IE users: please get the latest browser version at Microsoft Internet Explorer Home Page
if yot already have the latest browser version please verify that your browser settings is not in compatibility mode
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.