Fig. 8

Suspension culture of DEOs. a–d DEOs grown in Matrigel for 2 weeks with +NFDNiA isolated from the gel and cultured in suspension with additionally supplemented with smoothened agonist (SAG). Bright-field image of a DEO (a, left panel) and magnified image of the area indicated by red lined box (a, right panels, red and blue arrows indicate crystals growing and being shed from the organoid, respectively). The shed crystals were collected and attached on slide glass for immunohistochemistry (b and c) or subjected into inductively coupled plasma (ICP) mass spectrometry for analysis of metal ion composition (d). Bright image of attached crystals (b). Confocal image of crystals costained for amelogenin (c, green) and ameloblastin (c red). Nucleus were counterstained with TO-PRO-3 (c, blue). Scale bar = 20 μm (c)