Collagenase

Samples from twenty patients who underwent partial esophagectomy were immediately washed in 0.9% NaCl solution, removed, under aseptic conditions, from hemorrhagic necrotic tissue and adipose tissue by 5-min washing in 0.05% chlorhexidine followed by 20-min washing in D-Hank’s solution (containing penicillin 500 units/ ml, streptomycin 500 μg/ ml, amphotericin B 5 μg/ ml). This washing step was repeated 6 times, then the tissue samples were digested in 10% collagenase (Amresco, Solon, OH, United States) in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Carlsbad, CA, United States). After neutralizing the collagenase with culture medium, cells were spun down for 1 min at 200 g_av. The cell pellet was seeded in regular DMEM supplemented with 10% fetal bovine serum, 100 units/ ml penicillin, 100 μg/ml streptomycin, cultured at 37°C, 5% CO₂ changing medium every 48–72 h.

Atrial fibroblasts were isolated from the atrial tissues of sham and SP rats, as described previously (6 (link)). Briefly, tissues were digested with 100 U/mL collagenase (Type II) and 0.1% trypsin (Amresco) for 8 consecutive 7- to 10-minute treatment periods at 37°C. Atrial fibroblasts were pelleted at 1000 rpm for 10 minutes and resuspended in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin (Servicebio), and 100 μg/mL streptomycin (Servicebio). Experiments were performed with cells from the first and second passages. Before cells were treated with different chemical interventions, the FBS in the medium was deprived for 24 hours to induce growth arrest. The cells were then treated with TRPV4 antagonist GSK2193874 (300 nM) or TRPV4 agonist GSK1016790A (300 nM, Sigma-Aldrich) for an additional 24 hours in DMEM with 10% FBS in the presence of TGF-β1 (10 ng/mL, PeproTech). In another group of experiments, cells were preincubated with AKT specific inhibitor LY294002 (40 μM, Selleckchem), STAT3 specific inhibitor S3I-201 (50 μM, Selleckchem), P38 inhibitor SB 203580 (10 μM, Selleckchem), or SMAD3 inhibitor SIS3 (3 μM, Selleckchem) for 30 minutes before GSK1016790A treatment.