Ultra low attachment culture dishes

hESC colonies were lifted from the MEF feeder layer enzymatically with 1.5 mg/mL collagenase type IV (Gibco) and isolated by sedimentation and washing 3 times with wash medium. Colonies were suspended in fibroblast medium and grown in Ultra-Low Attachment Culture Dishes (Corning) for formation of embryoid bodies (EBs). Medium was replenished every 3 days by sedimentation and resuspension of EBs. After 9 days, EBs were transferred to tissue culture dishes to attach. Seven days later, EBs and fibroblast-like cells were passaged using Trypsin-EDTA (0.25%) (Gibco), triturated to single-cell suspension, and plated on tissue culture dishes. Cultures were maintained in fibroblast medium on plates treated with gelatin (Sigma-Aldrich) and were passaged every 5–7 days.

Cells were resuspended in “spheroid media” comprised of DMEM/F12 containing epidermal growth factor (EGF, 20 ng/mL; Sigma-Aldrich), basic fibroblast growth factor (bFGF, 20 ng/mL; Sigma-Aldrich), N-2 supplement (20 ng/mL; ThermoFisher Scientific), and B27 (20 ng/mL; Sigma-Aldrich) and then plated on Ultra-Low Attachment culture dishes (Corning Life Sciences). Spheroids were collected after 5–7 days except when noted otherwise. Protein was extracted for analysis, or cells were dissociated with Accutase (Innovative Cell Technologies) and used for other experiments. To count the number of spheroids, the number of spheroids >50 μm in diameter was counted in five fields after image processing using Imaris 7.6 (Bitplane).

hiPSCs (414C2 line) were cultured for 12 days in adhesion cultures with mouse embryonic fibroblasts and then allowed to form embryonic bodies in floating culture for 30 days. Aggregated cells were differentiated into NS/PCs derived from hiPSC-NS/PCs using various factors during each day of the incubation period (Okada et al., 2008 (link)).
hiPSCs (201B7 line) were pretreated for 6 days with 3 μM SB431542 (Tocris, 301836-41-9) and 150 nM LDN193189 (StemRD, 1062368-24-4). The cells were then dissociated and seeded at a density of 1×10⁵ cells per milliliter in ultra-low-attachment culture dishes (Corning) in neuronal induction medium consisting of medium hormone mix (MHM) (Okada et al., 2008 (link)) supplemented with 2% B27 supplement without vitamin A (Thermo Fisher, 17504-044), 20 ng/mL FGF-2, 10 μM Y27632 (Nacalai Tesque, 08945-71), 1 μM retinoic acid (RA; Sigma, R2625-1G), 3 μM CHIR 99021 (Reprocell, 04-0004) and 10 μM SB431542 (Calbiochem, 301836-41-9) in a hypoxic and humidified atmosphere (4% O₂, 5% CO₂) for 6 days. The formed neurospheres were passaged by dissociation into single cells and then cultured in slightly modified neuronal induction medium, MHM supplemented with 2% B27 without vitamin A, 20 ng/mL FGF-2, 10 μM Y27632, and 1 μM RA for 6 days under 4% O₂ (hypoxic) conditions.

Spheres of DU145 cells were formed as previously described [23 (link)]. Briefly, DU145 cells were plated on ultralow attachment culture dishes (Corning, NY, USA) (1 × 10³ cells/well in 6-well plates and 1 × 10⁵ cells/dish in 10 cm dishes) and cultured in DMEM/F-12 (Gibco, NY, USA) supplemented with B27 (Gibco), 4 μg/mL insulin (Sigma, MO, USA), 20 ng/mL epithelial growth factor (EGF; Gibco), and 20 ng/mL basic fibroblast growth factor (bFGF) (ORF, Kopavogur, Iceland) for 10 days at 37 °C and 5% CO₂.