Three-dimensional (3D) cell culture systems, such as tumor organoids and multicellular tumor spheroids (MCTS), 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 due 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. Here, 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 Promegas’ CellTiter-Glo®, CellTiter-Glo® 3D, and CellTiter 96® MTS Assay along with Thermo Fishers’ 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.

中文翻译：

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三维水凝胶中的细胞活力测定：准确性的比较研究

三维 (3D) 细胞培养系统，如肿瘤类器官和多细胞肿瘤球体 (MCTS)，部分是由于组织工程和生物制造技术的重大进步。由于其生理准确性，3D 细胞培养在药物开发、筛选、测试和精准医学方面提供了强大的功能。然而，自 3D 系统问世以来，很少有方法成功地定量分析水凝胶结构中的细胞活力。在这里，我们描述和比较了为二维 (2D) 应用开发的商用可行性测定，用于由有机、合成或混合水凝胶配方组成的 3D 构造。我们使用了 Promegas 的 CellTiter-Glo®、CellTiter-Glo® 3D、和 CellTiter 96® MTS 分析以及 Thermo Fishers 的 PrestoBlue™ 分析，以确定这些分析是否可以在 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，在不同水凝胶配方中，每种检测的细胞健康输出不准确。我们的结果应告知研究人员在 3D 培养中使用细胞活力测量时的潜在错误，并得出结论，应结合使用显微成像进行验证。