Department of Chemistry, University of North Carolina at Chapel Hill, Kenan and Caudill Laboratories, 125 South Road, Chapel Hill, NC 27599-3290, USA.
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 450 West Davis Drive, Chapel Hill, NC 27599-7295, USA.
Analyst. 2023 May 16;148(10):2245-2255. doi: 10.1039/d3an00283g.
Cellular viability measurements quantify decreased proliferation or increased cytotoxicity caused by drug candidates or potential environmental toxins. Direct viability measures count each cell to provide an accurate readout. This approach can prove analytically challenging and time-consuming when cells are maintained in 3D structures akin to tissues or solid tumors. While less labor-intensive, indirect viability measures can be less accurate due to the heterogeneous structural and chemical microenvironment that arises when cells are maintained in tissue-like architectures and in contact with extracellular matrices. Here we determine the analytical figures of merit of five indirect viability assays in the paper-based cell culture platform we continue to develop in our laboratory: calcein-AM staining, the CellTiter-Glo assay, imaging fluorescent protein expression, propidium iodide staining, and the resazurin assay. We also determined the compatibility of each indirect assay with hypoxic conditions, intra-experimental repeatability, inter-experimental reproducibility, and ability to predict a potency value for a known antineoplastic drug. Our results show that each assay has benefits and drawbacks to consider when choosing the appropriate readout to answer a particular research question. We also highlight that only one indirect readout is unaffected by hypoxia, a commonly overlooked variable in cell culture that likely yields inaccurate viability measures.
细胞活力测量定量减少增殖或增加细胞毒性由候选药物或潜在的环境毒素引起。直接的活力测量计算每个细胞，提供准确的读数。当细胞在类似于组织或实体瘤的 3D 结构中维持时，这种方法在分析上可能具有挑战性且耗时。虽然间接活力测量的劳动力强度较小，但由于细胞在组织样结构中维持并与细胞外基质接触时产生的异质结构和化学微环境，可能不太准确。在这里，我们确定了我们实验室继续开发的基于纸张的细胞培养平台中的五种间接活力测定方法的分析优点：钙黄绿素 AM 染色、CellTiter-Glo 测定、荧光蛋白表达成像、碘化丙啶染色和 Resazurin 测定。我们还确定了每种间接测定与低氧条件的兼容性、实验内可重复性、实验间可重复性以及预测已知抗肿瘤药物效力值的能力。我们的结果表明，在选择适当的读数来回答特定的研究问题时，每种测定方法都有其优点和缺点需要考虑。我们还强调，只有一种间接读数不受低氧影响，这是细胞培养中经常被忽视的一个变量，可能导致不准确的活力测量。
