Neutralized rat tail collagen type 1

Cultured human cardiac myofibroblasts from passage 4–8 were serum-starved for 24 hours. Each experiment utilized cells from the same passage. Myofibroblasts were trypsinized and added to a liquid form of neutralized rat-tail type I collagen (1.8 mg/mL, BD Biosciences) at high density (2.5 × 10⁵ cells/mL). Solutions were incubated at 37°C to allow for gel polymerization. Immediately after polymerization, 500 μL of IMDM either alone (serum-free medium [SFM]) or containing 10 ng/mL human recombinant TGF-β1 (Gibco-Invitrogen, Frederick MD), with or without 20 ng/mL LMW-FGF-2 (Invitrogen, Camarillo, CA, USA) was added to the culture wells and plates were further incubated overnight. To initiate ECM contraction, the cell-ECM constructs were released from the well wall using a sterile micro-spatula (Corning®). Serial images of the ECM dimensions were obtained from the time of release (baseline) and at 24 hours. ImageJ analysis software (NIH, USA) was used to measure the area of ECM contraction as a quantitative measure of ECM remodeling.