Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
Toxicol Sci. 2012 Jun;127(2):403-11. doi: 10.1093/toxsci/kfs122. Epub 2012 Mar 27.
Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this study was to investigate whether this observation could be extended to the determination of LD(50) values and whether 3D data could be correlated to in vivo observations. We developed a noninvasive means to estimate the amount of protein present in a 3D spheroid from it is planar area (± 21%) so that a precise dose can be provided in a manner similar to in vivo studies. This avoided correction of the actual dose given based on a protein determination after treatment (when some cells may have lysed). Conversion of published in vitro LC(50) data (mM) for six common drugs (acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid) to LD(50) data (mg compound/mg cellular protein) showed that the variation in LD(50) values was generally less than that suggested by the original LC(50) data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented here) demonstrated similar LD(50) values. Although in vitro 2D HepG2 data showed a poor correlation, the primary hepatocyte and 3D spheroid data resulted in a much higher degree of correlation with in vivo lethal blood plasma levels. These results corroborate that 3D hepatocyte cultures are significantly different from 2D cultures and are more representative of the liver in vivo.
许多出版物已经证明，在三维（3D）培养中生长的不朽细胞具有生理行为，这比使用经典二维（2D）培养技术培养相同细胞时更能让人联想到其亲本器官。本研究的目的是研究这种观察结果是否可以扩展到 LD（50）值的确定，以及 3D 数据是否可以与体内观察结果相关联。我们开发了一种非侵入性方法，可以根据 3D 球体的平面面积（±21%）来估计球体中存在的蛋白质量，从而可以以类似于体内研究的方式提供精确的剂量。这避免了根据治疗后蛋白质测定值（当一些细胞可能已经裂解时）校正实际给予的剂量。将六类常见药物（对乙酰氨基酚、胺碘酮、双氯芬酸、二甲双胍、苯乙双胍和丙戊酸）的已发表的体外 LC（50）数据（mM）转换为 LD（50）数据（mg 化合物/mg 细胞蛋白）表明，LD（50）值的变化通常小于原始 LC（50）数据所暗示的变化。对这六种化合物在 3D 球体培养中的毒理学分析（已发表或此处呈现）表明，LD（50）值相似。尽管体外 2D HepG2 数据相关性较差，但原代肝细胞和 3D 球体数据与体内致死性血浆水平的相关性更高。这些结果证实 3D 肝细胞培养与 2D 培养有很大的不同，并且更能代表体内肝脏。
