title: Cryo-isolation: a novel method for enzyme-free isolation of pancreatic islets involving in situ cryopreservation of islets and selective destruction of acinar tissue.
abstract: BACKGROUND
  A critical component of treating type I diabetes by transplantation is the availability of sufficient high-quality islets. Currently, islets can be obtained only by reliance on an expensive, inconsistent, and toxic enzyme digestion process. As an alternative, we hypothesize that cryobiologic techniques can be used for differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ.
  
  
  METHODS
  Pancreases were procured from juvenile pigs with the use of approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water (or saline solution) to fully distend the gland; 3) freezing the entire pancreas to -160°C, and stored in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen pancreas into small fragments; and 5) thawing, filtering and washing the frozen fragments with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and samples were taken for static glucose-stimulated insullin release assessment.
  
  
  RESULTS
  As predicted the cryo-isolated contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells, was variable. Islets were typically larger than their counterparts isolated from juvenile pigs with conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3).
  
  
  CONCLUSIONS
  An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a novel method that avoids the problems associated with conventional collagenase digestion methods.
authors.affiliation: Cell and Tissue Systems, North Charleston, South Carolina 29406, USA. mtaylor@celltissuesystems.com
authors.forename: M J
authors.initials: MJ
authors.lastname: Taylor
authors.forename: S
authors.initials: S
authors.lastname: Baicu
mesh_terms: D061354:Acinar Cells / Q000166:cytology*; D000818:Animals; D018929:Cell Culture Techniques; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry; D004230:Dithizone / Q000494:pharmacology; D004798:Enzymes / Q000737:chemistry*; D005615:Freezing; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation / Q000295:instrumentation / Q000379:methods*; D010179:Pancreas / Q000378:metabolism; D013552:Swine; D014867:Water / Q000378:metabolism
chemical_list: D003451:Cryoprotective Agents; D003470:Culture Media; D004798:Enzymes; D007328:Insulin; D014867:Water; D004230:Dithizone; D005947:Glucose