Aggrewell 400 24 well plate

A commercially available microwell culture plate was used to generate spheroids (AggreWell 400 24-well plate, 34411, StemCell Technologies) following the manufacturer's instructions. The microwells were first coated with an anti-adherence rinsing solution (07010, StemCell Technologies) and centrifuged at 2,000 g for 5 min. The solution was then replaced with fresh NBD + RI + laminin and left at 37°C until use. Glial cells were detached from their flask and iNeurons were thawed as detailed above. iNeurons were seeded to form spheroids of 250 cells on average (relying on the statistical distribution of the cells in the microwell). Glial cells were added to two thirds of the iNeuron-containing microwells, at a ratio of either 1:5 or 1:2. Moreover, spheroids purely comprised of glial cells were generated to confirm the lack of electrical activity in the absence of neurons. The microwells were then placed in an incubator for 2 days.

CI-hnESCs were treated with Accutase at 37 °C for 5 min and neutralized with DMEM/F-12, followed by gentle mechanical dissociation with a pipette. After centrifugation, the cell pellet was resuspended in 5iLAF-I medium and then transferred onto gelatin-coated plate and incubated at 37 °C for one hour to remove feeder cells. After feeder cell adhesion, the supernatant was collected and the cell number was determined using Countstar Mira FL with AOPI fluorescence staining to assess cell viability. 3.5 × 10⁴ cells were resuspended in 1 ml of 5iLAF-I medium and seeded into one well of an AggreWell 400 24-well plate (Stemcell,34411) pretreated with anti-adherence rinsing solution (Stem Cell Technologies, 07010) following the instructions and cultured under 20% O₂ and 5% CO₂ at 37 °C. Aggregates formed the other day, and the medium was replaced every day. Usually, spontaneous cavitation took place gradually from day3. To block OXPHOS when generated blastoids, 5 μM OXPHOS inhibitor IACS-010759(Selleck, 8731) was applied once cells were seeded in AggreWell. To induce HYP-like cells, SB590885 and PD0325901 were removed from day 3 or change medium to HDM^{19 (link)} or IVC^{64 (link)}.