FIG. 5.

Micropatterned PEG–fibrinogen hydrogels support three-dimensional polarization of primary hepatocytes and the formation of a functional bile canalicular network. Cells were plated and cultured in hepatocyte growth medium without EGF as previously described and then fixed and stained for the indicated epitopes. Single confocal cross-sections taken within the centermost 25% of the structure are depicted. (A, B) Cells cultured for 3 days and stained for CD26 (green), nuclei (blue), and the actin cytoskeleton (orange); samples are imaged at 10 × (A) and 63 × (B). (C, D) Cells cultured for 7 days and stained for CD26 (orange), FN (green), and nuclei (blue); samples are imaged at 20 × (C) and 40 × (D). (E) Visualization of functional bile canaliculi by confocal microscopy on day 6 using 5(and 6)-carboxy-2′,7′-dichlorofluorescein (CDF) diacetate at 20 × (with digital zoom of red highlighted region in top right inset). For reference, support scaffold channels have d = 340 μm. Scale bar = 50 μm in each image. Note regions of plasma membrane showing CD26 and FN colocalization (arrows), and concentrated CDF staining in the three-dimensional tissue structure, which is similar to CD26 staining in A and B (arrowheads = bile canaliculi; dotted arrows = intracellular uptake of CDF). Color images available online at www.liebertonline.com/tea.