Stem-cell-based in vitro models offer promising potential to elucidate pathomechanisms underlying neurological diseases such as Alzheimer’s disease. However, harnessing this potential is currently impaired by low reproducibility, maturity, and cell-type diversity of models and lack of AD-relevant pathologies. Therefore, we developed a novel 3D cortical tissue model containing neurons, astrocytes, and microglia with high reproducibility, maturity, and viability. Upon knock-in of AD-causing APP mutations we found typical pathologies such as time- and cell-type-dependent accumulation and aggregation of Aβ, increased phosphor-tau levels, and microglia activation. To get an unbiased overview of pathology-related changes, we performed proteome analysis of our cultures containing different cell types at different timepoints.