Skip to main content
SpringerLink
Log in

Assaying Proliferation Characteristics of Cells Cultured Under Static Versus Periodic Conditions

  • Protocol
  • First Online:
Cell Viability Assays

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2644))

  • 1181 Accesses

Abstract

Two-dimensional in vitro culture models are widely being employed for assessing a vast variety of biological questions in different scientific fields. Common in vitro culture models are typically maintained under static conditions, where the surrounding culture medium is replaced every few days—typically every 48 to 72 h—with the aim to remove metabolites and to replenish nutrients. Although this approach is sufficient for supporting cellular survival and proliferation, static culture conditions do mostly not reflect the in vivo situation where cells are continuously being perfused by extracellular fluid, and thus, create a less-physiological environment. In order to evaluate whether the proliferation characteristics of cells in 2D culture maintained under static conditions differ from cells kept in a dynamic environment, in this chapter, we provide a protocol for differential analysis of cellular growth under static versus pulsed-perfused conditions, mimicking continuous replacement of extracellular fluid in the physiological environment. The protocol involves long-term life-cell high-content time-lapse imaging of fluorescent cells at 37 °C and ambient CO2 concentration using multi-parametric biochips applicable for microphysiological analysis of cellular vitality. We provide instructions and useful information for (i) the culturing of cells in biochips, (ii) setup of cell-laden biochips for culturing cells under static and pulsed-perfused conditions, (iii) long-term life-cell high-content time-lapse imaging of fluorescent cells in biochips, and (iv) quantification of cellular proliferation from image series generated from imaging of differentially cultured cells.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ritter P, Bye LJ, Finol-Urdaneta RK, Lesko C, Adams DJ, Friedrich O, Gilbert DF (2020) A method for high-content functional imaging of intracellular calcium responses in gelatin-immobilized non-adherent cells. Exp Cell Res 395(2):112210. https://doi.org/10.1016/j.yexcr.2020.112210

    Article  CAS  PubMed  Google Scholar 

  2. Gilbert DF, Boutros M (2016) A protocol for a high-throughput multiplex cell viability assay. Methods Mol Biol 1470:75–84. https://doi.org/10.1007/978-1-4939-6337-9_6

    Article  CAS  PubMed  Google Scholar 

  3. Gilbert DF, Erdmann G, Zhang X, Fritzsche A, Demir K, Jaedicke A, Muehlenberg K, Wanker EE, Boutros M (2011) A novel multiplex cell viability assay for high-throughput RNAi screening. PLoS One 6(12):e28338. https://doi.org/10.1371/journal.pone.0028338

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gilbert DF, Friedrich O (2017) Cell viability assays. Springer New York, New York

    Google Scholar 

  5. Kuenzel K, Mofrad SA, Gilbert DF (2017) Phenotyping cellular viability by functional analysis of ion channels: GlyR-targeted screening in NT2-N cells. In: Gilbert DF, Friedrich O (eds) Cell viability assays: methods and protocols. Springer New York, New York, pp 205–214. https://doi.org/10.1007/978-1-4939-6960-9_16

    Chapter  Google Scholar 

  6. Walzik MP, Vollmar V, Lachnit T, Dietz H, Haug S, Bachmann H, Fath M, Aschenbrenner D, Abolpour Mofrad S, Friedrich O, Gilbert DF (2015) A portable low-cost long-term live-cell imaging platform for biomedical research and education. Biosens Bioelectron 64:639–649. https://doi.org/10.1016/j.bios.2014.09.061

    Article  CAS  PubMed  Google Scholar 

  7. Gu MB, Mitchell RJ, Kim BC (2004) Whole-cell-based biosensors for environmental biomonitoring and application. Adv Biochem Eng Biotechnol 87:269–305

    CAS  PubMed  Google Scholar 

  8. Wiest J, Brischwein M, Ressler J, Otto AM, Grothe H, Wolf B (2005) Cellular assays with multiparametric bioelectronic sensor chips. CHIMIA Int J Chem 59(5):243–246. https://doi.org/10.2533/000942905777676623

    Article  CAS  Google Scholar 

  9. Alexander F Jr, Eggert S, Wiest J (2017) A novel lab-on-a-chip platform for spheroid metabolism monitoring. Cytotechnology. https://doi.org/10.1007/s10616-017-0152-x

  10. Bhise NS, Ribas J, Manoharan V, Zhang YS, Polini A, Massa S, Dokmeci MR, Khademhosseini A (2014) Organ-on-a-chip platforms for studying drug delivery systems. J Control Release 190:82–93. https://doi.org/10.1016/j.jconrel.2014.05.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cho S, Yoon JY (2017) Organ-on-a-chip for assessing environmental toxicants. Curr Opin Biotechnol 45:34–42. https://doi.org/10.1016/j.copbio.2016.11.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Schmidt C, Markus J, Kandarova H, Wiest J (2020) Tissue-on-a-chip: microphysiometry with human 3D models on transwell inserts. Front Bioeng Biotechnol 8. https://doi.org/10.3389/fbioe.2020.00760

  13. Wiest J (2022) Systems engineering of microphysiometry. Organs-on-a-Chip 4:100016. https://doi.org/10.1016/j.ooc.2022.100016

    Article  CAS  Google Scholar 

  14. Pamies D, Leist M, Coecke S, Bowe G, Allen DG, Gstraunthaler G, Bal-Price A, Pistollato F, de Vries RBM, Hogberg HT, Hartung T, Stacey G Guidance document on good cell and tissue culture practice 2.0 (GCCP 2.0). https://doi.org/10.14573/altex.2111011

  15. Fang Y (2007) Non-invasive optical biosensor for probing cell signaling. Sensors (Basel, Switzerland) 7(10):2316–2329

    Article  CAS  PubMed  Google Scholar 

  16. Liu L, Cash TP, Jones RG, Keith B, Thompson CB, Simon MC (2006) Hypoxia-induced energy stress regulates mRNA translation and cell growth. Mol Cell 21(4):521–531. https://doi.org/10.1016/j.molcel.2006.01.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Weiss D, Brischwein M, Grothe H, Wolf B, Wiest J (2013) Label-free monitoring of whole cell vitality. Conf Proc IEEE Eng Med Biol Soc 2013:1607–1610. https://doi.org/10.1109/embc.2013.6609823

    Article  CAS  Google Scholar 

  18. Kuenzel K, Friedrich O, Gilbert DF (2016) A recombinant human pluripotent stem cell line stably expressing halide-sensitive YFP-I152L for GABAAR and GlyR-targeted high-throughput drug screening and toxicity testing. Front Mol Neurosci 9. https://doi.org/10.3389/fnmol.2016.00051

  19. Menzner AK, Abolpour Mofrad S, Friedrich O, Gilbert DF (2015) Towards in vitro DT/DNT testing: assaying chemical susceptibility in early differentiating NT2 cells. Toxicology 338:69–76. https://doi.org/10.1016/j.tox.2015.10.007

    Article  CAS  PubMed  Google Scholar 

  20. Menzner A-K, Gilbert DF (2017) A protocol for in vitro high-throughput chemical susceptibility screening in differentiating NT2 stem cells. In: Gilbert DF, Friedrich O (eds) Cell viability assays: methods and protocols. Springer New York, New York, pp 61–70. https://doi.org/10.1007/978-1-4939-6960-9_5

    Chapter  Google Scholar 

  21. Balansa W, Islam R, Fontaine F, Piggott AM, Zhang H, Webb TI, Gilbert DF, Lynch JW, Capon RJ (2010) Ircinialactams: subunit-selective glycine receptor modulators from Australian sponges of the family Irciniidae. Bioorg Med Chem 18(8):2912–2919. https://doi.org/10.1016/j.bmc.2010.03.002

    Article  CAS  PubMed  Google Scholar 

  22. Balansa W, Islam R, Fontaine F, Piggott AM, Zhang H, Xiao X, Webb TI, Gilbert DF, Lynch JW, Capon RJ (2013) Sesterterpene glycinyl-lactams: a new class of glycine receptor modulator from Australian marine sponges of the genus Psammocinia. Org Biomol Chem 11(28):4695–4701. https://doi.org/10.1039/c3ob40861b

    Article  CAS  PubMed  Google Scholar 

  23. Balansa W, Islam R, Gilbert DF, Fontaine F, Xiao X, Zhang H, Piggott AM, Lynch JW, Capon RJ (2013) Australian marine sponge alkaloids as a new class of glycine-gated chloride channel receptor modulator. Bioorg Med Chem 21(14):4420–4425. https://doi.org/10.1016/j.bmc.2013.04.061

    Article  CAS  PubMed  Google Scholar 

  24. Chung SK, Vanbellinghen JF, Mullins JG, Robinson A, Hantke J, Hammond CL, Gilbert DF, Freilinger M, Ryan M, Kruer MC, Masri A, Gurses C, Ferrie C, Harvey K, Shiang R, Christodoulou J, Andermann F, Andermann E, Thomas RH, Harvey RJ, Lynch JW, Rees MI (2010) Pathophysiological mechanisms of dominant and recessive GLRA1 mutations in hyperekplexia. J Neurosci 30(28):9612–9620. https://doi.org/10.1523/jneurosci.1763-10.2010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gebhardt FM, Mitrovic AD, Gilbert DF, Vandenberg RJ, Lynch JW, Dodd PR (2010) Exon-skipping splice variants of excitatory amino acid transporter-2 (EAAT2) form heteromeric complexes with full-length EAAT2. J Biol Chem 285(41):31313–31324. https://doi.org/10.1074/jbc.M110.153494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gilbert D, Esmaeili A, Lynch JW (2009) Optimizing the expression of recombinant alphabetagamma GABAA receptors in HEK293 cells for high-throughput screening. J Biomol Screen 14(1):86–91. https://doi.org/10.1177/1087057108328017

    Article  CAS  PubMed  Google Scholar 

  27. Gilbert DF, Islam R, Lynagh T, Lynch JW, Webb TI (2009) High throughput techniques for discovering new glycine receptor modulators and their binding sites. Front Mol Neurosci 2:17. https://doi.org/10.3389/neuro.02.017.2009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gilbert DF, Mofrad SA, Friedrich O, Wiest J (2018) Proliferation characteristics of cells cultured under periodic versus static conditions. Cytotechnology. https://doi.org/10.1007/s10616-018-0263-z

  29. Gilbert DF, Wilson JC, Nink V, Lynch JW, Osborne GW (2009) Multiplexed labeling of viable cells for high-throughput analysis of glycine receptor function using flow cytometry. Cytometry A 75(5):440–449. https://doi.org/10.1002/cyto.a.20703

    Article  CAS  PubMed  Google Scholar 

  30. Kahl M, Gertig M, Hoyer P, Friedrich O, Gilbert DF (2019) Ultra-low-cost 3D bioprinting: modification and application of an off-the-shelf desktop 3D-printer for biofabrication. Front Bioeng Biotechnol 7(184). https://doi.org/10.3389/fbioe.2019.00184

  31. Talwar S, Lynch JW, Gilbert DF (2013) Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells. PLoS One 8(3):e58479. https://doi.org/10.1371/journal.pone.0058479

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Abolpour Mofrad S, Kuenzel K, Friedrich O, Gilbert DF (2016) Optimizing neuronal differentiation of human pluripotent NT2 stem cells in monolayer cultures. Develop Growth Differ 58(8):664–676. https://doi.org/10.1111/dgd.12323

    Article  CAS  Google Scholar 

  33. Dakhil H, Gilbert DF, Malhotra D, Limmer A, Engelhardt H, Amtmann A, Hansmann J, Hübner H, Buchholz R, Friedrich O, Wierschem A (2016) Measuring average rheological quantities of cell monolayers in the linear viscoelastic regime. Rheol Acta 55(7):527–536. https://doi.org/10.1007/s00397-016-0936-5

    Article  CAS  Google Scholar 

  34. Demmel F, Brischwein M, Wolf P, Huber F, Pfister C, Wolf B (2015) Nutrient depletion and metabolic profiles in breast carcinoma cell lines measured with a label-free platform. Physiol Meas 36(7):1367–1381. https://doi.org/10.1088/0967-3334/36/7/1367

    Article  CAS  PubMed  Google Scholar 

  35. Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE (2010) Reconstituting organ-level lung functions on a chip. Science 328(5986):1662–1668. https://doi.org/10.1126/science.1188302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Inamdar NK, Borenstein JT (2011) Microfluidic cell culture models for tissue engineering. Curr Opin Biotechnol 22(5):681–689. https://doi.org/10.1016/j.copbio.2011.05.512

    Article  CAS  PubMed  Google Scholar 

  37. Khademhosseini A, Langer R (2016) A decade of progress in tissue engineering. Nat Protoc 11(10):1775–1781. https://doi.org/10.1038/nprot.2016.123

    Article  CAS  PubMed  Google Scholar 

  38. Liu Q, Wu C, Cai H, Hu N, Zhou J, Wang P (2014) Cell-based biosensors and their application in biomedicine. Chem Rev 114(12):6423–6461. https://doi.org/10.1021/cr2003129

    Article  CAS  PubMed  Google Scholar 

  39. Mahto SK, Yoon TH, Rhee SW (2010) A new perspective on in vitro assessment method for evaluating quantum dot toxicity by using microfluidics technology. Biomicrofluidics 4(3). https://doi.org/10.1063/1.3486610

  40. McGillicuddy N, Floris P, Albrecht S, Bones J (2017) Examining the sources of variability in cell culture media used for biopharmaceutical production. Biotechnol Lett. https://doi.org/10.1007/s10529-017-2437-8

  41. Pfister C, Bozsak C, Wolf P, Demmel F, Brischwein M (2015) Cell shape-dependent shear stress on adherent cells in a micro-physiologic system as revealed by FEM. Physiol Meas 36(5):955–966. https://doi.org/10.1088/0967-3334/36/5/955

    Article  CAS  PubMed  Google Scholar 

  42. van der Valk J, Bieback K, Buta C, Cochrane B, Dirks WG, Fu J, Hickman JJ, Hohensee C, Kolar R, Liebsch M, Pistollato F, Schulz M, Thieme D, Weber T, Wiest J, Winkler S, Gstraunthaler G (2017) Fetal Bovine Serum (FBS): past - present - future. ALTEX. https://doi.org/10.14573/altex.1705101

  43. van Midwoud PM, Janse A, Merema MT, Groothuis GM, Verpoorte E (2012) Comparison of biocompatibility and adsorption properties of different plastics for advanced microfluidic cell and tissue culture models. Anal Chem 84(9):3938–3944. https://doi.org/10.1021/ac300771z

    Article  CAS  PubMed  Google Scholar 

  44. Yao T, Asayama Y (2017) Animal-cell culture media: history, characteristics, and current issues. Reprod Med Biol 16(2):99–117. https://doi.org/10.1002/rmb2.12024

    Article  PubMed  PubMed Central  Google Scholar 

  45. McConnell HM, Owicki JC, Parce JW, Miller DL, Baxter GT, Wada HG, Pitchford S (1992) The cytosensor microphysiometer: biological applications of silicon technology. Science 257(5078):1906–1912

    Article  CAS  PubMed  Google Scholar 

  46. Eklund SE, Taylor D, Kozlov E, Prokop A, Cliffel DE (2004) A microphysiometer for simultaneous measurement of changes in extracellular glucose, lactate, oxygen, and acidification rate. Anal Chem 76(3):519–527. https://doi.org/10.1021/ac034641z

    Article  CAS  PubMed  Google Scholar 

  47. Marx U, Andersson TB, Bahinski A, Beilmann M, Beken S, Cassee FR, Cirit M, Daneshian M, Fitzpatrick S, Frey O, Gaertner C, Giese C, Griffith L, Hartung T, Heringa MB, Hoeng J, de Jong WH, Kojima H, Kuehnl J, Leist M, Luch A, Maschmeyer I, Sakharov D, Sips AJ, Steger-Hartmann T, Tagle DA, Tonevitsky A, Tralau T, Tsyb S, van de Stolpe A, Vandebriel R, Vulto P, Wang J, Wiest J, Rodenburg M, Roth A (2016) Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing. ALTEX 33(3):272–321. https://doi.org/10.14573/altex.1603161

    Article  PubMed  PubMed Central  Google Scholar 

  48. Weltin A, Slotwinski K, Kieninger J, Moser I, Jobst G, Wego M, Ehret R, Urban GA (2014) Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem. Lab Chip 14(1):138–146. https://doi.org/10.1039/c3lc50759a

    Article  CAS  PubMed  Google Scholar 

  49. Wolf B, Brischwein M, Baumann W, Ehret R, Kraus M (1998) Monitoring of cellular signalling and metabolism with modular sensor-technique: the PhysioControl-Microsystem (PCM). Biosens Bioelectron 13(5):501–509

    Article  CAS  PubMed  Google Scholar 

  50. Weber T, Wiest J, Oredsson S, Bieback K (2022) Case studies exemplifying the transition to animal component-free cell culture. Altern Lab Anim:02611929221117999. https://doi.org/10.1177/02611929221117999

  51. Gilbert DF, Meinhof T, Pepperkok R, Runz H (2009) DetecTiff: a novel image analysis routine for high-content screening microscopy. J Biomol Screen 14(8):944–955. https://doi.org/10.1177/1087057109339523

    Article  CAS  PubMed  Google Scholar 

  52. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Sabatini DM (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7(10):R100. https://doi.org/10.1186/gb-2006-7-10-r100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Medical Biotechnology, Department of Chemical and Biological Engineering (CBI), Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany

    Daniel F. Gilbert & Oliver Friedrich

  2. cellasys GmbH, Kronburg, Germany

    Joachim Wiest

Authors
  1. Daniel F. Gilbert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Oliver Friedrich
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joachim Wiest
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel F. Gilbert .

Editor information

Editors and Affiliations

  1. Institute of Medical Biotechnology (MBT), Department of Chemical and Biological Engineering (CBI), Friedrich-Alexander-University Erlangen-Nüremberg, Erlangen, Germany

    Oliver Friedrich

  2. Institute of Medical Biotechnology (MBT), Department of Chemical and Biological Engineering (CBI), Friedrich-Alexander-University Erlangen-Nüremberg, Erlangen, Germany

    Daniel F. Gilbert

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Gilbert, D.F., Friedrich, O., Wiest, J. (2023). Assaying Proliferation Characteristics of Cells Cultured Under Static Versus Periodic Conditions. In: Friedrich, O., Gilbert, D.F. (eds) Cell Viability Assays. Methods in Molecular Biology, vol 2644. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3052-5_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3052-5_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3051-8

  • Online ISBN: 978-1-0716-3052-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation