Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell-cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.tissue mechanics | microfluidics | tumor growth | mechanotransduction

The applicability of micro fluid segments for studying the behaviour of multicellular systems, in particular embryonic development, has been investigated. It was found that eggs from the zebrafish Danio rerio can be introduced into micro fluid segments without serious damage by using perfluoromethyldecalin (PP9) as the carrier liquid and Teflon (PTFE) as the tube material. The development processes of fish embryos were observed over a time period of 80 hours, until hatching time. After five days, the fish larvae were brought out of the micro fluid segments and transferred into breeding reservoirs. Effects of the membrane-damaging anionic surfactant sodium dodecyl sulfate (SDS) alone and SDS with the addition of CuCl(2) (copper(II) chloride) were investigated. By analyzing different end points, we found inhibiting and also supporting effects on the development of the embryos. Low SDS concentrations with and without copper(II) ions were supportive, while higher SDS concentrations led to negative impacts on the development of the embryos. The results showed that automated micro screening processes with complex biological systems can be performed using microfluidic systems and are applicable for future toxicological and drug screening studies.

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Liquid core capsules having a hydrogel membrane are becoming a versatile tool for three-dimensional culture of micro-organisms and mammalian cells. Making sub-millimeter capsules at a high rate, via the breakup of a compound jet in air, opens the way to high-throughput screening applications. However, control of the capsule size monodispersity, especially required for quantitative bioassays, was still lacking. Here, we report how the understanding of the underlying hydrodynamic instabilities that occur during the process can lead to calibrated core-shell bioreactors. The requirements are: i) damping the shear layer instability that develops inside the injector arising from the co-annular flow configuration of liquid phases having contrasting viscoelastic properties; ii) controlling the capillary instability of the compound jet by superposing a harmonic perturbation onto the shell flow; iii) avoiding coalescence of drops during jet fragmentation as well as during drop flight towards the gelling bath; iv) ensuring proper engulfment of the compound drops into the gelling bath for building a closed hydrogel shell. We end up with the creation of numerous identical compartments in which cells are able to form multicellular aggregates, namely spheroids. In addition, we implement an intermediate composite hydrogel layer, composed of alginate and collagen, allowing cell adhesion and thus the formation of epithelia or monolayers of cells.