However, perfusion with smooth muscle cell medium did not significantly affect any of these parameters.
Primary human bladder smooth muscle cells (pBSMCs) were cultured in smooth muscle cell medium (SMCM, Sciencell Research Laboratories, Carlsbad, CA) at 37°C in a humidified incubator with 5% CO2.
Then, cells were incubated in serum-free smooth muscle cell medium (ScienCell Research Laboratories) in the presence or absence of 10 nmol/L human recombinant relaxin-2 (H2-relaxin, PepRocky Hillcky Hill, NJ, USA) for 24 hours.
Cells were cultured as monolayers in smooth muscle cell medium (ScienCell) containing essential and nonessential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals, and 2%% (v/ v) fetal bovine serum at 37 °C in a humidified 5%% CO2 atmosphere.
Human brain vascular smooth muscle cells (HBVSMC) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in smooth muscle cell medium, which consists of 500 mL basal medium, 10 mL fetal bovine serum, 5 mL smooth muscle cell growth supplement, and 5 mL penicillin/streptomycin solution.
Each biopsy was dissected into 1 mm pieces, washed in HBSS to remove excess blood, placed in Smooth Muscle cell medium containing collagenase XI (1 mg/ml), collagenase IA (1 mg/ml) and fatty acid free bovine serum albumin (0.5%) (Sigma-Aldrich, Oakville, ON, Canada), and digested for 60 minutes at 37oC.
Trypsin digestion was terminated using smooth muscle cell medium-basal (Sciencecell, San Diego, CA, USA).
The cell pellet was resuspended using smooth muscle cell medium-basal.
In order to induce differentiation of myoblasts into myotubes, cultures with a confluence of up to 80% were not split and proliferation medium was replaced by differentiation medium composed of Skeletal Muscle Cell Basal Medium, 5% horse serum (Invitrogen) and 5 mg Insulin (PromoCell).
The proliferation medium consisted of Skeletal Muscle Cell Basal Medium (Cat. No. C-23260, PromoCell), Skeletal Muscle Cell Growth Medium Supplement Pack (Cat. No. C-39360, PromoCell), 10% FBS and 1.5% L-alanyl-L-glutamine (Invitrogen).
HAoSMCs (Lonza, Walkersville, MD) were cultured in basal medium Clonetics smooth muscle cell (SMC) medium (SmGM-2, CAT# CC-3181, Lonza) enriched with the supplied SMC growth additives (Insulin, CAT# CC-4021D; hFGF-B, CAT# CC-4068D; GA-1000, CAT# CC-4081D, FBS, CAT# CC-4102D, hEGF, CAT# CC-4230D, Lonza).
