title: Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes.
abstract: BACKGROUND
  The aim of this study was to evaluate in vitro and in vivo functions of isolated hepatocytes after immortalization, cryopreservation, encapsulation and xenotransplantation into mice with fulminant liver failure (FLF).
  
  
  METHODS
  Rat and human hepatocytes were isolated from normal liver tissue by collagenase digestion. Human hepatocytes were immortalized using lentiviral vectors coding for SV 40 large T antigen, Bmi-1 and telomerase. Rat and immortalized human hepatocytes (IHH) were encapsulated in 400 micron alginate-PLL-alginate membranes and cryopreserved using a computerized device. In vitro, encapsulated hepatocytes (cryopreserved or freshly isolated) were cultured in albumin-free medium and albumin production was measured by enzyme-linked immunosorbent assay (ELISA). In vivo, a model of FLF was established in C57/BL6 mice by acetaminophen administration (700 mg/kg i.p.) followed 15 h later by a 30% hepatectomy. Microencapsulated (cryopreserved or freshly isolated) hepatocytes were transplanted intraperitoneally to mice with FLF and the following experimental groups were performed: group 1 (n = 10) Tx of empty capsules; group 2 (n = 12) Tx of free primary rat hepatocytes; group 3 (n = 12) Tx of cryopreserved encapsulated rat hepatocytes; group 4 (n = 10) Tx of fresh encapsulated rat hepatocytes; group 5 (n = 9) Tx of cryopreserved encapsulated IHH; group 6 (n = 10) Tx of fresh encapsulated IHH. Animals were killed at regular intervals and histopathology of microcapsules and liver tissue was obtained.
  
  
  RESULTS
  In vitro, cryopreserved or fresh encapsulated rodent hepatocytes showed a progressively decreasing albumin secretion over 1 week in culture. In contrast, cryopreserved or fresh encapsulated IHH showed minimal, but stable albumin secretion. In vivo, FLF was achieved by combination of acetaminophen with 30% hepatectomy, resulting in a reproducible survival of 23% +/- 5%. In groups 1 and 2, survival rates were not improved significantly compared with untreated mice. In groups 3 and 4, Tx of cryopreserved or fresh encapsulated rat hepatocytes significantly increased survival rate to 66% and 80%, respectively (P < 0.01). In groups 5 and 6, Tx of cryopreserved or fresh encapsulated IHH improved survival to 50% and 55%, respectively (P < 0.05). Histopathology revealed that encapsulated hepatocytes were viable up to 2 weeks post-Tx.
  
  
  CONCLUSIONS
  Primary rodent hepatocytes maintained synthetic functions after encapsulation and cryopreservation short-term. IHH showed minimal albumin secretion in the absence of encapsulation and cryopreservation, suggesting that hepatocytes loose specific functions after immortalization. After induction of FLF in mice, intraperitoneal Tx of encapsulated (primary or immortalized, fresh or cryopreserved) xenogeneic hepatocytes significantly improved survival. These results indicate that naïve and genetically modified hepatocytes can successfully be encapsulated, stored using cryopreservation, and be transplanted into xenogeneic recipients with liver failure and sustain liver metabolic functions.
authors.affiliation: Surgical Research Unit, Department of Surgery, University Hospital Geneva, Switzerland.
authors.forename: Gang
authors.initials: G
authors.lastname: Mai
authors.forename: Tuan Huy
authors.initials: TH
authors.lastname: Nguyen
authors.forename: Nguyen Tuan
authors.initials: NT
authors.lastname: Huy
authors.forename: Philippe
authors.initials: P
authors.lastname: Morel
authors.forename: Jie
authors.initials: J
authors.lastname: Mei
authors.forename: Axel
authors.initials: A
authors.lastname: Andres
authors.forename: Domenico
authors.initials: D
authors.lastname: Bosco
authors.forename: Reto
authors.initials: R
authors.lastname: Baertschiger
authors.forename: Christian
authors.initials: C
authors.lastname: Toso
authors.forename: Thierry
authors.initials: T
authors.lastname: Berney
authors.forename: Pietro
authors.initials: P
authors.lastname: Majno
authors.forename: Gilles
authors.initials: G
authors.lastname: Mentha
authors.forename: Didier
authors.initials: D
authors.lastname: Trono
authors.forename: Leo H
authors.initials: LH
authors.lastname: Buhler
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation*; D006801:Humans; D017114:Liver Failure, Acute / Q000601:surgery*; D008297:Male; D051379:Mice; D051381:Rats; D015996:Survival Rate; D014183:Transplantation, Heterologous
chemical_list: D000418:Albumins