Gibco™Neurobasal™ Medium

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	Culture in orbital shaker

Protocol tips
Culture in orbital shaker

Publication protocol

Brain organoids were generated using a protocol modified from Pasca, et al., 2015. In brief, stem cell colonies were grown until they were approximately 3-4 mm in diameter, after which they were detached by incubation with collagenase type IV for 45- 60 minutes at 37oC. The detached colonies were washed once with hES media and then placed in ultra-low attachment 6-well plates (Corning, Corning, NY) in Induction Media consisting of hES media supplemented with 100 nM LDN193189 (Stemgent, Cambridge, MA), 10 μM SB431542 (Stemgent), 2 μM XAV939 (Stemgent), and 10 μM Y-27632 (Tocris Biosciences, Bristol, United Kingdom). From differentiation day (dd) 1-6, the developing embryoid bodies were maintained in Induction Media without Y-27632 with daily media changes. From dd6 onwards, embryoid bodies were cultured on an orbital shaker. From dd6-25, the developing organoids were maintained in Neuronal Media consisting of Neurobasal media (Invitrogen) supplemented with 1:50 B27 without vitamin A (Invitrogen), 1X Glutamax (Invitrogen), 100 U/mL penicillin/100 μg/mL streptomycin (Invitrogen), 20 ng/mL bFGF (R&D Systems), and 20 ng/mL EGF (R&D Systems) with daily media changes until dd17 and then every other day afterwards. From dd25-43, every other day media changes with Neuronal Media supplemented with 20 ng/mL NT3 (R&D Systems) and 20 ng/ML BDNF (R&D Systems) were performed. After dd43, every other day media changes with Neuronal Media without growth factors were performed. Developing iPSC-derived organoids were cut at dd30-35 with sterile fine-tip forceps to a diameter less than 500 μm. Organoids derived from ESC and iPSC cells used for μTENN construction were between dd100-150 and dd62-79, respectively.

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Manufacturer protocol

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