A neural stem cell-derived 3D spheroid model that recapitulates prion infection and pathology.

Prion diseases are fatal, transmissible neurodegenerative disorders marked by the accumulation of misfolded prion protein (PrPSc) in the central nervous system. Most in vitro models, largely 2D cultures, have been used to study prion biology but often fail to capture the brain's structural and cellular complexity. Here, we present a 3D spheroid model of neurons and astrocytes derived from mouse neural stem cells (NSCs) to study prion pathogenesis. NSCs were induced to form spheroids and then infected with Rocky Mountain Laboratory prions. Infected spheroids exhibited apoptosis and gliosis, recapitulating aspects of a neuron-astrocyte microenvironment and enabling the observation of neurodegenerative processes. We also show that the spheroids propagate prions, retain infectivity, and preserve pathological and cellular features after cryopreservation. This model supports sustained observation of prion dynamics over the tested culture period and assessment of therapeutic agents, bridging the gap between simple in vitro systems and in vivo models. Our findings highlight this 3D platform as a useful tool for prion research.