Isolated bovine pancreatic islets as an alternate in vitro model for diabetes research

Abstract

Background & objectives Isolation of functional pancreatic islets for diabetes research and clinical islet transplantation stands as a big challenge despite the advancements in the field. In this context, the non-availability of human/animal tissues is one of the major impediments to islet-based research, which has tremendous scope for translation. The current study explores the feasibility of using the bovine pancreas as an alternative source to isolate pancreatic islets and assess its functionality for in vitro studies. Methods The bovine pancreas was collected from a registered slaughterhouse and transported in an ice-cold medium – Hank’s Balanced Salt Solution (HBSS) to the laboratory. Islets were isolated by sequential collagenase digestion followed by a two-step filtration and purification by density gradient separation method. After isolation, islets were identified with dithizone staining and the islet function was assayed in vitro for assessing the dynamic insulin secretory function by monitoring the glucose-stimulated insulin secretion (GSIS), in response to low and high glucose. Staining techniques were also used to understand the cytoarchitecture of the bovine pancreas. Results The islet yield was 157±23 islets per gram of pancreas and was viable. The cold ischaemia time was reduced to 60-75 min. The islets released insulin with glucose stimulation. The insulin release was observed more with high glucose (28 mM) than with low glucose (2.8 mM). Dithizone staining confirmed the presence of islets after isolation and the size of islets ranged from 50 to 600 µm size. The mantled islets (islets with acinar tissue) were also noted with the pure islets in culture. Hematoxylin and eosin (H&E) and aldehyde- fuchsin showed islets interspersed in the acinar tissue of the bovine pancreas. Special stain defined the islets better than regular staining. Fluorescent and diaminobenzidine (DAB) staining with insulin, glucagon and somatostatin revealed the arrangement of the cells in each islet. The beta cells were majorly found in the islet core with alpha cells interspersed with the delta cells in the periphery. Interpretation & conclusions The isolation procedure described in this study yielded viable islets for in vitro studies which showed a differential response to glucose challenge, confirming their viability. We provide a simple and reproducible method for small-scale isolation of functional islets from the bovine pancreas. This model proffers the beginner a hands-on in islet experiments and helps to re-iterate the process that could be extrapolated to other pancreatic tissues as well as to expand on diabetes research.