Gibco™Neurobasal™ Medium

Protocol tips

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	Change media every other day

Protocol tips
Change media every other day

Publication protocol

Culture of Midbrain Organoids from Human iPSCs: To generate midbrain-specific organoids (Figure 6A), human iPSC colonies were detached with Collagenase Type IV 7 days after passage and washed with fresh stem cell medium in a 15 ml conical tube. On day 1, the detached and washed iPSC colonies were transferred to an Ultra-Low attachment 6-well plate containing EB medium, consisting of DMEM:F12, 15% Knockout Serum Replacer, 1X Glutamax, 1X 2-Mercaptoenthanol, 100 nM LDN-193189, 10 M SB-431542, 100 ng/ml SHH (Peprotech), 2 M Purmorphamine (Stemgent), 100 ng/ml FGF-8 (Peprotech). On day 5, EB medium was gradually switched to SHH medium, consisting of DMEM:F12, 1X N2 Supplement, 1X Glutamax, 100 nM LDN-193189, 3 M CHIR99021, 100 ng/ml SHH, 2 M Purmorphamine, 100 ng/ml FGF-8. On day 7, SHH medium was replaced with induction medium, consisting of DMEM:F12, 1X N2 Supplement, 1X Glutamax, 100 nM LDN-193189, 3 M CHIR99021. On day 14, 10-20 organoids were transferred to SpinΩ with differentiation medium, consisting of Neurobasal, 1X B27 Supplement, 1X Glutamax, 1X 2-Mercaptoenthanol, 20 ng/ml BDNF, 20 ng/ml GDNF, 0.2 mM Ascorbic Acid, 1 ng/ml TGF, and 0.5 mM c-AMP. All media were changed every other day.

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Manufacturer protocol

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