Islet Metabolism Core, DERC - UCSF

DERC - UCSF Islet Metabolism Core :

Although islet transplantation has become a clinical and experimental reality, the challenge to investigators has been that human pancreatic tissue processing is a very complex, highly specialized and costly procedure that requires technical expertise and a properly equipped facility. This Core provides FDA approved human islets for use in clinical islet transplant trials and provides human and mouse islets to investigators working on basic and applied research projects.

Services :

• Islet Production
• Islet Assessment

People :

Jeffrey A. Bluestone, Ph.D. : Director, Islet Metabolism Core, . Phone: 415-476-4451

Allison Xu, Ph.D. : Co-Director, Islet Metabolism Core, . Phone: 415-476-2467

Islet Cell Biology, DERC - Columbia University

DERC - Columbia Islet & Immunology :

The Cell Bank/Immunology Core will provide services and reagents to support the work of DERC investigators in several different research areas. The Core will provide both rodent and human islets to investigators who use them for experimental purposes. The Core will isolated islets from transgenic animals, many of which may express transgenes in the islet cells. These islets will be characterized metabolically by Stimulation Index, and in the future, by perifusion studies. Analysis of islet cells as well as purified populations will also be provided by fluorescence activated cell sorting. Through interactions with the Islet Cell Resource Center, the Core will provide human islets for investigators. We anticipate that this will be an important reagent in bringing new discoveries from the laboratory to clinical applications. The Core will acquire and maintain a Flow Cytometer for analysis use and provide support for cell sorting through interactions with the Flow Cytometry Core in the Irving Cancer Center. Finally, to give investigators reagents to directly study human disease pathogenesis including genetic studies to identify genes involved in the cause of the disease and complications, the Core will maintain and characterize a cell bank of peripheral blood mononuclear cells from patients. These will include patients with Type 1 and Type 2 diabetes as well as individuals at high risk for development of diabetes. B lymphocyte cell lines will be prepared and used in genetic studies as well as functional assays.

Services :

People :

Domenico Accili, M.D. : Center Director and Director, Islet & Immunology Core, Russ Berrie Pavilion, Room 238, 1150 St.Nicholas Avenue, New York, NY 10032. Phone: 212-851-5332

Islet Procurement & Analysis, DRTC - Vanderbilt University

DRTC Vanderbilt Islet Procurement & Analysis Core :

The Islet Procurement and Analysis Core (IPA Core) is supported by the VDRTC to provide access to isolated pancreatic islets from normal and diabetic models.

Services :

People :

Marcela Brissova : Islet Procurement and Analysis Core Director, . Phone: 615-936-1672

Anastasia Golovin : Research Assistant, Islet Procurement & Analysis Core, . Phone: 615-936-2858

Islet Cell Biology, DERC - University of Washington

DERC U Washington Islet Cell Biology Core :

The Islet Cell and Functional Analysis Core (IFAC) is a broad-based resource for research involving pancreatic islets. Services include preparing isolated islets from rodents and primates, chronic and acute treatment and culture of isolated islets, and experimental analysis of islets and islet cells in regard to their metabolic, functional and morphological state. Flow culture combined with sophisticated assessment techniques using optical detection have been developed specifically for islets and provide a unique set of investigational tools (1-2). Parameters that can be measured feature those reflecting mitochondrial function (NADH, oxygen consumption, cytochrome c reduction, CO2 production, mitochondrial membrane potential, reactive oxygen species), as well as lactate production, insulin secretion, cytosolic calcium and calcium uptake. The real time analytical tools that were developed for islets can also accommodate most cell and tissue types; those that have been analyzed in the past include cultured cells, lymphocytes, endothelial cells, fibroblasts, hepatocytes, brain slices, retina, and muscle. For all core users, consultation regarding experimental design and data analysis is provided. Classes of studies carried out have involved control of insulin secretion (3-4), cell death, mitochondrial metabolism, beta cell surface monoclonal antibody production, phenotyping genetically altered mice, transplantation studies(5), and binding and uptake studies (6).

Services :

Islet Preparation

Preparation of Rodent Islets:
The IFAC has experience with Fischer, Sprague-Dawley, Wistar and BB rat islets, and C57 Black mouse islets. Other strains have also been successfully used. Investigators provide animals for rodent islet isolations if specific strains are desired. Although it is strain-dependent, rodents yield about 700 islets/rat, and 200 islets/mouse.

Preparation of Monkey Islets:
Primate pancreata are obtained by the IFAC for islet isolation and Affiliate use. Macaca nemestrina (pigtail monkey) pancreata are available an average of 1-2 times monthly for islet isolations from the Northwest Primate Research Center. These islets have similarities with human islets not shared by rodent islets. The yield is quite variable but averages about 20,000 IE/preparation.

Human Islets:
Human islets are not prepared by the IFAC. Arrangements can be made for the IFAC to receive human islets in order to carry out studies for investigators. To do this, investigators requesting human islets should contact one or more of the following laboratories.

Southern California Islet Cell Resource Center
City of Hope National Medical Center
http://icr.coh.org/ Contact Autumn Tate

Islet Isolation Core
Children's Hospital of Pittsburgh
University of Pittsburgh
Division of Immunogenetics
Contact Dr. Rita Bottino

University of California
San Francisco, Diabetes Center
Contact Dr. Gregory Szot

Dispersed Islet Cells:
To facilitate experiments that require dispersed islet cells, islets are treated with trypsin and then mechanically disrupted. Cells can be attached to glass slides allowing for binding, secretion or imaging studies.

Islet and Cell Assessment

Flow Culture/Assessment of Islets:
A flow culture system has been developed that allows continuous and concomitant assessment of hormone secretion, metabolite release (lactate, pyruvate etc.), oxygen consumption, and cytochrome redox state of islets cells or tissue for up to 48 hours (1-3). In addition, it can temporally control the culture conditions of the islets including chemical and gaseous composition of media and temperature, and can accommodate non-recirculating, recirculating and co-culture modes. Studies have typically involved the study of the acute and chronic effects of fuels, pharmacologic agents, hormones, neurotransmitters, metabolic poisons, cytokines, as well as studies of hypoxia. A sample data set is shown for the effect of glucose, diazoxide and glibenclamide on islet metabolism.

Static Culture and Assessment of Islets:
As a complement to flow culture studies, culture and assessment of islets maintained statically are carried out in response to specific needs determined by the Affiliate. More conditions can be handled statically so this mode is ideal for dose responses, particularly when cells need to be harvested for acquisition of sample. Examples are insulin secretory studies (7), uptake and efflux of radioactively labeled compounds, particularly for evaluation of beta cell imaging agents (6), effects of oxygen tension on cell viability and metabolic rates, production of low levels of metabolites or signaling molecules in the media, and phenotyping functional characteristics of islets from genetically altered mice. Sample data sets are shown at illustrating the dose response of -ketoisocaproate on insulin secretion, and on the kinetics of binding of radiolabeled glibenclamide to INS-1 cells.

Single Islet or Cell Functional Imaging of Calcium and NAD(P)H:
Measurement of calcium is done after loading the islets with FURA1-AM (4 µM) for 30 min. Islets (5) are loaded into a Bioptechs temperature-controlled perifusion chamber (Bioptechs Inc, Butler, PA) and perifused using a Gilson peristaltic pump. The perifusion chamber is placed onto the stage of a Nikon TE200 Eclipse inverted fluorescence microscope; images of the emitted fluorescence at 510 nm are captured using a CoolSnap EZ digital camera (Roper Scientific GmbH, Germany) every minute during excitation at alternatively 340 nm and 380 nm (pulse duration = 40 ms) accomplished using a Lambda 10-B filter wheel (AutoMate Scientific, Inc. San Francisco, CA) and a Nikon Xenon lamp. Data acquisition is driven using a Dell computer loaded with Metafluor software (Molecular Devices, Sunnyvale CA). NADH is measured similarly to calcium, except there is no need for loading with dye and the excitation wavelength is 360 nm and emission wavelength is 460 nm. Sample data sets are shown for the effect of glucose and acetylcholine on calcium in islets, and the effect of glucose on NAD(P)H in HMECs.

* Protocols for all services may be requested by email.

People :

Ian Sweet, Ph.D. : Director, Islet Cell Biology Core, Campus Mailbox: 357710. Phone: 206-685-4775

Denise Kuok : Research Scientist, . Phone:

Drew Couron : Research Scientist, . Phone: 206-616-2482

Islet Cell Biology, DERC - University of Pennsylvania

DERC - U Pennsylvania Islet Cell Biology Core :

It is the purpose of this core to assist investigators who currently study or have plans to study independently or collaboratively various aspects of pancreatic islet cell biology

Services :

In order to accomplish the above stated objectives the Core provides four services:

• isolate, culture, and functionally assess pancreatic islets of rat and mouse including batch incubations, perifusions, respirometry, measurements of Ca i ++, the P-potential (ATP, ADP, AMP and P i) and other metabolites, hormone contents and release.

• Will perform extra corporal phenotyping of the endocrine pancreas of mouse and rat using the intact isolated perfused or minced perifused pancreas (both of these techniques to be fully established).

• maintains a broad and well characterized stock of transformed islet cells, grows large batches of such cells or generates pseudo islets by embedding such cells into agarose beads for dynamic studies of metabolism and hormone release.

• provides in depth consultation and helps develop strategies how to use the services of the core optimally or will attempt to modify available technologies to solve particular problems.

People :

Franz M. Matschinsky, M.D. : Proffesor of Biochemistry and Biophysics ,Director of Islet Biology Core , University of Pennsylvania 605 CRB 15 Curie Boulevard Philadelphia, PA 19104-6015. Phone: (215)-898-4368

Nicolai Doliba, Ph.D., D.Sc. : Senior Research Investigator , Technical Director of Islet Biology Core , University of Pennsylvania 605 CRB 15 Curie Boulevard Philadelphia, PA 19104-6015. Phone: (215)-898-4368

Carol Buettger : Senior Research Specialist, . Phone: 215-898-5879

Habiba Najafi : Research Specialist, islet cell isolation team, . Phone: 215-898-4368

Qin Wei : Research Specialist, islet cell isolation, culture and functional testing, . Phone: 215-898-4368

Islet Cell Biology, DRTC - University of Chicago

DRTC U Chicago Islet Cell Biology Core :

The Islet Cell Biology Core Laboratory provides independently funded investigators with rodent and human pancreatic islets from normal and diabetic animals for their studies. It also maintains insulinoma cell lines for distribution. The Core also provides service and training in methods for studying islet and beta-cell biology including insulin secretion, immunohistochemistry of insulin- and other hormone-secreting cells, and calcium imaging and other biophysical methods for studying pancreatic islet and beta-cell function.

The overall objective of this Core is to provide high quality islet and insulin–secreting cells to investigators together with basic and advanced methods to evaluate islet function. Specific objectives are:
 

• Provide mouse, rat and human pancreatic islets and insulinomas from normal and diabetic sources
• Maintain insulinoma cell lines such as HIT-T15, RINm5F, betaTC-3, INS-1 and MIN6 for distribution
• Provide training in isolation and culture of pancreatic islets from mice, rats and humans
• Provide service and training in methods for studying insulin synthesis and secretion using perifused islet and beta cells
• Provide service and training in methods for immunohistochemical analysis of islets and insulin-secreting cells
• Provide service and training in calcium imaging and other biophysical methods for studying islet and beta cell physiology

Services :

• Provide rat, mouse and human islets from normal and diabetic sources.The staff obtains the appropriate animals and using approved protocols, prepares islets and initiates cultures or other special handling as requested. They also supervise or teach the assays of hormone secretion that are part of the quality control protocols.
• Maintain insulinomas and insulinoma cell lines for distributionDr. Steiner’s laboratory maintains a large number of insulinoma, neuroendocrine, and other relevant cell lines. The laboratory directly confers with Investigators and provides flasks of cells or frozen cells at particular passage numbers as requested, and from time to time test the viability and quality of specific lots. The laboratory maintains logs of cell lines available, storage and growth conditions, passage number, and quality control aspects, such as glucose-stimulated insulin secretion and mycoplasma monitoring. A partial listing of available material includes HIT, RINm5f, BTC3, BTC6, INS-1 (several kinds), MIN-6, HEK, COS-7, CHO, HeLa and SF9. A large number of mouse, rat, hamster and human insulinoma samples are also available.
• Provide training in isolation and culture of pancreatic islets from mice, rats and humans.A major function of the laboratory is to provide training in islet isolation, culture and biology. Interested faculty, fellows, and students are provided with hands-on training from experienced technicians so that they can gain first hand knowledge of these important skills.
• Provide service and training in methods for studying insulin synthesis and secretion using a variety of methods.Insulin biosynthesis has been a main focus of Dr. Steiner’s laboratory. A variety of approaches have been provided for studying islet hormone biosynthesis in great detail. Insulin secretion can be studied in a variety of ways. Both static and dynamic approaches to insulin secretion measurements are available. Direct and indirect, biophysical measures are also provided. These studies are carried out in close coordination with the requesting investigator.
• Provide service and training in methods for immunohistochemical analysis of islets and insulin-secreting cells.Investigators desiring these services consult with the Director and determine the appropriate reagents and conditions for particular studies. Histochemical studies are coordinated with the assistance of the Pathology section of the Animal Resource Center at the University of Chicago, the Electron Microscopy Core, and the Digital Imaging Core. Digital images are prepared for publication. Archival data storage of key images, reconstructions, and movie files are also maintained.
• Provide service and training in calcium imaging and other biophysical methods for studying islet and beta-cell physiologyInvestigators have available a number of techniques, including optical measurement of NAD(P)H, Rh123, TMRE, fura-2, fluo-3, MgGreen, FuraRed, and MgFura. Also included is detection of FRET (fluorescence resonance energy transfer) substrates and use of targeted GFP-based fusion proteins. The emphasis of the Core is on specialized live-cell techniques and dedicated islet perfusion systems. The Core also provides an interface to appropriate Divisional Cores, such as the Digital Imaging Core and Electron Microscopy Core of the Division of Biological Sciences. Through collaboration with Dr. Norbert Scherer in the Department of Chemistry, multiphoton imaging analysis is also available.

People :

Donald F. Steiner, M.D. : Center Associate Director and Co-Director, Islet Cell Biology Core, . Phone: 773-702-1334

Louis H. Philipson, M.D., Ph.D. : Center Associate Director and Director, Islet Cell Biology Core, . Phone: 773-702-9180

Islet Cell Biology, DERC - University of Colorado

DERC University of Colorado Islet & Animal Resources Core :

The Bioresources Core provides users with high-quality animal husbandry and facilities for animal manipulation and production of pancreas and islet tissue are fundamental requisites for a wide variety of projects studying the pathogenesis of Type 1 and Type 2 diabetes.

Services :

Animal Resources: Provision for the stringent husbandry and analysis of specific pathogen free (SPF) rodents with special attention to unique congenic/knockout/immune-deficient strains relevant to diabetes research. Generation of gene-targeted and transgenic mice through collaboration between this Core, the DERC Molecular Resources Core and the Cancer Center Transgenic Core.

Diagnostic Services: Training of investigators in a variety of animal manipulations such as tail DNA collection, intravenous/intraperitoneal injection, survival and non-survival surgery, timed pregnancy and islet transplantation. More commonly requested animal manipulations and diagnostic testing are be offered as a fee-for-service.

Pancreatic Islet Resources: Rodent (mouse/rat) islets are generated by experienced technical staff from the DERC-maintained animal colonies. Human islets are obtained from the Rocky Mountain Islet Transplantation Program / Islet Cell Resources Center under FDA-approved, GMP conditions. The resource is backed up by routine stringent quality control of islet viability and function. The service can provide fetal/neonatal pancreas or pro-islet tissue for investigators studying pancreatic/islet development and maintain a bank of frozen islet tissue and low passage tissue culture cell lines commonly used by islet researchers (e.g. Min6, INS-1, PANC-1, AR42J).

Genotyping: Molecular genotyping to ensure genetic integrity of established lines and to track mutant alleles in investigator-derived animal strains. The service will be conducted jointly with the Molecular Core, using microsatellite marker analyses and SNP typing for the generation of "speed congenic" progeny and to facilitate other selective animal breeding projects.

Location: Barbara Davis Center at Fitzsimons, 3rd floor and CLAC RC1North basement.
 

People :

Danny Zipris, Ph.D. : Director, BioResources Core, Barbara Davis Center for Childhood Diabetes, UCDHSC. Assistant Professor, Department of Pediatrics. Phone: 303-724-0108