| Project/Area Number |
12470251
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Digestive surgery
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| Research Institution | University of Tsukuba |
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Principal Investigator |
YANAGI Kennichi Institute of Basic Medical Sciences, Lecturer, 基礎医学系, 講師 (70239797)
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| Co-Investigator(Kenkyū-buntansha) |
TAKADA Yasutsugu University of Kyoto, School of Medicine, Associate professor, 医学部, 助教授 (10272197)
MIYOSHI Hirotoshi Institute of Basic Medical Sciences, Assistant professor, 基礎医学系, 講師 (70292547)
OHSHIMA Norio Institute of Basic Medical Sciences, Professor, 基礎医学系, 教授 (50015971)
KANAMORI Toshiyuki National Institute of Advanced Industrial Science and Technology, Institute for Materials and Chemical Process, Chief Researcher, 物質プロセス研究部門, 主任研究官
TANIGUCHI Hideki Yokohama City University, School of Medicine, Professor, 医学部, 教授 (70292555)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2002: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 2000: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Keywords | Bioartificial liver / Porcine / Fulminant hepatic failure / Packed-bed type reactor / α-amanitine / lipopolisaccharide / High-density culture of hepatocytes / Ammonium / 生存時間 / 動物モデル / 充填層型リアクタ / ポリビニルホルコール樹脂多孔質体 / 肝細胞 |
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| Research Abstract |
The authors have developed a packed-bed type bioreactor using porous polyvinyl formal resin as cell supporting material tor use as a bioartificial liver. Large scale and high density culture of hepatocytes was achieved using the reactor. In the present study, the reactor volume was scaled up from a 10 cm^3 of the prototype to 1,000 cm^3, and the metabolic performance of the reactor was evaluated in in vitro culture experiments and ex vivo perfusion experiments using a fulminant hepatic failure model of a porcine.
[Methods] A PVF resin with a mean pore size of 250 μm cut into cubes (2 mm or 3 mm) was used as a cell supporting material. Packed-bed reactors with the culture volume 1,000 cm^3 were used. Hepatocytes obtained from pigs were inoculated onto the PVF resin. Culture medium (Williams' E medium supplemented with 10% fetal bovine serum, hormones and antibiotics) was perfused from the reservoir into the reactor through an oxygenator. Oxygen concentration and pH were measured at the o
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utlet of the reactor, and were maintained at 40% and 7.4, respectively. In in vitro culture experiments performed for a week, metabolic activities of the cultured hepatocytes were evaluated in terms of a few liver-specific functions, i.e., ammonium metabolism, urea synthesis, lidocaine metabolism and albumin secretion. The immobilized cell density was measured by the DNA quantity in the PVF resin cubes. Morphology of the immobilized hepatocytes was observed. In ex vivo perfusion experiments, acute hepatic failure was induced by injecting alpha-amanitine (0.15 mg/kg) and lipopolysaccharide (1 μg/kg) in the portal vein of pigs (8 - 12 kg). Twenty-two hours after injection of the drugs, porcine blood was dialyzed against the perfusate of the packed-bed type reactor using a hollow-fiber type dialyzer. Blood samples were drawn periodically for later analysis. Extracorporeal perfusions using the module without hepatocytes were performed as a control.
[Results and Conclusion] Hepatocytes were well immobilized in the PVF. The maximum cell count in the reactor reached a value of 1.2 x 10^<10> cells (120 g) on Day 2. The cell density gradually decreased with culture time. Hepatocytes immobilized in the packed-bed reactor showed satisfactory and stable metabolic activities comparable to those in vivo liver. Morphological observation revealed that the immobilized cells showed well-differentiated morphologies similar to those observed in vivo. In ex vivo perfusion experiments, following the administration of α-amanitine and LPS, pigs showed a diseased state of fulminant hepatic failure, and died at an average of 50 hours after injection of the drugs. In the pigs that received hepatic support from the bioartificial liver, the rise in the blood ammonium concentration was suppressed, and there was a tendency for an extension in survival times. In conclusion, the packed-bed type bioreactor is a promising module of importance in developing a bioartificial liver. Less
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