a Bright-field images of HEK293T spheroids obtained at four subsequent days inside agarose mold. Initial seeding density is ∼30 cells per micro well (left). Exponential fit (solid line) to the mean cell number (black circles) per spheroid yields a doubling time of ~32 h. Time series has been recorded twice. Distribution of cell number per spheroid indicated in (c, center panel) is represented in blue box plot showing median (center line), 25 and 75 percentiles (lower and upper box bound) as well as 10 and 90 percentiles (whiskers). b Representative image of a HEK293T cell in virtual channel of 16 µm diameter (sample 114 µM MC at Qsa = 20 nl s−1, sheath 5 mM PEG40000 at Qsh = 1000 nl s−1, left). Corresponding scatter plots of area strain (center) and Young’s modulus (right) versus HEK293T cell size. Young’s modulus is calculated from area strain assuming an interfacial stress of 66 Pa. Shaded area indicates cells not confined inside the virtual channel. c Representative image of HEK293T spheroid in virtual channel of 190 µm width (sample 114 µM MC at Qsa = 330 nl s−1, sheath 5 mM PEG40000 at Qsh = 200 nl s−1, left). Projected line plots in (b) and (c) indicate the squared intensity gradient (arb. units) perpendicular to the flow direction while centers of the intensity maxima identify the virtual channel interfaces (dashed white lines). Corresponding scatter plots of deformation (center) and Young’s modulus (right) versus HEK293T spheroid size. Inset in right graph compares Young’s modulus of single cells (E = 0.79 ± 0.06 kPa) and spheroids (E = 84 ± 78 Pa) from three independent biological replicates consisting of n = 2433 single-cell and n = 762 spheroid measurements (p = 0.0003). Scale bar is 20 µm. Color in scatter plots indicates a linear density scale from min (blue) to max (red). Statistical data analysis is performed using linear mixed models and data are represented as mean ± standard error of the mean (***p < 0.001).