshii Fukumitsu
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Takamichi Ishii
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Satoshi Ogiso
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Yuya Miyauchi
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Ryoya Yamaoka
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Takayuki Kawai
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Hokahiro Katayama
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | | | - Sadahiko Kita
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.,Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Katsutaro Yasuda
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Naoya Sasaki
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.,Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Junji Komori
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Shinji Uemoto
- Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
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3
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Ogoke O, Oluwole J, Parashurama N. Bioengineering considerations in liver regenerative medicine. J Biol Eng 2017; 11:46. [PMID: 29204185 PMCID: PMC5702480 DOI: 10.1186/s13036-017-0081-4] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2017] [Accepted: 09/25/2017] [Indexed: 12/19/2022] Open
Abstract
Background Liver disease contributes significantly to global disease burden and is associated with rising incidence and escalating costs. It is likely that innovative approaches, arising from the emerging field of liver regenerative medicine, will counter these trends. Main body Liver regenerative medicine is a rapidly expanding field based on a rich history of basic investigations into the nature of liver structure, physiology, development, regeneration, and function. With a bioengineering perspective, we discuss all major subfields within liver regenerative medicine, focusing on the history, seminal publications, recent progress within these fields, and commercialization efforts. The areas reviewed include fundamental aspects of liver transplantation, liver regeneration, primary hepatocyte cell culture, bioartificial liver, hepatocyte transplantation and liver cell therapies, mouse liver repopulation, adult liver stem cell/progenitor cells, pluripotent stem cells, hepatic microdevices, and decellularized liver grafts. Conclusion These studies highlight the creative directions of liver regenerative medicine, the collective efforts of scientists, engineers, and doctors, and the bright outlook for a wide range of approaches and applications which will impact patients with liver disease.
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Affiliation(s)
- Ogechi Ogoke
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Furnas Hall, Buffalo, NY 14260 USA.,Clinical and Translation Research Center (CTRC), University at Buffalo (State University of New York), 875 Ellicott St., Buffalo, NY 14203 USA
| | - Janet Oluwole
- Clinical and Translation Research Center (CTRC), University at Buffalo (State University of New York), 875 Ellicott St., Buffalo, NY 14203 USA.,Department of Biomedical Engineering, University at Buffalo (State University of New York), Furnas Hall, 907 Furnas Hall, Buffalo, NY 14260 USA
| | - Natesh Parashurama
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Furnas Hall, Buffalo, NY 14260 USA.,Clinical and Translation Research Center (CTRC), University at Buffalo (State University of New York), 875 Ellicott St., Buffalo, NY 14203 USA.,Department of Biomedical Engineering, University at Buffalo (State University of New York), Furnas Hall, 907 Furnas Hall, Buffalo, NY 14260 USA
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4
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Giri S, Acikgöz A, Pathak P, Gutschker S, Kürsten A, Nieber K, Bader A. Three dimensional cultures of rat liver cells using a natural self-assembling nanoscaffold in a clinically relevant bioreactor for bioartificial liver construction. J Cell Physiol 2011; 227:313-27. [PMID: 21437901 DOI: 10.1002/jcp.22738] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Till date, no bioartificial liver (BAL) procedure has obtained FDA approval or widespread clinical acceptance, mainly because of multifactorial limitations such as the use of microscale or undefined biomaterials, indirect and lower oxygenation levels in liver cells, short-term undesirable functions, and a lack of 3D interaction of growth factor/cytokine signaling in liver cells. To overcome preclinical limitations, primary rat liver cells were cultured on a naturally self-assembling peptide nanoscaffold (SAPN) in a clinically relevant bioreactor for up to 35 days, under 3D interaction with suitable growth factors and cytokine signaling agents, alone or combination (e.g., Group I: EPO, Group II: Activin A, Group III: IL-6, Group IV: BMP-4, Group V: BMP4 + EPO, Group VI: EPO + IL-6, Group VII: BMP4 + IL-6, Group VIII: Activin A + EPO, Group IX: IL-6 + Activin A, Group X: Activin A + BMP4, Group XI: EPO + Activin A + BMP-4 + IL-6 + HGF, and Group XII: Control). Major liver specific functions such as albumin secretion, urea metabolism, ammonia detoxification, phase contrast microscopy, immunofluorescence of liver specific markers (Albumin and CYP3A1), mitochondrial status, glutamic oxaloacetic transaminase (GOT) activity, glutamic pyruvic transaminase (GPT) activity, and cell membrane stability by the lactate dehydrogenase (LDH) test were also examined and compared with the control over time. In addition, we examined the drug biotransformation potential of a diazepam drug in a two-compartment model (cell matrix phase and supernatant), which is clinically important. This present study demonstrates an optimized 3D signaling/scaffolding in a preclinical BAL model, as well as preclinical drug screening for better drug development.
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Affiliation(s)
- Shibashish Giri
- Department of Cell Techniques and Applied Stem Cell Biology, Centre for Biotechnology and Biomedicine, University of Leipzig, Leipzig, Germany.
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Yang H, Roth CM, Ierapetritou MG. Analysis of Amino Acid Supplementation Effects on Hepatocyte Cultures Using Flux Balance Analysis. OMICS-A JOURNAL OF INTEGRATIVE BIOLOGY 2011; 15:449-60. [DOI: 10.1089/omi.2010.0070] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Affiliation(s)
- Hong Yang
- Department of Chemical and Biochemical Engineering, Rutgers, the State University of New Jersey, Piscataway, New Jersey
| | - Charles M. Roth
- Department of Chemical and Biochemical Engineering, Rutgers, the State University of New Jersey, Piscataway, New Jersey
- Department of Biomedical Engineering, Rutgers, the State University of New Jersey, Piscataway, New Jersey
| | - Marianthi G. Ierapetritou
- Department of Chemical and Biochemical Engineering, Rutgers, the State University of New Jersey, Piscataway, New Jersey
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Novik E, Maguire TJ, Chao P, Cheng KC, Yarmush ML. A microfluidic hepatic coculture platform for cell-based drug metabolism studies. Biochem Pharmacol 2009; 79:1036-44. [PMID: 19925779 DOI: 10.1016/j.bcp.2009.11.010] [Citation(s) in RCA: 158] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2009] [Revised: 11/02/2009] [Accepted: 11/10/2009] [Indexed: 11/29/2022]
Abstract
Within the global pharmaceutical and biotech industries, there is significant interest in identifying in vitro screening systems that are more human-relevant-i.e., that offer greater utility in predicting subcellular and cellular physiological responses in humans in vivo-and that thereby allow investigators to reduce the incidence of costly late-stage failures during pharmaceutical clinical trials, as well as to reduce the use of animals in drug testing. Currently incumbent in vitro screening methods, such as culturing human hepatocytes in suspension, while useful, are limited by a lack of long term cellular function. In order to address this limitation, we have established an integrated, microfluidic, in vitro platform that combines the patented HmuREL((R)) microdevice with a hepatic coculture system. In the present report, we use this platform to study clearance and metabolite generation of a battery of molecular entities. The results show that the flow-based coculture system is capable of clearing, with improved resolution and predictive value, compounds with high, medium, and low clearance values. In addition, when coculture is coupled with flow, higher metabolite production rates are obtained than in static systems.
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Affiliation(s)
- Eric Novik
- Hurel Corporation, Beverley Hills, California, CA, USA
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Yang H, Roth CM, Ierapetritou MG. A rational design approach for amino acid supplementation in hepatocyte culture. Biotechnol Bioeng 2009; 103:1176-91. [PMID: 19422042 DOI: 10.1002/bit.22342] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Improvement of culture media for mammalian cells is conducted via empirical adjustments, sometimes aided by statistical design methodologies. Here, we demonstrate a proof of principle for the use of constraints-based modeling to achieve enhanced performance of liver-specific functions of cultured hepatocytes during plasma exposure by adjusting amino acid supplementation and hormone levels in the medium. Flux balance analysis (FBA) is used to determine an amino acid flux profile consistent with a desired output; this is used to design an amino acid supplementation. Under conditions of no supplementation, empirical supplementation, and designed supplementation, hepatocytes were exposed to plasma and their morphology, specific cell functions (urea, albumin production) and lipid metabolism were measured. Urea production under the designed amino acid supplementation was found to be increased compared with previously reported (empirical) amino acid supplementation. Not surprisingly, the urea production attained was less than the theoretical value, indicating the existence of pathways or constraints not present in the current model. Although not an explicit design objective, albumin production was also increased by designed amino acid supplementation, suggesting a functional linkage between these outputs. In conjunction with traditional approaches to improving culture conditions, the rational design approach described herein provides a novel means to tune the metabolic outputs of cultured hepatocytes.
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Affiliation(s)
- Hong Yang
- Department of Chemical and Biochemical Engineering, Rutgers, State University of New Jersey, Piscataway, New Jersey 08854, USA
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8
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Nagrath D, Xu H, Tanimura Y, Zuo R, Berthiaume F, Avila M, Yarmush R, Yarmush ML. Metabolic preconditioning of donor organs: defatting fatty livers by normothermic perfusion ex vivo. Metab Eng 2009; 11:274-83. [PMID: 19508897 PMCID: PMC2814076 DOI: 10.1016/j.ymben.2009.05.005] [Citation(s) in RCA: 123] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2008] [Revised: 04/26/2009] [Accepted: 05/22/2009] [Indexed: 12/12/2022]
Abstract
Fatty liver is a significant risk factor for liver transplantation, and accounts for nearly half of the livers rejected from the donor pool. We hypothesized that metabolic preconditioning via ex vivo perfusion of the liver graft can reduce fat content and increase post-transplant survival to an acceptable range. We describe a perfusate medium containing agents that promote the defatting of hepatocytes and explanted livers. Defatting agents were screened on cultured hepatocytes made fatty by pre-incubation with fatty acids. The most effective agents were then used on fatty livers. Fatty livers were isolated from obese Zucker rats and normothermically perfused with medium containing a combination of defatting agents. This combination decreased the intracellular lipid content of cultured hepatocytes by 35% over 24h, and of perfused livers by 50% over 3h. Metabolite analysis suggests that the defatting cocktail upregulated both lipid oxidation and export. Furthermore, gene expression analysis for several enzymes and transcription factors involved in fatty acid oxidation and triglyceride clearance were elevated. We conclude that a cocktail of defatting agents can be used to rapidly clear excess lipid storage in fatty livers, thus providing a new means to recondition donor livers deemed unacceptable or marginally acceptable for transplantation.
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Affiliation(s)
- Deepak Nagrath
- Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA 02114, USA
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9
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Nagaki M, Naito T, Ohnishi H, Akaike T, Muto Y, Moriwaki H. Effects of plasma from patients with fulminant hepatic failure on function of primary rat hepatocytes in three-dimensional culture. Liver Int 2005; 25:1010-7. [PMID: 16162161 DOI: 10.1111/j.1478-3231.2005.01127.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
BACKGROUND/AIM As biotechnology continues to advance, a bioartificial liver is expected to be developed for the treatment of patients with fulminant hepatic failure (FHF) whose liver dysfunction is potentially reversible or for providing liver support as a bridge to liver transplantation. While monolayer-cultured hepatocytes rapidly lose their capacity to express many liver-specific functions over time when cultured, spherical-shaped hepatocytes in three-dimensional culture with the use of extracellular matrix components sustain long-term survival by maintaining differentiated hepatocyte functions. The aim of this study was to investigate whether sufficient functions of viable spherical-shaped hepatocytes could be maintained in plasma of patients with FHF in order to use these cells in an extracorporeal system. METHODS Hepatocyte functions were evaluated under monolayer or three-dimensional culture in FHF plasma. RESULTS Primary rat hepatocytes on poly-N-p-vinylbenzyl-D-lactonamide (PVLA) formed spheroids even in FHF plasma and maintained their spherical shapes in FHF plasma as long as in medium. Spherical-shaped hepatocytes on PVLA cultured in FHF plasma showed higher activity in albumin secretion, urea formation, and gluconeogenesis than those in normal human plasma or medium. As being cultured in medium, hepatocytes on PVLA cultured in plasma were also superior to cells on collagen in regard to albumin secretion, amino acid metabolism, and gluconeogenesis. CONCLUSIONS These findings demonstrated that FHF plasma is not toxic to rat hepatocyte spheroids and that hepatocyte spheroids have potential use in the development of a bioartificial liver.
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Affiliation(s)
- Masahito Nagaki
- First Department of Internal Medicine, Gifu University School of Medicine, Yanagido 1-1, Gifu 501-1194, Japan.
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Gan JH, Zhou XQ, Qin AL, Luo EP, Zhao WF, Yu H, Xu J. Hybrid artificial liver support system for treatment of severe liver failure. World J Gastroenterol 2005; 11:890-894. [PMID: 15682488 PMCID: PMC4250604 DOI: 10.3748/wjg.v11.i6.890] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/09/2004] [Revised: 04/11/2004] [Accepted: 05/13/2004] [Indexed: 02/06/2023] Open
Abstract
AIM To construct a novel hybrid artificial liver support system (HALSS) and to evaluate its efficacy in patients with severe liver failure. METHODS Hepatocytes were isolated from suckling pig by the modified Seglen's method. Isolated hepatocytes were cultured in a spinner flask for 24 h to form spheroids before use and the functions of spheroids were detected. HALSS consisted of a plasma separator, a hemo-adsorba and a bioreactor with hepatocytes spheroids in its extra-fiber space. HALSS was applied to 10 patients with severe liver failure. The general condition and the biochemical indexes of the patients were studied just before and after the treatment. RESULTS The number of cells per liver was about 2-4 x 10(10) (mean, 3.1+/-1.5 x 10(10)). The cell viabilities were more than 95%. After 24 h of spheroid culture, most hepatocytes formed spheroids. The levels of albumin and urea in the medium of spheroid culture were higher than those in supernatant of petri dish culture (P = 0.0015 and 0.0001, respectively). The capacity of albumin production and urea synthesis remained stable for more than one wk and declined rapidly after two weeks in vitro. In HALSS group, the duration of HALSS treatment was 6-10 h each time. All patients tolerated the treatment well without any fatal adverse reaction. After HALSS treatment, the general condition, psychic state, encephalopathy and hepatic function of the patients were improved. The survival rate of the HALSS group, Plasmapheresis group and control group was 30% (3/10), 20% (2/10) and 0% (0/10), respectively (P = 0.024). Two weeks after treatment, Tbil and ALT decreased and the PTA level elevated in HALSS group and pasmapheresis group (P value: 0.015 vs 0.020, 0.009 vs 0.012 and 0.032 vs 0.041, respectively). But there was no significant change of blood albumin concentration before and after treatment in HALSS group and Plasmapheresis group. CONCLUSION The HALSS established by us is effective in supporting liver function of patients with severe liver failure.
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Affiliation(s)
- Jian-He Gan
- Department of Infectious Diseases, First Hospital affiliated to Suzhou University, Suzhou 215006, Jangsu Province, China.
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Chen Z, Ding Y, Xu Q, Yu D. Bioartificial liver inoculated with porcine hepatocyte spheroids for treatment of canine acute liver failure model. Artif Organs 2003; 27:613-622. [PMID: 12823416 DOI: 10.1046/j.1525-1594.2003.07140.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The aim of this study was to evaluate a novel bioartificial system in a canine model of acute liver failure. An acute liver failure model in canines was induced by an end-side portocaval shunt combined with common bile duct ligation and transection. The bioartificial liver system, which utilized blood perfusion through a hollow fiber bioreactor from BIOLIV A3A inoculated with 1.0 - 3.1 x 1010 porcine hepatocyte spheroids, was developed for the treatment of acute liver failure. Sixteen acute liver failure model canines were divided between a group treated with bioartificial liver (n=8) and a control group (n=8) for 5 h. Blood alanine aminotransferase (ALT), alkaline phosphatase (AKP), total bilirubin (TBi), direct bilirubin (DBi), prothrombin time (PT), ammonia levels, and the ratio of branched chain to aromatic amino acids (Fischer's ratio) were determined. ALT, AKP, TBi, DBi, and ammonia levels were significantly elevated, PT was significantly prolonged, and Fischer's ratio decreased significantly in the canine model of the two groups on day 14 after operation compared to baseline. There were no significant differences between the two groups in laboratory data before treatment. In canines treated with the bioartificial liver system, ALT, AKP, TBi, DBi, and ammonia levels decreased significantly, PT was significantly shortened, Fischer's ratio was significantly elevated after treatment, and the survival rate by day 7 after treatment was 100%. In canines in the control group, on the other hand, there were no significant differences in ALT, AKP, TBi, DBi, PT, and ammonia levels between pretreatment and posttreatment, though these indices decreased to a slight degree after treatment. The survival rate by day 7 after treatment was 62.5% in the control group. Fischer's ratio decreased after treatment. ALT, AKP, TBi, DBi, PT, and ammonia levels in the bioartificial liver system group were lower, and Fischer's ratio and survival rate were higher than those in the control group after treatment. These results indicate that the novel bioartificial liver system we developed has a significant impact on the course of canine acute liver failure model and has potential advantages for clinical use in patients with acute liver failure.
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Affiliation(s)
- Zhong Chen
- Department of General Surgery, Affiliated Hospital, Nantong Medical College, Nantong, Jiangsu Province, P.R. China.
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12
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Chan C, Berthiaume F, Lee K, Yarmush ML. Metabolic flux analysis of cultured hepatocytes exposed to plasma. Biotechnol Bioeng 2003; 81:33-49. [PMID: 12432579 DOI: 10.1002/bit.10453] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution. The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure. Furthermore, supplementing the plasma with amino acids restored hepatospecific functions. In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation. We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis. These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes. The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma. Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma. The increase in fatty acid oxidation reduced intracellular triglyceride accumulation. Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture.
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Affiliation(s)
- Christina Chan
- Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, GRB 1401, 55 Fruit Street, Boston, USA
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Chan C, Berthiaume F, Lee K, Yarmush ML. Metabolic flux analysis of hepatocyte function in hormone- and amino acid-supplemented plasma. Metab Eng 2003; 5:1-15. [PMID: 12749840 DOI: 10.1016/s1096-7176(02)00011-3] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells. Previous attempts to culture hepatocytes in plasma yielded poor functional results. Recently we reported that hormone (insulin and hydrocortisone) and amino acid supplementation reduces intracellular lipid accumulation and restores liver-specific function in hepatocytes exposed to heparinized human plasma. In the current study, we performed metabolic flux analysis (MFA) using a simplified metabolic network model of cultured hepatocytes to quantitively estimate the changes in lipid metabolism and relevant intracellular pathways in response to hormone and amino acid supplementation. The model accounts for the majority of central carbon and nitrogen metabolism, and assumes pseudo-steady-state with no metabolic futile cycles. We found that beta-oxidation and tricarboxylic acid (TCA) cycle fluxes were upregulated by both hormone and amino acid supplementation, thus enhancing the rate of lipid oxidation. Concomitantly, hormone and amino acid supplementation increased gluconeogenic fluxes. This, together with an increased rate of glucose clearance, caused an increase in predicted glycogen synthesis. Urea synthesis was primarily derived from ammonia and aspartate generated through transamination reactions, while exogenous ammonia removal accounted for only 3-6% of the urea nitrogen. Amino acid supplementation increased the endogenous synthesis of oxaloacetate, and in turn that of aspartate, a necessary substrate for the urea cycle. These findings from MFA provide cues as to which genes/pathways relevant to fatty acid oxidation, urea production, and gluconeogenesis may be upregulated by plasma supplementation, and are consistent with current knowledge of hepatic amino acid metabolism, which provides further credence to this approach for evaluating the metabolic state of hepatocytes under various environmental conditions.
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Affiliation(s)
- Christina Chan
- Center For Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and The Shriners Hospitals for Children, 55 Fruit Street, Bigelow 1401, Boston, MA 02114, USA.
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14
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Chan C, Berthiaume F, Washizu J, Toner M, Yarmush ML. Metabolic pre-conditioning of cultured cells in physiological levels of insulin: generating resistance to the lipid-accumulating effects of plasma in hepatocytes. Biotechnol Bioeng 2002; 78:753-60. [PMID: 12001167 DOI: 10.1002/bit.10275] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Understanding the regulation of hepatocyte lipid metabolism is important for several biotechnological applications involving liver cells. During exposure of hepatocytes to plasma, as is the case in extracorporeal bioartificial liver assist devices, it has been reported that hepatic-specific functions, e.g., albumin and urea synthesis and diazepam removal, are dramatically compromised and hepatocytes progressively accumulate cytoplasmic lipid droplets. We hypothesized that the composition of hepatocyte culture medium significantly affects lipid metabolism during subsequent plasma exposure. Rat hepatocytes were cultured in medium containing either physiological (50 microU/mL) or supra-physiological (500 mU/mL) insulin levels for 1 week and then exposed to human plasma supplemented with or without amino acids. We found that insulin's anabolic effects, such as stimulation of triglyceride storage, were carried over from the pre-conditioning to the plasma exposure period. While hepatocytes cultured in high insulin medium accumulated large quantities of triglycerides during subsequent plasma exposure, culture in low insulin medium largely prevented lipid accumulation. Urea and albumin secretion, as well as the ammonia removal rate, were largely unaffected by insulin but increased with amino acid supplementation. Thus, hepatocyte metabolism during plasma exposure can be modulated by medium pre-conditioning and supplements added to plasma.
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Affiliation(s)
- Christina Chan
- Center for Engineering in Medicine/Surgical Services, Boston, Massachusetts 02114, USA
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15
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Morsiani E, Pazzi P, Puviani AC, Brogli M, Valieri L, Gorini P, Scoletta P, Marangoni E, Ragazzi R, Azzena G, Frazzoli E, Di Luca D, Cassai E, Lombardi G, Cavallari A, Faenza S, Pasetto A, Girardis M, Jovine E, Pinna AD. Early experiences with a porcine hepatocyte-based bioartificial liver in acute hepatic failure patients. Int J Artif Organs 2002; 25:192-202. [PMID: 11999191 DOI: 10.1177/039139880202500305] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Orthotopic liver transplantation (OLT) is the only effective therapeutic modality in severe acute hepatic failure (AHF). The scarcity of organs for transplantation leads to an urgent necessity for temporary liver support treatments in AHF patients. A hepatocyte-based bioartificial liver (BAL) is under investigation with the main purpose to serve as bridging treatment until a liver becomes available for OLT, or to promote spontaneous liver regeneration. We developed a novel radial-flow bioreactor (RFB) for three-dimensional, high-density hepatocyte culture and an integrated pumping apparatus in which, after plasmapheresis, the patient's plasma is recirculated through the hepatocyte-filled RFB. Two hundred thirty grams of freshly isolated porcine hepatocytes were loaded into the RFB for clinical liver support treatment. The BAL system was used 8 times in supporting 7 AHF patients in grade III-IV coma, all waiting for an urgent OLT Three patients with no history of previous liver diseases were affected by fulminant hepatic failure (FHF) due to hepatitis B virus, 3 by primary non-function (PNF) of the transplanted liver, and one by AHF due to previous abdominal trauma and liver surgery. Six out of 7 patients underwent OLT following BAL treatment(s), which lasted 6-24 hours. All patients tolerated the procedures well, as shown by an improvement in the level of encephalopathy, a decrease in serum ammonia, transaminases and an amelioration of the prothrombin time, with full neurological recovery after OLT Our initial clinical experience confirms the safety of this BAL configuration and suggests its clinical efficacy as a temporary liver support system in AHF patients.
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Affiliation(s)
- E Morsiani
- Department of Surgery, Sant'Anna University Hospital, University of Ferrara School of Medicine, Italy.
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16
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Washizu J, Berthiaume F, Mokuno Y, Tompkins RG, Toner M, Yarmush ML. Long-term maintenance of cytochrome P450 activities by rat hepatocyte/3T3 cell co-cultures in heparinized human plasma. TISSUE ENGINEERING 2001; 7:691-703. [PMID: 11749727 DOI: 10.1089/107632701753337654] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Little information on the effect of plasma on hepatocyte cytochrome P450 (CYP) activities is currently available. We characterized the effect of plasma on CYPs of hepatocyte-mesenchymal cell co-cultures, which exhibit stable liver specific functions and may be potentially useful for bioartificial liver design. Rat hepatocyte-mouse 3T3-J2 cell co-cultures were maintained for 6 days in medium, and then switched to heparinized human plasma containing 3-methylcholanthrene (3MC; 2 microM), phenobarbital (PB; 1 mM), or no inducer for up to 7 days. CYP activities were measured in situ based on the o-dealkylation of ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD), or benzyloxy- (BROD) resorufin. Plasma alone increased PROD/BROD but not EROD/MROD. The endogenous inducer was in the high molecular weight fraction (>5 kD) of plasma and inhibited by >5 nM okadaic acid and >10 microM dibutyryl cyclic AMP, two inhibitors of PB-inducible CYPs. Furthermore, plasma increased CYP1A1 and CYP2B1/2 mRNA levels. In plasma, 3MC induced EROD/MROD to about 60% of the level induced in culture medium while PB induced PROD/BROD that were three- to 10-fold above levels induced in medium. CYP activities decreased between days 2 and 7 of plasma exposure, but were enhanced by plasma supplementation with amino acids, insulin, glucagon, and hydrocortisone.
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Affiliation(s)
- J Washizu
- Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA
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Shito M, Kim NH, Baskaran H, Tilles AW, Tompkins RG, Yarmush ML, Toner M. In vitro and in vivo evaluation of albumin synthesis rate of porcine hepatocytes in a flat-plate bioreactor. Artif Organs 2001; 25:571-8. [PMID: 11493279 DOI: 10.1046/j.1525-1594.2001.025007571.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Several configurations of extracorporeal bioartificial liver devices have been developed for the potential treatment of fulminant hepatic failure or as a bridge to liver transplantation. Recently, we developed a microchannel flat-plate bioreactor with an internal membrane oxygenator in which porcine hepatocytes are cultured as a monolayer on the bottom glass surface. In the present study, we investigated synthetic function of porcine hepatocytes in the bioreactor in both in vitro and in vivo flow circuit models. In vitro, albumin synthesis was stable in the bioreactor for up to 4 days of perfusion. In vivo, with the extracorporeal connection of the bioreactor to rat vasculature, porcine albumin was detectable for 24 h in the rat plasma. We also developed a simple mathematical model to predict the in vivo porcine albumin concentration in rat plasma. These results indicate that this configuration of a microchannel flat-plate bioreactor has potential as a liver support device and warrants further investigation.
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Affiliation(s)
- M Shito
- Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, U.S.A
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18
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Washizu J, Chan C, Berthiaume F, Tompkins RG, Toner M, Yarmush ML. Amino acid supplementation improves cell-specific functions of the rat hepatocytes exposed to human plasma. TISSUE ENGINEERING 2000; 6:497-504. [PMID: 11074936 DOI: 10.1089/107632700750022143] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Maintaining hepatocyte function during plasma exposure is critical for the successful development of hepatocyte-based bioartificial liver assist systems. Past attempts to culture hepatocytes in plasma yielded discouraging results. Using a stable culture model based on sandwiching hepatocytes between two layers of collagen gel, we investigated the effect of hormone and amino acid supplementation during exposure of rat hepatocytes to heparin-treated human plasma for 1 week. Morphology and hepatocyte-specific functions were evaluated for hepatocytes cultured in Dulbecco's Modified Eagle medium (DMEM), nonsupplemented plasma, plasma supplemented with hormones, or with hormones plus amino acids. Amino acids were supplemented at four-fold concentration of Basal Medium Eagle with 4 mM glutamine, whereas hormones included 7.5 microg/mL of hydrocortisone and 50 microU/mL of insulin. Cuboidal structure and bile canaliculi formation were observed throughout the 1-week exposure period for control hepatocytes in DMEM and for hepatocytes cultured in hormone supplemented plasma. Albumin and urea synthesis rates of hepatocytes in hormone plus amino acid supplemented plasma during the last day of plasma exposure were 60.4 +/- 13.7 and 75.6 +/- 6.5 (microg/day per 1 x 10(6) cells, mean +/- SD), respectively, comparable to cultures in standard culture medium. On the other hand, hepatocytes exposed to nonsupplemented plasma suffered significant morphological and functional damage. The results of this study indicate that hormone plus amino acid supplementation help to restore function in hepatocytes exposed to plasma.
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Affiliation(s)
- J Washizu
- Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospital for Children, Boston, Massachusetts 02114, USA
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Washizu J, Berthiaume F, Chan C, Tompkins RG, Toner M, Yarmush ML. Optimization of rat hepatocyte culture in citrated human plasma. J Surg Res 2000; 93:237-46. [PMID: 11027466 DOI: 10.1006/jsre.2000.5986] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
BACKGROUND Maintenance of liver-specific functions in hepatocyte cultures during plasma exposure is critical for the clinical application of bioartificial liver assist systems. Sodium citrate is a common anticoagulant but has been shown to be cytotoxic to hepatocytes. We have tested the effect of various supplements on the viability and function of adult primary rat hepatocytes exposed to citrated plasma. MATERIALS AND METHODS Freshly isolated rat hepatocytes were cultured in the collagen gel sandwich configuration in culture medium for 6 days followed by exposure to citrated human plasma with various supplements for 1 week. Controls were left in culture medium throughout. Viability and synthetic functions were evaluated. RESULTS Hepatocytes exposed to unsupplemented citrated plasma lost significant viability and function within the first 2 days. Cells cultured in plasma supplemented with a fivefold concentrate of standard hepatocyte culture medium maintained urea (1. 2-2.1 micromol/day/10(6) cells) and albumin (51-62 microg/day/10(6) cells) synthesis rates equal to or higher than those of controls. Among the various components of the concentrated medium supplement, calcium chloride (1.8 mM), magnesium sulfate (0.8 mM), amino acids (fourfold Basal Medium Eagle amino acids including 4 mM glutamine), and glucagon (14 ng/ml) were found to be essential in maintaining urea synthesis. Maintenance of a high albumin synthesis rate also required the addition of hydrocortisone (7.5 microg/ml) and insulin (0.5 U/ml). CONCLUSIONS Appropriate metabolic and hormonal supplementation of citrated human plasma prevents its cytotoxic effects and may be used in conjunction with in vivo use of bioartificial liver assist systems.
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Affiliation(s)
- J Washizu
- Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
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20
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Ledezma GA, Folch A, Bhatia SN, Balis UJ, Yarmush ML, Toner M. Numerical model of fluid flow and oxygen transport in a radial-flow microchannel containing hepatocytes. J Biomech Eng 1999; 121:58-64. [PMID: 10080090 DOI: 10.1115/1.2798043] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The incorporation of monolayers of cultured hepatocytes into an extracorporeal perfusion system has become a promising approach for the development of a temporary bioartificial liver (BAL) support system. In this paper we present a numerical investigation of the oxygen tension, shear stress, and pressure drop in a bioreactor for a BAL composed of plasma-perfused chambers containing monolayers of porcine hepatocytes. The chambers consist of microfabricated parallel disks with center-to-edge radial flow. The oxygen uptake rate (OUR), measured in vitro for porcine hepatocytes, was curve-fitted using Michaelis-Menten kinetics for simulation of the oxygen concentration profile. The effect of different parameters that may influence the oxygen transport inside the chambers, such as the plasma flow rate, the chamber height, the initial oxygen tension in the perfused plasma, the OUR, and K(m) was investigated. We found that both the plasma flow rate and the initial oxygen tension may have an important effect upon oxygen transport. Increasing the flow rate and/or the inlet oxygen tension resulted in improved oxygen transport to cells in the radial-flow microchannels, and allowed significantly greater diameter reactor without oxygen limitation to the hepatocytes. In the range investigated in this paper (10 microns < H < 100 microns), and for a constant plasma flow rate, the chamber height, H, had a negligible effect on the oxygen transport to hepatocytes. On the contrary, it strongly affected the mechanical stress on the cells that is also crucial for the successful design of the BAL reactors. A twofold decrease in chamber height from 50 to 25 microns produced approximately a fivefold increase in maximal shear stress at the inlet of the reactor from 2 to 10 dyn/cm2. Further decrease in chamber height resulted in shear stress values that are physiologically unrealistic. Therefore, the channel height needs to be carefully chosen in a BAL design to avoid deleterious hydrodynamic effects on hepatocytes.
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Affiliation(s)
- G A Ledezma
- Center for Engineering in Medicine, Massachusetts General Hospital, Shriners Burns Hospital, Harvard Medical School, Boston 02114, USA
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Balis UJ, Behnia K, Dwarakanath B, Bhatia SN, Sullivan SJ, Yarmush ML, Toner M. Oxygen consumption characteristics of porcine hepatocytes. Metab Eng 1999; 1:49-62. [PMID: 10935754 DOI: 10.1006/mben.1998.0105] [Citation(s) in RCA: 101] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Oxygen uptake rate (OUR) of hepatocytes is an important parameter for the design of bioartificial liver assist (BAL) devices. Porcine hepatocytes were cultured in a specially constructed measurement chamber with an incorporated mixing system and a Clark polarographic oxygen electrode. Signal noise associated with conventional Clark electrode implementations was circumvented by the combination of real time digital numerical averaging and subsequent finite impulse response (FIR) spectral filtering. Additional software allowed for the automated generation of cellular oxygen consumption coefficients, namely, Vmax and K0.5, adding a high degree of objectivity to parameter determination. Optimization of the above numerical techniques identified a 0.1 Hz/200 data point sample size and a 0.004 Hz cutoff frequency as ideal parameters. Vmax values obtained for porcine hepatocytes during the first two weeks of culture showed a maximal consumption of 0.9 nmole/sec/10(6) cells occurring on Day 4 post seeding, and a gradual decrease to 0.31 nmole/sec/10(6) cells by Day 15. K0.5 values increased from 2 mm Hg on Day 2 to 8 mm Hg by Day 8, with gradual subsequent decrease to 4 mm Hg by Day 15. The Vmax and K0.5 values measured for porcine cells were higher than maximal values for rat hepatocytes (Vmax: 0.43 nmole/sec/10(6) cells, K0.5: 5.6 mmHg) and thus may necessitate significantly altered BAL device design conditions to ensure no oxygen limitations. Finally, these results highlight the need for species specific characterization of cellular function for optimal BAL device implementations.
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Affiliation(s)
- U J Balis
- Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA
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