Dabos K J, Nelson L J, Bradnock T J, Parkinson J A, Sadler I H, Hayes P C, Plevris J N
Liver Cell Biology Laboratory, Department of Internal Medicine, University of Edinburgh, Edinburgh, UK.
Biochim Biophys Acta. 2001 May 3;1526(2):119-30. doi: 10.1016/s0304-4165(01)00097-6.
The high aspect ratio vessel allows the culture of primary porcine hepatocytes in an environment of low shear stress and simulated microgravity. Primary porcine hepatocytes have been difficult to maintain in culture long term while preserving their metabolic functions. This study was carried out in order to characterise key metabolic functions of cell aggregates formed by primary porcine hepatocytes cultured in a high aspect ratio vessel for a predetermined period of 21 days. 10(8) porcine hepatocytes were loaded into the high aspect ratio vessel and continuously rotated during the experiments. 0.7 ml of the culture medium was sampled on days 1, 2, 4, 7, 10, 14 and 21. 1H nuclear magnetic resonance spectroscopy of the culture medium, using the presaturation technique, assessed the following: glucose metabolism, glutamine synthesis and ketogenesis. There was glucose breakdown anaerobically during the first 10 days as manifested by lactate production and pyruvate and threonine consumption. After day 10 there was significantly smaller lactate production (day 1 vs day 10 P < 0.01), and significantly smaller pyruvate (day 1 vs day 14 P < 0.03) and threonine consumption (day 1 vs day 10 P < 0.002), indicative of an aerobic metabolic pattern. Significantly more glutamate was produced after day 10 (day 1 vs day 10 P < 0.031), and more glutamine was consumed after day 14. There was a steadily diminishing production of acetate which reached a minimum on day 14 (day 2 vs day 14 P < 0.00014). After an initial 10 day period of acclimatisation cell aggregates formed in the high aspect ratio vessel switched from the anaerobic pattern of metabolism to the more efficient aerobic pattern, which was exhibited until the experiments were terminated. The high aspect ratio vessel is suitable for long-term culture of porcine hepatocytes and it is worthwhile carrying out scale-up feasibility studies.
高纵横比容器允许在低剪切应力和模拟微重力环境中培养原代猪肝细胞。原代猪肝细胞长期维持培养并保持其代谢功能一直很困难。本研究旨在表征在高纵横比容器中培养21天预定时间的原代猪肝细胞形成的细胞聚集体的关键代谢功能。将10⁸个猪肝细胞加载到高纵横比容器中，并在实验过程中持续旋转。在第1、2、4、7、10、14和21天采集0.7 ml培养基。使用预饱和技术对培养基进行¹H核磁共振波谱分析，评估以下内容：葡萄糖代谢、谷氨酰胺合成和生酮作用。在最初的10天内存在无氧葡萄糖分解，表现为乳酸生成以及丙酮酸和苏氨酸消耗。第10天后，乳酸生成显著减少（第1天与第10天，P < 0.01），丙酮酸（第1天与第14天，P < 0.03）和苏氨酸消耗显著减少（第1天与第10天，P < 0.002），表明为有氧代谢模式。第10天后产生的谷氨酸显著增多（第1天与第10天，P < 0.031），第14天后谷氨酰胺消耗增多。乙酸盐产量稳步下降，在第14天达到最低值（第2天与第14天，P < 0.00014）。经过最初10天的适应期后，在高纵横比容器中形成的细胞聚集体从无氧代谢模式转变为更高效的有氧代谢模式，这种模式一直持续到实验结束。高纵横比容器适用于猪肝细胞的长期培养，值得进行放大可行性研究。
