Webinar - Cell density monitoring in cell culture – a comparison of different methods.
This Webinar is titled - “Cell density monitoring in cell culture – a comparison of different methods.”
The webinar’s speaker isProfessor Mike Butler, distinguished professor of Animal cell technology, University of Manitoba.
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Cell density monitoring in cell culture - a comparison of different methods
Several methods are available to monitor the growth of mammalian cells in culture. This webinar will describe the principles behind 5 of these: particle counting, image analysis, in situ capacitance measurement, fluorescence-based flow cytometry and dielectrophoretc cytometry (DEP). All of these methods were used to monitor the progress of a culture of monoclonal antibody-producing mammalian cells (CHO). This included measurements of cell density as well as viability which is a key parameter for protein productivity.
The various techniques gave similar values during the exponential growth phase. However, beyond the point of highest cell density the measurements diverged. Flow cytometry with a range of fluorescent markers was used to investigate this divergence and to establish the progress of cell apoptosis. The cell density estimates by the intermediate stage apoptosis assay agreed with those obtained by the bulk capacitance probe and the early stage apoptosis assay viability measurements correlated well with the DEP cytometer. The trypan blue assay had much higher estimates for cell density and viability that did not correlate with either the capacitance probe or the DEP cytometer.
The DEP cytometer measures the dielectric properties of individual cells and was able to identify at least two populations of cells with distinct polarizability. As verified by comparison with Nexin assay, one population was associated with viable (non-apoptotic) cells and the other with apoptotic cells. From the end of the exponential through the stationary and decline phases there was a gradual shift of cell count from the viable into the apoptotic population. However, the two populations maintained their individual dielectric properties throughout this shift. This leads to the conclusion that changes in bulk dielectric properties of cultures might be better modeled as shifts in cells between different dielectric populations, rather than assuming a homogeneous dielectric population. Therefore, this finding shows how dielectric probes are sensitive to the early apoptotic changes in cells.
During the early stage of apoptosis there is a rapid decrease in the ATP content of cells which leads to a decline in the energetic status and a malfunction of the ATP-dependent ion pumps. We have shown that these events can be closely monitored in cells treated with oligomycin which inhibits oxidative phosphorylation and ATP generation from the mitochondria. DEP cytometry offers a novel and unique technology for analyzing and characterizing mammalian cells based on their dielectric properties, and suggests a potential application of the device as a low-cost, label-free, online electronic monitor of physiological changes in cells.