At 60d and 90d of culture, we subjected organoids from a healthy control (Ctrl) and a familial (fAD) patient (APP duplication, APPDp1-1, reported in Israel et al., 2012) to a number of assays for AD-relevant phenotypes. We also examined additional fAD lines in (S1 Table). As the organoid continues to increase in size, the deeper regions of the tissue show evidence of necrosis, which is likely due to the absence of vasculature and lack of nutrients and oxygen penetration in the deeper layers. As the presence of necrosis could confound our measurements of AD-like phenotypes. We used immunohistochemistry for cleaved caspase 3 (CC3; S3A Fig) as an indicator of apoptotic cells and calculated the average distance between the surface and regions of increasing CC3 immunoreactivity. We found that the region of neuron-rich, CC3-sparse tissue extended an average of 250 μm from the surface into the interior of the organoid (S3A Fig). In addition, we incubated sections with secondary antibody alone (after blocking) to assess the presence of nonspecific binding. We observed that the inner tissue region also gave rise to
