Extracellular vesicles are released by various cell types, particularly tumor cells, and may be potential targets for blood-based cancer diagnosis. However, studies performed on blood-borne vesicles to date have been limited by lack of effective, standardized purification strategies. Using in situ prepared nanoporous membranes, we present a simple strategy employing a microfluidic filtration system to isolate vesicles from whole blood samples. This method can be applied to purify nano-sized particles from blood allowing isolation of intact extracellular vesicles, avoiding the need for laborious and potentially damaging centrifugation steps or overly specific antibody-based affinity purification. Porous polymer monoliths were integrated as membranes into poly(methyl methacrylate) microfluidic chips by benchtop UV photopolymerization through a mask, allowing precise positioning of membrane elements while preserving simplicity of device preparation. Pore size could be manipulated by changing the ratio of porogenic solvent to prepolymer solution, and was tuned to a size proper for extraction of vesicles. Using the membrane as a size exclusion filter, we separated vesicles from cells and large debris by injecting whole blood under pressure through the microfluidic device. To enhance isolation purity, DC electrophoresis was employed as an alternative driving force to propel particles across the filter and increase the separation efficiency of vesicles from proteins. From the whole blood of melanoma-grown mice, we isolated extracellular vesicles and performed RT-PCR to verify their contents of RNA. Melan A mRNA derived from melanoma tumor cells were found enriched in filtered samples, confirming the recovery of vesicles via their cargo. This filtration system can be incorporated into other on-chip processes enabling integrated sample preparation for the downstream analysis of blood-based extracellular vesicles.

We introduce a device that uses centrifugal force and a filter with micro-sized pores to generate a large quantity of cell-derived nanovesicles.

Artificial exosomes of ~100 nm diameter, enclosed with lipid bilayers, are fabricated from living cells and transfer biological signal components such as encapsulated RNAs and proteins, plasma membrane proteins, or both.

Extracellular vesicles (EVs) such as exosomes and microvesicles released from cells are potential biomarkers for blood-based diagnostic applications. To exploit EVs as diagnostic biomarkers, an effective pre-analytical process is necessary. However, recent studies performed with blood-borne EVs have been hindered by the lack of effective purification strategies. In this study, an efficient EV isolation method was developed by using polyethylene glycol/dextran aqueous two phase system (ATPS). This method provides high EV recovery efficiency (~70%) in a short time (~15 min). Consequently, it can significantly increase the diagnostic applicability of EVs.

Extracellular vesicles (EVs) are secreted nano‐sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal‐reflection microscopy and analyzes their co‐localization to investigate EV subpopulations. The single‐vesicle imaging and co‐localization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.

We propose a microfluidic system that generates nanovesicles (NVs) by slicing living cell membrane with microfabricated 500 nm-thick silicon nitride (SixNy) blades. Living cells were sliced by the blades while flowing through microchannels lined with the blades. Plasma membrane fragments sliced from the cells self-assembled into spherical NVs of ~100-300 nm in diameter. During self-assembly, the plasma membrane fragments enveloped exogenous materials (here, polystyrene latex beads) from the buffer solution. About 30% of beads were encapsulated in NVs, and the generated NVs delivered the encapsulated beads across the plasma membrane of recipient cells, but bare beads could not penetrate the plasma membrane of recipient cells. This result implicates that the NVs generated using the method in this study can encapsulate and deliver exogenous materials to recipient cells, whereas exosomes secreted by cells can deliver only endogenous cellular materials.