Figure 4.

In vitro generation of millimeter-scale, colonic epithelium with a polarized architecture from primary tissue. (A) Culture of colonoids in a PDMS microwell. A crypt was loaded into a microwell on the array. By 48 h, a 3D colonoid formed that filled the microwell. By 96 h, a 2D/3D hybrid culture was formed. The top panels are brightfield images while the bottom panels are overlaid red/green fluorescence images. (B) Overlaid fluorescence image of the 2D/3D hybrid culture at 96 h (green = EGFP, red = Muc2 stained by immunofluorescence, blue = nuclei stained with Hoechst 33342). (C–D) SEM image of colonic epithelial tissues while still on the PDMS microwell array. The 2D/3D hybrid culture was imaged at 96 h (C) and 144 h (D). (E) SEM images of a 1.7 mm² section of the in vitro-generated colonic epithelial tissue transferred to a tape so that the tissue is viewed from its underside. (F) A close-up of the underside of the tissue shown in (E). Scale bars = 200 μm. The Matrigel plug is not visualized in the SEM images since its protein concentration (~4 mg/mL) is too low to maintain a solid structure during fixation.