Impact of islet size on pancreatic islet transplantation and potential interventions to improve outcome

Abstract

Better results have been recently reported in clinical pancreatic islet transplantation (ITX) due mostly to improved isolation techniques and immunosuppression; however, some limitations still exist. It is known that following transplantation, 30% to 60% of the islets are lost. In our study, we have investigated 1) the role of size as a factor affecting islet engraftment and 2) potential procedural manipulations to increase the number of smaller functional islets that can be transplanted. C57/BL10 mice were used as donors and recipients in a syngeneic islet transplant model. Isolated islets were divided by size (large, >300 μm; medium 150-300 μm; small, <150 μm). Each size was transplanted in chemically induced diabetic mice as full (600 IEQ), suboptimal (400 IEQ), and marginal mass (200 IEQ). Control animals received all size islets. Engraftment was defined as reversal of diabetes by day 7 posttransplantation. When the superiority of smaller islets was observed, strategies of overdigestion and fragmentation were adopted during islet isolation in the attempt to reduce islet size and improve engraftment. Smaller islets were significantly superior in engraftment compared to medium, large, and control (all sizes) groups. This was more evident when marginal mass data were compared. In all masses, success decreased as islet size increased. Once islets were engrafted, functionality was not affected by size. When larger islets were fragmented, a significant decrease in islet functionality was observed. On the contrary, if pancreata were slightly overdigested, although not as successful as small naive islets, an increase in engraftment was observed when compared to the control group. In conclusion, smaller islets are superior in engraftment following islet transplantation. Fragmentation has a deleterious effect on islet engraftment. Islet isolations can be performed by reducing islet size with slight overdigestion, and it can be safely adopted to improve clinical outcome.

Figure 1

A. Stainless steel filtration mesh used to separate islets according to their size. B. 150 µm pore size to separate small islets. C. 300 µm pore size to separate medium from large islets.

Figure 2

Isolated islets divided by size using stainless steel mesh filtration. A. Small islets (50–150 µm), B. Medium islets (150–300 µm), C. Large islets (>300 µm). Aliquots stained with dithizone and observed at light microscopy (×4).

Figure 3. Reversal of diabetes

A. Overall reversal by size; in the small islet group 86% of the animals reversed diabetes (25/29) as compared to control animals that reversed in 77% of the cases (23/31); a statistically significant difference in engraftment was observed with reduced engraftment as the islet size increased (p=0.0003) [medium islets 56% (14/25) and large islets 33% (9/27)]. B. When the data were analyzed by size and mass, the same trend was observed with a confirmed increase in engraftment as the islet size decreased (p=0.015). This trend was highly represented in the marginal mass group where small islets reversed in 75% of the cases (9/12) as compared to 37.5% (3/8), 10% (1/10) and 36% (4/11) in medium, large, and control groups, respectively.

Figure 4. Time of diabetes reversal for each study group (Kaplan-Meier). Log-rank test < 0.0001

Figure 5. Comparison of reversal of diabetes in marginal mass

When pancreata were over-digested (n=14), islets engrafted better than control (n=11) (57% vs. 36%); when medium and large islets were fragmented (n=14), islets performed significantly worse than naïve medium and large (n=18) (14% vs. 24%). Small naïve islets (n=12) remains the most successful group with 75% of animals reversing diabetes. Freeman-Halton test, p=0.0067.

Figure 6. IPGTT curves (short term)

BGL value at different time points was calculated as average among each study group. A. Full mass group included 16 control, 5 large, 6 medium, and 9 small islet animal recipients; B. Suboptimal mass group included 3 control, 3 large, 5 medium, and 7 small islet animal recipients. C. Marginal mass group included 4 control, 1 large, 3 medium, and 9 small animal recipients. IPGTT from naïve (non-transplanted) animals were included for comparison (n=2). The two-factor ANOVA test did not show a difference in IPGTT when small, medium, large, control, and naïve (non-transplanted) groups were compared among marginal (p=0.1063), suboptimal (p=0.4152) and full islet mass (p=0.1242).

Figure 7. IPGTT curves (short term): comparison in marginal mass

BGL value at different time points was calculated as average among each study group. Groups included are control (n=4), fragmented (n=2), over-digested (n=8), and small (n=8). IPGTT from naïve (non-transplanted) animals were included for comparison (n=2). The two-factor ANOVA test did not show a difference in IPGTT when naïve (non-transplanted) animals and marginal mass recipients of fragmented, over-digested, small, and control islets were compared (p=0.1469); The one-factor ANOVA at 120 minutes showed a statistically significant higher value of BGL in the fragmented group as compared to all the other groups (p<0.0001).