A–D: At 2 weeks post-engraftment, large clusters of HFH (white arrows) were observed in gels containing HFH, Bcl-2-HUVEC and rCI/hFN alone (A), or with additional mLBM (B), additional HGF-HUVEC (C), or additional mLBM and HGF-HUVEC (D). HFH clusters were surrounded by multiple layers of endothelial cells when cast in gels containing mLBM (white boxes in B and D). E–F: TEM analyses confirmed the presence of a Disse-like space in vivo, bordered by HFH and endothelial cells and containing matrix proteins (letter D, H, and E, respectively, in panels E and F and letter M in panel F). Size bars: 10 µm (A–D), 2 µm (E), and 500 nm (F). G–I: Analysis of mRNA expression in gel tissue revealed loss of hepatocyte phenotype in HFH-containing gels, based on lower levels of albumin (G) and HNF4α (H), and higher levels of CK7 (I), compared to Huh-7.5-containing gels. Albumin and HNFα expression (G and H, respectively) was slightly better maintained by inclusion of LBM and/or HGF-HUVEC. The presence of continued HGF expression in vivo was confirmed by HGF qRT-PCR (J) and most prominent in gels containing mLBM. Data in panels G–I is expressed as a ratio of target gene mRNA to GAPDH mRNA internal control.