Skip to main content
SpringerLink
Log in

Comparative studies of cellular viability levels on 2D and 3D in vitro culture matrices

  • Original Article
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

In this study, the cellular viability and function of immortalized human cervical and dermal cells are monitored and compared in conventional 2D and two commercial 3D membranes, Collagen and Geltrex, of varying working concentration and volume. Viability was monitored with the aid of the Alamar Blue assay, cellular morphology was monitored with confocal microscopy, and cell cycle studies and cell death mechanism studies were performed with flow cytometry. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to 3D environment causing alterations to effective resazurin concentration, uptake and conversion rates, which was dependent on exposure time, but also due to the effect of the membrane itself on cellular function. These effects were verified by flow cytometry, in which no significant differences in viable cell numbers between 2D and 3D systems were observed after 24 h culture. The results showed the observed effect was different after shorter exposure periods, was also dependent on working concentration of the 3D system and could be mediated by altering the culture vessel size. Cell cycle analysis revealed cellular function could be altered by growth on the 3D substrates and the alterations were noted to be dependent on 3D membrane concentration. The use of 3D culture matrices has been widely interpreted to result in “improved viability levels” or “reduced” toxicity or cellular “resistance” compared to cells cultured on traditional 2D systems. The results of this study show that cellular health and viability levels are not altered by culture in 3D environments, but their normal cycle can be altered as indicated in the cell cycle studies performed and such variations must be accounted for in studies employing 3D membranes for in vitro cellular screening.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Al-Nasiry S, Geusens N, Hanssens M, Luyten C, Pijneuborg R (2007) The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Hum Reprod 22:1304–1309

    Article  CAS  Google Scholar 

  • Annabi N, Tamayol A, Uquillas JA, Akbari M, Bertassoni LE, Cha C, Camci-unal G, Dokmeci MR, Peppas NA (2014) 25th anniversary article: rational design and applications of hydrogels in regenerative medicine. Adv Mater 26:85–124

    Article  CAS  Google Scholar 

  • Antoni D, Burckel H, Josset E, Noel G (2015) Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci 16:5517–5527

    Article  CAS  Google Scholar 

  • Bonnier F, Keating ME, Wróbel T, Majzner K, Baranska M, Garcia A, Blanco A, Byrne HJ (2015) Cell viability assessment using the Alamar Blue assay: a comparison of 2D and 3D cell culture models. Toxicol In Vitro 29:124–131

    Article  CAS  Google Scholar 

  • Breslin S, O’Driscoll L (2013) Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today 18:240–249

    Article  CAS  Google Scholar 

  • Cartmell SH, Porter BD, Garcia AJ, Guldberg RE (2003) Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro. Tissue Eng 9:1197–1203

    Article  CAS  Google Scholar 

  • Casey A, Gargotti M, Bonnier F, Byrne HJ (2016) Chemotherapeutic efficiency of drugs in vitro: comparison of doxorubicin exposure in 3D and 2D culture matrices. Toxicol In Vitro 33:99–104

    Article  CAS  Google Scholar 

  • Cody D, Casey A, Naydenova I, Mihaylova E (2013) A comparative cytotoxic evaluation of acrylamide and diacetone acrylamide to investigate their suitability for holographic photopolymer formulations. Int J Polym Sci 2013. doi:10.1155/2013/564319

  • Drife JO (1986) Breast development in puberty. Ann N Y Acad Sci 464:58–65

    Article  CAS  Google Scholar 

  • European Union—Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010

  • Freshney RI (2005) Culture of animal cells. A manual of basic technique, 5th edn. Wiley, Hoboken

    Book  Google Scholar 

  • Gilbert TW, Sellaro TL, Badylak SF (2006) Decellularization of tissues and organs. Biomaterials 27:3675–3683

    CAS  Google Scholar 

  • Gorbsky GJ (2001) The mitotic spindle checkpoint. Curr Biol 11:R1001–R1004

    Article  CAS  Google Scholar 

  • Han Z, Chatter D, He DM, Pantazis P, Wyche JH, Hendrickson EA (1995) Evidence for a G2 checkpoint in p53-independent apoptosis induction by X-irradiation. Mol Cell Biol 15:5849–5857

    Article  CAS  Google Scholar 

  • Herzog E, Casey A, Lyng FM, Chambers G, Byrne HJ, Davoren M (2007) A new approach to the toxicity testing of carbon-based nanomaterials—the clonogenic assay. Toxicol Lett 174:49–60

    Article  CAS  Google Scholar 

  • Hutmacher DW (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials 21:2529–2543

    Article  CAS  Google Scholar 

  • Kim JB (2005) Three-dimensional tissue culture models in cancer biology. Semin Cancer Biol 15:365–377

    Article  Google Scholar 

  • Kuda T, Yano T (2003) Colorimetric alamarBlue assay as a bacterial concentration and spoilage index of marine foods. Food Control 14:455–461

    Article  Google Scholar 

  • Kutys ML, Doyle AD, Yamada KM (2013) Regulation of cell adhesion and migration by cell-derived matrices. Exp Cell Res 319:2434–2439

    Article  CAS  Google Scholar 

  • Lee J, Cuddihy MJ, Kotov NA (2008) Three-dimensional cell culture matrices: state of the art. Tissue Eng Part B Rev 14:61–86

    Article  CAS  Google Scholar 

  • Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    Article  CAS  Google Scholar 

  • Mukherjee SG, O’Claonadh N, Casey A (2011) Comparative in vitro cytotoxicity study of silver nanoparticle on two mammalian cell lines. Toxicol In Vitro 26:238–251

    Article  Google Scholar 

  • O’Brien J, Wilson I, Orton T, Pognan F (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267:5421–5426

    Article  Google Scholar 

  • Padmalayam I, Suto MJ (2012) 3D cell cultures. Mimicking in vivo tissues for improved predictability in drug discovery. Annu Rep Med Chem 47:367–378

    Article  CAS  Google Scholar 

  • Parker WB (2009) Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev 109:2880–2893

    Article  CAS  Google Scholar 

  • Peck Y, Wang D-A (2013) Three-dimensionally engineered biomimetic tissue models for in vitro drug evaluation: delivery, efficacy and toxicity. Expert Opin Drug Deliv 10:369–383

    Article  CAS  Google Scholar 

  • Pettit RK, Weber CA, Kean MJ, Hoffmann H, Pettit GR, Tan T, Franks KS, Horton ML (2005) Microplate Alamar Blue assay for Staphylococcus epidermidis biofilm susceptibility testing. Antimicrob Agents Chemother 49(7):2612–2617

    Article  CAS  Google Scholar 

  • Place ES, George JH, Williams CK, Stevens MM (2009) Synthetic polymer scaffolds for tissue engineering. Chem Soc Rev 38:1139–1151

    Article  CAS  Google Scholar 

  • Rampersad SN (2012) Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Switzerland) 12:12347–12360

    Article  CAS  Google Scholar 

  • Ravi M, Paramesh V, Kaviya SR, Anuradha E, Solomon FD (2015) 3D cell culture systems: advantages and applications. J Cell Physiol 230:16–26

    Article  CAS  Google Scholar 

  • Rimann M, Graf-Hausner U (2012) Synthetic 3D multicellular systems for drug development. Curr Opin Biotechnol 23:803–809

    Article  CAS  Google Scholar 

  • Riss T (2014) Overview of 3D Cell culture model systems validating cell-based assays for use with 3D cultures [PowerPoint slides]. Retrieved from https://worldwide.promega.com/-/media/files/promega-worldwide/north-america/promega-us/webinars-and-events/2014/3d-cell-culture-webinar-march-2014.pdf?la=en

  • Seluanov A, Hine C, Azpurua J, Feigenson M, Bozzella M, Mao Z, Catania KC, Gorbunova V (2009) Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat. Proc Natl Acad Sci 106:19352–19357

    Article  CAS  Google Scholar 

  • Stacey G, Bar P, Granville R (2009) Primary cell cultures and immortal cell lines. Encycl Life Sci, pp. 1–6. http://orca.cf.ac.uk/24620/

  • Vega-Avila E, Pugsley MK (2011) An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. Proc West Pharmacol Soc 54:10–14

    CAS  Google Scholar 

  • White MJ, DiCaprio MJ, Greenberg DA (1996) Assessment of neuronal viability with Alamar Blue in cortical and granule cell cultures. J Neurosci Methods 70:195–200

    Article  CAS  Google Scholar 

  • Worthington P, Pochan DJ, Langhans SA (2015) Peptide hydrogels—versatile matrices for 3D cell culture in cancer medicine. Front Oncol 2015:92

    Google Scholar 

Download references

Acknowledgements

This study were funded by the Government of Libya for M. Gargotti and Consejo Nacional de Cienciay Tecnología, Mexico for U. Lopez-Gonzalez and this work has been enabled by Science Foundation Ireland Principle Investigator Award 11/PI/1108.

Author information

Authors and Affiliations

  1. School of Physics, Nanolab Research Centre, FOCAS Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland

    M. Gargotti, U. Lopez-Gonzalez & A. Casey

  2. FOCAS Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland

    M. Gargotti, U. Lopez-Gonzalez, H. J. Byrne & A. Casey

Authors
  1. M. Gargotti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. U. Lopez-Gonzalez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. J. Byrne
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Casey
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Gargotti.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gargotti, M., Lopez-Gonzalez, U., Byrne, H.J. et al. Comparative studies of cellular viability levels on 2D and 3D in vitro culture matrices. Cytotechnology 70, 261–273 (2018). https://doi.org/10.1007/s10616-017-0139-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-017-0139-7

Keywords

Search

Navigation