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In the pancreatic islet transplantation for curing diabetes mellitus, reaggregation of pancreatic islet single cells for making ‘islet spheroid’ is considered as a useful strategy in terms of islet viability and gene delivery into islet. However, during aggregation of islet spheroid, their insulin secretion pattern was not recovered to that of intact islets. We have developed a new cell-culture system using poly(dimethylsiloxane) (PDMS)-based concave-patterned film for aggregating islet spheroid. Here we optimized the effective formation of islets spheroid by using the concave-patterned film with different microwell diameter (300, 500, and 700-μm). Aggregated islet spheroids in different concave microwells showed same morphology with a limited size range and diameter dependency of microwell, which was similar to that of intact islet. However, islet spheroid in flat-bottomed dish (control) showed random aggregation with broad size distribution. Interestingly, insulin secretion pattern of islet spheroids in 300 and 500-μm concave patterned-film was same to that of intact islets, which was caused by the improved cell-to-cell interaction that was confirmed by electron microscope and strong phalloidin immunostain. However, islet spheroids in concave patterned-film with 700-μm diameter and flat-dish did not show normal insulin secretion in accordance with different glucose concentration, which might be caused by weak cell-to-cell interaction. On the other hand, the viability of islet spheroids in concave patterned-film was not affected during cultivation due to biocompatibility of PDMS polymer. Collectively, we demonstrated that PDMS-based concave-patterned film could be used for designing islet spheroids with improved cellular functionality and size uniformity to cure diabetes mellitus. Open image in new window

Transplantation of insulin-secreting pancreatic islets can provide real-time regulation of blood glucose in patients with type 1 diabetes mellitus. Currently, noninvasive and repetitive monitoring of islet engraftment and function is an emerging and promising modality for successful islet transplantation. Here we report a new technique for highly sensitive in vivo magnetic resonance (MR) imaging of transplanted pancreatic islets. To this end, heparinized superparamagnetic iron oxide (heparin-SPIO) nanoparticle was newly synthesized for chemical conjugation onto islet surface. Compared to typical cellular labeling of Feridex® via random endocytosis, chemical conjugation of heparin-SPIO was stable and improved the hypointensity of transplanted islets due to surface modification of every islet. These heparin-SPIO-conjugated islets showed normal viability and insulin secretion, and were quantified by spin echo T2-weighted MR imaging with linear correlation depending on transplanted islet mass in vitro and in vivo for 30 days. Also, from the immunohistochemistry, we confirmed the existence of heparin-SPIO and insulin biosynthesis in transplanted islets. However, Feridex-uptake islets showed late glucose responsiveness according to changing glucose concentration although they could normally control the blood glucose levels in diabetic mouse. Thus, we anticipate that this surface labeling with heparin-SPIO can be directly applicable for MR imaging of transplanted islets.

Pancreatic islet transplantation is an ideal therapy for type I diabetes. Interestingly, small size of islets may exhibit superior performance in transplantation in terms of function and hypoxia. To this end, large size of islets was dissociated to generate islet single cells and they were re-aggregated to make small size of islet-cell-cluster (ICC). However, the function of insulin secretion from ICCs was damaged due to weak cell-to-cell interaction. To overcome this limitation, here the dispersed islet single cells were re-aggregated at the concave microwell array that was fabricated with poly(dimethylsiloxane) (PDMS) by using soft-lithography and mold-replication technology. The aggregated ICCs had uniform size, and their insulin secretion in response to the change of low and high glucose concentration was similar to that of intact islets since the cell-cell interaction of islet single cells was strongly induced due to the shape of concave microwell. Also, after cultivation for 2 weeks, the expression of hypoxia-induced factor- 1α(HIF-1α; a hypoxia marker protein) in the ICCs was rare; meaning that hypoxic state in the central area of ICCs was attenuated. On the other hand, the ICCs generated in flat-bottomed wells had abnormal insulin secretion caused by weak cell-to-cell interaction. In addition, after cultivation for 2 weeks, the generated ICCs in flat-bottomed well had weak signal of HIF-1α, while intact islets had its strong signal. Therefore, new culture system using concave microwell array may be a promising alternative to success fully prepare functional ICCs. Open image in new window

Subcutaneous site is ideal for clinical islet transplantation because it has the advantage of simple operation procedure under local anesthesia and can be biopsied when needed. However, the transplantation outcomes at subcutaneous site have been disappointing due to hypoxia-induced oxidative stress by poor vascularization. We hypothesized that subcutaneously transplanted islets would have hypoxia resistance by using internalization of metallothionein (MT), an antioxidant scavenging enzyme, which was mediated by fusion between MT and cell penetrating Tat peptide. The Tat-MT was dose-dependently transduced into islets without any damage. Tat-MT-treated islets could be protected from oxidative stress induced by intracellular nitric oxide donor, sodium nitroprusside (SNP). When Tat-MT-treated islets were subcutaneously transplanted into diabetic nude mice, they normally controlled the blood glucose levels without severe fluctuation (median survival time (MST): > 30 days), whereas most untreated islets were rejected (MST 17 days). From the intraperitoneal glucose tolerance test 5 days after posttransplantation, glucose responsiveness of Tat-MT-treated islets was similar to that of normal healthy mice, while untreated islets had delayed glucose responsiveness. From the results of immunohistochemical stain, Tat-MT-treated islets had strong anti-insulin positive cells and lower anti-HIF-1α positive cells. However, untreated islets had rare anti-insulin positive cells and strong anti-HIF-1α-positive cells. Collectively, these findings demonstrated that Tat-MT delivery into islet could offer a new strategy for successful islet transplantation under subcutaneous space.