Isolation Of Exosomes From Extracellular Vesicles By Microfluidic Viscoelastic Flows

Exosomes,molecular cargos secreted by almost all mammalian cells,are considered as promising biomarkers to identify many diseases including cancers.However,the small size of exosomes poses serious challenges in their isolation from complex media containing a variety of extracellular vesicles(EVs)of different sizes,especially in small sample volumes.A microfluidic device consisting of a high-aspect-ratio straight microchannel,two inlets,and three outlets is designed for viscoelastic separation.Two Inlets are used for the introduction of sample and sheath fluids containing a low concentration of PEO(0.1%),respectively.The addition of PEO makes the fluids highly viscoelastic and consequently generates elastic lift forces on suspended nanoparticles in a Poiseuille flow to control their lateral positions.The suspended nanoparticles with different diameters from inlet I are first aligned around the microchannel sidewalls,by controlling the flow rate ratio of inlet II to inlet I and laterally driven toward the microchannel centerline with size-dependent lateral speeds controlled by the elastic lift forces.Large nanoparticles that migrate faster to the microchannel centerline are collected from the middle outlet,whereas small nanoparticles with lower migration speed are collected from two-sided outlets.In this manner,a label-free and continuous separation of nanoparticles of different sizes is achieved.We further apply the present technique to isolate exosomes from fetal bovine serum without any presample preparation to demonstrate its feasibility for realworld applications.The untreated serum samples are processed in 0.1 wt % PEO at sheath/sample flow rates of 1200/200 μL/h.The characterization of initial samples by NTA measurement shows two mode diameters at 100 nm(corresponding to exosomes)and 400 nm(corresponding to large EVs),respectively.After viscoelastic separation,the samples collected from the side and center outlets are also characterized by NTA,showing two distinct particle populations with a size cutoff of 200 nm.The recovery rate and purity of exosomes(<200 nm)are determined to be 93.6% and 96% based on the NTA data,respectively,showing a high-efficient separation of exosomes from serum.In comparison with conventional exosome separation techniques,the present method is able to obtain comparable recovery and purity of exosomes with easy operation and low cost.Specifically,the viscoelastic separation in this work has a much higher recovery(>80%)of exosomes than that of ultracentrifugation(5-25%),which is one of the most accessible batch techniques for exosome isolation.The remarkable merit of this microfluidic systemlies in its speed,low cost,and ready accommodation for handling the small volume of EVs(100 μL).The passage time of exosomes through the microchannel is less than 0.1 s(vs 2-5 h for ultracentrifugation),minimizing their physical damage during the process.Comparing with nanoscale deterministic lateral displacement(DLD),another lab-on-achip system for field-free and label-free exosome separation,our system allows for a much higher processing speed(200 μL/h in our work vs 12 nL/h in nano DLD),as well as the use of a much larger channel structure(20 μm in our work vs 235 nm in nano DLD)to minimize the risk of clogging.The present sample throughput is still limited compared to conventional macroscale separation methods,such as ultracentrifugation and membrane filtration.However,its handling capability could be largely enhanced by parallelization or using a slit-shaped microchannel design,with an improved sample throughput up to 3 orders of magnitude in particle separation.In this work,we have developed a viscoelasticity-based microfluidic system for field-free,label-free,and continuous separation of exosomes from large EVs based on their sizes.A high separation purity and recovery of exosomes are achieved by adding a small amount of PEO(0.1 wt %)into the cell culture medium or pure serum,without any complex or time-consuming operations.The size cutoff in viscoelasticity-based microfluidics can be easily controlled using different PEO concentrations.On the basis of this sizedependent viscoelastic separation strategy,we envision the handling of diverse nanoscale objects,such as gold nanoparticles,DNA origami structures,and quantum dots.