FIGURE 3.

Propagation of HS‐1 from the biopsy‐PDO. (A) Effect of culture conditions. Phase contrast images of HS‐1 cultured under various conditions. Organoid culture medium is supplemented with 10% FBS or defined growth factors (DGF) without serum. Scale bars, 250 μm. (B) Cell proliferation of HS‐1 in culture. The cell numbers were counted using the trypan blue dye exclusion method. (C) Phase contrast images of HS‐1 in the medium with DGF in the absence of Matrigel. Small aggregates are distributed on the surface of the well, while large aggregates expand upward (arrow). Scale bars, 50 μm. (D) Dual staining of cell aggregates. Semi‐floating and floating cells are stained. Dead cells (red) and viable cells (green) are differentially stained. (E) Schematic illustration of the HS‐1 propagation. Cells grew in a spherical shape in Matrigel, whereas the aggregate grew larger and eventually detached from the dish (arrow) in the absence of Matrigel. (F) Western blotting of the lysate and supernatant of HS‐1. Ponceau S staining of the membrane serves as a loading control. (G) Western blotting of the culture supernatant. Time‐lapse samples are analyzed for secreted trypsin.