Microfluidic Hydrogel Hanging-Drop Network for Long-Term Culturing of 3D Microtissues and Simultaneous High-Resolution Imaging

Three-dimensional (3D) microtissues, cultured in microfluidic platforms, enable to study complex biological mechanisms that cannot be replicated in two-dimensional cell cultures. Deeper insights can be obtained if these 3D culture systems are rendered compatible with high-resolution time-lapse imaging systems, which requires precise placement and immobilization of the specimen while ensuring high viability and functionality of the 3D cell constructs. This article presents a versatile microfluidic platform for longterm culturing and analysis of 3D microtissues. The platform is compatible with time-lapse high-resolution confocal microscopy. Hanging hydrogel drops enable the precise placement and stable immobilization of the microtissues in the microfluidic chip. The chip includes perfusion capability to apply drugs, staining and clearing solutions. The features of the chip are demonstrated by studying (i) colon cancer microtissues to monitor tissue growth and cell death; on-chip clearing was used to augment the penetration depth for endpoint imaging; (ii) primary human liver microtissues were exposed to cytochalasin D to observe its effect on the bile canaliculi. The results obtained with both sample types demonstrate the suitability of the system for investigating complex processes in organotypic 3D microtissues, down to single-cell level, and for observation of physiologically relevant processes at subcellular scale.