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For the isolation and purification of extracellular vesicles/exosomes from large volumes of cell culture

MassivEV™ EV Purification Column PS / MassivEV™ Purification Buffer Set

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Extracellular vesicles (EVs), such as exosomes, are small vesicles consisting of a lipid bilayer and are released by cells. EVs encapsulate nucleic acids, proteins, and lipids, serving as mediators of intercellular communication in living organisms. EVs are the subject of active research in various fields including immunology, cancer, neurology, and metabolism.

EVs have diverse applications, and are expected to be used as therapeutics, biomarkers, and drug delivery systems (DDS). In particular, EVs secreted by mesenchymal stem cells (MSCs) have attracted attention not only in the medical field but also in the cosmetics and food industries because of their anti-inflammatory, anti-fibrotic, and tissue repair effects.1)

The practical application of EVs requires technology capable of efficiently isolating and purifying high-purity EVs in large quantities. In collaboration with Prof. Hanayama of the Department of Immunology at the Graduate School of Medical Sciences, Kanazawa University, Fujifilm Wako has developed the MassivEV™ EV Purification Column PS, which utilizes our proprietary EV isolation and purification technology known as the PS affinity method.2) Used in conjunction with the specialized MassivEV™ Purification Buffer Set, this system enables convenient isolation and purification of EVs, including exosomes, from liter-scale cell culture supernatants.

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    MassivEV™ EV Purification Column PS

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    MassivEV™ Purification Buffer Set

Product Overview

The MassivEV™ EV Purification Column PS and MassivEV™ Purification Buffer Set have been specifically designed for the isolation and purification of extracellular vesicles (EVs), including exosomes, from large volumes of cell culture supernatants. This is achieved by utilizing the PS affinity method, which employs the Tim4 protein. Tim4 binds to phosphatidylserine (PS) on the surface of EV membranes in a calcium-dependent manner, making the purification of high-purity EVs a straightforward exercise.

Features

  • High-purity EVs can be efficiently isolated and purified from large volumes of cell culture supernatants (from 10 mL to several liters)
  • Eliminates the need for expensive equipment like tangential flow filtration (TFF) systems.* Only a peristaltic pump is required.

Table 1 Comparison of isolation and purification of EVs from 200 mL of MSC cell culture supernatant (evaluated by Fujifilm Wako)

MassivEV™ TFF + anion-exchange chromatography TFF + size-exclusion chromatography
EVs that can be purified PS-positive EVs Varies depending on fractions Varies depending on fractions
Purity High Low Low
Number of steps for purification 1 step
- PS affinity method
2 steps
- TFF system
- Anion-exchange chromatography
2 steps
- TFF system
- Size-exclusion chromatography
Yield of EV particles (reference value) 1.7x1011 particles 1.1×1011 particles 0.7×1011 particles
Time required for purification 8 hr 10 hr 10 hr
[Reference] Suggested dose of EVs for one mouse3-5): 1.0x109 particles/mouse

Sample

Cell culture supernatant (e.g., MSC): From 10 mL to several liters

Note: Please use the MagCapture™ Exosome Isolation Kit PS Ver. 2 (Product No. 290-84103) when isolating EVs from 10 mL or less of cell culture supernatant.

Processing Capacity

1 mL (Product No. 131-19491) 5 mL (Product No. 137-19493)
Sample volume*1 200 mL 1 L
Dynamic binding capacity*2 5×1011 particles/mL resin 2.5×1012 particles/5 mL resin
*1: The volume mentioned is an approximate amount of MSC culture supernatant that can be processed in a single purification. The sample volume required may vary depending on the concentration of EV particles present in the cell culture supernatant. Note that the same column can be reused multiple times for processing the same sample. Fujifilm Wako has confirmed that the column can be used repeatedly up to five times, comprising one initial use and four subsequent repeat uses.
*2: This information is based on studies using EVs derived from MSCs. Results may vary depending on the cell type and other conditions.

Principle

The PS affinity method, Fujifilm Wako’s proprietary EV isolation technique, utilizes the Tim4 protein, which specifically binds to phosphatidylserine (PS) present on the surface of EVs. The high specificity of the PS-Tim4 interaction, coupled with the gentle elution by a chelating agent, enables the isolation of high-purity EVs in their intact state.

  • Capture

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  • Elution

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Figure 1 Principle of isolation and purification of EVs using the MassivEV™ EV Purification Column PS (PS affinity method)

Protocol

Cell Culture
Step
01
Cell Culture
Culture the desired cells, facilitate cell proliferation and EV production, and collect the cell culture supernatant.
Sample preparation
(>2 hrs)
Step
02
Sample Preparation
Add EV Binding Enhancer to the cell culture supernatant.
Step
03
Pretreatment of Cell Culture Supernatant
Remove impurities by centrifugation or filtration.
Step
04
Degassing
Heat the cell culture supernatant so that it exceeds room temperature (25-28°C) and filter it through a filter.
Processing with a column
(>7 hrs)
Step
05
First Washing
Bring the column to room temperature and pass the Washing Buffer through the column.
Step
06
Sample Reaction
Pass the cell culture supernatant through the column, allowing the PS on EVs to react with the Tim4 protein on the resin.
Step
07
Second Washing
Pass EV Binding Enhancer/Washing Buffer through the column to wash the resin.
Step
08
Elution
Replace 50% of the EV Binding Enhancer/Washing Buffer in the column with EV Elution/EV-Stabilizer Buffer. Then, place an EV collection tube below the column and allow the EV Elution/EV-Stabilizer Buffer to flow through it to collect the EVs.
Step
09
Column Storage
After flushing the column with Washing Buffer, circulate 20% ethanol/Storage Buffer through the column. Secure the top of the column with Parafilm and store in a refrigerator at 2-10°C.
Buffer exchange
(30 min to 1 hr)
Step
10
Buffer Exchange
Exchange the buffer using either gel filtration or size-exclusion filtration techniques.
ManualFor a more detailed protocol, please refer to the instruction manual.

Data

Performance Data

Comparison with conventional methods

Bone marrow-derived MSCs were cultured in the proliferation medium (MSCulture™/10% FBS) and then in the EV production medium (EV-Up™). The cell culture supernatant was collected and filtered through a 0.22 μm filter. EVs were isolated and purified from 200 mL of the filtered cell culture supernatant using the four methods described below. The solutions containing the purified EVs were subsequently analyzed using Nanoparticle Tracking Analysis (NTA) and ELISA.

[Isolation and purification method]

MassivEV™
MassivEV™ EV Purification Column PS / MassivEV™ Purification Buffer Set (This product, PS affinity method)
TFF
Only Tangential flow filtration (500 kDa)
TFF+AEX
Tangential flow filtration (500 kDa)+Anion-exchange chromatography
TFF+SEC
Tangential flow filtration (500 kDa)+Size-exclusion chromatography

(1) Particle analysis by NTA

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[Result]
MassivEV™ yielded a greater number of particles compared to TFF+AEX or TFF+SEC, but fewer particles were obtained compared to TFF alone.

(2) Comparison of EV recovery as measured by CD63 ELISA and CD81 ELISA (n=3)

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[Result]
MassivEV™ achieved a higher rate of recovery of EVs compared to conventional methods.

About commercial or for-profit purposes

Please use this product for research purposes only. If you wish to use it for commercial or for-profit purposes, please contact Fujifilm Wako at wkuslabchem@fujifilm.com.

References

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Please use this product with MassivEV™ Purification Buffer Set.

Please use this product with MassivEV™ EV Purification Column PS.

Related Product List

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EV production medium for MSC

Please use the basic medium in conjunction with the supplement.

EV/Exosome Isolation Kit

Suitable for EV isolation and purification from body fluid samples and cell culture supernatants up to 10 mL.

For research use or further manufacturing use only. Not for use in diagnostic procedures.

Product content may differ from the actual image due to minor specification changes etc.

If the revision of product standards and packaging standards has been made, there is a case where the actual product specifications and images are different.

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