After various trials of fusion strategies, we found spontaneous fusion of two closely-positioned hMGEO and hCO was the most efficient and convenient approach. Briefly, on day 18 of organoids culture, single hMGEO and single hCO were transferred into one well of ultra-low-attachment 96-well plate and cultured statically for 3 days to allow spontaneous fusion sufficiently. Half of the neural differentiation media was carefully changed 2 days after fusion, without agitating the organoids on the bottom. 3 days later, the fused organoids (hfMCOs) were transferred to ultra-low-attachment 6-well plate for spinning culture, and the following culture conditions were the same with cultures of hMGEOs and hCOs. To generate hfMCOs for neuronal calcium imaging, H1 hESCs-derived hMGEOs and hCOs were incubated with media containing AAV1.syn.GCaMP6s.WPRE.SV40 and Lenti-hSyn-RFP virus overnight, respectively. The next day, Gcamp6s-labeled hMGEOs and RFP-labeled hCOs were fused as described above.
