Human-induced pluripotent stem cell culture

Dear Stephanie,

we carried out most work using smNPCs, which require different media than iPSCs. For the small amount of iPSC work, we followed our methods listed in the eLife paper exactly. I will paste them below again for your convenience. As a note: For organoid work, a lot depends on the cell line, the cell density, the media before aggregation, the lots of the ingredients, etc etc. You may be better off buying one of the organoid generation kits from the usual sellers (not sure I am allowed to advertise here), which have stringent QC for their ingredients. This will eliminate a lot of the guess work from getting all the right lot numbers with the right bioactivity. 

Hope this helps. If not, contact me via LinkedIn.

Best,

Jan Bruder

from our eLife paper methods:

For cortical organoid generation (see method section below), human induced Pluripotent Stem Cells (hiPSCs) (parental line of AMO line 2 smNPCs) were generated and characterized during a previous study (Reinhardt et al., 2013b). Cells were cultured in Vitronectin (Thermo Fisher)-coated 6-well plates in mTeSR Plus medium (Stemcell technologies) supplemented with 1% penicillin/streptomycin. The medium was changed every other day and cells were passaged at a ratio of 1:10 to 1:15, using accutase when they reached 80–90% confluency. After splitting, the medium was supplemented with the ROCK inhibitor Y-27632 (10 μM, tebu-bio) until the first media exchange.

For the generation of iPSC-based cerebral organoids (see method section below and Figure 5—figure supplement 1) hiPSC culture was performed feeder-free using modified FTDA medium (Frank et al., 2012) in 1% (v/v) Matrigel-coated 6-well plates with iPSCs from the same line as AMO line 2. FTDA medium consisted of DMEM-F12 supplemented with 1% human serum albumin (Biological Industries), 1% Chemically Defined Lipid Concentrate (Life Technologies), 0.1% Insulin-Transferrin-Selenium (BD), 1% penicillin/streptomycin/glutamine. We fed the iPSCs daily and added 10 ng/mL FGF2 (PeproTech GmbH), 0.2 ng/mL TGFβ3 (PeproTech GmbH), 50 nM Dorsomorphin (Enzo Life Sciences), 5 ng/mL Activin A (eBioscience), 20 nM C59 (Tocris) before each media exchange. We split the iPSCs as single cells every 3–5 days using accutase for approximately 10 min at 37°C. We transferred 600,000 cells per well of a 6-well plate to be seeded to DMEM-F12 with 0.1% BSA and centrifuged at 220 g for 2 min. We resuspended the cell pellet in fresh FTDA medium supplemented with 1:2000 ROCK inhibitor Y-27632 (tebu-bio) and plated the iPSCs on Matrigel-coated 6-well plates.