Asymmetric depth‐filtration: A versatile and scalable method for high‐yield isolation of extracellular vesicles with low contamination

Abstract

We developed a novel asymmetric depth filtration (DF) approach to isolate extracellular vesicles (EVs) from biological fluids that outperforms ultracentrifugation and size‐exclusion chromatography in purity and yield of isolated EVs. By these metrics, a single‐step DF matches or exceeds the performance of multistep protocols with dedicated purification procedures in the isolation of plasma EVs. We demonstrate the selective transit and capture of biological nanoparticles in asymmetric pores by size and elasticity, low surface binding to the filtration medium, and the ability to cleanse EVs held by the filter before their recovery with the reversed flow all contribute to the achieved purity and yield of preparations. We further demonstrate the method's versatility by applying it to isolate EVs from different biofluids (plasma, urine, and cell culture growth medium). The DF workflow is simple, fast, and inexpensive. Only standard laboratory equipment is required for its implementation, making DF suitable for low‐resource and point‐of‐use locations. The method may be used for EV isolation from small biological samples in diagnostic and treatment guidance applications. It can also be scaled up to harvest therapeutic EVs from large volumes of cell culture medium.

Current advances in non‐viral gene delivery systems: Liposomes versus extracellular vesicles

“…This approach is simple, fast, inexpensive and does not require specialized equipment. This method was first proposed by Chernyshev et al [63] . In a single‐step procedure, plasma was diluted in 1:50 Phosphate‐buffered saline, centrifuged at 4500 g for 30 min at 4°C, and the supernatant was collected and filtered through a 0.8 μm filter.…”

“…The collected sample was then concentrated by centrifugation at 3500 g using a 100 kDa MWCO filter to isolate EVs. The team demonstrated that DF outperforms ultracentrifugation and SEC in terms of isolation purity and yield [63] . Therefore, this new technique may be used for both diagnostics and EV research.…”

Recent developments in isolating methods for exosomes

“…Furthermore, asymmetric depthfiltration (DF) can isolate extracellular vesicles by immobilizing extracellular vesicles on the surface and within the depth of porous medium, then recovering them by reversing the carrier flow through the filter. Compared to the optimized three-step isolation procedure, DF is more suitable for the separation of plasma extracellular vesicles (Chernyshev et al, 2022).…”

The molecular mechanism of human stem cell-derived extracellular vesicles in retinal repair and regeneration

“…While the ISEV's position statement in the 2018 update has yet to endorse any isolation method capable of maintaining both high yields and high purity, new isolation techniques in recent years offer hope. Two new technologies, tangential flow filtration [ 93 ] and deep filtration [ 94 ], have been reported to simultaneously maintain product purity while increasing yield, improving the separation efficiency of target EVs. These novel technologies may become the mainstream methods of choice for EV treatment in the future.…”