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Optimization of exosome isolation protocols for mouse serum and rabbit primary lacrimal gland acinar cells
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Optimization of exosome isolation protocols for mouse serum and rabbit primary lacrimal gland acinar cells
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i
Optimization of Exosome Isolation Protocols for
Mouse Serum and Rabbit Primary Lacrimal Gland Acinar Cells
by
Benjamin Cooperman
A thesis presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
AUGUST 2018
Copyright 2018 Benjamin Cooperman
ii
ACKNOWLEDGMENTS
I am extremely grateful to my mentors and committee members, Dr. Sarah
Hamm-Alvarez and Dr. Curtis Okamoto. They believed in my ability to take on my own
project and allowed me the freedom to follow my interests. My path towards earning
this degree would have been far less rewarding without their encouragement and
guidance. They provided me with all the tools that I will need to grow into a successful
scientist and I will never forget the lessons that I learned.
I am also thankful to my committee member Dr. Yong Zhang for his help in
reviewing and editing my thesis.
Additionally, I would like to thank all the wonderful people in Dr. Hamm-
Alvarez’s lab for their friendship and help over the course of the past two years. This
especially applies to Shruti Kakan, who was not only a great friend, but was instrumental
in helping me bring this project to fruition.
Finally, I would like to thank my parents for always believing in me. Without
them, I never would have been able to follow my passion for discovery and complete my
master’s degree.
iii
TABLE OF CONTENTS
Acknowledgements.............................................................................................................ii
List of Tables/Figures…........................................................................................................v
Abbreviations ....................................................................................................................vi
Abstract ............................................................................................................................vii
CHAPTER ONE:
Introduction........................................................................................................................1
1.1 Exosome Biogenesis................................................................................................1
1.2 Exosomes in Disease...............................................................................................4
1.3 Trafficking Proteins…………………………………………………………………………………………….7
1.3 Mouse and Cell Models...........................................................................................8
1.4 Exosome Isolation Protocols……………………………………………………………………………….9
CHAPTER TWO:
Materials and Methods.....................................................................................................10
2.1. Mice......................................................................................................................10
2.2. Cell Culture............................................................................................................10
2.3. SDS-PAGE and Western Blot.................................................................................11
2.4. ZetaView Nanoparticle Tracking Analysis.............................................................12
2.5. Nanosight Nanoparticle Tracking Analysis............................................................13
2.6. Dynamic Light Scattering…………………………………………………………………………………..13
2.7. Transmission Electron Microscopy…………………………………………………………….……..14
iv
2.8. Differential Ultracentrifugation………………………………………………………….…………….14
2.9. iZON Size Exclusion Column………………………………………………………………..…………….17
CHAPTER THREE:
Results...............................................................................................................................18
3.1. Comparison of Early and Optimized Differential Ultracentrifugation Protocols…18
3.2. The Effects of Trehalose on Particle Size and Total Particle Count.......................22
3.3. iZON Size Exclusion Column Optimization…………………………………………………….…..25
3.4. Quality Control Testing of Ultracentrifugation and Column Protocols……………...28
3.5. Negative Stain Transmission Electron Microscopy, ZVNTA, and DLS Sizing Suggest
the Presence of Exosomes………………………………………………………………………………..31
CHAPTER FOUR:
Discussion.........................................................................................................................34
4.1. Differential Ultracentrifugation…………………………………………………………………………34
4.2. Column……………………………………………………………………………………………………..………37
CHAPTER FIVE:
Conclusion.........................................................................................................................40
References…..................................................................................................................43
v
LIST OF TABLES/FIGURES
Table 1: List of Primary Antibodies.……………………………………..……………………..………12
Table 2: List of Secondary Antibodies …..…………………………….……………………..………12
Figure 1: Exosome Biogenesis………………………………………………………………………………..3
Figure 2: Isolation protocol for rabbit LGAC media.……………..……………………..……….15
Figure 3: Isolation protocol for mouse serum…..……………………………………..…..………16
Figure 4: Comparison of exosomes isolated using the initial and the optimized
ultracentrifugation protocols...............................................................19-21
Figure 5: Effects of 25 mM trehalose dihydrate (TRE) on isolated exosome
characteristics…………………………………..…………………………………………..………23
Figure 6: Particle count from iZON qEV column isolation of exosomes from C57 and
3DKO mice……………………………………………………………………………………….26-27
Figure 7: Quality controls of ultracentrifugation and column protocols…..…..…29-30
Figure 8: TEM image of exosomes isolated from BALB/c mice……..……………..………32
Figure 9: DLS and ZVNTA sizing of exosomes isolated from mice…..…………………....33
vi
ABBREVIATIONS
EV: Extracellular Vesicle
ILV: Intraluminal Vesicle
MVB: Multivesicular Body
ESCRT: Endosomal Sorting Complexes Required for Transport
SS: Sjögren's Syndrome
CTSS: Cathepsin S
PD: Parkinson’s Disease
3DKO: Rab3DKO
27bKO: Rab27b knockout
27KO: Ashen/27bKO
LGAC: Lacrimal Gland Acinar Cell
PBS: Phosphate Buffered Saline
NTA: Nanoparticle Tracking Analysis
ZVNTA: ZetaView Nanoparticle Tracking Analysis
NSNTA: Nanosight Nanoparticle Tracking Analysis
TRE: Trehalose Dihydrate
TEM: Transmission Electron Microscope
BCA: Bicinchroninic Acid
BSA: Bovine Serum Albumin
HCE: Human Corneal Epithelial
vii
Abstract
Exosomes are small, cell-derived vesicles that are thought to regulate a multitude of
biological processes. Through transmission of their miRNA and protein content,
exosomes are capable of transmitting phenotypic information from cell to cell under
both normal and pathological conditions. Recently, the study of exosomes has expanded
into nearly all fields of translational research. In this project, I developed a robust set of
protocols capable of isolating exosomes from a variety of starting materials and
volumes. Two commonly used methodologies, ultracentrifugation and size-exclusion
chromatography, were optimized for the isolation of exosomes from mouse serum and
rabbit lacrimal gland acinar cell culture medium. These protocols were adapted from the
existing literature and modified to work with our samples. Characterization of the
membrane fraction thought to be enriched in exosomes by Western blotting,
nanoparticle tracking analysis, and dynamic light scattering demonstrates the success of
these protocols in isolating and characterizing exosomes from samples of interest.
1
CHAPTER ONE:
Introduction
1.1. Exosome Biogenesis
Exosomes are a type of extracellular vesicle (EV) known to be involved in cell-to-cell
communication (Mulcahy et al., 2014). Although only ~30-150 nm in diameter, exosomes are
capable of transmitting phenotypic information from cell to cell through delivery of their miRNA
and protein content (Romano and Kwong, 2017; Vestad et al., 2017). The initial step of
exosome biogenesis begins with the budding of early endosomes from the plasma membrane
(Théry et al., 2002). Trafficking molecules, such as the tetraspanin CD9, cause invagination of
the endosomal membrane forming intraluminal vesicles (ILVs) and lead to the formation of
multivesicular bodies (MVBs). Further activation by additional trafficking molecules then
determines whether an MVB will undergo lysosomal fusion, form an amphisome, or fuse back
into the cell membrane (Figure 1). Studies have also shown that the composition of the MVB
membrane determines its destination (Raposo and Stoorvogel, 2013). MVBs rich in cholesterol
but lacking lysobisphosphatidic acid become exosomes while MVBs poor in cholesterol but
containing lysobisphosphatidic acid fuse with the lysosome (Möbius et al., 2002; Wubbolts et
al., 2003). When fusion occurs between MVBs and the cell membrane fusion, the ILVs inside of
MVBs are released from the cell as exosomes (Hessvik and Llorente, 2018).
The endosomal sorting complexes required for transport (ESCRT) are a set of proteins that
associate together and assist in the development of exosomes. While ESCRT proteins are often
associated with the formation of ILVs in MVBs, there is an alternative mechanism of ILV
invagination which is independent of ESCRT proteins. This pathway relies on sphingomyelinase,
2
a ceramide producing enzyme, to generate self-organizing lipid rafts. Although inefficient,
MVBs can use these lipid domains to form ILVs spontaneously, without the need for ESCRT
machinery (Babst, 2011). The small monomeric GTPase Rab7 is another protein known to
associate with MVBs. Rab7 binds to a variety of partners ranging from motor proteins to
lysosomes (Beer and Wehman, 2016). Related Rab proteins Rab27a and Rab27b, are
responsible for the final steps involved in docking and fusion of MVBs to the plasma membrane.
During this step, soluble N-ethylmaleimide-sensitive factor attachment protein receptors such
as VAMP-7 bind and dock the MVB to the plasma membrane (Beer and Wehman, 2016).
Many trafficking molecules involved in exosome biogenesis are either embedded in or
associated with endosomes. Inward budding of MVBs captures these proteins and they become
part of the new exosomal membrane (Ostrowski et al., 2010). This mechanism partially explains
why exosomal content is specific to the originating cell. In the same vein, inward budding of the
MVB also captures cytosolic proteins and RNA (Tkach and Théry, 2016). Conversely, miRNA
packaging is targeted and does not occur at random. Instead, miRNA binding motifs associate
with the MVB membrane as it buds inward and sorts specific miRNAs inside the new vesicles
(Villarroya-Beltri et al., 2013). These sorting processes only occur in the ESCRT-dependent
exosome pathway because of the stability needed for packaging. Studies have shown that
ESCRT proteins bind to the neck of budding ILVs, which is thought to physically hold the vesicle
open for packaging of cargo (Babst, 2011).
3
“Molecules shown to affect exosome biogenesis and/or release” by Hessvik, N.P. & Llorente is
licensed under CC BY 4.0.
Figure 1. Exosome Biogenesis. Early endosomes are activated by trafficking molecules causing
budding in of the membrane. The resulting multivesicular body (MVB) can then fuse with the
cell membrane and release exosomes.
4
1.2. Exosomes in Disease
Sjögren's Syndrome (SS) is a chronic autoimmune condition commonly associated with
lymphocytic infiltration of the salivary and lacrimal glands. While the initial mechanism of onset
is unclear, hormonal factors are thought to be involved as SS occurs most frequently in women
(Nair and Singh, 2017). Beyond the glandular effects, SS affects nearly every organ, causing a
wide range of symptoms such as fatigue, hepatomegaly, bronchial swelling, and Non-Hodgkin
lymphoma. There is no cure for SS and treatment only mitigates the symptoms. Furthermore,
diagnosis is a difficult process requiring a subjective evaluation of multiple criteria (Mavragani
and Moutsopoulos, 2014). This highlights the need for identification of non-invasive, disease-
specific biomarkers. Recent studies suggest that elevated activity levels of tear cathepsin S
(CTSS), a cysteine protease involved in maturation of MHC class II molecules and in multiple
inflammatory processes, are associated with SS (Hamm-Alvarez et al., 2014; Shi et al., 1999). It
is thought that autoimmunity is enhanced by the presence of high levels of CTSS due to the
dysregulation of antigen presentation caused by accelerated maturation of MHC class II
molecules as well as by activation of other inflammatory pathways such as protease-activated
receptor 2 (Driessen et al., 1999; Elmariah et al., 2014; Hamm-Alvarez et al., 2014).
Interestingly, it has also been shown that CTSS plays a role in the regulation of MVBs, a
common progenitor of both exosomes and lysosomes (Huang et al., 2016). High levels of CTSS
activity might therefore affect the characteristics of tear exosomes from SS patients. This
hypothesis is supported by a previous study characterizing exosomes isolated from the saliva of
SS patients which were found to contain a significantly different miRNA profile compared to
5
healthy control subjects (Gallo et al., 2016). Together, these data indicate the need for further
study of exosomes in the search for SS biomarkers.
Parkinson’s disease (PD) is a chronic neurodegenerative condition known for the motor
symptoms caused by the death of dopaminergic neurons in the substantia nigra (Massano and
Bhatia, 2012). While this is the most visible symptom, PD is not a motor syndrome, but rather a
multisystem disease affecting the entire body (Jellinger, 2011). The mechanism of onset is
unknown, and the most common type of PD is idiopathic but there are a variety of heredity
forms which reveal clues about the complex nature of the condition. Mutations in the genes for
SNCA, UCHL1, LRRK2, Parkin, Pink1, and DJ-1 are frequently found among patients with familial
PD (Massano and Bhatia, 2012). One of the most commonly studied genes is SNCA, which
encodes the protein alpha-synuclein. The function of this gene is hotly contested as it is an
intrinsically disordered protein that behaves differently depending on the surrounding
environment. In some conditions it has been found to stabilize membranes and in others it has
been found to disrupt membranes (Rocha et al., 2018; Ysselstein et al., 2016). Much of this has
to do with the different oligomers and fibrils that alpha-synuclein can form, each of which
possesses a unique function and level of toxicity (Rocha et al., 2018).
Regardless of its function, alpha-synuclein is known to aggregate inside of cells and form large
particles called Lewy bodies. These protein aggregates build up over time and eventually either
kill the cell or disrupt their ability to function (Bendor et al., 2013). In a prion-like manner,
alpha-synuclein is thought to spread throughout the body and induce conformational change in
harmless isoforms, converting them into toxic aggregates (Recasens and Dehay, 2014). Studies
have shown that exosomes might be responsible for the transport of these toxic alpha
6
synuclein aggregates. While alpha-synuclein is normally cleared by the unfolded protein
response or the autophagy-lysosome pathway, a breakdown in these disposal systems can lead
to intracellular buildup of the protein (Bellucci et al., 2011; Longhena et al., 2017). Cells have
been observed utilizing MVBs to remove protein waste via membrane fusion rather than
through lysosomal fusion (Verweij et al., 2012). In one study, bafilomycin A1 was used to induce
lysosomal dysfunction in alpha-synuclein over-expressing cells, leading to an increase in
exosomal alpha-synuclein (Alvarez-Erviti et al., 2011). This indicates that when degradation
pathways break down, exosomes might act as an emergency waste removal system to expel the
alpha-synuclein deposits. These exosomes are then taken up by nearby cells; releasing their
cargo and spreading toxic aggregates throughout the body.
A large challenge with PD is the difficulty in diagnosis. The characteristic motor symptoms
associated with PD often occur late in the progression of the disease (Frank et al., 2006). While
treatment for PD exists, it cannot reverse or stop the spread of the disease, especially once
motor symptoms start to present. There is a desperate need for the development of diagnostic
biomarkers capable of identifying Parkinson’s during the early, asymptomatic stage, when
treatment is still effective (Oertel, 2017). Prophylactic diagnosis that a patient will not refuse, is
most likely feasible with noninvasive testing of a biofluid, such as saliva and tears, the
diagnostic potential of which has been known for years (Haeckel and Hänecke, 1993). As with
SS, PD is associated with a dysregulation of intracellular trafficking which might lead to a change
in exocrine gland secretion (Dehay et al., 2012). A previous study found no difference in
exosomal alpha-synuclein of salivary samples between health control patients and PD patients
(Kang et al., 2016). Conversely, tear fluid of PD patients has been found to contain elevated
7
levels of oligomeric alpha-synuclein compared to health control patients (Hamm-Alvarez et al.,
2018). This highlights a need for the investigation of exosomal alpha synuclein in tears of PD
patients.
1.3. Trafficking Proteins
Rab27a and Rab27b are monomeric GTPases involved in membrane transport and fusion (Izumi
et al., 2003). They have been shown to promote the stability of MVB docking with the cell
membrane before the release of contained ILVs. This is a vital step in the secretion of exosomes
and silencing of the Rab27 isoforms has been shown to decrease the number of released
exosomes by 50% (Ostrowski et al., 2010). Similarly, the Rab27 molecules assist in the
exocytosis of lytic granules by cytotoxic cells (Ménasché et al., 2003). Although they are thought
to be compensatory for each other, only Rab27b was found to regulate exocytosis of secretory
cells in lacrimal gland acinar cells (Chiang et al., 2011).
Rab3D, a small GTPase similar to Rab27a and Rab27b is known to mediate exocytosis of
secretory granules (Evans et al., 2008). Previous work has shown that an imbalance between
levels of Rab3D, Rab27a, and Rab27b leads to an increase in CTSS secretion in the NOD mouse
model of SS compared to BALB/c controls (Meng et al., 2016). Furthermore, the Rab3 and
Rab27 isoforms are known to aid in the maturation of secretory granules in PC12 cells (Kögel et
al., 2013). While there is little published literature attesting to this, it has been reported that
increased secretion of exosomes was observed in a Rab3D overexpression model of breast
cancer and melanoma cell lines (Yang et al., 2015). On the other hand, Rab3D was not found to
be associated with MVBs in alveolar epithelial type II cells (van Weeren et al., 2004). It is
possible that alterations in cancer cell trafficking systems explain why Rab3D was associated
8
with exosome secretion in one study and not found to be associated with MVBs in another.
Alternatively, interactions between the Rab3D and the Rab27 isoforms suggest the possibility of
a more indirect relationship between Rab3D and the exosomal pathway, although little
evidence exists in the literature.
1.4. Mouse and Cell Models
To elucidate the interactions between different Rab proteins and the exosomal secretion
system, knockout mouse models were created. Rab3D (3DKO) and Rab27b (27bKO) mice were
created as knockout models using C57BL/6 mice (Riedel et al., 2002; Tolmachova et al., 2007).
Mice deficient in Rab27a (Ashen) spontaneously arose and were bred with 27bKO mice to
produce mice deficient in both Rab27a and Rab27b (27KO) (Tolmachova et al., 2007). These
mice demonstrate unique inflammatory and secretory phenotypes related to the knocked-out
gene(s). In general, exosome release is controlled by similar trafficking machinery throughout
the body. While there are some Rab proteins which are only expressed in specific locations,
such as the synaptic vesicle-associated Rab3A, many Rab proteins are omnipresent (Fischer von
Mollard et al., 1990). For this reason, serum exosomes are a reasonable surrogate for a pilot
study of exosome secretion in mouse models.
Lacrimal gland acinar cells (LGACs) are more physiologically relevant secretory cells which can
be maintained in primary culture and used to study secretory pathways (Chiang et al., 2011).
The activity of the widely used secretagogue carbachol has been extensively documented in
LGACs, although not in the context of exosome production or secretion (Gierow et al., 1995).
Previous studies have demonstrated the successful use of an adenovirus construct to knock out
Rab27b in LGAC which will be useful for exosome secretion studies (Meng et al., 2016). This
9
knockout might also be used to determine if release of exosomes from secretory cells is
polarized. In one experiment, mouse pancreatic acinar cells were used to show apical
localization of Rab27B and Rab3D (Hou et al., 2015).
1.5. Exosome Isolation Protocols
Although discovered in 1983, interest has only recently begun to grow as exosomes have been
found to play a role in nearly all biological functions. This sudden growth has led to
heterogeneity among exosome isolation protocols. Without a commonly accepted definition of
what an exosome is, it is difficult to determine which new technique is the best (Willis et al.,
2017). Among these, the most widespread technique is differential ultracentrifugation. Multiple
spins of increasing sedimentation force are used to remove vesicles of decreasing size and
density. Importantly, it is not possible to completely purify a specific subtype of vesicles;
however, it is possible to produce a sample enriched in exosomes using this technique. Another
commonly used method of isolation relies on size exclusion chromatography. This technique
was adopted only recently but is quickly gaining popularity because of the apparent increased
purity of isolated particles (Gardiner et al., 2016). With this method, samples are run through a
column which separates particles into a specific fraction based on their size. The proper choice
of technique depends on both the composition of the starting sample and the eventual goal of
the study (Helwa et al., 2017). In this thesis, I aim to demonstrate the benefits and drawbacks
of each method, as well as the reasons behind using ultracentrifugation and column-based
exosome isolation techniques.
10
CHAPTER TWO:
Materials and Methods
2.1. Mice
Blood from different mouse strains, segregated by experiment, was removed by cardiac
puncture and used to isolate exosomes. Strains used in the study included: BALB/c, NOD,
Ashen, Rab27b knockout (27bKo), Ashen/27bKO (27KO), and Rab3DKO (3DKO). BALB/cJ and
NOD/ShiLtJ were obtained from Jackson Laboratories (Sacramento, CA) or bred in house from
existing breeding pairs. Ashen, 27bKO, 27KO, and 3DKO mice were generated as previously
described and bred in house (Meng et al., 2016). Male and female mice of varying ages were
used depending on availability at the time of exosome isolation. All animal procedures were in
accordance with the Guiding Principles for the Use of Animals in Research and approved by USC
Institutional Animal Care and Use Committee. Serum was isolated in MiniCollect .8 mL gold cap
Z Serum Separator tubes (Greiner Bio-One, Kremsmünster, Austria) or in 2 mL Eppendorf tubes
by centrifuging blood at 2,000 x g for 15 minutes at 4 °C.
2.2. Cell Culture
Female New Zealand White rabbits (2.0-3.0 kg) were obtained from Western Oregon Rabbit
Company (Philomath, OR). Rabbit lacrimal gland acinar cells (LGAC) were isolated as previously
described with or without Matrigel and seeded in 150 mm cell culture dishes at a density of 2.0
x 10
6
cells/mL or 4.0 x 10
6
cells/mL. Serum-free media was used to prevent contamination by
unwanted vesicles. Cells were incubated at 37 °C in a humidified incubator with 5% CO 2 and
allowed to grow for 3 days before removal of media for isolation of exosomes (Meng et al.,
2016).
11
2.3. SDS-PAGE and Western Blot
Equal volumes of sample were mixed with 6x Laemmli Sample Buffer in the presence of
absence of DTT depending on the marker in use. Samples were boiled for 5 minutes at 95 °C
then run on one of two gels. 20 µL of sample was run on 10% PAGEr EX Gels (Lonza, Basel,
Switzerland) for 2 hours at 100 volts while up to 40 µL of sample was run on 8-16% Novex
WedgeWell Tris-Glycine Gels (ThermoFisher, Waltham, MA) for 75 minutes at 100 volts. Gels
were then transferred to nitrocellulose using an iBLOT 2 device and Invitrogen iBLOT 2 NC
stacks (ThermoFisher, Waltham, MA) followed by blocking in Blocking Buffer for Fluorescent
Western Blotting (Rockland, Pottstown, PA) for 1 hour at room temperature. Next, membranes
were incubated with primary antibody overnight at 4 °C. Following six five-minute washes with
tris-buffered saline + 0.1% Tween-20, secondary antibodies were added and allowed to
incubate for 1 hour at room temperature. After repeating the previous wash cycle, membranes
were imaged on a LI-COR Odyssey Fluorescent Imager using the accompanying software version
2.1.15. Primary and secondary antibodies dilutions were made in blocking buffer. Antibodies
used can be found on Table 1 and Table 2. Although unpublished, both antibodies were found
to have reactivity with exosomes isolated from rabbit lacrimal gland acinar cell medium.
12
Table 1. List of Primary Antibodies
1° Antibodies Host Published
Reactivity
Product No. Company
TSG101 Rabbit Mouse
Rat
Human
ab125011 Abcam
CD9 Mouse Mouse
Human
MA1-80307 ThermoFisher
Table 2. List of Secondary Antibodies.
2° Antibodies Product No. Company
IRDye goat anti-rabbit 800 926-32211 LI-COR
IRDye goat anti-mouse 680 926-68070 LI-COR
2.4. ZetaView Nanoparticle Tracking Analysis
The size and concentration of exosomes were measured with ZetaView nanoparticle tracking
analysis (ZVNTA). Samples were shipped to Alpha Nano Tech LLC (Chapel Hill, NC) for analysis by
a ZetaView S/N 17-332 (Particle Metrix, Meerbusch, Germany) running the software ZetaView
8.04.02. After calibration with 100 nm standards (Applied Microspheres, The Netherlands),
samples were diluted in varying amounts of phosphate buffered saline (PBS) to reach an
optimal concentration for analysis then loaded into the device for measurement. 11 positions
were sampled for two cycles each with outliers automatically removed by the software.
Measurements were taken using a temperature of 22 °C, a sensitivity of 75, a frame rate of 30,
and a shutter speed of 100. These measurements were analyzed using a minimum brightness of
13
20, a maximum size of 500 pixels, and a minimum size of 10 pixels. As it is widely known to be
the best determinant of particle size, the mode was selected as the main sizing parameter
(Helwa et al., 2017). Total Particle count was calculated to account for varying resuspension
volumes. Figures without error bars represent the results of one experiment.
2.5. Nanosight Nanoparticle Tracking Analysis
In some samples, Nanosight nanoparticle tracking analysis (NSNTA) was used to measure the
size and concentration. Samples were shipped to Alpha Nano Tech LLC (Chapel Hill, NC) for
analysis by a Nanosight LM10-HS (Malvern Instruments, Amesbury, UK) running the software
NTA 3.2 Dev Build 3.2.16. After calibration with 100 nm standards (Applied Microspheres, The
Netherlands), samples were diluted in varying amounts of PBS to reach an optimal
concentration for analysis then loaded into the device for measurement. The device was
equipped with a sCMOS camera, a 638 nm laser, and a syringe pump. Measurements were
taken using a temperature of 21 °C, a camera level of 15, and a frame rate of 25. 374 frames
were examined per sample using the viscosity of water. Analysis settings include a detection
threshold of 3, blur size on auto, and max jump distance set to 6.9 – 8.4 pixels. Figures without
error bars represent the results of one experiment.
2.6. Dynamic Light Scattering
DLS was performed in the USC School of Pharmacy Translational Research Laboratory using a
Wyatt DynaPro Plate Reader and analyzed with the accompanying software. Samples of 60 µL
were run in triplicate at 25 °C and diluted in PBS when necessary. The hydrodynamic radii of
isolated exosomes was then measured and presented as a normalized radius or diameter.
Figures without error bars represent the results of one experiment.
14
2.7. Transmission Electron Microscopy
Exosome samples stored at -80 °C were unfrozen and applied to 150 mesh copper carbon
formvar grids (VWR, Radnow, PA). Using high precision negative forceps (Electron Microscopy
Sciences, Hatfield, PA), 10 µL aliquots were incubated on the grid for 5 minutes, then blotted
using filter paper. Ultrapure water was used to wash the grid before further incubation in 1%
aqueous uranyl acetate (VWR, Radnow, PA) for 5 minutes. After another wash in ultrapure
water, the grid was allowed to air dry for 30 minutes before storage or immediate viewing in a
JEM1400 transmission electron microscope operating at 100 kV.
2.8. Differential Ultracentrifugation
Different spin cycles were utilized to optimize the collection of exosomes from culture media
and serum. All low-speed spins were performed on a tabletop centrifuge and
ultracentrifugation was done on a Beckman Coulter Optima LE-80k using a Beckman Coulter
Type 50.2 Fixed Angle Rotor. Optimized ultracentrifugation protocols for media and serum can
be found in Table 3 and Table 4 respectively.
15
Figure 2. Isolation protocol for rabbit LGAC media.
Lacrimal Gland Acinar Cells
1. Centrifuge at 2000 x g for 5 min at 4°C. Collect Supernatant.
2. Centrifuge at 2000 x g for 20 min at 4°C. Collect Supernatant.
3. Centrifuge at 12,000 x g for 30 min at 4°C. Collect Supernatant.
4. Centrifuge at 110,000 x g for 70 min at 4°C.
Resuspend pellet in PBS + 25 mM Trehalose.
Resuspend pellet in PBS + 25 mM Trehalose
5. Centrifuge at 110,000 x g for 60 min at 4°C.
Collect Conditioned Media
Plate at 4x10^6 cells/mL for 3 days in 150 mm dish
16
Figure 3. Isolation protocol for mouse serum.
Remove blood by cardiac puncture
1. Centrifuge at 2000 x g for 15 min at 4°C. Collect Supernatant.
2. Centrifuge at 2000 x g for 30 min at 4°C. Collect Supernatant.
3. Centrifuge at 12,000 x g for 45 min at 4°C. Collect Supernatant.
4. Centrifuge at 110,000 x g for 120 min at 4°C.
Resuspend pellet in PBS + 25 mM Trehalose.
Resuspend pellet in PBS + 25 mM Trehalose
5. Centrifuge at 110,000 x g for 70 min at 4°C.
Let blood clot for 20 minutes
Collect blood in serum separator tubes
17
2.9. iZON Size Exclusion Column
iZON qEVoriginal (iZON, Christchurch, New Zealand) were used as an alternative method to
isolate exosomes. Serum samples were processed by spinning at 10,000 x g for 30 minutes, 4 °C
then brought to 500 µL by either adding PBS or concentrating with Amicon Ultra - 0.5 mL
Ultracel – 10k Centrifugal filters (Millipore Sigma, Burlington, MA). PBS was filtered through 200
nm filters then centrifuged at 3,300 x g for 3 minutes. Before addition to the column, samples
were vortexed then lightly centrifuged to remove bubbles. Column was equilibrated with 15 mL
PBS then capped and excess buffer was removed. Without allowing the column to dry, sample
was carefully added to the top of the filter without allowing bubbles to form. The bottom cap
was then removed, and sample was allowed to enter into the column. Once the sample fully
entered the column, up to 2 mL of PBS was added to keep the filter from drying out. The void
volume of 3 mL (6 fractions) was collected and measured in a 15 mL tube. As soon as 3 mL
liquid exited the column, the following 1.5 mL (fractions 7, 8, and 9) was collected in a 2 mL
Eppendorf tube. The sample was then either taken for immediate analysis, mixed with 6x
Laemmli sample buffer, further concentrated down to 220 µL, or frozen at -80 °C for storage.
18
CHAPTER THREE:
Results
3.1. Comparison of Early and Optimized Differential Ultracentrifugation Protocols
The original protocol for isolation of exosomes using differential ultracentrifugation differed
from the optimized final protocol at several steps. A commonly used protocol was adapted and
used to isolate exosomes from rabbit LGAC media (Théry et al., 2006). This original protocol
was followed as published aside from the use of an initial 2,000 x g, 5-minute spin used for
pelleting of cells rather than 300 x g, 10-minutes as originally published. The major differences
in the optimized protocol compared to the original protocol are in steps 3, 4, and 5 which are
increased by 2,000 x g, 10,000 x g, and 10,000 x g, respectively. Changes in this protocol may
have led to a decrease in average particle size and a probable increase in concentration of the
exosomal preparation. 160 million rabbit LGACs were incubated on 150 mm cell culture dishes
and allowed to produce/secrete exosomes. After three days, the media was removed and
subjected to either the initial exosome isolation protocol or the optimized exosome isolation
protocol. As shown in Figure 4A and Figure 4B, the TSG101 signal was stronger in samples
obtained from the optimized protocol compared to the TSG101 signal from samples isolated
using the initial protocol. Samples from the optimized protocol also produced a CD9 signal
(Figure 4B). Unfortunately, samples from the initial protocol were never blotted for CD9 so we
do not know if a signal would have been seen. Comparison by DLS in Figure 4C demonstrates an
improvement in sample purity as measured by a decrease in the normalized hydrodynamic
radius. This suggests a decrease in the co-isolation of unwanted, larger particles with the
optimized protocol.
19
Figure 4A. Comparison of exosomes isolated using the initial and the optimized
ultracentrifugation protocols. Western blot of isolated exosomes for TSG101 from the initial
experimental protocol. Rabbit LGAC were plated on 150 mm cell culture dishes and either
treated with 100 µM carbachol (C) for 30 minutes or left untreated (U). Cells were seeded on
four dishes per group at a density of 2.0 x 10
6
cells/mL for a total of 160 million cells. Exosomes
were isolated from the media after three days of incubation using the initial differential
ultracentrifugation protocol. Western blotting for TSG101 shows a noticeable band around the
expected size of ~45 kDa with no signal in the secondary control.
50
37
75
100
150
250
25
20
15
10
50
37
75
100
150
250
25
20
15
10
kDa kDa
C U
C U
Anti-TSG101 Secondary
Control
A Carbachol and Untreated RB LGAC
20
Figure 4B. Comparison of exosomes isolated using the initial and the optimized
ultracentrifugation protocols. Western blot of isolated exosomes for TSG101 and CD9 from the
optimized experimental protocol. Rabbit LGAC were plated on 150 mm cell culture dishes and
seeded on two dishes at a density of 4.0 x 10
6
cells/mL for a total of 160 million cells. Exosomes
were isolated from the media after three days of incubation using the optimized differential
ultracentrifugation protocol. Western blotting for TSG101 shows a strong band around the
expected size of ~45 kDa with no signal in the secondary control. Blotting for CD9 also produces
a signal at the expected size of ~23 kDa. Blots were run by Shruti Kakan.
kDa kDa
Anti-TSG101 Secondary
Control
No Treatment RB LGAC B
50
37
75
100
150
250
25
20
15
50
37
75
100
150
250
25
20
15
50
37
75
100
150
250
25
20
15
10
50
37
75
100
150
250
25
20
15
10
Anti-CD9 Secondary
Control
kDa kDa
21
Figure 4C. Comparison of exosomes isolated using the initial and the optimized
ultracentrifugation protocols. Normalized radii of exosomes isolated from rabbit LGAC in
experiments A and B. DLS was used to calculate the hydrodynamic radii of particles in each
sample and run in triplicate. Exosomes enriched by the optimized protocol, experiment B, are
of a smaller size compared to exosomes enriched by the initial protocol.
C
A
B
5 0
6 0
7 0
8 0
9 0
In it ia l v s . O p tim iz e d U C P r o to c o l
Is o la t e d E x o s o m e S a m p le
N o r m a liz e d R a d ii in n m
22
3.2. The Effects of Trehalose on Particle Size and Total Particle Count
Although it is not widely used in current protocols, trehalose dihydrate (TRE) has been shown to
prevent the aggregation of exosomes (Bosch et al., 2016). TRE is added at a 25 mM
concentration to the PBS used for dilution of sample and for resuspension of the pellet after
steps 4 and 5. The effects of TRE were tested on rabbit LGAC media using the optimized
ultracentrifugation spin protocol. Two groups of 240 million cells were plated on six 150 mm
cell culture dishes. One group was treated with 25 mM TRE in PBS during dilution and
resuspension steps and the other was treated with just PBS. Samples were flash frozen, stored
at -80 °C, and shipped out for ZVNTA analysis the following week. As shown in Figure 5A, the
mode of the particle size of TRE treated exosomes was noticeably lower than the untreated
group. This smaller size might be due to a reduction in exosome aggregation and is consistent
with the higher total particle count. In Figure 5B, the particle count of TRE treated exosome
samples is nearly twice as high as in the untreated group. Because of these results, nearly all
further ultracentrifugation experiments for isolation of exosomes were performed using TRE.
23
R B L G A C
R B L G A C + 2 5 m M T R E
0
5 1 0
1 0
1 1 0
1 1
1 .5 1 0
1 1
E ffe c t o f T R E o n P a r tic le C o u n t
T o t a l P a r t ic le C o u n t
B
R B L G A C
R B L G A C + 2 5 m M T R E
0
2 0
4 0
6 0
8 0
1 0 0
E ffe c t s o f T R E o n P a r t ic le S iz e
P a r t ic le D ia m e t e r M o d e in n m
A
24
Figure 5. Effects of 25 mM trehalose dihydrate (TRE) on isolated exosome characteristics.
Rabbit LGAC were plated on 150 mm cell culture dishes and either isolated in the presence or
absence of 25 mM TRE. Cells were seeded on three dishes per experimental group at a density
of 4.0 x 10
6
cells/mL for a total of 240 million cells. Exosomes were isolated from the medium
after three days of incubation using the optimized differential ultracentrifugation protocol.
ZVNTA was used to determine the size and total particle count of both samples. A: The mode of
particle sizes in the TRE treated group is lower than that in the untreated group. B: The total
particle count in the TRE treated group is around twice as high than in the untreated group.
25
3.3. iZON Size Exclusion Column Optimization
An alternative to the differential ultracentrifugation protocol was developed to isolate
exosomes through the use of size exclusion chromatography. iZON qEVoriginal columns are
capable of quickly isolating large amounts of exosomes from small samples sizes around 500 µL.
Although we do not yet have Western blot data confirming the presence of exosomes in these
samples, ZVNTA and DLS were used to analyze samples generated from materials prepared
using these columns. 500 µL of processed serum from two C57 and two 3DKO mice was run
through the iZON column then repeated with new mice in a second run. Mouse strains were
chosen to validate earlier NSNTA data from ultracentrifugation of 3DKO, C57, and 27KO mice.
Total particle count of exosomes from knockout mice showed a decreased exosome
concentration compared to C57 controls. As shown in Figure 6A, Run 1 ZVNTA data showed no
difference between 3DKO and C57 particle counts. Although we failed to validate the previous
experiment, the absolute number of particles was larger than that obtained from any previous
mouse ultracentrifugation experiment. This experiment was repeated with some success in Run
2 in which twice as many exosomes were recovered from C57 as 3DKO (Figure 6B). The ratio of
total particles recovered from C57 compared to 3DKO was plotted in Figure 6C to compensate
for potential column use errors, improvement in skill over time, and the use of different
methodologies.
26
3 D K O
C 5 7
0
2 1 0
9
4 1 0
9
6 1 0
9
T o ta l P a r tic le C o u n t R u n 1
T o t a l P a r t ic le C o u n t
A
3 D K O
C 5 7
0
1 1 0
1 0
2 1 0
1 0
3 1 0
1 0
4 1 0
1 0
T o ta l P a r tic le C o u n t R u n 2
T o t a l P a r t ic le C o u n t
B
27
Figure 6. Particle count from iZON qEV column isolation of exosomes from C57 and 3DKO mice.
A: Total particle count of Run 1 as measured by ZVNTA. Run 1 was performed with blood
collected from two male 3DKO mice aged 16-19 weeks and two male C57 mice aged 14-15
weeks. Serum was isolated and then centrifuged at 10,000 x g for 30 minutes at 4 °C and 500 µL
was then used for Run 1 of the iZON column. B: Total particle count of Run 2 as measured by
ZVNTA. Run 2 was performed using blood collected from two male 3DKO and two male C57
mice aged 12-14 weeks. Samples were processed the same as in Run 1. C: The ratio of the total
particle count obtained from C57 versus 3DKO mice. This ratio is compared to a similar ratio
obtained from earlier ultracentrifugation exosome isolation of blood (UC) from 16 male 3DKO,
15 male 27KO, and 15 male C57 mice aged 12-16 weeks. Particle Count was measured by
NSNTA.
R u n 1
R u n 2
U C R u n
0
2
4
6
R a t io o f C 5 7 t o 3 D K O P a r t ic le C o u n t
R a t io o f
P a r t ic le C o u n t
C
28
3.4. Quality Control Testing of Ultracentrifugation and Column Protocols
As a quality control test of these protocols, samples were scaled up and recovered particles
were measured with ZVNTA. Furthermore, as iZON columns are meant to be reused, we tested
the particle recovery after the first and second use. 15 male C57 mice aged 14-17 weeks were
split into two groups of five and ten. Serum was removed, and exosomes were isolated using
the optimized ultracentrifuge protocol without TRE. NSNTA analysis of these samples showed
an increase in total particle count similar to the increase in starting material (Figure 7A).
Similarly, serum from six C57 female mice aged nine weeks was processed and concentrated
into a 750 µL sample. 125 µL was mixed with 375 PBS and exosomes were isolated from an
iZON column. Following this, 500 µL was run through the same column to isolate exosomes
from four times the starting material. Scaling up using the column provided less than a
proportionate increase in exosomes, but this might be due to exceeding the capacity of the
column or ZVNTA experimental margin of error. To ensure that exosomes were not being lost
or gained through reusing the column, total particle count was measured from a column’s first
and second use. Serum from four C57 females aged 15-20 weeks was collected and processed
for use in the column. Total particle counts from both first and second use were close, but
repetition of this experiment is necessary to confirm the effects of reuse.
29
C 5 7 1 2 5 µ L
C 5 7 5 0 0 µ L
0
2 1 0
1 0
4 1 0
1 0
6 1 0
1 0
T o ta l P a r t ic le C o u n t F r o m
1 2 5 µ L v s . 5 0 0 µ L C 5 7 S e r u m
T o t a l P a r t ic le C o u n t
B
5 C 5 7 M ic e
1 0 C 5 7 M ic e
0
5 1 0
8
1 1 0
9
1 .5 1 0
9
2 1 0
9
U ltr a c e n tr ifu g a tio n o f
5 v s 1 0 M ic e
T o t a l P a r t ic le C o u n t
A
30
Figure 7. Quality controls of ultracentrifugation and column protocols. A: Serum from a group
of five and a group of 10 male C57 mice aged 14-17 weeks was used. Exosomes were isolated
using ultracentrifugation without TRE. Increasing the starting material produced a similar, but
not necessarily proportional, increase in the total particle count. Particle count was measured
by NSNTA. B: Serum from six C57 female mice aged nine weeks was processed and
concentrated. Exosomes were first isolated from 125 µL concentrated serum mixed with 375 µL
PBS. After washing the column, it was reused to isolate exosomes from 500 µL concentrated
serum. Measured particle count increased when starting material increased. C: Serum from
four C57 females aged 15-20 weeks was collected and processed. 500 µL of serum was run
through an iZON column which was then reused to run another 500 µL of serum. Total particle
count of both samples was similar in number.
C 5 7 R u n 1
C 5 7 R u n 2
0
5 1 0
9
1 1 0
1 0
1 .5 1 0
1 0
T o ta l P a r t ic le C o u n t F r o m
F ir s t U s e v s . S e c o n d U s e C o lu m n
T o t a l P a r t ic le C o u n t
C
31
3.5. Negative Stain Transmission Electron Microscopy, ZVNTA, and DLS Sizing Suggest the
Presence of Exosomes
Although the most common method of exosome identification is Western blotting, other
methods are also used. Because our lab has had difficulty producing high quality blots of
exosomes due the limited protein content of the samples, transmission electron microscopy
(TEM), ZVNTA, and DLS were used extensively for further characterization. Exosomes were
produced using the optimized ultracentrifugation protocols from the serum of 5 BALB/c mice
aged 12 weeks. Samples were then transferred to 150 mesh copper carbon Formvar grids and
treated with 1% uranyl acetate for negative staining. When viewed under the microscope,
multiple exosomes were seen (Figure 8). These particles demonstrate the classical cup shaped
morphology commonly seen with TEM (Wu et al., 2017). In another experiment serum was
collected from two 3DKO, C57, and 27KO male mice and processed according to the iZON
column isolation technique. DLS and ZVNTA sizing of isolated exosomes suggest a population of
particles ~80-100 nm in diameter (Figure 9).
32
Figure 8. TEM image of exosomes isolated from BALB/c mice. Serum from five male BALB/c
mice aged 12 weeks old was collected used to isolate exosomes using the optimized
ultracentrifugation protocol.
33
Figure 9. DLS and ZVNTA sizing of exosomes isolated from mice. Serum from two 3DKO, C57,
and 27KO mice aged 12-14 weeks was collected and processed. Exosomes were isolated using
separate iZON columns. Samples were used for both DLS and ZVNTA analysis. Both sizing
techniques produced measurements of a similar size.
3 D K O
C 5 7
2 7 K O
0
5 0
1 0 0
1 5 0
C o r r o b o r a t io n B e t w e e n D L S a n d N T A M e a s u r e m e n ts
P a r t ic le D ia m e t e r in n m
D L S
N T A
34
CHAPTER FOUR:
Discussion
4.1. Differential Ultracentrifugation
The first protocol that our lab chose to develop was a differential ultracentrifugation that would
be possible with the equipment on hand. This protocol needed to allow for efficient isolation of
exosomes from both small and large volume formats. While smaller samples sizes may be used
with column-based methods, differential ultracentrifugation has allowed us to work with a
larger range of samples sizes ranging from 500 µL of mouse serum to 70 mL of cell culture
media.
Our biggest challenge in developing these protocols stemmed from the difficulty involved in
validating the success of the purification. We had no access to an NTA device and eventually
were forced to outsource our samples for analysis. Early attempts at protein quantification of
exosomes utilizing the Pierce bicinchoninic acid (BCA) assay with bovine serum albumin (BSA) as
a standard protein gave inconsistent results unrelated to the amount of starting material. This
might be due to the impact of protein contamination from the overlying supernatant that
centrifugation is unable to completely remove. Serum especially suffers from this drawback as
albumin is present in large volumes and may make it impossible to interpret protein assay
results. It is also possible that lysis conditions were not sufficient for breakdown of exosomal
membranes, preventing the release of cargo necessary for measurement (Gallart-Palau et al.,
2015). For this reason, our initial confirmation of the relative purification of exosomes was
entirely based on Western blotting of equal volumes of sample.
35
Once we were confident that our methodology was correct we extended the verification to DLS
and then NTA, which provided a far more powerful method of analysis. Being able to
characterize the size and relative quantity of particles in our isolations gave us an idea of how
to further optimize the protocols. For example, a larger average particle size might suggest the
need for filtration, an increased centrifugation speed in early steps, or possibly the need for
better storage conditions to prevent aggregation.
Another drawback to the differential ultracentrifugation protocol is the need for reliable
centrifuges. Because of how expensive such large machines are, they are rarely replaced, which
means that many of the centrifuges we used were old. While an expensive service contract
might help prevent a total shutdown of this equipment in the lab, it is still inconvenient and
often costly when a vital piece of equipment breaks down. Before we found the large
preparative ultracentrifuge that our lab currently uses, we were relying on a small, unreliable
mini-ultracentrifuge. The machine would often fail to start spinning and sometimes we were
forced to give up on the experiment and lose our samples.
Furthermore, we had no access to a swinging bucket rotor which drastically reduced the yield
of our experiments. In a fixed angle rotor, the pellet adheres to the side of the tube and is often
difficult to find. On the other hand, swinging bucket rotors pellet membranes at the bottom of
the tube, which enables easier resuspension of the pellet. In addition, the final resuspension
step requires small volumes relative to tube size, and with the difficulty involved in locating a
tiny pellet, it is possible to only recover a small proportion of what might have adhered to the
tube.
36
In a more general sense, ultracentrifugation protocols also have the potential to damage
exosomes (Lobb et al., 2015). Repeated spin cycles cause damage to vesicles and lower the final
yield. Although spin cycles were kept to a minimum, our ultracentrifugation protocols isolate
significantly less exosomes than our column-based method. One step we took to reduce the
damage suffered from repeated spins is the addition of TRE. TRE is a natural sugar known for its
bioprotective capabilities. Recently, it was reported that the presence of TRE during
ultracentrifugation reduces particle size while increasing total particle count (Bosch et al.,
2016). While our results suggest that this is correct, it is impossible for us to determine which
step was affected by the TRE. It is likely that TRE prevented aggregation during the spin cycle,
however it is also possible that the effect of TRE was in preventing free-thaw damage.
Unfortunately, we cannot determine the true mechanism of action because we are unable to
perform NTA analysis on-site. Regardless of the reason, our results show that that 25 mM TRE
helped to reduce aggregation and we are currently determining if it will be possible to also use
TRE with the column method.
Even with its many flaws, differential ultracentrifugation is not an outdated method that we
should entirely abandon. For many reasons, it is still the most commonly used technique in
exosome isolation (Gardiner et al., 2016). When the starting material is large enough to ignore
the issue of recovery yield, ultracentrifugation can provide consistent results across multiple
samples. Multiple replicates can be run at the same time and under similar conditions,
removing possible sources of experimental bias. Many ultracentrifuges are also capable of
operating at a defined temperature, allowing samples to be kept at 4 °C for as long as possible
and minimizing the potential effect of protease activity.
37
Aside from the issue of protein contamination, the vesicle purity of differential
ultracentrifugation is very high and can potentially be combined with the use of density
gradient purification for the isolation of extremely pure exosome samples. This might be
necessary for population profiling or treatment-based experiments where the presence of
different types of vesicles would be problematic (Willms et al., 2016; Zhu et al., 2017). Besides
this, the characteristics of isolated exosomes may change depending on the method of
isolation, so using the most common technique makes it easier to compare results to what is
published (Mateescu et al., 2017).
4.2. Column
Compared to the ultracentrifugation method, the iZON column appears to be the better option
for isolation of exosomes from our samples. There is no need for time-consuming spin cycles or
the skill and experience needed for pellet resuspension. As seen in the results, exosomes
isolated from the column appear to be of higher quality in each comparable metric. Sizing by
DLS and NTA as well as particle quantification all suggest that the column is the superior choice.
Although, no Western blot results have yet been generated from column isolated exosomes, we
are confident that thick bands for CD9 and TSG101 will be found. We are also looking into the
identification of other putative exosome markers, such as ALIX and CD81, in our preparations.
One drawback to using the iZON columns is the issue of reuse. Due to their cost, it is desirable
for the columns to be used five times, before being discarded. A single use column option is
available, but the cost is high, and only 200 µL of starting material can be used. While we saw
that total particle counts from both first and second use were close, repetition of the effects of
multiple uses still must be tested. This in and of itself will be expensive as we will likely need to
38
use at least three columns, five times each, and then send these samples our for costly NTA
analysis.
Results from an ultracentrifugation exosome isolation of C57 and 3DKO serum suggested that
there was a large increase in the total particle count from the C57 sample compared to the
3DKO sample (Figure 6C). When repeated using the column isolation protocol, this difference
was either greatly reduced, or not found at all. Unfortunately, the NTA data from the
ultracentrifugation experiment was obtained using NSNTA while the column data was obtained
from ZVNTA. While the measurements should be similar, it is impossible to determine the
impact of using different machines on the resulting particle sizes. This is further complicated
because we have not been isolating the same number of exosomes from the same number of
mice through the column. While the ultracentrifugation method always produces similar total
particle counts, we have observed a difference of an order of magnitude between samples from
a similar starting size. An alternative explanation might be that our starting material was
sufficiently different to explain this difference. For pilot studies, we used differently aged and
gendered mice between experiments, but it is possible that their age and gender affected their
production of exosomes. It is certainly known that gender can affect exosomal content in
humans, but the quantity of exosomes is not known to change (Chen et al., 2018). If the
differences between the mice caused the varying particle counts, then it is more likely that age
was the deciding factor. Age has been shown to Increase internalization of exosomes which
might lead to a reduction of particles in the serum (Eitan et al., 2017). Our results support this
hypothesis as more exosomes were isolated in an experiment using younger mice (Figure 7B)
than from an experiment using older mice (Figure 7A). To confirm this, the iZON column should
39
be used for further exosome isolations of homogenous samples to determine if similar particle
counts can be consistently obtained.
40
CHAPTER FIVE:
Conclusion
In recent years the study of exosomes has expanded dramatically. Exosomes have been
implicated in a wide range of roles ranging from tumor-mediated immune suppression to the
formation of memory (Whiteside; Pastuzyn et al., 2018). As the field expands, it is important to
remember the impact of the method of isolation used for the exosomes under study. According
to a recent survey of exosome researchers, there are more than six different techniques
currently in use, many of which are often combined (Gardiner et al., 2016). As previously
mentioned, the techniques used for isolation have an impact on the size, morphology, and
specific population of recovered exosomes (Mateescu et al., 2017). Because of this, it is
important for a laboratory to develop a variety of isolation protocols, both to corroborate
results and to ensure a thorough analysis.
Although I do not have the data yet, I have already begun to analyze the contents of exosomes
isolated by the optimized ultracentrifugation protocol. To identify possible biomarkers of
interest in our mouse model of Sjögren’s syndrome, I have isolated RNA from NOD and BALB/c
serum exosomes. This RNA was then sent for library preparation and miRNA sequencing.
Because of the time required for sequencing, I have not yet received the data from this
experiment. It is my hope that the subsequent analysis of this data will provide clues as to what
systems in our disease model are being affected by the transmission of exosomal miRNA.
Before using the iZON column for larger experiments, we must first confirm the presence of
exosomal markers and investigate the potential effects of using a column for multiple
exosomes isolations. Once this is accomplished, there are multiple studies that we would like to
41
perform. One such study involves the data collected from 3DKO mouse serum. We would like to
further investigate the connection between Rab3D and exosomes by transduction of human
corneal epithelial (HCE) cells with a dominant negative Rab3D construct. While we have
conflicting data collected from the serum of knockout mice, it is possible that the role of Rab3D
is more complicated than merely increasing or decreasing exosome release. It is known that the
function of Rab27a and Rab27b are tied to the trafficking of exosomes and, given the
relationship between Rab3D and Rab27 in the lacrimal gland, it is likely that Rab3D also plays a
role (Meng et al., 2016; Ostrowski et al., 2010). Creating this cell model will allow for a closer
analysis of the connection between these deeply intertwined trafficking pathways and the
formation/release of exosomes.
Another potential study of interest involves isolating exosomes from tears. We are currently
conducting a study investing that use of Parkinson’s patient tears as a biomarker. The focus of
our study is oligomeric alpha-synuclein, which appears to be elevated in the patient tears as
compared to healthy control subjects (Hamm-Alvarez et al., 2018). Interestingly, previous
studies have identified pathogenic species of alpha-synuclein in exosomes isolated from
cerebrospinal fluid of Parkinson’s patients (Stuendl et al., 2016). As exosomes have been
isolated from tear fluid in volumes as low as 10 µL, it might be possible to isolate exosomes
from the tears of Parkinson’s patients using the iZON column (Aqrawi et al., 2017).
Unfortunately, the levels of alpha-synuclein and its oligomeric protein, while present in tears,
are found at very low levels. Measurement of exosomal alpha-synuclein is almost certainly
lower than what will be found in whole tears, unless all the alpha-synuclein is contained within
exosomes. If the amount of alpha-synuclein and other proteins of interest in tear exosomes are
42
at or below the limit of detection with current assays, the use of complicated and costly
proteomics-based analysis might be required. Even so, however, the study of tear exosomes
from Parkinson’s patients has to the potential to shine a light on the presently unknown
mechanisms of disease pathogenesis.
43
References
Alvarez-Erviti, L., Seow, Y., Schapira, A.H., Gardiner, C., Sargent, I.L., Wood, M.J.A., and Cooper,
J.M. (2011). Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and
transmission. Neurobiol. Dis. 42, 360–367.
Aqrawi, L.A., Galtung, H.K., Vestad, B., Øvstebø, R., Thiede, B., Rusthen, S., Young, A., Guerreiro,
E.M., Utheim, T.P., Chen, X., et al. (2017). Identification of potential saliva and tear biomarkers
in primary Sjögren’s syndrome, utilising the extraction of extracellular vesicles and proteomics
analysis. Arthritis Res. Ther. 19.
Babst, M. (2011). MVB Vesicle Formation: ESCRT-Dependent, ESCRT-Independent and
Everything in Between. Curr. Opin. Cell Biol. 23, 452–457.
Beer, K.B., and Wehman, A.M. (2016). Mechanisms and functions of extracellular vesicle release
in vivo—What we can learn from flies and worms. Cell Adhes. Migr. 11, 135–150.
Bellucci, A., Navarria, L., Zaltieri, M., Falarti, E., Bodei, S., Sigala, S., Battistin, L., Spillantini, M.,
Missale, C., and Spano, P. (2011). Induction of the unfolded protein response by α-synuclein in
experimental models of Parkinson’s disease. J. Neurochem. 116, 588–605.
Bendor, J., Logan, T., and Edwards, R.H. (2013). The Function of α-Synuclein. Neuron 79.
Bosch, S., de Beaurepaire, L., Allard, M., Mosser, M., Heichette, C., Chrétien, D., Jegou, D., and
Bach, J.-M. (2016). Trehalose prevents aggregation of exosomes and cryodamage. Sci. Rep. 6.
Chen, L., Chen, R., Kemper, S., Cong, M., You, H., and Brigstock, D.R. (2018). Therapeutic effects
of serum extracellular vesicles in liver fibrosis. J. Extracell. Vesicles 7, 1461505.
44
Chiang, L., Ngo, J., Schechter, J.E., Karvar, S., Tolmachova, T., Seabra, M.C., Hume, A.N., and
Hamm-Alvarez, S.F. (2011). Rab27b regulates exocytosis of secretory vesicles in acinar epithelial
cells from the lacrimal gland. Am. J. Physiol. - Cell Physiol. 301, C507–C521.
Dehay, B., Martinez-Vicente, M., Ramirez, A., Perier, C., Klein, C., Vila, M., and Bezard, E. (2012).
Lysosomal dysfunction in Parkinson disease. Autophagy 8, 1389–1391.
Driessen, C., Bryant, R.A.R., Lennon-Duménil, A.-M., Villadangos, J.A., Bryant, P.W., Shi, G.-P.,
Chapman, H.A., and Ploegh, H.L. (1999). Cathepsin S Controls the Trafficking and Maturation of
Mhc Class II Molecules in Dendritic Cells. J. Cell Biol. 147, 775–790.
Eitan, E., Green, J., Bodogai, M., Mode, N.A., Bæk, R., Jørgensen, M.M., Freeman, D.W., Witwer,
K.W., Zonderman, A.B., Biragyn, A., et al. (2017). Age-Related Changes in Plasma Extracellular
Vesicle Characteristics and Internalization by Leukocytes. Sci. Rep. 7, 1342.
Elmariah, S.B., Reddy, V.B., and Lerner, E.A. (2014). Cathepsin S Signals via PAR2 and Generates
a Novel Tethered Ligand Receptor Agonist. PLOS ONE 9, e99702.
Evans, E., Zhang, W., Jerdeva, G., Chen, C.-Y., Chen, X., Hamm-Alvarez, S.F., and Okamoto, C.T.
(2008). DIRECT INTERACTION BETWEEN RAB3D AND THE POLYMERIC IMMUNOGLOBULIN
RECEPTOR AND TRAFFICKING THROUGH REGULATED SECRETORY VESICLES IN LACRIMAL GLAND
ACINAR CELLS. Am. J. Physiol. Cell Physiol. 294, C662–C674.
Fischer von Mollard, G., Mignery, G.A., Baumert, M., Perin, M.S., Hanson, T.J., Burger, P.M.,
Jahn, R., and Südhof, T.C. (1990). rab3 is a small GTP-binding protein exclusively localized to
synaptic vesicles. Proc. Natl. Acad. Sci. U. S. A. 87, 1988–1992.
Frank, C., Pari, G., and Rossiter, J.P. (2006). Approach to diagnosis of Parkinson disease. Can.
Fam. Physician 52, 862–868.
45
Gallart-Palau, X., Serra, A., Wong, A.S.W., Sandin, S., Lai, M.K.P., Chen, C.P., Kon, O.L., and Sze,
S.K. (2015). Extracellular vesicles are rapidly purified from human plasma by PRotein Organic
Solvent PRecipitation (PROSPR). Sci. Rep. 5, 14664.
Gallo, A., Jang, S.-I., Ong, H.L., Perez, P., Tandon, M., Ambudkar, I., Illei, G., and Alevizos, I.
(2016). Targeting the Ca2 + Sensor STIM1 by Exosomal Transfer of Ebv-miR-BART13-3p is
Associated with Sjögren’s Syndrome. EBioMedicine 10, 216–226.
Gardiner, C., Vizio, D.D., Sahoo, S., Théry, C., Witwer, K.W., Wauben, M., and Hill, A.F. (2016).
Techniques used for the isolation and characterization of extracellular vesicles: results of a
worldwide survey. J. Extracell. Vesicles 5, 32945.
Gierow, J.P., Lambert, R.W., and Mircheff, A.K. (1995). Fluid phase endocytosis by isolated
rabbit lacrimal gland acinar cells. Exp. Eye Res. 60, 511–525.
Haeckel, R., and Hänecke, P. (1993). The application of saliva, sweat and tear fluid for diagnostic
purposes. Ann. Biol. Clin. (Paris) 51, 903–910.
Hamm-Alvarez, S.F., Janga, S.R., Edman, M.C., Madrigal, S., Shah, M., Frousiakis, S.E.,
Renduchintala, K., Zhu, J., Bricel, S., Silka, K., et al. (2014). Tear Cathepsin S–A Candidate
Biomarker for Sjögren’s Syndrome. Arthritis Rheumatol. Hoboken NJ 66, 1872–1881.
Hamm-Alvarez, S.F., Lew, M., Feigenbaum, D., Janga, S.R., Shah, M., Freire, D., Cooperman, B.,
Ghanshani, R., Mack, W., Okamoto, C. (2018). Tear Proteins as Possible Biomarkers for
Parkinson’s Disease. ARVO E-Abstract 4909.
Helwa, I., Cai, J., Drewry, M.D., Zimmerman, A., Dinkins, M.B., Khaled, M.L., Seremwe, M.,
Dismuke, W.M., Bieberich, E., Stamer, W.D., et al. (2017). A Comparative Study of Serum
46
Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents. PLOS
ONE 12, e0170628.
Hessvik, N.P., and Llorente, A. (2018). Current knowledge on exosome biogenesis and release.
Cell. Mol. Life Sci. CMLS 75, 193–208.
Hou, Y., Ernst, S.A., Stuenkel, E.L., Lentz, S.I., and Williams, J.A. (2015). Rab27A Is Present in
Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion. PLoS ONE 10.
Huang, C.-C., Lee, C.-C., Lin, H.-H., and Chang, J.-Y. (2016). Cathepsin S attenuates endosomal
EGFR signalling: A mechanical rationale for the combination of cathepsin S and EGFR tyrosine
kinase inhibitors. Sci. Rep. 6, 29256.
Izumi, T., Gomi, H., Kasai, K., Mizutani, S., and Torii, S. (2003). The Roles of Rab27 and Its
Effectors in the Regulated Secretory Pathways. Cell Struct. Funct. 28, 465–474.
Jellinger, K.A. Neuropathology of sporadic Parkinson’s disease: Evaluation and changes of
concepts. Mov. Disord. 27, 8–30.
Kang, W., Chen, W., Yang, Q., Zhang, L., Zhang, L., Wang, X., Dong, F., Zhao, Y., Chen, S., Quinn,
T.J., et al. (2016). Salivary total α-synuclein, oligomeric α-synuclein and SNCA variants in
Parkinson’s disease patients. Sci. Rep. 6, 28143.
Kögel, T., Rudolf, R., Hodneland, E., Copier, J., Regazzi, R., Tooze, S.A., and Gerdes, H.-H. (2013).
Rab3D Is Critical for Secretory Granule Maturation in PC12 Cells. PLOS ONE 8, e57321.
Lobb, R.J., Becker, M., Wen Wen, S., Wong, C.S.F., Wiegmans, A.P., Leimgruber, A., and Möller,
A. (2015). Optimized exosome isolation protocol for cell culture supernatant and human
plasma. J. Extracell. Vesicles 4.
47
Longhena, F., Faustini, G., Missale, C., Pizzi, M., Spano, P., and Bellucci, A. (2017). The
Contribution of α-Synuclein Spreading to Parkinson’s Disease Synaptopathy. Neural Plast. 2017.
Massano, J., and Bhatia, K.P. (2012). Clinical Approach to Parkinson’s Disease: Features,
Diagnosis, and Principles of Management. Cold Spring Harb. Perspect. Med. 2.
Mateescu, B., Kowal, E.J.K., van Balkom, B.W.M., Bartel, S., Bhattacharyya, S.N., Buzás, E.I.,
Buck, A.H., de Candia, P., Chow, F.W.N., Das, S., et al. (2017). Obstacles and opportunities in the
functional analysis of extracellular vesicle RNA – an ISEV position paper. J. Extracell. Vesicles 6.
Mavragani, C.P., and Moutsopoulos, H.M. (2014). Sjögren syndrome. CMAJ Can. Med. Assoc. J.
186, E579–E586.
Ménasché, G., Feldmann, J., Houdusse, A., Desaymard, C., Fischer, A., Goud, B., and Basile, G.
de S. (2003). Biochemical and functional characterization of Rab27a mutations occurring in
Griscelli syndrome patients. Blood 101, 2736–2742.
Meng, Z., Edman, M.C., Hsueh, P.-Y., Chen, C.-Y., Klinngam, W., Tolmachova, T., Okamoto, C.T.,
and Hamm-Alvarez, S.F. (2016). Imbalanced Rab3D versus Rab27 increases cathepsin S
secretion from lacrimal acini in a mouse model of Sjögren’s Syndrome. Am. J. Physiol. - Cell
Physiol. 310, C942–C954.
Möbius, W., Ohno-Iwashita, Y., van Donselaar, E.G., Oorschot, V.M.J., Shimada, Y., Fujimoto, T.,
Heijnen, H.F.G., Geuze, H.J., and Slot, J.W. (2002). Immunoelectron microscopic localization of
cholesterol using biotinylated and non-cytolytic perfringolysin O. J. Histochem. Cytochem. Off.
J. Histochem. Soc. 50, 43–55.
Mulcahy, L.A., Pink, R.C., and Carter, D.R.F. (2014). Routes and mechanisms of extracellular
vesicle uptake. J. Extracell. Vesicles 3, 24641.
48
Nair, J.J., and Singh, T.P. (2017). Sjogren’s syndrome: Review of the aetiology, Pathophysiology
& Potential therapeutic interventions. J. Clin. Exp. Dent. 9, e584–e589.
Oertel, W.H. (2017). Recent advances in treating Parkinson’s disease. F1000Research 6.
Ostrowski, M., Carmo, N.B., Krumeich, S., Fanget, I., Raposo, G., Savina, A., Moita, C.F., Schauer,
K., Hume, A.N., Freitas, R.P., et al. (2010). Rab27a and Rab27b control different steps of the
exosome secretion pathway. Nat. Cell Biol. 12, 19–30.
Pastuzyn, E.D., Day, C.E., Kearns, R.B., Kyrke-Smith, M., Taibi, A.V., McCormick, J., Yoder, N.,
Belnap, D.M., Erlendsson, S., Morado, D.R., et al. (2018). The Neuronal Gene Arc Encodes a
Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer. Cell 172,
275-288.e18.
Raposo, G., and Stoorvogel, W. (2013). Extracellular vesicles: Exosomes, microvesicles, and
friends. J Cell Biol 200, 373–383.
Recasens, A., and Dehay, B. (2014). Alpha-synuclein spreading in Parkinson’s disease. Front.
Neuroanat. 8.
Riedel, D., Antonin, W., Fernandez-Chacon, R., Alvarez de Toledo, G., Jo, T., Geppert, M.,
Valentijn, J.A., Valentijn, K., Jamieson, J.D., Südhof, T.C., et al. (2002). Rab3D Is Not Required for
Exocrine Exocytosis but for Maintenance of Normally Sized Secretory Granules. Mol. Cell. Biol.
22, 6487–6497.
Rocha, E.M., De Miranda, B., and Sanders, L.H. (2018). Alpha-synuclein: Pathology,
mitochondrial dysfunction and neuroinflammation in Parkinson’s disease. Neurobiol. Dis. 109,
249–257.
49
Romano, G., and Kwong, L. (2017). miRNAs, Melanoma and Microenvironment: An Intricate
Network. Int. J. Mol. Sci. 18, 2354.
Shi, G.-P., Villadangos, J.A., Dranoff, G., Small, C., Gu, L., Haley, K.J., Riese, R., Ploegh, H.L., and
Chapman, H.A. (1999). Cathepsin S Required for Normal MHC Class II Peptide Loading and
Germinal Center Development. Immunity 10, 197–206.
Stuendl, A., Kunadt, M., Kruse, N., Bartels, C., Moebius, W., Danzer, K.M., Mollenhauer, B., and
Schneider, A. (2016). Induction of α-synuclein aggregate formation by CSF exosomes from
patients with Parkinson’s disease and dementia with Lewy bodies. Brain 139, 481–494.
Théry, C., Zitvogel, L., and Amigorena, S. (2002). Exosomes: composition, biogenesis and
function. Nat. Rev. Immunol. 2, 569–579.
Théry, C., Amigorena, S., Raposo, G., and Clayton, A. (2006). Isolation and Characterization of
Exosomes from Cell Culture Supernatants and Biological Fluids. In Current Protocols in Cell
Biology, J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds.
(Hoboken, NJ, USA: John Wiley & Sons, Inc.), p.
Tkach, M., and Théry, C. (2016). Communication by Extracellular Vesicles: Where We Are and
Where We Need to Go. Cell 164, 1226–1232.
Tolmachova, T., Åbrink, M., Futter, C.E., Authi, K.S., and Seabra, M.C. (2007). Rab27b regulates
number and secretion of platelet dense granules. Proc. Natl. Acad. Sci. U. S. A. 104, 5872–5877.
Verweij, F.J., Middeldorp, J.M., and Pegtel, D.M. (2012). Intracellular signaling controlled by the
endosomal-exosomal pathway. Commun. Integr. Biol. 5, 88–93.
50
Vestad, B., Llorente, A., Neurauter, A., Phuyal, S., Kierulf, B., Kierulf, P., Skotland, T., Sandvig, K.,
Haug, K.B.F., and Øvstebø, R. (2017). Size and concentration analyses of extracellular vesicles by
nanoparticle tracking analysis: a variation study. J. Extracell. Vesicles 6.
Villarroya-Beltri, C., Gutiérrez-Vázquez, C., Sánchez-Cabo, F., Pérez-Hernández, D., Vázquez, J.,
Martin-Cofreces, N., Martinez-Herrera, D.J., Pascual-Montano, A., Mittelbrunn, M., and
Sánchez-Madrid, F. (2013). Sumoylated hnRNPA2B1 controls the sorting of miRNAs into
exosomes through binding to specific motifs. Nat. Commun. 4, 2980.
van Weeren, L., de Graaff, A.M., Jamieson, J.D., Batenburg, J.J., and Valentijn, J.A. (2004).
Rab3D and Actin Reveal Distinct Lamellar Body Subpopulations in Alveolar Epithelial Type II
Cells. Am. J. Respir. Cell Mol. Biol. 30, 288–295.
Whiteside, T.L. Exosomes and tumor-mediated immune suppression. J. Clin. Invest. 126, 1216–
1223.
Willis, G.R., Kourembanas, S., and Mitsialis, S.A. (2017). Toward Exosome-Based Therapeutics:
Isolation, Heterogeneity, and Fit-for-Purpose Potency. Front. Cardiovasc. Med. 4.
Willms, E., Johansson, H.J., Mäger, I., Lee, Y., Blomberg, K.E.M., Sadik, M., Alaarg, A., Smith,
C.I.E., Lehtiö, J., Andaloussi, S.E., et al. (2016). Cells release subpopulations of exosomes with
distinct molecular and biological properties. Sci. Rep. 6, 22519.
Wu, M., Ouyang, Y., Wang, Z., Zhang, R., Huang, P.-H., Chen, C., Li, H., Li, P., Quinn, D., Dao, M.,
et al. (2017). Isolation of exosomes from whole blood by integrating acoustics and microfluidics.
Proc. Natl. Acad. Sci. 114, 10584–10589.
Wubbolts, R., Leckie, R.S., Veenhuizen, P.T.M., Schwarzmann, G., Möbius, W., Hoernschemeyer,
J., Slot, J.-W., Geuze, H.J., and Stoorvogel, W. (2003). Proteomic and Biochemical Analyses of
51
Human B Cell-derived Exosomes POTENTIAL IMPLICATIONS FOR THEIR FUNCTION AND
MULTIVESICULAR BODY FORMATION. J. Biol. Chem. 278, 10963–10972.
Yang, J., Liu, W., Lu, X., Fu, Y., Li, L., and Luo, Y. (2015). High expression of small GTPase Rab3D
promotes cancer progression and metastasis. Oncotarget 6, 11125–11138.
Ysselstein, D., Mishra, V., McCabe, G., and Rochet, J.-C. (2016). Effect of Stabilizing Alpha
Synuclein-Membrane Interactions on the Protein’s Aggregation and Neurotoxicity. FASEB J. 30,
518.7-518.7.
Zhu, L., Kalimuthu, S., Gangadaran, P., Oh, J.M., Lee, H.W., Baek, S.H., Jeong, S.Y., Lee, S.-W.,
Lee, J., and Ahn, B.-C. (2017). Exosomes Derived From Natural Killer Cells Exert Therapeutic
Effect in Melanoma. Theranostics 7, 2732–2745.
Abstract (if available)
Abstract
Exosomes are small, cell-derived vesicles that are thought to regulate a multitude of biological processes. Through transmission of their miRNA and protein content, exosomes are capable of transmitting phenotypic information from cell to cell under both normal and pathological conditions. Recently, the study of exosomes has expanded into nearly all fields of translational research. In this project, I developed a robust set of protocols capable of isolating exosomes from a variety of starting materials and volumes. Two commonly used methodologies, ultracentrifugation and size-exclusion chromatography, were optimized for the isolation of exosomes from mouse serum and rabbit lacrimal gland acinar cell culture medium. These protocols were adapted from the existing literature and modified to work with our samples. Characterization of the membrane fraction thought to be enriched in exosomes by Western blotting, nanoparticle tracking analysis, and dynamic light scattering demonstrates the success of these protocols in isolating and characterizing exosomes from samples of interest.
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Cooperman, Benjamin
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Optimization of exosome isolation protocols for mouse serum and rabbit primary lacrimal gland acinar cells
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School of Pharmacy
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Master of Science
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Molecular Pharmacology and Toxicology
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07/29/2018
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