Background. The procedure of human islet isolation needs further optimization and standardization. Here, we describe techniques to enhance enzymatic digestion and minimize mechanical forces during the digestion process. The isolation protocol has also been modified to meet current GMP (cGMP) standards. Moreover, the impact of donor- and process-related factors was correlated to the use of islets for clinical transplantation. Methods. One hundred twelve standardized consecutive islet isolations were evaluated. Metyltioninklorid and indermil (topical tissue adhesive) were applied to detect leakage of collagenase injected and to repair the damaged pancreatic glands. The effects of dye and glue were evaluated in terms of islet yield, islet function using the perifusion assay, and success rate of the isolation. To analyze key factors for successful isolations, both univariate and multivariate regression analysis were performed. Results. Both Metyltioninklorid and Indermil were effective to prevent leakage of enzyme solutions from the pancreatic glands. Both islet yield and success rate were higher when these tools were applied (4,516.1±543.0 vs. 3,447.7±323.5, P=0.02; 50.0% vs. 21.3%, P=0.02, respectively). No adverse effects on islet function or collagenase activity were observed. Multivariate regression analysis identified the maximal recorded amylase >100 U/L (P=0.026), BMI (P=0.03), and the use of catecholamine (P=0.04) as crucial donor-related factors. In addition, cold ischemia time (P=0.005), the dissection procedure using whole glands with duodenum (P=0.02), and the local procurement team (P=0.03) were identified as crucial isolation-related variables. Conclusions. A standardized technique of islet isolation is presented applying novel means to improve enzymatic digestion and to meet cGMP standards.

One of the main obstacles to clinical islet xenotransplantation is the injurious instant blood-mediated inflammatory reaction (IBMIR) that causes rapid binding of platelets to the islet surface, activation of the coagulation and complement systems, and leukocyte infiltration of the islets when the islets are exposed to blood. This study assesses the effect of low molecular weight dextran sulfate (LMW-DS) on IBMIR induced by porcine islets in an in vitro tubing loop assay using human blood and in an in vivo model using diabetic athymic mice. In vitro experiments demonstrated that platelet consumption, coagulation, and complement activation were already reduced in the presence of LMW-DS at 0.01 mg/mL, and that at 0.1 mg/mL, LMW-DS prevented IBMIR. Immunohistochemical investigation showed that the leukocyte infiltration was abrogated at the highest dose. In vivo experiments showed that the transplanted pig islets survived for a significantly longer period in recipients treated with LMW-DS, and morphologic examination of transplanted islets showed a reduction in IBMIR analogous to that demonstrated by in vitro studies. Given that LMW-DS has been used in clinical studies without serious adverse reactions, it has potential as a drug candidate that can control the strong innate immune response induced by pig islets when transplanted through the portal vein.

Aims Immunosuppressive drugs could be crucial factors for a poor outcome after islet allotransplantation. Unlike rapamycin, the effects of tacrolimus, the current standard immunosuppressant used in islet transplantation, on graft revascularization remain unclear. We examined the effects of tacrolimus on islet revascularization using a highly sensitive imaging system, and analyzed the gene expression in transplanted islets by introducing laser microdissection techniques. Methods Islets isolated from C57BL/6-Tg (CAG-EGFP) mice were transplanted into the nonmetallic dorsal skinfold chamber on the recipients. Balb/c athymic mice were used as recipients and were divided into two groups: including a control group (n = 9) and tacrolimus-treated group (n = 7). The changes in the newly-formed vessels surrounding the islet grafts were imaged and semi-quantified using multi-photon laser-scanning microscopy and a Volocity system. Gene expression in transplanted islets was analyzed by the BioMark dynamic system. Results The revascularization process was completed within 14 days after pancreatic islet transplantation at subcutaneous sites. The newly-formed vascular volume surrounding the transplanted islets in the tacrolimus-treated group was significantly less than that in the control group (p<0.05). Although the expression of Vegfa (p<0.05) and Ccnd1 (p<0.05) was significantly upregulated in the tacrolimus-treated group compared with that of the control group, no differences were observed between the groups in terms of other types of gene expression. Conclusions The present study demonstrates that tacrolimus inhibits the revascularization of isolated pancreatic islets without affecting the characteristics of the transplanted grafts. Further refinements of this immunosuppressive regimen, especially regarding the revascularization of islet grafts, could improve the outcome of islet allotransplantation.

The role(s) of collagenase G (ColG) and collagenase H (ColH) during pancreatic islet isolation remains controversial, possibly due to the enzyme blends used in the previous studies. We herein examined the role of ColG and ColH using highly pure enzyme blends of recombinant collagenase of each subtype. Rat pancreases were digested using thermolysin, together with ColG, ColH, or ColG/ColH ( n = 9, respectively). No tryptic-like activity was detected in any components of the enzyme blends. The efficiency of the collagenase subtypes was evaluated by islet yield and function. Immunohistochemical analysis, in vitro collagen digestion assay, and mass spectrometry were also performed to examine the target matrix components of the crucial collagenase subtype. The islet yield was highest in the ColG/ColH group (4,101 ± 460 islet equivalents). A substantial number of functional islets (2,811 ± 581 islet equivalents) was obtained in the ColH group, whereas no islets were retrieved in the ColG group. Mass spectrometry demonstrated that ColH reacts with collagen I and III. In the immunohistochemical analysis, both collagen I and III were located in exocrine tissues, although collagen III expression was more pronounced. The collagen digestion assay showed that collagen III was more effectively digested by ColH than by ColG. The present study reveals that ColH is crucial, while ColG plays only a supporting role, in rat islet isolation. In addition, collagen III appears to be one of the key targets of ColH.