Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
Division of Surgical Oncology, Department of Surgery, Wake Forest Baptist Health, Winston Salem, North Carolina, USA.
Tissue Eng Part C Methods. 2021 Jul;27(7):401-410. doi: 10.1089/ten.TEC.2021.0060.
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 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.
三维（3D）细胞培养系统，如肿瘤类器官和多细胞肿瘤球体，部分是由于组织工程和生物制造技术的重大进步而发展起来的。3D 细胞培养由于其生理准确性，在药物开发、筛选、测试和精准医学方面具有巨大的能力。然而，自从 3D 系统出现以来，很少有方法被报道能够成功地定量分析水凝胶结构内的细胞活力。在这项研究中，我们描述并比较了市售的适用于二维（2D）应用的细胞活力测定法，用于由有机、合成或混合水凝胶配方组成的 3D 结构。我们使用了 Promega 的 CellTiter-Glo、CellTiter-Glo 3D 和 CellTiter 96 MTS 测定法以及 Thermo Fisher 的 PrestoBlue 测定法，以确定这些测定法是否可以在 3D 系统中准确使用。与 2D 细胞培养中常用的直接细胞活力相比，我们的结果显示，每种测定法在不同水凝胶配方中的细胞健康输出都存在不准确的情况。我们的结果应该让研究人员了解在 3D 培养物中使用细胞活力测量时可能存在的误差，并得出结论，应该结合显微镜成像进行验证。
