A practical guide to rodent islet isolation and assessment

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.

Figure 1

Islet morphology following isolation. a A freshly isolated islet shown among pancreatic acinar tissue on the day of isolation. b Islets purified from acinar tissue, after incubation at 37°C and 5% CO₂ for 18–20 h. c Islet incubated 18–20 h with darkened, hypoxic center.

Figure 2

Assessment of islet viability by fluorescence intensity of propidium iodide. a, b Untreated (healthy) islets shown in brightfield (a, top panel) display only trace amounts of PI fluorescence (a, bottom panel). Islets treated overnight with a combination of cytokines (100 pg/mL TNF-α, 50 pg/mL IL-1β, 100 pg/mL IFN-γ) shown in brightfield (b, top panel), in contrast, display substantial PI fluorescence (b, bottom panel).

Figure 3

A comparison of glucose-stimulated calcium (GSCa) response for islets cultured overnight in 5.5 mM glucose (5 G) and 11 mM glucose (11 G). a GSCa for islets cultured in 5.5 mM glucose (solid, mean of n = 8) and 11 mM glucose (dashed, mean of n = 9). Arrows indicate the three phases of GSCa; stimulation with 11 mM glucose is indicated by the horizontal bar. b Mean amplitude of the calcium response during each phase (phase 0, 1, and 2) for islets cultured in 5.5 mM glucose (solid, n = 37) or 11 mM glucose (stripes, n = 34); * indicates p < 0.05. Note that although a difference in latency is apparent in (a), this not a consistent or significant effect in the larger data set.

Figure 4

Anatomy of the mouse upper intraperitoneal (IP) cavity. NOTE: The top is the caudal portion of the mouse, bottom is the rostral portion, ie, this is from the perspective of the mouse lying on its back tail away from the surgeon and nose toward the surgeon.

Figure 5

Injection site and clamping the Common Bile Duct (CBD) at the duodenum.

Figure 6

Anatomy of the mouse upper intraperitoneal cavity. The top is the caudal portion of the mouse, bottom is the rostral portion, i.e., this is from the perspective of the mouse lying on its back tail away from the surgeon and nose toward the surgeon.

Figure 7

Injection site and clamping the common bile duct (CBD) at the duodenum.