EV-Save (TM) Extracellular Vesicle Blocking Reagent for TFF

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.

Features

• Suppresses EV damage and adsorption to the filtration membrane during EV purification, concentration, and buffer exchange using TFF
• Simple protocol: add directly to the sample and equilibration buffer
• Prevents EV loss due to adsorption to tubes and protects against freeze-thaw damage, enabling direct storage of EV solutions after TFF. (Note: This effect is achieved only when this product is added during buffer exchange.)

Sample (Recommended)

• Purified EV
• Cell culture supernatant
  Note: The anti-adsorption effect may be reduced in serum, plasma an samples containing high levels of interfering substances.

TFF modules (Recommended)

MicroKros mPES 100 kDa (Repligen, Product Number: C02-E100-05-N)

Compatibility has also been confirmed with the following TFF modules:

• Pellicon XL UF Mod Biomax 100 kD 0.005m2 (Millipore)
• Single-Use Discover 12” 100kD 0.5 mm (Sartorius)

Please note that the following TFF modules are not compatible with this product:

• Minimate TFF Capsules with Omega Membrane - 100K (Cytiva)
• TFF-Easy - tangential flow filtration EV concentrator (HansaBio)

Performance Data

Improved Recovery during Buffer Exchange of Purified EVs

EVs were purified from the cell culture supernatants of bone marrow-derived mesenchymal stem cells (BMMSC) and adipose-derived mesenchymal stem cells (ADMSC) using the MassivEV™ EV Purification Column PS (Product Number: 131-19491). 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) (Product Number: 298-80601), 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

• Input
• Without EV-Save™
• With EV-Save™

[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 of BMMSC 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 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.

Anti-adsorptive and Cryoprotective Effects on Purified EVs

(1) Anti-adsorptive Effects

Either the conventional EV-Save™ Extracellular Vesicle Blocking Reagent (Code No. 058-09261) or the EV-Save™ Extracellular Vesicle Blocking Reagent for TFF (this product) was added at a 1:100 dilution to EV solutions purified from cell culture supernatants of BMMSC. The solutions were transferred to new microtubes and stored for 48 hours. EV quantities were evaluated using ELISA with anti-CD63 and anti-CD81 antibodies. For the figure, the value immediately after tube storage is set to 100.

Cryoprotective Effects

EVs purified from BMMSC cell culture supernatants using TFF were mixed with this product at a 1:100 dilution, then subjected to three freeze-thaw cycles (-20 °C to room temperature). EV levels before and after freeze-thaw were assessed using ELISA with anti-CD63 and anti-CD81 antibodies. For the figure, the value before freeze-thaw is set to 100.

[Result]
These results demonstrate that this product provides both anti-adsorptive and cryoprotective effects, making it ideal for use as an EV storage solution.