Methods for assessing the viability of cells cultured in vitro in 2D and 3D structures

Abstract

Over the past decades, cell viability tests have been an essential research tool in cell biology, tissue engineering, and regenerative medicine. Assessment of cell viability is mandatory in the production and quality control of cell products for biomedical applications.

Methods of viability assessment can be broadly classified according to the underlying mechanism and how the results obtained are evaluated. This article presents variants of the most commonly used tests and protocols for assessing cell viability. Their advantages and disadvantages are presented, which should be considered when planning experiments, e.g., when developing cell preparations for regenerative medicine.

The authors point out the main factors influencing the choice of viability assessment method: efficiency, speed, safety, reproducibility, sample integrity, compatibility with biomaterial, and cell line type. Finally, the authors discuss separately cell viability tests that can be applied not only to 2D cell structures but also to 3D cell structures, which have recently become widespread due to more accurate modeling of biological processes.

Keywords

viability analysis of cells / 2D cell structures / 3D cell structures / dye exclusion assay / colorimetric method / fluorescent dyes / luminometric assays / flow cytometry

References

[1] Kumar P, Nagarajan A, Uchil PD. Analysis of cell viability by the MTT assay. Cold Spring Harb Protoc. 2018;2018(6). doi: 10.1101/pdb.prot095505

[2] Kumar P., Nagarajan A., Uchil P.D. Analysis of cell viability by the MTT assay // Cold Spring Harb Protoc. 2018. Vol. 2018, N 6. doi: 10.1101/pdb.prot095505

[3] Castro-concha LA, Escobedo RM, Miranda-Ham Mde L. Measurement of cell viability. Methods Mol Biol. 2012;877:49–56. doi: 10.1007/978-1-61779-818-4_5

[4] Castro-concha L.A., Escobedo R.M., Miranda-Ham Mde L. Measurement of cell viability // Methods Mol Biol. 2012. Vol. 877. P. 49–56. doi: 10.1007/978-1-61779-818-4_5

[5] Kabakov AE, Gabai VL. Cell death and survival assays. Methods Mol Biol. 2018;1709:107–127. doi: 10.1007/978-1-4939-7477-1_9

[6] Kabakov E., Gabai V.L. Cell death and survival assays // Methods Mol Biol. 2018. Vol. 1709. P. 107–127. doi: 10.1007/978-1-4939-7477-1_9

[7] Kamiloglu S, Sari G, Ozdal T, Capanoglu E. Guidelines for cell viability assays. Food Frontiers. 2020;1(3):332–349. doi: 10.1002/fft2.44

[8] Kamiloglu S., Sari G., Ozdal T., Capanoglu E. Guidelines for cell viability assays // Food Frontiers. 2020. Vol. 1, N 3. P. 332–349. doi: 10.1002/fft2.44

[9] Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2001; Suppl. 3:3B. doi: 10.1002/0471142735.ima03bs21

[10] Strober W. Trypan Blue exclusion test of cell viability // Curr Protoc Immunol. 2001. Suppl 3. P. 3B. doi: 10.1002/0471142735.ima03bs21

[11] Huang CN, Cornejo MJ, Bush DS, Jones RL. Estimating viability of plant protoplasts using double and single staining. Protoplasma. 1986;135:80–87. doi: 10.1007/BF01277001

[12] Huang C.N., Cornejo M.J., Bush D.S., Jones R.L. Estimating viability of plant protoplasts using double and single staining // Protoplasma. 1986. Vol. 135. P. 80–87. doi: 10.1007/BF01277001

[13] Babakhanova G, Zimmerman SM, Pierce LT, et al. Quantitative, traceable determination of cell viability using absorbance microscopy. PLoS One. 2022;17(1):e0262119. doi: 10.1371/journal.pone.0262119

[14] Babakhanova G., Zimmerman S.M., Pierce L.T., et al. Quantitative, traceable determination of cell viability using absorbance microscopy // PLoS One. 2022. Vol. 17, N 1. P. e0262119. doi: 10.1371/journal.pone.0262119

[15] Alamoudi WA, Ahmad F, Acharya S, et al. A simplified colorimetric method for rapid detection of cell viability and toxicity in adherent cell culture systems. J BUON. 2018;23(5):1505–1513.

[16] Alamoudi W.A., Ahmad F., Acharya S. A simplified colorimetric method for rapid detection of cell viability and toxicity in adherent cell culture systems // J BUON. 2018. Vol. 23, N 5. P. 1505–1513.

[17] Boncler M, Rózalski M, Krajewska U, et al. Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells. J Pharmacol Toxicol Methods. 2014;69(1):9–16. doi: 10.1016/j.vascn.2013.09.003

[18] Boncler M., Rózalski M., Krajewska U., et al. Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells // J Pharmacol Toxicol Methods. 2014. Vol. 69, N 1. P. 9–16. doi: 10.1016/j.vascn.2013.09.003

[19] Bopp SK, Lettieri T. Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line. BMC Pharmacol. 2008;8:8. doi: 10.1186/1471-2210-8-8

[20] Bopp S.K., Lettieri T. Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line // BMC Pharmacol. 2008. Vol. 8. P. 8. doi: 10.1186/1471-2210-8-8

[21] Cory AH, Owen TC, Barltrop JA, Cory JG. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun. 1991;3(7):207–212. doi: 10.3727/095535491820873191

[22] Cory H., Owen T.C., Barltrop J.A., Cory J.G. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture // Cancer Commun. 1991. Vol. 3, N 7. P. 207–212. doi: 10.3727/095535491820873191

[23] Goodwin CJ, Holt SJ, Downes S, Marshall NJ. Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS. J Immunol Methods. 1995;179(1):95–103. doi: 10.1016/0022-1759(94)00277-4

[24] Goodwin J., Holt S.J., Downes S., Marshall N.J. Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS // J Immunol Methods. 1995. Vol. 179. P. 95–103. doi: 10.1016/0022-1759(94)00277-4

[25] Lutter AH, Scholka J, Richter H, Anderer U. Applying XTT, WST-1, and WST-8 to human chondrocytes: a comparison of membrane-impermeable tetrazolium salts in 2D and 3D cultures. Clin Hemorheol Microcirc. 2017;67(3-4):327–342. doi: 10.3233/CH-179213

[26] Lutter A.H., Scholka J., Richter H., Anderer U. Applying XTT, WST-1, and WST-8 to human chondrocytes: a comparison of membrane-impermeable tetrazolium salts in 2D and 3D cultures // Clin Hemorheol Microcirc. 2017. Vol. 67, N 3-4. P. 327–342. doi: 10.3233/CH-179213

[27] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4

[28] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays // J Immunol Methods. 1983. Vol. 65. P. 55–63. doi: 10.1016/0022-1759(83)90303-4

[29] Präbst K, Engelhardt H, Ringgeler S, Hübner H. Basic colorimetric proliferation assays: MTT, WST, and Resazurin: basic color prolif assays MTT, WST, Resazurin. Methods Mol Biol. 2017;1601:1–17. doi: 10.1007/978-1-4939-6960-9_1

[30] Präbst K., Engelhardt H., Ringgeler S., Hübner H. Basic colorimetric proliferation assays: MTT, WST, and Resazurin: basic color prolif assays MTT, WST, Resazurin // Methods Mol Biol. 2017. Vol. 1601. P. 1–17. doi: 10.1007/978-1-4939-6960-9_1

[31] Hall MD, Martin C, Ferguson DJ, et al. Comparative efficacy of novel platinum (IV) compounds with established chemotherapeutic drugs in solid tumour models. Biochem Pharmacol. 2004;67(1):17–30. doi: 10.1016/j.bcp.2003.07.016

[32] Hall M.D., Martin C., Ferguson D.J., et al. Comparative efficacy of novel platinum (IV) compounds with established chemotherapeutic drugs in solid tumour models // Biochem Pharmacol. 2004. Vol. 67, N 1. P. 17–30. doi: 10.1016/j.bcp.2003.07.016

[33] Berridge MV, Tan AS. Characterization of the cellular reduction of (MTT) subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys. 1993;303(2):474–482. doi: 10.1006/abbi.1993.1311

[34] Berridge M.V., Tan A.S. Characterization of the cellular reduction of (MTT) subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction // Arch Biochem Biophys. 1993. Vol. 303, N 2. P. 474–482. doi: 10.1006/abbi.1993.1311

[35] Bernas T, Dobrucki J. Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes. Cytometry. 2002;47(4):236–242. doi: 10.1002/cyto.10080

[36] Bernas T., Dobrucki J. Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes // Cytometry. 2002. Vol. 47, N 4. P. 236–242. doi: 10.1002/cyto.10080

[37] Chakrabarti R, Kundu S, Kumar S, Chakrabarti R. Vitamin A as an enzyme that catalyzes the reduction of MTT to formazan by vitamin C. J Cell Biochem. 2000;80(1):133–138. doi: 10.1002/1097-4644(20010101)80:1<133::AID-JCB120>3.0.CO;2-T

[38] Chakrabarti R., Kundu S., Kumar S., Chakrabarti R. Vitamin A as an enzyme that catalyzes the reduction of MTT to formazan by vitamin C // J Cell Biochem. 2000. Vol. 80, N 1. P. 133–138. doi: 10.1002/1097-4644(20010101)80:1<133::AID-JCB120>3.0.CO;2-T

[39] Collier AC, Pritsos CA. The mitochondrial uncoupler dicumarol disrupts the MTT assay. Biochem Pharmacol. 2003;66(2):281–287. doi: 10.1016/S0006-2952(03)00240-5

[40] Collier C., Pritsos C.A. The mitochondrial uncoupler dicumarol disrupts the MTT assay // Biochem Pharmacol. 2003. Vol. 66, N 2. P. 281–287. doi: 10.1016/S0006-2952(03)00240-5

[41] Ulukaya E, Colakogullari M, Wood EJ. Interference by anti-cancer chemotherapeutic agents in the MTT-tumor chemosensitivity assay. Chemotherapy. 2004;50(1):43–50. doi: 10.1159/000077285

[42] Ulukaya E., Colakogullari M., Wood E.J. Interference by anti-cancer chemotherapeutic agents in the MTT-tumor chemosensitivity assay // Chemotherapy. 2004. Vol. 50, N 1. P. 43–50. doi: 10.1159/000077285

[43] Vistica DT, Skehan P, Scudiero D, et al. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res. 1991;51(10):2515–2520.

[44] Vistica D.T., Skehan P., Scudiero D., et al. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production // Cancer Res. 1991. Vol. 51, N 10. P. 2515–2520.

[45] Promega Corporation. CellTiter 96® Non-Radioactive Cell Proliferation Assay. Insructions use of products G4000 and G41000. Technical Bulletin. USA, 2012.

[46] Lü L, Zhang L, Sen M, et al. Toxicology in vitro exocytosis of MTT formazan could exacerbate cell injury. Toxicol In Vitro. 2012;26(4):636–644. doi: 10.1016/j.tiv.2012.02.006

[47] Lü L., Zhang L., Mun Wai M., et al. Toxicology in vitro exocytosis of MTT formazan could exacerbate cell injury // Toxicol In Vitro. 2012. Vol. 26, N 4. P. 636–644. doi: 10.1016/j.tiv.2012.02.006

[48] Riss TL, Moravec RA, Niles AL, et al. Cell viability assays. Assay Guid Man. 2004:1–25.

[49] Riss T.L., Moravec R.A., Niles A.L., et al. Cell viability assays // Assay Guid Man. 2004. C. 1–25.

[50] Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev. 2005;11:127–152. doi: 10.1016/S1387-2656(05)11004-7

[51] Berridge M.V., Herst P.M., Tan A.S. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction // Biotechnol Annu Rev. 2005. Vol. 11. P. 127–152. doi: 10.1016/S1387-2656(05)11004-7

[52] Repetto G, del Peso A, Zurita JL. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc. 2008;3(7):1125–1131. doi: 10.1038/nprot.2008.75

[53] Repetto G., del Peso A., Zurita J.L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity // Nat Protoc. 2008. Vol. 3, N 7. P. 1125–1131. doi: 10.1038/nprot.2008.75

[54] Gomez-Gutierrez JG, Bhutiani N, Mcnally MW, et al. During autophagy or in an acidic microenvironment in vitro. Biotech Histochem. 2022;96(4):302–310. doi: 10.1080/10520295.2020.1802065

[55] Gomez-gutierrez J.G., Bhutiani N., McNally M.W., et al. During autophagy or in an acidic microenvironment in vitro // Biotech Histochem. 2022. Vol. 96, N 4. P. 302–310. doi: 10.1080/10520295.2020.1802065

[56] Altman SA, Randers L, Rao G. Comparison of Trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations. Biotechnol Prog. 1993;9(6):671–674. doi: 10.1021/bp00024a017

[57] Altman S.A., Randers L., Rao G. Comparison of Trypan Blue dye exclusion and fluorometric assays for mammalian cell viability determinations // Biotechnol Prog. 1993. Vol. 9, N 6. P. 671–674. doi: 10.1021/bp00024a017

[58] Shum D, Radu C, Kim E, et al. A high density assay format for the detection of novel cytotoxic agents in large chemical libraries. J Enzyme Inhib Med Chem. 2008;23(6):931–945. doi: 10.1080/14756360701810082

[59] Shum D., Radu C., Kim E., et al. A high density assay format for the detection of novel cytotoxic agents in large chemical libraries // J Enzyme Inhib Med Chem. 2008. Vol. 23, N 6. P. 931–945. doi: 10.1080/14756360701810082

[60] O’Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000;267(17):5421–5426. doi: 10.1046/j.1432-1327.2000.01606.x

[61] O’Brien O., Wilson I., Orton T., Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity // Eur J Biochem. 2000. Vol. 267, N 17. P. 5421–5426. doi: 10.1046/j.1432-1327.2000.01606.x

[62] Luzak B, Siarkiewicz P, Boncler M. An evaluation of a new high-sensitivity PrestoBlue assay for measuring cell viability and drug cytotoxicity using EA.hy926 endothelial cells. Toxicol In Vitro. 2022;83:105407. doi: 10.1016/j.tiv.2022.105407

[63] Luzak B., Siarkiewicz P., Boncler M. An evaluation of a new high-sensitivity PrestoBlue assay for measuring cell viability and drug cytotoxicity using EA.hy926 endothelial cells // Toxicol In Vitro. 2022. Vol. 83. P. 105407. doi: 10.1016/j.tiv.2022.105407

[64] Ahmed SA, Gogal RM, Walsh JE. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H] thymidine incorporation assay. J Immunol Methods. 1994;170(2):211–224. doi: 10.1016/0022-1759(94)90396-4

[65] Ahmed S.A., Gogal R.M., Walsh J.E. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H] thymidine incorporation assay // J Immunol Methods. 1994. Vol. 170, N 2. P. 211–224. doi: 10.1016/0022-1759(94)90396-4

[66] Lavogina D, Lust H, Tahk MJ, et al. Revisiting the Resazurin-based sensing of cellular viability: widening the application horizon. Biosensors (Basel). 2022;12(4):196. doi: 10.3390/bios12040196

[67] Lavogina D., Lust H., Tahk M.J., et al. Revisiting the Resazurin-based sensing of cellular viability: widening the application horizon // Biosensors (Basel). 2022. Vol. 12, N 4. P. 196. doi: 10.3390/bios12040196

[68] Patent USA N 5501959/26.03.1996. Lancaster MV, Fields RD. Antibiotic and cytotoxic drug susceptibility assays using resazurin and poising agents. Available from: https://patentimages.storage.googleapis.com/e5/b8/74/fadfc8c037ebbc/US5501959.pdf

[69] Patent USA N 5501959/26.03.1996. Lancaster M.V., Fields R.D. Antibiotic and cytotoxic drug susceptibility assays using resazurin and poising agents. Режим доступа: https://patentimages.storage.googleapis.com/e5/b8/74/fadfc8c037ebbc/US5501959.pdf

[70] https://www.thermofisher.com/ [Internet]. Invitrogen Corporation. AlamarBlue™ HS cell viability reagent. Available from: https://www.thermofisher.com/order/catalog/product/A50100

[71] https://www.thermofisher.com/ [Internet]. Invitrogen Corporation. AlamarBlue™ HS cell viability reagent. Доступ по ссылке: https://www.thermofisher.com/order/catalog/product/A50100

[72] Voytik-Harbin SL, Brightman AO, Waisner B, et al. Application and evaluation of the alamarblue assay for cell growth and survival of fibroblasts. In Vitro Cell Dev Biol Anim. 1998;34(3):239–246. doi: 10.1007/s11626-998-0130-x

[73] Voytik-Harbin S.L., Brightman A.O., Waisner B., et al. Application and evaluation of the alamarblue assay for cell growth and survival of fibroblasts // In Vitro Cell Dev Biol Anim. 1998. Vol. 34, N 3. P. 239–246. doi: 10.1007/s11626-998-0130-x

[74] Nakayama GR, Caton MC, Nova MP, Parandoosh Z. Assessment of the Alamar Blue assay for cellular growth and viability in vitro. J Immunol Methods. 1997;204(2):205–208. doi: 10.1016/S0022-1759(97)00043-4

[75] Nakayama G.R., Caton M.C., Nova M.P., Parandoosh Z. Assessment of the Alamar Blue assay for cellular growth and viability in vitro // J Immunol Methods. 1997. Vol. 204, N 2. P. 205–208. doi: 10.1016/S0022-1759(97)00043-4

[76] Hamid R, Rotshteyn Y, Rabadi L, et al. Comparison of Alamar Blue and MTT assays for high through-put screening. Toxicol In Vitro. 2004;18(5):703–710. doi: 10.1016/j.tiv.2004.03.012

[77] Hamid R., Rotshteyn Y., Rabadi L., et al. Comparison of alamar blue and MTT assays for high through-put screening // Toxicol in Vitro. 2004. Vol. 18, N 5. P. 703–710. doi: 10.1016/j.tiv.2004.03.012

[78] Shahan TA, Siegel PD, Sorenson WG, et al. A sensitive new bioassay for tumor necrosis factor. J Immunol Methods. 1994;175(2):181–187. doi: 10.1016/0022-1759(94)90361-1

[79] Shahan T.A., Siegel P.D., Sorenson W.G., et al. A sensitive new bioassay for tumor necrosis factor // J Immunol Methods. 1994. Vol. 175, N 2. P. 181–187. doi: 10.1016/0022-1759(94)90361-1

[80] Dayeh VR, Bols NC, Schirmer K. The use of fish-derived cell lines for investigation of environmental contaminants: an update following OECD’s fish toxicity testing framework No. 171. Curr Protoc Toxicol. 2013;Chapter 1:Unit1.5. doi: 10.1002/0471140856.tx0105s56

[81] Dayeh V.R., Bols N.C., Schirmer K. The use of fish-derived cell lines for investigation of environmental contaminants: an update following OECD’s fish toxicity testing framework No. 171 // Curr Protoc Toxicol. 2013. Chapter 1. Unit1.5. doi: 10.1002/0471140856.tx0105s56

[82] White MJ, DiCaprio MJ, Greenberg DA. Assessment of neuronal viability with Alamar blue in cortical and granule cell cultures. J Neurosci Methods. 1996;70(2):195–200. doi: 10.1016/S0165-0270(96)00118-5

[83] White M.J., DiCaprio M.J., Greenberg D.A. Assessment of neuronal viability with Alamar blue in cortical and granule cell cultures // J Neurosci Methods. 1996. Vol. 70, N 2. P. 195–200. doi: 10.1016/S0165-0270(96)00118-5

[84] Yu SY, Sales KM, Fuller BJ, et al. Inducing apoptosis of human colon cancer cells by an IGF-I D domain analogue peptide. Mol Cancer. 2008;7:17. doi: 10.1186/1476-4598-7-17

[85] Yu S.Y., Sales K.M., Fuller B.J., et al. Inducing apoptosis of human colon cancer cells by an IGF-I D domain analogue peptide // Mol Cancer. 2008. Vol. 7. P. 17. doi: 10.1186/1476-4598-7-17

[86] Yajko DM, Madej JJ, Lancaster MV, et al. Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis. J Clin Microbiol. 1995;33(9):2324–2327. doi: 10.1128/jcm.33.9.2324-2327.1995

[87] Yajko M., Madej J.J., Lancaster M.V., et al. Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis // J Clin Microbiol. 1995. Vol. 33, N 9. P. 2324–2327. doi: 10.1128/jcm.33.9.2324-2327.1995

[88] Xu M, Mccanna DJ, Sivak JG. Use of the viability reagent PrestoBlue in comparison with alamarBlue and MTT to assess the viability of human corneal epithelial cells. J Pharmacol Toxicol Methods. 2015;71:1–7. doi: 10.1016/j.vascn.2014.11.003

[89] Xu M., Mccanna D.J., Sivak J.G. Use of the viability reagent PrestoBlue in comparison with alamarBlue and MTT to assess the viability of human corneal epithelial cells // J Pharmacol Toxicol Methods. 2015. Vol. 71. P. 1–7. doi: 10.1016/j.vascn.2014.11.003

[90] Yu HG, Chung H, Yu YS, et al. A new rapid and non-radioactive assay for monitoring and determining the proliferation of retinal pigment epithelial cells. Korean J Ophthalmol. 2003;17(1):29–34. doi: 10.3341/kjo.2003.17.1.29

[91] Yu H.G., Chung H., Yu Y.S., et al. A new rapid and non-radioactive assay for monitoring and determining the proliferation of retinal pigment epithelial cells // Korean J Ophthalmol. 2003. Vol. 17, N 1. P. 29–34. doi: 10.3341/kjo.2003.17.1.29

[92] Niles AL, Moravec RA, Hesselberth P, et al. A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal Biochem. 2007;366:197–206. doi: 10.1016/j.ab.2007.04.007

[93] Niles L., Moravec R.A., Hesselberth P., et al. A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers // Anal Biochem. 2007. Vol. 366. P. 197–206. doi: 10.1016/j.ab.2007.04.007

[94] https://assets.thermofisher.com/ [Internet]. Invitrogen. PrestoBlue® cell viability reagent. Available from: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018370-PrestoBlueCellViabilityReagent-PI.pdf

[95] https://assets.thermofisher.com/ [Internet]. Invitrogen. PrestoBlue® Cell Viability Reagent. Режим доступа: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018370-PrestoBlueCellViabilityReagent-PI.pdf

[96] Richards R, Honeywell ME, Lee MJ. FLICK: an optimized plate reader-based assay to infer cell death kinetics. STAR Protoc. 2021;2(1):100327. doi: 10.1016/j.xpro.2021.100327

[97] Richards R., Honeywell M.E., Lee M.J. FLICK: an optimized plate reader-based assay to infer cell death kinetics // STAR Protoc. 2021. Vol. 2, N 1, P. 100327. doi: 10.1016/j.xpro.2021.100327

[98] Yang N, Hui W, Dong S, et al. Temperature tolerance electric cell-substrate impedance sensing for joint assessment of cell viability and vitality. ACS Sens. 2021;6(10):3640–3649. doi: 10.1021/acssensors.1c01211

[99] Yang N., Hui W., Dong S., et al. Temperature tolerance electric cell-substrate impedance sensing for joint assessment of cell viability and vitality // ACS Sens. 2021. Vol. 6, N 10. P. 3640–3649. doi: 10.1021/acssensors.1c01211

[100] Lundin A. Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites. Methods Enzymol. 2000;305(1975):346–370. doi: 10.1016/s0076-6879(00)05499-9

[101] Lundin A. Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites // Methods Enzymol. 2001. Vol. 305, N 1975. P. 346–370. doi: 10.1016/s0076-6879(00)05499-9

[102] Auld DS, Zhang YQ, Southall NT, et al. A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution. J Med Chem. 2009;52(5):1450–1458. doi: 10.1021/jm8014525

[103] Auld S., Zhang Y.Q., Southall N.T., et al. A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution // J Med Chem. 2009. Vol. 52, N 5. P. 1450–1458. doi: 10.1021/jm8014525

[104] Promega Corporation. CellTiter-Glo® luminescent cell viability assay. [Internet]. Madison, USA, 2023. Available from: https://worldwide.promega.com/-/media/files/resources/protocols/technical-bulletins/0/celltiter-glo-luminescent-cell-viability-assay-protocol.pdf?rev=0d95c232094b41daa06d0c110d1d916f&sc_lang=en

[105] Promega Corporation. CellTiter-Glo® luminescent cell viability assay. [Internet]. Madison, USA, 2023. Доступ по ссылке: https://worldwide.promega.com/-/media/files/resources/protocols/technical-bulletins/0/celltiter-glo-luminescent-cell-viability-assay-protocol.pdf?rev=0d95c232094b41daa06d0c110d1d916f&sc_lang=en

[106] https://worldwide.promega.com/ [Internet]. RealTime-GloTM MT Cell Viability Assay. Available from: https://no.promega.com/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/realtime_glo-mt-cell-viability-assay/?catNum=G9711

[107] https://worldwide.promega.com/ [Internet]. RealTime-GloTM MT Cell Viability Assay. Доступ по ссылке: https://no.promega.com/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/realtime_glo-mt-cell-viability-assay/?catNum=G9711

[108] Duellman SJ, Zhou W, Meisenheimer P, et al. Bioluminescent, nonlytic, real-time cell viability assay and use in inhibitor screening. Assay Drug Dev Technol. 2015;13(8):456–465. doi: 10.1089/adt.2015.669

[109] Duellman S.J., Zhou W., Meisenheimer P., et al. Bioluminescent, nonlytic, real-time cell viability assay and use in inhibitor screening // Assay Drug Dev Technol. 2015. Vol. 13, N 8. P. 456–465. doi: 10.1089/adt.2015.669

[110] Ibrahim SF, van den Engh G. Flow cytometry and cell sorting. Adv Biochem Eng Biotechnol. 2007;106:19–39. doi: 10.1007/10_2007_073

[111] Ibrahim S.F., van den Engh G. Flow cytometry and cell sorting // Adv Biochem Eng Biotechnol. 2007. Vol. 106. P. 19–39. doi: 10.1007/10_2007_073

[112] Mckinnon KM. Flow cytometry: an overview. Curr Protoc Immunol. 2018;120:5.1.1–5.1.11. doi: 10.1002/cpim.40

[113] Mckinnon K.M. Flow cytometry: an overview // Curr Protoc Immunol. 2018. Vol. 120. P. 5.1.1–5.1.11. doi: 10.1002/cpim.40

[114] Wagner T, Guber SE, Stubenrauch ML, et al. Low propidium iodide intensity in flow cytometric white blood cell counting as a marker of cell destruction? Transfusion. 2005;45(2):228–233. doi: 10.1111/j.1537-2995.2004.04213.x

[115] Wagner T., Guber S.E., Stubenrauch M.L. Low propidium iodide intensity in flow cytometric white blood cell counting as a marker of cell destruction? // Transfusion. 2005. Vol. 45, N 2. 228–233. doi: 10.1111/j.1537-2995.2004.04213.x

[116] Moravvej H, Memariani H, Memariani M, et al. Evaluation of fibroblast viability seeded on acellular human amniotic membrane. Biomed Res Int. 2021;2021:5597758. doi: 10.1155/2021/5597758

[117] Moravvej H., Memariani H., Memariani M., et al. Evaluation of fibroblast viability seeded on acellular human amniotic membrane // Biomed Res Int. 2021. Vol. 2021. P. 5597758. doi: 10.1155/2021/5597758

[118] https://www.thermofisher.com/ [Internet]. ThermoFisher Scientific. DAPI (4´,6-diamidino-2-phenylindole). Available from: https://www.thermofisher.com/ru/ru/home/life-science/cell-analysis/fluorophores/dapi-stain.html

[119] https://www.thermofisher.com/ [Internet]. ThermoFisher Scientific. DAPI (4´,6-diamidino-2-phenylindole). Доступ по ссылке: https://www.thermofisher.com/ru/ru/home/life-science/cell-analysis/fluorophores/dapi-stain.html

[120] Richards R, Schwartz HR, Honeywell ME. Drug antagonism and single agent dominance result from differences in death kinetics. Nat Chem Biol. 2020;16(7):791–800. doi: 10.1038/s41589-020-0510-4

[121] Richards R., Schwartz H.R., Honeywell M.E. Drug antagonism and single agent dominance result from differences in death kinetics // Nat Chem Biol. 2020. Vol. 16, N 7. P. 791–800. doi: 10.1038/s41589-020-0510-4

[122] Masuda S, Shimizu S, Matsuo J, et al. Measurement of NET formation in vitro and in vivo by flow cytometry. Cytom Part A. 2017;91(8):822–829. doi: 10.1002/cyto.a.23169

[123] Masuda S., Shimizu S., Matsuo J., et al. Measurement of NET formation in vitro and in vivo by flow cytometry // Cytom Part A. 2017. Vol. 91, N 8. P. 822–829. doi: 10.1002/cyto.a.23169

[124] Zhong J, Yang D, Zhou Y, et al. Multi-frequency single cell electrical impedance measurement for label-free cell viability analysis. Analyst. 2021;146(6):1848–1858. doi: 10.1039/d0an02476g

[125] Zhong J., Yang D., Zhou Y., et al. Multi-frequency single cell electrical impedance measurement for label-free cell viability analysis // Analyst. 2021. Vol. 146, N 6, P. 1848–1858. doi: 10.1039/d0an02476g

[126] https://d1spbj2x7qk4bg.cloudfront.net/ [Internet]. BioLegend Corporation. Zombie NIR™ Fixable Viability Kit. Available from: https://d1spbj2x7qk4bg.cloudfront.net/fr-lu/products/zombie-nir-fixable-viability-kit-8657?pdf=true&displayInline=true&leftRightMargin=15&topBottomMargin=15&filename=Zombie%20NIR%E2%84%A2%20Fixable%20Viability%20Kit.pdf&v=20220831123135

[127] https://d1spbj2x7qk4bg.cloudfront.net/ [Internet]. BioLegend Corporation. Zombie NIR™ Fixable Viability Kit. Доступ по ссылке: https://d1spbj2x7qk4bg.cloudfront.net/fr-lu/products/zombie-nir-fixable-viability-kit-8657?pdf=true&displayInline=true&leftRightMargin=15&topBottomMargin=15&filename=Zombie%20NIR%E2%84%A2%20Fixable%20Viability%20Kit.pdf&v=20220831123135

[128] https://www.thermofisher.com/ [Internet]. ThermoFisher Scientific. Live/Dead cell viability assays. Available from: https://www.thermofisher.com/ru/ru/home/life-science/cell-analysis/cell-viability-and-regulation/cell-viability/live-dead-cell-viability-assays.html

[129] https://www.thermofisher.com/ [Internet]. ThermoFisher Scientific. Live/Dead cell viability assays. Доступ по ссылке: https://www.thermofisher.com/ru/ru/home/life-science/cell-analysis/cell-viability-and-regulation/cell-viability/live-dead-cell-viability-assays.html

[130] https://www.bdbiosciences.com/ [Internet]. BD Biosciences. Fixable Viability Stain 780. Available from: https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.au.565388.pdf

[131] https://www.bdbiosciences.com/ [Internet]. BD Biosciences. Fixable Viability Stain 780. Доступ по ссылке: https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.au.565388.pdf

[132] Madorran E, Stožer A, Arsov Z, et al. A promising method for the determination of cell viability: the membrane potential cell viability assay. Cells. 2022;11(15):2314. doi: 10.3390/cells11152314

[133] Madorran E., Stožer A., Arsov Z., et al. A promising method for the determination of cell viability: the membrane potential cell viability assay // Cells. 2022. Vol. 11, N 15. P. 2314. doi: 10.3390/cells11152314

[134] Zhang R, Wei M, Chen S, et al. A cell viability assessment method based on area-normalized impedance spectrum (ANIS). Biosens Bioelectron. 2018;110:193–200. doi: 10.1016/j.bios.2018.03.041

[135] Zhang R., Wei M., Chen S., et al, A cell viability assessment method based on area-normalized impedance spectrum (ANIS) // Biosens Bioelectron. 2018. Vol. 110. P. 193–200. doi: 10.1016/j.bios.2018.03.041

[136] Bonnier F, Keating ME, Wróbel TP, et al. Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models. Toxicol In Vitro. 2015;29(1):124–131. doi: 10.1016/j.tiv.2014.09.014

[137] Bonnier F., Keating M.E., Wróbel T.P., et al. Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models // Toxicol In Vitro. 2015. Vol. 29, N 1. P. 124–131. doi: 10.1016/j.tiv.2014.09.014

[138] Walzl A, Unger C, Kramer N, et al. The resazurin reduction assay can distinguish cytotoxic from cytostatic compounds in spheroid screening assays. J Biomol Screen. 2014;19(7):1047–1059. doi: 10.1177/1087057114532352

[139] Walzl A., Unger C., Kramer N., et al. The resazurin reduction assay can distinguish cytotoxic from cytostatic compounds in spheroid screening assays // J Biomol Screen. 2014. Vol. 19, N 7. P. 1047–1059. doi: 10.1177/1087057114532352

[140] Gantenbein-Ritter B, Potier E, Zeiter S, et al. Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds. Tissue Eng Part C Methods. 2008;14(4):353–358. doi: 10.1089/ten.tec.2008.0313

[141] Gantenbein-Ritter B., Potier E., Zeiter S., et al. Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds // Tissue Eng Part C Methods. 2008. Vol. 14, N 4. P. 353–358. doi: 10.1089/ten.tec.2008.0313

[142] Sanfilippo S, Canis M, Ouchchane L, et al. Viability assessment of fresh and frozen/thawed isolated human follicles: reliability of two methods (Trypan blue and Calcein AM/ethidium homodimer-1). J Assist Reprod Genet. 2011;28(12):1151–1156. doi: 10.1007/s10815-011-9649-y

[143] Sanfilippo S., Canis M., Ouchchane L., et al. Viability assessment of fresh and frozen/thawed isolated human follicles: reliability of two methods (Trypan blue and Calcein AM/ethidium homodimer-1) // J Assist Reprod Genet. 2011. Vol. 28, N 12. P. 1151–1156. doi: 10.1007/s10815-011-9649-y

[144] Dittmar R, Potier E, Van Zandvoort M, Ito K. Assessment of cell viability in three-dimensional scaffolds using cellular auto-fluorescence. Tissue Eng Part C Methods. 2012;18(3):198–204. doi: 10.1089/ten.tec.2011.0334

[145] Dittmar R., Potier E., Van Zandvoort M., Ito K. Assessment of cell viability in three-dimensional scaffolds using cellular auto-fluorescence // Tissue Eng Part C Methods. 2012. Vol. 18, N 3. P. 198–204. doi: 10.1089/ten.tec.2011.0334

[146] Kessel S, Cribbes S, Bonasu S, et al. Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer. Cytom Part A. 2017;91(9):883–892. doi: 10.1002/cyto.a.23143

[147] Kessel S., Cribbes S., Bonasu S., et al. Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer // Cytom Part A. 2017. Vol. 91, N 9. P. 883–892. doi: 10.1002/cyto.a.23143

[148] Chabert C, Laporte C, Fertin A, et al. New automatized method of 3D multiculture viability analysis based on confocal imagery: application to islets and mesenchymal stem cells co-encapsulation. Front Endocrinol (Lausanne). 2018;9:272. doi: 10.3389/fendo.2018.00272

[149] Chabert C., Laporte C., Fertin A., et al. New automatized method of 3D multiculture viability analysis based on confocal imagery: application to islets and mesenchymal stem cells co-encapsulation // Front Endocrinol (Lausanne). 2018. Vol. 9. P. 272. doi: 10.3389/fendo.2018.00272

[150] Forsythe SD, Devarasetty M, Shupe T, Bishop C. Environmental toxin screening using human-derived 3d bioengineered liver and cardiac organoids. Front Public Heal. 2018;6:1–10. doi: 10.3389/fpubh.2018.00103

[151] Forsythe S.D., Devarasetty M., Shupe T., Bishop C. Environmental toxin screening using human-derived 3D bioengineered liver and cardiac organoids // Front Public Heal. 2018. Vol. 6. P. 1–10. doi: 10.3389/fpubh.2018.00103

[152] https://worldwide.promega.com/ [Internet]. 3D Cell Viability Assay. Available from: https://www.promega.co.uk/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/celltiter-glo-3d-cell-viability-assay/?catNum=G9681&gclid=CjwKCAjw6raYBhB7EiwABge5KvC1d1YNEzYbQfDa-H3Uh_Jjlc72nBKIq9QbNN4Jdh3Ap5HO79TG9RoCY1EQAvD_BwE

[153] https://worldwide.promega.com/ [Internet]. 3D Cell Viability Assay. Доступ по ссылке: https://www.promega.co.uk/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/celltiter-glo-3d-cell-viability-assay/?catNum=G9681&gclid=CjwKCAjw6raYBhB7EiwABge5KvC1d1YNEzYbQfDa-H3Uh_Jjlc72nBKIq9QbNN4Jdh3Ap5HO79TG9RoCY1EQAvD_BwE

[154] Dominijanni AJ, Devarasetty M, Forsythe SD, et al. Cell viability assays in three-dimensional hydrogels: a comparative study of accuracy. Tissue Eng Part C Methods. 2021;27(7):401–410. doi: 10.1089/ten.tec.2021.0060

[155] Dominijanni A.J., Devarasetty M., Forsythe S.D., et al. Cell viability assays in three-dimensional hydrogels: a comparative study of accuracy // Tissue Eng Part C Methods. 2021. Vol. 27, N 7. P. 401–410. doi: 10.1089/ten.tec.2021.0060

[156] Spreda M, Hauptmann N, Lehner V, et al. Porous 3D scaffolds enhance msc vitality and reduce osteoclast activity. Molecules. 2021;26(20):6258. doi: 10.3390/molecules26206258

[157] Spreda M., Hauptmann N., Lehner V., et al. Porous 3D scaffolds enhance msc vitality and reduce osteoclast activity // Molecules. 2021. Vol. 26, N 20. P. 6258. doi: 10.3390/molecules26206258

[158] Eremeev AV, Zubkova OA, Ruchko ES, et al. Key parameters of autologous biomedical product for cartilage tissue repair. Medicine of Extreme Situations. 2020;22(4):59–66. doi: 10.47183/mes.2020.014

[159] Еремеев A.В., Зубкова О.А., Ручко Е.С., и др. Ключевые характеристики аутологичного биомедицинского продукта для коррекции дефекта хрящевой ткани // Медицина экстремальных ситуаций. 2020. Т. 22, № 4. С. 59–66. doi: 10.47183/mes.2020.014

RIGHTS & PERMISSIONS

Eremeev A.V., Pikina A.S., Vladimirova T.V., Bogomazova A.N.