Non adherent 24 well culture plates

Primary free-floating mOSE spheres were collected and washed in PBS. Spheres were dissociated by first incubating in trypsin/PBS (Invitrogen) at 37 °C for 10 min, then by passing cells through a 25 gauge needle to obtain a single cell suspension. Single cell suspension was verified using phase contrast microscopy. Cells were washed in PBS, counted using a hemocytometer, and plated in stem cell media at 5 × 10⁴ cells/mL. Cells were incubated in non-adherent 24-well culture plates (Corning) at 37 °C, 5% CO₂ for 14 days. Spheres were quantified using ImageJ using a pixel cutoff of >500 pixels and a circularity limit of 0.5–1.0.

For free-floating spheres, mOSE cells were cultured in stem cell media [Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (Sigma) supplemented with 1 X B27 supplement (Invitrogen), 0.02 µg/mL EGF (R&D Systems), 0.04 µg/mL fibroblast growth factor (FGF; R&D Systems), 4 µg/mL heparin (Sigma) and 0.01 mg/mL ITSS (Roche), and 2 µg/mL EGF (R&D Systems)] at 5 × 10⁴ cells/mL in non-adherent 24-well culture plates (Corning) and incubated at 37 °C, 5% CO₂ for 14 days. For TGFB1-treated spheres, cells were pre-treated with recombinant TGFB1 (10 ng/mL) for 4 days prior to being placed in spheroid culture and was replenished when plating cells in spheroid culture. Spheres were quantified using ImageJ using a pixel cutoff of >1000 pixels and a circularity limit of 0.5–1.0. For spheres cultured in methylcellulose, mOSE cells were placed in a 1:1 mixture of methylcellulose and stem cell media at 5 × 10⁴ cells/mL in 24-well culture plates (Corning), and incubated at 37 °C, 5% CO₂ for 28 days. Methylcellulose-embedded spheres were quantified using ImageJ using a pixel cutoff of >500 pixels and a circularity limit of 0.5–1.0. For each experiment, a minimum of 3 replicates were performed, each replicate was performed in three independent wells, spheres were counted in 4 fields per well, and the average count was reported.