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.
The Rogosin Institute-Xenia Division, Xenia, OH.
Robert W
RW
Holdcraft
VitaCyte, LLC, Indianapolis, IN.
Michael L
ML
Green
VitaCyte, LLC, Indianapolis, IN.
Andrew G
AG
Breite
The Rogosin Institute-Xenia Division, Xenia, OH.
Lisa
L
Circle
The Rogosin Institute-Xenia Division, Xenia, OH.
Eric D
ED
Meyer
The Rogosin Institute-Xenia Division, Xenia, OH.
Hollie
H
Adkins
The Rogosin Institute-Xenia Division, Xenia, OH.
Steven G
SG
Harbeck
The Rogosin Institute, New York, NY.
Barry H
BH
Smith
The Rogosin Institute, New York, NY.
Lawrence S
LS
Gazda