Catapano G, De Bartolo L
Department of Chemical and Materials Engineering, University of Calabria, I-87036 Arcavacata di Rende (CS), Italy.
Biotechnol Prog. 1998 May-Jun;14(3):500-7. doi: 10.1021/bp9800182.
In this paper, we report on the development of a technique for the kinetic characterization of the metabolic reactions of liver cells in adhesion culture. The technique is based on the use of a continuous-flow bioreactor which is designed and operated in such a way as to ensure a uniform distribution of metabolite at the cell site: hence, the metabolite concentration at the surface of cells cultured in adhesion at the bottom of the bioreactor equals that in the stream leaving the bioreactor. Under steady conditions, the rate of a given cell reaction is directly estimated from the metabolite concentration difference in the streams entering and leaving the bioreactor and can be correctly related to the actual concentration at the cell surface. Such a technique was used for a preliminary investigation of the kinetics of ammonia elimination, urea synthesis, and phenolsulfonphthalein (PSP) elimination by primary rat hepatocytes cultured in adhesion on collagen, with respect to ammonia and PSP concentration, respectively. The rate at which the hepatocytes eliminated ammonia increased with increasing ammonia concentrations according to a Michaelis-Menten kinetics. The hepatocytes synthesized urea also in the absence of ammonia in the medium: as ammonia concentration increased, the cells synthesized urea at a rate that increased according to a saturation kinetics. In the concentration range investigated, the hepatocytes eliminated PSP at a rate that increased linearly with the actual PSP concentration in the medium. Such kinetic information can be coupled to the mechanism of metabolite transport in a hybrid liver support device to yield an effective device design for the treatment of acute liver failure.
在本文中，我们报告了一种用于贴壁培养的肝细胞代谢反应动力学表征技术的开发情况。该技术基于一种连续流生物反应器的使用，该反应器的设计和操作方式可确保代谢物在细胞位点处均匀分布：因此，在生物反应器底部贴壁培养的细胞表面的代谢物浓度与离开生物反应器的流中的代谢物浓度相等。在稳定条件下，给定细胞反应的速率可直接根据进入和离开生物反应器的流中的代谢物浓度差来估算，并且可以与细胞表面的实际浓度正确关联。这种技术用于初步研究原代大鼠肝细胞在胶原蛋白上贴壁培养时氨的消除、尿素合成以及酚红（PSP）消除的动力学，分别针对氨和PSP浓度。肝细胞消除氨的速率根据米氏动力学随氨浓度的增加而增加。即使培养基中不存在氨，肝细胞也会合成尿素：随着氨浓度的增加，细胞合成尿素的速率根据饱和动力学增加。在所研究的浓度范围内，肝细胞消除PSP的速率与培养基中实际PSP浓度呈线性增加。这种动力学信息可与混合肝支持装置中的代谢物转运机制相结合，以产生用于治疗急性肝衰竭的有效装置设计。
