that were immunopositive for both Aβ antibodies (Fig 1B), while 4G8 immunoreactivity appeared against intracellular amyloid that co-localizes with MAP2 as well as putative extracellular aggregates (Fig 1B, S4A Fig and S1 and S2 Movies). We then used the D54D2 antibody to quantify the size and number of Aβ aggregates. The 90d fAD organoids contained numerous Aβ aggregates compared to the control organoid tissue (Fig 1C). Analysis at 60d and 90d of culture identified a progressive increase in number and size of Aβ aggregates in the fAD organoids compared to controls (Fig 1D and S4B Fig). Additionally, we performed Western blot on the 90d whole organoids lysates and observed increased levels of Aβ oligmers in fAD organoids compared to controls (data not shown). These data demonstrate the presence of robust and spontaneous Aβ aggregation in AD patient-derived organoid culture models that appears to develop in a time-dependent manner.
