Smooth muscle growth supplement smgs

HASMCs were cultured in Medium 231 (Life Technologies, Carlsbad, CA, USA) supplemented with a Smooth Muscle Growth Supplement (SMGS, Life Technologies, Carlsbad, CA, USA) and 1% of 100 units/mL penicillin and 100 mg/mL streptomycin (Sigma Aldrich, St. Louis, MO, USA) in tissue culture flasks at 37 °C in a humidified atmosphere and 5% CO₂, as previously described [36 (link),37 (link)]. Cells were split 1:2 twice a week and used until passage 18.

Samples from aortic tunica media, stored in serum-free Medium 231 (Life Technologies, Carlsbad, CA, USA), were washed with Phosphate Buffer Saline (PBS), minced and digested O/N at 37 °C in a solution of 2 mg/mL collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ, USA) in complete Medium 231, supplemented with Smooth Muscle Growth Supplement (SMGS, Life Technologies, Carlsbad, CA, USA). Then, the result of tissue digestion was filtered with 100 μm cell strainer, pelleted and plated in complete Medium 231. To improve cell growth, the day after isolation the medium was changed to remove the residual erythrocytes. Regarding MFS samples, cells used for the in vitro experiments were those isolated only by non-dilated zone.

Studies were conducted under ethics approval obtained from the West of Scotland Research Ethics Service (WS/12/0294) and all subjects provided written informed consent. Small arteries were dissected from surplus surgical tissue of patients receiving elective craniofacial surgeries at the Craniofacial/Oral and Maxillofacial Unit, Queen Elizabeth University Hospital, Glasgow.
Primary human VSMCs (hVSMCs) were isolated from 12 individuals as we previously described [34 (link)] and were cultured in DMEM supplemented with Smooth Muscle Growth Supplement (SMGS) (Life Technologies, Paisley, U.K.) and antibiotics. Confluent cells were rendered quiescent overnight using DMEM containing 0.5% FBS before experiments. hVSMCs were studied up to passages 5–8.