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: In clinical islet transplantation, inflammatory responses initiated by the transplanted islets and by the host immune system cause acute and chronic graft loss. The resolution of acute inflammation is an active process mediated by specific signals and mediators such as resolvin E1 (RvE1). We investigated the effect of RvE1 on i) the inflammatory status of human pancreatic islets, ii) islet viability and apoptosis, and iii) the instant blood-mediated inflammatory reaction (IBMIR) IN VITRO. Pro-inflammatory cytokines and tissue factor (TF) in isolated human islets were determined by real-time RT-qPCR (mRNA levels), CBA and Gyrolab bioaffy (protein levels) after lipopolysaccaride (LPS) stimulation. Islet viability was measured using insulin secretion in a dynamic model, ADP/ATP ratio and total ATP content. Apoptosis was measured using commercial kits after stimulation with proinflammatory cytokines. To assess effect on IBMIR, human islets were mixed with non-anticoagulated, RvE1 or vehicle pretreated ABO-compatible blood in heparin-coated tubing loops. Treatment of human islets with RvE1 (500 nM) for 24 h reduced LPS-induced increase in mRNA and protein levels of selected pro-inflammatory markers (IL-8, MCP-1, and TF). RvE1 lowered the ADP/ATP ratio, but had no effect on insulin secretion. RvE1 reduced the apoptotic effect of proinflammatory cytokines. Additionally, RvE1 reduced platelet consumption and TAT complex formation during the first 5 min after islet-blood contact. RvE1 suppresses proinflammatory markers and lowers the ADP/ATP ratio in human islets IN VITRO. RvE1 demonstrates anti-apoptotic effects in a proinflammatory milieu. Additionally, RvE1 has modest dampening effects on IBMIR. We conclude that RvE1 may have potential in clinical islet transplantation.Experimental and Clinical Endocrinology & Diabetes 04/2010; 118(4):237-44. DOI:10.1055/s-0029-1241825 · 1.76 Impact Factor
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ABSTRACT: Type 1 diabetes is a chronic disease characterized by the selective destruction of insulin-producing cells in the pancreas. Enterovirus (EV) is the prime candidate to initiate this destruction and several inflammatory chemokines are induced by EV infection. Nicotinamide has been shown to protect isolated human islets, and to modulate chemokine expression. The aim of this study was to evaluate the effect of nicotinamide on EV replication and EV-induced chemokine secretion and cytolysis of human islets. Two EV strains were used to infect human islets in vitro, one lytic (Adrian) isolated from a child at onset of type 1 diabetes, and one non-lytic (VD2921). Secretion of the chemokines IP-10 and MCP-1, viral replication, and virus-induced cytopathic effect (CPE), were measured at different time points post-infection. Addition of nicotinamide to the culture medium reduced viral replication and virus-induced islet destruction/CPE, significantly. Both EV strains increased secretion of IP-10 and MCP-1, when measured days 2-3, and days 5-7 post infection, compared to mock-infected control islets. IP-10 was not produced by uninfected isolated islets, whereas a basal secretion of MCP-1 was detected. Interestingly, addition of nicotinamide blocked completely (Adrian), or reduced significantly (VD2921), the virus-induced secretion of IP-10. Secretion of MCP-1 was also reduced in the presence of nicotinamide, from infected and uninfected islets. The reported antiviral effects of nicotinamide could have implications for the treatment/prevention of virus- and immune-mediated disease. Also, this study highlights a possible mechanism of virus-induced type 1 diabetes through the induction of MCP-1 and IP-10 in pancreatic islets.Journal of Medical Virology 06/2009; 81(6):1082-7. DOI:10.1002/jmv.21476 · 2.22 Impact Factor
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ABSTRACT: Enterovirus infections, in particular those with Coxsackieviruses, have been linked to the development of type 1 diabetes (T1D). Although animal models have demonstrated that interferons (IFNs) regulate virus-induced T1D by acting directly on the beta cell, little is known on the human pancreatic islet response to IFNs. Here we show that human islet cells respond to IFNs by expressing signature genes of antiviral defense. We also demonstrate that they express three intracellular sensors for viral RNA, the toll like receptor 3 (TLR3) gene, the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene-5 (MDA-5), which induce type I IFN production in infected cells. Finally, we show for the first time that the IFN-induced antiviral state provides human islets with a powerful protection from the replication of Coxsackievirus. This may be critical for beta cell survival and protection from virus-induced T1D in humans.Virology 11/2007; 367(1):92-101. DOI:10.1016/j.virol.2007.05.010 · 3.28 Impact Factor