Table_1_Spheroid Size Does not Impact Metabolism of the β-blocker Propranolol in 3D Intestinal Fish Model.docx

Compared to two-dimensional (2D) cell culture, cellular aggregates or spheroids (3D) offer a more appropriate alternative in vitro system where individual cell-cell communication and micro-environment more closely represent the in vivo organ; yet we understand little of the physiological conditions at this scale. The relationship between spheroid size and oxygen microenvironment, an important factor influencing the metabolic capacity of cells, was first established using the fish intestine derived RTgutGC cell line. Subsequently, pharmaceutical metabolism (Propranolol), as determined by high performance liquid chromatography, in this intestinal model was examined as a function of spheroid size. Co-efficient of variation between spheroid size was below 12% using the gyratory platform method, with the least variation observed in the highest cell seeding density. The viable, high oxygen micro-environment of the outer rim of the spheroid, as determined by electron paramagnetic resonance (EPR) oximetry, decreased over time, and the hypoxic zone increased as a function of spheroid size. Despite a trend of higher metabolism in smaller spheroids, the formation of micro-environments (quiescent, hypoxic or anoxic) did not significantly affect metabolism or function of an environmentally relevant pharmaceutical in this spheroid model.

相较于二维（2D）细胞培养，细胞聚集体或细胞球体（3D）是更合适的体外培养体系，其个体细胞间通讯与微环境更贴近体内器官的真实状态；然而目前我们对该尺度下的生理状态仍知之甚少。作为影响细胞代谢能力的关键因素，球体尺寸与氧微环境之间的关联，最初是通过源自鱼类肠道的RTgutGC细胞系得以阐明的。随后，本研究以该肠道模型为对象，采用高效液相色谱法（high performance liquid chromatography）检测了普萘洛尔（Propranolol）的药物代谢情况，并分析其与球体尺寸的相关性。采用旋转平台法制备的细胞球体，其尺寸的变异系数低于12%，且在最高细胞接种密度下变异程度最小。通过电子顺磁共振（EPR）血氧测定法检测发现，球体外层存活区域的高氧微环境随时间推移逐渐减弱，而缺氧区域则随球体尺寸增大而扩张。尽管较小的球体呈现出更高的代谢趋势，但各类微环境（静息、缺氧或无氧）的形成，并未对该球体模型中环境相关药物的代谢与功能产生显著影响。