A PROSPECTIVE COMPARISON OF DISCONTINUOUS EUROFICOLL AND EURODEXTRAN GRADIENTS FOR ISLET PURIFICATION

Density gradient separation of islets from exocrine tissue is usually performed with Ficoll, However, this reagent adds significantly to the cost of the isolation. The aim of this study was to evaluate the perf ormance of Dextran as a potential low-cost substitute for Ficoll and t o evaluate the effects of cold storage of the pancreatic digest prior to purification. Pancreases were procured from mongrel dogs, loaded wi th collagenase and mechanically dissociated. Washed pancreatic digest was collected and divided into two fractions that were purified using discontinuous gradients on the Cobe 2991 processor using identically p repared EuroFicoll (EF) or EuroDextran (ED) gradients. Alternate group s were suspended in EC and stored on ice, while the other fraction wer e resuspended in the 1.108-g/mL gradient layer (either EF or ED) and l oaded into the COPE. This tissue layer was overlaid with layers of den sities 1.096 and 1.037 g/mL and a HBSS cap, and centrifuged for 5 min at 800 x g, Purified islets were collected from the interface between the 1.037 and 1.096 layers and islet recovery, purity, and function we re assessed. From a series of eight isolations, 72.9 +/- 8.2% (mean +/ - SEM) of the islets were recovered from the EF purified gradients com pared with 62.6 +/- 8.3% from ED gradients (p = NS, paired t-test), Gr adients of ED that were run following hypothermic storage of the diges t in cold EC solution (stored ED) had reduced islet recovery when comp ared with islet recovery from gradients prepared in EF(stored EF) (51. 6 +/- 9.6% for ED stored vs. 72.9 +/- 11.9 for EF stored, p < 0.05), I slet recovery from EF gradients was equivalent between the stored and nonstored groups. The purity of preparations from the stored ED gradie nts was also reduced (71.3 +/- 4.3%) when compared with islets that we re immediately purified after dissociation (82.5 +/- 4.8%, p < 0.05), Static glucose stimulation assay showed equivalence between the islets from ED and EF gradients. The stimulation index (SI) was 9.3 +/- 0.9 for EF islets compared with 7.9 +/- 1.4 for ED islets for digest purif ied immediately. However, if the digest was hypothermically stored in EC solution, a decrease in functional viability was observed in both t he EF and the ED groups (7.7 +/- 1.4 and 5.9 +/- 0.8, respectively). O ut of five alloxan-induced diabetic nude mice transplanted under the k idney capsule with 2000 islets isolated from the nonstored groups, thr ee remained euglycemic >50 days posttransplant with either EF or ED is lets, These experiments demonstrate effective recovery of equivalent n umbers of canine islets using discontinuous gradients of ED or EF imme diately following enzymatic digestion. However, following storage of t he digest in cold EC solution results in a reduction in both islet rec overy and function when gradients of ED are utilized. (C) 1998 Elsevie r Science Inc.