Kino Y, Sawa M, Kasai S, Mito M
Second Department of Surgery, Asahikawa Medical College, 4-5, Nishikagura, Asahikawa, Hokkaido, 078-8510, Japan.
J Surg Res. 1998 Sep;79(1):71-6. doi: 10.1006/jsre.1998.5389.
This study was aimed at developing an optimal method for immobilizing isolated hepatocytes in cellulose multiporous microcarriers (MCs) and evaluating the metabolic activity of MC-immobilized hepatocytes.
Hepatocytes isolated from the livers of male Wistar rats were immobilized in collagen-coated MCs by intermittent stirring (30 rpm for 2 min per 15 min) for 180 min or accumulation methods. The accumulation method was performed by pouring aliquots of hepatocyte suspension (8 x 10(5)) and MC suspension (1 mg) in turn onto a nylon mesh (pore size: 100 micron). The metabolic activity of MC-immobilized hepatocytes in floating culture and in a newly developed bioreactor was evaluated. The metabolic activity of MC-immobilized hepatocytes in the bioreactor was also evaluated in in vitro perfusion of a hollow-fiber-based hybrid artificial liver support system.
The accumulation method immobilized 20 times more hepatocytes in collagen-coated MCs than the intermittent stirring method (P < 0.01). Morphological observation of hepatocyte-immobilized MCs revealed that many hepatocytes were immobilized deep within the MCs maintaining a spherical shape and normal microvilli on their surface. MC-immobilized hepatocytes in floating culture revealed similar NH3 metabolism and glucose synthesis to monolayer-cultured hepatocytes, and this metabolic activity was maintained during 9h of floating culture. MC-immobilized hepatocytes in a bioreactor also showed similar NH3 metabolism to monolayer-cultured hepatocytes. The NH3 metabolism of MC-immobilized hepatocytes in in vitro perfusion of a hybrid artificial liver support system was 241.5 microg/h/mg protein/m2 membrane surface.
The results of this study indicate that the accumulation method was optimal for immobilizing isolated rat hepatocytes in MCs and that MC-immobilized hepatocytes maintained their metabolic activity for a long period.
本研究旨在开发一种将分离的肝细胞固定于纤维素多孔微载体（MCs）中的最佳方法，并评估固定于MCs的肝细胞的代谢活性。
从雄性Wistar大鼠肝脏分离的肝细胞，通过间歇搅拌（每15分钟以30转/分钟搅拌2分钟）180分钟或累积法固定于胶原包被的MCs中。累积法是将肝细胞悬液（8×10⁵）和MC悬液（1毫克）的等分试样依次倾注到尼龙网（孔径：100微米）上。评估了固定于MCs的肝细胞在悬浮培养和新开发的生物反应器中的代谢活性。还在基于中空纤维的混合人工肝支持系统的体外灌注中评估了生物反应器中固定于MCs的肝细胞的代谢活性。
累积法在胶原包被的MCs中固定的肝细胞数量比间歇搅拌法多20倍（P<0.01）。对固定有肝细胞的MCs进行形态学观察发现，许多肝细胞固定于MCs内部深处，保持球形且表面有正常微绒毛。悬浮培养中固定于MCs的肝细胞显示出与单层培养肝细胞相似的氨代谢和葡萄糖合成，并且这种代谢活性在悬浮培养9小时期间得以维持。生物反应器中固定于MCs的肝细胞也显示出与单层培养肝细胞相似的氨代谢。在混合人工肝支持系统的体外灌注中，固定于MCs的肝细胞的氨代谢为241.5微克/小时/毫克蛋白/平方米膜表面积。
本研究结果表明，累积法是将分离的大鼠肝细胞固定于MCs的最佳方法，并且固定于MCs的肝细胞长时间维持其代谢活性。
