title: Isoprostanes as markers of oxidative stress-induced cell damage in porcine islet cell isolation.
abstract: BACKGROUND
  Mechanical stress and reagents used during the isolation and purification process as well as digestion time and temperature can alter the success of porcine islet cell (PIC) isolation. This study aimed to characterize the occurrence of isoprostanes during PIC isolation using a modified automated Ricordi method and to evaluate their influence on PIC isolation outcome.
  
  
  METHODS
  Porcine pancreatic tissue was harvested at the local slaughter house, and 10 PIC isolations were performed using a modified automated Ricordi method. As positive controls for tissue damage-associated oxidative stress, six consecutive PIC isolations were performed in the presence of 1 mug lipopolysaccharide (LPS). PIC were purified by density gradient centrifugation using the Lymphoprep density gradient. Isoprostane measurement was performed using enzyme-linked immunosorbent assay.
  
  
  RESULTS
  The final yield of viable and pure PICs in the experimental group was 3479 +/- 542 IEQ/g pancreas, and the LPS group yielded lower cell numbers compared to the experimental group. Isoprostane levels were significantly elevated in the LPS group as compared to the experimental group at all time points during the isolation from the beginning of the digestion process.
  
  
  CONCLUSIONS
  PIC isolation and purification results significantly differed in the two experimental groups, underlining the negative effects of oxidative stress on PIC viability and purity, which impact negatively on PIC transplantation success.
authors.affiliation: Department of Surgery, Division for Transplantation Surgery, Medical University Graz, Graz, Austria. philipp.stiegler@meduni-graz.at
authors.forename: P
authors.initials: P
authors.lastname: Stiegler
authors.forename: S
authors.initials: S
authors.lastname: Schaffellner
authors.forename: F
authors.initials: F
authors.lastname: Hackl
authors.forename: F
authors.initials: F
authors.lastname: Iberer
authors.forename: R
authors.initials: R
authors.lastname: Aigner
authors.forename: B
authors.initials: B
authors.lastname: Christine
authors.forename: K
authors.initials: K
authors.lastname: Tscheliessnigg
authors.forename: V
authors.initials: V
authors.lastname: Stadlbauer
mesh_terms: D000003:Abattoirs; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000473:pathology*; D028421:Isoprostanes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D018384:Oxidative Stress; D013314:Stress, Mechanical; D013552:Swine
chemical_list: D015415:Biomarkers; D028421:Isoprostanes; D008070:Lipopolysaccharides