Next Generation Aqueous Two-Phase System for Gentle, Effective, and Timely Extracellular Vesicle Isolation and Transcriptomic Analysis

Abstract

The isolation of extracellular vesicles (EVs) using currently available methods frequently compromises purity and yield to prioritize speed. Here, we present a next-generation aqueous two-phase system (next-gen ATPS) for the isolation of EVs regardless of scale and volume that is superior to conventional methods such as ultracentrifugation (UC) and commercial kits. This is made possible by the two aqueous phases, one rich in polyethylene glycol (PEG) and the other rich in dextran (DEX), whereby fully encapsulated lipid vesicles preferentially migrate to the DEX-rich phase to achieve a local energy minimum for the EVs. Isolated EVs as found in the DEX-rich phase are more amenable to biomarker analysis such as nanoscale flow cytometry (nFC) when using various pre-conjugated antibodies specific for CD9, CD63 and CD81. TRIzol RNA isolation is further enabled by the addition of dextranase, a critical component of this next-gen ATPS method. RNA yield of next-gen ATPS-isolated EVs is superior to UC and other commercial kits. This negates the use of specialized EV RNA extraction kits. The use of dextranase also enables more accurate immunoreactivity of pre-conjugated antibodies for the detection of EVs by nFC. Transcriptomic analysis of EVs isolated using the next-gen ATPS revealed a strong overlap in microRNA (miRNA), circular RNA (circRNA) and small nucleolar RNA (snoRNA) profiles with EV donor cells, as well as EVs isolated by UC and the exoRNeasy kit, while detecting a superior number of circRNAs compared to the kit in human samples. Overall, this next-gen ATPS method stands out as a rapid and highly effective approach to isolate high-quality EVs in high yield, ensuring optimal extraction and analysis of EV-encapsulated nucleic acids.

Keywords: EV isolation; RNA extraction; biomarker analysis; circRNA; dextranase; extracellular vesicles (EVs); miRNA; nanoscale flow cytometry; next‐generation aqueous two‐phase system (next‐gen ATPS); snoRNA.

FIGURE 1

Workflow of a two‐step ATPS and analysis of extracellular vesicles (EV) partition. (A) Schematic diagram of the first step ATPS to isolate EVs from cell culture conditioned media (CM). CM of BPH‐1‐zsGreen cells is mixed with polyethylene glycol and dextran. Mixture is centrifuged at 200 × g’s for 15 mins. nFC of CM, 1st PEG‐rich phase and 1st DEX‐rich phase revealed partition and enrichment of EVs in the 1st DEX‐rich phase (left three cytograms). (B) Schematic diagram of the second step ATPS. 1st PEG is removed, and additional polyethylene glycol is mixed with the 1st DEX‐rich phase, followed by centrifugation at 200 × g’s for 15 mins. nFC of 2nd PEG‐rich and 2nd DEX‐rich phase revealed partition and greater enrichment of EVs in the 2nd DEX‐rich phase (right two cytograms). (C) EV concentration and recovery efficiency in all phases of two‐step ATPS compared to UC (n = 6). (D) Confocal microscopy of the 1st DEX‐rich phase. Scale bars 25 µm (top panel) and 1 µm (bottom left panel). Scan line analysis revealed the size of captured EVs (bottom right panel). (E) TEM analysis of EVs isolated by ATPS (left panel, scale bar 400 nm) and UC (400 nm).

FIGURE 2

Removal of dextran with dextranase prevents polysaccharide coagulation and leads to efficient RNA extraction. (A) Dextran‐alcohol precipitate is formed during TRIzol RNA isolation with EVs isolated with ATPS (top panel, white arrow). Absence of dextran‐alcohol precipitate during TRIzol RNA isolation after dextranase treatment (bottom panel). (B) Volume of dextran‐alcohol precipitate in EV‐containing DEX‐rich phase treated with dextranase at various concentrations during TRIzol RNA isolation (n = 6). (C) Bioanalyzer analysis of EV RNA with dextran‐alcohol precipitate. (D) Bioanalyzer analysis of EV RNA without dextran‐alcohol precipitate after dextranase treatment. (E) NanoDrop measurements of EV RNA isolated from DEX‐rich phase pre‐treated with dextranase at various concentrations (n = 3). Dash line represents the expected RNA yield/concentration retrieved from bioanalyzer analysis. (F) Bioanalyzer analysis of concentrations of EV RNA isolated from DEX‐rich phase pre‐treated with dextranase at various concentrations (n = 3). (G) Recovery efficiency of RNA isolated from DEX‐rich phase pre‐treated with dextranase at various concentrations (n = 3). (H) RT‐qPCR analysis on GAPDH mRNA copy number in RNA isolated from DEX‐rich phase pre‐treated with dextranase at various concentrations (n = 3). (I) Bioanalyzer analysis of RNA isolated from CM (black); cfRNA mimicry (blue); RNA isolated from CM with additional cfRNA mimicry (red); RNA isolated from RNase‐treated CM with additional cfRNA mimicry (green); RNA isolated from RNase‐treated CM (purple); RNase‐treated cfRNA mimicry (orange).

FIGURE 3

Removal of dextran with dextranase leads to accurate detection of purified EVs by nFC. (A) nFC scatterplots of PC3‐zsGreen CM (top row), ATPS‐isolated PC3‐zsGreen EVs (middle row) and ATPS‐isolated PC3‐zsGreen EVs after dextranase treatment (bottom row). Analysis revealed zsGreen‐positive EV subpopulations (first lane), biomarker‐positive EV subpopulations (second lane to fourth lane) and zsGreen/biomarker double‐positive EV subpopulations (fifth lane to seventh lane). (B) Large EV (lEVs, larger than 100 nm) subpopulation concentration in CM and ATPS‐isolated EVs after dextranase treatment (n = 7). (C) Small EV (sEVs, smaller than 100 nm) subpopulation concentration in CM‐ and ATPS‐isolated EVs after dextranase treatment (n = 7). (D) zsGreen/biomarker subpopulation concentration in CM‐ and ATPS‐isolated EVs after dextranase treatment (n = 7). (E) Recovery of EV subpopulations of ATPS‐isolated EVs with (right) or without (left) dextranase treatment (n = 7).

FIGURE 4

Presence of dextran improves EV internalization in recipient cells. (A) Confocal microscopy images and orthogonal views of BPH‐1 cells co‐cultured with ATPS‐isolated PC3‐zsGreen EVs, ATPS‐isolated PC3‐zsGreen EVs after dextranase treatment, UC‐isolated PC3‐zsGreen EVs, and UC‐isolated PC3‐zsGreen EVs mixed with dextran for 24 h (top panel) and 48 h (bottom panel). EVs were labelled with zsGreen (green). Recipient BPH‐1 cells were labelled with Hoechst 33,342 for nucleus (blue) and WGA Alexa Flour 647 for the plasma membrane (red). (B) Proportion of recipient cells showed EV internalization after 24‐h co‐culturing (n = 3). (C) Proportion of recipient cells showed EV internalization after 48 h co‐culturing (n = 3).

FIGURE 5

Transcriptomic profiles of CM EV RNA isolated by various methods. (A) The amount of total small RNA isolated from conditioned media of PC3 and MDA‐MB‐231 cells (n = 3). (B). Average number of miRNA species detected from next‐gen ATPS, UC and Qiagen exoRNeasy kit isolated EVs from conditioned media of PC3 (light grey bars, n = 2) and MDA‐MB‐231 cells (dark grey bars, n = 2). (C) Bioanalyzer profile of RNA extracted from EVs isolated by next‐gen ATPS (left), UC (middle) and Qiagen exoRNAeasy kit (right). EVs isolated from conditioned media of PC3 cells. (D) Venn diagrams describing the number of uniquely expressed and overlapped miRNA (left), circRNA (middle) and snoRNA (right) species across all three methods. (E) Venn diagrams describing the number of uniquely expressed and overlapped miRNA (top), circRNA (middle) and snoRNA (bottom) of all three methods compared to the small RNA species detected from the EV donor cells, PC3.

FIGURE 6

Comparison of EV isolation kit based on single event analysis with human prostate cancer plasma samples. (A) Appearance of a pellet in the DEX‐rich phase when using the 1.2‐mL version of ATPS. (B) Treatment of the DEX‐rich phase reduces pellet size. (C) Recovery ratios of EVs from both the DEX‐rich phase and the pellet when using 1.2‐, 6‐ and 12‐mL versions of the ATPS (n = 3). (D) Scatterplots showing single‐event analysis of isolated EVs from each isolation method using nFC. Isolation methods are shown across the left to right columns. Single event analysis for detection of CD9 (top row), CD63 (middle row) and CD81 (bottom row) +ve EVs. IEV, large‐size EVs (100–1400 nm). sEV, small EVs (<100 nm), dark grey area denotes LALS of 110‐nm calibration beads. Light grey area denotes LALS of 220‐nm calibration beads. Dark blue area denotes LALS of 800‐nm calibration beads. Light blue area denotes the LALS of 1400‐nm calibration beads. (E) Graph of EV concentration of CD9‐+ve EVs across all four EV isolation methods (n = 5). (F) Graph of EV concentration of CD63‐+ve EVs across all four EV isolation methods (n = 5). (G) Graph of EV concentration of CD81‐+ve EVs across all four EV isolation methods (n = 5). (H) Amount of small RNA from EVs isolated from the next‐gen ATPS method (n = 8), exoRNeasy kit (n = 8), total exosome isolation reagent (n = 5) and ultracentrifugation (n = 6). * denotes p < 0.01, Student's t‐test. (I) Transcriptomic profiles of miRNA (left panel), circular RNA (circRNA, middle panel) and small non‐coding RNA (snoRNA; right panel) present in EVs isolated via next‐gen ATPS versus exoRNeasy. The extent of transcriptomic overlap between the two methods is depicted in the beige intersection.