In an attempt to reduce the variability in the yields of human islets isolations and to identify donor factors that were potentially deleterious, we retrospectively reviewed 153 human islets isolations in our center over a 3-year period. Isolations were performed using controlled collagenase perfusion via the duct, automated dissociation, and Ficoll purification. Factors leading to successful isolations (recovery of >100,000 islet equivalents at a purity >50%) were analyzed retrospectively using univariate and multivariate analysis. Critical factors in the multiorgan cadaveric donors that were identified using univariate analysis included donor age (P<0.01), body mass index (BMI)(P<0.01), cause of death (P<0.01), and prolonged hypotensive episodes (systolic blood pressure <90 mmHg or mean arterial pressure <60 mmHg for > 15 min) requiring high vasopressors (>15 microgram/kg/min dopamine or >5 microgram/kg/min Levophed) (P>0.01). Independent analysis of 19 donor variables using multivariate logistic stepwise regression showed six factors were statistically significant. Odds ratio (OR) showed that donor age (OR 1.1, P<0.01), local procurement team (OR 10.9, P<0.01), and high BMI (OR 1.4, P<0.01) had a positive correlation with islet recovery. In contrast, hyperglycemia (all blood glucose >10 mmol/L) (OR 0.63, P<0.01), frequency and duration of cardiac arrest (OR 0.7, P<0.01), and increased duration of cold storage before islet isolation (OR 0.83, P<0.01) had negative correlation. Using these combinations of factors, the prediction of success was 85% accurate. By donor age, success was 13% for 2.5- to 18-year-old donors (n=23), 37% for 19- to 28-year-old donors (n=30), 65% for 29- to 50-year-old donors (n=70), and 83% for 51- to 65-year-old (n=29) donors. However, when vitro function was assessed by perifusion, the insulin secretory capabilities of islets isolated from the >50-year-old donor group was significantly reduced as compared with the 2.5- to 18-year-old group (P<0.02). Multiple regression analysis using postdigestion and postpurification islet recovery as outcome variables identified BMI, procurement team, pancreas weight, and collagenase digestion time factors tht can affect the recovery of human islets. Locally procured pancreases and donors with elevated minimum blood glucose levels were identified as factors that affect the insulin secretory capabilities of the isolated islets. This review of parameters suggests an improved approach to the prediction of successful islet isolation from human pancreases. Selection of suitable pancreases for processing may improve consistency in human islet isolation and thereby decrease costs.

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4 degrees C) Liberase-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35 degrees C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32 degrees C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 +/- 2.6 g vs. 4.6 +/- 2.1 g, mean +/- SEM, p < 0.05). Postdigestion recovery of islets was 471 +/- 83 x 10(3) IE in the perfusion group compared with 391 +/- 57 x 10(3) IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 +/- 45 vs. 251 +/- 28 x 10(3) IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 +/- 0.6 for the perfusion group and 4.2 +/- 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.

In this paper we report that curing at 120 degrees C can be used to improve the in vivo durability of a miniaturized glucose sensor with an outer coating of the Dupont perfluorinated ionomer, Nafion. Sensors based on glucose oxidase trapped in an albumin/glutaraldehyde matrix were able to withstand curing at 120 degrees C without noticeable change in electrode sensitivity (+/- 22% SD). Curing above 120 degrees C caused a gradual decline in sensitivity, with no sensitivity seen at 170 degrees C. Curing Nafion at 120 degrees C eliminated ascorbic acid and urea interferences and improved selectivity for glucose against uric acid and acetaminophen, compared to room temperature-cured Nafion coatings. The Nafion film reduced O2 demand by the sensor, so the signal was O2 independent across a partial pressure range of 8-140 mmHg. Several of the fully assembled, heat-cured, needle-type glucose sensors remained functional for at least 10 days after subcutaneous implantation in dogs, without degradation of their sensitivity (average 3 nA/mM in vivo at 37 degrees C and 6 nA/mM in vitro at 37 degrees C).

Inhibition of intrinsic protease activity within pancreases after prolonged cold storage improves isolation of viable islets.

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