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Current Drug Discovery Technologies


ISSN (Print): 1570-1638
ISSN (Online): 1875-6220

Review Article

Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays

Author(s): Abdel-Baset Halim*

Volume 17 , Issue 1 , 2020

Page: [2 - 22] Pages: 21

DOI: 10.2174/1570163815666180925095433

Price: $65


Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated.

A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.

Keywords: Cell proliferation, apoptosis, cytotoxicity, high-throughput screen, cell-based assays, cell lines, PBMC, drug discovery, biomedical research, cytochemistry.

Graphical Abstract
Iorio F, Knijnenburg TA, Vis DJ, et al. A Landscape of pharmacogenomic interactions in cancer. Cell 2016; 166(3): 740-54.
[] [PMID: 27397505]
Thomas DW, Burns J, Audette J, Carroll A, Dow-Hygelund C, Hay M. Clinical Development Success Rates 2006-2015 - BIO, Biomedtracker, Amplion 2016. Available at : https: // Development%20Success%20Rates%202006-2015%20-%20BIO,%20Biomedtracker,%20Amplion%202016.pdf
Barretina J, Caponigro G, Stransky N, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012; 483(7391): 603-7.
[] [PMID: 22460905]
Fox JT, Myung K. Cell-based high-throughput screens for the discovery of chemotherapeutic agents. Oncotarget 2012; 3(5): 581-5.
[] [PMID: 22653910]
Nichols AC, Black M, Yoo J, et al. Exploiting high-throughput cell line drug screening studies to identify candidate therapeutic agents in head and neck cancer. BMC Pharmacol Toxicol 2014; 15: 66.
[] [PMID: 25428177]
Allen DD, Caviedes R, Cárdenas AM, Shimahara T, Segura-Aguilar J, Caviedes PA. Cell lines as in vitro models for drug screening and toxicity studies. Drug Dev Ind Pharm 2005; 31(8): 757-68.
[] [PMID: 16221610]
Seashore-Ludlow B, Rees MG, Cheah JH, et al. Harnessing connectivity in a large-scale small-molecule sensitivity dataset. Cancer Discov 2015; 5(11): 1210-23.
[] [PMID: 26482930]
Tighe S. Recent trends in cell-based assays. J Biomol Tech 2010; 21(3)(Suppl.): S5.
An WF, Tolliday NJ. Introduction: cell-based assays for high-throughput screening. Methods Mol Biol 2009; 486: 1-12.
[] [PMID: 19347612]
An WF, Tolliday N. Cell-based assays for high-throughput screening. Mol Biotechnol 2010; 45(2): 180-6.
[] [PMID: 20151227]
Wong AHH, Li H, Jia Y, et al. Drug screening of cancer cell lines and human primary tumors using droplet microfluidics. Sci Rep 2017; 7(1): 9109.
[] [PMID: 28831060]
Astashkina A, Mann B, Grainger DW. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity. Pharmacol Ther 2012; 134(1): 82-106.
[] [PMID: 22252140]
Collins FS, Tabak LA. Policy: NIH plans to enhance reproducibility. Nature 2014; 505(7485): 612-3.
[] [PMID: 24482835]
Begley CG, Ioannidis JP. Reproducibility in science: improving the standard for basic and preclinical research. Circ Res 2015; 116(1): 116-26.
[] [PMID: 25552691]
Freedman LP, Cockburn IM, Simcoe TS. The economics of reproducibility in preclinical research. PLoS Biol 2015; 13(6)e1002165
[] [PMID: 26057340]
Hughes P, Marshall D, Reid Y, Parkes H, Gelber C. The costs of using unauthenticated, over-passaged cell lines: how much more data do we need? Biotechniques 2007; 43((5)) 575-, 577-578, 581- 582 passim.
[] [PMID: 18072586]
Lorsch JR, Collins FS, Lippincott-Schwartz J. Cell biology. Fixing problems with cell lines. Science 2014; 346(6216): 1452-3.
[] [PMID: 25525228]
Horbach SPJM, Halffman W. How cell line misidentification contaminates the scientific literature. PLoS One 2017; 12(10)e0186281
Dockrill P. Over 30,000 Published Studies Could Be Wrong due to contaminated cells-this is very, very bad. Science Alert 2017
Kenny L. The use of novel PET tracers to image breast cancer biologic processes such as proliferation, DNA Damage and Repair, and Angiogenesis. J Nucl Med 2016; 57(Suppl. 1): 89S-95S.
[] [PMID: 26834108]
Jao CY, Salic A. Exploring RNA transcription and turnover in vivo by using click chemistry. Proc Natl Acad Sci USA 2008; 105(41): 15779-84.
[] [PMID: 18840688]
Madhavan H. Simple Laboratory methods to measure cell proliferation using DNA synthesis property. J Stem Cells Regen Med 2007; 3(1): 12-4.
[PMID: 24693014]
Duque A, Rakic P. Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate. J Neurosci 2011; 31(42): 15205-17.
[] [PMID: 22016554]
Uddin M, Altmann GG, Leblond CP. Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine. J Cell Biol 1984; 98(5): 1619-29.
[] [PMID: 6202697]
Roberts JD, Kunke TA. Fidelity of DNA replication DNA replication in eukaryotic cells Cold Spring Harbor Laboratory Press. 1996; pp. 217-47. [Internet Cited 2018 Jan 1]
Thomas MJ, Platas AA, Hawley DK. Transcriptional fidelity and proofreading by RNA polymerase II. Cell 1998; 93(4): 627-37.
[] [PMID: 9604937]
Cooper GM. The Cell: A Molecular Approach 2nd Ed. Sunderland CMAJ: Sinaves Associates 2000
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 2002. From DNA to RNA
Wang B, Opron K, Burton ZF, Cukier RI, Feig M. Five checkpoints maintaining the fidelity of transcription by RNA polymerases in structural and energetic details. Nucleic Acids Res 2015; 43(2): 1133-46.
[] [PMID: 25550432]
Neef AB, Luedtke NW. Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proc Natl Acad Sci USA 2011; 108(51): 20404-9.
[] [PMID: 22143759]
Kuan CY, Schloemer AJ, Lu A, et al. Hypoxia-ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain. J Neurosci 2004; 24(47): 10763-72.
[] [PMID: 15564594]
Darzynkiewicz Z, Traganos F, Zhao H, Halicka HD, Li J. Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on “click chemistry”. Cytometry A 2011; 79(5): 328-37.
[] [PMID: 21425239]
Tani H, Akimitsu N. Genome-wide technology for determining RNA stability in mammalian cells: historical perspective and recent advantages based on modified nucleotide labeling. RNA Biol 2012; 9(10): 1233-8.
[] [PMID: 23034600]
Marguerat S, Bähler J. Coordinating genome expression with cell size. Trends Genet 2012; 28(11): 560-5.
[] [PMID: 22863032]
Lee LG, Chen CH, Chiu LA. Thiazole orange: A new dye for reticulocyte analysis. Cytometry 1986; 7(6): 508-17.
[] [PMID: 2430763]
Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol 1991; 7: 663-98.
[] [PMID: 1809356]
Steller H. Mechanisms and genes of cellular suicide. Science 1995; 267(5203): 1445-9.
[] [PMID: 7878463]
Guzińska-Ustymowicz K, Pryczynicz A, Kemona A, Czyzewska J. Correlation between proliferation markers: PCNA, Ki-67, MCM-2 and antiapoptotic protein Bcl-2 in colorectal cancer. Anticancer Res 2009; 29(8): 3049-52.
[PMID: 19661314]
Arends MJ, Morris RG, Wyllie AH. Apoptosis. The role of the endonuclease. Am J Pathol 1990; 136(3): 593-608.
[PMID: 2156431]
Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119(3): 493-501.
[] [PMID: 1400587]
Tong JS, Zhang QH, Huang X, et al. Icaritin causes sustained ERK1/2 Activation and induces apoptosis in human endometrial cancer cells. PLoS One 2011; 6(3) e16781
Croom E. Metabolism of xenobiotics of human environments. Prog Mol Biol Transl Sci 2012; 112: 31-88.
[] [PMID: 22974737]
Collins JA, Schandi CA, Young KK, Vesely J, Willingham MC. Major DNA fragmentation is a late event in apoptosis. J Histochem Cytochem 1997; 45(7): 923-34.
[] [PMID: 9212818]
Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte-Hermann R. In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 1995; 21(5): 1465-8.
[PMID: 7737654]
Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun 2005; 73(4): 1907-16.
[] [PMID: 15784530]
Toné S, Sugimoto K, Tanda K, et al. Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis. Exp Cell Res 2007; 313(16): 3635-44.
[] [PMID: 17643424]
Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol 2000; 182(3): 311-22.
[<311:AID-JCP1>3.0.CO;2-9] [PMID: 10653597]
van Oijen MG, Medema RH, Slootweg PJ, Rijksen G. Positivity of the proliferation marker Ki-67 in noncycling cells. Am J Clin Pathol 1998; 110(1): 24-31.
[] [PMID: 9661919]
Endl E, Gerdes J. Posttranslational modifications of the KI-67 protein coincide with two major checkpoints during mitosis. J Cell Physiol 2000; 182(3): 371-80.
[<371:AID-JCP8>3.0.CO;2-J] [PMID: 10653604]
Bruno S, Darzynkiewicz Z. Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells. Cell Prolif 1992; 25(1): 31-40.
[] [PMID: 1540682]
Strober W. Trypan blue exclusion test of cell viability Curr Protoc Immunol 2015; 111 A3B1-3
Repetto G, del Peso A, Zurita JL. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 2008; 3(7): 1125-31.
[] [PMID: 18600217]
Karimi MA, Lee E, Bachmann MH, et al. Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay. PLoS One 2014; 9(2)e89357
[] [PMID: 24586714]
Riss TL, Moravec RA, Niles AL, et al. Cell Viability Assays. In: Sittampalam GS, Coussens NP, Brimacombe K, et al., Eds., Assay Guidance Manual: Eli Lilly Company and the National Center for Advancing Translational Sciences. Bethesda, MD 2016; pp. 1-31.
Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 2005; 11: 127-52.
[] [PMID: 16216776]
Hiebl B, Peters S, Gemeinhardt O, Niehues SM, Jung F. Impact of serum in cell culture media on in vitro lactate dehydrogenase (LDH) release determination. J Cellular Biotechnology 2017; 3: 9-13.
Valvona CJ, Fillmore HL, Nunn PB, Pilkington GJ. The regulation and function of lactate dehydrogenase A: Therapeutic potential in brain tumor. Brain Pathol 2016; 26(1): 3-17.
[] [PMID: 26269128]
Gstraunthaler G, Seppi T, Pfaller W. Impact of culture conditions, culture media volumes, and glucose content on metabolic properties of renal epithelial cell cultures. Are renal cells in tissue culture hypoxic? Cell Physiol Biochem 1999; 9(3): 150-72.
[] [PMID: 10494029]
Chan FKM, Moriwaki K, De Rosa MJ. Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol Biol 2013; 979: 65-70.
[] [PMID: 23397389]
Fukami T, Yokoi T. The emerging role of human esterases. Drug Metab Pharmacokinet 2012; 27(5): 466-77.
[] [PMID: 22813719]
McIlwain DR, Berger T, Mak TW. Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol 2013; 5(4) a008656
[] [PMID: 23545416]
McStay GP, Salvesen GS, Green DR. Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ 2008; 15(2): 322-31.
[] [PMID: 17975551]
Fischer U, Jänicke RU, Schulze-Osthoff K. Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ 2003; 10(1): 76-100.
[] [PMID: 12655297]
Essmann F, Engels IH, Totzke G, Schulze-Osthoff K, Jänicke RU. Apoptosis resistance of MCF-7 breast carcinoma cells to ionizing radiation is independent of p53 and cell cycle control but caused by the lack of caspase-3 and a caffeine-inhibitable event. Cancer Res 2004; 64(19): 7065-72.
[] [PMID: 15466201]
Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J 1999; 341(Pt 2): 233-49.
[] [PMID: 10393078]
Lin CJ, Lee CC, Shih YL, et al. Inhibition of mitochondria- and endoplasmic reticulum stress-mediated autophagy augments temozolomide-induced apoptosis in glioma cells. PLoS One 2012; 7(6)e38706
[] [PMID: 22745676]
Solaini G, Baracca A, Lenaz G, Sgarbi G. Hypoxia and mitochondrial oxidative metabolism. Biochim Biophys Acta 2010; 1797(6-7): 1171-7.
[] [PMID: 20153717]
Tiede LM, Cook EA, Morsey B, Fox HS. Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins Cell Death Des 2011; 2: e246
Lee SH, Meng XW, Flatten KS, Loegering DA, Kaufmann SH. Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm. Cell Death Differ 2013; 20(1): 64-76.
[] [PMID: 22858544]
van Genderen H, Kenis H, Lux P, et al. In vitro measurement of cell death with the annexin A5 affinity assay. Nat Protoc 2006; 1(1): 363-7.
[] [PMID: 17406257]
Fadeel B, Gleiss B, Högstrand K, et al. Phosphatidylserine exposure during apoptosis is a cell-type-specific event and does not correlate with plasma membrane phospholipid scramblase expression. Biochem Biophys Res Commun 1999; 266(2): 504-11.
Clarke RG, Lund EK, Johnson IT, Pinder AC. Apoptosis can be detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, but not by TUNEL assay or sub-G0 DNA content. Cytometry 2000; 39(2): 141-50.
[<141:AID-CYTO7>3.0.CO;2-O] [PMID: 10679732]
Bundscherer A, Malsy M, Lange R, et al. Cell harvesting method influences results of apoptosis analysis by annexin V staining. Anticancer Res 2013; 33(8): 3201-4.
[PMID: 23898079]
Boyd V, Cholewa OM, Papas KK. Limitations in the use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and cell permeable nucleic acid stains for viability measurements of isolated islets of langerhans. Curr Trends Biotechnol Pharm 2008; 2(2): 66-84.
[PMID: 20814586]

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