Islet transplantation 30 years after the first transplants.

First clinical islet allotransplantation in patients affected by type 1 diabetes mellitus was performed about 30 years ago. Despite the progressive improvement of the success rate, the clinical indication to the islet allotransplantation remains limited to selected patients affected by brittle type 1 diabetes mellitus. The burden of the immunosuppression therapy still represents the main critical issue but other areas might be subject to further improvements, such as the islet production, islet engraftment and long-term function.  Several strategies have been proposed to increase the success rate of pancreas digestion and islet purification or to facilitate islet engraftment by reducing islet hypoxia and the inflammatory reaction occurring in the site of transplantation. The co-transplantation of progenitors of beta cell together with the islets has expected to contribute to prolong graft function. Clinical trials are expected soon. Scientific advances, as well as economical efforts, are required to make this procedure a real therapeutical option for patients with type 1 diabetes mellitus.

Formulation and characterization of silk fibroin films as a scaffold for adipose-derived stem cells in skin tissue engineering.

Skin substitutes are epidermal, dermal or complete bilayered constructs, composed by natural or synthetic scaffolds and by adherent cells such as fibroblasts, keratinocytes or mesenchymal stem cells. Silk fibroin is a promising polymer to realize scaffolds, since it is biocompatible, biodegradable, and exhibits excellent mechanical properties in terms of tensile strength. Moreover, fibroin can be added of others components in order to modify the biomaterial properties for the purpose. The aim of this work is to prepare silk fibroin films for adipose-derived stem cell (ADSCs) culture as a novel feeder layer for skin tissue engineering. Pectin has been added to promote the protein conformational transition and construct strength, while glycerol as plasticizer, providing biomaterial flexibility. Eighteen formulations were prepared by casting method using fibroin, pectin (range 1-10% w/w), and glycerol (range 0-20% w/w); films were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry assay, to select the optimal composition. A stable fibroin conformation was obtained using 6% w/w pectin, and the best mechanical properties were obtained using 12% w/w glycerol. Films were sterilized, and human ADSCs were seeded and cultured for 15 days. Cells adhere to the support assuming a fibroblastic-like shape and reaching confluence. The ultrastructural analysis evidences typical active-cell features and adhesion structures that promote cell anchorage to the film, thus developing a multilayered cell structure. This construct could be advantageously employed in cutaneous wound healing or where the use of ADSCs scaffold is indicated either in human or veterinary field.

Culture medium modulates proinflammatory conditions of human pancreatic islets before transplantation.

A portion of transplanted islets is lost during engraftment as a result of stressful events, involving hypoxia and production of proinflammatory molecules by islets. Two of these molecules (monocyte chemoattractant protein-1, CCL2/MCP-1 and tissue factor, TF) are directly correlated with reduced graft function. We evaluated which factors reduce islet proinflammatory conditions. In particular the effects of different culture media supplemented with proteins or antioxidant agents on CCL2/MCP-1 and TF human islet release were evaluated. We observed that human islets after culture in final wash culture medium (FW) significantly decreased CCL2/MCP-1 release and TF production compared with CMRL and M199. These effects were independent from the type of protein added to the media (human serum, human albumin, fetal calf serum). Glutathione in FW further decreased CCL2/MCP-1 in a dose-dependent manner. Culture conditions can modulate the proinflammatory state of islets, and could be used in clinical islet transplantation to reduce inflammation during engraftment.

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function.

AIMS/HYPOTHESIS: Efficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation. METHODS: Since 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome. RESULTS: By univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p=0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p=0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p=0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p=0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p=0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071). CONCLUSIONS/INTERPRETATION: These data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.

In vitro modulation of monocyte chemoattractant protein-1 release in human pancreatic islets.

Islet transplantation is a new approach to treat type 1 diabetic patients. Despite its great potential and progressively increasing success rate, islet engraftment still represents an unsolved problem. Only part of the transplanted beta-cell mass survives after infusion due to hypoxia and inflammatory reactions, principally mediated by macrophages. We have demonstrated that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of a transplant. In this study we have attempted to modulate in vitro MCP-1 release by human islets. Human islets isolated using the automated method were cultured in CMRL or M199 standard culture media alone or supplemented with (1) two intracellular kinase inhibitors (10 micromol/L RO8220, a protein kinase C inhibitor, and rcAMP 20 micromol/L, a protein kinase A inhibitor) or (2) two antioxidant and cell-protective agents (vitamin E, vitamin B); or (3) immunosuppressive drugs (0.001 to 10 ng/mL cyclosporine, 0.1 to 100 ng/mL rapamycin, 0.1 to 10 ng/mL tacrolimus, 0.001 to 10 ng/mL mycophenolate acid). We observed that the only culture condition that significantly decreased MCP-1 in human islets were CMRL (31 +/- 12 in CMRL vs 539 +/- 184 pg/mL, in M199, P <.05) or cyclosporine (514 +/- 83 pg/mL in control islet vs 307 +/- 13, 231 +/- 44, 192 +/- 4, 242 +/- 113, 169 +/- 15 pg/mL in islet plus cyclosporine ranging from 0.001 to 10 ng/mL, respectively, P >.05). The capacity of in vitro factors to decrease human islet MCP-1 release suggests strategies to increase the success of islet transplantation.

Identification of in vitro parameters predictive of graft function: a study in an animal model of islet transplantation.

The quality of human islets is one of the factors decisive for the success of human islet transplantation. Several parameters have been proposed to characterize islet quality, but none of them has been able to predict the fate of a transplant. The aim of our study was to correlate a panel of in vitro parameters for islet viability with their in vivo function after transplantation in nude mice. Islets were obtained after enzymatic digestion of a human pancreas; they were purified from exocrine tissue using a continuous-density gradient. Two aliquots of islets (1000 and 2000 islets) were transplanted under the kidney capsule of diabetic nude mice. The animals were followed for 1 month with repeated measurements of blood glucose and body weight. One month after transplantation, mice were killed and their graft harvested for histologic analysis. In parallel we studied in vitro islet viability with propidium iodide and fura-2, their insulin content, their purity, and their insulin response to glucose upon static incubation. Ten islet preparations were transplanted: 3 out of 10 preparations did not restore normoglycemia; 4 out of 10 normalized glycemia only in mice receiving 2000 islets, and 3 out of 10 fully restore normoglycemia in all mice. The purity of preparations (R(2) = 0.63 and 0.85, respectively, with 1000 and 2000 islets) and the insulin content (R(2) = 0.75 with 2000 IE) correlated with transplant success. These data show that purity of islet preparations and their insulin content should be useful parameters for the selection of islet preparations for transplant purposes.

Islet allotransplantation in type 1 diabetic patients.

Islet transplantation was proposed more than 10 years ago as treatment for normalising glucose homeostasis in type 1 diabetic patients. Since the beginning it has aroused great interest among diabetic patients being an easy procedure, burdened by minor complications: islet transplantation in fact consists on a transhepatic percutaneous injection under local anaesthesia. The initial clinical outcomes not came up to expectations, being low the insulin independence rate and the long term graft function in recipients. Recently, thanks to the introduction of new immunosuppression strategies, clinical data greatly improved: insulin independence was reached in all recipients and maintained in more than 70% of them 2 years from the transplant. The need of an immunosuppression therapy limits the indication of islet transplantation to diabetic patients already immunosuppressed for a previous organ transplant or to patients with brittle diabetes, that is not controlled also with the new strategies of insulin treatment, with a poor quality of life and an increased rate of diabetic complications. Other problems are represented by the progressive decrease of graft function during long term follow up, and by the low number of organ donors that limits the number of transplantation feasible per year.