Two‐Layer Droplet Arrays Enable Dynamic Manipulation of Cell Microenvironment During High‐Throughput Bacterial Cultivation

Arrays of pico-to-microliter droplets, organized on a surface, enable chemical and biological workflows at high throughput. Here, a platform employing two-layer droplets is presented to enable flexible manipulation of the droplets' microenvironment for dynamic biological cultivation. Arrays of 6784 agarose droplets (≈2.0 nL per droplet) encapsulating and immobilizing bacterial cells are generated. After that, aqueous droplets (≈3.7 nL) with a defined composition are deposited atop to form a thin liquid layer surrounding the agarose droplets. Chemical exchange between the two layers is extremely fast (equilibrium within 15 s for fluorescein). Moreover, the aqueous layer can be removed, opening the possibility to extract substances from the agarose droplets. Indeed, repeated addition and aspiration of a buffer successfully remove dyes or drugs previously added to the agarose droplets. Therefore, antibiotic drug testing can be performed under both static and transient exposure profiles. The latter reveals that bacterial responses such as bacterial killing and resuscitation are both heterogeneous at the single-cell level. Last, it is exemplified how such droplet manipulation strategy can also be use in long-term experimentation, where medium replenishment, performed at 12-h intervals during a 72-h experiment, enables the cultivation of a slow-growing microorganism in nanoliter droplets.