HMSCs were harvested with TrypLE at 60 to 70% confluence, and a solution containing 6 × 105 cells in 70 μl of medium was mixed with 30 μl of a 3% (w/v) liquid low-melting agarose solution (i.e., stored at 37°C) (Sigma-Aldrich, Saint Quentin Fallavier, France) diluted in culture medium containing gentamicin (50 μg/ml; Sigma-Aldrich) (1:3, v/v), resulting in a 100-μl solution of 6 × 106 cells/ml in 0.9% (w/v) agarose.

HMSCs and agarose were loaded into a 100-μl glass syringe (SGE, Analytical Science, France), while Fluorinert FC-40 oil (3M, Paris, France) containing 1% (w/w) PEG-di-Krytox surfactant (RAN Biotechnologies, Beverly, USA) was loaded into a 1- and 2.5-ml glass syringes (SGE, Analytical Science). Droplets of cell-liquid agarose were generated in the FC-40 containing PEG-di-Krytox, at the flow-focusing junction, by controlling the flow rates using syringe pumps (neMESYS Low-Pressure Syringe Pump, Cetoni GmbH, Korbussen, Germany) (table S1). After complete loading, the chips were immersed in PBS, and the cells were allowed to settle down and to organize as MBs overnight in the CO2 incubator. Then, the agarose was gelled at 4°C for 30 min, after which the PEG-di-Krytox was extensively washed in flushing pure FC-40 in the culture chamber. After washing, cell culture medium was injected to replace the FC-40. All flow rates are indicated in table S1. Further operations were allowed by gelling the agarose in the droplets, such that the resulting beads were retained mechanically in the traps rather than by capillary forces (Fig. 2G). This step allowed the exchange of the oil surrounding the droplets by an aqueous solution, for example, to bring fresh medium for long-term culture, chemical stimuli, or the different solutions required for cell staining.

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