1999 Fiscal Year Final Research Report Summary
A study of long term cryopreservation of a bio-artificial liver for clinical applications.
| Project/Area Number |
09557093
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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 | 展開研究 |
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| Research Field |
General surgery
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
MATSUSHITA Michiaki Hokkaido Univ., School of Med., Asso. Pro., 医学部, 助教授 (20250425)
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| 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)
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| Project Period (FY) |
1997 – 1999
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| Keywords | spheroid / rapid / bio-aritificial liver / cryoreservation / urea synthesis / albumin / culture condition / collagen gel |
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| 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
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