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Page 34 Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10

pregnancies (4 pups/dam). We assessed milk EVs and microRNAs, gut development, barrier function,
mRNA expression profile in the jejunum, postnatal weight gain, and milk quality and intake. Statistics:
unpaired t-test (Tsg101/Dicer vs. control); P < 0.05.
Results: KO of TSG101 and Dicer caused an 80% and 60% decrease of EVs and microRNAs, respectively,
in milk. The loss of milk EVs and microRNAs led to an up to 20% shorter length of the gut, 20% decrease
of villi height and 15% crypt depth, 50% increase in leakiness of the gut (appearance of FITC-dextran in
blood), and a 50% loss of postnatal weight gain in pups. Approximately 400 mRNAs were differentially
expressed in the jejunums of pups fostered to TSG101 KO dams or WT dams. Nutritional quality of milk
and milk intake were not study confounders.
Conclusions: Mothers communicate with their offspring through EVs and microRNAs in milk, and the
maternal message plays a role in optimal growth and gut health in neonate mice.
Funding: NIFA/USDA 2016-67001-25301 and 2020-67017-30834, NIH P20GM104320, USDA Hatch and
W-40022 and Gates Foundation OPP1200494. J.Z. is a consultant for PureTech Health, Inc.

18. High-capacity membranes for simple, rapid extracellular vesicle isolation with high yield
and purity

Authors: Yi Zhao*, Brenda Huang, Boris Levitan, Michael Haugwitz, Andrew A. Farmer
E-mail: yi_zhao@takarabio.com
Affiliations: Takara Bio USA, Inc., Mountain View, CA 94043, USA
Abstracts:
Introduction: Despite their small size, extracellular vesicles (EVs), such as exosomes, play important
roles in normal physiological processes and diseases. A critical bottleneck in EV research is the isolation
of the vesicles, which has historically been accomplished via ultracentrifugation (UC). However, UC is
time consuming, is not scalable, requires specialized equipment, may damage vesicles during the high-
speed spins, can pull down non-EV proteins and nucleic acids, and suffers from low yield. More recently,
precipitation solutions have been utilized to simplify EV isolation protocols, but these techniques are often
inconsistent, provide low yields, and reduce purity. Thus, there is a strong need for a rapid EV isolation
method that does not compromise purity or yield.
In order to overcome these shortcomings, we developed a new method of purifying EVs using a membrane-
column-based approach. This method comprises a novel membrane conjugated to a lectin-based compound
that selectively binds EVs. The membrane is chemically modified to have increased surface area, allowing
higher binding capacity and yield, while also providing a highly pure and concentrated EV preparation.
Additionally, the membrane is assembled into benchtop-centrifuge- compatible spin columns, which can
be used to isolate EVs in under 30 minutes, improving on lengthy UC protocols. These kits provide a new
and reliable method to rapidly enrich EVs from biological fluids for downstream analyses.
Results: Capturem-isolated EVs exhibited a more uniform and smaller particle size distribution, with an
average particle size of 81 nm and a D90 value of 110 nm. Conversely, UC-isolated EVs were larger, with
an average particle size of 135 nm and a D90 value of 203 nm. Additionally, the particle sizes isolated from
UC were more variable than those from Capturem isolation. EVs isolated from plasma using the Capturem
kit or UC were labeled with fluorescent dye and subjected to fNTA analysis. The results showed that UC-
isolated EVs were highly contaminated with other particles and only 20% exhibited EV-specific labeling. In
contrast, Capturem-isolated EVs demonstrated more than 4X this enrichment, with over 84% EV-specific
labeling. Thus, Capturem columns consistently provide a pure, intact, and concentrated EV population in
less than 30 minutes.