Fig 1: Long-term stable passage of organoids. (A) Workflow for the isolation of human endometrial epithelial glands and culture in a 3D extracellular Matrigel plug. Collect human endometrial tissue, obtain human endometrial primary cells, and construct EEGOs. Scale bar: 100 μm. (B) Immunofluorescence microscopy of MUC1 (red), E-Cadherin (red), EpCAM (red), and DNA (blue) in human primary endometrial cells. Scale bar: 20 μm. (C) Brightfield images of organoids formed within 8 days. Scale bar: 200 μm. (D) Brightfield images of organoids under 500 ng/ml and 250 ng/ml R-spondin conditions. Scale bar: 200 μm. (E) Diameter of organoids on days 3, 5, and 7. (F) Number of organoids per drop of Matrigel on days 3, 5, and 7. (G) Brightfield images of different passages demonstrating the long-term expansion of EEGOs. Scale bar: 200 μm. The results are shown as the mean ± SEMs of triplicate samples and are representative of three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.