Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular...

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Main Authors:	Stull, Natalie D., Breite, Andrew, McCarthy, Robert, Tersey, Sarah A., Mirmira, Raghavendra G.
Format:	Online
Language:	English
Published:	MyJove Corporation 2012
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490250/

id	pubmed-3490250
recordtype	oai_dc
spelling	pubmed-34902502012-11-09 Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease Stull, Natalie D. Breite, Andrew McCarthy, Robert Tersey, Sarah A. Mirmira, Raghavendra G. Cellular Biology The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains. MyJove Corporation 2012-09-07 /pmc/articles/PMC3490250/ /pubmed/22987198 http://dx.doi.org/10.3791/4137 Text en Copyright © 2012, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Stull, Natalie D. Breite, Andrew McCarthy, Robert Tersey, Sarah A. Mirmira, Raghavendra G.
spellingShingle	Stull, Natalie D. Breite, Andrew McCarthy, Robert Tersey, Sarah A. Mirmira, Raghavendra G. Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease
author_facet	Stull, Natalie D. Breite, Andrew McCarthy, Robert Tersey, Sarah A. Mirmira, Raghavendra G.
author_sort	Stull, Natalie D.
title	Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease
title_short	Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease
title_full	Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease
title_fullStr	Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease
title_full_unstemmed	Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease
title_sort	mouse islet of langerhans isolation using a combination of purified collagenase and neutral protease
description	The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.
publisher	MyJove Corporation
publishDate	2012
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490250/
_version_	1611921724672049152

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

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