Large-Scale Purification of Human Islets Utilizing Discontinuous Albumin Gradient on IBM 2991 Cell Separator

Lake, S. P.; Bassett, P. D.; Larkins, A. P.; Revell, John; Walczak, K.; Chamberlain, J.; Rumford, G M; London, N.J.M.; Veitch, P. S.; Bell, P. R. F.; James, R. F. L.

doi:10.2337/diab.38.1.s143

Cited by 190 publications

(98 citation statements)

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“…Islets are purified based on their differential density compared to the surrounding pancreatic exocrine tissue. The development of the COBE 2991 (COBE Laboratories, Inc., Lakewood, CO), originally designed for blood separation, has made this separation more reproducible, shortened isolation times, and increased islet purity (Lake et al 1989). Originally, most density gradients were composed of Ficoll, a synthetic polymer of sucrose, or a derivative of it.…”

Section: Islet Purificationmentioning

confidence: 99%

Update on Islet Transplantation

McCall

Shapiro²

2012

Cold Spring Harbor Perspectives in Medicine

191

147

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Clinical islet transplantation has progressed considerably over the past 12 years, and .750 patients with type 1 diabetes have received islet transplants internationally over this time. Many countries are beginning to accept the transition from research to accepted and funded clinical care, especially for patients with brittle control that cannot be stabilized by more conventional means. Major challenges remain, including the need for more than one donor, and the requirement for potent, chronic immunosuppression. Combining immunological tolerance both to allo-and autoantigens, and a limitless expandable source of stem cell-or xenograft-derived insulin-secreting cells represent remaining hurdles in moving this effective treatment to a potential cure for all those with type 1 or 2 diabetes.

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Section: Islet Purificationmentioning

confidence: 99%

Update on Islet Transplantation

McCall

Shapiro²

2012

Cold Spring Harbor Perspectives in Medicine

191

147

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“…Pancreatic islets were isolated after ductal distension of the pancreata and digestion of the tissue with Lib-erase (Roche Diagnostics; Meylan, France) according to the automated method of Ricordi et al (1988), with modifications (Kerr-Conte et al 1994). Semipurification was achieved with Histopaque (Sigma; Saint-Quentin Fallavier, France) discontinuous density gradients using a cell separator (Lake et al 1989). Islet number and diameter were determined on triplicate samples of each preparation after dithizone (DTZ) staining (Latif et al 1988) and expressed in islet equivalents (IEs) corresponding to islets with a diameter of 150 m (Ricordi et al 1990).…”

Section: Human Islet Processingmentioning

confidence: 99%

Identification and Purification of Functional Human β-cells by a New Specific Zinc-fluorescent Probe

Lukowiak

Vandewalle

Riachy

et al. 2001

J Histochem Cytochem.

144

119

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S U M M A R Y Pancreatic ␤ -cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-sensitive fluorescent probe able to selectively label labile zinc in viable ␤ -cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number ( r ϭ 0.98) and between zinc fluorescence and insulin content ( r ϭ 0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resulted in a clear separation of ␤ -cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the visible spectrum, appears very promising for ␤ -cell experimentation, both clinically and for basic research. P ancreatic islets contain a much higher concentration of zinc than other tissues. Zinc plays an important role in packaging insulin because it is firmly established as an integral part of the insulin crystal as a 2-Zn-insulin hexamer. In addition, free ionized zinc is found in the extragranular space of ␤ -cells, where it may act as a reservoir for granular zinc (Figlewicz et al. 1984). These pools of free and loosely bound zinc can be visualized histochemically by most cytological stains, unlike zinc tightly complexed to proteins such as metalloproteins, including transcription factors and metalloenzymes. Our study was designed to search for a new non-toxic probe that was able to specifically stain living ␤ -cells. Our aim was threefold: (a) to visualize ␤ -cells in pancreatic islets by confocal microscopy or image analysis; (b) to isolate these ␤ -cells for physiological studies; and (c) to rapidly estimate, in allograft management, the amount of viable ␤ -cells in semipurified preparations containing contaminating exocrine cells. The latter pretransplantation evaluation of the quality of the graft has become a crucial prerequisite to ensure successful engraftment. Here we describe the use of Newport Green, a new zinc-sensitive probe with an excitation wavelength in the visible spectrum, which fulfills all the criteria required for efficient staining of human ␤ -cells in clinical and research applications. Materials and Methods Human Islet ProcessingHuman pancreata ( n ϭ 11; mean age 35 Ϯ 12 years) were harvested from adult brain-...

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“…Pancreatic islets were isolated after ductal distension of the pancreas and digestion of the tissue with Liberase (Roche Diagnostics, Meylan, France) according to the automated method of Ricordi et al (1988) with modifications (Kerr-Conte et al 1994). Semi-purification was achieved with Euro-Ficoll discontinuous density gradient using a cell separator (Lake et al 1989). Islet number was determined on samples of each preparation after dithizone staining and expressed as equivalent number of islets (IE) (Ricordi et al 1990).…”

Section: Human Islet Processingmentioning

confidence: 99%

Beneficial effect of 1,25 dihydroxyvitamin D3 on cytokine-treated human pancreatic islets

Riachy¹,

Vandewalle²,

Belaïch³

et al. 2001

Journal of Endocrinology

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We examined whether 1,25 dihydroxyvitamin D 3 (1,25 D 3 ), the active form of vitamin D involved in the regulation of the immune system, may also protect human pancreatic islet cells from destruction induced by cytokines. In this study, we specifically investigated the effect of 1,25 D 3 on oxidative stress and major histocompatibility complex (MHC) induction, both implicated in cytokineinduced islet cell dysfunction and destruction. We also investigated the effects of 1,25 D 3 on interleukin (IL)-6, a pleiotropic cytokine implicated in the pathogenesis of immunoinflammatory disorders.Human pancreatic islets, isolated from heart-beating donors, were treated with a combination of three cytokines, IL-1 +tumor necrosis factor +interferon , in the presence or absence of vitamin D, and compared with with untreated control cells. Metabolic activity was assessed by cell viability and insulin content. Oxidative stress was estimated by heat shock protein 70 (hsp70) expression, cell manganese superoxide dismutase (MnSOD) activity and nitrite release, a reflexion of nitric oxide (NO) synthesis. Variation of immunogenicity of islet preparations was determined by analysis of the MHC class I and class II transcripts. Inflammatory status was evaluated by IL-6 production. After 48 h of contact with cytokines, insulin content was significantly decreased by 40% but cell viability was not altered. MHC expression significantly increased six-to sevenfold as well as NO and IL-6 release (two-to threefold enhancement). MnSOD activity was not significantly induced and hsp70 expression was not affected by the combination of cytokines.The addition of 1,25 D 3 significantly reduced nitrite release, IL-6 production and MHC class I expression which then became not significantly different from controls. These results suggest that the effect of 1,25 D 3 in human pancreatic islets cells may be a reduction of the vulnerability of cells to cytotoxic T lymphocytes and a reduction of cytotoxic challenge. Hence, 1,25 D 3 might play a role in the prevention of type 1 diabetes and islet allograft rejection.

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Large-Scale Purification of Human Islets Utilizing Discontinuous Albumin Gradient on IBM 2991 Cell Separator

Cited by 190 publications

References 0 publications

Update on Islet Transplantation

Update on Islet Transplantation

Identification and Purification of Functional Human β-cells by a New Specific Zinc-fluorescent Probe

Beneficial effect of 1,25 dihydroxyvitamin D3 on cytokine-treated human pancreatic islets

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