Islet isolation assessment in man and large animals

Recent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity and in vitro and in vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 μ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason, in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers. © 1990 Casa Editrice il Ponte

Critical Role of Gap Junction Coupled K(ATP) Channel Activity for Regulated Insulin Secretion

Pancreatic β-cells secrete insulin in response to closure of ATP-sensitive K(+) (K(ATP)) channels, which causes membrane depolarization and a concomitant rise in intracellular Ca(2+) (Ca(i)). In intact islets, β-cells are coupled by gap junctions, which are proposed to synchronize electrical activity and Ca(i) oscillations after exposure to stimulatory glucose (>7 mM). To determine the significance of this coupling in regulating insulin secretion, we examined islets and β-cells from transgenic mice that express zero functional K(ATP) channels in approximately 70% of their β-cells, but normal K(ATP) channel density in the remainder. We found that K(ATP) channel activity from approximately 30% of the β-cells is sufficient to maintain strong glucose dependence of metabolism, Ca(i), membrane potential, and insulin secretion from intact islets, but that glucose dependence is lost in isolated transgenic cells. Further, inhibition of gap junctions caused loss of glucose sensitivity specifically in transgenic islets. These data demonstrate a critical role of gap junctional coupling of K(ATP) channel activity in control of membrane potential across the islet. Control via coupling lessens the effects of cell–cell variation and provides resistance to defects in excitability that would otherwise lead to a profound diabetic state, such as occurs in persistent neonatal diabetes mellitus

Long Term Follow-Up of the Endovascular Trans-Vessel Wall Technique for Parenchymal Access in Rabbit with Full Clinical Integration

OBJECTIVE: Endovascular techniques are providing options to surgical/percutaneous cell transplantation methods. Some cells, e.g. insulin producing cells, are not suitable for intra-luminal transplantation and for such cells, other options must be found. We have constructed a "nanocatheter" with a penetrating tip for vessel perforation, thereby creating a working channel for parenchymal access by endovascular technique. To finish the procedure safely, the distal tip is detached to provide a securing plug in the vessel wall defect. MATERIALS AND METHODS: We have performed interventions with full clinical integration in the superior mesenteric artery (SMA), the subclavian artery and the external carotid artery in rabbits. No hemorrhagic- or thromboembolic events occurred during the procedure. Stenosis formation and distal embolisation were analyzed by angiography and macroscopic inspection during autopsy at five, 30 and 80 days. All animals and implanted devices were also evaluated by micro-dissections and histochemical analysis. RESULTS: In this study we show safety data on the trans-vessel wall technique by behavioral, angiographical and histological analysis. No stenosis formation was observed at any of the follow-up time points. No animals or organs have shown any signs of distress due to the intervention. Histological examination showed no signs of hemorrhage, excellent biocompatibility with no inflammation and a very limited fibrous capsule formation around the device, comparable to titanium implants. Further, no histological changes were detected in the endothelia of the vessels subject to intervention. CONCLUSIONS: The trans-vessel wall technique can be applied for e.g. cell transplantations, local substance administration and tissue sampling with low risk for complications during the procedure and low risk for hemorrhage, stenosis development or adverse tissue reactions with an 80 days follow-up time. The benefit should be greatest in organs that are difficult or risky to reach with surgical techniques, such as the pancreas, the CNS and the heart

Report from IPITA-TTS opinion leaders meeting on the future of β-cell replacement

Peer reviewe

Enumeration of islets by nuclei counting and light microscopic analysis

Author Manuscript 2011 May 1.Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with (1) visual counting in a hemocytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D. DNA content averaged 6.5 and 6.9 pg of DNA per cell for rat and human islets, respectively, in agreement with literature estimates. With pure rat islet preparations, precision improved with increasing counts, and samples with about greater than or equal to 160 islets provided a coefficient of variation of about 6%. Aliquots of human islet preparations were processed for LM analysis by stereological point counting. Total nuclei counts and islet volume fraction from LM analysis were combined to obtain the number of islet equivalents (IEs). Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared with LM/nuclei counting for 12 freshly isolated human islet research preparations. Nuclei counting combined with islet volume fraction measurements from LM is a novel method for achieving accurate islet enumeration.National Institutes of Health (U.S.) (Grant NCRR ICR U4Z 16606)National Institutes of Health (U.S.) (Grant R01-DK063108-01A1)National Institutes of Health (U.S.) (Grant NCRR ICR U42 RR0023244-01)Joslin Diabetes and Endocrinology Research Center (Grant DK36836)Diabetes Research & Wellness FoundationJuvenile Diabetes Research Foundation International (Islet Transplantation, Harvard Medical School

Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.Leona M. and Harry B. Helmsley Charitable Trust (3-SRA-2014-285-M-R)United States. National Institutes of Health (EB000244)United States. National Institutes of Health (EB000351)United States. National Institutes of Health (DE013023)United States. National Institutes of Health (CA151884)United States. National Institutes of Health (P41EB015871-27)National Cancer Institute (U.S.) (P30-CA14051