All-in-one generation and multiomic profiling of human whole brain organoid on a millifluidic plate

Human brain organoids (hBOs) have been recently regarded as neurobiologically relevant brain models and exponentially exploited in a variety of neuroscience research. However, the current gold-standard method for generating hBOs is intricate and laborious, resulting in hBOs with morphological variability and inconsistent batch-to-batch reproducibility. Despite several studies reporting simplified hBO culture methods, few of those methods was biologically validated with multiomic profiling, which is crucial for neurobiological studies. Here, we demonstrate an all-in-one millifluidic plate (AIOMP) with individually perfusable microchambers for hBOs, simplifying the culture process, improving the uniformity and reproducibility, and enabling long-term cultivation and real-time morphogenesis observation. Additionally, our comprehensive transcriptomic and proteomic analyses revealed that AIOMP increases neurogenesis and corticogenesis of hBOs, suggesting a stronger correlation between the AIOMP-generated hBOs and human fetal brain than those generated through conventional method. Metabolomic and neurophysiological results further support the maturation-enhancing effects of AIOMP on hBOs, showing improved neurotransmitter synthesis and electrophysiological functionality. Overall, the AIOMP approach offers a simplified, reproducible, and biologically validated method for hBOs generation and maturation, with potential applications in neurobiology, neurological disease research, and central nervous system drug assessment.