Flexercell strain unit

Human aortic smooth muscle cells (HASMCs), purchased from ScienCell (U.S.A.), were cultured in SMC medium (ScienCell, U.S.A.) with 5% CO₂ at 37°C. Passages 4–7 cells were seeded (10⁵ cells/well) onto six-well Flexcell plates coated with collagen I, and when they reached 90% confluence, serum-free medium was added to induce quiescence for 24 h and replaced with fresh complete medium, then physiological stretch (10% elongation, 1 Hz) was applied by use of a computer-controlled Flexcell 5000-Tension apparatus (Flexercell Strain Unit, FlexCell International). The static control cells subjected to the same conditions as the experimental cells, except that they were not exposed to stretch. For inhibition of stretch-induced activation of transcription factors and signaling pathways, pharmacological inhibitors were added to the culture media 1 h before stretch treatment.

HCM were cultured for 48 h in static conditions (standard cell culture incubator) or upon mechanical stretch (with the use of FlexerCell Strain Unit placed in the standard cell culture incubator). In order to achieve different iron availability conditions, the cell culture medium was supplemented with 100 μM deferoxamine (DFO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or 200 μM ammonium ferric citrate (AFC; Sigma-Aldrich; Merck KGaA) (Figure 1). Specifically, for mimicking the physiological work of the cardiac cells, HCM were cultured on the type 1 collagen-coated 6-well plates and the mechanical stretch was introduced with the use of FlexerCell Strain Unit (Flexcell®, biaxal cyclic stretch (15%, 0.5 Hz)). Both DFO and AFC were chosen and applied based on available data from cell culture studies [11 (link),12 (link),13 (link)]. After being diluted by a factor of 1000, the compounds were added to the cells.

When cells were approximately 80% confluent, the cells were plated onto six-well, flexible-bottom culture plates precoated with collagen type I in 2 mL of DMEM medium for stretch. After 24–48 hours, the cells were subjected to a static stretch of 11% using a Flexercell strain unit (for more details, please see http://www.flexcellint.com/; Flexcell International Corp). The cells were stretched for a total of 30 hours. After being stretched for 24 hours, the cells were incubated with and without forskolin (100 μΜ) in the absence and presence of IL-1β (1 ng/mL). The cells were then stretched again for 6 hours. Unstretched cells grown and treated similarly were used as controls.