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Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10 Page 35

Conclusions: Research and clinical studies could greatly benefit from technology that provides rapid
and simple isolation of EVs from various biofluids-especially as it concerns methods that generate pure,
concentrated EV samples with enough yield for subsequent proteomic, genomic, and transcriptomic
analyses. We have designed the Capturem™ Extracellular Vesicle Isolation Kit to meet these high standards,
enabling researchers to consistently obtain pure EVs. These tools have the potential to enable faster drug
discovery and better diagnostics for a variety of diseases.

19. Isolation and characterization of extracellular vesicles in saliva of children with asthma

Authors: Nicole Comfort, Tessa Bloomquist, Amparito Cunningham, Marissa Hauptman, Wanda
Phipatanakul, Andrea Baccarelli
E-mail: nc2710@cumc.columbia.edu
Affiliations:
Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York,
NY, USA.
Harvard Medical School, Boston, MA, USA.
Division of Allergy and Immunology, Boston Children's Hospital, Boston, MA, USA.
Abstracts:
Asthma is the most common chronic disorder in children and is a heterogeneous disease, making
identification of objective biomarkers of its subtypes and their underlying pathology a research priority.
Identifying biomarkers that can profile clinical subtypes early in the course of asthma is critical in applying
tailored therapy, yet the clinical utility of current biomarkers is very limited because they are either invasive or
non-specific and unable to identify clinical subtypes. Non- invasive methods to study and monitor childhood
asthma severity and distinguish pathophysiological subgroups is a critical research gap. Extracellular vesicles
(EVs) and their bioactive cargo are attractive candidates for biomarkers of asthma endotypes.
Saliva, regarded as the “mirror of the body”, harbors constituents that provide sources for monitoring
of health and disease states. Thus, we hypothesize that saliva EVs and their cargo may serve as a novel
biomarker for asthma subtypes. Here, we first confirm the presence of EVs in saliva of children with asthma
and characterize the EV population isolated from saliva supernatant using a high- throughput EV isolation
method that can be scaled to large epidemiological studies of childhood asthma such as the School Inner
City Asthma Intervention Study (SICAS-2).
Objective: To characterize the population of EVs isolated from saliva supernatant of children with asthma.
Methods: EVs were isolated from saliva supernatant of 209 SICAS-2 study participants using ExoQuick-TC
(System Biosciences) following a modified protocol. Pelleted EVs were re-suspended in filtered 1X dPBS.
5 μL of re-suspended EVs were aliquoted from each sample and combined to create a pooled sample for
downstream analyses. EVs were visualized by morphological analyses (TEM). Capillary immunoassays
confirmed the presence of EV-associated proteins. Microfluidic Resistive Pulse Sensing (MRPS) and
Nanoparticle Tracking Analysis (NTA) were performed to determine EV concentration and size
distribution. The concentration and size distribution of specific tetraspanin subpopulations (CD9, CD63,
CD81) was determined using the ExoView R100 (NanoView Biosciences).
Results: Rounded, membrane-bound structures (25-350 nm) identified by TEM were positive for CD9, CD63,
CD81, ANXA5, and ICAM-1 with limited cellular contamination (negative for CANX). MRPS detected a broad
6
distribution of particle sizes with a significant population at 800 nm (concentration of peak population: 5.9 × 10
particles/mL, 0.01× stock). Further analysis of small (< 200 nm) EVs with fluorescent NTA revealed a bi-modal
distribution of EVs at 90 nm and 150 nm. 28% of EVs were positive for CD9, 35% were double positive for CD9
and CD63, 25% expressed CD9 and CD81, and 12% expressed all three tetraspanins.
Conclusions: Our findings characterize saliva EVs from children with asthma and show that saliva EVs can
be isolated in a high-throughput method, opening a new avenue for asthma epidemiology studies.