Computational fluid dynamic analysis of physical forces playing a role in brain organoid cultures in two different multiplex platforms

Livia Goto-Silva,Fernanda Tovar-Moll,Sidarta Ribeiro,Magno Junqueira,Bernard Lamien,Nilton P Silva,Iasmin L Herzog,Gilberto B Domont,Annie Da Costa Souza,Michele Martins,Stevens K Rehen,Nadia M E Ayad,Helcio R B Orlande

doi:10.1186/s12861-019-0183-y

Abstract

BackgroundOrganoid 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.ResultsHere 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.ConclusionOur 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.

Highlights

Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure
Improved embryoid body (EB) formation and analysis of organoid growth To improve the first step of EB formation, we introduced some variations to the protocol published by Lancaster and Knoblich (2014) [9]
Changes in the protocol include the addition of Rho-associated protein kinase inhibitor (RO CKi) for cell survival at cell dissociation [13] and postplating centrifugation [14]. induced pluripotent stem cells (iPSCs) cultivated in mTeSR1 medium derived from manual passages showed better EB formation compared with Ethylenediaminetetraacetic acid (EDTA)-passaged iPSCs

Summary

Introduction

Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Cerebral organoids have been cultured in spinner flasks [9] These flasks have the advantage of providing a low-shear environment [10], which is important because hPSCs have been shown to be sensitive to shear stress [10, 11]. Spinner flasks have the disadvantage of requiring a high volume of cell culture media for cultivation, increasing the costs of experiments. They are limited to drug testing and other multiplex experiments including comparison of multiple patients and controls. Qian et al (2016) [12] proposed the use of a 3D-printed scalable mini-biore actor, the SpinΩ, which would be cost effective and

Results

Discussion

Conclusion