Self-assembly of hepatic tissue equivalents and improvement of their functions through gene delivery.

摘要

Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids, where they exhibit ultrastructural characteristics of the native liver tissue and enhanced liver-specific functions. Therefore, hepatocyte spheroids represent an ideal model system for studies related to liver tissue architecture and function.; The spheroid formation process involves redistribution of cell-cell and cell-substratum adhesion forces and reorganization of the cytoskeleton, which is the major apparatus for force generation. To elucidate the function of the cytoskeleton, hepatocytes in static cultures, which promote spheroid formation, were treated with cytoskeleton-disrupting drugs. Disorganization of actin filaments caused inhibition of spheroid formation whereas perturbation of microtubules had minimal effects on spheroid assembly. Spheroids formed in the absence of drugs or in the presence of taxol or nocodazole were morphologically and functionally similar.; For hepatic tissue engineering, aside from the mechanism that cells employ to form tissue-like structures, maintenance of differentiated functions within these structures is also important. Cytochrome P450 2B1/2 (CYP2B1/2), which mediate phase I biotransformation of xenobiotics in the rat liver, exhibit low constitutive expression, which is highly inducible by phenobarbital. CYP2B1/2 activities were assessed in situ in cultured hepatocytes via 7-pentoxyresorufin-O-dealkylation (PROD) and confocal laser scanning microscopy. Compared to hepatocyte monolayers cultured on collagen, spheroids exhibited higher levels of CYP2B1/2 activity with maximum PROD activity at the aggregate center, and retained their ability for PB induction.; Hepatocyte CYP2B1/2 activity was boosted by transferring the CYP2B1 gene with the cytomegalovirus promoter, via recombinant adenovirus. Spheroids of transduced hepatocytes displayed CYP2B1/2 activity above its constitutive level for more than 3 weeks. In parallel studies, spheroid-forming hepatocytes were transfected with an adenovirus carrying the gene for green fluorescence protein (GFP). GFP fluorescence was detected for more than 33 days. Prolonged transgene expression in hepatocyte spheroids had minimal effects on other liver-specific functions. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to understand how to control the cellular assembly and function in tissue engineering and clinical applications.