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Running title: Computational fluid dynamics in multiplex brain organoid cultures 23 24 Highlights 41  Improvements to organoid preparation protocol 42  Multiplex suspension culture protocol successfully generate brain organoids 43  Computational fluid dynamics (CFD) reveals emerging properties of suspension 44 cultures 45  CFD of steering plates is equivalent to that of spinner flask cultures 46 Keywords: brain organoids, multiplex culture, stem cell research, 47 computational fluid dynamics analysis 48 Abbreviations: Three dimensional (3D), human pluripotent stem cells (hPSC), 49 induced pluripotent stem cells (iPSC), embryoid bodies (EBs) 50 GlutaMAX, and 1:100 P/S in DMEM/F12) and left for 4 days in static culture. 126 Subsequently, cerebral organoids were grown in suspension using two different 127 platforms: 1) steering plates on a standard orbital shaker (six-well culture plates), 128 agitated at 90 rpm [as proposed by Lancaster and Knoblich (2014)(Lancaster and 129 Knoblich, 2014b)]; and 2) SpinΩ system developed by Qian et al. (Qian et al., 2016), 130which was 3D printed by the company DelthaThinkers using the blueprints provided in 131 the manuscript and coupled to 12-well culture plates, agitated at 60 rpm. In both cases, 132 10 organoids were placed in 3 ml neurodifferentiation medium with vitamin A (day 14). 133