Tissue-like structures of cells organized in vitro have a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1 transgene did not impair these liver specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices.
Bibliographical noteFunding Information:
This work was supported in part by grants from the National Institute of Health (DK45371 to W.S.H. and CA49248 to D.J.W.) and the National Aeronautics Space Administration (NAG8-1349). E.S.T. was supported by a NIGMS Biotechnology traineeship (T2GM-083478). We thank Dr. B. Massie for providing us with the AdGFP vector. The resources support from the Center for Interfac ial Engineering, the University of Minnesota, and the Minnesota Superc omputer Institute is gratefully ac knowledged. Kristine Groehler and Diane Tobolt are also gratefully acknowledged for their technical support.