Proliferation and cell death in hypoxic and normoxic 3D-cultures in ECM-derived substrate. A

<p>Ki-67 immunofluorescence (green) and actin (red) staining of primary human breast epithelial cells in 3D-culture in ECM-derived substrate at 21% and 1% oxygen for 12, and 21 days. Representative images from one of three independent experiments with breast epithelial cells isolated from different healthy individuals are shown. Size bars 20 µm. <b>B.</b> MCF-10A cells stained for Ki-67 (green) and actin (red) after 3, 6, 12, and 21 days of 3D-culture in ECM-derived substrate under normoxic (21%) or hypoxic (1%) conditions. Representative images from one of three independent experiments are shown. Size bars 20 µm. <b>C.</b> Percentage of cells with Ki-67 positive nuclei in normoxic and hypoxic MCF-10A cell organoids 3, 12, and 21 days post-seeding, in three independent experiments. Statistical analysis was performed with Student’s paired t-test (p). In each experiment at least 200 cells were included in the calculation. <b>D.</b> Cell death in MCF-10A cells grown in 3D-culture under normoxic and hypoxic conditions for 12 days, by <i>in situ</i> cell death detection (red), nuclear staining with DAPI (blue). All confocal micrographs were acquired at the Z-plane where the depictured acini-like structure had the widest circumference. Size bars 20 µm. <b>E.</b> Percentage of cells with nuclei positive for <i>in situ</i> cell death detection in normoxic (21%) and hypoxic (1%) 3D-cultures at 9 and 12 days post-seeding. Data from four experiments are shown. In each experiment at least 200 cells were included in the calculation.