Subscriber access provided by Universiteit Utrecht
Article
Rapid Acoustic Enrichment of Extracellular Vesicles from Biological Fluids anson Ku, Hooi Ching Lim, Mikael Evander, Hans Lilja, Thomas Laurell, Stefan Scheding, and Yvonne Ceder Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00914 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Rapid Acoustic Enrichment of Extracellular Vesicles from Biological Fluids Anson Ku †,‡ , Hooi Ching Lim ‡,§, Mikael Evander∥, Hans Lilja†, ¶, Thomas Laurell∥, Stefan Scheding§,Þ, Yvonne Ceder*,ç ‡
Equal contribution authors Department of Translational Medicine, Lund University, Malmö, Sweden § Division of Molecular Hematology and Lund Stem Cell Center, Lund University, Lund, Sweden ∥Department of Biomedical Engineering, Lund University, Lund, Sweden ¶ Urology Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. ¶ Genitourinary Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA ¶ Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA ¶ Nuffield Department of Surgical Sciences, University of Oxford, Oxford, U.K. Þ Department of Hematology, Skåne University Hospital, Lund, Sweden. ç Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden. *E-mail:
[email protected] †
ABSTRACT: Extracellular vesicles (EVs) have emerged as a rich source of biomarkers providing diagnostic and prognostic information in diseases such as cancer. Large-scale investigations into the contents of EVs in clinical cohorts are warranted, but a major obstacle is the lack of a rapid, reproducible, efficient, and low-cost methodology to enrich EVs. Here, we demonstrate the applicability of an automated acoustic-based technique to enrich EVs, termed acoustic trapping. Using this technology, we have successfully enriched EVs from cell culture conditioned media, and urine and blood plasma from healthy volunteers. The acoustically trapped samples contained EVs ranging from exosomes to microvesicles in size and contained detectable levels of intravesicular microRNAs. Importantly, this method showed high reproducibility and yielded sufficient quantities of vesicles for downstream analysis. The enrichment could be obtained from a sample volume of 300 µL or less, an equivalent to 30 mins of enrichment time – depending on the sensitivity of downstream analysis. Taken together, acoustic trapping provides a rapid, automated, low-volume compatible, and robust method to enrich EVs from biofluids. Thus, it may serve as a novel tool for EV enrichment from large number of samples in a clinical setting with minimum sample preparation.
Extracellular vesicles (EVs) are small, cell-derived phospholipid membrane enclosed vesicles that constitute important cell-to-cell messengers, regulating diverse cellular functions of recipient cells. EVs are not only constitutively released by mammalian cells, but are also secreted by prokaryotes and eukaryotes, suggesting vital and evolutionarily-conserved functions across domains. There are three main classes of EVs; exosomes (30-120 nm), microvesicles (also referred to as ectosomes or microparticles) (100-1000 nm) and apoptotic bodies (1-5 µm), which are classified based on their origin and biophysical properties.1-5 It has become increasingly clear that EVs contain bioactive molecules that reflect the status of their cellular origin. These include, but are not restricted to, various proteins, lipids, mRNAs and non-coding RNAs.6-8 EVs play pivotal roles in regulating physiological and pathophysiological processes, as documented for disease progression in solid cancers, leukemia, and atherosclerosis.9-15 As such, EVs have a considerable potential to serve as biomarkers in the diagnosis and prognosis of various diseases.16,17
EVs can be isolated from cell culture conditioned media as well as a variety of biological fluids such as plasma, urine, cerebrospinal fluid and saliva.18-20 However, advancement in EV research has been partly hampered by the lack of a rapid and convenient method for isolation and characterization. Currently, the gold standard for EV isolation is differential ultracentrifugation.18,19 However, the ultracentrifugation method is laborious and gives inconsistent results across the reported studies due to the differences in protocols, types of rotor used as well as different biophysical properties of the samples.19,21-23 Furthermore, ultracentrifugation requires large sample volumes, thus making it an onerous method that is difficult to apply in the clinical setting.24 Advancement in microfluidic technologies has led to the development of several microfluidic based EV-isolation methods, such as immuneaffinity capture implemented at the microchip level, sieving and trapping with porous microstructures.25-29 There are several drawbacks to some of these microfluidic-based EV isolation methods, e.g. they require extra reagents such as antibodies
1 ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(immuno-chip), there is the risk of vesicle damage due to shear stress (sieving), and time consuming using porous structure. As an alternative, we recently reported the successful isolation of platelet-derived microvesicles from plasma of healthy donors and patients with myocardial infarction using a prototype microfluidic-based acoustic trapping device. Acoustic trapping is a non-contact platform that makes use of ultrasonic waves scattering between micron-size seeding particles and nanoparticles of interest in a resonant cavity for EV enrichment. The technique is dependent on size, density, and compressibility of both particles and fluids, which results in an attractive force between the EVs and pre-seeded with microns-size polystyrene beads. Briefly, a local λ/2 acoustic standing wave was created in a glass capillary by a piezoelectric transducer and seeding polystyrene beads were aspirated and trapped in the capillary by the primary acoustic forces (Figure 1a,b). 30,31 It was followed by aspiration of sample containing EVs. The EVs were attracted and trapped together with the seeding cluster by secondary acoustic forces (Figure 1c). The cluster of EVs and seeding particles were washed and released into the 96-well plate when ultrasound was deactivated (Figure 1d). A typical acoustic trapping system (Figure 1e). Here we show that acoustic trapping can be used to isolate EVs, including microvesicles and exosome-size vesicles, from cell culture conditioned media, human urine and plasma samples. The acoustic trapping method presented herein is automated and enables label-free EV isolation from small sample volumes with minimal sample preparation. Thus, acoustic trapping represents a novel EV isolation tool with the potential to be used in clinical settings for rapid enrichment of large number of biological samples.
EXPERIMENTAL SECTION Polystyrene beads model. Fluorescently labeled 0.1, 0.2, 0.29 µm (Bangs Laboratory, Indiana), 0.49, 0.87 µm (Spherotech
Page 2 of 10
Inc., Illinois), and 1 µm (Sigma Aldrich, Missouri) polystyrene beads with carboxyl, amine or no surface modifications and density of ~1.06 g/mL were diluted with phosphate buffer saline (PBS) to different particle concentrations (Table S3, Supporting Information) for acoustic trapping. The fluorescent intensities were measured after acoustic trapping with a fluorescent microplate reader (Synergy2, BioTek, Vermont, or Wallac 1420 Victor2, Perkin Elmer, Massachusetts) and the recovery was calculated by dividing fluorescent intensity of trapped polystyrene beads with fluorescent intensity of polystyrene beads before trapping after adjusting for dilutions and volume. Olympus CKX41 fluorescent microscope equipped with a Lumenera Infinity 1 camera was used to image the trapping of 100 nm beads. Conditioned media, plasma and urine preparation. Human neuroblastoma cells, SHSY5Y were grown in Dulbecco’s modified Eagle’s Media (Gibco, Massachusetts) supplemented with 15% fetal bovine serum (Life technologies, California) and 1% of antibiotic-antimycotic solution (Sigma Aldrich, Missouri) at 37oC and 5% CO2. For EV isolation, the cells were grown in serum free media for 72 h and conditioned media was collected. Urine and blood samples were collected from consenting healthy donors. The collection and use of human samples was approved by the Regional Ethics committee. Urine samples were immediately centrifuged at 2000 × g for 10 min at room temperature to remove large particles and cells followed by storage at 4oC. Human whole blood was collected in sodium citrate vacutainers (Becton Dickinson, New Jersey) and platelet-poor-plasma was obtained after two serial centrifugations at 1600 × g for 15 min at room temperature. The platelet-poor-plasma samples were aliquoted and kept frozen at -80oC. For EV enrichment by acoustic trapping or ultracentrifugation, plasma samples were thawed in a 37oC water bath for 2 min before dilution (1:1) with PBS. Prepared biological samples prior to acoustic or ultracentrifugation enrichment were defined as “Input” samples.
Figure 1. Schematic illustration of acoustic trapping for EV isolation. a. A piezoelectric transducer generates a local λ/2 acoustic standing wave in a fluidic channel. Seeding particles are aspirated after trapping in the acoustic field. The excess seeding particles are washed away. b. Seeding particles are retained by the acoustic standing wave. c. Sample containing EVs is aspirated and the EVS are attracted and trapped with seeding cluster by the secondary acoustic forces as a result of particle-particle interaction. d. Seeding cluster and trapped vesicles are washed and released after the acoustic wave is turned off. e. Photograph of the automated trapping device, AcouTrap
2 ACS Paragon Plus Environment
Page 3 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Extracellular vesicle isolation by acoustic trapping. Acoustic trapping was performed using the AcouTrap instrument (AcouSort AB, Lund, Sweden) as previously described.31,32 A borosilicate capillary channel was coupled to the acoustic transducer with a thin layer of glycerol (Figure 1). The transducer was operated at approximately 4MHz, 10 Volts peak-topeak sinusoidal wave. Samples were loaded onto a 96 wellplate where enrichment was performed automatically through user-defined scripts. Briefly, 12 µm polystyrene beads serving as seeding particles (Sigma Aldrich) were trapped and washed with 50 µL PBS buffer followed by aspiration and extracellular vesicle trapping of 300 µL – 5 mL of conditioned media, urine or plasma, at 15 µL/min at room temperature. The size of EVs are too small to enable direct acoustic trapping by the primary acoustic radiation force, however, as the EVs come in proximity of the seed particles, sound scattering between the acoustically trapped seed particles and the EVs induce a force, inversely dependent of the distance, d, between the particles to the fourth power, d-4, that attract the EVs to the stationary seed particles. After sample aspiration and EV enrichment, the seed particle and EV cluster were washed in the trap with 200 µL PBS buffer before releasing the cluster by turning off the transducer and eluting the samples in 110 µL PBS buffer. Extracellular vesicle isolation by ultracentrifugation. All high speed centrifugation steps were performed at 4oC with a Beckman Coulter Optima XE-90 centrifuge and SW-41 Ti swing bucket rotor. Polyallomer tubes (Beckman Coulter) were used. Differential centrifugation of conditioned media was performed at 500 × g for 15 min then 10 000 × g for 20 min to remove cells and large particles before two centrifugations at 100 000 × g for 1.5 h with a pellet wash in-between. For urine and plasma samples, 300 µL – 5 mL samples were diluted with sufficient PBS in polyallomer tubes and spun twice at 100 000 × g for 1.5 h with a pellet wash with PBS inbetween. All ultracentrifugation pellets were resuspended with the same volume of PBS as acoustically trapped samples. Extracellular vesicle size measurements and transmission electron microscopy. The size distribution of EVs were measured using the NanoSight LM10 instrument (Malvern Inc., UK) equipped with a 488 nm laser. 1 mL of sample was injected into the trapping chamber by continuous-flush mode using the manufacturer-provided syringe pump. Screen gain was set to 1 and camera level was set to 13, 13, and 10 respectively for conditioned media, urine and plasma samples in the acquisition software. All samples were measured 5 times for 2 min, totaling in 10 min per sample. Nanoparticle tracking analysis (NTA) was performed using NTA version 3.2. Detection threshold was set to 2, 10, and 10 respectively for conditioned media, urine, and plasma samples to obtain the sample particle size distribution. For transmission electron microscopy (TEM), enriched samples were prepared using the protocol described by previous publication.18 Briefly, enriched vesicles were loaded onto Formvar carbon-coated grids and incubated at room temperature for 20 min. The grids were dried with filter paper and fixed with 2% paraformaldehyde for 20 min followed by washing with PBS and fixed with 1% glutaraldehyde for 5 min. Specimens were washed with water before being stained with 2% uranyl acetate and subsequently examined with a Tecnai Spirit BioTWIN transmission electron microscope (TEI). CD9, CD63 and CD81 enzyme-linked immunosorbent assay (ELISA). The presence of CD9, CD63, and CD81 in the
enriched samples were evaluated using commercially available ELISA kits as per manufacturer’s protocol (Cell Guidance, Montana). Briefly, cross-linked streptavidin 96-well plates were incubated with 100 µL of the provided biotinylated-antiCD9, CD63 or CD81 antibody for 1 h on an orbital shaker at room temperature before rinsing with wash buffer for three times. Next, 100 µL of enriched samples or serial-diluted exosome standards derived from LnCaP cell lines (provided by manufacturer) or PBS blanks were pipetted onto a 96-well plate in triplicates, incubated for 1 h at room temperature on an orbital shaker, followed by washing three times with wash buffer. The 96-well plates were then incubated with manufacturer-supplied europium-labeled anti- CD9, CD63 or CD81 antibody for 1 h on an orbital shaker at room temperature, washed and finally incubated with europium fluorescence intensifier (Kiavogen, Turku, Finlnd) for 10 min. Fluorescence measurements were obtained using a Wallac 1420 Victor2 microplate reader (Perkin Elmer, Massachusetts) RNase A treatment, RNA isolation and qRT-PCR of enriched extracellular vesicles. Enriched samples from conditioned media were treated with either 0.5 µg/µL of RNase A (Thermo Scientific, Massachusetts) or PBS as control for 10 min at 37oC. RNA from conditioned media was isolated using the miRCURY RNA isolation kit (Exiqon, Vedbæk, Denmark) following the manufacturer’s protocol and eluted in nuclease free water. Reverse transcription was performed using Universal cDNA synthesis kit II (Exiqon). The expression levels of selected miRNAs were quantified using the SYBR Green PCR Master Mix (Applied Biosystems, California) with microRNA LNTTM primers for let-7b, miR-103a and miR-191 (Exiqon). For enriched urine and plasma samples, RNA was isolated using the mirVana Paris RNA and Protein isolation kit (Ambion, Massachusetts) and eluted with nuclease free water. RNA products were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, California) and specific miRNA primers. The level of individual miRNAs was quantified using TaqMan Universal PCR master mix (Applied Biosystems, California), miR-16, miR-21, and miR-24 specific primers (TaqMan microRNA Assay) by QuantStudio 7K system (Life Technologies, Massachusetts). Analyses were performed using the 2-∆Ct method. No-templatecontrol was run to exclude contamination. RNA from enriched samples were profiled by Bioanalyzer 2100 with RNA 6000 Pico Kit (Agilent) in accordance to the manufacturer’s instruction. Statistics. The cumulative size distribution and concentration of the particle distribution were calculated using R (version 3.3.2) and RStudio (1.04). Trapping efficiency was calculated by dividing the concentration of the trapped sample after adjusting for dilutions and volume by the input concentration after adjusting for dilution and volume. Linear regression analysis was performed with α
