Pancreatic Islet Isolation and Purification from Lewis Rats Using Enzymatic Digestion and Density-Gradient Separation

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Abstract
Pancreatic islets are essential for both clinical islet transplantation and preclinical studies aimed at understanding the mechanisms and progression of diabetes. However, progress in these fields is often limited by the availability of high-quality islets, as emerging therapeutic strategies require larger, cleaner, and more standardized preparations. This article presents a high-throughput, standardized protocol for surgical harvesting, enzymatic digestion, and density-gradient purification of pancreatic islets from Lewis rats. The method combines intraductal pancreas perfusion with a defined enzyme blend, pooled digestion of multiple pancreases, and density-based separation to maximize yield while minimizing fragmentation and eliminating the need for routine hand-picking. Islet yield is quantified using two complementary approaches: a traditional aliquot-based counting method commonly used in clinical settings, and a semi-automated whole-preparation imaging-based method. In a representative isolation from 12 pancreases, manual counting yielded approximately 3,300 islet equivalents (IEQ) and 2,700 absolute islets per pancreas (~39,600 total IEQ), with a size distribution of 62% (50-100 µm), 17% (100-150 µm), 10% (150-200 µm), 5% (200-250 µm), and 6% (>250 µm). In comparison, whole-prep automated analysis reported 16,350 total IEQ (1,362 IEQ/pancreas), highlighting inherent variability in aliquot-based quantification. Preparations had a purity of 98% by dithizone staining, and islet viability was maintained at 98% in vitro on days 3 and 7 after isolation. Functional assessment by static glucose-stimulated insulin secretion demonstrated a 10-fold increase in insulin release following a 1-h stimulation with 16.7 mM glucose. Overall, this protocol provides a reproducible and efficient approach for generating high-purity, functionally intact islet preparations that can be used immediately without additional cleanup. Islets retain robust function in culture for up to seven days, enabling flexibility for large-scale in vitro assays as well as in vivo transplantation studies.

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