Microfluidic filtration system to isolate extracellular vesicles from blood

Abstract

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.

Therapeutic biomaterials based on extracellular vesicles: classification of bio‐engineering and mimetic preparation routes

“…Other methods, such as immunoaffinity isolations, are only used for small amounts of original sample because of the high price of the reagents required. Finally, microfluidic methods [45,53] are promising, with the possibility of being coupled to online analysis [54]. …”

Microfluidics‐based on‐a‐chip systems for isolating and analysing extracellular vesicles

“…In their pioneering work, Davies et al developed two kinds of Mf-F devices – pressure- and electrophoresis-driven – that separate cells, debris and small EVs via a nanoporous membrane with an adjustable pore size [87]. The limitation of pressure-driven Mf-F is that the pores become blocked after obtaining approximately 4 μ L of filtrate.…”

“…The limitation of pressure-driven Mf-F is that the pores become blocked after obtaining approximately 4 μ L of filtrate. Using electrophoresis avoids this problem and increases the separation efficiency and purity [87]. Cho et al isolated EVs using electrophoretic migration across a dialysis membrane with 30-nm pores [88].…”

Field-Free Isolation of Exosomes from Extracellular Vesicles by Microfluidic Viscoelastic Flows

“…Viscoelastic microfluidics has demonstrated itself as an efficient technology to separate tumor cells, 15 blood cells, 16,17 bacteria, 15 droplets, 18 and microspheres. 11,19−21 In comparison with other microfluidic separators resorting to acoustics, 22−24 electrophoresis, 25 magnetics, 26,27 and so forth, 28,29 the viscoelastic manipulation of particles can be continuously performed without any externally applied fields, dramatically simplifying the design and fabrication of microfluidic devices. Most importantly, viscoelastic microfluidics enables the precise manipulation of submicrometer particles in small sample volumes, which is difficult to achieve by inertial microfluidics.…”