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.

Lab on a rod: Size-based particle separation and sorting in a helical channel.

Size-based particle separation using inertial microfluidics in spiral channels has been well studied over the past decade. Though these devices can effectively separate particles, they require a relatively large device footprint with a typical outer channel radius of approximately 15 mm. In this paper, we describe a microfluidic device with a footprint diameter of 5.5 mm, containing a helical channel capable of inertial particle separation fabricated using abrasive jet micromachining. The separation of particles in several channel geometries was studied using wide-field fluorescence microscopy. A maximum separation efficiency of approximately 90% was achieved at a flow rate of 1.5 ml/min with a purity of approximately 95% at the outlet, where large particles were collected. An accompanying computational fluid dynamics model was developed to allow researchers to quickly assess the separation capability of their helical or spiral devices.

Magnetic polyelectrolyte microcapsules via water-in-water droplet microfluidics.

Polyelectrolyte microcapsules (PEMCs) have biocompatible microcompartments. Therefore, PEMCs are useful for applications in cosmetics, food, pharmaceutics, and other industries. The fabrication of PEMCs often involves the use of harsh chemicals or cytotoxic organic phases that make biomedical applications of the microcapsules challenging. In this report, we present an all-aqueous droplet microfluidics platform for the generation of magnetic PEMCs. In the platform, we use an aqueous-two-phase system (ATPS) of polyethylene glycol (PEG) and dextran (Dex), to generate water-in-water droplets, which are magnetically functionalized with ferrofluid. Strong polyelectrolytes (PEs) with opposite charges are used in each ATPS phase. We make emulsion templates of magnetic Dex, containing the polycations, in a continuous phase of PEG. We then apply a magnetic field to move the magnetic droplets to a second PEG phase, which contains the polyanions. By careful tuning of the fluxes of the two PEs in their respective phases, we trigger the formation of a shell at the droplet interface. Owing to the presence of the ferrofluid, the resulting microcapsules are magnetically responsive. We show that the magnetic PEMCs are capable of passive release of large pseudo-drugs as well as triggered release using external stimuli such as osmotic shock and pH change. We expect that magnetic PEMCs from this biocompatible all-aqueous platform will find utility in the fabrication of functionalized drug carriers for targeted drug delivery.

Magnetic water-in-water droplet microfluidics: Systematic experiments and scaling mathematical analysis.

A major barrier to the clinical utilization of microfluidically generated water-in-oil droplets is the cumbersome washing steps required to remove the non-biocompatible organic oil phase from the droplets. In this paper, we report an on-chip magnetic water-in-water droplet generation and manipulation platform using a biocompatible aqueous two-phase system of a polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer (PEG-PPG-PEG) and dextran (DEX), eliminating the need for subsequent washing steps. By careful selection of a ferrofluid that shows an affinity toward the DEX phase (the dispersed phase in our microfluidic device), we generate magnetic DEX droplets in a non-magnetic continuous phase of PEG-PPG-PEG. We apply an external magnetic field to manipulate the droplets and sort them into different outlets. We also perform scaling analysis to model the droplet deflection and find that the experimental data show good agreement with the model. We expect that this type of all-biocompatible magnetic droplet microfluidic system will find utility in biomedical applications, such as long-term single cell analysis. In addition, the model can be used for designing experimental parameters to achieve a desired droplet trajectory.

AC Electrothermal Effect in Microfluidics: A Review.

The electrothermal effect has been investigated extensively in microfluidics since the 1990s and has been suggested as a promising technique for fluid manipulations in lab-on-a-chip devices. The purpose of this article is to provide a timely overview of the previous works conducted in the AC electrothermal field to provide a comprehensive reference for researchers new to this field. First, electrokinetic phenomena are briefly introduced to show where the electrothermal effect stands, comparatively, versus other mechanisms. Then, recent advances in the electrothermal field are reviewed from different aspects and categorized to provide a better insight into the current state of the literature. Results and achievements of different studies are compared, and recommendations are made to help researchers weigh their options and decide on proper configuration and parameters.

Controlled generation of spiky microparticles by ionic cross-linking within an aqueous two-phase system.

Microparticles are used in a variety of different fields, such as drug delivery. Recently, non-spherical microparticle generation has become desirable. The high surface-to-volume ratio of non-spherical microparticles allows for enhanced targeting, and attachment to cells and tissue. Current non-spherical microparticle generation techniques require complicated setup, and utilizing natural micrograins, such as pollen grains, as non-spherical delivery vehicles, requires extensive post-processing. Here, we describe a unique and facile chemical synthesis approach, for controlled generation of pollen-like microparticles, based on ionic cross-linking of alginate and calcium chloride (CaCl2), within an all-biocompatible aqueous two-phase system (ATPS) of dextran (DEX) and polyethylene glycol (PEG). Our technique controls the length of spikes that emerge on the surface of these microparticles. We anticipate that these pollen-like spiky microparticles may be used as drug delivery vehicles, and this new chemical synthesis approach may be used for generating other biomaterials.

Microfluidic diamagnetic water-in-water droplets: a biocompatible cell encapsulation and manipulation platform.

Droplet microfluidics enables cellular encapsulation for biomedical applications such as single-cell analysis, which is an important tool used by biologists to study cells on a single-cell level, and understand cellular heterogeneity in cell populations. However, most cell encapsulation strategies in microfluidics rely on random encapsulation processes, resulting in large numbers of empty droplets. Therefore, post-sorting of droplets is necessary to obtain samples of purely cell-encapsulating droplets. With the recent advent of aqueous two-phase systems (ATPS) as a biocompatible alternative of the conventional water-in-oil droplet systems for cellular encapsulation, there has also been a focus on integrating ATPS with droplet microfluidics. In this paper, we describe a new technique that combines ATPS-based water-in-water droplets with diamagnetic manipulation to isolate single-cell encapsulating water-in-water droplets, and achieve a purity of 100% in a single pass. We exploit the selective partitioning of ferrofluid in an ATPS of polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer (PEG-PPG-PEG) and dextran (DEX), to achieve diamagnetic manipulation of water-in-water droplets. A cell-triggered Rayleigh-Plateau instability in the dispersed phase thread results in a size distinction between the cell-encapsulating and empty droplets, enabling diamagnetic separation and sorting of the cell-encapsulating droplets from empty droplets. This is a simple and biocompatible all-aqueous platform for single-cell encapsulation and droplet manipulation, with applications in single-cell analysis.

Microfluidic Generation of Particle-Stabilized Water-in-Water Emulsions.

Herein, we present a microfluidic platform that generates particle-stabilized water-in-water emulsions. The water-in-water system that we use is based on an aqueous two-phase system of polyethylene glycol (PEG) and dextran (DEX). DEX droplets are formed passively, in the continuous phase of PEG and carboxylated particle suspension at a flow-focusing junction inside a microfluidic device. As DEX droplets travel downstream inside the microchannel, carboxylated particles that are in the continuous phase partition to the interface of the DEX droplets due to their affinity to the interface of PEG and DEX. As the DEX droplets become covered with carboxylated particles, they become stabilized against coalescence. We study the coverage and stability of the emulsions, while tuning the concentration and the size of the carboxylated particles, downstream inside the reservoir of the microfluidic device. These particle-stabilized water-in-water emulsions showcase good particle adsorption under shear, while being flowed through narrow microchannels. The intrinsic biocompatibility advantages of particle-stabilized water-in-water emulsions make them a good alternative to traditional particle-stabilized water-in-oil emulsions. To illustrate a biotechnological application of this platform, we show a proof-of-principle of cell encapsulation using this system, which with further development may be used for immunoisolation of cells for transplantation purposes.

Developing Health-Related Indicators of Climate Change: Australian Stakeholder Perspectives.

Climate-related health indicators are potentially useful for tracking and predicting the adverse public health effects of climate change, identifying vulnerable populations, and monitoring interventions. However, there is a need to understand stakeholders' perspectives on the identification, development, and utility of such indicators. A qualitative approach was used, comprising semi-structured interviews with key informants and service providers from government and non-government stakeholder organizations in South Australia. Stakeholders saw a need for indicators that could enable the monitoring of health impacts and time trends, vulnerability to climate change, and those which could also be used as communication tools. Four key criteria for utility were identified, namely robust and credible indicators, specificity, data availability, and being able to be spatially represented. The variability of risk factors in different regions, lack of resources, and data and methodological issues were identified as the main barriers to indicator development. This study demonstrates a high level of stakeholder awareness of the health impacts of climate change, and the need for indicators that can inform policy makers regarding interventions.

Coal seam gas water: potential hazards and exposure pathways in Queensland.

The extraction of coal seam gas (CSG) produces large volumes of potentially contaminated water. It has raised concerns about the environmental health impacts of the co-produced CSG water. In this paper, we review CSG water contaminants and their potential health effects in the context of exposure pathways in Queensland's CSG basins. The hazardous substances associated with CSG water in Queensland include fluoride, boron, lead and benzene. The exposure pathways for CSG water are (1) water used for municipal purposes; (2) recreational water activities in rivers; (3) occupational exposures; (4) water extracted from contaminated aquifers; and (5) indirect exposure through the food chain. We recommend mapping of exposure pathways into communities in CSG regions to determine the potentially exposed populations in Queensland. Future efforts to monitor chemicals of concern and consolidate them into a central database will build the necessary capability to undertake a much needed environmental health impact assessment.