Highlights d Microfluidic droplet pairs sequentially trapped in capillary anchors before merging d 1 spheroid/droplet, with microenvironment modulations driven by droplet merging d A wide range of drug concentrations tested on hepatic-like spheroids in a single chip d Data-driven approach unravels 3D tissue-level dynamic drug response Authors Raphaë l F.

The relevance of traditional cell cultures to cellular behavior in vivo is limited, since the two-dimensional (2D) format does not appropriately reproduce the microenvironment that regulates cell functions. In this context, spheroids are an appealing 3D cell culture format to complement standard techniques, by combining a high level of biological relevance with simple production protocols. However the methods for spheroid manipulation are still labor intensive, which severely limits the complexity of operations that can be performed on statistically relevant numbers of individual spheroids. Here we show how to apply hundreds of different conditions on spheroids in a single microfluidic chip, where each spheroid is produced and immobilized in an anchored droplet. By using asymmetric anchor shapes, a second drop can be merged with the spheroid-containing drop at a later time. This time-delayed merging uniquely enables two classes of applications that we demonstrate: (1) the initiation of cell-cell interactions on demand, either for building micro-tissues within the device or for observing antagonistic cell-cell interactions with applications in immuno-therapy or host-pathogen interactions, (2) a detailed dose-response curve obtained by exposing an array of hepatocyte-like spheroids to droplets containing a wide range of acetaminophen concentrations. The integrated microfluidic format allows time-resolved measurements of the response of hundreds of spheroids with a single-cell resolution. The data shows an internally regulated evolution of each spheroid, in addition to a heterogeneity of the responses to the drug that the single-cell analysis correlates with the initial presence and location of dead cells within each spheroid.droplet microfluidics | spheroids | tissue engineering | screening | liver toxicity R ecent years have seen the emergence of many new cell culture approaches to improve the relevance of ex vivo experiments to the behavior of the cells residing within living tissues. One of the main objectives of these methods is to recapitulate the native cells' microenvironment, including biochemical signaling delivered from blood stream or from neighboring cells, the formation of intercellular junctions, interactions with the endogenous extra cellular matrix (ECM), mechano-transduction, and other effects such as diffusion gradients (1). The three-dimensional (3D) culture formats that have emerged range from the culture of individual cells in hydrogel matrices (2) or de-cellularized scaffolds (3), making functional aggregates such as spheroids (4) or organoids (5), or building more complex engineered structures that involve multiple cell types on a microfluidic device (6). Indeed the combination of microfluidics and 3D cell culture has allowed the emergence of a wide range of "organ on a chip" approaches that include many of these different strategies (7).These formats are not meant to replace two-dimensional (2D) culture. Instead they will allow specific questions to be posed on more physiologically relevant ...