Embryoid Body Generation for 3D Spontaneous Differentiation

Embryoid bodies (EBs) of 9000 cells each were created in 96-well round-bottom plates coated with 1% agarose prepared in deionized water to confer a non-adherent surface which promotes iPSC self-aggregation (Friedrich et al., 2009; Dahlmann et al., 2013). A single-cell iPSC suspension was created via Accutase dissociation as described above and re-suspended in Essential 6, which lacks the essential pluripotency factors TGFβ and FGF2 (Cat# A1516401, ThermoFisher), supplemented with 10 μM Y-27632 to promote cell survival (Lin and Chen, 2014). Essential 6 media was replenished every other day to promote spontaneous differentiation.