Hiinduced Neural Progenitor Cell Spheres

On day 4 after thawing, the hiNPCs were singularized with Accutase for 10 min at 37 °C and 5% CO₂ (#07920, Stemcell Technologies, Vancouver, BC, Canada) and centrifuged (300× g, 10 min). After the cell pellet was resuspended in the respective neural progenitor medium with 10 µM Y-27632 (#HB2297, Hello Bio, Bristol, UK), 2 × 10⁶ cells were transferred into one well of a new 6-well plate (#83.3920, Sarstedt, Nürmbrecht, Germany) in 4 mL medium. Sphere formation took place in a gyrical shaking incubator (#LT-XC, Kuhner Shaker GmbH, Basel, Switzerland) at 140 rpm, 12.5 mm diameter, 37 °C, 5% CO₂, and 85% humidity for 7 days. On day 7, to equalize the size of the hiNPC spheres, they were chopped to 250 µm (McIlwain tissue chopper, Mickle Laboratory Engineering Co. LTD., Guildford, UK) as described previously [71 (link)].