Synchronized, Spontaneous, and Oscillatory Detachment of Eukaryotic Cells: A New Tool for Cell Characterization and Identification

Abstract Despite the importance of cell characterization and identification for diagnostic and therapeutic applications, developing fast and label‐free methods without (bio)‐chemical markers or surface‐engineered receptors remains challenging. Here, we exploit the natural cellular response to mild thermal stimuli and propose a label‐ and receptor‐free method for fast and facile cell characterization. Cell suspensions in a dedicated sensor are exposed to a temperature gradient, which stimulates synchronized and spontaneous cell‐detachment with sharply defined time‐patterns, a phenomenon unknown from literature. These patterns depend on metabolic activity (controlled through temperature, nutrients, and drugs) and provide a library of cell‐type‐specific indicators, allowing to distinguish several yeast strains as well as cancer cells. Under specific conditions, synchronized glycolytic‐type oscillations are observed during detachment of mammalian and yeast‐cell ensembles, providing additional cell‐specific signatures. These findings suggest potential applications for cell viability analysis and for assessing the collective response of cancer cells to drugs.

. Spontaneous and irreversible cell detachment -Correlative fluorescence analysis, temperature, and surface dependence. a-c, Long-term R th plots as function of time for different temperatures. All data show that the R th recovery due to cell detachment is maintained over long time scales. Set up for simultaneous HTM and microscopy experiment, which allows to monitor the chip-to-liquid interface from underneath using an inverted fluorescence microscope with a meander system as a heater (d). The cells were fluorescently labelled yeast cells, expressing the green fluorescent protein (GFP). The outcome of the experiment in which the drop in the thermal resistance signal at t d50 ≈ 52 min coincides with a sudden drop in the fluorescence intensity (e). Noteworthy, the fluorescence signal does not drop back to its baseline: This indicates that cells, after detachment, are still in the proximity of the chip surface where they contribute to the intensity since the focus depth of the microscope reaches into the liquid. This reference experiment was performed several times and, while the drop of R th was observed consistently, the decrease in the fluorescence signal could not always be observed sharply, confirming that the upward displacement of cells is limited. The absolute t d50 value of 52 min agrees well to data obtained for the laboratory S. cerevisiae S288C at the same concentration of 5  10 6 cells/ml and chip temperature of T 1 = 33 o C in Figure 5b. f, Linear scaling model: The inverse of t d50 displays a linear trend with the chip temperature. g, Temperature difference between T 1 and T 2 as a function of chip temperature. We note that the values reported are not the absolute values across single cells, since we measure the gradient macroscopically (see Methods). h, SEM image of surface- imprinted polyurethane displaying surface-imprinted cavities, geometrically matching with yeast cells in shape and size. i, Time dependent R th responses for repeated measurements at 20 mg/ml and 4 mg/ml yeast concentrations showing a higher R th response for the former compared to the latter.
Supplementary Figure   Comparison of liquid temperature, T 2 signals from two HeLa cell measurements showing recoveries that being at the same time, t = 24.5 min (or 14.5 min after injection) but evolve faster for test 2 because of a higher environment temperature due to microscope warming. The average t d50 value is 22.5. c, Correlation between heater voltage and the temperature of the microscope chamber for two measurements.

Estimation of shear rate and its effect on cell lift-off
The accuracy of the numerical result, f depends on the spacing h of the grid and the order of convergence p (ℎ) = (0) + ℎ + (ℎ ) , > Where f(0) is the 'exact' value and the coefficient a is independent of the grid spacing h.
The error E is given by the difference between the f(0) and the approximate solution f(h) =  Applying Richardson extrapolation using the two finest grids: The grid convergence index (GCI) amounts then = 2.49%, = 1.01% The solutions lie within the asymptotic range of convergence as = 0.995 According to Krishnan et al. (reference [73] in the main manuscript), the onset of lifting depends on the relation Reynolds numbers / , with =̇ / and = / .
is the Reynold number, which is given by the ratio between the inertia of a particle with radius and the drag force due to the viscosity of the solvent . In this case the particle has a velocity due to the local shear rate . the velocity is given by the sedimentation velocity = of a (spherical) particle. Therefore, The onset of lifting is around = 10 according to ref. [73]. Assuming that for a cell Δ = 0.1 / and = 10 , we required that ̇> 500 to obtain convection-driven lifting, which is certainly not the case here.