Morsiani E, Puviani A C, Brogli M, Pazzi P, Tosatti S, Valieri L, Gorini P, Fogli L, Galavotti D, Borra M, Bellini C, Squarzoni P, Ricci D, Reverberi R, Azzena G
Patologia Speciale Chirurgica dell'Università di Ferrara ed Azienda Ospedaliera Arcispedale Sant'Anna, Ferrara.
Ann Ital Chir. 2000 May-Jun;71(3):337-45.
Hepatocyte based artificial liver support systems are under investigation to support acute liver failure patients. The main purpose of such systems is to serve as a bridge to liver transplantation, or to promote spontaneous liver recovery. Limitation in mass-transfer capacity makes hollow-fiber bioreactors unsuited for long-term functioning of hybrid devices. We developed a novel radial-flow bioreactor in which the fluid perfuses the module from the center to the periphery, after having diffused through a space occupied by a three-dimensional structure filled with the hepatocytes. Five grams of freshly isolated porcine hepatocytes were seeded into uncoated, woven-non woven, hydrophilic polyester fabric, overlaid by two polyethersulfone membranes. Liver cells were perfused with 37 degrees C-warm, oxygenated, serum-free tissue culture medium, in which NH4Cl and Lidocaine were added at the final concentration of 1 mM and 60 micrograms/ml, respectively. Ammonium chloride removal, urea synthesis, monoethylglycinexylide (MEGX), pO2, pCO2, and pH were measured throughout the 14 day duration of the study. In a separate set of experiments, a scaled-up version of the radial flow bioreactor containing 150 grams of cells was perfused for 7 h with recirculating human plasma and MEGX production was monitored. During the 2 weeks of the study, an increasing production of urea was paralleled by constant ammonium removal. MEGX concentration after Lidocaine addition increased throughout the 14 days of perfusion with tissue culture medium, as well as after 7 hour perfusion with human plasma. Under transmission and scanning electron microscopy cells appeared attached to the polyester and one to each other, displaying ultrastructural features typical of functioning hepatocytes. Our study showed that liver cells were metabolically active when perfused into the radial-flow bioreactor. This configuration allowed close contact between media, or plasma, and cells at a physiological flow rate, by equalizing the concentration of the perfusing components, including O2, throughout the module. Our results suggest a potential use of this system for temporary extracorporeal liver support in acute hepatic failure patients.
基于肝细胞的人工肝支持系统正在进行研究，以支持急性肝衰竭患者。此类系统的主要目的是作为肝移植的桥梁，或促进肝脏自发恢复。传质能力的限制使得中空纤维生物反应器不适用于混合装置的长期运行。我们开发了一种新型的径向流生物反应器，流体在通过充满肝细胞的三维结构所占据的空间扩散后，从中心向周边灌注模块。将5克新鲜分离的猪肝细胞接种到未涂层的、编织-非编织的亲水性聚酯织物上，上面覆盖两层聚醚砜膜。肝细胞用37℃温热、充氧、无血清的组织培养基灌注，其中氯化铵和利多卡因的最终浓度分别为1 mM和60μg/ml。在整个14天的研究期间，测量氯化铵清除率、尿素合成、单乙基甘氨酰二甲苯胺（MEGX）、pO2、pCO2和pH。在另一组实验中，用含有150克细胞的放大版径向流生物反应器灌注循环人血浆7小时，并监测MEGX的产生。在研究的2周内，尿素产量增加的同时氯化铵清除量保持恒定。在组织培养基灌注14天期间以及用人血浆灌注7小时后，加入利多卡因后的MEGX浓度均升高。在透射电子显微镜和扫描电子显微镜下，细胞似乎附着在聚酯上且彼此相连，呈现出功能正常的肝细胞典型的超微结构特征。我们的研究表明，将肝细胞灌注到径向流生物反应器中时，肝细胞具有代谢活性。这种结构通过使包括O2在内的灌注成分在整个模块中的浓度均匀化，在生理流速下使培养基或血浆与细胞紧密接触。我们的结果表明该系统在急性肝衰竭患者的临时体外肝支持方面具有潜在用途。
