title: The nitric oxide donor S-nitrosoglutathione reduces apoptotic primary liver cell loss in a three-dimensional perfusion bioreactor culture model developed for liver support.
abstract: BACKGROUND
  Artificial extracorporeal support for hepatic failure has met with limited clinical success. In hepatocytes, nitric oxide (NO) functions as an antiapoptotic modulator in response to a variety of stresses. We hypothesized that NO administration would yield improved viability and hepatocellular restructuring in a four-compartment, hollow fiber-based bioreactor with integral oxygenation for dynamic three-dimensional perfusion of hepatic cells in bioartificial liver support systems.
  
  
  METHODS
  Isolated adult rat liver cells were placed in culture medium alone (control) or medium supplemented with various concentrations of an NO donor (S-nitrosoglutathione [GSNO]) in the bioreactors. Media samples were obtained from the cell perfusion circuit to monitor cellular response. After 24 and 72 h, histology biopsies were taken to investigate spontaneous restructuring of the cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to quantify apoptotic nuclei.
  
  
  RESULTS
  Control bioreactors exhibited 47.9 +/- 2.9% (mean +/- standard error of the mean) apoptotic nuclei. In contrast, NO-treated bioreactors exhibited a biphasic response. Fewer apoptotic nuclei were seen in the 200 and 500 microM GSNO groups (14.4 +/- 0.4%). No effect was observed in the 10 microM GSNO group (47.3%), and increased TUNEL staining was observed in the 1000 microM GSNO group (82.6%). Media lactate dehydrogenase levels were lower in bioreactor groups treated with 200 or 500 microM GSNO (310 +/- 38 IU/L) compared with the control group (919 +/- 188 IU/L; p < 0.05). Protein synthesis was not affected, as measured by albumin levels in the media (115 +/- 19 microg/day/cell inoculum in GSNO-treated bioreactors at 24 h vs. 110 +/- 13 in controls; p = 0.851). Histologically, all of the bioreactor groups exhibited liver cell aggregates with some attached to the bioreactor capillaries. Increased numbers of cells in the aggregates and superior spontaneous restructuring of the cells were seen at 24 and 72 h in the bioreactor groups treated with either 200 or 500 microM GSNO compared with the control groups.
  
  
  CONCLUSIONS
  Addition of an NO donor reduces adult rat liver cell apoptosis during the initial 24 h after cell inoculation within a three-dimensional perfusion bioreactor system for liver support and promotes liver cell aggregation and spontaneous restructuring of the cells at 24 and 72 h. GSNO-treated bioreactors remain metabolically active and show significantly lower levels of cellular injury as compared with controls. Further studies will be required to evaluate the impact of NO treatment of liver support bioreactors for clinical studies.
authors.affiliation: Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15219-3130, USA.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Prince
authors.forename: Yoram
authors.initials: Y
authors.lastname: Vodovotz
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Baun
authors.forename: Satdarshan Pal
authors.initials: SP
authors.lastname: Monga
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
authors.forename: Jörg C
authors.initials: JC
authors.lastname: Gerlach
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000473:pathology*; D008297:Male; D008954:Models, Biological; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D026422:S-Nitrosoglutathione / Q000494:pharmacology*; D013194:Staining and Labeling; D013997:Time Factors
chemical_list: D020030:Nitric Oxide Donors; D026422:S-Nitrosoglutathione; D007770:L-Lactate Dehydrogenase