Abstract
Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.
Highlights
Pancreatic islets are thought to play a key role in the pathophysiology of Type 1 and Type 2 diabetes through the failure of islet beta cells to secrete sufficient quantities of insulin to regulate blood glucose [1]
We have found that optimal collagenase formulations for rat islet isolations provide acceptable criteria for rating collagenase used in our mouse islet isolation procedures
Insulin secretion remained lower in islets cultured in five other types of culture media brought to comparable glucose concentrations [30]
Summary
Many published islet isolation protocols specific to mouse and rat, few provide the necessary details for researchers to successfully perform the complex procedures. The primary goal of isolating pancreatic islets, whether for in vivo transplantation or in vitro studies, is to obtain viable purified islets that respond in a manner consistent with their function in vivo. The key elements of a successful islet isolation procedure are: [1] enzymatically digesting the tissues connecting the islets to the exocrine tissue, [2] separating islets from non-islet tissue, and [3] culturing isolated islets in an environment that maintains cell viability. We review these key elements and provide methods for evaluating islet quality. By no means, the only successful method for isolating pancreatic islets; the additional details provided in this protocol are intended to provide the rationale for each step in the process in order to assist researchers in their efforts to obtain healthy islets for study
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