Selective Osmotic Shock for Islet Isolation in the Cadaveric Canine Pancreas

Abstract

Currently, islet isolation is performed using harsh collagenases that cause nonspecific injury to both islets and exocrine tissue, negatively affecting the outcome of cell transplantation. We evaluated a novel islet isolation protocol utilizing high concentrations of glucose to cause selective osmotic shock (SOS). Islets have a membrane glucose transporter that allows adaptation to changes in glucose concentrations while exocrine tissue can be selectively destroyed by these osmolar shifts. Canine pancreata were obtained within 15 min after euthanasia from animals (n = 6) euthanized for reasons unrelated to this study. Each pancreas was divided into 4 segments that were randomized to receive 300 mOsm glucose for 20 min (group 1), 600 mOsm for 20 min (group 2), 300 mOsm for 40 min (group 3), or 600 mOsm for 40 min (group 4). Islet yield, purity, and viability were compared between groups. Mean ± standard error of the mean islet yield for groups 1 to 4 was 428 ± 159, 560 ± 257, 878 ± 443, and 990 ± 394 islet equivalents per gram, respectively. Purity ranged from 37% to 45% without the use of density gradient centrifugation and was not significantly different between groups. Islet cell viability was excellent overall (89%) and did not differ between treatment protocol. Islet function was best in groups treated with 300 mOsm of glucose (stimulation index [SI] = 3.3), suggesting that the lower concentration of glucose may be preferred for use in canine islet isolation. SOS provides a widely available means for researchers to isolate canine islets for use in islet transplantation or in studies of canine islet physiology.

PEGDA microencapsulated allogeneic islets reverse canine diabetes without immunosuppression

“…While the canine islet purity achieved was within the published ranges [ 27 , 44 , 47 , 48 ], the islet cell viability was slightly lower compared to previously published ranges of 87% [ 48 ] to 95% [ 27 ]. However, our viability and purity values were within the acceptable range published by the Collaborative Islet Transplant Registry for humans with ranges [ 49 ].…”

Isolation of feline islets of Langerhans by selective osmotic shock produces glucose responsive islets

“…The SOS isolation method has been shown to yield higher islets per gram of tissue (13,423 islets/g) ( 26 ) compared to Liberase-treated porcine pancreata (4,210 islets/g) ( 46 ) and compared to human cadaveric pancreata (2,279 islets/g) ( 19 ). In dogs, the SOS protocol had lower yield (28×10 3 islet-like equivalents) compared to traditional methods (49 to 234×10 3 islet-like equivalents) ( 47 ), however, delay in quantification and warm ischemia are suggested to have affected yield ( 27 ). There is minimal information on feline islet yield, with a single publication showing a mean yield of islet-like cell clusters (±SD) to be 2,200 ± 1,400 from a mean 3.8 ± 1.0 g of pancreas ( 30 ).…”

“…While this protocol will be useful in obtaining feline islets for in vitro studies of feline islet physiology, it still requires significant improvements in islet purity and yield before clinical application of islet transplantation. Following SOS, Thompson et al ( 27 ) incorporated the use of a sieve and cell strainer prior to plating canine islets for cell culture. We found that, in feline islet isolation, this drastically compromised yield, therefore the only purification step performed was hand picking islets to separate them from acinar tissue.…”

“…Meanwhile, cells that do not express this membranal transporter are subjected to rapid changes in tonicity, cell membrane damage, and subsequent cell death. This selective osmotic shock technique has been successfully applied for islet isolation from porcine, canine, and human pancreata (26)(27)(28). Moreover, it is technically simpler and yields higher quantities compared to enzymatic digestions (26,27).…”

Understanding the Structural Arrangement of Islets in Chronic Pancreatitis

“…Cytoarchitecture of islet and its related functions was assessed by using static method [glucose-stimulated insulin release (GSIR)]. 27,28 The selected islets ( n =50) in each individual sample group were washed with Roswell Park Memorial Institute (RPMI) media, followed by Krebs–Ringer bicarbonate HEPES (NaCl, 120 mM; KCl, 5 mM; CaCl 2 , 2.5 mM; MgCl 2 , 1.1 mM; NaHCO 3 , 25 mM; and HEPES, 10 mM) containing 0.2% bovine serum albumin. The secreted insulin from isolated islets in the media in response to both basal (5.5 mmol/l) and high (16.5 mmol/l) glucose concentrations was measured by enzyme-linked immunosorbent assay method (Mercodia, Uppsala).…”