闭合型双循环生物人工肝支持系统的构建

Construction of closed bi-circulating bioartificial liver support system (CBC-BALSS)

目的 构建简便、安全和稳定的闭合型双循环生物人工肝支持系统 (closedbi -circulat ingbioartificialliversupportsystem ,CBC -BALSS)。方法 采用改良两步原位胶原酶灌注法分离猪肝细胞。利用肝细胞、细胞池和BiolivA3A生物反应器构建CBC -BALSS ,进行 8次闭合循环实验 ,监测双循环路压力 ,肝细胞密度和活率、生化全套和MEGX。结果 闭合循环过程中 ,双循环路压力于 30min后趋于平稳至 (10 5± 5 )mmHg。循环中肝细胞总数、活率和密度变化无显著性差异。细胞循环路ALT、LDH、TB、DB和ALB在各时间点间均不存在显著性差异 ;TB和DB显著性升高。结论 在闭合循环实验中 ,肝细胞活率和功能良好 ,双循环路压力平稳 ,生物膜通透性好。我们构建的CBC -BALSS安全、稳定、有效 。

Objective To construct stable, safe and efficacious CBC-BALSS. Methods Suckling swine hepatocytes were isolated by modified two-setp in situ collagenase perfusion method and cultured in serum-free medium at high density. CBC-BALSS was constructed with hepatocytes, cell kettle and Bioliv A3A bioreator. The hepatocytes in closed tube were circulated 8 times in this study. During every circulation, bi-circulating pressure were observed, while cell viability, density, biochemistrical index and MEGX were also evaluated. Results During circulation, bi-circulating pressure was elevated to 105 mmHg gradually with fluctuating around ±5 mmHg. The cell number, viability, density, ALT, TB, DB, ALT and LDH were stable in whole circulation. Change of MEGX in circulating hepatocytes were similar to those in stirring culture ones. There are no significant difference in ALT, LDH and ALB in RPMI1640 circulation at each time point, but TB and DB were elevated significantly. Conclusions The viability and function of hepatocytes and pressure in bicirculation is maintained well in closed circulating experiment, so as to the permeability of the membrane in bioreator. CBC-BALSS constructed with Bioliv A3A and cell kettle successfully is stable, safe and efficacious.