Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture.
ABSTRACT 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.
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ABSTRACT: Serotonergic biosynthesis in the endocrine pancreas, of which the islets of Langerhans is the major constituent, has been implicated in insulin release and β cell proliferation. In this study, we investigated the feasibility of quantitative noninvasive imaging of the serotonergic metabolism in the pancreas using the PET tracer (11)C-5-hydroxy-l-tryptophan ((11)C-5-HTP). Uptake of (11)C-5-HTP, and its specificity for key enzymes in the serotonergic metabolic pathway, was assessed in vitro (INS-1 and PANC1 cells and human islet and exocrine preparations) and in vivo (nonhuman primates and healthy and diabetic rats). In vitro tracer uptake in endocrine cells (INS-1 and human islets), but not PANC1 and exocrine cells, was mediated specifically by intracellular conversion into serotonin. Pancreatic uptake of (11)C-5-HTP in nonhuman primates was markedly decreased by inhibition of the enzyme dopa decarboxylase, which converts (11)C-5-HTP to (11)C-serotonin and increased after inhibition of monoamine oxidase-A, the main enzyme responsible for serotonin degradation. Uptake in the rat pancreas was similarly modulated by inhibition of monoamine oxidase-A and was reduced in animals with induced diabetes. The PET tracer (11)C-5-HTP can be used for quantitative imaging of the serotonergic system in the endocrine pancreas.Journal of Nuclear Medicine 02/2014; · 5.77 Impact Factor
- Transplantation 12/2013; 96(12):e91-3. · 3.78 Impact Factor
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ABSTRACT: Type 1 diabetes (T1D) is an autoimmune disease where local release of cytokines such as IL-1β and IFN-γ contribute to β-cell apoptosis. To identify relevant genes regulating this process we performed a meta-analysis of 8 datasets of β-cell gene expression after exposure to IL-1β and IFN-γ. Two of these datasets are novel and contain time-series expressions in human islet cells and rat INS-1E cells. Genes were ranked according to their differential expression within and after 24 hours from exposure, and characterized by function and prior knowledge in the literature. A regulatory network was then inferred from the human time expression datasets, using a time-series extension of a network inference method. The two most differentially expressed genes previously unknown in T1D literature (RIPK2 and ELF3) were found to modulate cytokine-induced apoptosis. The inferred regulatory network is thus supported by the experimental validation, providing a proof-of-concept for the proposed statistical inference approach.Genomics 01/2014; · 3.01 Impact Factor