Cardiosphere-derived cells are isolated from atrial or ventricular biopsy specimens of patients undergoing heart surgery. After tissue processing and culturing, a fibroblast-like cell layer forms. A number of cells begin to migrate onto this layer, and are further isolated and cultured to form cardiospheres. After expansion, the vast majority of CDCs were CD105+, with significant pluralities that were c-Kit+, CD90+, CD34+, and CD31+. These cells were also MDR1−, CD133−, and CD45−, as well as negative for a cocktail of blood lineage markers.
Cut isolated myocardial tissue into 1- to 2-mm pieces, wash with PBS, and digest with trypsin (0.2%) and collagenase IV (0.1%). Discard the obtained cells and wash the remaining tissue fragments with complete explant medium (CEM) (Iscove’s Modified Dulbecco’s Medium [IMDM]) supplemented with fetal calf serum (10%), penicillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (2 mM), and 2-mercaptoethanol (0.1 mM). Culture as explants at 37°C and 5% CO2. After a period ranging from 1 (embryo) to 3 (adult) weeks, a layer of fibroblast-like cells is generated from adherent explants over which small, phase-bright cells migrate. These phase-bright cells can be collected by pooling two washes with PBS, at room temperature under visual control. Seed the obtained cells at ≈0.5-2×10^5 cells/mL in poly-D-lysine-coated multiwell plates in cardiosphere-growing medium (CGM: complete IMDM (35%), DMEM–Ham F-12 (65%), B27 (2%), 2-mercaptoethanol (0.1 mM), epidermal growth factor [EGF; 10 ng/ml], basic fibroblast growth factor [bFGF; 20 ng/ml], cardiotrophin-1 (40 nmol/L), thrombin (40 nmol/L), antibiotics, and L-Glu, as in CEM). Isolation of the cardiosphere-forming cells can be performed from the same explant at least 4 times, at 6-10-day intervals. Passage cardiospheres every 2-3 days by partial changing of the medium and mechanical trituration of the larger clusters.