FIG. 3.

Morphological characterization by confocal microscopy of cysts formed by differentiated hiPSCs on a suspended mesh sheet. (A) A schematic of setup during confocal microscopy. Focus was shifted up and down to capture images of the cysts for 3D rendering. (B) A 3D reconstruction showing both the apical (i) and the basal (ii) surfaces of two different spherical cysts hanging downward from a mesh sheet. Both images illustrate that the cysts were closed spheres. To obtain the images, mesh sheet was turned upside down and imaged as shown in (A). (C) High resolution imaging of a representative cyst cultured continuously on mesh and showing vascularization. (i) A phase contrast image of a mesh generated cyst at day 40. (ii) A 3D reconstruction of a cyst stained with Calcein AM with a view window showing vascular-like cellular networks in the interior. “INT” denotes interior, “EXT” denotes exterior of a cyst. (iii) A 3D reconstruction based on maximum intensity projection showing vascularization (yellow arrows) in the same cyst as in the left image. Pseudo coloring was applied for easy visualization. Scale bar represent 200 μm in (i) and 100 μm in (ii) and (iii).