V bottomed conical wells

iPSC lines were dissociated into single cells with TrypLE™ Express (Life Technologies), and 10,000 cells/well were plated into low-cell-adhesion 96-well culture plates with V-bottomed conical wells (Sumitomo Bakelite) to form uniform EBs cultured in: DMEM media, 20% FBS, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, P/S. After 20 days, EBs were fixed with 4%PFA and stained for three markers: smooth muscle actin (mesoderm), beta-III Tubulin (ectoderm), alpha-fetoprotein (endoderm) by Molecular Probes® 3-Germ Layer Immunocytochemistry Kit (Cat. No. A25538).

This approach is a modified simplified version of the protocols published by Eiraku et al. and Nakano et al. 11, 41. Cells were harvested at 90% confluence. hESCs/hiPSCs were dissociated to a single cell suspension by treatment with Accumax (1 ml per well) for up to 3 minutes. TeSR1 was added to each well and dissociated cells were collected and spun at 1,000 rpm for 5 minutes. Cell pellets were resuspended in TeSR1 medium containing 10 μM Y‐27632, counted in a hemocytometer, and reaggregated by seeding 9,000 cells per well in 100 μl into low adhesion Sumilon PrimeSurface 96 well plates with V‐bottomed conical wells (Sumitomo Bakelite, Osaka, Japan) or V‐bottomed 96 well plates coated with 0.5 wt% Lipidure‐CM5206 (amsbio). After 48 hours, the media was changed to retinal differentiation media. Media was changed every 3 days. On day 12, the resulting EBs were collected using a sterile disposable plastic Pasteur pipette and transferred into a low attachment 6 well plate (Corning) or 9 cm petri dish.
Cultures were differentiated in retinal differentiation medium comprising of ventral neural induction media supplemented with recombinant human IGF‐1 (Sigma, Hartfordshire, U.K.) as previously described 3, 8.