Exosome-Specific Mesenchymal Stem Cell Medium (Serum-Free, Phenol Red-Free)

1. Culture MSCs under standard conditions using serum-containing medium, platelet lysate-supplemented medium, or serum-free medium containing multiple protein factors.

2. When MSCs reach approximately 25%-50% confluency under these conditions, discard the medium.

3. Rinse the cells 3-5 times with PBS (1×) to remove residual exogenous proteins and other potential contaminants.

4. Prepare the exosome-specific mesenchymal stem cell complete medium by mixing the basal medium and supplement at a ratio of 100:1.

Note: a. This product contains L-glutamine and sodium bicarbonate, additional glutamine supplementation is not required during use.

b. The complete medium should be freshly prepared before use, stored at 4°C protected from light, and used within one week. For repeated use, the supplement is recommended to be aliquoted and stored at -20°C.

5. Replace the culture medium with the prepared complete medium and continue culturing the MSCs for 24-48 h. When cell confluency reaches around 80%, collect the supernatant for exosome isolation.

Note: For higher yields, the complete medium can be refreshed every 24 h and supernatant collected, provided cell health is maintained.