Original Paper Cells Tissues Organs 2001;169:34–41 Optimization of Hepatocyte Spheroid Formation for Hepatic Tissue Engineering on Three-Dimensional Biodegradable Polymer within a Flow Bioreactor prior to Implantation É. Török a,d J.-M. Pollok a P.X. Ma e P.M. Kaufmann a M. Dandri b J. Petersen b,c M.-R. Burda b D. Kluth a F. Perner d X. Rogiers a a Department of Surgery, b Heinrich-Pette-Insititut and c Department of Internal Medicine, University of Hamburg, Germany; d Department of Transplantation and Surgery, Semmelweis Medical University, Budapest, Hungary; e Dental School, University of Michigan, Ann Arbor, Mich., USA Accepted after revision: August 30, 2000 Dr. med. Éva Török Department of Hepatobiliary Surgery University Hospital Hamburg-Eppendorf Martinistrasse 52, D–20246 Hamburg (Germany) Tel. +49 40 42803 8572, Fax +49 40 42803 2406, E-Mail toeroek@uke.uni-hamburg.de ABC Fax + 41 61 306 12 34 E-Mail karger@karger.ch www.karger.com © 2001 S. Karger AG, Basel 1422–6405/01/1691–0034$17.50/0 Accessible online at: www.karger.com/journals/cto Key Words Tissue engineering W Hepatocyte spheroids W Transplantation W Flow culture W Bioreactor W Rat Abstract We hypothesize that in vitro conditioning of hepatocytes within biodegradable poly-L-lactic acid (PLLA) polymer matrices prior to implantation may increase hepatocyte survival and function after transplantation. The purpose of this study was to optimize the culture conditions of hepatocytes in a pulsatile flow bioreactor. PLLA discs were seeded with rat hepatocytes in a concentration of 2.5, 5, 10, 20 and 40 ! 10 6 cells/ml. Seeded discs were exposed to recirculated perpendicular flow of 0, 7, 15, 24, 32, 52 ml/min of supplemented Williams’ Medium E and harvested after 6 days in flow culture. Only under flow conditions the hepatocytes formed spheroidal aggre- gates (SphA) of 50–260 Ìm in diameter with a liver-like morphology and active metabolic function. The number of SphA was examined by phase contrast microscopy and the reductive enzyme function of the hepatocytes was tested using MTT. Hematoxylin and eosin histology showed vital hepatocytes within the SphA less than 200 Ìm in diameter but central necrosis in the SphA exceeding this size. Immunohistochemical staining con- firmed albumin production of hepatocytes within the SphA. The optimal cell seeding concentration was 10 ! 10 6 cells/ml with a flow speed of 24 ml/min. SphA of hepatocytes cultured with this flow bioreactor method may prove useful as a functional unit for tissue engineer- ing of an in vivo liver substitute. Copyright © 2001 S. Karger AG, Basel Abbreviations used in this paper H&E hematoxylin and eosin MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PLLA poly(L-lactic acid) SphA spheroidal aggregates