Targeting regenerative exosomes to myocardial infarction using cardiac homing peptide

Vandergriff, Adam; Huang, Ke; Shen, Deliang; Hu, Shiqi; Hensley, Michael Taylor; Caranasos, Thomas G.; Qian, Li; Cheng, Ke

Exosomes were isolated as previously described [25, 28] using ultrafiltration. Briefly, cells were grown to confluency in fetal bovine serum (FBS; Corning; Corning, NY)-supplemented Iscove's Modified Dulbecco's Medium (Gibco; Waltham, MA) then the media was changed to media without FBS. The media was conditioned on the cells for an extended period of time: 14 days for cardiosphere-derived cells (CDCs) [18, 20] , 5 days for HT1080 [29] (MilliporeSigma; St. Louis, MO). The HT1080-XOs were only used for the ex vivo targeting experiment; all other experiments used CDC-XOs. The conditioned media was filtered through a 0.22 μm sterilization filter to remove any cell particulates or apoptotic bodies, then concentrated and buffer exchanged to PBS using a 100 kDa ultrafiltration column (EMD Millipore; Billerica, MA). Sizes and concentrations of the isolated exosomes were ascertained using nanoparticle tracking analysis (NTA; NanoSight, Malvern, Worcestershire, United Kingdom). For electron microscopy, exosomes were stained using uranyl oxalate following a previously described protocol [30] . SDS-PAGE and Western blotting was performed using 5 μg of protein on 4-15% gradient gels (Bio-rad; Hercules, CA) and transferred using a wet transfer method.