Abstract

Introduction Long-term cell
viability monitoring is of interest for many researchers in the fields of drug
development, cancer research and regenerative medicine. Cell viability is
currently measured using e.g. cell counting, live/dead viability staining and metabolic
activity assays. However, these methods are generally invasive and can thus not
be used for long-term viability monitoring. An alternative for this would be to
use the confluency as a measure of cell viability. By taking microscopic images
of the cultured cells at several timepoints viability can be followed over time.
When using the CytoSMART Omni, an automated live-cell imager,
multiple samples can be analyzed at the same time inside a regular CO₂-incubator.
This allows for noninvasive viability analysis at predefined intervals and at optimal
culture conditions. The aim of this study was to perform a long-term viability
study using confluency measurements obtained with the CytoSMART Omni.

Materials and Methods C6 cells (rat
glial tumor cells) were cultured in a 96-well plate for 24 h. Thereafter the
microtubule stabilizer Paclitaxel was added and a high-resolution scan of the well
plate was made every hour with the CytoSMART Omni (37°C
and 5% CO₂; n = 8 per group). The confluency of each individual well
was determined at each timepoint and normalized for the confluency at 0 h to
account for differences in confluency at the start of the experiment.

Results Immediately
after addition of 100 µM PX, the cells start to detach and aggregate, without
being able to reattach, leading to an approximately constant confluency between
36 and 70 hours. When 0.4 to
33 µM
PX was added to the samples, the cells lose their elongated morphology and
detach, with an increasing degree of detachment at increasing PX
concentrations. The majority
of cells treated with the lowest concentrations of PX (1.7 to 137 nM) keep
their elongated morphology, while only few cells were completely rounded. However,
at these concentrations of PX the cells seem to be unable to proliferate at the
same rate as the untreated cells. The untreated
cells have an elongated morphology and proliferate at a steady rate, reaching 100%
confluency approximately 42 hours after the start of the experiment.

Discussion In this
study, we showed that cell viability of C6 cells treated with PX could easily
be determined from images obtained every hour with the CytoSMART^TM
Omni. At all timepoints, the normalized confluency of the untreated samples was
significantly higher than that of the PX treated samples. Since PX is a microtubule
stabilizer it causes defects in mitotic spindle formation, thus inhibiting chromosome
segregation and subsequently cell division. This mechanism of action explains
the lower normalized confluency of all groups treated with PX compared to the untreated
group, at all timepoints.

The use of
real-time live-cell imaging to monitor cell viability not only provides
quantitative data on confluency but also qualitative information about cell
morphology. This information is lacking when analyzing cell viability by using
metabolic activity assays or cell counting. In our case, for example, PX not
only affects viability but also cell adhesion. The impaired cell adhesion would
be unnoticed when counting cells or analyzing metabolic activity.

Conclusion Summarizing, we
showed that long-term Paclitaxel treatment not only affects cell viability but
also cell morphology. Upon increase in Paclitaxel concentrations, the morphological
alterations increase and the confluency level decreases. Furthermore, the noninvasive
confluency measurements allowed for long-term monitoring