Artificial Organs 28(7):676–682, Blackwell Publishing, Inc. © 2004 International Center for Artificial Organs and Transplantation 676 Blackwell Science, LtdOxford, UKAORArtificial Organs0160-564X2004 International Society for Artificial Organs287676682Original Article HUMAN HEPATOCYTES FOR A BIOARTIFICIAL LIVERM. BARBICH Et al. Received June 2003; revised October 2003. Address correspondence and reprint requests to Dr. Pablo F. Argibay, Hospital Italiano de Buenos Aires, Potosí 4240, 8th floor, Buenos Aires C (1199 ACL), Argentina. E-mail: pablo.argibay@hospitalitaliano.org.ar Culture and Characterization of Human Hepatocytes Obtained after Graft Reduction for Liver Transplantation: A Reliable Source of Cells for a Bioartificial Liver *Mariana Barbich, *Alicia Lorenti, *Alejandra Hidalgo, *Marcelo Ielpi, *Martín de Santibáñez, †Eduardo de Santibáñez, *Vanina Morales, *María C. Marín, *Maria F. Callero, and *Pablo F. Argibay *Instituto de Ciencias Básicas y Medicina Experimental (ICBME); and †Department of Surgery, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina Abstract: This article describes results obtained when human liver cells obtained from reduced grafts are cultured in a chemically defined medium. Remnants of livers after reduction for pediatric transplantation were processed by a multiple cannulation system through the existing vascu- lature, which allowed the homogeneous perfusion of colla- genase. The graft weight ranged between 55 and 1000 g (median value: 145.6 g). The yield ranged between 0.13 ¥ 10 6 and 38 ¥ 10 6 cells/g of tissue (median value 14.73 ¥ 10 6 cells/g), and the viability was 61.17 ± 27.43%. The total number of cells ranged between 57.6 ¥ 10 6 and 12 150 ¥ 10 6 cells (median value: 740 ¥ 10 6 cells). Cells were cultured for 30 days. Albumin synthesis was observed during the first 2 weeks, with a peak value at day 6 (27.85 ± 1.77 mg/mL). Urea production was detected dur- ing the first week (peak value at day 6: 17.12 ± 2.11 mg/dL). Light microscopy showed the presence of cells in a mono- layer. Biliary pigments were observed at day 20. By immu- nohistochemistry, positive cells for albumin, for hepatocyte marker, cytokeratin 19, CD 34, CD 68, and for alpha actin for smooth muscle, were observed. Our results showed that hepatocytes obtained from reduced liver grafts are easily cultured and are able to maintain viability and functional- ity in vitro. This alternative source of human cells main- tained under controlled culture conditions may play an important role in the development of a bioartificial liver. Key Words: Human hepatocytes—Graft reduc- tion—Albumin synthesis—Urea production—Bioartificial liver—Primary culture. Although orthotopic liver transplantation is the elective therapy in acute liver failure, the scarcity of organs limits this procedure and demands a bridge therapy to sustain the patient until an organ becomes available. One of these therapies would be the use of a bio- artificial liver (BAL). The biological component of a BAL needs a number of hepatocytes performing all the array of detoxification functions characteristic of a normal liver, during the time when the patient can not perform these functions himself. Hence, swine cells are frequently used for this purpose, as human cells are not easily available. This problem makes important the efforts to improve the supply of human hepatocytes such as those from rejected or unused livers collected for orthotopic liver transplantation (1,2). In Argentina, approximately 20% of liver trans- plants are done in children with reduction proce- dures (3). The resulting liver fragments that are normally discarded, could be a potential source of hepatocytes for bioartificial liver devices. Culture of hepatocytes in vitro with maintenance of their differentiated specific functions would be useful for this application (4–6). Knowledge of the in vitro conditions that modulate physiological func- tions, such as culture media, growth factors, and attachment surfaces thus becomes of paramount importance. Here, we report on the isolation of cells from pieces of healthy liver arising from human hepatic