In Vitro Cell Death Determination for Drug Discovery: A Landscape Review of Real Issues

Méry, Benoite; Guy, Jean-Baptiste; Vallard, Alexis; Espenel, Sophie; Ardail, Dominique; Rodriguez-Lafrasse, Claire; Rancoule, Chloé; Magné, Nicolas

One of the most reliable methods for studying cell death is to measure the leakage of cellular components from compromised cultured cells when membrane integrity is altered, and especially measurement of intracellular proteins (most often enzymes) in cell culture supernatants. The presence of biomarker activity is typically proof positive for the presence of cytotoxicity, hence the frequent use of the assays for drug discovery experiments. For instance, lactate dehydrogenase (LDH) has long been used as a marker of cell death for in vitro models as LDH is released through the broken cell membrane after cell death. 23 Kits for the fluorometric or colorimetric detection of LDH are commercially available. Adenylate kinase and glucose-6-phosphate dehydrogenase are other cellular enzymes that can also be used as cell death markers even if a loss in their activity can occur during cell death assays, unlike LDH; that is why cell death assays based on LDH activity are more effective than other enzyme-based cell death assays. Lactate dehydrogenase activity can be indirectly measured by subjecting the sample to a coupled enzymatic chemistry reagent containing lactate, oxidized nicotinamide adenine dinucleotide, diaphorase, and an appropriate redox dye such as resazurin, which produces either a change in absorbance or a shift in the fluorescence profile. Methods have improved and a significantly easier assay has been described which allows same-well measurement of LDH without the need for sampling. This simplified assay suitable for high-throughput screening (HTS) protocols is now routinely used as it is inexpensive and can be easily implemented in 96-or 384-well plates. 24 However, a major inconvenience of these techniques is that physicochemical factors such as variations in the culture medium, ie, pH, may affect the activity of these enzymes. Subsequently, enzyme activity may decline with time in the extracellular milieu. 25 Besides, it is unable to discriminate between different cell death modes, and detection may be aggravated by morphologic changes in dying cells.