Extended Data Fig. 1: Characterization of the PiChip model.
a, 3D illustration of three different layers of the microfluidic-based PiChip system. Layer I consists of an orthogonal array of 400 microgrooves of 10 μm height and width and 500 μm length. Layer II consists of BRAIN and four PERIPHERAL compartments of 100 μm height. Layer III consists of a cylindrical chamber with 6000 μm length and radius and 2500 μm open hollow. The top and bottom panels show low and high magnification of each layer, respectively. b, Representative immunofluorescence staining of 3D-differentiated neurons (MAP-2; blue) and astrocytes (GFAP; red) in AD and CTRL cultures after four weeks of differentiation. Scale bar, 100 μm. c, d, Quantification of MAP-2 (c) and GFAP+ (d) expressing surface area in AD and CTRL cultures (n = 4 independent ROIs for each MAP2+ and GFAP+ CTRL, n = 5 independent ROIs for each MAP2+ and GFAP+ AD; P-values from unpaired, two-sided, t-test). e–h, Quantification of soluble Aβ38 (e), Aβ40 (f), Aβ42 (g) and Aβ42/40 ratio (h) in AD and CTRL cultures after four weeks of differentiation (n = 4 cell cultures from two independent experiments; P-values from unpaired, two-sided, t-test with Welch’s correction). i, Representative immunofluorescence staining of p-Tau (PHF-1; blue) in neuronal cell bodies and neurites (GFP; green) in AD and CTRL cultures. Scale bar, 50 μm. j, Quantification of PHF-1+ expressing surface area in AD and CTRL cultures (n=6 ROIs from at least 5 independent experiments; P-values from nonparametric, two-sided, Mann-Whitney test). k, UMAP visualization of a total of 20153 single cells from AD and CTRL cultures by single-cell transcriptome profiles. Distinct cell types are depicted with different colors. l, Volcano plot of differentially expressed genes in astrocytes in AD Neu/AC/iMGL versus AD cultures lacking iMGL (P-values from Wilcoxon Rank Sum test). Significantly upregulated (P < 0.05 and FC > 0.1) and downregulated (P < 0.05 and FC < 0.1) genes are shown in red and blue dots, respectively. m, Violin plot of highly enriched C3 gene in astrocytes in AD Neu/AC/iMGL compared to CTRL (n = 5 independent scRNA-seq data; P-values from nonparametric, two-sided, Mann-Whitney test). n, Vioin plots of selected significantly enriched genes associated with reactive astrocytes (VIM and GFAP) in AD Neu/AC/iMGL compared to AD cultures lacking iMGL (n = 5 independent single cell RNAseq data; P-values from nonparametric, two-sided, Mann-Whitney test). o, Violin plots of highly enriched genes associated with reactive astrocytes and interferon-γ in astrocytes in AD Neu/AC/iMGL compared to AD cultures lacking iMGL (n = 5 independent scRNA-seq data; P-values from nonparametric, two-sided, Mann-Whitney test). (c–h, j) In boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to minimum and maximum values. (m, n) White circles show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; polygons represent density estimates of data and extend to extreme values.
