Study of the Crosstalk Between Hepatocytes and Endothelial Cells Using a Novel Multicompartmental Bioreactor: A Comparison Between Connected Cultures and Cocultures Maria Angela Guzzardi, M.Sc., 1,2 Federico Vozzi, Ph.D., 1,3 and Arti Devi Ahluwalia, Ph.D. 3 The liver and other organs are connected to each other through the bloodstream. Therefore, the connection between tissues is generally mediated by soluble molecules able to cross the endothelial wall of capillaries. We developed a multicompartmental device, multicompartmental bioreactor (MCB), designed to mimic the con- nection between different tissues in which crosstalk is mediated by soluble molecules transported through the blood. A comparative study of the crosstalk between hepatocytes (HepG2) and endothelial cells (human um- bilical vein endothelial cells) in connected culture in the MCB and in a traditional static coculture system was performed by analyzing glucose consumption and secretion of albumin, urea, and nitric oxide. When hepato- cytes and endothelial cells were cultured together, the production of albumin and urea increased, and the increase was higher in the MCB than in traditional static coculture. In spite of this enhanced metabolic activity, the crosstalk between hepatocytes and endothelial cell leads to decreased glucose consumption with respect to hepatocytes alone, both in static and in dynamic conditions. However, the dynamic connected culture has a higher rate of metabolite synthesis and secretion with respect to cocultures. This means a more efficient use of energetic substrates and enhanced hepatocyte function in the MCB. Introduction T raditional static cell cultures are suitable for studying homotypic and heterotypic cell–cell interac- tions between different cells belonging to the same tissue, but they are not a suitable model to mimic interaction be- tween tissues belonging to different organs and compart- ments. In the past decade several studies have been published regarding improved function of hepatocytes co- cultured with fibroblasts or other nonparenchymal cells. Cocultivation of parenchymal and mesenchymal cells has been widely used to preserve hepatic phenotype in vitro. 1–5 The metabolic function of hepatocytes in vitro is of great in- terest in several fields given the variety of functions that the liver has in human physiology and pathology. For example, hepatocytes are employed in the fields of drug discovery and toxicology 6 as well as for the development of bioartificial liver, a cell-based therapeutic system for the treatment of hepatic failure. 7,8 The loss of function in isolated hepatocytes is one of the main problems that must be overcome in these applications. However, besides coculture of hepatocytes with nonparenchymal cells, the implementation of in vitro models for the study of the biochemical and molecular pathways involved in the development of metabolic disorders is still scarce, primarily due to the difficulty of reproducing the physiological interaction between tissues connected in the body by the bloodstream and the molecules transported through it. To address this gap, we developed a multicompartmental bioreactor (MCB) that represents a simplified downscaled in vitro human body. Our aim was to better understand the interaction between different tissues, with particular refer- ence to the metabolic system. The device was designed using biomedical engineering concepts based on biomimetics, as described elsewhere. 9 It is composed of cell culture chambers connected in series and in parallel to form a closed loop. The cell culture chambers are connected to each other through the flow of the culture medium that acts as the bloodstream, transporting molecules to and from the compartments. In the MCB, each chamber can be addressed and interrogated separately, and different cell types can be added stepwise to the system. In a previous report 10 we described the design of the system through allometric scaling, focusing on glucose pro- cessing for its relevance to diabetes and metabolic disorders. We also described a connected culture of murine hepatocytes 1 Institute of Clinical Physiology-CNR, Pisa, Italy. 2 Sector of Medicine, Scuola Superiore Sant’Anna, Pisa, Italy. 3 Faculty of Engineering, Interdepartmental Research Center ‘‘E.Piaggio,’’ University of Pisa, Pisa, Italy. TISSUE ENGINEERING: Part A Volume 15, Number 00, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tea.2008.0695 1