Rodent Islet ApplicationsRecommended Product
Gotoh Procedure Recommended Method
A recent review summarized development of methods used to isolate islets from rodent pancreata.(1) The initial rodent islet isolation method used was described by Lacy and Kosatianovsky (Lacy Procedure)(2). It was later modified by Gotoh et al. (Gotoh Procedure)(3). Both procedures inject Hanks Balanced Salt Solution (HBSS) into the pancreas through the common bile duct that connects to the main pancreatic duct. The injection of this fluid swells the pancreas, resulting in additional mechanical damage to the tissue structure. The primary difference in the procedures is summarized in the table below.
| Lacy Procedure | Gotoh Procedure | |
|---|---|---|
| Fluid Injected into Common Bile Duct | HBSS alone | HBSS containing collagenase-protease enzymes |
| Pancreas Preparation | Finely mince tissue | No treatment |
| Tissue Dissociation | Incubate minced tissue in HBSS-enzyme solution; mechanically shake vigorously during incubation at 37°C in water bath | Static incubation of pancreas in HBSS-enzyme solution at 37°C in water bath; after incubation, vigorously shake digest tubes manually for < 30 seconds |
Gotoh’s modification to the Lacy procedure simplifies the digestion of the pancreas by eliminating the need for mincing tissue and continual shaking of the tissue digest during the incubation period. It also avoids the requirement to pretreat all glassware used in the procedure with silicone before use. Islets are separated from the acinar cells by density gradient centrifugation handpicking, or by using both methods: picking islets from an enriched islet preparation.
Traditional crude or enriched collagenase products were used for rodent islet isolation for many years. There are limitations of using these products for islet isolation instead of purified defined enzymes.
Crude Collagenase
Consistency
Most labs pre-qualify a new lot before routine use
Purity
> 90% of dry weight is other biochemical components
Variability in endotoxin contamination that can impact islet function(4)
Composition
Composition of the collagenase and protease enzymes unknown
CIzyme RI
Consistency
Formulated using purified-defined collagenase and protease enzymes
Purity
> 95% purity
Enzyme purification minimizes endotoxin contamination
Composition
Enzyme compositions for collagen degradation and protease activity reported on Certificate of Analysis
A leading east coast diabetes and training center has been using CIzyme RI for rat and mouse islet isolations for many years. Several years ago, they provided a summary of their rat islet isolation yields from 220-250 g Sprague-Dawley rats. Over a six year period they used 12 different lots of CIzyme RI and found that no adjustments were needed to the dose or timing to the pancreatic digestion procedure.
Reconstitution and preparation of the digestion solution for using CIzyme RI in the Gotoh method is described in the CIzyme RI package insert.
Expected Cell Yield
Cell yield varies by sex, age, strain, and nutritional state of the animal. Consult VitaCyte’s white paper entitled “What is an acceptable rodent islet yield?” that provides information from each strain on the average islet yield per pancreas, method used, product and dose used in isolation, digest time, and purification method. These data are derived from those laboratories using purified collagenase and protease enzymes to avoid collagenase lot variability confounding the results.
Perspectives
A key component of a successful diabetes research program is consistent and cost-effective isolation of high-quality rodent islets. Most laboratories continue to use traditional collagenase products, accepting the unknown risk that contaminants in these products (e.g., variable endotoxin) may impact the functional characteristics of islets used in downstream experiments and the increased costs required to qualify a new lot of collagenase when a new lot is needed. There are several benefits to using a purified-defined collagenase-protease product designed for rodent islet isolation:
Avoidance of lot qualification, minimizing unproductive work
Improved control of the isolation process
Benchmark your isolation results to others using similar enzymes and isolation procedures
Confidentially share results with others since using defined enzyme mixtures enables others to replicate your results
Resources & Reviews
Four method videos reviewing the Gotoh method are published in the Journal of Visualized Experiments (JoVE) (5-8) and a published protocol for mouse islet isolation. (9)
Separate reviews describe the Lacy(10) and Gotoh(1, 11) procedures in more detail.
References
- Corbin KL, West HL, Brodsky S, Whitticar NB, Koch WJ, Nunemaker CS. A Practical Guide to Rodent Islet Isolation and Assessment Revisited. Biological Procedures Online. 2021;23:7.
- Lacy PE, Kostianovsky M. Method for the isolation of intact Islets of Langerhans from the rat pancreas. Diabetes. 1967;16:35-9.
- Gotoh M, Maki T, Kiyoizumi T, Satomi S, Monaco AP. An improved method for isolation of mouse pancretic islets. Transplant. 1985;40:437-8.
- Berney T, Molano RD, Cattan P, Pileggi A, Vizzardelli C, Oliver R, et al. Endotoxin-mediated delayed islet graft function is associated with increased intra-islet cytokine production and islet cell apoptosis. Transplant. 2001;71:125-32.
- Szot GL, Koudria P, Bluestone JA. Murine pancreatic islet isolation. JoVE. 2007; (7):255.
- Zmuda EJ, Powell CA, Hai T. A method for murine islet isolation and subcapsular kidney transplantation. JoVE. 2011; (50).
- Stull ND, Breite A, McCarthy R, Tersey SA, Mirmira RG. Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease. JoVE. 2012; (67).
- Neuman JC, Truchan NA, Joseph JW, Kimple ME. A method for mouse pancreatic islet isolation and intracellular cAMP determination. JoVE. 2014; (88):e50374.
- Graham K, Fynch S, Pappas E, Tan C, Kay T, Thomas HE. Isolation and culture of the Islets of Langerhans from mouse pancreas. Bio-protocol. 2016;6:1-12.
- Kelly CB, Blair LA, Corbett JA, Scarim AL. Isolation of islets of Langerhans from rodent pancreas. Meth Mol Med. 2003;83:3-14.