Recent breakthroughs in organ-on-a-chip and related technologies have highlighted the extraordinary potential for microfluidics to not only make lasting impacts in the understanding of biological systems but also to create new and important in vitro culture platforms. Adipose tissue (fat), in particular, is one that should be amenable to microfluidic mimics of its microenvironment. While the tissue was traditionally considered important only for energy storage, it is now understood to be an integral part of the endocrine system that secretes hormones and responds to various stimuli. As such, adipocyte function is central to the understanding of pathological conditions such as obesity, diabetes, and metabolic syndrome. Despite the importance of the tissue, only recently have significant strides been made in studying dynamic function of adipocytes or adipose tissues on microfluidic devices. In this critical review, we highlight new developments in the special class of microfluidic systems aimed at culture and interrogation of adipose tissue, a sub-field of microfluidics that we contend is only in its infancy. We close by reflecting on these studies as we forecast a promising future, where microfluidic technologies should be capable of mimicking the adipose tissue microenvironment and provide novel insights into its physiological roles in the normal and diseased states. Graphical abstract This critical review focuses on recent developments and challenges in applying microfluidic systems to the culture and analysis of adipocytes and adipose tissue.

A fully automated droplet generation and analysis device based on pressure driven push-up valves for precise pumping of fluid and volumetric metering has been developed for high resolution hormone secretion sampling and measurement. The device consists of a 3D-printer templated reservoir for single cells or single tissue culturing, a Y-shaped channel for reagents and sample mixing, a T-junction channel for droplet formation, a reference channel to overcome drifts in fluorescence signal, and a long droplet storage channel allowing incubation for homogeneous immunoassays. The droplets were made by alternating peristaltic pumping of aqueous and oil phases. Device operation was automated, giving precise control over several droplet parameters such as size, oil spacing, and ratio of sample and reference droplets. By integrating an antibody-oligonucleotide based homogeneous immunoassay on-chip, high resolution temporal sampling into droplets was combined with separation-free quantification of insulin secretion from single islets of Langerhans using direct optical readout from the droplets. Quantitative assays of glucose-stimulated insulin secretion were demonstrated at 15 second temporal resolution while detecting as low as 10 amol per droplet, revealing fast insulin oscillations that mirror well-known intracellular calcium signals. This droplet sampling and direct optical analysis approach effectively digitizes the secretory time record from cells into droplets, and the system should be generalizable to a variety of cells and tissue types.

Current methods for fabricating microparticles offer limited control over size and shape. Here, we demonstrate a droplet microfluidic method to form polyhedral microparticles with controlled concavity. By manipulating Laplace pressure, buoyancy, and particle rheology, we generate microparticles with diverse shapes and curvatures. Additionally, we demonstrate the particles provide increased capture efficiency when used for particle-templated emulsification. Our approach enables microparticles with enhanced chemical and biological functionality.

Suitable micropumping methods for flow control represent a major technical hurdle in the development of microfluidic systems for point-of-care testing (POCT). Passive micropumping for point-of-care microfluidic systems provides a promising solution to such challenges, in particular, passive micropumping based on capillary force and air transfer based on the air solubility and air permeability of specific materials. There have been numerous developments and applications of micropumping techniques that are relevant to the use in POCT. Compared with active pumping methods such as syringe pumps or pressure pumps, where the flow rate can be well-tuned independent of the design of the microfluidic devices or the property of the liquids, most passive micropumping methods still suffer flow-control problems. For example, the flow rate may be set once the device has been made, and the properties of liquids may affect the flow rate. However, the advantages of passive micropumping, which include simplicity, ease of use, and low cost, make it the best choice for POCT. Here, we present a systematic review of different types of passive micropumping that are suitable for POCT, alongside existing applications based on passive micropumping. Future trends in passive micropumping are also discussed.

Our understanding of adipose tissue biology has steadily evolved. While structural and energy storage functionalities have been in the forefront, a key endocrine role for adipocytes was revealed only over the last few decades. In contrast to the wealth of information on dynamic function of other endocrine tissues, few studies have focused on dynamic adipose tissue function or on tool development toward that end. Here, we apply our unique droplet-based microfluidic devices to culture, perfuse, and sample secretions from primary murine epididymal white adipose tissue (eWAT), and from predifferentiated clusters of 3T3-L1 adipocytes. Through automated control, oil-segmented aqueous droplets (∼2.6 nL) were sampled from tissue or cells at 3.5 second temporal resolution (including sample and reference droplets), with integrated enzyme assays enabling real-time quantification of glycerol (down to 1.9 fmol per droplet). This high resolution revealed previously unreported oscillations in secreted glycerol at frequencies of 0.2 to 2.0 min^-1 (∼30-300 s periods) present in the primary tissue but not in clustered cells. Low-level bursts (∼50 fmol) released in basal conditions were contrasted with larger bursts (∼300 fmol) during stimulation. Further, both fold changes and burst magnitudes were decreased in eWAT of aged and obese mice. These results, combined with immunostaining and photobleaching analyses, suggest that gap-junctional coupling or nerve cell innervation within the intact ex vivo tissue explants play important roles in this apparent tissue-level, lipolytic synchronization. High-resolution, quantitative sampling by droplet microfluidics thus permitted unique biological information to be observed, giving an analytical framework poised for future studies of dynamic oscillatory function of adipose and other tissues.

In this work, we expand upon our recently developed droplet-based sample chopping concepts by introducing a multiplexed fluidic micro-chopper device (μChopper). Six aqueous input channels were integrated with a single oil input, and each of these seven channels was controlled by a pneumatic valve for automated sampling through software control. This improved design, while maintaining high precision in valve-based droplet generation at bandwidths of 0.03 to 0.05 Hz, enabled a variety of analytical modes to be employed on-chip compared to previous devices limited to sample/reference alternations. The device was analytically validated for real-time, continuous calibration with a single sample and five standards; multiplexed analysis during calibration using a mixed mode; and standard addition through spiking of six sample droplets with varying amounts of standard. Finally, the standard addition mode was applied to protein quantification in human serum samples using on-chip, homogeneous fluorescence immunoassays. Ultimately, with only ~1.2 μL of total analyzed solution volume- representing 100-fold and 75-fold reductions in reagent and serum volumes, respectively-we were able to generate full, six-point standard addition curves in only 1.5 min, and results correlated well with those from standard plate-reader equipment. This work thus exploited microfluidic valves for both their automation and droplet phase-locking capabilities, resulting in a micro-analytical tool capable of complex analytical interrogation modes on sub-microliter sample volumes while also leveraging drastic noise rejection via lock-in detection. The multichannel μChopper device should prove particularly useful in analyzing precious biological samples or for dynamic analyses at small volume scales.

While genome databases are nearing a complete catalog of species commonly inhabiting the human gut, their representation of intraspecific diversity is lacking for all but the most abundant and frequently studied taxa. Statistical deconvolution of allele frequencies from shotgun metagenomic data into strain genotypes and relative abundances is a promising approach, but existing methods are limited by computational scalability. Here we introduce StrainFacts, a method for strain deconvolution that enables inference across tens of thousands of metagenomes. We harness a "fuzzy" genotype approximation that makes the underlying graphical model fully differentiable, unlike existing methods. This allows parameter estimates to be optimized with gradient-based methods, speeding up model fitting by two orders of magnitude. A GPU implementation provides additional scalability. Extensive simulations show that StrainFacts can perform strain inference on thousands of metagenomes and has comparable accuracy to more computationally intensive tools. We further validate our strain inferences using single-cell genomic sequencing from a human stool sample. Applying StrainFacts to a collection of more than 10,000 publicly available human stool metagenomes, we quantify patterns of strain diversity, biogeography, and linkage-disequilibrium that agree with and expand on what is known based on existing reference genomes. StrainFacts paves the way for large-scale biogeography and population genetic studies of microbiomes using metagenomic data.

The human immunodeficiency virus (HIV) integrates its genome into that of infected cells and may enter an inactive state of reversible latency that cannot be targeted using antiretroviral therapy. Sequencing such a provirus and the adjacent host junctions in individual cells may elucidate the mechanisms of the persistence of infected cells, but this is difficult owing to the 150-million-fold higher amount of background human DNA. Here we show that full-length proviruses connected to their contiguous HIV-host DNA junctions can be assembled via a high-throughput microfluidic assay where droplet-based whole-genome amplification of HIV DNA in its native context is followed by a polymerase chain reaction (PCR) to tag droplets containing proviruses for sequencing. We assayed infected cells from people with HIV receiving suppressive antiretroviral therapy, resulting in the detection and sequencing of paired proviral genomes and integration sites, 90% of which were not recovered by commonly used nested-PCR methods. The sequencing of individual proviral genomes with their integration sites could improve the genetic analysis of persistent HIV-infected cell reservoirs.

Patterned surfaces can enhance the sensitivity of laser desorption ionization mass spectrometry by segregating and concentrating analytes, but their fabrication can be challenging. Here, a simple method to fabricate substrates patterned with micrometer-scale wells that yield more accurate and sensitive mass spectrometry measurements compared to flat surfaces is described. The wells can also concentrate and localize cells and beads for cell-based assays.