Quantifying Mesenchymal Stem Cell Colony Formation

After trypsinization, MSCs derived from both monolayer and spheroid cultures were seeded on a 10-cm-diameter Petri dish at a density of 10 cells/cm² (Banfi et al., 2000 (link)). Cells were maintained in culture for 15 days in the complete medium, with refreshment every 3 days. Cells were then fixed in 10% buffered formalin (Bio Optica, Milan, Italy) for 10 min and stained with 1% methylene blue for 45 min. Stained dishes were scanned using the Epson Perfection 1260 scanner, and colony-forming units (CFUs) were analyzed using the ImageJ ColonyArea plugin (Guzmán et al., 2014 (link)). Colonies’ cell morphology was evaluated using a Leica DMi1 inverted phase microscope (Leica).