Gibco™Advanced DMEM/F-12

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Publication protocol

hESC and hiPSC culture: hESC (H9) and hiPSC (SB-Ad3) on Matrigel hESC-qualified matrix (BD; www.bd.com) were cultured in mTeSR1® medium (StemCell Technologies; www.stemcell.com). hESC and hiPSC colonies were treated with 0.02% EDTA (Lonza; www.lonza.com) and picked out by mechanical disruption using a tip to give rise to small clumps and plated on the Matrigel®-plate. hESC and hiPSC were maintained under mTeSR1® medium and seeded on a feeder layer of mouse embryonic fibroblasts (MEF) (mitotically inactivated by irradiation) for experimentation until passage 60. hiPSCs were used before passage number 50. Medium for the cells on MEF is knockout-Dulbecco’s modified Eagle’s medium (DMEM), 1 mM l-glutamine, 100 mM nonessential amino acids (NEAA), 20% knockout serum replacement (KOSR, Gibco; www.lifetechnologies.com), 1% penicillin–streptomycin (Gibco) and 8 ng/ml bFGF (Invitrogen; www.lifetechnologies.com). The medium was changed daily. hESC and hiPSC on MEF were dissociated with Accutase (Gibco; www.lifetechnologies.com) for 3 min, resuspended in differentiation medium and plated 100 μl per well (9000 cells/well) on 96-well low-cell-adhesion U-bottom plates (Lipidure Coat, NOF; www.nofamerica.com). Differentiation medium was G-MEM (Life Tech; www.lifetechnologies.com) supplemented with 1.5% KOSR (Invitrogen; www.lifetechnologies.com), 0.1 mM NEAA, 1 mM sodium pyruvate, 1 mM penicillin/streptomycin and 0.1 mM 2-mercaptoethanol (Life Tech). On day 1, half of the medium in each well was exchanged for fresh differentiation medium containing Growth Factor Reduced Matrigel (2% (v/v) final concentration, BD; www.bd.com). On day 3 of the protocol, BMP4 (10 ng/ml, Gibco) and SB-431542 (1 μM, TOCRIS; www.tocris.com) were added to each well at 5× concentration in 25 μl of fresh media. On days 5, FGF2 (25 ng/ml, Gibco) and LDN-193189 (1 μM, STEMGENT; www.stemgent.com) were added to each well at 6X concentration in 25 μl of fresh media. The concentration of Y-27632 (Chemdea) was maintained at 20 μM throughout days 0–8. The concentration of Matrigel was maintained at 2% (v/v) throughout days 1–8. On day 8 of differentiation, organoids were transferred to 6 or 12 well plates (Lipidure Coat, NOF) in N2 medium containing 1% (v/v) Matrigel. N2 medium contained Advanced DMEM/F12 (Gibco), 1X N2 Supplement (Life Tech), 50 μg/ml Normocin (Invitrogen) and 1 mM GlutaMAX. After 48 h the medium was changed completely with new N2 medium. Beginning on day 10, half of the medium was changed every other day during long-term floating culture for up to 90 days.

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