title: Influence of collagenase loading on long-term preservation of pig pancreas by the two-layer method for subsequent islet isolation.
abstract: BACKGROUND
  The introduction of the two-layer method (TLM) for long-term human pancreas preservation revealed the enormous potential of TLM to improve graft function of isolated islets. It is still unclear whether pig islets can be successfully isolated from pancreases after prolonged cold ischemia. To clarify this question, pig pancreases were subjected to 7-hour preservation by University of Wisconsin solution (UWS) storage or TLM. Another aim was to verify whether TLM can be synergistically combined with intraductal collagenase injection before cold storage.
  
  
  METHODS
  After intraductal flush with UWS, organs were distended with 4.4 PZ-U/g of UWS-dissolved collagenase NB-8 and neutral protease adjusted to respectively 1.1, 0.2, 0.5, or 0.8 DMC-U/g for pancreases freshly procured (n=6) or distended with enzymes before (TLM preloaded, n=7) or after cold storage (UWS storage, n=4; TLM postloaded, n=10).
  
  
  RESULTS
  Purified islet yield decreased from 429,200+/-86,700 islet equivalents (IEQ) in unstored pancreases to respectively 37,670+/-19620, 210,400+/-22900 and 238,000+/-26600 IEQ in UWS-stored (P<0.01), TLM-preloaded, or postloaded organs (P<0.05). Purity (>90%), viability (>95%), and insulin content were not different between groups. Islets from UWS-stored pancreases fragmented extensively, preventing further assessment of in vivo function. Compared with other experimental groups, islets from TLM-preloaded organs were characterized by enhanced basal and stimulated insulin release. Sustained normoglycemia was observed in diabetic nude mice transplanted with islets from TLM-postloaded or unstored pancreases in contrast with transient function in TLM-preloaded islets.
  
  
  CONCLUSIONS
  This study demonstrates that significant amounts of intact pig islets can be isolated after prolonged pancreas preservation by TLM. Enzyme administration before TLM preservation decreases islet graft function.
authors.affiliation: Third Medical Department, Justus-Liebig-University, Giessen, Germany. Daniel.Brandhorst@innere.med.uni-giessen.de
authors.forename: Daniel
authors.initials: D
authors.lastname: Brandhorst
authors.forename: Marcus
authors.initials: M
authors.lastname: Iken
authors.forename: Yasuki
authors.initials: Y
authors.lastname: Tanioka
authors.forename: Mathias D
authors.initials: MD
authors.lastname: Brendel
authors.forename: Reinhard G
authors.initials: RG
authors.lastname: Bretzel
authors.forename: Heide
authors.initials: H
authors.lastname: Brandhorst
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D017364:Collagenases / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000166:cytology* / Q000502:physiology; D016381:Islets of Langerhans Transplantation; D051379:Mice; D009926:Organ Preservation / Q000379:methods*; D013552:Swine
chemical_list: D017364:Collagenases