A novel affinity-based method for the isolation

of highly purified extracellular vesicles

Wataru Nakai1, Takeshi Yoshida1,2, Diego Diez3, Yuji Miyatake1,2, Takahiro Nishibu4, Ryo Ukekawa4, Naoko Imawaka4, Ken Naruse4, Yoshifusa Sadamura4 & Rikinari Hanayama1,2,5

1 Laboratory of Immune Network, WPI Immunology Frontier Research Center (IFReC), Osaka University, Japan 2 Department of Immunology, Kanazawa University Graduate School of Medical Sciences, Japan 3 Quantitative Immunology Research Unit, WPI Immunology Frontier Research Center (IFReC), Osaka University, Japan 4 Life Science Research Laboratories, Wako Pure Chemical Industries Ltd, Japan 5 PRESTO, Japan Science and Technology Agency (JST), Japan

Cell Culture

Supertanant

Serum Urine

EDTA

Tim4 binds EV surface phosphatidylserine (PS)

(Ca2+ dependent)

Native Elution of EV with Ca2+ chelators

(such as EDTA)

Biotinylated Tim4

Streptavidin

Magnetic Bead

Small EV (sEV)

MagCaptureTM Exosome Isolation Kit PS

Ca2+

Method

Tim4-affinity method (MagCaptureTM Exosome Isolation Kit PS)

Ultracentrifu-gation

Polymeric precipitation

Antibody-based affinity purification

EV’s Purity ■■■ ■■ ■ ■■■

State of vesicles

Intact Intact Intact Not Intact

Operability Easy and Stable Easy Easy and Fast Easy and Stable

Recovery amount

■■■ ■■ ■■■■ ■■

Code No. Product Name Package Size

299-77603 MagCapture™ Exosome Isolation Kit PS 2 tests

293-77601 MagCapture™ Exosome Isolation Kit PS 10 tests

MagCpatureTM Exosome Isolation Kit PS

Sample Type: cell culture supernatant,

serum, plasma, urine, etc. ◆ MagCpatureTM Exosome Isolation Kit PS

can purify any EVs which expose phosphatidylserine on the outer surface of their lipid bilayer. It has been confirmed that human, mouse, and bovine EVs can be purified by this isolation kit.

Comparison of recovery yield and purity of sEVs Particle analysis of sEVs purifeid by the Tim4-affinity methods sEVs from K562 culture sup.

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Lamp-1

Flotillin-2

CD63

Silver stain

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CD9

CD63

Silver stain

sEVs from human serum

CD9

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sEVs from human urine

Silver stain

sEVs from K562 culture supernatant, human serum, or human urine were purified by the Tim4-affinity method or conventional methods (Ultracentrifugation, Polymeric precipitation). Purified sEVs were analyzed by Silver stain or Western blotting.

Tim4-affinity is an ideal method to isolate sEVs containing exosome with much higher purity than that by using other conventional methods. Tim4-affinity method is suitable for isolating sEVs from biofluids including serum and urine.

(A)

(A)

(A)

(B) (A) Same volume of sEVs fraction were loaded (comparison of sEVs recovery)

(B) Equal amount of protein were loaded (comparison of sEVs purity)

W

The Uptake of sEV

W

Microarray analysis of miRNA

Linearity-of-Dilution Assessments Western blotting vs Tim4-based ELISA

Ultracentrifugation

200 nm

Size Distribution Mean 136±3.5 nm

Polymeric Precipitation

200 nm

Size Distribution Mean 183±4.4 nm

Tim4-affinity

200 nm

Size Distribution Mean 106±4.1 nm

sEVs from K562 cells were examined by transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) using NanoSight

The appearance of sEVs isolated by the Tim4-affinity method

matched the typical saucer-like shape as previous reported*.

The mean size of sEVs purified by the conventional methods was

larger than that by the Tim4-affinity method due to increased

populations of aggregated or fused sEVs larger than 200nm.

Tim4-affinity method could isolate sEVs with higher quality than

that by using conventional methods.

*Raposp, G. et al. (1996)

Hoechst33342 PKH67

Phase

Tim4-affinity (5μg, 2.8x1010 particles sEVs)

Ultracentrifugation (5μg sEVs)

Ultracentrifugation (2.8x1010 particles sEVs)

sEVs from K562 cells were labeled by the fluorescence dye and the uptake of these sEVs by HeLa cells was examined.

sEVs purified by the Tim4-affinity method were more efficiently

incorporated into the HeLa cells than that by the ultracentrifugation.

R² = 0.7582

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50000

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R² = 0.6925

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50000

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Tim4-affinity

R² = 0.821

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50000

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UC

Tim4-affinity W

Conclusion

miRNA microarray analysis of sEVs from COLO201 cells was performed

by the 3D-Gene (TORAY) Tim4-affinity method

・ Tim4-affinity method can isolate high purity and

quality extracellular vesicles from cell culture

supernatant and biofluid.

・ Tim4-affinity method can purify intact extracellular

vesicles.

・ High correlation of microRNA profiles between

Tim4-affinity method and ultracentrifugation was

revealed.

・sEVs purified by the Tim4-affinity method were

efficiently taken up by the recipient cells. miRNA microarray analysis of

sEVs from COLO201 cells

revealed high correlation of

miRNA profiles between Tim4-

affinity method and

ultracentrifugation method.

2

1 2

993

284

2

0 Tim4-affinity

Polymer UC

1. sEV binds to the Tim4 immobilized on the 96-well plate

2. Detect the sEV with sEV surface antigen antibody

and POD-labeled Anti-Mouse Antibody

sEV Surface Antigen Antibody

POD-labeled Anti-mouse IgG Antibody

Abs. 450 nm

Tim4-immobilized 96-well plate

sEV

Substrate

PS CaptureTM Exosome ELISA Kit

Code No. Product Name Package Size

297-79201 PS CaptureTM Exosome ELISA Kit (Anti-mouse IgG POD) 96 Reactions

sEV standard : purified sEVs from K562 or COLO201 Culture supernatnat (sEVs were purified by the MagCaptureTM Exosome Isolation Kit PS)

Sample : K562 or COLO201 cell culture supernatant.

y = 1351.7x + 0.113 R² = 0.9996

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0 0.002 0.004 0.006 0.008 0.01 0.012

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Dilution factor (×)

Cell culture sup. of COLO201 Cell culture supernatant of COLO201

CM volume (μL)

Dilution Assay value

Expected value % of

expected Ratio Factor (×) ng/mL ng/mL

0.0625 1：1600 0.000625 0.89 0.91 98.4

0.125 1：800 0.00125 1.82 1.72 105.6

0.25 1：400 0.0025 3.44 3.52 97.8

0.5 1：200 0.005 7.04 6.78 103.9

1 1：100 0.01 13.6 - -

Cell culture supernatant of K562

CM volume (μL)

Dilution Assay value

Expected value % of

expected Ratio Factor (×) ng/mL ng/mL

0.125 1：800 0.00125 3.02 3.07 98.2

0.25 1：400 0.0025 6.15 6.19 99.3

0.5 1：200 0.005 12.4 12.7 97.5

1 1：100 0.01 25.4 26.1 97.2

2 1：50 0.02 52.2 - -

The linearity-of-dilution was good over the wide range of dilution of cell culture supernatant. Tim4-based ELISA Kit could detect sEVs included in the 0.1μL Culture Supernatant.

y = 2627.4x - 0.5317 R² = 0.9998

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Dilution factor (×)

Cell culture sup. of K562

Primary Antibody : Anti-human CD63 Antibody

Samples : Purified sEVs from COLO201 cell culture supernatant (sEVs were purified by the MagCaptureTM Exosome Isolation Kit PS)

Limit of detection

(Blank＋3.3SD)

Limit of quantification (Blank＋10SD)

0.11 ng/mL (Total 11 pg)

0.34 ng/mL (Total 34 pg)

The sensitivity of the Tim4-based ELISA was 50 to 1,000 times higher than that of western blotting.

Limit of Detection (COLO201) was 11pg

Anti-CD63 antibody (company A)

←CD63

Limit of Detection was about 75 ng or 0.5ng

Western blotting

200n

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150n

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ng

75n

g

50n

g

Reducing

4n

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1n

g

0.5

ng

0.2

5ng

0.12

5ng

Non-reducing

Anti-CD63 antibody (Wako ; Code No. 012-27063)

0.0000

0.5000

1.0000

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Ab

s.4

50n

m

Purifed exosome (ng/mL)

Standard curve of COLO201

Tim4-based ELISA

(kDa)

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(kDa)

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(kDa)

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Signal Intensity

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Lipid staining by PKH67

Negative Control

Tim4-affinity (5μg, 2.8x1010 particles) Ultracentrifugation (5μg, 3.4x1010 particles)

Ultracentrifugation (4.1μg, 2.8x1010 particles)

Signal Intensity

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RNA staining by SYTO RNASelect

Negative Control

Tim4-affinity (5μg, 1.8x1010 particles)

Ultracentrifugation (5μg, 1.6x1010 particles)

Ultracentrifugation (5.6μg, 1.8x1010 particles)

Direct staining of the cells with fluorescence dye

Ca2+, EDTA

Ca2+