Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry
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The determination of cell viability is essential to many areas of life sciences and biotechnology. Typically, cell viability measurements are based on the optical analysis of stained cells, which requires additional labeling steps and is hard to implement online. Frequency-dependent impedance flow cytometry (IFC) provides a label-free, fast, and reliable alternative to determine cell viability at the single cell level based on the Coulter principle. Here, we describe the application of IFC to eukaryotic cell cultures and compare the results to commonly used staining methods. Yeast cell parameters were assessed in normal and heat-inactivated cells as well as in alcoholic fermentation and long-term batch cultures providing a precise and fast determination of the cell viability and further quantitative measures of the cell culture status. As an important new application, we have investigated recombinant protein production in the widely used baculovirus insect cell expression system. The IFC analysis revealed the presence of a subpopulation of cells, which correlates with the protein expression yield, but it is not detectable with conventional optical cell counters. We tentatively identify this subpopulation as cells in the late phase of infection. Their detection can serve as a predictor for the optimal time point of harvest. The IFC technique should be generally applicable to many eukaryotic cell cultures in suspension, possibly also implemented online.
KeywordsInsect cells Yeast Viability Cell density Microfluidics Single cell Fermentation Bioprocessing Label-free Recombinant expression
We thank Alexandra Meng for valuable suggestions on the manuscript and Doppelleu Boxer AG for providing yeast samples from their large-scale production tank.
Christian Opitz, Grit Schade, Marcel Ottiger, and Stephan Grzesiek designed the study. Christian Opitz, Grit Schade, and Silvan Kaufmann carried out data acquisition, analysis, and interpretation. Marco Di Berardino, Marcel Ottiger, and Stephan Grzesiek participated in data interpretation. Christian Opitz, Grit Schade, Silvan Kaufmann, Marco Di Berardino, Marcel Ottiger, and Stephan Grzesiek wrote the manuscript.
This work was supported by the Swiss National Science Foundation (grant 31-173089 to S.G.).
Compliance with ethical standards
Conflict of interest
Grit Schade, Silvan Kaufmann, Marco Di Berardino, and Marcel Ottiger are employees of Amphasys AG, Root, Switzerland. Christian Opitz and Stephan Grzesiek declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Barbau J (2017) Continuous suspension cell culture monitoring in bioreactors using quantitative phase imaging. 1–1Google Scholar
- Cadena-Herrera D, Esparza-De Lara JE, Ramírez-Ibañez ND, López-Morales CA, Pérez NO, Flores-Ortiz LF, Medina-Rivero E (2015) Validation of three viable-cell counting methods: manual, semi-automated, and automated. Biotechnol Rep (Amst) 7:9–16. https://doi.org/10.1016/j.btre.2015.04.004 CrossRefGoogle Scholar
- Crocetti S, Beyer C, Unternährer S, Benavides Damm T, Schade-Kampmann G, Hebeisen M, Di Berardino M, Fröhlich J, Franco-Obregón A (2014) Impedance flow cytometry gauges proliferative capacity by detecting TRPC1 expression. Cytometry A 85:525–536. https://doi.org/10.1002/cyto.a.22461 CrossRefPubMedGoogle Scholar
- Di Berardino M (2010) The Microflow Cytometer. In: Ligler FS, Kim JS (eds) Electrical detection in microfluidic flow cytometers. Pan Stanford, p. 379Google Scholar
- Küttel C, Nascimento E, Demierre N, Silva T, Braschler T, Renaud P, Oliva AG (2007) Label-free detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer. Acta Trop 102:63–68. https://doi.org/10.1016/j.actatropica.2007.03.002 CrossRefPubMedGoogle Scholar
- O’Reilly DR, Miller LK, Luckow VA (1994) Baculovirus expression vectors: a laboratory manual. Oxford University PressGoogle Scholar
- Stewart GG (2017) Yeast viability and vitality. In: Brewing and distilling yeasts. Springer, pp 147–165Google Scholar
- Wasilko DJ, Lee SE, Stutzman-Engwall KJ, Reitz BA, Emmons TL, Mathis KJ, Bienkowski MJ, Tomasselli AG, Fischer HD (2009) The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus. Protein Expr Purif 65:122–132. https://doi.org/10.1016/j.pep.2009.01.002 CrossRefPubMedGoogle Scholar
- Wei N, Sommer B (2013) Chapter 7: Cell viability measurements. In: Flickinger MC (ed) Upstream industrial biotechnology, Volume Set. Wiley, p 1854Google Scholar