Synaptic Assembloid Fusion Protocol

Between D90 and D120, TCF7L2-tdTomato⁺ hThOs were paired with VGLUT1-tdTomato⁺ hCOs of similar age. Each pair was transferred to a well of a low-attachment, 24-well plate in 500 mL Fusion media (BrainPhys supplemented with 1× N2, 50× B27 without vitamin A, 1× glutamax, 1× NEAA, 1× antibiotic-antimycotic, 200 µM ascorbic acid, 100 µM cAMP, 1% ES-FBS, 10 µM DAPT, 20 ng/mL BDNF, 20 ng/mL GDNF, and the CEPT cocktail (50 nM Chroman 1 [HY-15392, MedChem Express], 5 µM emricasan (S7775, Selleckchem), 0.7 µM trans-ISRIB (#5284, Tocris), and polyamine supplement (#P8483, Sigma-Aldrich^{105 (link)}) supplemented with 0.5% v/v GFR-Matrigel. The plate was left tilted and undisturbed in the incubator. After 3 days, 60% of the media in each well was replaced with fusion media. This was done slowly, while keeping the plate tilted with minimal disturbance to the “fused” organoid pair in each well. The same was done on D6 and D9. Subsequently, 80% of the media was replaced every 3 days. On D4, the plate was transferred to an orbital shaker at 80 rpm. The shaker speed was increased to 90 rpm on D5, 100 rpm on D6, and starting D7, the assembloids were kept at 110 rpm. Between 5 and 10 weeks postfusion, assembloids were harvested for electrophysiological experiments.