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.
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.
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.
A standardized technique of islet isolation is presented applying novel means to improve enzymatic digestion and to meet cGMP standards.
"Islets from human donors were obtained from the Nordic Network for Clinical Islet Transplantation in Uppsala, Sweden, via the Human Tissue Laboratory at Lund University Diabetes Center, Malmö, Sweden. In Uppsala, human islets were isolated using a modification of the semi-automated digestion–filtration method as previously described  and, followed by purification on a continuous density gradient in a refrigerated COBE 2991 centrifuge (COBE Blood Component Technology, Lakewood, CO). The islet preparations were placed in untreated petri dishes Sterilin (Tamro Med., Lab., AB) and kept at 37 • C in an atmosphere of 5% CO 2 in humidified air in culture medium, CMRL 1066 (Gibco-BRL, Invitrogen ) supplemented with 10 mM nicotinamide (Sigma Chemicals), 10 mM Hepes buffer (Gibco-BRL, Invitrogen), 0.25 g/ml fungizone (Gibco-BRL, Invitrogen), 50 g/ml gentamicin (Gibco-BRL, Invitrogen), 2 mM l-gLutamine (Gibco-BRL, Invitrogen), 10 g/ml ciprofloxacin (Bayer), and 10% (v/v) heat-inactivated human serum. "
"In all cases informed written consent was obtained from all donors or donor relatives. Islets of Langerhans were isolated in Uppsala, Sweden, using a protocol approved by the local ethics committee, as described previously . Isolated human islets had a purity ranging from 40% to 95%, and available for research only because the total islet volume was too low for clinical transplantation. "
[Show abstract][Hide abstract] ABSTRACT: Three large-scale Echovirus (E) epidemics (E4,E16,E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets.
PLoS ONE 11/2013; 8(11):e77850. DOI:10.1371/journal.pone.0077850 · 3.23 Impact Factor
"Pancreatic islet autotransplantation, despite the use of a lower beta-cell mass, shows good results and improves insulin-independence rates in comparison to the islet allotransplantation . Pancreatic islet allografts are affected by various factors, including relatively long cold ischemia times before pancreatic islet isolation , , the need for a purification procedure, and the use of immunosuppressive drugs to regulate the alloimmune responses to the islet grafts. "
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
PLoS ONE 04/2013; 8(4):e56799. DOI:10.1371/journal.pone.0056799 · 3.23 Impact Factor
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