Abstract
Brain organoids are a promising model for studying human neurodevelopment and disease. Despite the potential, their 3D structure exhibits high variability during differentiation across batches and cell lines, presenting a significant challenge for biomedical applications. During development, organoids are exposed to fluid flow shear stress (fFSS) generated by the flow of culture media over the developing tissue. This stress is thought to disrupt cellular integrity and morphogenesis, leading to variation in organoids architecture, ultimately affecting reproducibility. Understanding the interplay between tissue morphology, cell identity and organoid development is therefore essential for advancing the use of brain organoids. Here, we demonstrate that reducing fFSS, by employing a vertically rotating chamber during neuronal induction, a critical phase for organoid morphogenesis, along with an extended cell aggregation phase to minimize fusions, significantly improves the reproducibility of brain organoids. Remarkably, reducing fFSS minimizes morphological structure variation and preserves transcriptional signature fidelity across differentiation batches and cell lines. This approach could enhance the reliability of brain organoid models, with important implications for neurodevelopmental research and preclinical studies.