| Co-Investigator(Kenkyū-buntansha) |
OKUBO Hisashi Hokkaido Univ., Medical Hosp., Physician., 医学部・附属病院, 医員
MURABAYASHI Shun Hokkaido Univ., Grad.School of Eng., Asso.Pro., 大学院・工学研究科, 助教授 (30200306)
TODO Satoru Hokkaido Univ., Grad.School of Med., Pro., 大学院・医学研究科, 教授 (60136463)
KAMACHI Hirofumi Hokkaido Univ., Medical Hosp., Physician, 医学部・附属病院, 医員
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| Research Abstract |
This project was aimed to make artificial liver using porcine mixed liver cell sphroids, and to evaluate its function.
(1)Mass production of porcine mixed liver cell spheroid
Without chelating agent in cell dispersion, population of non-parenchymal cells were increased, on the contrary, cell viability and yield were decreased. In the collagenase solution, addition of disperse increased the yields and viability of hepatocytes and suppressed the spheroid formation. Production of spheroids could be acheved up to 2.5 x 10^7 cells/ml concentration of hepatocytes in the 1L size spinner flask. Suitable method for porcine liver cell spheroid formation is consisted of rinse with chelating agents, enzyme digestion by collagenase, and spinning culture in 1L sppiner flask with 2.5 x 10^10hepatocytes.
(2)Basic experiments : application of artificial blood for artificial liver
Evaluation of oxygen transport ability of artificial blood was studied in comparison with red blood cells and culture medium in
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vitro. Oxygen supply capability of artificial blood was equal to that of RBC.In addition, oxygen consumption rate of hepatocyte using artificial blood was the highest of all. Artificial blood could be a way to oxygen supply in artificial liver using hepatocytes.
(3)Fabrication and function of artificial liver module
Two channnel hollow fiber module, we have already developed, contained 2g amount of spheroids. The one channnel was for oxygenation and the other was for plasma circulation. Pyricoxalated-Hemoglobin-Polyoxyethylene conjugate : PHP was used as an oxygen carrier in the oxygenation circuit and was compared to the culture medium. Anmonium cloride clearance were higher in the PHP group(32% / 2hr)compared to the control group(7.4% / 2hr), and urea and albumin production were also observed. However, PHP easily leaked from the hollow fiber and transit to the other channnel, so the membrane of the fiber must be changed to fiber with smaller pore size. In addition, as the spheroids enclosed in the extra-capillary space aggregated each other, the function of the module was decreased. Therefore, coating or encapsulation of spheroids are considered to be needed for the maintenance of function. Less
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