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

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EV-Up™ is a culture medium specifically designed for the production of exosomes (EVs) from mesenchymal stem cells. The medium is prepared by combining "EV-Up™ MSC EV Production Supplement, AF" and "EV-Up™ EV-Production Basal Medium for MSC, AF", resulting in a serum and animal free medium applicable to any growth media.

Features

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

Applicable Cells

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

MSC EVs Production Protocol

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*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).

Application Data

Nano 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

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  • EV-Up™

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  • (A) Number of EVs particles

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    *3: Exosome-depleted FBS

  • (B) EVs diameter

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MSC cultured in EV-Up™ released 2.6 times more EVs than MSC cultured in DMEM + 10% EV-depleted FBS. The diameter of EVs was comparable.

 

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.

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  • Collagen Ⅲ

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  • α-SMA

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EVs produced in EV-Up™ significantly decreased the gene expression of fibrotic markers, such as Collagen Ⅲ (A) and αSMA (B).

 

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.

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Both conditions showed similar survival rates.

 

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

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  • CD63

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  • CD81

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EV-Up™ produced EVs expressing various EV marker proteins.

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Exosome Blocking Reagent

Isolation/Purification of Exosome

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