Self-assembly and 3D Bioprinting of Neurospheres and Evaluation of Caffeine and Photobiomodulation Effects in an Alzheimer’s Disease In Vitro Model

Several in vitro models of Alzheimer’s disease (AD) rely on 2D cell culture, and, more recently, 3D cultures represented by free-floating neurospheres have been used as models for the disease. The advantage of 3D over 2D cell culture is that cell-extracellular matrix and cell-cell interactions can be assessed, better representing the molecular and cellular hallmarks of the disease. In the current study, we developed two complementary 3D neurosphere models using SH-SY5Y human neuroblastoma cells to investigate AD pathology and evaluate potential therapies. First, self-assembled neurospheres were exposed to hydrogen peroxide (H₂O₂) and amyloid-beta oligomers (AβOs), inducing AD-like features such as increased production of reactive oxygen species (ROS), amyloid aggregation, and apoptosis. Treatment with caffeine or photobiomodulation (PBM) using LED irradiation significantly reduced Aβ1−42 accumulation, ROS generation, and decreased apoptosis markers. Second, 3D bioprinting of SH-SY5Y cells resulted in neurospheres with enhanced cellular organization and differentiation. These findings emphasize the advantages of 3D models for studying neurodegeneration and evaluating therapeutic strategies, bridging the gap between traditional 2D cultures and complex in vitro systems.Graphical AbstractDevelopment of two 3D neurosphere models, using self-assembly and bioprinting of SH-SY5Y cells, to replicate Alzheimer’s disease (AD) hallmarks. Treatment with 1 mM caffeine or 660 nm LED photobiomodulation (PBM) effectively prevent amyloid-beta aggregation, ROS production, and apoptosis, showcasing the potential of these therapies for AD.