Primary rat cardiac fibroblast isolation and culture

Rat primary cardiac fibroblasts were isolated, as previously described [32]. Male Sprague–Dawley rats weighing 101–125 g were anaesthetized with a ketamine–xylazine cocktail (100 mg/kg ketamine; 10 mg/kg xylazine) administered intraperitoneally. After verifying the loss of limb reflexes, heparin (6 mg/kg) was administered intravenously via the saphenous artery. Hearts were immediately excised and placed in 1:1 Dulbecco’s Modified Eagle’s medium/Ham’s F12 nutrient mixture (DMEM/F12, Gibco # 11,320-033). The hearts were cannulated via the aorta on a Langendorff apparatus and then subject to retrograde perfusion with DMEM/F12 for 5 min, followed by Minimum Essential Medium, Spinner’s Modification (S-MEM, Gibco # 11,380-037) for another 5 min to flush out calcium and promote cell dissociation. To digest the tissue, the hearts were then perfused with S-MEM supplemented with 600 U/mL collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ; #CLS-2) with recirculation for 25 min at 37 °C.

For quiescent fibroblast culture, the digested tissue was incubated at 37 °C, 5% CO₂ for 10 min, and then neutralized with 10 mL of Ham’s F-10 medium (F-10, Gibco, # 11,550-043) supplemented with 2% fetal bovine serum (FBS) and 100 U/mL penicillin–streptomycin. The tissue was further dissociated by trituration and the final cell suspension was gravimetrically passed through a 40 μm sterile cell strainer (Thermo Fisher Scientific, Waltham, MA). The cells were pelleted by centrifugation at 200×g for 5 min, and re-suspended in complete cell culture medium. Fibroblasts were allowed to adhere to prepared elastic surfaces for 3 h at 37 °C, 5% CO₂. Adherent cells were then briefly washed twice with 1X PBS supplemented with penicillin–streptomycin, and fresh complete culture medium was added. For the following 3 days, the cultures were once again washed, and the growth medium was replaced. Cells were used for experimentation 4-day post-plating.

For activated myofibroblast culture, digested tissue was incubated as described above, but was neutralized with DMEM/F12 supplemented with 10% FBS and penicillin–streptomycin. Cells were allowed the adhere for 2 h at 37 °C, 5% CO₂ on conventional plastic surfaces prior to brief washing in 1X PBS and replacing the complete culture medium. Cells were allowed to proliferate until ~ 75–80% confluence prior to passaging. Experiments using activated rat myofibroblasts were passaged once (P1).