Chung Yu Jeong, Lee Hyuk Joon, Koh Young Taeg, Kim Sang Beom, Kim Seong Hoon, Choi Seok Ho, Yi Nam Joon, Chang Seong Hwan, Yang Eun Lan, Suh Kyung Suk, Lee Yoon Shin, Lee Kuhn Uk
Department of Surgery, Seoul National University College of Medicine, Seoul, Korea.
Taehan Kan Hakhoe Chi. 2002 Sep;8(3):249-55.
BACKGROUND/AIMS: Acute hepatic failure is a serious problem. Its mortality reaches up to 80%. Only liver transplantation has been accepted as a definite treatment for patients with hepatic failure but shortage of donor organs is the main obstacle of this approach. A possible solution to this problem is a bioartificial liver system, perfusion of patients blood to isolated hepatocyte. In this study, we performed the isolation and culture of pig hepatocyte in large scale for the application of bioartificial liver system.
Hepatocyte isolation was performed by two-step collagenase method via portal vein perfusion in 10 kg female pigs. After that, we compared the functional differences of the spheroid culture to the monolayer culture of hepatocyte. The viability and the function of hepatocyte were assessed using trypan-blue exclusion test and the measurement of the rate of ureagenesis and ammonia removal.
The average viability and yield of hepatocyte were 86.8 +/- 8.0 % and 7.8 +/- 5.4 X 10(9), respectively. The spheroid culture was superior to the monolayer culture in functional aspect of hepatocyte, and their differences, especially for ammonia removal, were more apparent in parallel with culture time.
For hepatocyte isolation, we obtained sufficient viability and yield of hepatocyte for clinical usage of bioartificial liver system. The function of hepatocyte seems to be better in the spheroid culture than in the monolayer culture. Further studies are needed for application of bioartificial liver system in clinical setting.
背景/目的：急性肝衰竭是一个严重的问题。其死亡率高达80%。目前只有肝移植被公认为是肝衰竭患者的确定性治疗方法，但供体器官短缺是这种治疗方法的主要障碍。解决这一问题的一个可能办法是生物人工肝系统，即将患者血液灌注到分离的肝细胞中。在本研究中，我们为生物人工肝系统的应用进行了大规模猪肝细胞的分离和培养。
采用两步胶原酶法经门静脉灌注对10kg雌性猪进行肝细胞分离。之后，我们比较了肝细胞球状体培养与单层培养的功能差异。使用台盼蓝排斥试验以及尿素生成率和氨清除率的测定来评估肝细胞的活力和功能。
肝细胞的平均活力和产量分别为86.8±8.0%和7.8±5.4×10⁹。在肝细胞功能方面，球状体培养优于单层培养，并且随着培养时间的延长，它们之间的差异，尤其是氨清除方面的差异更加明显。
对于肝细胞分离，我们获得了足够的活力和产量，可用于生物人工肝系统的临床应用。肝细胞在球状体培养中的功能似乎比单层培养更好。生物人工肝系统在临床环境中的应用还需要进一步研究。
