Uniaxial Stretch Protocol for Cell Culture

Static stretch was administered (FX-6000 Tension System, Flexcell International Corporation) as previously described (Hornberger et al., 2005 (link); Baccam et al., 2019 (link)) with the following modifications. Cells were subjected to a static 5% uniaxial stretch in the last 4 or 24 h of CM treatment (as described above). Control cells (0% stretch) were grown under identical conditions but left unstretched and placed beside the incubator’s baseplate. For administration of stretch, culture plates were removed from the incubator, rapidly placed onto the stretch device’s baseplate, and then placed back in the incubator. The Flexcell Arctangle^® loading station was used for all experiments, which consists of six rigid posts covered in a thin layer of silicone lubricant, centered beneath the six-well Uniflex plate. Applied vacuum pressure deforms the membrane across the loading post in a single direction (e.g., north and south poles), creating a uniaxial stretch (Figure 3B). A custom ramp protocol, described here, was designed to ensure cell adherence to the membrane while vacuum pressure is applied: the Flexcell tension system was set to increase vacuum pressure gradually, and thus % elongation in increments of 1% every 2 s, up to 5%, which is then maintained for the duration of the experiment (e.g., 24 h), followed by a ramp-down to 0% at the conclusion of the experiment.