The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.

We report a microvortex manipulator (MVM) that is a passive, scalable system with great potential for the manipulation and separation of particulate samples in microfluidic environments. The movement of particles is determined by a unique combination of helical flow, buoyant, and gravitational forces. Helical flows are induced by topographically patterned microchannel surfaces, which have previously been used for molecular mixing in microfluidic devices. Here we exploit the behavior of particles in the generated microvortices and present a generic strategy to focus, guide and sort particles. Particles can be focused and guided along arbitrary continuous patterns in a microchannel, which can be easily scaled up to focus multiple streams of particles in parallel. We theoretically illustrate the mechanism of our technique and experimentally demonstrate its applications in passive focusing of beads and cells into parallel streams and guiding of particles and cells. We also explore the application of the unique density-selectivity of microvortex focusing and successfully sort a mixture of two bead populations whose density difference is as small as 0.1 g/cm3.

Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles that serve a means of cell-cell communication. Studying EVs at a single-particle level is important because EVs are inherently heterogeneous. Novel micro- and nanotechnological tools have open opportunities for realizing single-EV measurements exploiting their biochemical, electrical, mechanical, and/or optical properties. This review summarizes the recent development of technologies toward sorting and analyzing single EVs. Sorting EVs into a more homogeneous subset relaxes the sensitivity and throughput required on the EV detection, and hence related techniques are also included in this review. These exciting technologies are on the rise and will expand our understanding of EVs and their applications in the near future.

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CD4+ T cell counts are important tests used to stage HIV-postive patients, enabling clinicians to make informed antiretroviral treatment decisions and to monitor the therapeutic outcomes. However, state-of-the-art CD4 counting methods based on flow cytometry are not applicable in resource-limited settings, due to their high cost and technical requirements. In previous work, we reported the development of a cell isolation microchip that can be used at the point of care for CD4 counts. In that microfluidic chip, CD4+ T cells were separated from 10 μL of whole blood, and enumerated via either light microscopy or impedance sensing. The microchip counts matched flow cytometry results in the intermediate CD4 count range, between 200–800 cells/μL, but displayed a positive bias at absolute CD4 counts below 200 cells/μL, due largely to monocyte contamination. To enhance the performance in the low CD4 count range, we report here an improved design of a two-stage microfluidic device to deplete monocytes from whole blood, followed by CD4+ T cell capture. Using the double-stage device combined with a high viscosity rinsing solution, we obtained microchip CD4 counts comparable to flow cytometry results in the full clinically relevant range. In addition to CD4 counting, the strategy of contaminant depletion prior to target cell isolation can be easily adapted to immunoaffinity capture of other cell types that lack a unique surface marker from a complex biological fluid.

We demonstrate the fabrication and operation of "microcanals"(i.e. open-air microfluidic channels without a roof), which enable micropipette manipulation and probing of cells within a microfluidic environment. The microcanal devices are fabricated in PDMS on glass substrates using a PDMS membrane transferring technique. Here we show patch-clamp electrophysiological recording and intracellular dye injection performed on cells seeded in microcanals.

The ability to produce three-dimensional (3D) microstructures is of increasing importance in the miniaturization of mechanical or fluidic devices, optical elements, self-assembling components, and tissue-engineering scaffolds, among others. Traditional photolithography, the most widely used process for microdevice fabrication, is ill-suited for 3D fabrication, because it is based on the illumination of a photosensitive layer through a ''photomask'' (a transparent plate that contains opaque, unalterable solid-state features), which inevitably results in features of uniform height. We have devised photomasks in which the light-absorbing features are made of fluids. Unlike in conventional photomasks, the opacity of the photomask features can be tailored to an arbitrary number of gray-scale levels, and their spatial pattern can be reconfigured in the time scale of seconds. Here we demonstrate the inexpensive fabrication of photoresist patterns that contain features of multiple and͞or smoothly varying heights. For a given microfluidic photomask, the developed photoresist pattern can be predicted as a function of the dye concentrations and photomask dimensions. For selected applications, microfluidic photomasks offer a low-cost alternative to present gray-scale photolithography approaches. Photolithography is used to define critical feature size in the fabrication of the vast majority of microdevices including microelectronic circuits, microelectromechanical systems (MEMS), microfluidic devices, and nucleic acid͞protein microarrays (1). Essentially, photolithography consists of selectively illuminating a thin photosensitive layer (''photoresist'') with UV light through a mask containing opaque features (e.g., metal or ink emulsion) on a transparent background (e.g., glass or plastic). The photomasks impose two fundamental limitations on the features that can be produced. (i) The exposure is an all-or-none illumination process that results in photoresist features of uniform height; thus, the fabrication of threedimensional (3D) microstructures by traditional photolithography requires multiple exposure͞alignment steps. (ii) Photomask features are permanent, and thus design changes require the (costly and slow) fabrication of a new photomask, a major hurdle in research settings requiring fast-turnaround microdevice prototyping.To overcome the all-or-none illumination constraint of conventional photolithography, a number of ''gray-scale'' approaches capable of generating ranges of exposure levels have been developed. Presently, gray-scale photolithography can be realized with scanning lasers (2, 3), micromirror projection displays (4), high-energy beam-sensitive glass photomasks (www. canyonmaterials.com), ultra-high-resolution ''halftone'' photomasks (5), or metal-on-glass photomasks [where each gray-scale level is determined by a different metal thickness (6)]. Although extremely useful for producing 3D structures, these gray-scale approaches either (i) exacerbate the costs͞turnaround times of standard photolithography ...