Department of Chemistry "G. Ciamician", University of Bologna, Bologna, Italy.
Department of Chemistry, Connecticut College, New London, Connecticut.
Photochem Photobiol. 2017 Mar;93(2):531-535. doi: 10.1111/php.12718. Epub 2017 Feb 28.
Bioluminescent (BL) cell-based assays based on two-dimensional (2D) monolayer cell cultures represent well-established bioanalytical tools for preclinical screening of drugs. However, cells in 2D cultures do not often reflect the morphology and functionality of living organisms, thus limiting the predictive value of 2D cell-based assays. Conversely, 3D cell models have the capability to generate the extracellular matrix and restore cell-to-cell communications; thus, they are the most suitable model to mimic in vivo physiology. In this work, we developed a nondestructive real-time BL imaging assay of spheroids for longitudinal studies on 3D cell models. A high-throughput BL 3D cell-based assay in micropatterned 96-well plate format is reported. The assay performance was assessed using the transcriptional regulation of nuclear factor K beta response element in human embryonic kidney (HEK293) cells. We compared concentration-response curves for tumor necrosis factor-α with those obtained using conventional 2D cell cultures. One of the main advantages of this approach is the nonlysing nature of the assay, which allows for repetitive measurements on the same sample. The assay can be implemented in any laboratory equipped with basic cell culture facilities and paves the way to the development of new 3D bioluminescent cell-based assays.
基于二维（2D）单层细胞培养的生物发光（BL）细胞测定法是用于药物临床前筛选的成熟生物分析工具。然而，2D 培养中的细胞通常不能反映生物体内的形态和功能，从而限制了 2D 细胞测定法的预测价值。相反，3D 细胞模型具有生成细胞外基质和恢复细胞间通讯的能力；因此，它们是模拟体内生理学的最合适模型。在这项工作中，我们开发了一种用于 3D 细胞模型的球体的非破坏性实时 BL 成像测定法，用于纵向研究。报道了一种在微图案 96 孔板格式中的高通量 BL 3D 细胞测定法。使用人胚肾（HEK293）细胞中的核因子 K β反应元件的转录调控来评估测定法的性能。我们比较了肿瘤坏死因子-α的浓度-反应曲线与使用传统 2D 细胞培养获得的曲线。该方法的主要优点之一是测定法的非裂解性质，这允许在同一样品上进行重复测量。该测定法可以在配备基本细胞培养设施的任何实验室中实施，并为开发新的 3D 生物发光细胞测定法铺平了道路。
