Cell Viability Assays in Three-Dimensional Hydrogels: A Comparative Study of Accuracy

Three-dimensional (3D) cell culture systems, such as tumor organoids and multicellular tumor spheroids, have been developed in part as a result of major advances in tissue engineering and biofabrication techniques. 3D cell culture offers great capabilities in drug development, screening, testing, and precision medicine owing to its physiological accuracy. However, since the inception of 3D systems, few methods have been reported to successfully analyze cell viability quantitatively within hydrogel constructs. In this study, we describe and compare commercially available viability assays developed for two-dimensional (2D) applications for use in 3D constructs composed of organic, synthetic, or hybrid hydrogel formulations. We utilized Promega's CellTiter-Glo^®, CellTiter-Glo 3D, and CellTiter 96^® MTS Assay along with Thermo Fisher's PrestoBlue^™ assay to determine if these assays can be used accurately in 3D systems. Compared with direct cell viability commonly used in 2D cell culture, our results show cellular health output inaccuracies among each assay in differing hydrogel formulations. Our results should inform researchers of potential errors when using cell viability measurements in 3D cultures and conclude that microscopic imaging should be used, in combination, for validation. Impact statement Three-dimensional (3D) tissue organoids models are a valuable tool not only for studying drug toxicity but also for understanding human embryonic development, intra-tissue morphogenesis, and mechanisms of disease. In cancer organoids, such 3D models may be used for preclinical chemotherapy screening and for understanding cell death and viability mechanisms under physiologically relevant conditions. Cell viability assays are necessary for assessing the effect of biological reagents on cellular health and have been used on in vitro cell cultures for many years. With the increase of 3D systems in cellular biology research to determine therapeutic efficacy, two-dimensional assays that measure cell viability are being used outside their intended use on 3D constructs. In this study, we assess the accuracy of using various commercially available cell viability assays on different 3D hydrogel constructs to help researchers understand expected variability in their experimentation along microscopic imaging validation.