3D Culture of Differentiated Cells

On day 26, differentiated cells were dissociated with Accutase, detached single cells were collected, and cells were cultured in three dimensions in Matrigel (Corning, Corning, NY), as described [36 (link)], with modifications. In brief, Matrigel at 80 μL/cm² was spread and incubated at 37°C for 30 minutes. Detached cells in SAGM culture medium (Lonza, Verviers, Belgium) containing 2% Matrigel (vol/vol) were seeded on solidified gel. Y27632, an inhibitor of Rho-associated protein kinase, and SB431542 were added as additional factors [11 (link), 37 (link), 38 (link)]. Cells were seeded at a density of 2.0 × 10⁵ cells/well in 6-well plates or 1.5 × 10⁶ cells/dish in 10 cm dishes. The SAGM culture medium was changed every 2 days. JQ1 at a concentration of 0.2 μM was also added on days 28 and 30 to remove residual undifferentiated iPSCs. Cultured cells were harvested on day 40. mRNA expression levels were determined on days 26 and 40.