DMEM/F-12

Protocol tips

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	Keep organoids under static culture conditions with media changes every third day for 10, 35, or 70 days

Protocol tips
Keep organoids under static culture conditions with media changes every third day for 10, 35, or 70 days

Publication protocol

Differentiation of mfNPCs: For the differentiation into mDANs, 350,000 mfNPCs were seeded into a 12-well. Since SB and LDN are detrimental for differentiation, both small molecules were left out when seeding the cells. mfNPC expansion medium (without SB and LDN) was changed 2 days after seeding to N2B27 medium with 0.5 µM SAG, 0.7 µM CHIR, and 200 µM AA. After 4 days of patterning, maturation of the neurons was induced by changing the medium to N2B27 medium with 10 ng/ml brain-derived neurotrophic factor (BDNF, Peprotech), 10 ng/ml glial cell-derived neurotrophic factor (GDNF, Peprotech), 200 µM AA, 500 µM dibutyryl camp (Sigma), 1 ng/ml TGF-β3 (Peprotech), and 2.5 ng/ml ActivinA (Peprotech). When cultures became over-confluent during maturation, they were split into single cells using Accutase, or as small clumps using a cell scraper. To increase the maturation of differentiating cultures, the maturation medium was supplemented with 5–10 µM dual antiplatelet therapy (DAPT) (Cayman). Cultures were analyzed after 8–10 days under maturation conditions, unless otherwise indicated. N2B27 medium consists of Dulbecco’s modified Eagle’s medium/Nutrient Mixture F12 (DMEM-F12) (Invitrogen)/Neurobasal (Invitrogen) 50:50 with 1:200 N2 supplement (Invitrogen), and 1:100 B27 supplement lacking vitamin A (Invitrogen) with 1% penicillin/streptomycin/glutamine (Biochrome).
To generate midbrain-specific organoids, 3,000 cells were seeded per well to an ultra-low attachment 96-well round bottom plate and kept under maintenance conditions for 7 days. To start the pre-patterning, LDN and SB were withdrawn and after 3 additional days, the concentration of CHIR was reduced to 0.7 µM similar to 2D culturing. On day 9 of differentiation, the medium was changed to neuronal maturation medium including 10 µM DAPT, as described above. The organoids were kept under static culture conditions with media changes every third day for 10, 35, or 70 days.


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