Ehab Rafael

Optimising islet engraftment is critical for successful clinical islet transplantation

Clinical islet transplantation is currently being explored as a treatment for persons with type 1 diabetes and hypoglycaemia unawareness. Although ‘proof-of-principle’ has been established in recent clinical studies, the procedure suffers from low efficacy. At the time of transplantation, the isolated islets are allowed to embolise the liver after injection in the portal vein, a procedure that is unique in the area of transplantation. A novel view on the engraftment of intraportally transplanted islets is presented that could explain the low efficacy of the procedure.

Survival of macroencapsulated allogeneic parathyroid tissue one year after transplantation in nonimmunosuppressed humans.

The use of immunoisolation devices may allow transplantation without need for immunosuppression and could widen the indications for cell transplantation. In this study, we evaluated the survival of encapsulated parathyroid tissue in nonimmunosuppressed humans. Autologous parathyroid implants: Seven patients under-going parathyroidectomy had devices containing small pieces of their own parathyroid tissue implanted SC. These devices were explanted after 2–4 weeks for histological evaluation. Allogeneic parathyroid implants: Four patients with chronic hypoparathyroidism were transplanted with one to three large (40 μl) and one small (4.5 μl) device filled with meshed parathyroid tissue and implanted SC. The small devices were explanted at 4 weeks, while the large ones were explanted 8.5 to 14 months after implantation. In both studies, control implants were placed in nude mice. Autologous study results: At explantation, the grafts consisted of 22 ± 6% endocrine tissue and 63 ± 7% fibrosis, while 15 ± 5% of the grafts were necrotic. Allogeneic study results: In devices explanted from the patients at 4 weeks, fibrosis dominated and only 1%, 5%, and 23% of the grafts consisted of endocrine tissue. A similar histological appearance was found in grafts from nude mice. In devices explanted at 8.5–14 months, histologically intact endocrine tissue was found in all patients. However, nearly all the tissue consisted of fibrosis. There was no detectable increase in the parathormone (PTH) level in all patients. Macroencapsulated human allogeneic parathyroid tissue can survive up to 1 year after transplantation into nonimmunosuppressed patients. However, marked fibroblast overgrowth occurred, especially in the allogeneic implant study, using meshed parathyroid tissue. This was probably not related to the allo-response, because similar findings were observed in the nude mouse implants. In future studies, better tissue preparation and improvements in the physiological milieu inside the device may help to reduce fibroblast overgrowth and increase survival of the parathyroid cells.

Improved Survival of Macroencapsulated Islets of Langerhans by Preimplantation of the Immunoisolating Device: A Morphometric Study:

Encapsulation of cells in a semipermeable membrane may in the future provide an opportunity to treat a variety of endocrine and neurological disorders, without the need for lifelong immunosuppression. The physiological conditions in the device are crucial factors for graft survival. Previously, we have shown that the exchange across the immunoisolating membrane and the microcirculation around the TheraCyte™ device increase around 3 months after implantation. The aim of this study was to determine whether preimplantation of the TheraCyte™ device would improve the survival of a later transplanted islet graft. A TheraCyte™ device was implanted SC on one side of the back of a nondiabetic SD rat. After 3 months, 1500 islets isolated from SD rats were transplanted via the device port. At the same time, another device, loaded with the same number of islets, was implanted on the other side of the back. Both devices were explanted 2 weeks after islet transplantation (i.e., 3.5 months and 0.5 month after device implantation, respectively). Six pairs of devices were evaluated by morphometery. The volume densities of viable islets were 0.22 ± 0.04 in the preimplanted device vs. 0.06 ± 0.03 in the nonpreimplanted one (p < 0.05). The corresponding volume densities of fibrosis and necrosis were 0.64 ± 0.13 vs. 0.85 ± 0.08 (p < 0.05) and 0.11 ± 0.14 vs. 0.09 ± 0.07 (ns), respectively. When the absolute volumes (mm3) were calculated, preimplanted devices contained 1.1 ± 0.7 endocrine cells while nonpreimplanted ones contained 0.4 ± 0.2 (p < 0.05). The percentages of insulin-positive β-cells in the preimplanted versus nonpreimplanted device were 80 ± 5% and 67 ± 6%, respectively (p < 0.01). The corresponding volumes of fibrotic tissue were 3.0 ±1.8 vs. 5.2 ± 1.2 (p < 0.05), while the amount of necrotic tissue did not differ significantly (0.42 ± 0.5 vs. 0.50 ± 0.3). Preimplantation of the TheraCyte™ device seems to improve the survival of an encapsulated islet graft and reduce fibroblast outgrowth in the device.

In vivo evaluation of glucose permeability of an immunoisolation device intended for islet transplantation: a novel application of the microdialysis technique.

Immunoisolation devices consist of semipermeable membranes chosen to protect the islets from the immune system but still allow sufficient passage of nutrients, oxygen, and the therapeutic products, insulin. The exchange between the device and the microcirculation will influence the survival of the graft as well as the metabolic efficacy of the islet implant. Glucose is the important trigger factor for insulin secretion. In this study, we evaluate the in vivo glucose permeability of the Theracyte™ immunoisolation device at various times after implantation. Empty devices were implanted SC in rats. The glucose kinetics in the device was compared to that in the SC tissue during IV glucose tolerance tests (IVGTTs), using the microdialysis technique. In rats studied on day 1, or 1, 2, and 4 weeks after implantation, the peak glucose levels (Cmax) were significantly lower, the times-to-peak (TTP) were significantly longer, and the areas under the curve during the first 40 min (AUC0–40) were significantly smaller in the device than in the SC fat. However, at 3 months all parameters improved and Cmax, TTP, and AUC0–40 in the device did not differ significantly from those measured in the SC fat. Thus, during the first 4 weeks the device constitutes a significant diffusion barrier, but at 3 months the exchange between the lumen of devices and the blood stream improves. Our data indicate that implantation of the device several months before transplantation of the cellular graft would improve the exchange across the membrane during the early posttransplant period. This should have positive effects on graft survival and function. We also suggest that microdialysis is a useful tool for evaluating the in vivo performance of macroencapsulation devices.

Longitudinal studies on the microcirculation around the TheraCyte immunoisolation device, using the laser Doppler technique.

Encapsulation of cellular grafts in an immunoisolation membrane device may make it possible to perform transplantation without having to give immunosuppressive drugs. A common problem is the development of an avascular fibrotic zone around the implants, leading to impaired graft survival. The TheraCyte™ macroencapsulation device has therefore been designed to facilitate neovascularization of the devices surface. In this study, we evaluated the microcirculation around empty TheraCyte™ devices implanted SC in rats at various times after implantation, using a laser Doppler probe introduced via the device port. Studies were performed on day 1 or at 1, 2, and 4 weeks or at 2, 3, and 12 months after implantation. The mean flow was 158 ± 42, 148 ± 50, 133 ± 28, 72 ± 17, 138 ± 41, 165 ± 43, and 160 ± 29 perfusion units (PU), respectively. Thus, the microcirculation around the device was significantly reduced at 4 weeks after implantation (p < 0.01) while, from 2 months onwards the circulation had improved and did not differ significantly from that on day 1. The present study shows time-related changes in the microcirculatory flow around TheraCyte™ macroencapsulation devices that agree with our previous microdialysis studies on in vivo exchange of insulin and glucose between the device and the circulation. Laser Doppler flowmetry seems to provide a reliable technique for screening blood perfusion around macroencapsulation devices.

In vivo Studies on Insulin Permeability of an Immunoisolation Device Intended for Islet Transplantation Using the Microdialysis Technique

In this study, insulin was injected into TheracyteTM immunoisolation devices to analyze changes in the permeability of the device over time after implantation. The recovery of insulin was studied after subcutaneous implantation of the devices in rats, using the microdialysis technique. The area under the insulin cocnetration vs. time curves (AUC) after insulin injection in devices implanted 1 day previously did not differ significantly from the AUC after subcutaneous injection. At 1, 2 and 4 weeks after implantation, the recovery of insulin was significantly reduced, but at 3 months, the AUC was not significantly different from that in the control group. Histological examination showed that the number of vascular profiles within 15 μm of the device were significantly higher at 2, 4 weeks and 3 months after transplantation when compared to numbers at 1 week. The design of the device allows transplantation of cells at a chosen time point after its implantation. Delayed filling of the device would allow neovascularization of the device surface before graft implanatation and we suggest that such a schedule might improve function of the encapsulated graft.

Evaluation of Perfluorohexyloctane/Polydimethylsiloxane for Pancreas Preservation for Clinical Islet Isolation and Transplantation

This study aimed to evaluate a 50:50 mix of perfluorohexyloctane/polydimethylsiloxane 5 (F6H8S5) preservation of pancreases in a clinical setting compared with standard solutions for 1) cold ischemia time (CIT) <10 h and 2) an extended CIT >20 h. Procured clinical-grade pancreases were shipped in either F6H8S5 or in standard preservation solutions, that is, University of Wisconsin (UW) or Custodiol. F6H5S5 was preoxygenated for at least 15 min. Included clinical-grade pancreases were procured in UW or Custodiol. Upon arrival at the islet isolation laboratory, the duodenum was removed followed by rough trimming while F6H8S5 was oxygenated for 15-20 min. Trimmed pancreases were immersed into oxygenated F6H8S5 and stored at 4°C overnight followed by subsequent islet isolation. Pancreas preservation using F6H8S5 proved as effective as UW and Custadiol when used within CIT up to 10 h, in terms of both isolation outcome and islet functionality. Preservation in F6H8S5 of pancreases with extended CIT gave results similar to controls with CIT <10 h for both isolated islet functionality and isolation outcome. This study of clinically obtained pancreases indicates a clear benefit of using F6H8S5 on pancreases with extended CIT as it seems to allow extended cold ischemic time without affecting islet function and islet numbers.

Calcium: A Crucial Potentiator for Efficient Enzyme Digestion of the Human Pancreas

BACKGROUND Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5-10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. METHODS Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. RESULTS Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. CONCLUSIONS Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.Background: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5–10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. Methods: Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. Results: Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. Conclusions: Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.