Article

Refinement of the Automated Method for Human Islet Isolation and Presentation of a Closed System for In Vitro Islet Culture

Department of Radiology, Oncology and Clinical Immunology, Division of clinical Immunology, University Hospital, Uppsala, Sweden.
Transplantation (Impact Factor: 3.83). 11/2004; 78(9):1367-75. DOI: 10.1097/01.TP.0000140882.53773.DC
Source: PubMed

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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    • "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 [21] 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. "
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    ABSTRACT: Aims/hypothesis: The Gq-coupled 5-hydroxytryptamine 2B (5-HT2B) receptor is known to regulate the proliferation of islet beta cells during pregnancy. However, the role of serotonin in the control of insulin release is still controversial. The aim of the present study was to explore the role of the 5-HT2B receptor in the regulation of insulin secretion in mouse and human islets, as well as in clonal INS-1(832/13) cells. Methods: Expression of HTR2B mRNA and 5-HT2B protein was examined with quantitative real-time PCR, RNA sequencing and immunohistochemistry. α-Methyl serotonin maleate salt (AMS), a serotonin receptor agonist, was employed for robust 5-HT2B receptor activation. Htr2b was silenced with small interfering RNA in INS-1(832/13) cells. Insulin secretion, Ca(2+) response and oxygen consumption rate were determined. Results: Immunohistochemistry revealed that 5-HT2B is expressed in human and mouse islet beta cells. Activation of 5-HT2B receptors by AMS enhanced glucose-stimulated insulin secretion (GSIS) in human and mouse islets as well as in INS-1(832/13) cells. Silencing Htr2b in INS-1(832/13) cells led to a 30% reduction in GSIS. 5-HT2B receptor activation produced robust, regular and sustained Ca(2+) oscillations in mouse islets with an increase in both peak distance (period) and time in the active phase as compared with control. Enhanced insulin secretion and Ca(2+) changes induced by AMS coincided with an increase in oxygen consumption in INS-1(832/13) cells. Conclusions/interpretation: Activation of 5-HT2B receptors stimulates GSIS in beta cells by triggering downstream changes in cellular Ca(2+) flux that enhance mitochondrial metabolism. Our findings suggest that serotonin and the 5-HT2B receptor stimulate insulin release.
    No preview · Article · Jan 2016 · Diabetologia
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    • "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 [21] 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. "
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    ABSTRACT: Islet produced 5-hydroxy tryptamine (5-HT) is suggested to regulate islet hormone secretion in a paracrine and autocrine manner in rodents. Hitherto, no studies demonstrate a role for this amine in human islet function, nor is it known if 5-HT signaling is involved in the development of beta cell dysfunction in type 2 diabetes (T2D). To clarify this, we performed a complete transcriptional mapping of 5-HT receptors and processing enzymes in human islets, and investigated differential expression of these genes in non-diabetic and T2D human islet donors. We show the expression of fourteen 5-HT receptors as well as the processing enzymes involved in the biosynthesis of 5-HT at the mRNA level in human islets. Two 5-HT receptors (HTR1D and HTR2A) were over-expressed in T2D islet donors and while 5-HT inhibited both insulin and glucagon secretion in non-diabetic islet donors, 5-HT significantly increased the release of insulin in response to glucose in islets isolated from T2D donors. Attempting to understand the consequences of altered 5-HT receptor expression in the diabetic islet, we investigated the specific function of receptors 5-HT1d and 5-HT2a in non-diabetic islets. We found that a 5-HT1d receptor agonist inhibited insulin release while a 5-HT1d antagonist potentiated insulin release. Similarly, a 5-HT2a receptor agonist significantly increased insulin release, and an antagonist blunted the insulinotropic response to glucose. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Peptides
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    • "Pig islet isolation Pancreatic glands were removed from several pigs at a slaughterhouse that handles young market weight pigs (Large White/ Landrace x Duroc, 6 months old, 100 kg). Isolation of porcine islets was performed using the Islet Isolation Technique (Goto et al. 2004), with minor modifications. Purified islet fractions were pooled and cultured at 37°C in a humidified atmosphere with 5% CO 2 in CMRL1066 medium (Biochrom, Berlin, Germany) supplemented with 20% heat inactivated porcine serum, 2 mm N-acetyl-L-alanyl-L-glutamine, 10 mM N-2-hydroxyethylpiper- azine-N1-2-ethanesulfonic acid, 100 IU/mL penicillin, 100 µg/mL streptomycin (Biochrom) and 20 µg/mL ciprofloxacin (Bayer, Leverkusen, Germany). "
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    ABSTRACT: After producing α1-3-galactosyltransferase knockout (GKO) pigs, most of the organs of these pigs showed less antigenicity to the human body. However, wild-type adult pig islets (API) that originally contained negligible levels of α-galactosidase now showed a clear antigenicity to human serum. In this study, N-glycans were isolated from both APIs and human islets. Their structures were then analyzed by a mapping technique based on their high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric data. Both preparations contained substantial amounts of high-mannose structures. The N-glycans from human islets were separated into 17 neutral, 8 mono-sialyl and 4 di-sialyl glycans, and the API glycans were comprised of 11 neutral, 8 mono-sialyl, 3 di-sialyl, 2 mono-sulfated, 3 mono-sialyl-mono-sulfated and 1 di-sulfated glycans. Among them, the API preparation contained one neutral, five mono-sialyl glycans and six sulfated glycans that were not detected in human islets. The structures of 9 of these 12 could be clearly determined. In addition, a study of the sulfate-depleted API suggests that sulfate residues could be antigenic to humans. The data herein will be helpful for future studies of the antigenicity associated with API.
    Preview · Article · Feb 2014 · Glycobiology
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