P050

Purified tissue dissociating enzyme performance in isolating mouse islets

Andrew Breite¹, Natalie Stull², Robert McCarthy¹, Raghavendra Mirmira², Francis Dwulet¹

¹VitaCyte LLC; ²Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine; Indianapolis, IN, United States

Isolated rodent islets continue to serve as the most practical model to study β cell biology in vitro. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Crude or partially purified tissue dissociating enzymes (TDE) continue to be used by most laboratories isolating rodent islets. However, inconsistencies in islet yield and quality often limit the extent and feasibility of primary islet studies. Variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure, which frequently exhibit lot-to-lot variations in activity and often require protocol modifications upon acquiring a new lot. While reports exist of purified TDEs used to isolate rodent islets, the practice is still not widespread and has not been fully optimized. We developed and evaluated a purified, defined TDE composition to isolate islets from CD1 mice using the method described by Gotoh where TDEs are directly infused into the main pancreatic duct in situ. Once a favorable enzyme composition was identified, a number of isolations were performed and compared to a partially purified collagenase. The purified TDEs gave islet yields of 215 ± 42 islets per mouse (n=18) versus 206 ± 77 islets per mouse (n=13) using the partially purified collagenase. A total of two lots of purified collagenase and three lots of purified protease were used in the 18 isolations reported above suggesting the consistency in performance of using purified TDE. Use of purified TDEs eliminate the need for labor intensive lot qualification process and allow for optimization of enzyme activity for specific applications or to address differences between mice strains. Defined and purified TDE also enable concise reporting of optimal TDE compositions which lead to improved productivity and confident comparing of results between laboratories.