Gibco™Neurobasal™ Medium

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	Use a different protocol for the stationary culture

Protocol tips
Use a different protocol for the stationary culture

Publication protocol

To generate forebrain-specific organoids (Figure 1B), human iPSC colonies were detached 7 days after passage with Collagenase Type IV, washed with fresh stem cell medium and cultured in a 15 ml conical tube. On day 1, detached and washed iPSC colonies were transferred to an Ultra-Low attachment 6-well plate (Corning Costar), containing 3 ml of stem cell medium (without FGF-2), plus 2 M Dorsomorphine (Sigma) and 2 M A83-01 (Tocris). On days 5-6, half of the medium was replaced with induction medium consisting of DMEM:F12, 1X N2 Supplement (Invitrogen), 10 g/ml Heparin (Sigma), 1X Penicillin/Streptomycin, 1X Non-essential Amino Acids, 1X Glutamax, 4 ng/ml WNT-3A (R&D Systems), 1 M CHIR99021 (Cellagentech), and 1 M SB-431542 (Cellagentech). On day 7, organoids were embedded in Matrigel (BD Biosciences) and continued to grow in induction medium for 6 more days. On day 14, embedded organoids were mechanically dissociated from Matrigel by pipetting up and down onto the plate with a 5 ml pipette tip. Typically, 10 - 20 organoids were transferred to each well of a 12-well spinning bioreactor (SpinΩ) containing differentiation medium, consisting of DMEM:F12, 1X N2 and B27 Supplements (Invitrogen), 1X Penicillin/Streptomycin, 1X 2-Mercaptoenthanol, 1X Non-essential Amino Acids, 2.5 g/ml Insulin (Sigma). At day 71, differentiation medium was exchanged with maturation medium, consisting of Neurobasal (Gibco), 1X B27 Supplement, 1X Penicillin/Streptomycin, 1X 2-Mercaptoenthanol, 0.2 mM Ascorbic Acid, 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 ng/ml TFG
(Peprotech), and 0.5 mM cAMP (Sigma). The organoids could grow beyond 110 days in maturation medium. All media were changed every other day. For the stationary culture, day 14 organoids were generated following the same protocol and then maintained in an Ultra-Low attachment 6 well plate (Corning Costar) with differentiation media. The “intrinsic protocol” for differentiation of human iPSCs into cerebral organoids followed the published protocol (Lancaster et al., 2013).


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Manufacturer protocol

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