Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 May;101(5):1025-1035.
doi: 10.1097/TP.0000000000001454.

Effects of Composition of Alginate-Polyethylene Glycol Microcapsules and Transplant Site on Encapsulated Islet Graft Outcomes in Mice

Affiliations
Free PMC article

Effects of Composition of Alginate-Polyethylene Glycol Microcapsules and Transplant Site on Encapsulated Islet Graft Outcomes in Mice

Chiara Villa et al. Transplantation. .
Free PMC article

Abstract

Background: Understanding the effects of capsule composition and transplantation site on graft outcomes of encapsulated islets will aid in the development of more effective strategies for islet transplantation without immunosuppression.

Methods: Here, we evaluated the effects of transplanting alginate (ALG)-based microcapsules (Micro) in the confined and well-vascularized epididymal fat pad (EFP) site, a model of the human omentum, as opposed to free-floating in the intraperitoneal cavity (IP) in mice. We also examined the effects of reinforcing ALG with polyethylene glycol (PEG). To allow transplantation in the EFP site, we minimized capsule size to 500 ± 17 μm. Unlike ALG, PEG resists osmotic stress, hence we generated hybrid microcapsules by mixing PEG and ALG (MicroMix) or by coating ALG capsules with a 15 ± 2 μm PEG layer (Double).

Results: We found improved engraftment of fully allogeneic BALB/c islets in Micro capsules transplanted in the EFP (median reversal time [MRT], 1 day) versus the IP site (MRT, 5 days; P < 0.01) in diabetic C57BL/6 mice and of Micro encapsulated (MRT, 8 days) versus naked (MRT, 36 days; P < 0.01) baboon islets transplanted in the EFP site. Although in vitro viability and functionality of islets within MicroMix and Double capsules were comparable to Micro, addition of PEG to ALG in MicroMix capsules improved engraftment of allogeneic islets in the IP site, but resulted deleterious in the EFP site, probably due to lower biocompatibility.

Conclusions: Our results suggest that capsule composition and transplant site affect graft outcomes through their effects on nutrient availability, capsule stability, and biocompatibility.

Figures

FIGURE 1
FIGURE 1
Optimizing fabrication of ALG Micro capsules to minimize the volume of encapsulated islet grafts. A, Schematic of our approach to determine the effects of capsule composition and transplant site on encapsulated graft outcomes. B, Diameter distribution (n = 300) of cell-free 1.2% UP-MVG microcapsules (ALG) fabricated with the optimized parameters (Table 1, bold). C-D, Phase contrast images (C) and confocal images of live (green)/dead (red) stained (D) ALG microcapsules fabricated with optimized fabrication parameters (Table 1, bold) and loading density of pancreatic islets from Lewis Rats equal to 5 k, 15 k, and 30 k IEQ/mL and compared to Naked islets; scale bars 100 μm; nuclei: blue. E-F, GSIR of islets encapsulated using optimized fabrication parameters (Table 1, bold). Micro capsules loaded with 15 k IEQ/mL rat islets (green) are compared to naked islets (black). N = 3 aliquots of 100 IEQ per conditions from a minimum of n = 3 independent experiments. Absolute values of insulin concentration in supernatants after incubation in low glucose (L1), high glucose (HG) and low glucose (L2) (E), and stimulation indexes (F) are indicated. G, Diameter distribution (n = 253 capsules from n = 7 independent experiments) of islet-containing 1.2% UP-MVG microcapsules (ALG) fabricated with the optimized parameters (Table 1, bold).
FIGURE 2
FIGURE 2
Effects of transplantation site on the outcome of islet allografts encapsulated in optimized ALG microcapsules (Micro) without immunosuppression. The free-floating intraperitoneal (IP) site is compared with the confined and vascularized EFP site. A, Blood glucose of STZ-induced diabetic C57BL/6 mice transplanted with 750 IEQ naked in the EFP (black, n = 15) or IP (grey, n = 8) sites or 750 IEQ microencapsulated (Micro) in the EFP (green, n = 7) or IP (orange, n = 5) sites; all islets from fully MHC-mismatched BALB/c mice donors. B, Percentage of mice that reversed diabetes after transplantation. C, Percentage survival of allografts that reversed diabetes after transplantation. Tables below graphs indicate P values. D-F, Histological evaluation of EFP grafts and of capsules retrieved from the IP site by intraperitoneal lavage, fixed in formalin, embedded in paraffin, and thin sliced (5 μm). Shown are grafts that reversed diabetes and maintained euglycemia for more than 100 days. In H&E-stained sections (D) arrows point at areas of islet central necrosis. Scale bars, 100 μm. Confocal images: host vessels (CD31+, red), macrophages (MAC2+, green) and beta cells (INS+, cyan) are shown in panel (E); T cells (CD3+, red), B cells (B220+, green) and beta cells (INS+, cyan) are shown in panel (F). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (grey). Scale bar 150 μm; (G) Phase contrast images of baboon islets encapsulated in Micro capsules fabricated with optimized fabrication parameters (Table 1, bold) and loading density of 15 k IEQ/mL and compared to Naked islets. Scale bars, 200 μm; (H) Blood glucose of STZ-induced diabetic NOD-scid mice transplanted with 1000 IEQ naked (black, n = 5) or 750 IEQ microencapsulated (Micro, green, n = 4) islets in the EFP and compared to 1000 IEQ (light grey) and to 2000 IEQ (dark grey) naked islets transplanted in the kidney capsule (KD) controls; all islets from baboon nonhuman primate donors. I, Percentage of mice that reversed diabetes after transplantation of baboon islets. J, Histological evaluation of EFP grafts of naked versus Micro encapsulated in the EFP site analyzed 30 days after transplantation in diabetic NOD-scid mice. Scale bars 200 μm.
FIGURE 3
FIGURE 3
Design, fabrication and in vitro evaluation of PEG-ALG hybrid MicroMix and Double capsules compared to ALG Micro capsules. A-B, Osmotic pressure resistance of cell-free ALG capsules (Micro) compared to PEG capsules and ALG-PEG capsules (MicroMix). Percentage of intact Micro (n = 30) versus PEG (n = 30) versus ALG-PEG (n = 30) capsules after exposure to 2 hrs ddH2O followed by saline buffer for 60 minutes (A) and % intact capsule dependence on time exposure to saline (B). C, Schematic of ALG Micro versus hybrid ALG-PEG Micromix capsules, where PEG and ALG are interlaced, and Double capsules, where PEG is added to the ALG Micro capsule as a thin external layer. D, Schematic of the emulsion procedure for fabrication of Double capsules. E, Representative phase contrast (top) and confocal images (bottom) of Lewis rat islets enclosed in Micro, Micromix and Double capsules stained with anti-PEG antibodies (green). Nuclei are counterstained with Hoechst (blue). Thickness of double capsules was quantified on n = 12 capsules and was found to be 15 ± 2 μm. Scale bars 100 μm. F-H, Diameter distribution of islet-containing MicroMix (blue, F) and Double (purple, G) capsules and direct comparison with Micro capsules (H, P > 0.05). In panel H, statistical analysis of the measured values is presented in the table next to the graph. I, Viability assessment by live (green) and dead (red) staining via confocal imaging of Lewis rat islets enclosed in Micro, Micromix, and Double capsules 48 hours after encapsulation. Nuclei are counterstained with Hoechst (blue). Scale bar 100 μm. J, GSIR of Lewis rat islets encapsulated in Micro (green), MicroMix (blue), Double (purple) capsules and compared to naked islets (black). Absolute insulin secretion and stimulation index are indicated. N = 3 aliquots of 100 IEQ per conditions from a minimum of n = 3 independent experiments. K, Oxygen consumption rate (OCR) normalized to total DNA content of Lewis rat islets encapsulated in Micro (green), MicroMix (blue), Double (purple) capsules and compared to naked islets (black); n = 3 per condition.
FIGURE 4
FIGURE 4
Effects of composition of PEG-ALG capsules on the outcome of islet allografts in the IP site without immunosuppression. ALG Micro capsules are compared to PEG-ALG hybrid MicroMix and Double capsules containing islets and to naked islets. A, Blood glucose of STZ-induced diabetic C57BL/6 mice transplanted with 750 IEQ naked (grey, n = 7) islets or encapsulated in Micro (orange, n = 4), or MicroMix (blue, n = 4), or Double (purple, n = 6) capsules in the IP; all islets from fully MHC-mismatched BALB/c mice donors. B, Percentage of mice that reversed diabetes after transplantation. C, Percentage survival of allografts that reversed diabetes after transplantation. Tables below graphs indicate P values. D-F, Histological evaluation of grafts retrieved from the IP site by intraperitoneal lavage, fixed in formalin, embedded in paraffin, and thin sliced (5 μm). Shown are grafts that reversed diabetes and maintained euglycemia for more than 100 days. In H&E stained sections (D) arrows point at areas of islet necrosis. Scale bars 100 μm. Confocal images: host vessels (CD31+, red), macrophages (MAC2+, green) and beta cells (INS+, cyan) are shown in panel E; T cells (CD3+, red), B cells (B220+, green) and beta cells (INS+, cyan) are shown in panel F. Nuclei are counterstained with DAPI (grey). Scale bar, 150 μm.
FIGURE 5
FIGURE 5
Effects of composition of PEG-ALG capsules on the outcome of islet allografts in the EFP site without immunosuppression. ALG Micro capsules are compared to PEG-ALG hybrid MicroMix and Double capsules and to naked islets. A, Blood glucose of STZ-induced diabetic C57BL/6 mice transplanted with 750 IEQ naked (black, n = 15) islets or encapsulated in Micro (green, n = 7), or MicroMix (blue, n = 4), or Double (purple, n = 6) capsules in the EFP; all islets from fully MHC-mismatched BALB/c mice donors. B, Percentage of mice that reversed diabetes after transplantation. C, Percentage survival of allografts that reversed diabetes after transplantation. Tables below graphs indicate P values. D-F, Histological evaluation of EFP grafts fixed in formalin, embedded in paraffin, and thin sliced (5 μm). Shown are grafts that reversed diabetes and maintained euglycemia for more than 100 days. In H&E stained sections (D) arrows point at area of host reactivity. Scale bars 100 μm. Confocal images: host vessels (CD31+, red), macrophages (MAC2+, green) and beta cells (INS+, cyan) are shown in panel E, T cells (CD3+, red), B cells (B220+, green) and beta cells (INS+, cyan) are shown in panel (F). Nuclei are counterstained with DAPI (grey). Scale bar 150 μm. G-I, Biocompatibility of cell-free Micro (G), MicroMix (H) and Double (I) capsules in the EFP site: H&E staining (top) and Masson’s Trichrome (bottom), 7 days after implantation. Scale bar 100 μm.

Similar articles

See all similar articles

Cited by 11 articles

See all "Cited by" articles

References

    1. Barton FB, Rickels MR, Alejandro R, et al. Improvement in outcomes of clinical islet transplantation: 1999-2010. Diabetes Care. 2012;35:1436–1445. - PMC - PubMed
    1. Basta G, Calafiore R. Immunoisolation of pancreatic islet grafts with no recipient's immunosuppression: actual and future perspectives. Curr Diab Rep. 2011;11:384–391. - PubMed
    1. O'Sullivan ES, Vegas A, Anderson DG, et al. Islets transplanted in immunoisolation devices: a review of the progress and the challenges that remain. Endocr Rev. 2011;32:827–844. - PMC - PubMed
    1. Tuch BE, Keogh GW, Williams LJ, et al. Safety and viability of microencapsulated human islets transplanted into diabetic humans. Diabetes Care. 2009;32:1887–1889. - PMC - PubMed
    1. Faleo G, Lee K, Nguyen V, et al. Assessment of immune isolation of allogeneic mouse pancreatic progenitor cells by a macroencapsulation device. Transplantation. 2016;100:1211–1218. - PMC - PubMed
Feedback