br Introduction Transplantation of hepatocytes

Introduction Transplantation of hepatocytes (HTx) has been shown to be useful for patients with chronic or acute liver failure or genetic defects in liver function (Strom et al., 1997; Fisher et al., 2000; Fisher and Strom, 2006; Strom et al., 2006; Fox et al., 1998; Horslen et al., 2003; Muraca et al., 2002; Dhawan et al., 2006; Stephenne et al., 2006; Lee et al., 2007; Meyburg et al., 2009a; Mitry et al., 2004). The most common indications of HTx are liver-based inborn metabolic disorders. Because of large redundancies of function, the entire hepatic capacity is not normally required to maintain homeostasis. The main source of purchase MK-571 sodium salt hydrate for HTx is livers rejected for transplantation (Strom et al., 2006; Meyburg et al., 2009b; Hughes et al., 2006). Steatosis is the most common cause for rejection of a tissue for transplantation. Low cell viability, yield and drug-metabolizing enzyme activity has been reported in hepatocytes obtained from fatty livers (Donato et al., 2006). Domino liver transplantation (DLT) of organs from patients with metabolic diseases has been used to increase the number of organs available for transplant (Ericzon et al., 2008; Mazariegos et al., 2012; Popescu and Dima, 2012). The world registry lists 790 such transplants through 2009 (Roels and Rahmel, 2011). The metabolic disease in the donor liver either should not induce the disease in the recipient, or would be expected to produce symptoms of the disease only after many years. Unlike DLT, where the entirety of the liver is rapidly replaced with one with a metabolic disease, HTx generally results in replacement of <10% of the recipient liver with donor cells. Since most metabolic disease hepatocytes would not induce the symptoms of the disease in the recipient, hepatocytes from donors with metabolic diseases might be useful for transplantation if they could be isolated in useful numbers and retained sufficient viability and function. Here we examined the viability, cell yield and metabolic activity of hepatocytes isolated from organ donors and tissues obtained from patients receiving OLT for metabolic and other types of liver diseases. The results indicate that hepatocytes with high viability and function can be isolated from organs removed at the time of OLT from patients with diseased livers. If the diseases of the donor and recipient are carefully considered, these organs could provide a useful source of cells for clinical transplantation.
Materials and methods Human liver tissues were collected under IRB protocol 0411142 and hepatocytes were isolated from 35 organ donors (OD) and from 35 liver tissues obtained from patients receiving OLT for metabolic and other liver diseases (MD). Liver tissue from organ donors was flushed with either Belzer\'s UW solution or HTK depending on the solution preferred by the procurement agency. Metabolic disease cases were recovered in the OR and flushed with either Belzer\'s UW solution if stored for more than 90min or ice-cold Eagle\'s MEM if the tissues were taken to the lab immediately for isolation. Hepatocyte isolation and culture were performed as previously described (Gramignoli et al., 2012; Kostrubsky et al., 1999). Methods to assess cell viability, plating efficiency, or ammonia, testosterone and 7-ethoxyresorufin metabolism, resorufin conjugation, media and culture conditions were as previously described (Gramignoli et al., 2012). Cell-based assays for specific CYP (1A2; 2C9; 3A7; 3A4, Promega Corporation, Madison, WI, U.S.A.) were used according to the manufacturer\'s instructions. Results were expressed as Luminescent Counting Unit (LCU)/min and normalized to a million of viable cells (suspension cultures) or to the double strand DNA content (adherent cultures) measured, after the Glo™-assays are complete, by Quant-iT™ PicoGreen® dsDNA kit according to the manufacturer\'s instructions (Molecular Probes, Invitrogen, Camarillo, CA).