Gibco™ DMEM/F-12, GlutaMAX™ supplement

Protocol tips

Upstream tips	Protocol tips	Downstream tips

Publication protocol

Differentiation of 3D Ros: Three colonies, from the patient and control each were subjected to ROs generation. ROs were generated from iPSCs following a published method (Nakano et al., 2012) with slight modification. Briefly, iPSCs were dissociated into single cells with TrypLE Express (Cat. #12563-011; Gibco, Denmark) containing 0.05 mg/ml DNase I (Cat. #11284932001; Roche) and 20 μM Y-27632 resuspended in retinal differentiation medium I, and G-MEM medium (Cat. #11710-035; Gibco, United States) supplied with 20% KSR, 3 μM IWR1e (Cat. #681669; Merck Millipore, United States), 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin. Approximately 12,000 cells in 100 μl were added to each well and reaggregated in a V-bottom low-cell-adhesion 96-well plate (Cat. #MS-9096V; Sumitomo Bakelite, Japan). The differentiation starting day was defined as day 0; 20 μM Y-27632 was added to the retinal differentiation medium I on day 0, and half the medium was exchanged with fresh retinal differentiation medium I on day 6. From day 2 to day 18, Matrigel was added in a final proportion of 1% v/v. On day 12, the cell aggregates were transferred to petri dishes in retinal differentiation medium II, G-MEM medium supplied with 10% (v/v) FBS, 100 nM SAG (Cat. #ALX-270-426-M001; Enzo Life Sciences, United States), 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin. From day 18, each cell aggregate was cut into 3–5 small pieces and were maintained in neural retina culture medium, containing DMEM/F12-GlutaMAX medium (Cat. #10565-018; Gibco, United States) supplied with 10% (v/v) FBS, N2 supplement (Cat. #17502-048; Gibco, United States), 0.5 μM retinoic acid (Cat. #R2625; Sigma, United States), 12.5 μg/ml taurine (Cat. #T0625; Sigma, Japan), 100 U/ml penicillin and 100 mg/ml streptomycin. From days 18 to 30, about 50 organoids were maintained in a 100 mm Petri dish with 15–20 ml medium. After day 45, number of organoid in each dish was reduced to 30 with 15–20 ml medium. The neural retina culture medium was used from day 18 since after and changed weekly.

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-derived retinal organoids using Gibco™ DMEM/F-12, GlutaMAX™ supplement from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Gibco™ DMEM/F-12, GlutaMAX™ supplement below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media hiPSC-derived retinal organoids using Gibco™ DMEM/F-12, GlutaMAX™ supplement 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.