MassivEV EV Purification Column PS Data

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 mouse ^2-4): 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 (Catalog ID 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.

PS Affinity Method Performance Data

Capture

Elution

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

Protocol

For guidance on connecting the tubing, please refer the support document titled: Example of a purification system using the MassivEV EV Purification Column PS.

Data

Performance Data

EV isolation and purification from MSCs cell culture supernatant

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), ELISA and BCA method.

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

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)

Result: MassivEV achieved a higher rate of recovery of EVs compared to conventional methods.

(3) Number of particles per protein

The ratio of protein:particle is a useful indicator of EV purity⁵⁾. The total protein content in the collected EV solution was measured using the BCA method, and the particle count was determined by NTA. The number of particles per 1 μg of protein was then compared.

Result: Using MassivEV resulted in a higher number of particles per 1 μg of protein compared to traditional methods, indicating that EVs with higher purity can be recovered.