title: An optimized protocol for purification of functional islets of Langerhans.
abstract: Islets of Langerhans and β-cell isolation constitute routinely used cell models for diabetic research, and refining islet isolation protocols and cell quality assessment is a high priority. Numerous protocols have been published describing isolate of islets, but often rigorous and systematic assessment of their integrity is lacking. Herein, we propose a new protocol for optimal generation of islets. Pancreases from mice and rats were excised and digested using a low-activity collagenase solution and islets were then purified by a series of sedimentations and a Percoll gradient. Islets were maintained in culture for 5 days, during which viability, pro/antiapoptotic, and islet-specific genes, glucose-stimulated calcium entry, glucose uptake, and insulin secretion were assessed. The commonly used islet isolation technique by collagenase injection through the common bile duct (CBD) was also performed and compared with the present approach. This new protocol produced islets that retained a healthy status as demonstrated by the yield of stable living cells. Furthermore, calcium oscillation, glucose uptake, and insulin secretion remained intact in the islet cultures. This was reproducible when many rodent species were used, and neither sex nor age affected the cells behavior. When compared with the CBD technique, islet physiology was similar. Finally, this approach was used to uncover new ion channel candidates implicated in insulin secretion. In conclusion, this study outlines an efficient protocol for islet preparation that may support research into new therapeutic targets in diabetes research.
authors.affiliation: Laboratoire de Recherche en Physiologie et Physiopathologie LRPP, Pôle Technologie Santé, Faculté de Médecine, Université Saint Joseph, Beirut, Lebanon.
authors.forename: Youakim
authors.initials: Y
authors.lastname: Saliba
authors.affiliation: Laboratoire de Recherche en Physiologie et Physiopathologie LRPP, Pôle Technologie Santé, Faculté de Médecine, Université Saint Joseph, Beirut, Lebanon.
authors.forename: Jules-Joel
authors.initials: JJ
authors.lastname: Bakhos
authors.affiliation: Laboratoire Rodolphe Mérieux, Pôle Technologie Santé, Faculté de Pharmacie, Université Saint Joseph, Beirut, Lebanon.
authors.forename: Tarek
authors.initials: T
authors.lastname: Itani
authors.affiliation: Laboratoire de Recherche en Physiologie et Physiopathologie LRPP, Pôle Technologie Santé, Faculté de Médecine, Université Saint Joseph, Beirut, Lebanon.
authors.forename: Nassim
authors.initials: N
authors.lastname: Farès
mesh_terms: D000367:Age Factors; D000818:Animals; D017209:Apoptosis; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D005260:Female; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism* / Q000493:pharmacokinetics; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D034622:RNA Interference; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000379:methods*
chemical_list: D007328:Insulin; D050052:TRPC Cation Channels; D005947:Glucose