Biotechnol. J. 2012, 7, 117126

DOI 10.1002/biot.201100177

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Research Article

An in vitro model of glucose and lipid metabolism in a multicompartmentalbioreactor3 2 Bruna Vinci1,*, Ellen Murphy2,*, Elisabetta Iori2, FrancescoMeduri , Silvia Fattori4, Maria Cristina Marescotti , 4 2 1 Maura Castagna , Angelo Avogaro and Arti Ahluwalia1 2

Centro Interdipartimentale di Ricerca E. Piaggio, Faculty of Engineering, University of Pisa, Pisa, Italy Dipartimento di Medicina Clinica e Sperimentale, Divisione di Malattie del Metabolismo, University of Padua, Padua, Italy 3 Dipartimento di Chirurgia Generale Patologia Speciale, Azienda Ospedaliera di Padova, Padua, Italy 4 Dipartimento di Chirurgia, Anatomia Patologica III, Azienda Ospedaliero Universitaria Pisana, Pisa, Italy

The energy balance in vivo is maintained through inter-organ cross-talk involving several different tissues. As a first step towards recapitulating the metabolic circuitry, hepatocytes, endothelial cells and adipose tissue were connected in a multicompartmental modular bioreactor to reproduce salient aspects of glucose and lipid metabolism in vitro. We first examined how the two-way cellular interplay between adipose tissue and endothelial cells affects glucose and lipid metabolism. The hepatocyte cell line HepG2 was then added to the system, creating a three-way connected culture, to determine whether circulating metabolite concentrations were normalized, or whether metabolic shifts, which may arise when endothelial cells and adipose tissue are placed in connection, were corrected. The addition of hepatocytes to the system prevented the drop in the concentrations of glucose, L-alanine and lactate, and the rise in that of free fatty acids. There was no significant change in glycerol levels in either of the connected cultures. The results show that connected cultures recapitulate complex physiological systemic processes, such as glucose and lipid metabolism, and that the HepG2 hepatocytes normalize circulating metabolites in this in vitro environment in the presence of other cell types.

Received 26 March 2011 Revised 24 June 2011 Accepted 18 July 2011

Keywords:Adipose tissue Endothelial cells Hepatocytes In-vitro model Metabolism

1 IntroductionIt is now widely accepted that current in-vitro cell culture systems are poorly representati ve of human or animal physiolog y. This has been generally attributed to the fact that the compl exity of the physiological environment is not replicated in con-

Correspondence Professor Arti Ahluwalia, Centro Interdipartimentale di : Ricerca E. Piaggio, Faculty of Engineering, University of Pisa, Via Diotisalvi 2, 56125 Pisa, Italy E-mail: arti.ahluwalia@centropiaggio.unipi.it Abbreviations: AT, adipose tissue; ECG M endothelia cell growth medium; , FBS, fetal bovine serum; FFA, free fatty acid; HepG2, human hepatocellular liver carcinoma cell line; HUVEC human umbilical vein endothelial cell; , LFC, laminar flow chamber; MCmB, multicompartmental modular bioreactor; PLG A poly(lactic-co-glycolic) acid ,

ventional culture s. In fact, all cells are exquisitely sensiti ve to their micro-e nvironment, which is rich with 3-D cues from the extracellular matrix, other cells and from mechanical stimuli due to flow, concentration gradients and mov ement. A variety of methods have been proposed to refine the simplistic in-vitro representations that are currently used in cell-culture laboratories worl dwid e. For example, since the cross-talk between different tissues is important in modulating and enhancing cell func- tion, the use of conditioned media or the addition of growth factors generated by stromal or other cells is increasingly common, as are coculture s. Recent approaches use microscale device s, combining cell

* These authors contributed equally to this work.

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culture and microfluidics to fabricate cells and or- gans-on-chips [1]. We have developed a modular multicompartmental bioreactor (MCmB) system that enables mi- crowell protocols to be transferred directly to the bioreactor modules without redesign of cellcul- ture experiment s. Each module can be addressed and interrogated separately and different cell types can be added stepwise to the system. As explained previously [2], the MCmB design principles are based on allometric scaling of cell numbers and the mean residence time of molecules in metabolic tis- sue s, as well as considerations on oxygen tension and shear stres s, which together can be combined to establish organ and system model s. Here we use the MCmB to construct an in-vitro model of glucose and lipid metabolism starting from three tissue types known to play a fundamental role in metabolic homeostasis: liver, endothelium and adipose tissue (AT). Over 40 years ago, it was proposed that free fatty acids (FFAs) might compete with glucose as the main energy substrate in some tissue s, leading to decreased glucose oxidation when FFAs are el evated [3, 4]. Clinical studies have suggested that circulating metabolites display a reciprocal relationship between hepatic FFA uptake and glucose/lactate flux, which may deri ve from intrahepatic substrate competition [5]. AT releases FFAs into the circulation through lipolysi s. The liver is important in processing FFAs; therefor e, adequate liver function is critical in shuttling excess FFAs out of the circulation to limit consequent damage to the endothelium. This compl ex interpl ay between tissues is impossible to recapitulate in current cell-culture systems. Indeed, while a great deal of attention has been paid to identifying path ways and mechanisms in single cells, our understanding of inte r-organ cross-talk lags far behind. Besides the coculture of hepatocytes with non-parenc hymal cells, the implementation of integrated in-vitro models to stu dy the biochemical and molecular path ways involved in the development of metabolic disorders is still scarc e, 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. In a previous paper [6] we cultured the three different cell/tissue types separately in the MCmB bioreactors and demonstrated that flow plays an important role in modulating their metabolic profiles. Her e, endothelial cells (HUVECs), AT, and he- patocytes are connected together to investigate metabolic cross-talk between AT and endothelium and the role of hepatic cells in regulating circulat- ing metabolite s.

2

Materialsand methods

2.1 ExperimentaldesignThe MCmB is a modular system in which different bioreactor chambers are connected together by the flow of media. The configuration described here is composed of three module s. The hepatic and adi- pose tissue modules are the low-shear stres s, high- flow MCmB 2.0 chambers described previously [7], while the module used for the endothelial cell cul- ture is a laminar flow chamber (LFC) [8]. All the chambers are connected in a closed loop as shown in Figs. 1A and B. In addition, there is a mixing chamber for oxygenation and for the addition or sampling of medium, as well as a peristaltic pump (Ismatech IPC-4, Zurich, Switzerland) and silicone tubing, which connects the cell chamber s. To establish an in-vitro model of metabolism, we focused on the visceral compartment of the human abdomen to determine physiologically relevant cell ratios and transit time s. The visceral region was chosen firstly for its rel evance to shortterm metabolism, and secondly because the three tissues used make up a substantial fraction of the total cell number in this compartment. Using data available from the literature and physiological databases [2], the final hepatocyte:adipocyte:endothelial cell ratio was estimated to be 10:2:1. The number of cells added to each chamber at the beginning of the experiments was calculated to reach these ratios by the completion of each experiment. Shear stress is particularly critical for hepatocyte s, which may suffer damage at high flow rates; the flow rate was therefore fixed at 250 L/min, which we have shown to be optimal for rat and human hepatocytes [7, 9], and represents a fluid residence time of about 10 min in each modul e, comparable with the mean organ perfusion time of about 37 min in the human liver [10]. At this flow rate the wall shear stres s, calculated using finite element method s, was 0.002 Pa in the LFC and 105 Pa in the MCmB 2.0.

2.2 Cell/tissue sourcesAll reagents were from Sigma-Aldrich (Milan, Italy) unless otherwise specified. Omental AT was obtained, with informed consent, from surgical inter ventions in non-diabetic subjects free of known metabolic diseas e. After treatment with collagenase type II in Hanks balanced salt solution (HBSS), the partially digested tissue was placed on a 200-m mesh filter and rinsed with DMEM to re- move blood vessel s. Floating partially digested AT was then divided into aliquots and transferred to

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Figure. 1. The multicompartmental bioreactor modules: 3-way connected culture of HepG2 hepatocytes and adipose tissue in the round chambers and endothelial cells in the rectangular LFC. The mixing chamber is a partially filled plastic or glass bottle into which media droplets are passively oxygenated as they return from the culture chambers. (A) Experimental set up; (B) Connection scheme showing chamber dimensions in mm.

DMEM:F12 with 20% fetal bovine serum (FBS) for subsequent experiment s. For each condition test- ed, 200 mg partially digested tissue containing ap- proximately 300 000 adipocytes was used. The par- tial digestion all owed us to concentrate adipocytes without destr oying the collagen matrix, which is important for preventing dedifferentiation in the chamber s. HUVECs from Promocell (Heidelberg, Germa ny) were used to m odel the endothelium. The cells were cultured in endothelial cell basal medium (ECGM, Promocell) supplemented with 10% FBS, 0.1 ng/mL epidermal growth facto r, 1.0 ng/mL basic fibroblast growth facto r, 0.4% endothelial cell growth supplement/heparin, 1.0 g/mL hydrocortisone (Promocell), 100 U/mL penicillin and 100 g/mL strepto mycin. HUVECs were used up to the 4th passag e. Hepatocytes were from the human hepatocellular liver carcinoma (HepG2) cell line. Although their xenobiotic metabolic functions are kn own to be compromised, their endogenous meta- bolic functions are largely intact. They were grown in Eagle s minimal essential medium (EMEM) with 1 g/L glucose supplemented with 5% FBS, 1% nonessential amino acid s, 1% EMEM vitamin s, 2 mM L-glutamin e, 100 U/mL penicillin and 100 g/mL strepto mycin and used up to passage 22.

architectur e. 3-D scaffolds of poly(lactic- co-glycolic) acid (PLGA 75:25, MW 270 000; Boehringer Ingelheim, Ingelheim, Germa ny) were microfabricated using a pressure assisted microsyringe (PAM) [11]. The scaffolds can host high-density function- al cultures of HepG2 hepatocytes for up to 1 week and their fabrication, preparation and characterization have been reported [12]. The PLGA scaffolds used in this work were composed of three layers of hexagonal elements with sides of 500 m and ov er- all dimensions of 1 cm 1 cm 100 m. Scaffolds were routinely assessed for degradation after the end of the experiment by measuring changes in mas s, morphology and elastic modulus [13]. No ob- servable changes were detected, as verified by sim- ilar studies on this copolymer [14].

2.4 Cell cultureAll cultures were carried out using ECGM containing 10% FBS. Preliminary experiments were performed to verify that conditioned medium in the bioreactor system does not contribute significantly to the metabolic profile of cells and that the vitali- ty and morphology of all three cell types were con- ser ved with respect to their standard media [15]. In the two-way connected cultur e, the AT chamber was placed in series with the LFC containing the HUVEC s. In the three- way connected cultur e, the liver chamber was added in serie s. The components of the bioreactor system were sterilized using H2O2 gas plasma before each use and assembled under a

2.3 3-D scaffolds for HepG2 hepatocyte cultureOne of the most important influence hepatocyte function in presence of a 3-D factors that vitro is the

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laminar flow hood, connecting the three cell chamber s, the pum p, and the mixing chamber via tubing as shown in Fig. 1. The AT was placed in the MCmB 2.0 and 1 mL medium was added to the chambe r. The top of the chamber was layered with a pr ewetted 200-m nylon mesh san dwiched with 240- m nylon mesh circles to pr event movement of adipocytes out of the chamber and into the tubing [6]. The HUVECs were seeded onto a glass coverslip and all owed to adher e. The coverslip was then placed in the bottom of the LFC. HepG2 hepatocytes were seeded on collagencoated PLGA scaffolds placed on 12-mm glass coverslips in 24-well microplates (BD Bioscience s, Buccinasc o, Italy) at a density of 100 000 cells per scaffold in 2 mL ECGM. At 24 h the scaffolds were moved to a new 24-multiwell plat e. After a further 48 h, when the cells had proliferated to about 250 000 cells per scaffold; the slides were carefully transferred to the MCmB 2.0 and coated with an al- ginate film consisting of 250 L 1% sodium alginate dissol ved in serum -free medium, cross-linked with 50 L 1% CaCl 2. Excess alginate was rem oved with a pipett e. The alginate coating was used to protect the cells from direct mechanical stres s, while allowing adequate nutrient diffusion [10]. When all cells had been added to their respective chamber s, the chambers were closed. Medium was added to the mixing chamber to bring the total medium volume to 15 mL and the pump was turned on. When medium had filled all chamber s, the flow was set to 250 L/min, after which the bioreactor was placed inside a 37C/5% CO2 incubator for 15, 24, or 48 h. After the designated time period, cells/tissues were obser ved under light and/or fluorescent microscopes to confirm cell viabilit y, and medium was collected and stored at 80C for even- tual metabolite dosin g. Each condition was run at least in triplicat e.

analyzed using a microscop e, and

2.5 Cell viabilityand metabolite dosingBefore and after incubation, an aliquot of AT was digested with collagenas e, 1 mg/mL, in HBSS at 37C for 30 min; DMEM medium containing 10% FBS was then added, and cells were centrifuged. Floating cells were suspended in PBS containing Hoechst 33258. Adipocytes with Hoechst-positi ve nuclei were obser ved using a fluorescent microscope Olympus AX70 (Olympus Italia, Milan), con- firming cell viability and cell numbe r. There was no visible change in the size or the appearance of the tissue before and after incubation, and cells re- mained free of contamination. HUVEC and HepG2 morphology was

the total cell number for every time point was eval- uated using a Burker chamber and trypan blue to exclude non-viable cells. The number of dead cells counted was always less than 2% of the total. Glycerol, D-lactat e, and L-alanine concentrations were determined by a modified Lloyd ass ay using an automated spectrophotometer Cobas Fara II (Roche) [16]. FFAs were measured by an enzymatic colorimetric method (NE FA C test- Wako Chemicals GmbH, Germa ny) and glucose was determined by a hexokinase-based commercial ass ay (Glucosio HK CP-HoribaABX, Italy). Finall y, albumin (a marker of hepatic function as well as a carrier for FFAs) was ass ayed for the con- nected culture conditions using an enzymelinked immunosorbent ass ay specific for human albumin (Bet hyl Laboratorie s, Montgomer y, TX, USA).

was performed in triplicat e. Statistical significance was set at p