Abstract 5531: A bioluminescent cell viability assay optimized for 3D microtissues.

Abstract

Abstract It has been well demonstrated that the morphology, gene expression, and overall biological response of cells assessed in 3-dimensional (3D) culture models are frequently more physiologically relevant than that of cells studied in standard 2D culture formats. In spite of rapidly growing interest, one hindrance to the use of 3D models for drug discovery and other research efforts is the lack of convenient and effective assays explicitly validated for application to 3D microtissues. Here we report on a bioluminescent ATP-detection assay comprised of both an improved formulation and an optimized protocol specifically designed to measure the viability of cells grown in 3D culture. The ability of the 3D-optimized reagent to effectively lyse cells from throughout the full thickness of 3D microtissues, including cancer cell spheroids, was clearly demonstrated by use of confocal laser fluorescent microscopy in conjunction with a fluorogenic, membrane-impermeant DNA-binding dye. Importantly, the assay's ability to accurately report the ATP content, and therefore viability, of various 3D microtissues (including a range of cancer cell line spheroids) was demonstrated by comparison to a trichloroacetic acid ATP extraction method. In scaffold-free as well as scaffold-dependent 3D models, recovery and detection of greater than 80% of ATP present was routinely observed. In contrast, another frequently used ATP-detection assay was found to have greatly reduced lytic and ATP detection properties (<50% recovery). This new, homogeneous 3D-assay format requires minimal sample manipulation, a brief incubation period (generally less than 30 min), and exhibits excellent sensitivity. It should be amenable to both low- and high-throughput applications and accurately report drug sensitivities of cells assayed in both 2D and 3D culture models. Citation Format: Michael P. Valley, Chad Zimprich, James J. Cali, Dan F. Lazar. A bioluminescent cell viability assay optimized for 3D microtissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5531. doi:10.1158/1538-7445.AM2013-5531