Stefanovich P, Matthew H W, Toner M, Tompkins R G, Yarmush M L
Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital and the Shriners Burns Institute, Boston, Massachusetts, 02114, USA.
J Surg Res. 1996 Nov;66(1):57-63. doi: 10.1006/jsre.1996.0372.
The most promising approaches to developing a temporary bioartificial liver support system involve incorporating cultured primary hepatocytes into an extracorporeal perfusion device. As a result, it is important to characterize both the phenotypic response of these cells during extracorporeal perfusion and the critical factors involved in maintaining differentiated cell function over extended periods of perfusion. In this study, hepatocytes cultured in a collagen sandwich configuration were connected to a rat via a hollow fiber plasma separator and perfused with plasma on line. Perfusions were either continuous for 48 hr or intermittent for up to 174 hr with 6 hr per day of extracorporeal plasma perfusion alternating with 18 hr of culture medium perfusion. During perfusion cell morphology was continuously monitored by time-lapse video microscopy. After the procedure, hepatocytes were returned to static culture and function was evaluated by measuring the rates of urea synthesis daily for 7 days. During plasma perfusion all hepatocytes accumulated cytoplasmic lipid droplets in a time dependent manner. Urea synthesis was maintained at initial levels for up to 20 hr of continuous plasma perfusion. However, urea synthesis rates were reduced by 31 and 52% after 30 and 48 hr of continuous plasma exposure, respectively. With intermittent perfusions, as well as with control cells perfused with culture medium only, urea synthesis rates did not decrease for at least 78 hr of total perfusion. There was no difference between the urea synthesis rates after 48 hr of cumulative plasma exposure time between cells subjected to continuous and intermittent plasma perfusion. These results suggest that cultured hepatocytes may be exposed to plasma for at least 20 hr with no significant reduction in liver-specific function. Furthermore, an intermittent plasma perfusion schedule can be used to divide the useful plasma perfusion time over several days with no adverse effects on cell function.
开发临时生物人工肝支持系统最有前景的方法包括将培养的原代肝细胞整合到体外灌注装置中。因此，表征这些细胞在体外灌注过程中的表型反应以及在长时间灌注过程中维持分化细胞功能所涉及的关键因素非常重要。在本研究中，将以胶原夹心结构培养的肝细胞通过中空纤维血浆分离器与大鼠相连，并在线用血浆进行灌注。灌注持续48小时或间歇性进行长达174小时，每天进行6小时的体外血浆灌注与18小时的培养基灌注交替。在灌注过程中，通过延时视频显微镜连续监测细胞形态。实验结束后，将肝细胞放回静态培养，并通过每天测量尿素合成速率，持续7天来评估其功能。在血浆灌注期间，所有肝细胞均以时间依赖性方式积累细胞质脂滴。在持续血浆灌注长达20小时时，尿素合成维持在初始水平。然而，在持续血浆暴露30小时和48小时后，尿素合成速率分别降低了31%和52%。对于间歇性灌注以及仅用培养基灌注的对照细胞，在至少78小时的总灌注时间内，尿素合成速率没有下降。在连续和间歇性血浆灌注的细胞之间，累积血浆暴露时间48小时后的尿素合成速率没有差异。这些结果表明，培养的肝细胞可以暴露于血浆中至少20小时，而肝脏特异性功能没有显著降低。此外，间歇性血浆灌注方案可用于将有效的血浆灌注时间分散在几天内，而对细胞功能没有不利影响。
