ARTICLE Radial Flow Hepatocyte Bioreactor Using Stacked Microfabricated Grooved Substrates Jaesung Park, Yawen Li, Franc ¸ois Berthiaume, Mehmet Toner, Martin L. Yarmush, Arno W. Tilles Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Shriners Hospitals for Children and Harvard Medical School, Boston, MA 02114; telephone: 617-371-4874; fax: 617-371-4950; e-mail: arno_tilles@hms.harvard.edu Shriners Hospitals for Children, 51 Blossom Street, Boston, MA 02114. Received 22 March 2007; revision received 18 June 2007; accepted 26 June 2007 Published online 11 July 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21572 ABSTRACT: Bioartificial liver (BAL) devices with fully func- tioning hepatocytes have the potential to provide temporary hepatic support for patients with liver failure. The goal of this study was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow bioreactor. Photolithographic techniques were used to microfabricate concentric grooves onto the underlying glass substrates. The microgrooves served to protect the seeded hepatocytes from the high shear stresses caused by the volumetric flow rates necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bior- eactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor, creating a channel height of 100 mm between each substrate. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of the hepatocytes remained stable over 5 days of perfusion, and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves. This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure. Biotechnol. Bioeng. 2008;99: 455–467. ß 2007 Wiley Periodicals, Inc. KEYWORDS: radial flow bioreactor; stacked microgrooved substrates; microfabrication; perfusion; hepatocyte; bioar- tificial liver Introduction The overall goal of a bioartificial liver (BAL) device is to provide temporary hepatic support to a patient with an acutely failing liver. Although there have been several clinical trials evaluating BAL devices (Demetriou et al., 2004; Ellis et al., 1996; Mazariegos et al., 2002; Sauer et al., 2003), none of the devices have been clearly shown to benefit long-term survival, and as a result, none have been clinically approved by the Food and Drug Administration for treating patients with liver failure. Most of the devices that have undergone clinical trials have incorporated hollow fiber technology. In some hollow fiber devices the fiber membrane serves as a scaffold for cell attachment, as well as a permeable selective barrier allowing bidirectional exchange of biomolecules between the device and the patient. Due to the relatively large diameter of the fibers as well as transport resistances associated with the fiber wall, these systems are prone to substrate transport limitations. Modeling studies have suggested that oxygen transport to the hepatocytes is limiting in these hollow fiber designs which may result in their decreased performance (Catapano, 1996; Hay et al., 2000, 2001). By selecting appropriate membrane properties (e.g., membrane material, membrane pore size, membrane thickness) it may be possible to prevent substrate transport limitations from occurring in these designs. Some BAL designs incorporate discrete fiber bundles (Gerlach et al., 1994), or integrate hollow fibers into a nonwoven matrix (Flendrig et al., 1997) which may improved oxygen delivery to the hepatocytes. In the design of a BAL device, the cell distribution and flow should be uniform to eliminate substrate limitations and to meet the metabolic demands of the seeded hepatocytes. A single layer monoculture where hepatocytes This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/0006-3592/suppmat. Correspondence to: A.W. Tilles Contract grant sponsor: National Institutes of Health Contract grant numbers: K08 DK66040; R01 DK43371; P41 EB02503 ß 2007 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 99, No. 2, February 1, 2008 455