Microfluidically-generated Encapsulated Spheroids (µ-GELS): An All-Aqueous Droplet Microfluidics Platform for Multicellular Spheroids Generation.

Spheroids are three-dimensional clusters of cells that serve as in vitro tumor models to recapitulate in vivo morphology. A limitation of many existing on-chip platforms for spheroid formation is the use of cytotoxic organic solvents as the continuous phase in droplet generation processes. All-aqueous methods do not contain cytotoxic organic solvents but have so far been unable to achieve complete hydrogel gelation on chip. Here, we describe an enhanced droplet microfluidic platform that achieves on-chip gelation of all-aqueous hydrogel multicellular spheroids (MCSs). Specifically, we generate dextran-alginate droplets containing MCF-7 breast cancer cells, surrounded by polyethylene glycol, at a flow-focusing junction. Droplets then travel to a second flow-focusing junction where they interact with calcium chloride and gel on chip to form hydrogel MCSs. On-chip gelation of the MCSs is possible here because of an embedded capillary at the second junction that delays the droplet gelation, which prevents channel clogging problems that would otherwise exist. In drug-free experiments, we demonstrate that MCSs remain viable for 6 days. We also confirm the applicability of this system for cancer drug testing by observing that dose-dependent cell death is achievable using doxorubicin.

Water-in-water droplet microfluidics: A design manual.

Droplet microfluidics is utilized in a wide range of applications in biomedicine and biology. Applications include rapid biochemical analysis, materials generation, biochemical assays, and point-of-care medicine. The integration of aqueous two-phase systems (ATPSs) into droplet microfluidic platforms has potential utility in oil-free biological and biomedical applications, namely, reducing cytotoxicity and preserving the native form and function of costly biomolecular reagents. In this review, we present a design manual for the chemist, biologist, and engineer to design experiments in the context of their biological applications using all-in-water droplet microfluidic systems. We describe the studies achievable using these systems and the corresponding fabrication and stabilization methods. With this information, readers may apply the fundamental principles and recent advancements in ATPS droplet microfluidics to their research. Finally, we propose a development roadmap of opportunities to utilize ATPS droplet microfluidics in applications that remain underexplored.

Phase transition modulation and biophysical characterization of biomolecular condensates using microfluidics.

Membraneless organelles (MLOs) formed through liquid-liquid phase separation (LLPS) are becoming increasingly relevant to understanding viral-host interactions, neurodegenerative disease, and cancer. The modulation of LLPS involves many parameters and components. To describe these modulators, typical in vitro studies require laborious, manual sample preparation of different concentrations and costly biological reagents. Here, we introduce a minimal reagent, microfluidic platform to systematically generate samples of different concentrations and trigger phase separation. We demonstrate the platform's utility by constructing phase diagrams describing the modulation of LLPS using an aqueous two-phase system (ATPS) and an MLO-based phase separating system. We also show on-chip biophysical characterization typical of in vitro studies. We expect that this platform will be utilized by scientists to study the growing number of MLOs and inform clinical treatments for pathology related to LLPS.