Use of methods for determining cell viability in cryobiological research
DOI:
https://doi.org/10.15407/cryo35.04.181Ключевые слова:
cryobiology, cell viability, cryopreservation, dye exclusion method, colorimetric method, fluorescent dyes, luminometric assays, cultivationАннотация
Assessment of cell viability is critically important in the context of cryobiological research, as it allows determining the effectiveness of freeze-thawing procedures and low-temperature storage of isolated cells. The review systematizes the methods for determining cell viability used in cryobiological research, with an emphasis on their practical applicability, accuracy, and sensitivity. Both traditional methods based on the assessment of cell membrane integrity and metabolic activity, such as trypan blue staining, MTT assay, fluorescent dye assays, and more modern approaches, including flow cytometry, confocal microscopy, and methods for assessing cell functional activity, are reviewed. For each method, a description of the mechanism of action, a protocol, specific advantages, disadvantages, as well as equipment requirements and limitations for cell types are provided. A comparative analysis was conducted, which revealed the lack of a universal approach, but demonstrated the effectiveness of combined methods that combine fluorescent staining and metabolic assessment. Such combinations allow for high accuracy in assessing cell viability after cryopreservation. In addition, the review discusses the prospects for the development of methods for determining cell viability in cryobiology, including the introduction of the latest high-resolution imaging technologies, multiparametric analysis, as well as integration with microfluidic platforms and artificial intelligence to automate and improve the accuracy of the assessment. The review can serve as a practical tool for researchers in choosing the most optimal approach for assessing cell viability depending on the purpose of the experiment, available resources and cell type.
Probl Cryobiol Cryomed. 2025; 35(4): 179—193
Библиографические ссылки
Akbari S, Anderson P, Zang H, et al. Non-invasive real-time monitoring of cell concentration and viability using Doppler ultrasound. SLAS Technol. 2022; 27: 368-75. CrossRef
Al-Nasiry S, Geusens N, Hanssens M, et al. Th e use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Hum Reprod. 2007; 22(5): 1304-9. CrossRef
Ali M, Benfante V, Basirinia G, et al. Applications of artifi cial intelligence, deep learning, and machine learning to support the analysis of microscopic images of cells and tissues. J Imaging. [Internet]. 2025 Feb 15 [cited 2025 Oct 10]; 11(2): 59. Available from: https://www.mdpi.com/2313-433X/11/2/59 CrossRef
Asadian E, Bahramian F, Siavashy S, et al. A review on recent advances of AI-integrated microfl uidics for analytical and bioanalytical applications. TRAC-Trend Anal Chem. [Internet]. 2024 Oct 9 [cited 2025 Oct 10]; 181 (Part B): 118004. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0165993624004874 CrossRef
Atale N, Gupta S, Yadav UC, Rani V. Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques. J Microsc. 2014; 255(1): 7-19. CrossRef
Baust JM, Van Buskirk, Baust JG. Cell viability improves following inhibition of cryopreservation-induced apoptosis. In Vitro Cell Dev Biol Anim. 2000;36(4):26270. CrossRef
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. CrossRef
Berridge MV, Tan AS. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys. 1993; 303(2): 474-82. CrossRef
Bhuyan BK, Loughman BE, Fraser TJ, Day KJ. Comparison of different methods of determining cell viability after exposure to cytotoxic compounds. Exp Cell Res. 1976; 97(2): 275-80. CrossRef
Cai Y, Prochazkova M, Kim YS, et al. Assessment and comparison of viability assays for cellular products. Cytotherapy. 2024; 26(2): 201-9. CrossRef
Chandler DE, Roberson RW. Bioimaging: Current concepts in light and electron microscopy. Sudbury (MA): Jones & Bartlett Publishers; 2009.
Chatterjee S. Artefacts in histopathology. J Oral Maxillofac Pathol. 2014; 18: S111-S116. CrossRef
Clark NA, Swain JE. Oocyte cryopreservation: searching for novel improvement strategies. J Assist Reprod Genet. 2013; 30(7):865-75. CrossRef
Cory A, Owen T, Barltrop J, Cory JG. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun. 1991; 3(7): 207-12. CrossRef
Datta R, Heaster TM, Sharick JT, et al. Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications. J Biomed Opt. 2020; 25(7): 1-43. CrossRef
Decker T, Lohmann-Matthes ML. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods. 1988; 115: 61-9. CrossRef
Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986; 89(2): 271-7. CrossRef
Di Santo M, Tarozzi N, Nadalini M, Borini A. Human sperm cryopreservation: update on techniques, effect on DNA integrity, and implications for ART. Adv Urol. [Internet]. 2012 Dec 13 [cited 2025 Oct 10]; 2012: 854837. Available from: https://onlinelibrary.wiley.com/doi/10.1155/2012/854837 CrossRef
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. CrossRef
Fischer F, Hamann A, Osiewacz HD. Mitochondrial quality control: an integrated network of pathways. Trends Biochem Sci. 2012; 37(7): 284-92. CrossRef
Gao D, Critser JK. Mechanisms of cryoinjury in living cells. ILAR J. 2000; 41(4): 187-96. CrossRef
Gao Z, Li Y. Enhancing single-cell biology through advanced AI-powered microfluidics. Biomicrofluidics. [Internet]. 2023 Oct 3 [cited 2025 Oct 10]; 17(5): 051301. Available from: https://pubs.aip.org/aip/bmf/article/17/5/051301/2914114/Enhancing-single-cell-biology-through-advanced-AI CrossRef
Gebreyesus ST, Muneer G, Huang C, et al. Recent advances in microfluidics for single-cell functional proteomics. Lab Chip. [Internet]. 2023 Feb 22 [cited 2025 October 10]; 23: 1726. Available from: https://pubs.rsc.org/en/content/articlelanding/2023/lc/d2lc01096h CrossRef
Georgakoudi I, Quinn KP. Optical imaging using endogenous contrast to assess metabolic state. Annu Rev Biomed Eng. 2012; 14: 351-67. CrossRef
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. CrossRef
Green A, McElroy WD. Function of adenosine triphosphate in the activation of luciferin. Arch Biochem Biophys. 1956; 64: 257-71. CrossRef
Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods. 1989; 119(2): 203-10. CrossRef
Hornberger K, Yu G, McKenna D, Hubel A. Cryopreservation of hematopoietic stem cells: emerging assays, cryoprotectant agents, and technology to improve outcomes. Transfus Med Hemother. 2019; 46(3): 188-96. CrossRef
Huang SA, Heikal AA, Webb WW. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys J. 2002; 82(5): 2811-25. CrossRef
Jones KH, Senft JA. An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J Histochem Cytochem. 1985; 33(1): 77-9. CrossRef
Kaja S, Payne AJ, Singh T, et al. An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience. J Pharmacol Toxicol Methods. 2015; 73: 1-6. CrossRef
Karimi E, Nikkhah M, Hosseinkhani S. Label-free and bioluminescence-based nano-biosensor for ATP detection. Biosensors (Basel). [Internet]. 2022 Oct 24 [cited 2025 Oct 10]; 2022; 12(11):918. Available from: https://www.mdpi.com/2079-6374/12/11/918 CrossRef
Keshavarzi S, Dokht Eftekhari A, Vahabzadeh H, et al. Post-warming survival rates and clinical outcomes of human cleavage stage embryos vitrified/warmed using CryoTouch and Cryotop methods. Middle East Fertil Soc J. [Internet]. 2021 Aug 13 [cited 2025 Oct 10]; 26: 24. Available from: https://mefj.springeropen.com/articles/10.1186/s43043-021-00068-1 CrossRef
Kim SI, Kim HJ, Lee HJ, et al. Application of a non-hazardous vital dye for cell counting with automated cell counters. Anal Biochem. 2016; 492: 8-12. CrossRef
Kolenc OI, Quinn KP. Evaluating cell metabolism through autofluorescence imaging of NAD(P)H and FAD. Antioxid Redox Signal. 2019; 30(6): 875-89. CrossRef
Kramer DN, Guilbault GG. A substrate for the fluorometric determination of lipase activity. Anal Chem. 1963; 35: 588-9. CrossRef
Krause AW, Carley WW, Webb WW. Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture. J Histochem Cytochem. 1984; 12(10): 1081-90. CrossRef
Ley-Ngardigal S, Bertolin G. Approaches to monitor ATP levels in living cells: where do we stand? FEBS J. 2022; 289(24): 7940-69. CrossRef
Lundin A. Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites. Methods Enzymol. 2000; 305: 346-70. CrossRef
Luo T, Fan L, Zhu R, Sun D. Microfluidic single-cell manipulation and analysis: methods and applications. Micromachines (Basel). [Internet]. 2019 Feb 1 [cited 2025 Oct 10]; 10(2):104. Available from: https://www.mdpi.com/2072-666X/10/2/104 CrossRef
Mazur P. Cryobiology: the freezing of biological systems. Science. 1970; 168: 939-49. CrossRef
Mehta N, Shaik S, Devireddy R, Gartia MR. Single-cell analysis using hyper spectral imaging modalities. J Biomech Eng. 2018; 140(2):0208021-02080216. CrossRef
Milosevic J, Storch A, Schwarz J. Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells. Stem Cells. 2005; 23(5): 681-8. CrossRef
Mingji W, Rongbiao Z, Fei Z, et al. An evaluation approach of cell viability based on cell detachment assay in a single-channel integrated microfluidic chip. ACS Sensors. 2019; 4(10): 2654-61. CrossRef
Morgenstern DA, Ahsan G, Brocklesby M, et al. Post-thaw viability of cryopreserved peripheral blood stem cells (PBSC) does not guarantee functional activity: important implications for quality assurance of stem cell transplant programmes. Br J Haematol. 2016; 174(6): 942-51. CrossRef
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(12): 55-63. CrossRef
Park S, Veluvolu V, Martin WS, et al. Label-free, non-invasive, and repeatable cell viability bioassay using dynamic fullfield optical coherence microscopy and supervised machine learning. Biomed Opt Express. 2022; 13(6):3187-94. CrossRef
Paull KD, Shoemaker RH, Boyd MR, et al. Th e synthesis of XTT: A new tetrazolium reagent that is bioreducible to a water-soluble formazan. J Heterocycl Chem. 1988; 25: 911-4. CrossRef
Petrenko AYu. [The current state and prospects for the development of cryobiology and cryomedicine]. Visn Nac Acad Nauk Ukr. 2023; (2): 75-8. Ukrainian. CrossRef
Petrenko YA, Gorokhova NA, Tkachova EN, Petrenko AY. [The reduction of Alamar Blue by peripheral blood lymphocytes and isolated mitochondria]. Ukr Biokhim Zh. 2005; 77(5): 100-5. Ukrainian. PubMed
Petrushko M, Piniaiev V, Yurchuk T. The history of cryotechnologies in reproductive medicine: From randomness to stability. Hist Sci Technol. 2024; 14(2): 401-18. CrossRef
Priest JH, Baxter LK, Priest RE. Cell attachment assay as a measure of thawed cell viability. Cryobiology. 1965; 1(5): 345-7. CrossRef
Qian L, Dong Z, Guo T. Grow AI virtual cells: three data pillars and closed-loop learning. Cell Res. 2025; 35: 319-21. CrossRef
Rampersad SN. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Basel). 2012; 12(9): 12347-60. CrossRef
Reers M, Smiley ST, Mottola-Hartshorn C, et al. Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol. 1995; 260: 406-14. CrossRef
Riss TL, Moravec RA, Niles AL, et al. Cell viability assays. In: Markossian S, Grossman A, Arkin M, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004. [Cited 2025 Oct 10]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK144065/ PubMed
Robbins E, Marcus PI. Dynamics of acridine orange-cell interaction: I. Interrelationships of acridine orange particles and cytoplasmic reddening. J Cell Biol. 1963; 18(2): 237-50. CrossRef
Scudiero DA, Shoemaker RH, Paull KD, et al. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 1988; 48(17): 4827-33. PubMed
Sengbusch GV, Couwenbergs C, Kuhner J, Muller U. Fluorogenic substrate turnover in single living cells. Histochem J. 1976; 8: 341-50. CrossRef
Sivagnanam S, Mahato P, Das P. An overview on the development of different optical sensing platforms for adenosine triphosphate (ATP) recognition. Org Biomol Chem. 2023; 21(19): 3942-83. CrossRef
Stockert JC, Horobin RW, Colombo LL, Blázquez-Castro A. Tetrazolium salts and formazan products in cell biology: viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochem. 2018; 120(3): 159-67. CrossRef
Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2015; 111: A3.B.1-A3.B.3. CrossRef
Sukach OM, Kovalenko IF, Vsevolodska SO, et al. Use of autofluorescence for visualization of viable neural cells in three-dimensional structures. Bull Problems Biol Med. 2024; (2): 154-8. CrossRef
Tada H, Shiho O, Kuroshima K, et al. An improved colorimetric assay for interleukin 2. J Immunol Methods. 1986; 93(2): 157-65. CrossRef
Taylor DL, Wang YL, editors. Fluorescence microscopy of living cells in culture. Part B. Quantitative fluorescence microscopy-imaging and spectroscopy. Methods in cell biology. Vol. 30. San Diego: Academic Press;1989. 498 p. PubMed
Trusk TC. 3D Reconstruction of confocal image data. In: Gray JW, Price RL, editors. Basic Confocal Microscopy. 2nd ed. Cham: Springer Nature; 2018. p. 279-307. CrossRef
Vajta G, Kuwayama M. Improving cryopreservation systems. Theriogenology. 2006; 65(1): 236-44. CrossRef
Valyi-Nagy K, Betsou F, Susma A, Valyi-Nagy T. Optimization of viable glioblastoma cryopreservation for establishment of primary tumor cell cultures. Biopreserv Biobank. 2021; 19(1): 60-6. CrossRef
van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays: the MTT assay. Methods Mol Biol. 2011;731: 237-45. CrossRef
Verrier S, Zoladek A, Notingher I. Raman micro-spectroscopy as a non-invasive cell viability test. Methods Mol Biol. 2011; 740: 179-89. CrossRef
Vinci M, Gowan S, Boxall F, et al. Advances in establishment and analysis of three-dimensional tumor spheroid based functional assays for target validation and drug evaluation. BMC Biol. [Internet]. 2012 March 22 [cited 2025 Oct 10]; 10(1): 29. Available from: https://link.springer.com/article/10.1186/1741-7007-10-29 CrossRef
Wragg NM, Tampakis D, Stolzing A. Cryopreservation of mesenchymal stem cells using medical grade ice nucleation inducer. Int J Mol Sci. [Internet]. 2020 Nov 13 [cited 2025 Oct 10]; 21(22):8579. Available from: https://www.mdpi.com/1422-0067/21/22/8579 CrossRef
Wu SZ, Roden DL, Al-Eryani G, et al. Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis. Genome Med. [Internet]. 2021 May 10 [cited 2025 Oct 10]; 13: 81. Available from: https://link.springer.com/article/10.1186/s13073-021-00885-z CrossRef
Wyckoff J, Gligorijevic B, Entenberg D, et al. High-resolution multiphoton imaging of tumors in vivo. Cold Spring Harb Protoc. 2011; 2011(10): 1167-84. CrossRef
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. CrossRef
Yamatoya K, Nagai Y, Teramoto N, et al. Cryopreservation of undifferentiated and differentiated human neuronal cells. Reg Th er. 2022; 19: 58-68. CrossRef
Yurchuk T, Likszo P, Witek K, et al. New approach to the cryopreservation of GV oocytes and cumulus cells through the lens of preserving the intercellular gap junctions based on the bovine model. Int J Mol Sci. [Internet]. 2024 May 31 [cited 2025 Oct 10]; 25(11):6074. https://www.mdpi.com/1422-0067/25/11/6074 CrossRef
Yao N, Eisfelder BJ, Marvin J, Greenberg JT. The mitochondrion An organelle commonly involved in programmed cell death in Arabidopsis thaliana. Plant J. 2004; 40: 596-610. CrossRef
Younes N, Alsahan BS, Al-Mesaifri AJ, et al. JC-10 probe as a novel method for analyzing the mitochondrial membrane potential and cell stress in whole zebrafish embryos. Toxicol Res (Camb). 2021; 11(1): 77-87. CrossRef
Zaikov VS, Tarusin DN, Petrenko AY. Effect of Encapsulation into alginate microspheres on viability of mesenchymal stromal cells aft er exposure with penetrating cryoprotectants. Probl Cryobiol Cryomed. 2016; 26(3): 213-20. CrossRef
Zanoni M, Piccinini F, Arienti C, et al. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep. [Internet]. 2024 Oct 9 [cited 2025 Oct 10]; 6: 19103. Available from: https://www.nature.com/articles/srep19103 CrossRef
Zipfel WR, Williams RM, Webb WW. Nonlinear magic: Multiphoton microscopy in the biosciences. Nat Biotechnol. 2003; 21: 1369-77. CrossRef
Загрузки
Опубликован
Как цитировать
Выпуск
Раздел
Лицензия
Это произведение доступно по лицензии Creative Commons «Attribution» («Атрибуция») 4.0 Всемирная.
Авторы, публикующие в данном журнале, соглашаются со следующим:
- Авторы сохраняют за собой авторские права на работу и предоставляют журналу право первой публикации работы на условиях лицензии Creative Commons Attribution License, которая позволяет другим распространять данную работу с обязательным сохранением ссылок на авторов оригинальной работы и оригинальную публикацию в этом журнале.
- Авторы сохраняют право заключать отдельные контрактные договоренности, касающиеся не-эксклюзивного распространения версии работы в опубликованном здесь виде (например, размещение ее в институтском хранилище, публикацию в книге), со ссылкой на ее оригинальную публикацию в этом журнале.
- Авторы имеют право размещать их работу в сети Интернет (например в институтском хранилище или персональном сайте) до и во время процесса рассмотрения ее данным журналом, так как это может привести к продуктивному обсуждению и большему количеству ссылок на данную работу (См. The Effect of Open Access).
