We evaluated HepaRG cells after 13-14 days of differentiation on the fibrous dECM inserts. As shown in Figure , although the number of cells on dECM appeared to be less than on flat, the difference was not significant. There were over 200,000 cells for both conditions. Figure shows confocal microscopic views (top and sides) of hepatocytes in the scaffold. Clearly, the cells could infiltrate by ~50 µm into the fibrous layer to form a 3D culture. Hepatocytes were characterized by the ability to synthesize albumin and produce urea, two key fundamental functions of the cells . As suggested by Figures and, cells on both culture substrates showed albumin and urea production, indicating successful differentiation of HepaRG progenitors to hepatocytes. Interestingly, we did not observe significant differences in albumin production from cells on flat vs. on dECM, suggesting that some biosynthesis pathways of HepaRG hepatocytes might not be affected by the ECM. In contrast, cells on dECM produced significantly more urea than on flat, indicating that the metabolic activities of the hepatocytes might be enhanced by the ECM. Further experiments aligned with this observation, where the metabolic activity as measured by the MTS assay (Figure ) was significantly higher from cells on dECM than on flat for up to 43 days. Between Day 13 and Day 20, cells on both substrates showed increased MTS metabolism (although not significant for cells on flat), and thus, cells within this time frame were used for the following studies.