Extended Data Fig. 1: Optimization of culture conditions for murine mammary gland Organoid.
From: Reconstruction of dynamic mammary mini gland in vitro for normal physiology and oncogenesis
a, Confocal images of immunostaining of mammary gland at E13.5, E15.5, E17.5 and P1 showing the cellular heterogeneity, polarity and architecture during mammary gland development (top). Model representation of embryonic mammary rudiment formation from E13.5 to birth (bottom). b, Immunofluorescence staining for the indicated markers of organoid. c, Mammary organoid culture for extended time. d, Representative images of organoids cultured for 20d in sphere formation media. e, Bright-field (top and middle rows) and immunofluorescence (bottom row) images of organoids cultured for 3d in the indicated medium, showing the essential factors for symmetry breaking. f, Representative images of organoids cultured for 3d in the indicated inhibitors. g, Representative images (top and middle rows) of organoids cultured for 3d in basic media with/without addition of FGF2 or the indicated inhibitors. Bottom row: Immunofluorescence staining of the frozen section of the organoids. h, Immunofluorescence staining for the indicated markers of the frozen section of the organoids. i, Quantification of Ki67+ cells in mammary organoids under various conditions. n = 3, biological replicates. Data are represented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. j, Motility trajectory plots of individual cells after treatment of indicated factors. X and Y represent spatial coordinates. The unit for X and Y axis is μm. k, Quantification of the speed of movement for the individual cells under indicated treatments. Each panel displays superimposed trajectories of 20 individual cells. n = 3, biological replicates. Data are represented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. l, Time-lapse movie of mammary organoid formation and initiation of symmetry breaking. The red dashed line indicates the formation and healing of the cavity. m, Representative images of organoids cultured in collagen I. n, Representative images of organoid outgrowths in ECM of different ratio of Matrigel and Collagen I. The scale bar represents 500 μm (black) in b, c, d, e, f, g, m, n, 50 μm in a, b, e, g, h (white), l.
