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1 The world leader in serving science
Webinar :
Exosome isolation and monitoring:
From cell culture to clinically relevant research samples
Alexander Vlassov, PhD
Sr. Staff Scientist,
Molecular Biology
Ketil Winther Pedersen, PhD
Staff Scientist,
Cell Biology
Axl Neurauter, PhD
Sr. Manager, R&D,
Cell Biology
This webinar deck was designed to view together with verbal
presentation. So we recommend viewing with audio here:
http://bit.ly/1JjnOpw
2
Agenda
3. Direct isolation of exosomes from cell
culture media and urine
1. Exosome identification
2. Critical factors for exosome isolation
and analysis
3
Answer questionnaire at end of webinar
And get a free
smartphone
card-holder Improve grip and secure
for 1-3 cards
4
Introduction
NOMENCLATURE IS
BEING WORKED OUT
Heterogeneous group
of vesicles
Multivesicular bodies
(MVB)
Nucleus
Proteins
Nucleic acids
Membrane
proteins
Lysosome
Degradation
Fusion with the
plasma membrane
Release of
extracellular vesicles
Plasma
membrane
• Nucleic acid and protein containing
vesicles secreted by all cells
• Found in most body fluid
Where
• Eradication of obsolete molecules
• Cell-to-cell communication
• Dissemination of oncogenes from tumor
cells
Function
5
Lötvall et al. Journal of Extracellular Vesicles 2014, 3: 26913
• Report several proteins
• Host cell comparison
• Semi quantitative analysis
• Proteins with membrane-binding capacity
How can exosomes be identified ?
Lipid-bound extracellular proteins
Cytosolic proteins
Intracellular proteins
• Absence or under-representation of ER, Golgi,
mitoch or nucleus markers
Size distribution
• Nano particle tracking analysis (NTA)
6
Characterization of the size distribution
• Nanoparticle tracking analysis,
• Dynamic light scattering,
• Resistive pulse sensing
Combine with microscopic techniques
Do not distinguish between membrane
and non-membrane structures of
similar size
Co
n/m
L E
6
Methods
Challenge
Recommendation
7
Electron microscopy – stereological analysis
d
I
Grid size 500 nm
Q
Dynabeads™ magnetic bead
Grid overlay
Total # of intersections 458
Total # of exosomes 359
Formula: # exosomes / boundary = Q/((π/4)xlxd)
2 exosomes / µm Dynabeads™ magnetic beads profile
(1200 vs. 8000 theoretical exosomes / bead)
d
I
Q
8
• exosome loading at RT for 15 min
• block with 0.5% BSA 10 min
• prim Ab labelling for 30 min
• wash 5 x PBS 10 min total
• R&M 30 min
• wash 5 x PBS 10 min total
• protein A Au 10nm 15 min
• wash 5 x PBS 10 min total
• wash 5 x H20 10 min total
• embed in 0.3% UA in MC on ice
Labeling protocol
B-cell
lymphoma
exosomes
CD63
SW480
exosomes
CD81
How can I detect surface markers on exosomes?
9
20
24
28
32
36
40
003-1 003-1 003-1 003-1
miR16 miR24 miR107 miR574-3p
Ct
50ul Input 10ul Input
SW480 exosomes captured with Dynabeads™ magnetic beads
10
14
18
22
26
30
34
38
003-1 003-1 003-1
18S ACTB GAPDH
Ct
50ul Input 10ul Input
mRNA miRNA
Analysis of exosome RNA cargo after bead isolation
10
Size
RNA
qRT-PCR
Protein
WB
TEM
Flow Size distribution measurement
How can exosomes be identified?
Isolation
from
extracellular
fluid Immunolabeling
Ultrastructure
Oksvold et al Methods in Molecular Biology, vol. 1218, 2015
11
Jurkat exosomes / CD81-beads
CD9-PE CD81-PE
Exosome-Human CD81 Flow Detection Reagent
(from cell culture) SW480 exosomes / CD9-beads
Exosome-Human CD9 Flow Detection Reagent
(from cell culture)
CD9-PE CD81-PE
Analysis of exosomes by flow cytometry
12
• Easy detection of Dynabeads™ magnetic beads
• Defined exosome population
• Easy handling
• Verification by EM
𝑅𝑒𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 =0.61 x
n x sinθ
(light) = 200 nm
(e-) = 0.002 nm
Theoretical resolution
Why exosomes on magnetic beads for flow analysis?
TM TM
30 - 100 nm 100 nm - 1µm 1µm – 5 µm 8 – 12 µm
exosome microvesicle apoptotic body cell
13
Pre-
enrichment
Magnetic
Capture Staining Cell
culture
How can exosomes be captured for flow analysis?
• Ultracentrifugation: ― Differential
― Cushion
― Gradient
• Precipitation
• Filtration
• Size exclusion
chromatography
14
How can exosomes be analysed on flow cytometry?
−sheat
−sheat
Neg.
contol
staining
Note:
log scale
Dynabeads™
magnetic beads
CD9-PE
staining
15
How can we make sure flow cytometry is suitable?
Three exosome batches tested
with three different Dynabeads™ magnetic beads
Method
validation
0
20
40
60
80
100
120
140
Exosome batch 1 Exosome batch 2 Exosome batch 3
Sig
na
l / n
ois
e
Reference bead Control bead "Poor" bead
• Repeatability
• Intermediate precision
• Limit of detection
• Limit of quantification
• Linearity (dynamic range)
16
0
120
80
40
160
Concentration of capture beads
1x 3x 9x Beads
alone
Capture time
16-21 h
combined
0,9
54,4
25,8
12,0
0,0
10,0
20,0
30,0
40,0
50,0
60,0
Beads alone 1x 3x 9x
S/N
CD9
S ignal/ N
ois
e
54
26
1
12
10
20
30
40
50
60
117 119 118 118
101
5 h 16 h 19 h 21 h
140
0
60
100
20
120
80
40
Sig
nal/ N
ois
e
Exosome staining
25 L 50 L 75 L 100 L
0
120
80
40
S ignal/ N
ois
e
0 L
R2 = 0,937
R2 = 0,975 CD9
CD81
How can we maximize the signal for flow cytometry?
110
118
114 127
151 141
S ignal/ N
ois
e
20 L 30 L 40 L 50 L
Exosome input
17
How can exosomes be compared in flow cytometry?
Exosome – Human CD81 Flow Detection
(from cell culture)
Exosome - Human CD9 Flow Detection
(from cell culture)
18
Why should exosomes be captured directly?
monitoring the exosome release during cell culture
detailed analysis of exosomes (potential subpopulations)
compatible with many downstream applications
19
100 µL exosome
containing cell culture
media
Harvest
Pre-
enrichment
Staining
Wash to remove
contaminants and add
staining
Capture
Add 20 µL beads.
Incubate over night
2-8OC
How can exosomes be captured directly?
20
Direct capture of
exosomes from SW480 cell
culture sup. with
Dynabeads™ CD9
beads
20
38
68
R² = 0,9771
25 L 50 L 100 L
0
20
40
60
80
100
S / N
SW480 cell culture sup. input
6
11
18
R² = 0,9972
0
5
10
15
20
25
25 L 50 L 100 L
Direct capture of
exosomes from Jurkat cell
culture sup. with
Dynabeads™ CD81
beads S / N
Jurkat cell culture sup. input
Experimental
setup
How can the signal be increased?
100
75
50
25
PBS Exosomes
Titration of exosomes
Constant number of beads (20 L)
1 2 3
L
75 50
100
50 25
21
R2 = 0.9231 (log)
20
38
68 R² = 0,9771
25 L 50 L 100 L
0
20
40
60
80
100
175 L 350 L 700 L
81
93 97
S / N
SW480 cell culture supernatent input
Capture volume =
100 L
Capture volume =
cell culture input
Direct capture of
exosomes using
Dynabeads™ magnetic beads
Can exosomes be captured from larger volumes?
22
How do pre-enriched and direct captured exosomes compare?
Direct
(no pre-enrich.)
0
20
40
50
70
80
60
30
10
S /
N
68
36
67
Ultra-
centrifugation
Precipitation
Experimental
setup
Pre-enrich Conc. factor Volume pr. test Comment
No 0 100 µL
Ultracentrifugation 42x 2.4 µL Equal to 100 µL cell culture
Precipitation 16x 6.25 µL Equal to 100 µL cell culture
Dynabeads™ magnetic beads capture CD9 positive exosomes
Exosome–Human CD9
Flow Detection Reagent
23
3,6
5,3 5,2
7,9
7,3
11,2
5,8
8,9
100 µl 100 µl 100 µl
100 L
0
2
6
12
8
S /
N
4
10
200 L
400 L
200 L
Pre-enriched
(precipitation)
100 L
200 L
400 L
Donor A Donor B
200 L
Pre-enriched
(precipitation) Direct capture Direct capture
Can exosomes be isolated directly from urine Dynabeads™ magnetic beads?
Direct vs. pre-enrich.
Direct vs. pre-enrich.
Total Exosome Isolation Reagent (from urine)
24
Which method should I choose?
Perform direct exosome capture when:
• monitoring exosome production during cell culture
• analysis of exosomes with limited-volume
• simplified workflow (automation)
• multiple samples
• application compatibility
Perform pre-enrichment when:
• low exosome concentration
• increased exosome yield
• storage
Perform pre-enrichment followed by capture when:
• monitoring the pre-enriched exosomes
• analysis of exosomes which requires a solid surface
High exosome
concentration
Low exosome
concentration
capture & flow analysis
Pre-enrichenment
capture & flow analysis
25
Analysis of exosomes by western blotting (WB)
Exosome-Human
CD81 Isolation
Reagent
(from cell culture)
Exosome-Human
CD9 Isolation
Reagent
(from cell culture)
26
Maximize the surface area for
exosome docking
Bead concentration
optimized for western blotting
Pre-enriched
exosomes
What is critical for exosome isolation for western blotting?
Dynabeads™
magnetic beads
TM TM
27
Lipid-bound extracellular proteins Cytosolic proteins
• Tetraspanins (CD9, CD63, CD81)
• Cell adhesion molecules
• Integrins
• Heterotrimeric G proteins
• Growth factor receptors
Presence of membrane Membrane or receptor
binding capacity
Lötvall et al. Journal of Extracellular Vesicles 2014, 3: 26913
• TSG101
• Annexins
• Rabs
• Signal transduction
• Scaffolding proteins
Intracellular proteins
Absent or
under-represented
• ER: HSP90B1, calnexin
• Golgi: GM130
• Mitochondria: Cyto c
• Nucleus: Histones
How should the presence of exosomes be confirmed?
28
Amount of beads
Exosome-Human CD9 Isolation
Reagent (from cell culture) Exosome-Human CD9
Flow Detection Reagent
(from cell culture)
Pre-
enrichment
Capture for
flow from sup. Staining Capture for
western blot
Sup. after
isolation
60
50
40
30
20
10
1x 5x 10x 15x 20x 25x
Fold
increase
CD81
CD9
S /
N
How can the right conditions for isolation be found?
29
Increased amount of Dynabeads™ CD9
beads
R² = 0,9021
0
1
2
3
4
5
6
7
1xCD9 5xCD9 10xCD9 25xCD91x 5x 10x 25x
CD9
CD63
How does bead concentration influence the result?
Relative amounts of CD9
Increased input of isolation beads increased signal
30
What do exosomes isolated with beads provide?
EpCAM
CD81
CD9
Lane 1 Isolated exosomes
Lane 2 Isolated exosomes
Lane 3 Isolated exosomes
Lane 4 Prior to isolation
Critical for WB:
detection antibody Exosome-Human CD9 Isolation Reagent
(from cell culture)
Exosome-Human CD81 Isolation Reagent
(from cell culture)
Exosome-Human EpCAM Isolation
Reagent (from cell culture)
Exosome-anti- CD9
(for western detection)
Exosome-anti- CD63
(for western detection)
Exosome-anti- CD81
(for western detection)
31
Some exosomal
markers:
• CD63 – 53 kDa (smear)
• CD9 – 28 kDa
• CD81 – 26 kDa
• HSP70 – 53-70 kDa
• TSG101 – 44 kDa
TrueBlot™
Which method should be used for detection?
x-ray film / scanner
WB immunological
detection
Chemiluminescence
detection
HRP AP
fg-pg 10 pg
Colorimetric
detection
© 2015 Thermo Fisher Scientific Inc. All rights reserved. TrueBlot is a trademark of eBioscience Corporation.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
32
WB detection
Isolation using Dynabeads™ CD9 beads
Dynabeads™ CD81 beads
How do isolated exosomes compare?
Ultracentrifugation vs. Precipitation
33
Exosome isolation with Dynabeads™ CD9 magnetic beads
How do pre-enriched and directly captured exosomes compare?
Good correlations between flow and WB
34
Can exosomes be captured directly from urine?
Exosome-Human CD9 Isolation Reagent
(from cell culture)
Exosomes isolated with with Dynabeads™ CD9
magnetic beads
1,2
1,0
0,8
0,6
0,4
0,2
Direct UC Precip.
Urine Cell culture
Relative amount of CD9
Direct
28 KDa CD9
Direct UC Prec
Urine Cell culture
Direct
35
Conclusion
© 2015 Thermo Fisher Scientific Inc. All rights reserved. All other trademarks are the property of Thermo Fisher
Scientific and its subsidiaries.
Perform direct exosome
capture when:
• monitoring exosome production
• analysis of exosomes with limited-volume
• simplified workflow (automation)
• multiple samples
• application compatibility
Perform pre-enrichment when:
• low exosome concentration
• increased exosome yield
• storage is required
Perform pre-enrichment followed by capture when:
• monitoring the pre-enriched exosomes
• analysis of exosomes which requires a solid surface
Pre-enrichment Capture
Direct capture
For Research Use Only. Not for use in diagnostic procedures.
Hang on for Q&A, announcements, product overview
36
Morten P. Oksvold, Anette Kullmann, Lise Forfang, Bente Kierulf, Mu Li,
Andreas Brech, Alexander V. Vlassov, Erlend B. Smeland, Axl
Neurauter, Ketil W. Pedersen
Publications
“Expression of B-cell surface antigens in
subpopulations of exosomes released from
B-cell lymphoma cells”
M. Oksvold, A. Neurauter and K. W. Pedersen
Book chapter accepted by Methods in Molecular Biology
“Magnetic bead-based isolation of exosomes”
37
Your questions please!
Joint the conversation and post questions on
www.ExosomesTalk.com
Q&A
38
Missed ISEV2015? Watch the key sessions
Herper Collins Rothman Sachs Breakefield Lötvall
Sr. Editor Director Nobel CSO Harvard Pres.
Forbes NIH 2013 Thermo- ISEV
Med/Sci Fisher
Francis Collins Director of NIH
James Rothman 2013 Nobel® laureate, Fergus F. Wallace Professor of Biomedical
Sciences at Yale University
Xandra Breakefield Professor, Harvard Medical School
And more!
including panel
discussion
Exclusively at www.lifetechnologies.com/exosomevideo
39
Available products p.1 of 2
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Available products p.2 of 2
More info at: lifetechnologies.com/exosomes