2D and 3D in vitro Alzheimer's disease model

Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive decline. The most well-known pathophysiology of AD is the accumulation of Aβ peptides and phosphorylated Tau protein. The accumulation of Aβ peptides results in senile plaques, while phosphorylated Tau leads to the formation of neurofibrillary tangles. Other pathophysiologies include neuroinflammation, cholinergic neuron degeneration, and genetic disorders. Reliable and realistic in vitro models are needed to fully elucidate the pathogenesis of AD, the most common neurodegenerative disease affecting millions of people worldwide, understand its stages, and develop new treatment strategies. This thesis aims to create innovative in vitro models to contribute to drug-active ingredient research for AD. In this regard, in vitro AD models were developed using both two-dimensional (2D) and three-dimensional (3D) micro-tissue techniques using the Aβ1-42 peptide of SHSY5Y and N9 cell lines. During the modeling process, both 2D and 3D in vitro models were created using the SHSY5Y cell line, which exhibits cholinergic neuron characteristics, using the Aβ peptide, a pathophysiological marker. Subsequently, to examine the relationship between AD and the immune system and to create a model more similar to the in vivo model, the N9 microglial cell line was added to the model. Lipopolysaccharide (LPS) was added to the medium to ensure microglial activation. Subsequent characterization of the generated AD models included cell viability with Alamar BlueTM, quantitative real-time polymerase chain reaction (qRT-PCR) to determine the expression levels of AD-related genes, acetylcholine esterase (AchE) activity and phosphorylated tau (pTau) levels. The findings suggest that the 3D in vitro AD model developed exclusively with SHSY5Y cells holds significant promise for future research. However, in 2D and 3D AD models based on the neuroinflammation hypothesis, created by co-culture of SHSY5Y and N9 cells, cell-cell interactions and proinflammatory activity were not observed at the expected levels. This finding demonstrates the critical role of cell type compatibility in modeling intercellular interactions.