Gibco™Neurobasal™ Medium

Protocol tips

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	From D20, culture with constant shaking

Protocol tips
From D20, culture with constant shaking

Publication protocol

Induction of Inner Ear Organoids: Induction followed the protocol of Koehler and Hashino (2014), but with modifications (Figure S7). In brief, ESCs were dissociated in Accutase (STEMCELL Technologies) and resuspended in differentiation medium (DMLK; Table S6). On D0, 1,500 cells in 100 μL of DMLK per well were plated in low binding 96-well U-bottomed plates (Thermo Fisher). On D1, half of the medium was exchanged with fresh DMLK containing Matrigel (Corning; 2% final concentration). Bone morphogenetic protein 4 (PromoKine) and SB-431542 (Reprocell) were added on D3. Later, basic fibroblast growth factor (STEMCELL Technologies) and LDN-193189 (Reprocell) were added (Figure S7). On D8, aggregates were washed twice in PBS before being transferred to new 96-well U-bottomed plates in 100 μL of N2 medium (Table S6) containing 1% Matrigel and 3 μM CHIR99021 (DeJonge et al., 2016). After 48 h, aggregates were transferred to 24-well low binding plates in fresh N2 medium until D20. On D20, aggregates were cultured in organoid medium (Table S6) with constant shaking. Half of the medium was changed every other day during the long-term culture period.

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Manufacturer protocol

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