Heparin affinity purification of extracellular vesicles

Abstract

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity.

Figure 1. Extracellular vesicles are efficiently isolated and purified using heparin-coated agarose beads.

(a) Heparin coated agarose beads are incubated with EVs released from a variety of cells lines, (i), to yield an EV/heparin complex, (ii). Free floating proteins and nucleic acids are washed away with PBS, (iii). Beads are the incubated overnight with 2.15 M NaCl and the EVs are released and collected by spinning down the beads and collecting the supernatant (iv). Collected EVs are used as a source of RNA (biomarker) or used in biological assays (v). (b) Nanoparticle tracking analysis (NTA) counts of heparin-purified human 293T-derived EVs eluted with 2.15 M NaCl overnight at 4 °C following 3 wash steps. (c) To show specific heparin affinity we incubated heparin beads overnight with EVs, then rinsed beads 3 times with PBS and treated with Bacteroides Heparinase I or incubation buffer without heparinase and fractions were analyzed by NTA. (d) EVs were mixed with heparin beads and one round of purification was performed. The unbound and eluted fractions from round one were separately incubated with a fresh batch of heparin beads and round 2 purification performed on these samples. NTA was performed on each fraction of round 2 purification.

Figure 2. Characterization of total proteins and EV markers from heparin-purified (HeP), sucrose gradient-isolated (SuC) ultracentrifuged (UC), and commercial kit (kit)-isolated EVs

. In two separate preparations from 293T cells, recovered EVs purified with each of these methods had EVs counted using NTA. (a) The EV number (2.1 × 10¹⁰ particles for each lane) was used to normalize protein loading on the SDS PAGE gel. Coomassie staining revealed EV associated and co-pelleting proteins in each sample. The samples were also probed by western blotting for the EV marker Alix, (a, bottom panel; band indicated with open arrow). (b) Particle to total protein ratio (c) RNA yields and bioanalyzer profiles extracted using the HeP, SuC, UC and the Kit methods. (d) The recovered RNA from each method was reverse transcribed into cDNA and used as input for the qRT-PCR. Levels of several mRNAs were determined. The data is represented as the average Ct values±s.d. (lower means higher levels of the mRNA sequence) and normalized to the housekeeping mRNA, GAPDH (n = 3). p-values were calculated using the two-tailed t-test (*p < 0.05, **p < 0.01, ***p < 0.001); n.s. = non-significant.

Figure 3. mRNA quantification of heparin-purified and ultracentrifuged human plasma- derived EVs

. (a) Total protein in equal volumes from each isolation method. Left gel, 5 μl of sample loaded; right gel, 26 μl of sample loaded. (b) Two ml of plasma samples from healthy controls were thawed on ice, passed through a 100 kDa Amicon filter (Millipore) and filter-retained EVs washed with PBS buffer. The sample was split into three aliquots; one aliquot of washed EVs was added to biotin heparin - streptavidin coated magnetic beads and incubated on a rotator overnight at +4 °C to allow binding. A second aliquot was added to mock treated streptavidin coated magnetic beads. The third aliquot was stored at +4 °C until day 2 and then ultracentrifuged at 100,000 x g to collect EVs. RNA was extracted from all three samples using the miRNeasy kit (Qiagen) and analyzed for the presence of 2 mRNA messages: GAPDH and RPL11. Data is shown in Ct values (lower Ct means higher levels) and normalized to initial input (n.d. = not detected). Note: The mean and standard deviations are calculated from 3 healthy blood donor samples.

Figure 4. Transmission electron microscopic examination of heparin-purified EVs

. All preparations were isolated from 1 ml of concentrated conditioned media from 293T cells. (a) Ultracentrifuged EVs. (b) Commercial kit-purified EVs. (c) Heparin-purified EVs. Scale bars = 100 nm. Arrows point to large EVs (~50-100 nm) and arrowheads point to small EVs (< ~ 50 nm). Note: It is unclear which of the structures observed with the commercial kit are EVs or if some are a component of the proprietary precipitating reagent.

Figure 5. Heparin-purified EVs are internalized into cells.

293T-derived extracellular vesicles from (a, b) ultracentrifuged and heparin-purified (c, d) samples were labeled with red fluorescent lipid dye (see methods) and incubated with recipient U87 glioma cells to visualize internalization. After 60 minutes of incubation at 37 °C cells were fixed in formaldehyde, nuclei stained with Dapi and imaged using a fluorescence microscope. For control, a dye-only sample with no EVs was added to cells (e, f). Scale bar = 110 μm.