The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs.
Clinical islet allotransplantation has become an increasingly efficient "routine" therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 +/- 720 islet equivalents per gram organ (IEQ/g); n = 25; 2-3 years old; RB] and also from young hybrid pigs [2868 +/- 260 IEQ/g; n = 65; 4-6 months old; HY] using LiberasePI and a modified version of Ricordi's digestion-filtration technique. As expected, isolations from RB showed significantly better results (p < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of organs from RB (80%) contained mainly large islets (diameter > 200 microm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RB(Large Islets): 5680 +/- 3,318 IEQ/g (n= 20); RB(Small Islets): 1353 +/- 427 IEQ/g (n = 5); p < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrep density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrep was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the isolation process. Under these conditions highly successful isolations can reliably be performed even from young market pigs.
Krickhahn
Mareike
M
Experimental Transplantation Immunology, Department of Surgery, University of Wuerzburg Hospital, Josef-Schneider-Strasse 2, D-97080 Wuerzburg, Germany.
Bühler
Christoph
C
Meyer
Thomas
T
Thiede
Arnulf
A
Ulrichs
Karin
K
D000367:Age Factors; D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D017364:Collagenases / Q000494:pharmacology; D005260:Female; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation; D013045:Species Specificity; D013552:Swine; D013820:Thermolysin / Q000494:pharmacology; D014019:Tissue Donors
D017364:Collagenases; C106635:Liberase; D013820:Thermolysin