Objective

The objective of the Islet Cell Biology Core is to provide DRC members with state of the art support including experimental design, islet isolation, and performance of and/or training in an expansive range of assays for physiological and morphometric assessment of pancreatic islet function and growth. We also enlist unique expertise of newly interested faculty to adapt existing technologies to solve unique problems that cannot be addressed by standard methodologies. For example, new ties with our Physiology Department promise an expansion of consultation and services to study membrane biophysics critical for understanding normal and diseased islet cells.

Core Functions

Failure of insulin secreting pancreatic beta cells characterizes the progression of all forms of diabetes. The ICBC is thus positioned to contribute in a significant manner to the basic and translational research activities of the Institute of Diabetes, Obesity and Metabolism (IDOM) at the Perelman School of Medicine of the University of Pennsylvania. The ICBC has developed exceptional expertise in working with human and rodent pancreatic islet tissue, acquiring instrumentation and establishing procedures that are not readily available to the averagelaboratory.

Services:

We offer a range of services that generally begin with islet isolation from rodent animal models and may be followed by a period of culture by the core for as long as 7 days. Islet hormone secretion can be assessed in static “batch” incubations or by more informative perifusions that require larger numbers of islets and expensive immunoassays, routinely for insulin and upon request for glucagon and cAMP. The core may provide these services in an ongoing manner or it may provide critical training to allow the investigator laboratory to perform the experiments independently over time. The core also has the advanced technology and expertise to perform individual islet and single cell fluorescence imaging (Cai 2+), respirometry with islet batches using a Seahorse Extracellular Flux Analyzer, perifusion coupled with respirometry, and “closed” respirometry experiments for our investigators.

The ICBC currently provides the following services:

Islet isolation and culture:
  • Culture of human pancreatic islets
  • Isolation and culture of mouse or rat pancreatic islets.
Hormone Secretion:
  • Islet perifusion to measure insulin, glucagon and cAMP release

  • Batch incubation experiments to measure insulin, glucagon and cAMP release
  • Note: Hormone assays are performed by the Radioimmunoassay/Biomarkers Core

Mitochondrial Function:

  • Quantitative oxygen consumption of perifused freshly isolated or cultured islet tissue using optical methods (requires 500-750 islets).
  • Electrode based closed cell respirometry for measuring oxygen consumption of isolated islets or other tissues (e.g. adipose)using a water-jacketed glass vessel (requires 150 islets).
  • Respiration and extracellular acidification rate (ECAR) measured by Seahorse Extracellular Flux Analyzer(recently upgraded from XFe24 to XFe96).

Intracellular calcium imaging:

Free intracellular Ca2+ measurements using new instrumentation for state of the art fluorescence imaging (Zeiss Observer.Z1 with AxioVision softwere).
  • Intact islets
  • Cell clusters/aggregates
  • Single cells

Morphometry:

  • Provide access and instruction for quantitative imaging of pancreatic islet mass.
New Electrophysiology Service Initiative:
  • Provide beta-cell Vm measurement and record the basic beta-cell electrogram in response to glucose.
  • Perform individual channel analysis in well defined applications

Consultation:

  • To optimize use of core services
  • To optimize murine models of beta cell specific gene ablation
  • To develop new strategies and technologies to solve particular problems.

Training and Education:

The ICBC provides training for graduate students, postdoctoral fellows and research staff in:

  • Pancreatic islet isolation and perfusion techniques (Wei Qin).
  • Basic principles of respiration and oxidative phosphorylation and measurement of oxygen consumption by different methods, including electrode based closed respirometry (Clark electrode),phosphorescence respirometry, and oxygen consumption and ECAR by Seahorse (Dr. Doliba).
  • All aspects of islet cell physiology and biochemistry (Drs. Doliba, Hoshi, Matschinsky, Stoffers).