Iron oxide nanoparticles have been used in preclinical studies to label stem cells for non-invasive tracking and homing. The search continues for novel particle candidates that are suitable for clinical applications. Since standard analyses to investigate cell-particle interactions and safety are labor-intensive, an efficient procedure is required to guide future particle development and to exclude adverse health effects. The application of combined Raman trapping microscopy with fluidic chips is reported for the analysis of single cells labeled with different types of aminated iron oxide particles. Multivariate data analysis revealed Raman signal differences that could be clearly assigned to cell-particle interactions and cytotoxicity, respectively. A validation dataset verified that more than 95 % of the spectra were correctly classified. Thus, our approach enables rapid discrimination of non-hazardous from cytotoxic nanoparticles as a prerequisite for safe clinical applications.

Cancer therapy via redirected lysis mediated by antibodies and antibody-derived agents relies on the availability of substantial numbers of sufficiently active immune effector cells. To monitor antitumor responses before and during therapy, sensitive methods are needed, capable of quantitating specific lysis of target cells. Here we present a chip-based single-cell cytometric assay, which uses adherent human target cells arrayed in structured micro-fields. Using a fluorescent indicator of cell death and time-lapse microscopy in an automated high-throughput mode, we measured specific target cell lysis by activated human NK cells, mediated by the therapeutic single chain triplebody SPM-2 (33-16-123). This antibody-derived tri-specific fusion protein carries binding sites for the myeloid antigens CD33 and CD123 and recruits NK cells via a binding site for the Fc-receptor CD16. Specific lysis increased with increasing triplebody concentration, and the single-cell assay was validated by direct comparison with a standard calcein-release assay. The chip-based approach allowed measurement of lysis events over 16 hours (compared to 4 hours for the calcein assay) and required far smaller numbers of primary cells. In addition, dynamic properties inaccessible to conventional methods provide new details about the activation of cytolytic effector cells by antibody-derived agents. Thus, the killing rate exhibited a dose-dependent maximum during the reaction interval. In clinical applications ex vivo monitoring of NK activity of patient's endogenous cells will likely help to choose appropriate therapy, to detect impaired or recovered NK function, and possibly to identify rare subsets of cancer cells with particular sensitivity to effector-cell mediated lysis.

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.

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