α minimum essential medium eagle α mem

The sterilized acellular cardiac scaffolds were repopulated with pATMSCs obtained from porcine pericardial adipose tissue, as previously reported^{13 (link)}, to generate both the engineered myocardial and pericardial grafts. Initially, the decellularized scaffolds were rehydrated with a mixture of 175 μL of peptide hydrogel RAD16-I (Corning, Corning, NY) and 175 μL of GFP⁺-pATMSCs (1.75 × 10⁶) in 10% sucrose (Sigma-Aldrich, St Louis, MO), added dropwise on top of the scaffold. To promote hydrogel jellification, α-minimum essential medium eagle (α-MEM) (Sigma-Aldrich) was added over the scaffold. The produced cell-enriched grafts were maintained over one week under standard culture conditions (37 °C, 95% air, 5% CO₂), changing medium every 2 days.

ASCs were isolated from adult Sprague–Dawley rats as previously reported^{15 (link)}. Visceral fat was carefully dissected and minced using a sterile razor blade. Later, tissue was enzymatically dissociated with 0.15% (w/v) collagenase type I (Invitrogen, UK) for 90 min at 37 °C under constant agitation. Collagenase was neutralized by the addition of α-Minimum Essential Medium Eagle (α-MEM, Sigma-Aldrich, Poole, UK) containing 10% (v/v) foetal bovine serum (FBS, LabTech, Uckfield, UK). Undissociated tissue was removed passing the solution through a 100μm filter and then centrifuged at 1200 rpm for 10 min. The stromal cell pellet was resuspended in α-MEM (Sigma-Aldrich, Poole, UK) containing 10% (v/v) FBS (LabTech, Uckfield, UK), 2mM L-glutamine (GE Healthcare UK, Little Chalfont, UK) and 1% (v/v) penicillin/streptomycin solution (Sigma-Aldrich, UK). Cultures were maintained at sub-confluent levels in a 37 °C incubator with 5% CO₂.