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.

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，部分由于组织工程和生物制造技术的重大进步而得到开发。3D 细胞培养因其生理准确性而在药物开发、筛选、测试和精准医学方面提供了强大的能力。然而，自 3D 系统问世以来，很少有方法能够成功地定量分析水凝胶结构中的细胞活力。在这项研究中，我们描述并比较了为二维 (2D) 应用开发的商业可行性分析，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 Promega 的 CellTiter-Glo ^®、CellTiter-Glo 3D 和 CellTiter 96 ^®MTS Assay 与 Thermo Fisher 的 PrestoBlue ^™测定一起确定这些测定是否可以在 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，我们的结果显示不同水凝胶配方中每种测定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。