HTS Core Facility, Molecular Pharmacology & Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, NY 10065, USA.
Expert Opin Drug Discov. 2010 Mar;5(3):223-33. doi: 10.1517/17460441003596685. Epub 2010 Jan 29.
Monitoring cell viability in vitro is critical in many areas of biomedical research, and the ultimate goal in drug discovery is the ability to predict the in vivo toxicology of drug candidates based on their toxicity profile in vitro. Over the last decade, the contribution of high-throughput screening toward this goal has been tremendous, providing the ability to screen compounds in parallel against multiple cell types. However, the toxic effects of drug candidates uncovered during clinical trials are by far the main reason for their failure. Over the same period, our understanding of programmed cell death has evolved dramatically with the identification of critical control points in the cell death pathways. As a result, cell viability should no longer be characterized solely on the basis of discrete end point measurements such as membrane permeability.
This review summarizes the traditional viability assays currently commercially available, focusing on methods amenable to high density format. Assays categorized into the following classes are discussed: dye exclusion assays, DNA condensation-based assays and assays monitoring a metabolic function.
We describe current approaches for assessing cell viability and, using case studies, emphasize their limitations. As an alternative, we propose the use of live, multiplexed readouts to accurately record cell death induction.
Current low-content methods based on single parameter readouts are prone to error due to the heterogeneity of cell populations and the multi-faceted nature of cell death. High-content approaches based on continuous, multiplexed readouts are becoming increasingly important for monitoring multiple markers of cell death induction simultaneously on a cell by cell basis. The use of such content-rich platforms is a necessity to predict the toxicology of drug candidates accurately.
监测体外细胞活力在许多生物医学研究领域至关重要，药物发现的最终目标是能够根据候选药物在体外的毒性特征预测其体内毒理学。在过去的十年中，高通量筛选在这一目标上的贡献是巨大的，它提供了同时平行筛选多种细胞类型化合物的能力。然而，候选药物在临床试验中发现的毒性作用是导致其失败的主要原因。在同一时期，我们对程序性细胞死亡的理解发生了巨大的变化，确定了细胞死亡途径中的关键控制点。因此，细胞活力的评估不应仅仅基于细胞膜通透性等离散终点测量。
本篇综述总结了目前商业上可用的传统细胞活力检测方法，重点介绍了适用于高密度格式的方法。讨论了以下几类检测方法：染料排除检测、基于 DNA 凝聚的检测和监测代谢功能的检测。
我们描述了评估细胞活力的当前方法，并通过案例研究强调了它们的局限性。作为替代方法，我们建议使用活的、多重读出方法来准确记录细胞死亡诱导。
目前基于单一参数读出的低内容方法由于细胞群体的异质性和细胞死亡的多方面性质容易出错。基于连续、多重读出的高内容方法对于同时在单细胞基础上监测多种细胞死亡诱导标志物的监测变得越来越重要。使用这种内容丰富的平台是准确预测候选药物毒理学的必要条件。
