Optimizing Porcine Islet Isolation to Markedly Reduce Enzyme Consumption Without Sacrificing Islet Yield or Function.

BACKGROUND: Human allogeneic islet transplantation for treatment of type 1 diabetes provides numerous clinical benefits, such as fewer episodes of hypoglycemic unawareness and tighter control of blood glucose levels. Availability of human pancreas for clinical and research use, however, is severely limited. Porcine pancreas offers an abundant source of tissue for optimization of islet isolation methodology and future clinical transplantation, thereby increasing patient access to this potentially lifesaving procedure. METHODS: Porcine islet isolations were performed using varying amounts of collagenase (7.5, 3.75, or 2.5 Wunsch units per gram tissue) and neutral protease activity (12 000, 6000, or 4000 neutral protease units per gram tissue) and perfusion volumes (1.7 or 0.85 mL/g tissue) to assess their effects on isolation outcomes. Retention of dissociative enzymes within the pancreas during perfusion and digestion was evaluated, along with distribution of the perfusion solution within the tissue. RESULTS: Reducing enzyme usage by as much as 67% and perfusion volume by 50% led to equally successful islet isolation outcomes when compared with the control group (48 ± 7% of tissue digested and 1088 ± 299 islet equivalents per gram of pancreas vs 47 ± 11% and 1080 ± 512, respectively). Using margin-marking dye in the perfusion solution to visualize enzyme distribution demonstrated that increasing perfusion volume did not improve tissue infiltration. CONCLUSIONS: Current protocols for porcine islet isolation consume excessive amounts of dissociative enzymes, elevating cost and limiting research and development. These data demonstrate that islet isolation protocols can be optimized to significantly reduce enzyme usage while maintaining yield and function and thus accelerating progress toward clinical application.

Enhancement of in vitro and in vivo function of agarose-encapsulated porcine islets by changes in the islet microenvironment.

The transplantation of porcine islets of Langerhans to treat type 1 diabetes may provide a solution to the demand for insulin-producing cells. Porcine islets encapsulated in agarose-agarose macrobeads have been shown to function in nonimmunosuppressed xenogeneic models of both streptozotocin-induced and autoimmune type 1 diabetes. One advantage of agarose encapsulation is the ability to culture macrobeads for extended periods, permitting microbiological and functional assessment. Herein we describe optimization of the agarose matrix that results in improved islet function. Porcine islets (500 IEQs) from retired breeding sows were encapsulated in 1.5% SeaKem Gold (SG), 0.8% SG, or 0.8% Litex (Li) agarose, followed by an outer capsule of 5% SG agarose. Insulin production by the encapsulated islets exhibited an agarose-specific effect with 20% (0.8% SG) to 50% (0.8% Li) higher initial insulin production relative to 1.5% SG macrobeads. Insulin production was further increased by 40-50% from week 2 to week 12 in both agarose types at the 0.8% concentration, whereas islets encapsulated in 1.5% SG agarose increased insulin production by approximately 20%. Correspondingly, fewer macrobeads were required to restore normoglycemia in streptozotocin-induced diabetic female CD(SD) rats that received 0.8% Li (15 macrobeads) or 0.8% SG (17 macrobeads) as compared to 1.5% SG (19 macrobeads). Islet cell proliferation was also observed during the first 2 months postencapsulation, peaking at 4 weeks, where approximately 50% of islets contained proliferative cells, including ß-cells, regardless of agarose type. These results illustrate the importance of optimizing the microenvironment of encapsulated islets to improve islet performance and advance the potential of islet xenotransplantation for the treatment of type 1 diabetes.

The use of pancreas biopsy scoring provides reliable porcine islet yields while encapsulation permits the determination of microbiological safety.

For clinical xenogenic islet transplantation to be successful, several requirements must be met. Among them is a sizeable and reliable source of fully functional and microbiologically safe islets. The inherent variability among porcine pancreases, with respect to islet yield, prompted us to develop a Biopsy Score technique to determine the suitability of each pancreas for islet isolation processing. The Biopsy Score consists of an assessment of five variables: warm ischemia time, pancreas color, fat content, islet size, and islet demarcation, each of which is assigned a value of -1 or +1, depending on whether or not the established criteria is met. For determination of islet size and demarcation, fresh biopsies of porcine pancreases are stained with dithizone (DTZ) solution and examined under a dissecting microscope. Based on the scoring of such biopsies in pancreases from 26-56-month-old sows, we report here that the presence of large (>100 microm diameter), well-demarcated islets in the pancreas biopsy is a reliable predictor of isolation success. Encapsulation of the isolated porcine islets within the inner layer of a 1.5% agarose and an outer layer of 5.0% agarose macrobead, containing 500 equivalent islet number (EIN), provides for extended in vitro functional viability (>6 months of insulin production in response to glucose), as well as for comprehensive microbiological testing and at least partial isolation of the xenogeneic islets from the host immune system. All microbiological testing to date has been negative, except for the presence of porcine endogenous retrovirus (PERV). Taken together, we believe that the Biopsy Score enhancement of our islet isolation technique and our agarose-agarose macroencapsulation methodology bring us significantly closer to realizing clinical porcine islet xenotransplantation for the treatment of insulin-dependent diabetic patients.