Crude or partially purified collagenases are the most common tissue dissociating enzyme (TDE) products used for isolating rodent islets. A small number of reports used purified TDEs for this procedure1,2 but the practice is not widespread despite the major advantages of using purified TDE products that are routinely used for human islet isolation3,4. This post summarizes results from an ongoing collaboration with Dr. Raghu Mirmira’s lab at the Wells Center for Pediatric Research at the Indiana University School of Medicine to define an optimal collagenase and neutral protease composition for use in mouse islet isolation procedures.
Several combinations of collagenase and neutral proteases were evaluated at different concentrations using a protocol based on that of Gotoh in which the TDEs are perfused directly into the pancreatic duct5. In all cases, the purified enzymes were compared to the yield and morphology of islets obtained with Sigma Collagenase Type XI (Type XI). As with islets isolated from other species, differences were observed between thermolysin and other neutral proteases. Smaller islets, lower yields and greater loss of morphology were observed after culture when thermolysin was used in the enzyme mixture. In contrast, islets isolated with BP Protease (analogous to Dispase®) were of similar size and morphology to those obtained after isolation with Type XI. The collagenase activity also impacts islet yield and morphology as the CIzyme Collagenase MA was comparable to Type XI and superior to those isolated with CIzyme Collagenase HA with average yields of 140, 130 and 87 islets per organ, respectively from C57/B6 mice.
Significant variability in average islet yields from different strains of rats and mice have been reported. The major advantages of using purified TDEs in this application are once the VitaCyte enzyme composition is defined, the lot to lot consistency is assured. Additional modifications of the composition can be made to improve the yield and quality of islets recovered from different mouse strains. The major benefit of using purified TDEs in this application is the elimination of the labor intensive process of lot qualification normally required for enriched collagenase products. Reporting optimal enzyme compositions for isolation of islets from different strains of mice can be confidently communicated, leading to more productive used of resources to address the needs of ongoing research programs.
1. Nikolova G, Jabs N, Konstantinova I, et al (2006) The vascular basement membrane: a niche for insulin gene expression and beta cell proliferation. Dev Cell 10:397-405.
2. Giraud S, Claire B, Eugene M, Debre P, Richard F, and Barrou B. (2007) A new preservation solution increases islet yield and reduces graft immunogenicity in pancreatic islet transplantation. Transplantation. 83:1397-400 .
3. Linetsky E, Bottino R, Lehmann R, Alejandro R, Inverardi L, and Ricordi C (1997) Improved human islet isolation using a new enzyme blend, liberase. Diabetes 46:1120-3.
4. Olack B.J., Swanson C.J., Howard T.K., and Mohanakumar T. (1999) Improved method for the isolation and purification of human islets of langerhans using Liberase enzyme blend. Hum Immunol 60:1303-9.
5. Gotoh M, Maki T, Satomi S, Porter J, Bonner-Weir S, O’Hara CJ, and Monaco AP (1987) Reproducible high yield of rat islets by stationary in vitro digestion following pancreatic ductal or portal venous collagenase injection. Transplantation. 43:725-30.
6 Carter JD, Dula SB, Corbin KL, Wu R, and Nunemaker CS (2009) A practical guide to rodent islet isolation and assessment. Biological Procedures Online. Proc. Online 11:3-31.
7 Li D-S, Yuan Y-H, Tu H-J, Liang Q-L and Dai L-J (2009) A protocol for islet isolation from mouse pancreas. (2009) Nature Protocols 4:1649-52.