Following successful reprogramming, growth factor reduced Matrigel (BD Biosciences, NJ, USA) was used as the substrate for the maintenance of the iPSCs culture in serum- and feeder-free conditioned medium (StemPro®, Life Technologies) following the manufacturer's instructions. Cells were split at a ratio of 1:6 every 3-4 days by Versene (Life Technologies) before experiments.
Similar to our cancer cell seeding protocol, hydrogels were sterilized under UV for 1 hour. Human iPSCs at 70–80% confluency were detached by Accutase (Innovative Cell Technologies) and resuspended in regular culture medium with 5uM ROCK inhibitor Y27632 (Stemgent). Cells were seeded at concentrations of 100 k or 400 k mL-1 into each of the well of a 24-well plate, which had an individual hydrogel array construct. The plates were spun at a speed of 50 g for 3 minutes and then incubated in a 37°C, 5% CO2 incubator. Maintenance medium was replaced everyday. EBs formed spontaneously within the center of each concave hydrogel structure, and were monitored and imaged using a Leica DIC microscope. Image analysis (e.g. EB diameter size) was performed on imageJ software.
Similar to our cancer cell seeding protocol, hydrogels were sterilized under UV for 1 hour. Human iPSCs at 70–80% confluency were detached by Accutase (Innovative Cell Technologies) and resuspended in regular culture medium with 5uM ROCK inhibitor Y27632 (Stemgent). Cells were seeded at concentrations of 100 k or 400 k mL-1 into each of the well of a 24-well plate, which had an individual hydrogel array construct. The plates were spun at a speed of 50 g for 3 minutes and then incubated in a 37°C, 5% CO2 incubator. Maintenance medium was replaced everyday. EBs formed spontaneously within the center of each concave hydrogel structure, and were monitored and imaged using a Leica DIC microscope. Image analysis (e.g. EB diameter size) was performed on imageJ software.