Recombinant human brain derived neurotrophic factor bdnf

On day 19 of the differentiation protocol, cells were gently detached from the plates and re-plated with 1:3 splitting in the same conditions, for neuronal differentiation and drug treatment. From day 19 to day 30 of differentiation CBD, Δ⁹-THC and two different SCs, EG-018 and THJ-018 were added to the medium at every medium change at a concentration of 10 μM in ethanol, except CBD, which was added at a 1–10 μM concentration. Untreated and vehicle (0.01% ethanol)-treated cultures were used as a control. At day 30 gentle replating was performed once again and the cultures were allowed to mature in complete BrainPhys^TM Neuronal Medium (StemCell Technologies)—supplemented with NeuroCult^TM SM1 Neuronal Supplement (StemCell Technologies), N2 Supplement-A (StemCell Technologies), Recombinant Human Brain-Derived Neurotrophic Factor (BDNF, PeproTech, 20 ng/ml), Recombinant Human Glial-Derived Neurotrophic Factor (GDNF, PeproTech, 20 ng/ml), dibutyryl cAMP (1 mM, Sigma), and ascorbic acid (200 nM, Sigma). One-third of the medium volume was changed every 3 days.

On day 13, the medium was half-changed to N2B27 (+Vitamin A) without any small molecule supplementation and was maintained in the presence of this medium until day 41. This medium was replaced every 2/3 days. On day 41 of differentiation, the organoids were cultured in BrainPhys^TM Neuronal Medium (StemCell Technologies), supplemented with NeuroCult^TM SM1 Neuronal Supplement (StemCell Technologies), N2 Supplement-A (StemCell Technologies), Recombinant Human Brain Derived Neurotrophic Factor (BDNF, PeproTech, 20 ng/mL), Recombinant Human Glial-Derived Neurotrophic Factor (GDNF, PeproTech, 20 ng/mL), dibutyryl cAMP (1 mM, Sigma), and ascorbic acid (200 nM, Sigma). One third of the total volume was replaced every 2–3 days.