In Vitro Scratch Wound Healing Assay

Migration in response to wounding was assessed in vitro using the scratch assay as previously described [²⁷]. Briefly, HC and LT1D MSCs (where significant gene expression differences in the gene ontology term [GO term] stress and wounding was detected by array; n = 4 donors per group) were seeded in duplicates into a 24-well plate at a density of 1.7 × 10⁵ cells per well in 1 ml culture medium (density previously optimized for 100% confluence upon adherence and spreading). Cells were allowed to adhere overnight before removal of the medium and introduction of a full-depth scratch using a pipette tip (average wound width 0.9 mm). Wounded cells were washed twice with phosphate-buffered saline (PBS) to remove cellular debris before replacing with 1 ml of fresh culture medium. Wound closure was visualized by acquiring transmitted light images (10-millisecond exposure) in three viewpoints per well at ×5 optical magnification at 37°C using a Leica DMI6000 wide-field microscope with an EM-CCD 16-bit camera (Evolve; Andor Technology, Windsor, CT, http://www.andor.com) at 30-minute intervals for 24 hours. Degree of wound closure was assessed by measuring wound width, using Image J 1.46r software (NIH, Bethesda, MD, http://imagej.nih.gov/ij), every 4 hours after scratching. Data are expressed as percentage wound closure compared to initial wound width. At the end of the 24-hour imaging, cell lysates were harvested with RLT buffer (Qiagen) for gene expression analysis with qRT-PCR. A parallel plate without scratch wounds, incubated in corresponding conditions, was used as a control.