Improved quality and reproducibility of cerebral cortical organoids under microfluidic culture conditions: a proof-of-concept study

Source abstract

Abstract Cerebral organoids have emerged as advanced three-dimensional in vitro models capable of recapitulating essential aspects of human brain development, thereby offering new opportunities for studying brain physiology and disease. Nonetheless, their use in preclinical settings remains limited by their high variability. Microfluidic devices offer a promising alternative to conventional culture supports by enabling improved recapitulation of the cellular microenvironment; however, most cerebral organoids-on-chips rely on custom-made academic prototypes, limiting their scalability and translatability. Here, we established a brain organoid-on-chip model by combining a regionalized cortical organoid differentiation protocol with a commercial, standardized and industrially-compatible microfluidic device (Duplex™, NETRI). Cortical organoid quality and reproducibility were evaluated using a previously established multi-criteria Quality Control (QC) framework based on a scoring system. To enhance objectivity and throughput, the non-invasive morphometric QC criteria were automated using deep learning-based image analysis. Using a single human induced pluripotent stem cell (hiPSC) line, we assessed whether microfluidic culture conditions could improve the quality and reproducibility of cortical organoids within a controlled genetic background. Compared with conventional cultures, on-chip cortical organoids demonstrated improved inter- and intra-batch reproducibility across multiple scales, including morphology, size, cytoarchitectural organization, and transcript expression, together with higher QC scores and reduced QC failure rates. Finally, the QC framework was adapted for neurotoxicity assessment using a decision tree to classify compounds according to their neurotoxic risk. While this study represents a proof-of-concept within this cellular background, the integrated brain organoid-on-chip platform and standardized QC evaluations provide a flexible and translatable framework for preclinical and industrial studies, and may facilitate future cross-line validation of reproducible cortical organoid cultures.