Background
Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids has not been determined.
Results
Here we used computational fluid dynamics (CFD) to simulate two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the computed variables of the steering plates were closer to those of the spinning flask.
Conclusion
Our protocol improves the initial steps of the standard brain organoid formation, and the produced organoids displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.
Electronic supplementary material
The online version of this article (10.1186/s12861-019-0183-y) contains supplementary material, which is available to authorized users.
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
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