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

Recent Advances in Electrical Impedance Sensing Technology for Single-Cell Analysis

“…Recent IFC devices applied in single-cell analysis are summarized in Table 1 . These applications, discussed in this subsection, are simply classified according to cell species, including blood cells [ 110 , 111 , 112 , 113 , 114 ], tumor cells [ 43 , 52 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ], stem cells [ 123 , 124 , 125 , 126 , 127 ], plant cells [ 60 , 128 , 129 , 130 , 131 , 132 ] and microbes [ 53 , 62 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 ]. In terms of blood cells, researchers focused on the identification and counting of normal or diseased blood cells.…”

Study of Oenological Fermentation: Which Strategy and Which Tools?

“…Flow cytometry is undoubtedly the most promising method to simultaneously obtain the quantification of yeast cells; the determination of their viability; and the assessment of their physiology, e.g., by morphological analysis of the budding division process [64,65]. The latter authors carried out online monitoring on a laboratory scale, thanks to automated sampling and sample processing (dilution and addition of a fluorochrome), but they considered that this monitoring is currently difficult to implement for process control.…”

Application of In-Situ and Soft-Sensors for Estimation of Recombinant P. pastoris GS115 Biomass Concentration: A Case Analysis of HBcAg (Mut+) and HBsAg (MutS) Production Processes under Varying Conditions

“…Dielectric properties of the cell suspension, such as capacitance (pF) and permittivity (pF/cm), proportionally relate to viable cell volume. This phenomenon is used by in-situ permittivity sensors [26,27] and Coulter-counters [28,29]. Modern instruments use frequency-scanning techniques in a range from 100 KHz to 20 MHz, capable for cell size and biomass concentration estimation (dielectric spectroscopy).…”