Significance In this study we show that pancreatic islets embedded in modules coated with endothelial cells and injected under the skin return streptozotocin-induced diabetic SCID/beige mice to normoglycemia. The transplanted islets became revascularized and directly integrated with host’s vasculature, a feature not seen previously in the subcutaneous space. These implants were also retrievable, an important clinical consideration. The success here means that islet transplantation can move away from inhospitable sites such as the peritoneal cavity or the liver.

Abstract The transplantation of pancreatic islets, following the Edmonton Protocol, is a promising treatment for type I diabetics. However, the need for multiple donors to achieve insulin independence reflects the large loss of islets that occurs when islets are infused into the portal vein. Finding a less hostile transplantation site that is both minimally invasive and able to support a large transplant volume is necessary to advance this approach. Although the s.c. site satisfies both these criteria, the site is poorly vascularized, precluding its utility. To address this problem, we demonstrate that modular tissue engineering results in an s.c. vascularized bed that enables the transplantation of pancreatic islets. In streptozotocin-induced diabetic SCID/beige mice, the injection of 750 rat islet equivalents embedded in endothelialized collagen modules was sufficient to restore and maintain normoglycemia for 21 days; the same number of free islets was unable to affect glucose levels. Furthermore, using CLARITY, we showed that embedded islets became revascularized and integrated with the host’s vasculature, a feature not seen in other s.c. studies. Collagen-embedded islets drove a small (albeit not significant) shift toward a proangiogenic CD206+MHCII−(M2-like) macrophage response, which was a feature of module-associated vascularization. While these results open the potential for using s.c. islet delivery as a treatment option for type I diabetes, the more immediate benefit may be for the exploration of revascularized islet biology.

Pancreatic islet transplantation is a promising treatment for those living with type 1 diabetes (1, 2), but this therapy has limitations (3, 4). Using current methods (portal vein infusion), up to 60% of transplanted islets are lost within the first 3 d (5), and thus islets from multiple donors are required to achieve normoglycemia (6). Islet engraftment is adversely affected by the blood-mediated inflammatory response (7) and a lack of vascularity, subjecting the islets to ischemia.

The s.c. site has been considered as an alternative site for islet transplantation because of its large (albeit not necessarily readily accessible) size, and s.c. injection could limit procedural risk compared with other sites. However, because s.c. vascularity is poor (8), various strategies to vascularize the site are being explored (9, 10), e.g., the use of mesenchymal stromal cells (MSC) (11). With the perspective that the underlying problem is hypoxia, Ludwig et al. (12) created an implantable chamber equipped with a refillable oxygen chamber to support s.c. islet transplantation. Alternatively, others have used a two-step protocol (13, 14) in which a cavity is prepared in the s.c. space; islets are then added after several weeks’ delay to minimize the impact of the inflammatory response while perhaps generating a prevascularized site.

Pancreatic islets are highly susceptible to apoptosis due to insufficient oxygen and nutrient diffusion (15). After transplantation it is critical that the pancreatic islets become rapidly revascularized and integrated with the host’s systemic vasculature to achieve glucose homeostasis (16). While revascularization has been observed in vascularized extrahepatic sites such as the small-bowel mesentery (10) and kidney capsule (17), revascularization and anastomosis with the host’s vasculature have not previously been shown in the s.c. space. Such integration is presumed to be necessary for long-term graft survival.

Modular tissue engineering is a bottom-up approach to create vascularized tissue implants that can be injected s.c. (18). Submillimeter collagen cylinders (“modules”) are coated with endothelial cells (EC) (18); the EC migrate and vascularize the s.c. site as the modules remodel. With MSC embedded in modules as support cells, a mature s.c. vascular bed is created within 14–21 d of transplantation in both immune-suppressed Sprague–Dawley rats (19) and immune-compromised SCID/beige (bg) mice (20).

The objective of this study was to show that pancreatic islets embedded in endothelialized modules and injected s.c. became revascularized and returned streptozotocin-induced diabetic SCID/bg mice to normoglycemic levels. The protocol is illustrated in Fig. S1. The effect of adding adipose-derived mesenchymal stromal cells (adMSC) or omitting human umbilical vein endothelial cells (HUVEC) was also tested. We used the CLARITY protocol (21, 22) to image the entire explant and show the anastomosis of the pancreatic islets with the host’s systemic vasculature. In addition, we used flow cytometry to characterize the inflammatory response, which is a key driver of vascularization but may also limit islet survival after transplantation.

Fig. S1. Schematic of the experimental setup. SCID/Bg mice were made diabetic using streptozotocin 7 d before s.c. implantation. Two days before the implantation, Wistar rat islets were isolated and were either embedded in modules (with other cells) or left as free islets. The islet modules (750 IEQ) were then cultured for 1 d before s.c. injection. Blood glucose monitoring was performed daily, and at day 14 an IPGTT was administered. At day 21, implants in animals that had returned to normoglycemia were removed without killing the animal, and animals were followed for an additional 3 d to confirm that the implant was responsible for returning the diabetic mouse to normoglycemia. Explants were analyzed at days 7, 14, and 21/24 using histology, CLARITY, and flow cytometry.

Conclusion The s.c. injection of rat pancreatic islets embedded in endothelialized modules was able to return streptozotocin-induced diabetic SCID/bg mice to normoglycemia. HUVEC seeded on the modules was important for vascularization at early time points and for preserving the structural integrity of the modular implants. Islets embedded in endothelialized modules became revascularized, and whole-explant imaging showed that the intraislet vasculature had integrated with the host’s vasculature by day 14, a feature not shown in previous s.c. studies. In addition, embedded islets experienced an inflammatory response that consisted of mainly M2-like macrophages, which was considered important for tissue remodeling and vascularization, although the high neutrophil response at day 7 suggests that there may be a need to modulate other aspects of early inflammation to protect islet viability. Module-based vascularization in which the transplanted islets become integrated with the host vasculature has enabled the s.c. site to be a suitable option for islet transplantation.

Materials and Methods Islet Isolation. Primary rat islets were harvested from 12-wk-old Wistar rats using a previously described protocol (50). IEQ were then calculated based on volumetric assumptions (51). Primary rat islets were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Pen/Strep) (Gibco). For the in vivo studies, eight independent islet isolations and transplantations were performed. Each isolation generated enough islets (2,800) for three implants, spanning multiple test groups. Module Fabrication. Modules were fabricated as before (20). HUVEC-coated islet modules were produced by embedding rat islets (750 IEQ) without adMSC. In addition, HUVEC-coated modules that contained adMSC (106 cells/mL collagen solution; Lonza) or no embedded cells were produced. For a complete list of the different cell compositions used for the modules in this study, please refer to Table S1. Table S1. Cell compositions of different transplant groups in the study Module Transplantation. All animal experiments and surgeries were performed at the University of Toronto and were approved by the Faculty of Medicine Animal Care Committee. SCID/bg mice (5–6 wk old; Charles River) were made diabetic through a single i.p. injection of streptozotocin (200 mg/kg) (Sigma-Aldrich) 1 wk before transplantation. Blood glucose was measured via the tail vein with a glucose meter (OneTouch UltraSmart; LifeScan), and animals were considered diabetic when their blood glucose levels exceeded 20 mM for two consecutive daily readings. The modules were rinsed with PBS (Gibco) and s.c. injected into the dorsum using an 18-G needle, as previously described (19, 20). Animals were housed individually in sterile conditions and were provided with irradiated food and water. Nonfasting blood glucose levels were monitored daily until the implants were harvested at days 7, 14, or 21. An IPGTT was performed at days 7 and 14. Mice that were hyperglycemic at day 21 (>11.1 mM) were killed, and the s.c. implants were removed for analysis. Mice that returned to normoglycemia by day 21 were anesthetized, the s.c. modular implant was removed, and the animals were allowed to recover. Blood glucose levels were monitored for the following 2–3 d before the animal was killed; this enabled confirmation that the implant was responsible for the return to normoglycemia. Explants were either sent for histological processing or analyzed using flow cytometry (Fig. S1). CLARITY Processing and Imaging. GSL-1 (Vector Laboratories) conjugated to Alexa-555 (Thermo Fisher Scientific) was injected via the tail vein of the SCID/bg mouse, and the whole body was perfused as previously described (39). The clarified tissue was imaged using light-sheet microscopy (Zeiss Z1 light-sheet microscope at the Sick Kids Imaging Facility) to generate a 1-mm3 image, which was then digitally processed using Bitplane IMARIS (version 8.1). Tissue Digestion and Flow Cytometry. For flow cytometry studies, explants were digested and stained with a panel of markers for inflammatory cells, following previous reports (52). Results were acquired using a five-laser LSR Fortessa X-20 flow cytometer (BD), and data were further analyzed using FlowJo software (v10.0.8). Detailed materials and methods for our protocols are available in Supporting Information.

SI Materials and Methods Islet Isolation. Briefly, the pancreas was cannulated via the bile duct with a 2.0-mg/mL collagenase XI solution (lot #SLBG3259V; Sigma). The pancreas was excised and incubated in a 37-°C water bath for 19 min, mechanically digested, and washed using islet wash buffer containing HBSS (Gibco) supplemented with 1% Pen/Strep (Gibco), 10 mM Hepes (Gibco), and 0.1% BSA (Gibco). The islets were purified using a discontinuous Ficoll gradient with Histopaque-1077 (Sigma) and Histopaque-1119 (Sigma). Finally, the islets were further purified by hand picking and were counted after being stained with Dithizone (Sigma). For in vivo transplantation studies, eight independent islet isolations were performed. Each isolation generated enough islets (2,800) for three implants, spanning multiple test groups. Module Fabrication. Briefly, bovine type I collagen (3 mg/mL; Cedarlane) was combined with 10× α-Minimum Essential Medium (α-MEM; Gibco) and neutralized using 0.8 M NaOH (Sigma Aldrich) to pH ∼7.4. adMSC (1.0 × 106 cells/mL of collagen) and/or pancreatic islets (750 IEQ) were mixed with the collagen before gelling and then were gelled at 37 °C for 45 min (packs were periodically inverted to prevent islets from settling) in sterile 0.71-mm i.d. polyethylene tubing (PE 60; Intramedic; BD) and were cut into small pieces (2 mm long × 0.6 mm diameter) using a custom automatic cutter. Modules were separated from the outer tubing by gentle vortexing and were cultured in the appropriate culture medium. In most cases, HUVEC (2.0 × 106 cells/mL collagen) (P3-6; Lonza) were seeded dynamically onto the surface of the modules for 60 min in an appropriate coculture medium. Modules contract overnight to a final dimension of 0.6 mm long × 0.4 mm diameter. There was an average of a single islet per individual module, and ≈750 modules were transplanted per implant. HUVEC (Lonza) were cultured in EGM-2 medium (Lonza) while adMSC (Lonza) were cultured in DMEM (D6046; Sigma) supplemented with 10% FBS (Gibco) and 1% Pen/Strep (Gibco). HUVEC/adMSC modules were cultured for 3 d before implantation in a 50/50 mixture of EGM-2 and low-glucose adMSC medium. HUVEC/islet modules were cultured overnight in a 50/50 mixture of EGM-2 and islet medium; the short time was used to minimize in vitro culture of islets. HUVEC/islet/adMSC modules were cultured overnight in a 50/50 mixture of EGM-2 and low-glucose adMSC medium; the latter is an acceptable medium for islets. Immediately before injection, different modules were mixed to create the experimental groups as summarized in Table S1. Implant volumes were all nominally 0.1 mL (albeit slightly larger when no HUVEC were present); implants were made up of 0.05 mL of islet-containing modules that had been cultured overnight (islet modules) and 0.05 mL of other modules (without islets) that had been cultured for 3 d (to effect HUVEC confluence). In the islet (+HUVEC) module group, empty modules coated with HUVEC were used to keep the dose of HUVEC constant while omitting the use of adMSC. IPGTT. Mice were fasted for 4 h before being challenged with a 2 g/kg glucose solution via i.p. injection. After injection, the blood glucose levels of the mice were measured at 15, 30, 60, and 120 min by tail-vein blood sampling. AUC blood glucose was calculated and compared among transplant groups. Histological Analysis. Some explants were fixed in 4% neutral buffered formalin (Sigma) for 48 h for processing by the Pathology Research Laboratory at the Toronto General Hospital. Tissue samples were cut in 5-μm sections at multiple levels 50–100 μm apart. Sections were stained for Masson’s trichrome, H&E, UEA-1, CD31, SMA, F4/80, and insulin. Sections were scanned (20×, Aperio ScanScope XT; Leica Microsystems) at the Advanced Optical Microscopy Facility of the University Health Network, Toronto. Scans were analyzed using ImageScope software version 11 (Aperio). CLARITY Processing and Imaging. In some animals, GSL-1 (Vector Laboratories) conjugated to Alexa-555 (Thermo Fisher Scientific) was injected into SCID/Bg mice via the tail vein, and the whole body was perfused using an acrylamide and paraformaldehyde monomer solution (2% and 4%, respectively) (22, 39). After perfusion the s.c. implant was removed and incubated in monomer solution at 4 °C for 1 wk before being incubated at 37 °C for 3 h to allow the monomers to crosslink to the proteins via a thermal initiator (VA-044; Wako Pure Chemical Industries) (21). The cross-linked implants were cleared in 8% SDS/1 M borate buffer for 1 wk at 50 °C to remove lipids and then were incubated with 1 M borate buffer for 2 d. The implants were stained using DAPI (200 pg/mg) (Thermo Fisher Scientific) for 2 d and then incubated in 67% thiodiethanol for 1 d to match the refractive index of the clarified tissue (53). The clarified tissue was imaged using light-sheet microscopy (Zeiss Z1 light-sheet microscope at the SickKids Imaging Facility of the Hospital for Sick Children, Toronto) to generate a 1-mm3 image, which was then digitally processed using Bitplane IMARIS (Version 8.1). Tissue Digestion and Flow Cytometry. For flow cytometry studies, explants were placed in digestion buffer containing collagenase I (Sigma), collagenase XI (Sigma), DNase I (Sigma), hyaluronidase (Sigma), and 1 M Hepes buffer (Gibco) in HBSS (Gibco). Samples were homogenized with the OctoMACS dissociator (Miltenyi) and incubated at 37 °C with shaking for 1 h. Digested samples were filtered with a 40-µm cell strainer (Fisher Scientific) and were counted, and red blood cells were lysed by suspension in 1 mL of 1× red blood cell lysis buffer (BD) for 3 min at 37 °C. Samples were neutralized with PBS supplemented with 0.5% BSA and 2 mM EDTA and then blocked (FC block; BD) to prevent nonspecific binding. The cell suspension was stained with a panel of markers for flow cytometry: Live/Dead, CD45, CD11b, Ly6G, F4/80, CD206, CD86, and MHCII, following previous reports (52). Results were acquired using a five-laser LSR Fortessa X-20 flow cytometer (BD), and data were further analyzed using FlowJo software (v10.0.8). Glucose-Stimulated Insulin Secretion. Three samples of 50 free islets or islets embedded in modules with or without HUVEC were preincubated in low-glucose (3.3 mM) Krebs–Ringer bicarbonate (KRB) (K4002; Sigma) buffer with 0.25% BSA for 40 min at 37 °C. Following the preincubation step, islets were incubated in fresh low-glucose KRB buffer for 1 h at 37 °C and then were incubated in high-glucose (16.7 mM) KRB buffer with 0.25% BSA for 1 h at 37 °C. Aliquots (400 µL) were taken after the low- and high-glucose incubation steps and were stored at −20 °C. Samples were analyzed for rat insulin using a Rat/Mouse Insulin ELISA kit (EZRMI-13K; EMD Millipore) per the manufacturer’s instructions. Insulin stimulation indexes were calculated by taking the ratio of the high-glucose insulin concentration to the low-glucose insulin concentration for each sample, which was then averaged and reported ±SEM (Fig. S11). Fig. S11. (A) Glucose-stimulated insulin secretion assay of free islets and islets embedded in modules with and without HUVEC. There were no significant differences (P = 0.076) in the insulin stimulation index among the three groups after 4 d of culture in vitro. (B) Absolute values of insulin secreted at basal and stimulated glucose solutions. n = 3 in each group; error bars in A indicate SEM. Intraislet Vasculature Analysis and Quantification. Intraislet vasculature quantification was performed using a published MATLAB algorithm (40). The proportion of voxels contributed by the GSL-1+–perfused intraislet vasculature relative to the total islet volume was estimated for eight islets from the day 21 islet module (+HUVEC) group. Our values were compared with those reported for freshly isolated rodent islets (40). Statistical Analysis. One-way ANOVA with a Games–Howell post hoc test (54) was used to compare the means among multiple groups unless otherwise stated in the figure captions. Data were considered statistically significant at a P value of 0.05. For the assessment of host inflammatory cell numbers, the data were log-transformed to correct for large differences in cell numbers and the corresponding absence of a normal distribution. All statistical analysis was performed using SPSS software version 22 (IBM).

Acknowledgments We thank Chuen Lo for his help and expertise with animal surgeries and islet isolations and Abdullah Muhammad Syed and Shrey Sindhwani of Warren Chan’s laboratory at the University of Toronto for helping with imaging our CLARITY-processed tissues. This work was supported by Canadian Institutes of Health Research (CIHR) Grant 142406. A.E.V. and N.C. were supported by scholarships from CIHR and the Province of Ontario. A.E.V. also received funding from the CIHR Training Program for Regenerative Medicine.