Cortical organoid diffusion MRI reveals protocol-associated spatial reproducibility and increasing apparent diffusivity with diffusion time

Early human cortical development involves rapid cytoarchitectural changes that are difficult to study non-invasively. Diffusion MRI can probe developing microstructure and membrane water exchange, but model assumptions are difficult to validate in human tissue. Cortical organoids provide a tractable platform for relating diffusion measurements to tissue architecture. We scanned six fixed cortical organoids at 9.4 T, three grown under directed and three under undirected differentiation protocols, at three diffusion times and b-values up to 7000 s/mm ² . Center-to-periphery profiles of diffusion kurtosis imaging (DKI) metrics were reproducible across directed organoids but more variable across undirected organoids, consistent with nuclear staining. Unexpectedly, DKI-derived apparent mean diffusivity (MD) increased with diffusion time in more than 80% of tissue voxels in both batches, opposite to previous organoid measurements. Across 12,348 analytically simulated two-compartment substrates and Monte Carlo diffusion simulations in sphere packings, this trend emerged in permeable substrates with a marked contrast between extracellular and apparent intracellular diffusivities. Detailed Monte Carlo trajectory analysis of a representative substrate showed that fitted MD increased despite decreases in displacement-derived and second-cumulant diffusivities, revealing an exchange-sensitive finite-b effect involving higher-order signal contributions. These findings support using cortical organoids to probe early microstructure, test diffusion models, and investigate potential emerging diffusion biomarkers.