EV-Up™ Exosomes Production Medium for MSC (animal component-free)

• Provides higher EV yield compared to conventional serum-containing media
• Enables production of EVs with high activity
• Maintains MSC viability in culture

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

*1: The complete media composed of “EV-Up™ MSC EV Production Supplement, AF” and "EV-Up™ EV-Production Medium for MSC, AF".

When culturing on a 6-well plate

1. Seed 5×10³/cm² MSCs and culture using growth media (with or without serum) until 80-90% confluent.
2. Replace growth media with 4mL of EV-Up™. Incubate for 3-5 days.
3. Transfer the culture supernatant to a tube and centrifuge at 2,000×g for 20 minutes. Collect the culture supernatant and transfer to a new tube.
   Optionally, add EV-Save™ Extracellular Vesicle Blocking Reagent (Code No. 058-09261) to the culture supernatant at a final dilution of 1:100, to suppress absorption of EVs to the tube wall.
4. Isolate EVs from the supernatant using MagCapture™ Exosome Isolation Kit PS Ver.2 (Code No. 290-84103).

Nanoparticle Tracking Analysis (NTA)

EVs isolated from various cell culture supernatant by PS affinity method*² were analyzed with NTA.

*2 EVs are captured by phosphatidylserine (PS)-binding proteins and metal ions, and eluted with a chelating reagent.

• DMEM + 10% EV-depleted FBS

  

• EV-Up™

  

• (A) Number of EVs particles

  

  *3: Exosome-depleted FBS

• (B) EVs diameter

  

　

Anti-fibrotic Effect

5×10⁸ particles/mL of EVs isolated by the PS affinity method from various cell culture supernatant were added to normal human fetal lung-diploid fibroblasts cells (TIG3) that were stimulated by TGFβ for subsequent quantification of the fibrotic marker expression (collagen Ⅲ and α-SMA) by RT-PCR.

• Collagen Ⅲ

  

• α-SMA

  

　

Cell Viability

The impact of EV-Up™ on cell viability was measured using human bone marrow-derived MSC. Cells were first expanded in serum-containing medium, and then cultured for in EV-Up™ for five days prior to survival rate estimation.

　

Protein Expression of Exosome Markers

ELISA was used to quantify the protein expression of markers CD9, CD63 and CD81 in EVs isolated from various cell culture supernatants.

• CD9

  

• CD63

  

• CD81