Proliferation characteristics of cells cultured under periodic versus static conditions
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In vitro culture models have become an indispensable tool for assessing a vast variety of biological questions in many scientific fields. However, common in vitro cultures are maintained under static conditions, which do not reflect the in vivo situation and create a non-physiological environment. To assess whether the growth characteristics of cells cultured at pulsed-perfused versus static conditions differ, we observed the growth of differentially cultured cells in vitro by life-cell time-lapse imaging of recombinant HEK293YFPI152L cells, stably expressing yellow fluorescent protein. Cells were grown for ~ 30 h at 37 °C and ambient CO2 concentration in biochips mounted into a custom-designed 3D printed carrier and were imaged at a rate of ten images per hour using a fluorescence microscope with environment control infrastructure. Cells in one chip were maintained under static conditions whereas cells in another chip were recurrently perfused with fresh media. Generated image series were quantitatively analyzed using a custom-modified cell detection software. Imaging data averaged from four biological replicates per culturing condition demonstrate that cells cultured under conventional conditions exhibit an exponential growth rate. In contrast, cells cultured in periodic mode exhibited a non-exponential growth rate. Our data clearly indicate differential growth characteristics of cells cultured under periodic versus static conditions highlighting the impact of the culture conditions on the physiology of cells in vitro.
KeywordsCell growth Biochip Microfluidics HEK293 YFPI152L Long-term time-lapse microscopy
The authors gratefully acknowledge funding of the Staedtler Stiftung and ongoing support from the Erlangen Graduate School in Advanced Optical Technologies (SAOT) by the German Research Foundation (DFG) in the framework of the German Excellence Initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
J.W. and D.F.G. conceived the study. S.A.M. and D.F.G. conducted imaging experiments. D.F.G. analyzed and displayed imaging data. D.F.G. and J.W. and wrote the paper. All authors commented and agreed on the manuscript.
Compliance with ethical standards
Conflict of interest
JW is CEO and shareholder of Cellasys GmbH.
- 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 30:9612–9620CrossRefGoogle Scholar
- Coecke S, Balls M, Bowe G, Davis J, Gstraunthaler G, Hartung T, Hay R, Merten OW, Price A, Schechtman L, Stacey G, Stokes W (2005) Guidance on good cell culture practice. a report of the second ECVAM task force on good cell culture practice. Altern Lab Anim 33:261–287Google Scholar
- Gu MB, Mitchell RJ, Kim BC (2004) Whole-cell-based biosensors for environmental biomonitoring and application. Adv Biochem Eng Biotechnol 87:269–305Google Scholar
- Hartung T, Gstraunthaler G, Coecke S, Lewis D, Blanck O, Balls M (2001) Good cell culture practice (GCCP)–an initiative for standardization and quality control of in vitro studies. The establishment of an ECVAM task force on GCCP. Altex 18:75–78Google Scholar
- 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 9:51. https://doi.org/10.3389/fnmol.2016.00051 CrossRefGoogle Scholar
- 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 in Molecular Biology 1601:205–214. Humana Press, New York, NYGoogle Scholar
- 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:pii:034111. https://doi.org/10.1063/1.3486610
- 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 33:272–321Google Scholar
- Pamies D, Bal-Price A, Simeonov A, Tagle D, Allen D, Gerhold D, Yin D, Pistollato F, Inutsuka T, Sullivan K, Stacey G, Salem H, Leist M, Daneshian M, Vemuri MC, Mcfarland R, Coecke S, Fitzpatrick SC, Lakshmipathy U, Mack A, Wang WB, Yamazaki D, Sekino Y, Kanda Y, Smirnova L, Hartung T (2017) Good cell culture practice for stem cells and stem-cell-derived models. Altex 34:95–132Google Scholar
- Russell WMS, Burch RL (1959) The principles of humane experimental technique. Methuen, LondonGoogle Scholar
- Shekar R, Ranganathan K (2012) Phenotypic and growth characterization of human mesenchymal stem cells cultured from permanent and deciduous teeth. Indian J Dent Res 23:838–839Google Scholar
- 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 (2018) Fetal Bovine Serum (FBS): Past - Present - Future. ALTEX 35:99–118. https://doi.org/10.14573/altex.1705101 CrossRefGoogle Scholar
- 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 2013:1607–1610Google Scholar