EV-Save™ Extracellular Vesicle Blocking Reagent

EV-Save™ Extracellular Vesicle Blocking Reagent is a blocking reagent that suppresses the adsorption of extracellular vesicles (EVs), such as exosomes, to microtubes, pipette tips, and TFF (tandem flow filtration) filter membranes. Simply adding it to samples or purified EV solutions will suppress adsorption. It also acts as a cryoprotectant, minimizing damage to EVs during freeze-thaw cycles. Fujifilm Wako has a lineup of EV-Save™ products specialized for each application.

Product Lineup

Suppress EV adsorption
to tubes, pipette tips, etc.
+
Protect EVs
from freeze-thaw damage

EV-Save™ Extracellular Vesicle
Blocking Reagent

Suppress EV adsorption
and administered to animals
+
Protect EVs
from freeze-thaw damage

EV-Save™ Extracellular Vesicle
Blocking Reagent for in vivo

suppress EV adsorption
on TFF filtration membranes
+
Protect EVs
from freeze-thaw damage

EV-Save™ Extracellular Vesicle
Blocking Reagent for TFF

EV-Save™ Extracellular Vesicle Blocking Reagent is a polymeric reagent that prevents EV from adsorbing to tubes, pipette tips, and other labware. It prevents loss of EVs due to adsorption during processing and storage, thereby increasing the yield. Addition of this product is highly recommended prior to ultrafiltration, purification, and storage.

Data

Anti-adsorption Effect

EV-Save™ was added to COLO201 cell-derived EVs purified by the PS affinity method and allowed to stand for 3 minutes. The sample was then transferred to another tube, and the transfer was repeated. The amounts of EV (CD63 signal) in the sample tubes were measured by PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD).

[Result]
CD63 signal decreased with transfer. The addition of EV-Save™ almost completely prevented the decrease in signal caused by the transfer.

Cryoprotection Effect

EV-Save™ was added to COLO201 cell-derived EVs purified by the PS affinity method and freeze-thawed. The amounts of EV (CD63 signal) in the sample tubes were measured by PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD), and their structure was analyzed by transmission electron microscopy (TEM).

(1) ELISA

(2) Transmission electron microscopy

[Result]
The CD63 signal decreased with freeze-thawing. The addition of EV-Save™ prevented the decrease in signal caused by freeze-thawing. Observation by electron microscopy confirmed that the reduction in particle numbers was suppressed.

EV-Save™ Extracellular Vesicle Blocking Reagent for in vivo applications contains only components that have been used as pharmaceutical additives. This EV anti-adsorption and cryoprotectant can be used for administration of EVs to laboratory animals.

Data

Anti-adsorption Effect

EV-Save™ and EV-Save™ for in vivo applications were added to COLO201 cell-derived EVs purified by the PS affinity method and stored at 4°C for 16 hours. The amounts of EV (CD9 signal) in the sample tubes were measured by PS Capture™ Exosome ELISA Kit (Streptavidin HRP).

[Result]
CD9 signal decreased without the addition of EV-Save™. The addition of EV-Save™ or EV-Save™ for in vivo applications prevented the decrease in signal.

Cryoprotection Effect

EV-Save™ or EV-Save™ for in vivo applications was added to COLO201 cell-derived EVs purified by the PS affinity method and freeze-thawed once or three times. The amounts of EV (CD9 signal) in the sample tubes were measured by PS Capture™ Exosome ELISA Kit (Streptavidin HRP).

[Result]
In the absence of EV-Save™, freeze-thawing reduced the CD9 signal. This signal reduction was prevented by the addition of EV-Save™ or EV-Save™ for in vivo applications.

Tangential flow filtration (TFF) is widely used as an effective method for concentrating extracellular vesicles (EVs) and performing buffer exchange. By flowing the sample parallel to the filtration membrane, TFF minimizes sample loss by preventing accumulation on the membrane surface. However, for samples such as EVs, which are prone to nonspecific adsorption, recovery may be reduced due to adsorption to the membrane and physical stress during filtration.
EV-Save™ Extracellular Vesicle Blocking Reagent for TFF is a specialized reagent developed to improve EV recovery by mitigating physical stress and suppressing membrane adsorption during TFF processing. Use of this product enables efficient and consistent recovery of EVs.

Data

Improved Recovery during Buffer Exchange of Purified EVs

EVs were purified from the culture supernatants of bone marrow-derived mesenchymal stem cells (BMMSC) and adipose-derived mesenchymal stem cells (ADMSC) using the MassivEV™ EV Purification Column PS. Buffer exchange was then performed using a Repligen TFF system with HBS buffer containing this product at a 1:100 dilution. EV recovery after buffer exchange was evaluated using the PS Capture™ Exosome ELISA Kit (Streptavidin-HRP), with anti-CD63 or anti-CD81 antibodies used as detection antibodies. Particle size distribution was assessed using nanoparticle tracking analysis (NTA).

(1) ELISA

[Result]
The addition of this product improved EV recovery during TFF-based buffer exchange.

(2) NTA

[Result]
No significant change in EV particle size distribution was observed by the addition of this product.

Improved Recovery during TFF-Based EV Purification from Cell Culture Supernatants

Cell culture supernatants were first concentrated 20-fold using TFF, followed by a 100-fold buffer exchange. This product was added at a 1:100 dilution to both the cell culture supernatant and the replacement buffer. EV recovery was measured using ELISA with anti-CD63 and anti-CD81 antibodies. Particle size distribution was analyzed using NTA.

(1) ELISA

[Result]
The addition of this product improved EV recovery during EV purification from cell culture supernatants.

(2) NTA

[Result]
No significant change in EV particle size distribution was observed by the addition of this product.

1. Welsh, J. A. et al.: J. Extracell. Vesicles, 13(2), e12404(2024).
   Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches
2. Evtushenko, E. G. et al.: PloS One, 15(12), e0243738(2020).
   Adsorption of extracellular vesicles onto the tube walls during storage in solution
3. 「決定版エクソソーム実験ガイド」, ed. by Yoshioka, Y. and Ochiya, T., Yodosha, Japan, (2004). (Japanese)