Alina Ostrowska zyxwvutsr Histological identification of purified and cryopreserved allogeneic hepatocytes following transplantation in a murine model without host immunosuppression Frederick M. Karrer Bahri M. Bilir zyxwvutsrqp Received: 10 August 1998 Received after revision: 2 March 1999 Accepted: 26 March 1999 A. Ostrowska zyxwvutsrqp ([XI) International Institute for the Advancement of Medicine, 814 Cedar Avenue, Scranton, PA 18505, U.S.A. Fax: +15703463455 Email: aostrow@voicenet.com F. M. Karrer Division of Transplantation Surgery, Department of Surgery, University of Colorado School of Medicine, 4200 E. Ninth Ave., Denver, CO 80262, U. S. A. B. M. Bilir Porter-Care Liver Transplantation Program, 2535 S. Downing Street, Suite 380, Denver, CO 80210, U. S. A. Abstract Hepatocyte transplanta- tion is a conceptually attractive al- ternative to whole organ grafting for some inborn metabolic errors and for fulminant liver failure. However, studies of the immunogenicity of transplanted allogeneic hepatocytes have yielded contradictory results. In these experiments, the effect of purification and cryopreservation of the hepatocytes on the ability of these cells to engraft in the mouse allogeneic recipients without immu- nosuppression was studied. BALB/ cByJ mouse crude (unpurified), modified (purified or cryopre- served), or dead (irradiated) hepa- tocyte preparations labeled with fluorescein dye CFSE were infused either into the portal vein or into the spleen parenchyma of the recipient CBA mice. A histological examina- tion revealed normal appearance of engrafted modified hepatocytes with no signs of acute rejection up to 21 days posttransplant. Many of the intrasplenically implanted hepato- cytes migrated into the hepatic si- nusoids. The modified hepatocytes showed intact ultrastructural ap- pearance 7 days after transplanta- tion. The numbers of inoculated crude hepatocytes rapidly declined with signs of dense infiltration of mononuclear cells in the graft indi- cating destructive response. The fluorescence of dead hepatocytes was undetectable. These results sug- gest that reduced immunogenicity may be responsible for the longer survival time of inoculated, purified or cryopreserved hepatocytes with no adverse morphological effects. Key words Allogeneic hepatocytes . Transplantation CFSE . Murine model Introduction Hepatocyte transplantation is being developed as a bridge to solid organ grafting. Potential advantages of the use of this method include long-term hepatocyte cryopreservation and in vitro cell manipulation to de- crease immunogenicity [l, 41. However, the use of hep- atocyte transplantation clinically has been hindered by limited survival of the cells in vivo. The ideal site for hepatocyte transplantation is the liver itself where the inoculated cells may function in their natural environ- ment, receiving the hepatotrophic factors important for cell differentiation. The disadvantage of intrahe- patic placement of the grafts is the difficulty of differen- tiating donor from host hepatocytes, unless the donor viable cells are labeled with specific markers [9, 161. Another site widely used for hepatocyte transplanta- tion is the spleen. There are many reports describing long-term survival of the hepatocyte grafts in the sple- nic pulp of syngeneic recipients, although the majority of inoculated cells translocate into the liver [8, 111. Re- jection of the transplanted allogeneic hepatocytes re- mains the major problem in several animal models. Ad- dition of immunosuppressive drugs has shown only lim- ited success [15, 171. The elimination of hepatocyte im- munogenicity would permit long-term graft survival