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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

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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

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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

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Q&A

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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!

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