Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry

Abstract

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.

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Acknowledgments

We thank Alexandra Meng for valuable suggestions on the manuscript and Doppelleu Boxer AG for providing yeast samples from their large-scale production tank.

Funding

This work was supported by the Swiss National Science Foundation (grant 31-173089 to S.G.).

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Authors

Contributions

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.

Corresponding authors

Correspondence to Marcel Ottiger or Stephan Grzesiek.

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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.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Opitz, C., Schade, G., Kaufmann, S. et al. Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry. Appl Microbiol Biotechnol 103, 8619–8629 (2019). https://doi.org/10.1007/s00253-019-10046-3

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Keywords

  • Insect cells
  • Yeast
  • Viability
  • Cell density
  • Microfluidics
  • Single cell
  • Fermentation
  • Bioprocessing
  • Label-free
  • Recombinant expression