Cross‐flow microfiltration for isolation, selective capture and release of liposarcoma extracellular vesicles

Abstract

We present a resource‐efficient approach to fabricate and operate a micro‐nanofluidic device that uses cross‐flow filtration to isolate and capture liposarcoma derived extracellular vesicles (EVs). The isolated extracellular vesicles were captured using EV‐specific protein markers to obtain vesicle enriched media, which was then eluted for further analysis. Therefore, the micro‐nanofluidic device integrates the unit operations of size‐based separation with CD63 antibody immunoaffinity‐based capture of extracellular vesicles in the same device to evaluate EV‐cargo content for liposarcoma. The eluted media collected showed ∼76% extracellular vesicle recovery from the liposarcoma cell conditioned media and ∼32% extracellular vesicle recovery from dedifferentiated liposarcoma patient serum when compared against state‐of‐art extracellular vesicle isolation and subsequent quantification by ultracentrifugation. The results reported here also show a five‐fold increase in amount of critical liposarcoma‐relevant extracellular vesicle cargo obtained in 30 min presenting a significant advance over existing state‐of‐art.

Microfluidic Approaches and Methods Enabling Extracellular Vesicle Isolation for Cancer Diagnostics

“…The implementation of this approach for microfluidic devices usually requires functionalization of the walls of a microfluidics device with immobilized antibodies [104]. Therefore, surface preparation for functionalization [106,107] plays an important role in tethering antibodies [96]. Physicochemical interactions leading to eventual binding or capture of antigens at the immobilized antibody site include hydrogen bonding, coulombic interactions, Van der Waals interactions, and hydrophobic interactions [104].…”

“…Label-free isolation methods used in filtration devices contribute to higher entrapment efficiency while maintaining the functionality of EVs so that they can be examined after isolation [111]. However, the main challenge for these EV isolation methods is the lack of specificity in isolating particles [96]. NeutrAvidin is used to coat the surface of the device, which helps in the incorporation of desthiobiotinconjugated antibodies required for recognition of surface markers of EVs.…”

“…Label-free isolation methods used in filtration devices contribute to higher entrapment efficiency while maintaining the functionality of EVs so that they can be examined after isolation [111]. However, the main challenge for these EV isolation methods is the lack of specificity in isolating particles [96].…”

“…Smaller, 83 nm lipid vesicles were trapped and recovered within the device with ~60% retention, while larger, 120 nm lipid vesicles observed a 15% retention decrease [95]. Casadei et al integrated the tasks of size-based separation in a crossflow arrangement with the CD-63 antibody immunoaffinitybased capture of liposarcoma-derived EVs in a single micro-nanofluidic device (Figure 5) and achieved ~76% and ~32% EV recovery for liposarcoma cell-conditioned media and dedifferentiated liposarcoma patient serum, respectively, when compared against ultracentrifugation [96]. They also reported a significant advance over existing state-of-the-art techniques with a five-fold enhancement in the quantity of liposarcoma-relevant EV-DNA obtained in 30 min [96].…”

Automated Parallel Pattern Search Optimisation of Microfluidic Geometry for Extracellular Vesicle Liquid Biopsies

“…10 min after bonding, microfluidic channels (n = 3 of each design) were functionalised with an antibody against CD63 (SantaCruz, sc-5275) by using a well-established linker chemistry 4,[28][29][30] . Briefly, 8% percent (3-Aminopropyl)triethoxysilane (APTES, Sigma) in ethanol was flowed at a rate of 20 µl/min for 15 min then left stagnant for 20 min, followed by a flush with pure ethanol.…”