EV-Up(TM) EV Production Basal Medium for MSC, AF

EV-Up™ EV Production Basal Medium for MSC, AF is a culture medium optimized for the production of extracellular vesicle (EV) from mesenchymal stem cell (MSC). The medium is free of animal-derived components and serum. When used together with the separately available EV-Up™ MSC EV Production Supplement, AF, it enhances EV production, including exosomes.

Features

• Increased EV production compared with conventional serum-supplemented media
• Enhanced biological activity of EVs
• High survival rate of MSCs is maintained

Applicable cells

Applicable to a wide range of MSCs, regardless of their species of origin.

Examples of cells

• Bone marrow-derived MSC
• Adipose-derived MSC
• Umbilical cord-derived MSC

Protocol

[Note]
For extracellular vesicle isolation, we recommend using PS affinity-based methods, such as the MagCapture™ Exosome Isolation Kit PS Ver.2 or the MassivEV™ EV Purification Column PS.

About MSC growth medium

This product is compatible with any growth medium. For EV production from MSCs, we recommend MSCulture™ High Growth Basal Medium/Supplement.

Example of Protocol (6-well plate)

1. Seed MSCs at 5 x 10³ cells/cm² and culture in growth medium until they reach 80-90 % confluence.
2. Remove the growth medium, add 4 mL of EV-Up™ MSC EV Production Supplement, AF with Supplement, and culture for 3-5 days.
3. Collect the cell culture supernatant and centrifuge at 2,000 x g for 20 minutes. Carefully transfer the supernatant to a new tube without disturbing the pellet.
   [Note] Adding EV-Save™ Extracellular Vesicle Blocking Reagent at 1/100 of the culture supernatant volume can reduce adsorption of EVs to tubes and pipette tips.
4. Purify EVs from the culture supernatant using the PS affinity method or other suitable techniques.

Comparison of EV Particle Counts

EVs were purified from the cell culture supernatant of human bone marrow-derived MSCs cultured in each medium using the PS affinity method and quantified by nanoparticle tracking analysis (NTA) with a NanoSight instrument.

[Result]
The number of EV particles released from MSCs cultured in EV-Up™ was approximately 2.6-fold higher than that from MSCs cultured in conventional medium.

Comparison of EV Marker Protein Expression

EVs were isolated from the cell culture supernatants of human bone marrow–derived MSCs cultured in each medium using the PS affinity method. Levels of EV marker proteins (CD9, CD63, CD81) were measured by ELISA.

[Result]
The cell culture supernatant from EV-Up™ contained higher levels of EV marker proteins than that from conventional medium.

Comparison of Cell Viability

Human bone marrow-derived MSCs were first expanded in a serum-containing medium and then cultured for 5 days in each medium. Cell viability was subsequently measured.

[Result]
MSCs cultured in EV-Up™ exhibited high viability, comparable to that of MSCs cultured in conventional medium.

Change in EV Production Yield Over Culture Duration

After expanding bone marrow-derived MSCs, the culture medium was replaced with supplemented EV-Up™ MSC EV Production Basal Medium, AF, and the cells were cultured for 5 days to produce EVs. Subsequently, EVs were isolated from the cell culture supernatant using the MagCapture™ Exosome Isolation Kit PS Ver.2. EV marker protein levels were then measured using the PS Capture™ Exosome ELISA Kit (Streptavidin-HRP).

[Result]
EV marker protein levels increased with culture duration. By day 5 of culture, EV-Up™ yielded higher levels of all markers than the conventional medium.

Antifibrotic Activity of EVs Produced in Different Media

EVs (5 x 10⁸ particles/mL) were purified from the cell culture supernatant of human bone marrow-derived MSCs using the PS affinity method. These EVs were then added to human fetal lung fibroblasts (TIG3 cells) stimulated with TGF-β. Antifibrotic activity was evaluated by quantifying mRNA levels of the fibrosis markers collagen III and α-SMA using RT-PCR.

[Result]
EVs produced from MSCs cultured in EV-Up™ exhibited stronger antifibrotic activity compared with those produced in conventional medium.

Comparison with Competitor’s EV Production Medium

Bone marrow-derived MSCs were expanded in MSCulture™ High Growth Basal Medium/Supplement, and then cultured in each EV production medium for 5 days to produce EVs. Subsequently, the cell culture supernatant was filtered through a 0.22 μm filter and EVs were isolated from 5 mL of the filtered supernatant using the PS affinity method. For the resulting EVs, particle number was measured by NTA; EV marker protein expression levels were measured by ELISA using the PS Capture™ Exosome ELISA Kit (Streptavidin-HRP); and anti-inflammatory activity was measured by a reporter assay using the LumiMAT™ Pyrogen Detection Kit.

(3) Anti-inflammatory Activity

[Result]
EV-Up™ outperformed competitor’s product in terms of EV particle yield, EV marker protein expression levels, and anti-inflammatory activity.