EV-Up™ Exosomes Production Medium for MSC (animal component-free)
EV-Up™ media is Exosomes (EVs) production media for mesenchymal stem cells (MSC). This media is adjusted as complete medium by adding "EV-Up™ MSC EV Production Supplement, AF" to "EV-Up™ EV-Production Basal Medium for MSC, AF". This is serum- and animal-free and applicable to any growth media.
- Better EVs yield than conventional serum containing media
- Able to produce EVs with high activity
- Able to maintain MSC viability
- Bone-marrow-derived MSC
- Adipose-derived MSC
- Umbilical cord-derived MSC
MSC EVs Production Protocol
*1: The complete media which “EV-Up™ MSC EV Production Supplement, AF” added to "EV-Up™ EV-Production Medium for MSC, AF".
When culturing on a 6-well plate
- 5×103 cells of various MSC seed and culture using Growth media (with or without serum) until 80-90% confluent.
- Growth media discard and change to 4mL of EV-Up™ media. Incubates for 3-5 days.
- The culture supernatant collect to a tube and centrifuge at 2,000×g for 20 minutes. Collect the culture supernatant of it in a new tube.
Able to suppress absorption of EVs to a microtube and chip by adding EV-Save™ Extracellular Vesicle Blocking Reagent (Code No. 058-09261) that 1/100 amount of the culture supernatant.
- EVs isolate from the supernatant using MagCapture™ Exosome Isolation Kit PS Ver.2 (Code No. 290-84103) etc..
Nano Tracking Analysis (NTA)
EVs isolated from various media supernatant by PS affinity method*2 were analyzed with NTA.
*2: EVs are captured by phosphatidylserine (PS)-binding proteins and metal ions, then captured EVs are eluted with a chelating reagent.
DMEM + 10% EV depleted FBS
(A) The number of EVs particles
*3: Exosome-Depleted FBS
(B) EVs diameter
MSC cultured in EV-Up™ media released 2.6 times more EVs than MSC cultured
in DMEM + 10% EV-depleted FBS. And the EV’s diameters were almost same.
5×107 particles/mL of EVs isolated from various media supernatant by PS affinity method were added to normal human fetal lung-diploid fibroblasts cells (TIG3) that were stimulated TGFβ. And fibrotic marker (Collagen Ⅲ, αSMA) gene expression was quantified by RT-PCR.
Significantly, MSC EVs produced in EV-Up™ media decreased
the gene expression of fibrotic markers such as Collagen Ⅲ (A) and αSMA (B).
After the expansion of human bone marrow-derived MSC in serum containing media, the media was switched with EV-Up™ media and cultured for five days to produce EVs. After that, cell viability was measured.
MSC that was cultured in EV-Up™ showed high survival rate like when cultured in DMEM + 10% EV.
Exosome Marker Protein Expression
EV marker protein expression (CD9, CD63, CD81) of Evs isolated from various media supernatant were confirmed by ELISA.
EV-Up™ media produced EVs that expressed various EV marker protein.
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