ARTICLE Perfusion of 3D Encapsulated Hepatocytes — A Synergistic Effect Enhancing Long-Term Functionality in Bioreactors Rui M. Tosto ˜ es, 1 Sofia B. Leite, 1 Joana P. Miranda, 1 Marcos Sousa, 1 Daniel I.C. Wang, 2 Manuel J.T. Carrondo, 1,3 Paula M. Alves 1 1 Animal Cell Technology Laboratory, IBET/ITQB-UNL, Oeiras, Portugal; e-mail: marques@itqb.unl.pt 2 Department of Chemical Engineering, Massachussets Institute of Technology, Cambridge, Massachussets 3 Faculdade de Cie ˆncia e Tecnologia, Departamento de Quı´mica, Universidade Nova de Lisboa (FCT-UNL), Caparica, Caparica, Portugal Received 14 May 2010; revision received 27 July 2010; accepted 26 August 2010 Published online 1 September 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.22920 ABSTRACT: Long-term primary cultures of hepatocytes are essential for bioartificial liver (BAL) devices and to reduce and replace animal tests in lead candidate optimization in drug discovery and toxicology tests. The aim of this work was to improve bioreactor cultures of hepatocyte spheroids by adding a more physiological perfusion feeding regime to these bioreactor systems. A continuous perfusion feeding was compared with 50% medium replacement (routinely used for in vitro tests) at the same dilution rate, 0.125 day 1 , for three operative weeks. Perfusion feeding led to a 10-fold improvement in albumin synthesis in bioreactors containing non-encapsulated hepatocyte spheroids; no significant improvement was observed in phase I drug metabolizing activity. When ultra high viscous alginate encapsulated spheroids were cultured in perfusion, urea synthesis, phase I drug metabolizing activity and oxygen consumption had a threefold improvement over the 50% medium replacement regime; albumin production was the same for both feeding regimes. The effective diffusion of albumin in the alginate capsules was 7.75.10 9 cm 2 s 1 and no diffusion limitation for this protein was observed using these alginate capsules under our operational conditions. In conclusion, perfusion feeding coupled with alginate encapsulation of hepatocyte spheroids showed a synergistic effect with a threefold improvement in three independent liver-specific functions of long-term hepatocyte spheroid cultures. Biotechnol. Bioeng. 2011;108: 41–49. ß 2010 Wiley Periodicals, Inc. KEYWORDS: hepatocytes; 3D; perfusion; bioreactor; long- term functionality; liver-specific functions Introduction The liver has a major role in maintaining physiological homeostasis and in detoxifying blood. In mammals, it is responsible for nitrogen excretion in the form of urea, albumin biosynthesis (Quinlan et al., 2005), glucose storage in the form of glycogen, gluconeogenesis (Pilkis and Granner, 1992), glutamine homeostasis (Watford et al., 2002), and xenobiotic detoxification (Gebhardt et al., 2003; Gomez-Lechon et al., 2003; LeCluyse, 2001; Maurel, 1996). Acute liver failure (ALF), at an approximate clinical rate of 2,000 cases per year in the US (Lee, 1993), leads to death in 33% of cases. Due to liver donor scarcity, bioartificial liver (BAL) devices and immunoisolated xenografts of hepato- cytes have been used to support these patients while waiting for a liver donor; nevertheless, the liver-specific functions of the hepatocytes are rapidly downregulated during support operation. This same problem affects primary cultured hepatocytes used in lead candidate optimization for drug development (Gomez-Lechon et al., 2003). ALF and drug development assays drive the need to have cells expressing liver-specific functions. Since the expression of several of the drug metabolizing isozymes cytochrome P450 (CYP450), as well as nitrogen excretion and albumin synthesis, is usually downregulated in hepatoma or immorta- lized cell lines (Park et al., 2008; Sinz et al., 2008), the main choice for BAL devices and drug development tests have been primary cultured hepatocytes. These cells maintain most of their liver-specific functions immediately after their isolation (Coecke et al., 1999; Maurel, 1996). However, primary cultured hepatocytes lose their liver-specific functions along culture time (Nakamura et al., 1983, 1985); this problem has been the subject of many studies whose main goal is to Joana P. Miranda’s present address is iMED, Faculty of Pharmacy, University of Lisbon (UL), Lisboa, Portugal Correspondence to: Paula M. Alves, Animal Cell Technology IBET/ITQB-UNL, Apartado 12, Oeiras 27801-901, Portugal. ß 2010 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 108, No. 1, January 1, 2011 41