Connected Culture of Murine Hepatocytes and HUVEC in a Multicompartmental Bioreactor Federico Vozzi, Ph.D., 1,2 Jan-Michael Heinrich, Ph.D., 3 Augustinus Bader, M.D., 3 and Arti D.Ahluwalia, Ph.D. 1 A multicompartmental bioreactor was conceived and designed to mimic cross talk between cells in different culture chambers connected only by flow, such that cell–cell interaction is mediated by soluble ligands as occurs in the body. The system was tested with a connected culture of murine hepatocytes and human umbilical vein endothelial cells. Metabolites such as albumin, urea, lactate and viability were monitored during the course of the experiments and compared with monoculture conditions in the bioreactor. When the two cell types are placed in connected culture, there is an increase in endothelial cell viability and hepatic glucose synthesis as well as albumin and urea production, while overall lactate production in the system is downregulated. The results show that the multicompartmental bioreactor enhances cell function, effectively combining both heterotypic interactions with increased nutrient availability. Introduction C ell biology has experienced a technical revolution in the past decade. Several new research tools to improve our understanding of cell function and to analyze the effects of different forces and stimuli have been produced. These tools or devices enable cells in culture to be exposed to conditions more representative of the physiological in vivo environment. The multicompartment connected culture bio- reactor (MCB) was designed with the aim of reproducing a downscaled in vitro ‘‘human body’’ to better understand in- teractions between different cell types in the presence of physical and biochemical stimuli, with particular reference to the metabolic system. This biotechnological tool has been developed using biomedical engineering concepts based on biomimetics, and is composed of cell culture chambers, con- nected in series and in parallel to form a closed loop bio- logical system in vitro. In particular, the MCB was designed using allometric laws that mathematically correlate nonlinear quantities (organ volume, blood flow, blood retention time, and metabolic rate). Using these laws, a system was realized in which, not only kinetic but also metabolic, volumetric, and exchange rate relationships between cells are conserved. 1 The complete MCB culture chamber and its corresponding equivalent circuit are illustrated in Figure 1a and b. Systems in which cells are cocultured with or without physical con- tact have been described in the literature. In fact, coculture studies have provided wide ranging evidence that hetero- typic interactions contribute positively to cell function, in- creasing, for example, expression of albumin and hepato- specific genes in hepatocytes as well as expression of cell surface receptors in endothelial cells. 2–5 Li et al. 6 reported an open-plan multiwell system in which various tissues are placed in wells bathed by a common medium. The micro- scale cell culture analog (mCCA) described by Shuler and coworkers is a silicon-based microfabricated device with interconnected compartments, designed using a physiologi- cally based pharmacokinetic model. 7–11 The mCCA has been used to analyze metabolic products of xenobiotics (in par- ticular, naphthalene), using hepatocytes, rat lung cells, and adipocytes, and has been shown to be a powerful tool for studying chemical toxicity using very small volumes—in all, the total volume of the device is a few hundreds of micro- liters. This implies that most type of quantitative protein expression or metabolite analysis in the system will require withdrawal of the entire contents of the device. The MCB is similar to the mCCA because cells in their individual cham- bers are connected by flow of the common medium although they differ in dimensions, fabrication method, design prin- ciples, and applications. The underlying philosophy behind the MCB is to recreate a downscaled metabolic system using allometric design principles in which each chamber can be addressed, interrogated, and viewed separately. The concept of ‘‘connected cultures’’ seeks to mimic the physiological milieu in which cells from different tissues or organs are ex- posed to soluble ligands through blood, lymph, or other fluids 1 MCB Group, Faculty of Engineering, Interdepartmental Research Center ‘‘E. Piaggio,’’ University of Pisa, Pisa, Italy. 2 Laboratory of Biomimetic Materials and Tissue Engineering, IFC-CNR, Pisa, Italy. 3 Laboratory of Cell Techniques and Applied Stem Cell Biology, Biocity, University of Leipzig, Leipzig, Germany. TISSUE ENGINEERING: Part A Volume 14, Number 00, 2008 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tea.2008.0066 1