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A study of long term cryopreservation of a bio-artificial liver for clinical applications.

Research Project

Project/Area Number 09557093
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field General surgery
Research InstitutionHOKKAIDO UNIVERSITY

Principal Investigator

MATSUSHITA Michiaki  Hokkaido Univ., School of Med., Asso. Pro., 医学部, 助教授 (20250425)

Co-Investigator(Kenkyū-buntansha) TODO Satoru  Hokkaido Univ., School of Med., Pro., 医学部, 教授 (60136463)
GANDO Satoshi  Hokkaido Univ., School of Med., Pro., 医学部, 教授 (30125306)
MURABAYASHI Shun  Hokkaido Univ., School of Med., Asso. Pro., 大学院・工学研究科, 助教授 (30200306)
高橋 学  北海道大学, 医学部附属病院, 医員
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥12,800,000 (Direct Cost: ¥12,800,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1997: ¥8,500,000 (Direct Cost: ¥8,500,000)
Keywordsspheroid / rapid / bio-aritificial liver / cryoreservation / urea synthesis / albumin / culture condition / collagen gel / ハイブリッド型人工肝 / 回旋培養 / 冷凍保存 / ハイブリッド型人工用
Research Abstract

1.An experimental study of rapid formation of hepatocyte spheroids.
We established methods of a rapid formation and a large scale production of hepatocyte spheroids as a bioreactor of bio-artificial liver. Isolated rat hepatocytes prepared by using perfusion buffers excluding EGTA and Ca2+. About 80% of isolated hepatocytes formed hepatocyte spheroids in 6 hours in rotation culture. In preliminary studies using pig hepatocytes, ten times concentrated pig hepatocytes compared with rat hepatocytes formed hepatocyte spheroids in 6 hours in rotation culture.
2..Cryopreservation of rat hepatocyte spheroids.
We examined cryopreservation of rat hepatocytes spheroids. Hepatocyte spheroids were suspended in cryoprotectant medium(L-15 + 10%(v/v) FCS + 10%(v/v) DMSO) for 20 minutes at room temperature. The suspension was supercooled at -5.5℃ and maintained for 15 minutes after ice formation. A freezing ratio of 1℃/min was applied until -40℃ and hepatocyte spheroids were stored at -80℃ for 7 to 10 da … More ys. Cryopreserved hepatocyte spheroids were rapidly thawed in a 37℃ water bath and stepwise dilutions of cryoprotectant medium were performed. Phasecontrast microscopic observations, an amount of LDL retention, urea synthesis and albumin productions were examined. Morphological examinations revealed hepatocyte spheroids forming a round shape after rotation culture with some blebs on the surface, and these blebs remained during submerging in cryoprotectant medium. Hepatocyte spheroids maintained the round shape after thawing, but those blebs were destroyed. The amount of LDH retention was about 27% of fresh spheroids after thawing and declined continuously. Urea synthesis was about 50% of fresh ones and declined to 18% after 24 hours in culture. Albumin productions were significantly impaired by cryopreservation, the amounts were 1/200 compared with fresh ones.
In this study, an unreversal death of hepatocyte spheroids occurred by cryopreservation. We speculated the destruction of blebs caused lethal insults in spheroids. But cryopreserved hepatocyte spheroids were available for short duration in themes of detoxification. We have to establish a more sophisticated method to make hepatocyte spheroids.
3..Culture conditions of hepatocyte spheroids
Urea synthesis of hepatocyte spheroids declined in a early period in culture, but can be maintained high levels for a long duration in the collagen gel culture conditions. Less

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] 松下通明: "肝移植と人工肝"組織培養工学. 23. 35-39 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 松下通明: "培養肝細胞を用いた、肝障害時に増加する血清胆汁酸の肝細胞障害性の検討"日本外科系連合学会誌. 22. 890-894 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 松下通明: "人工肝臓-この1年の進歩"人工臓器. 27. 804-805 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 松下通明: "バイオ人工肝臓"組織培養工学. 24・5. 188 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hiromitsu Noto: "Effect of High Concentrations of Bile Acids on Cultured Hepatocytes"Artificial Organs. 22(4). 300-307 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 大久保尚: "ハイブリッド型人工肝作製を目指した至適大量肝細胞単離法の検討"日本外科学会雑誌. 100. 558 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 松下通明: "人工臓器と再生医工学"北海道医報. 938. 6-9 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Michiaki Matsushita: "Liver transplantation and Bioartificial Liver"The Tissue Culture Engineering. 23. 35-39 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Michiaki Matsushita: "Cytotoxicity of Bileacid Elevated in Liver Failure on Cultured Hepatocytes"Journal of Japanese College of Surgeons. 22. 890-894 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Michiaki Matsushita: "Bioartificial Liver-progress in this year"The Japanese Journal of Artificial Organs. 27. 804-805 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Michiaki Matsushita: "Dioartificial Liver"The Tissue Culture Engineering. 24(5). 188 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hiromitsu Noto: "Effect of High Concentrations of Bile Acids on Cultured Hepatocytes"Artificial Organs. 22(4). 300-307 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Hisashi Okubo: "A Moderate isolation method of hepatocytes for Hybrid Bioartificral Liver"Journal of Japan Surgical Society. 100. 558 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Michiaki Matsushita: "Artificial Organ and Tissue Reconstruction"Hokkaido Medical Journal. 938. 6-9 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 松下 通明: "人工臓器と再生医工学"北海道医報. 938. 6-9 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 松下 通明: "人工肝臓-この1年の進歩" 人工臓器. 27. 804-805 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 松下 通明: "バイオ人工肝臓" 組織培養工学. 24・5. 188 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Hiromitsu Noto: "Effect of High Concentrations of Bile Acids on Cultured Hepatocytes" Artificial Organs. 22(4). 300-307 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 大久保 尚: "ハイブリッド型人工肝作製を目指した至適大量肝細胞単離法の検討" 日本外科学会雑誌. 100. 558 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] 松下 通明: "肝移植と人工肝" 繊維培養工学. 23. 35-39 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 能登 啓光: "培養肝細胞を用いた、肝障害時に増加する血清胆汁酸の肝細胞障害性の検討" 日本外科系連合学会誌. 22. 890-894 (1997)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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