LAUSR

Human midbrain organoids with functional dopaminergic (DA) neurons are invaluable for the therapeutic development of Parkinson's disease (PD). However, current methods face significant limitations, including challenges in generating pint‐sized organoids enriched with DA neurons and the lack of robust functional assays for efficiently evaluating neural networks over extended periods. Here we present an innovative approach that combines developmental patterning with mechanical cutting to produce small midbrain organoids, with diameters less than 300 μm, suitable for long‐term evaluation, along with a comprehensive functional assay system consisting of calcium transient assay, neurite extension assay, and multielectrode array (MEA) assay. Radial cutting of organoids into four to eight portions according to their sizes at the appropriate developmental stage significantly increases the yield of viable organoids while reducing necrotic cell regions. Using the functional assay system, we demonstrate that DA neurons within the organoids extend long projections, respond to dopamine stimulation, and form neural networks characterised by giant depolarising potential‐like events. Our approach supports the generation of midbrain organoids and PD models that can be used for long‐term functional testing.