Separation of U87 glioblastoma cell-derived small and medium extracellular vesicles using elasto-inertial flow focusing (a spiral channel)

Abstract

Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.

Recent microfluidic advances in submicron to nanoparticle manipulation and separation

“…In this work, medium EVs (mEVs) could be focused, but sEVs were too small to be focused and randomly dispersed. 250 They recovered 55% of sEVs with 6% contamination of mEVs and 80% of mEVs with 22% contamination of sEVs after one round of recycling.…”

“…Since then, this technique has attracted increasing attention for submicron particle focusing and separation. 193,246,248–250 For instance, Liu et al 193 proposed a viscoelastic microfluidic device for the size-based separation of exosomes from larger EVs. The device consists of a straight main channel, two inlets for the sample and sheath flow, and three outlets, Fig.…”

Viscoelastic microfluidics for enhanced separation resolution of submicron particles and extracellular vesicles

“…Viscoelastic microfluidic devices with wavy 27 and spiral 28 microchannel structures have been developed to focus larger submicron particles and filtrate them from their smaller counterparts. In both works, particles above a certain size ( i.e.…”

Electrochemical Approach for Specific Capture and Rapid Release of Nanoscale Placental Extracellular Vesicles Using Aptamer-Modified Conducting Terpolymer-Coated Carbon Cloth

“…This device integrated the use of CD63 antibody immunoaffinity for the specific capture of EVs from the liposarcoma cell-conditioned media . Apart from basic microfluidic techniques, a variety of other strategies have been developed for the efficient capture and release of extracellular vesicles, such as magnetic isolation methods using magnetic beads/nanoparticles, microstructure channels, and electric field-assisted techniques . Recently, Zhang et al fabricated an aptamer-based magnetic isolation system to capture and release intact EVs from the cell culture medium and human plasma .…”