Air-Liquid interface midbrain organoids model the pathological features of Parkinson's disease.

Human-induced pluripotent stem cell-derived midbrain organoids offer a promising platform for modeling Parkinson's disease (PD). Yet, their utility has been limited by the absence of microglia and the development of a necrotic core during maturation. Here, we present an air-liquid interface (ALI) slice culture system for extended cultivation of midbrain organoids (mORGs), enabling efficient microglial integration, improved neuronal viability, and enhanced functional maturation. Compared with conventional mORGs grown in suspension cultures, the ALI method supports more consistent engraftment of microglial progenitors and the development of astrocytes and oligodendrocyte progenitors, as revealed by single-cell RNA sequencing. Functionally, ALI-mORGs exhibited robust and reproducible neural network activity, with N-methyl-D-aspartate (NMDA) stimulation reliably inducing synchronous bursting, as measured by 3D microelectrode array recordings. Importantly, exposure to alpha-synuclein (αSyn) preformed fibrils triggered the progressive accumulation of phosphorylated αSyn inclusions, closely recapitulating key features of PD pathology. The ALI-mORG model addresses major limitations of existing systems and provides a more physiologically relevant platform for investigating cellular mechanisms of PD and for supporting future therapeutic strategies.