Figure 2. PHH spheroids can be maintained for at least 5 weeks in serum-free conditions.

(A) H&E staining of PHH spheroids. Note that spheroid sizes decreased coinciding with increased expression of E-cadherin (B), which has been previously shown to promote spheroid compaction⁵. (C) Levels of cleaved caspase-3, a marker for apoptosis, remained at low levels throughout the culture period as determined by immunohistochemistry. (D) MRP2 immunostaining revealed bile canaliculi at early as well as late stages of spheroid culture. (E) Staining for the perivenous marker CYP3A4 and the periportal marker albumin reveals that the zonation-identity of liver cells is maintained. (F) Cellular ATP levels remained constant throughout 5 weeks of culture (n = 20 spheroids from 3 donors per time point). Absolute ATP values were normalized to spheroid volume to compensate for compaction. All scale bars = 100 μm.