The development of 3D organoids has provided a valuable tool for studying human tissue and organ development in vitro. Cerebral organoids, in particular, offer a unique platform for investigating neural diseases. However, current methods for generating cerebral organoids suffer from limitations such as labor-intensive protocols and high heterogeneity among organoids. To address these challenges, we present a microfluidic device designed to automate and streamline the formation and differentiation of cerebral organoids. The device utilizes microwells with two different shapes to promote the formation of a single aggregate per well and incorporates continuous medium flow for optimal nutrient exchange. In silico simulations supported the effectiveness of the microfluidic chip in replicating cellular microenvironments. Our results demonstrate that the microfluidic chip enables uniform growth of cerebral organoids, significantly reducing the hands-on time required for maintenance. Importantly, the performance of the microfluidic system is comparable to the standard 96-well plate format even when using half the amount of culture medium, and the resulting organoids exhibit substantially developed neuroepithelial buds and cortical structures. This study highlights the potential of custom-designed microfluidic technology in improving the efficiency of cerebral organoid culture.

Core Facility Cellular Imaging CEITEC Masaryk University Brno Czech Republic

Faculty of Medicine Department of Histology and Embryology Masaryk University Brno Czech Republic

Institute for Chemical and Bioengineering Department of Chemistry and Applied Biosciences ETH Zürich Zürich Switzerland

Institute of Molecular Genetics of the Czech Academy of Sciences Laboratory of Genome Integrity Prague Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

Loschmidt Laboratories Institute of Experimental Biology and RECETOX Faculty of Science Masaryk University Brno Czech Republic

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