Isolation and Culture of Porcine Valve Interstitial Cells

Valve interstitial cells for in vitro culture were isolated from fresh porcine hearts obtained from a local abattoir (Cockrum’s Custom Meat Processing and Taxidermy, AR) using techniques published by us previously [14 (link), 19 (link), 20 (link)]. Briefly, the heart was transported to the laboratory in ice-cold, sterile PBS solution and quickly dissected in the laboratory using aseptic techniques. All three aortic valve leaflets were dissected and incubated in 1 mg/ml collagenase solution (Worthington, NJ) for 3 h at 37 °C with frequent agitation. After collagenase digestion, cold 10% fetal bovine serum (FBS)-containing Dulbecco’s Modified Eagle Media (DMEM) was added to arrest enzymatic activity. The solution was filtered with the cell strainer 100 μm pore size (Corning, NY) to remove any remaining tissue debris prior to centrifugation for 5 min at 200 g and 4 °C. The resulting cell pellet was re-suspended in 10% FBS-containing cell culture media, plated in a flask and maintained in a 37 °C incubator. Fresh media was changed at least every 3 days. Cells from passage 1–7 were used in all subsequent two-dimensional (2D) and three-dimensional (3D) culture studies.