Gibco™Neurobasal™ Medium

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The organoid Maturation Medium (OMM) contains a 50:50 mixture of Advanced DMEM:F12 (Gibco) and Neurobasal Medium (Gibco) with supplements

Protocol tips
The organoid Maturation Medium (OMM) contains a 50:50 mixture of Advanced DMEM:F12 (Gibco) and Neurobasal Medium (Gibco) with supplements

Publication protocol

hPSC differentiation: To start differentiation, hPSC cells were dissociated with StemPro Accutase (Invitrogen, cat. no. A1110501) and distributed, 5,000 cells per well, onto low-adhesion 96-well V-bottom plates in E8 medium containing 20 μM Y-27632 (Stemgent, cat. no. 04-0012-02) and Normocin. Following a 48 hour incubation, the aggregates were transferred to low-adhesion 96-well U-bottom plates in 100 μl of Chemically Defined Medium (CDM) containing 4 ng ml-1 FGF-2 (Peprotech, cat. no. 100-18B), 10 μM SB-431542 (Stemgent, cat. no. 04-0010-05), and, for some experiments, 2.5 ng ml-1 BMP4 (Stemgent, cat. no. 03-0007), and 2% Growth Factor Reduced (GFR) Matrigel (Corning, cat. no. 354230) to initiate non-neural induction—i.e. differentiation day 0. CDM contained a 50:50 mixture of F-12 Nutrient Mixture with GlutaMAX (Gibco) and Iscove's Modified Dulbecco's Medium with GlutaMAX (IMDM; Gibco) additionally supplemented with 0.5% Bovine Serum Albumin (BSA), 1X Chemically Defined Lipid Concentrate (Invitrogen), 7 μg ml-1 Insulin (Sigma), 15 μg ml-1 Transferrin (Sigma), 450 μM Mono-Thioglycerol, and Normocin (see Supplementary Table 1 for detailed formulation). After 4 days of incubation, 25 μl of CDM containing a 250 ng ml-1 FGF-2 (50 ng/ml final concentration) and 1 μM LDN-193189 (200 nM final concentration; Stemgent, cat. no. 04-0074-02) was added to the pre-existing 100 μl of media in each well. After an additional 4 days (8 days total), 25 μl of CDM was added to the media. For some experiments, CDM containing a 18 μM CHIR99021 (3 μM final concentration; Stemgent, 04-0004-02) was added to the pre-existing 125 μl of media in each well on day 8—we determined that this treatment is optional for inner ear organoid production, but may improve induction of otic placode-like cells. On differentiation day 12, the aggregates were pooled together and washed with freshly prepared Organoid Maturation Medium (OMM) containing a 50:50 mixture of Advanced DMEM:F12 (Gibco) and Neurobasal Medium (Gibco) supplemented with 0.5× N2 Supplement (Gibco), 0.5× B27 without Vitamin A (Gibco), 1× GlutaMAX (Gibco), 0.1 mM β-Mercaptoethanol (Gibco), and Normocin (see Supplementary Table 2 for detailed formulation). The aggregates were resuspended in ice cold undiluted GFR Matrigel and distributed in ∼25 μl droplets on the surface of a 100 mm culture plate. After at least 30 minutes of incubation at 37°C, the droplets were bathed in 10 ml of OMM containing 3 μM CHIR99021. For non-droplet otic induction, the aggregates were washed and plated individually into each well of a 24-well low cell adhesion plate in OMM containing 3 μM CHIR and 1% GFR Matrigel. For both culture formats, the medium was changed completely on day 15 with OMM containing fresh 3 μM CHIR. During days 12-18, aggregates were monitored using DIC imaging daily. Otic pit-like protrusions were identifiable as bright translucent bulges from the dark core of the aggregates. On day 15 or 16, a subset of at least three aggregates were fixed, cryosectioned, and immunostained using PAX2/PAX8 antibodies to positively identify otic pit-like structures. On day 18 of differentiation, the CHIR was removed from the medium by washing and the droplet aggregates were moved to a floating culture. Droplets were carefully dislodged using a wide-mouth P1000 tip and transferred to 75 ml of fresh OMM in a 125 ml disposable spinner flask (Corning). Spinner flasks were maintained on an in-incubator stir plate (Thermo Scientific) at 65 RPM for up to 180 days of differentiation. For some experiments, the aggregates were maintained in individual wells of 24-well low-cell adhesion plates in 1 ml of OMM on an in-incubator orbital shaker (Thermo Scientific) at 65 RPM for up to 140 days. Technical repeats entailed a different start date, different passage number, and typically a different lot of reagents (some experiments used the same reagent lots).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing 3D Cell Culture Media hiPSC-inner ear organoids using Gibco™Neurobasal™ Medium from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Gibco™Neurobasal™ Medium below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media hiPSC-inner ear organoids using Gibco™Neurobasal™ Medium from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.