Kemas Aurino M, Lauschke Volker M
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
Methods Mol Biol. 2022;2445:337-349. doi: 10.1007/978-1-0716-2071-7_21.
Organotypic and microphysiological culture of primary human tissues and cancers has emerged as a powerful set of technologies that allow to faithfully mimic cellular metabolism and functions ex vivo. The predominant 3D culture methods include spheroids and microfluidic chips. These cultures use low cell numbers and culture volumes, which, however, poses important limitations for the available amounts of sample for downstream analyses. Here, we describe a detailed method for the measurement of glucose consumption dynamics in organotypic culture using a bienzymatic colorimetric assay that accurately quantifies glucose levels using nanoliter input volumes. As an example we utilize spheroids consisting of primary human hepatocytes. The assay has been carefully optimized and benchmarked and is compatible with both longitudinal and high-throughput screening in both static and perfused conditions. The method is straightforward and only requires a microplate reader capable of running absorbance kinetic measurements.
原代人组织和癌症的器官型及微生理培养已成为一套强大的技术，能够在体外忠实地模拟细胞代谢和功能。主要的3D培养方法包括球体培养和微流控芯片。这些培养方法使用的细胞数量和培养体积较少，然而，这对用于下游分析的样本量造成了重要限制。在此，我们描述了一种详细的方法，用于通过双酶比色法测量器官型培养中的葡萄糖消耗动态，该方法使用纳升输入体积准确量化葡萄糖水平。作为示例，我们利用由原代人肝细胞组成的球体。该测定方法经过精心优化和基准测试，适用于静态和灌注条件下的纵向和高通量筛选。该方法简单直接，仅需要一台能够进行吸光度动力学测量的微孔板读数器。
