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Abstract
Cells vary in their dynamic response to external stimuli, due to stochastic fluctuations and non-uniform progression through the cell cycle. Hence, single-cell studies are required to reveal the range of heterogeneity in their responses to defined perturbations, which provides detailed insight into signaling processes. Here, we present a time-lapse study using arrays of micro-trenches to monitor the timing of cell division and apoptosis in non-adherent cells at the single-cell level. By employing automated cell tracking and division detection, we precisely determine cell cycle duration and sister-cell correlations for hundreds of individual cells in parallel. As a model application we study the response of leukemia cells to the chemostatic drug vincristine as a function of cell cycle phase. The time-to-death after drug addition is found to depend both on drug concentration and cell cycle phase. The resulting timing and dose-response distributions were reproduced in control experiments using synchronized cell populations. Interestingly, in non-synchronized cells, the time-to-death intervals for sister cells appear to be correlated. Our study demonstrates the practical benefits of micro-trench arrays as a platform for high-throughput, single-cell time-lapse studies on cell cycle dependence, correlations and cell fate decisions in general.
Highlights
Cell-to-cell variability in responses to external stimuli is a pervasive feature of cellular systems, which prevails even in isogenic cell populations[1,2]
We demonstrate that automated image analysis using automated cell tracking permits precise determination of the distribution of cell cycle duration and detection of sister-cell correlations
To facilitate tracking of non-adherent cells over several generations in a label-free manner, we designed arrays of micro-trenches made of the biocompatible hydrogel polyethylene(glycol) diacrylate (PEG-DA; the fabrication of these arrays is described in Materials and Methods)
Summary
Cell-to-cell variability in responses to external stimuli is a pervasive feature of cellular systems, which prevails even in isogenic cell populations[1,2]. To study the effect of cell cycle phase on stimulus response with high statistical power, large numbers of single cells must be observed continuously. Daunorubicin, an anthracycline aminoglycoside antineoplastic, intercalates into DNA and inhibits the function of the enzyme topoisomerase II during transcription and replication[27] Both drugs are routinely used to treat a number of neoplasms[28,29], including acute myeloid leukemia (AML)[30,31]. In the presence of the anti-mitotic agent vincristine, the time-to-death interval decreases as the cell cycle progresses No such effect is observed in the case of the topoisomerase II inhibitor daunorubicin. We find the time-to-death of sister cells to be strongly correlated in the unsynchronized population
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