Gerlach J C, Brombacher J, Klöppel K, Schnoy N, Neuhaus P
Surgical Department, Universitätsklinikum Rudolf Virchow, Freie Universität Berlin, Germany.
Transplantation. 1994 May 15;57(9):1318-22. doi: 10.1097/00007890-199405150-00005.
Using the pig liver, parameters for large scale hepatocyte isolation were studied in order to develop a technique suitable for human organs. These investigations led to a 5-step modification of the original 2-step method. Four groups were compared. A nonenzymatic EDTA perfusion technique has been shown to be inconvenient for mass cell isolation. The enzymatic 2-step perfusion, using 0.08% collagenase and 20-kg pigs, resulted in a mean hepatocyte viability of 61 +/- 1.9%, with a mean yield of 67 +/- 6.5% wet weight of the organ. The enzymatic 5-step method resulted in a mean hepatocyte viability of 74 +/- 1.7% with a mean yield of 80 +/- 1.8% wet weight. Five-step portal venous perfusion in combination with arterial perfusion resulted in 76 +/- 2.6% viability with a yield of 82 +/- 6.1%. The results were dependent on collagenase concentration and weight of the donors, improving with decreasing body weight. The 5-step method with combined arterial and portal vein perfusion developed for pig liver was used for mass human liver cell isolation with a minimum viability of 57% and a minimum yield of 58% wet weight.
为了开发一种适用于人体器官的技术，利用猪肝研究了大规模肝细胞分离的参数。这些研究导致了对原始两步法的五步改进。比较了四组。已证明非酶促乙二胺四乙酸灌注技术对于大规模细胞分离不方便。使用0.08%胶原酶和20千克猪的酶促两步灌注法，肝细胞平均活力为61±1.9%，平均产量为器官湿重的67±6.5%。酶促五步法肝细胞平均活力为74±1.7%，平均产量为湿重的80±1.8%。五步门静脉灌注联合动脉灌注的活力为76±2.6%，产量为82±6.1%。结果取决于胶原酶浓度和供体体重，随着体重减轻而改善。为猪肝开发的联合动脉和门静脉灌注的五步法用于大规模人肝细胞分离，最低活力为57%，最低产量为湿重的58%。
