Experience of islet isolation without neutral protease supplementation

Kin, Tatsuya; O’Gorman, Doug; Senior, Peter; Shapiro, AM James

doi:10.4161/isl.2.5.12602

Cited by 15 publications

(15 citation statements)

References 27 publications

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“…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 laboratories [1][2][3][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.…”

mentioning

confidence: 99%

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

Stull

Breite²,

McCarthy³

et al. 2012

JoVE

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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 laboratories [1][2][3][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 isolations 5,6 , 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 Gotoh 7,8 , in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient 9 , 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 a 6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate 6 . 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 assay 10 . 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.

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confidence: 99%

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

Stull

Breite²,

McCarthy³

et al. 2012

JoVE

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“…In fact, Collagenase NB1/NP made progress to advance the field; however, it has been reported that such enzyme may not be highly purified because trace proteolytic enzymes were present, influencing the isolation outcomes and thus lot-to-lot variability. 43 …”

Section: Discussionmentioning

confidence: 99%

The Choice of Enzyme for Human Pancreas Digestion Is a Critical Factor for Increasing the Success of Islet Isolation

Valiente

McFadden

et al. 2015

Transplantation Direct

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Background We evaluated three commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality post-isolation. Methods Retrospectively compared and analyzed islet isolations from pancreata using three different enzyme groups: Liberase HI (n=63), Collagenase NB1/Neutral Protease (NP) (n=43), and Liberase Mammalian Tissue Free Collagenase/Thermolysin (MTF C/T) (n=115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate (ΔOCR), and in vivo transplantation model in mice. Results Donor characteristics were not significantly different among the three enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index (BMI), hemoglobin A1c (HbA1c), cold ischemia time (CIT), and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the Liberase MTF C/T group (73.5 ± 1.5 %) when compared to the Liberase HI group (63.6 ± 2.3 %) (p<0.001) and the Collagenase NB1/NP group (61.7 ± 2.9%) (p<0.001). The stimulation index for GSIS was significantly higher in the Liberase MTF C/T group (5.3 ± 0.5) as compared to the Liberase HI (2.9 ± 0.2) (p<0.0001) and the Collagenase NB1/NP (3.6 ± 2.9) (p=0.012) groups. Furthermore, the Liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic NOD Scid mice (65%), which was significantly higher than the Liberase HI (42%, p=0.001) and the Collagenase NB1/NP enzymes (41%, p<0.001). Conclusions Liberase MTF C/T is superior to Liberase HI and Collagenase NB1/NP in terms of digestion efficacy and glucose-stimulated insulin secretion in vitro. Moreover, Liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to Liberase HI and Collagenase NB1/NP enzymes.

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“…The enzyme was prepared by dissolving in Hanks’ balanced salt solution with 3.6 mMol CaCl 2 with a final volume of 350 mL. Collagenase dose was 1 vial with an activity range of 2322–2453 PZ Units, haNP dosing range was 0.60–2.31 DMC/g of pancreas and thermolysin dose was <850 TU/g of pancreas 11 . Enzyme was delivered via pancreatic duct simultaneously under controlled temperature and pressure conditions.…”

Section: Methodsmentioning

confidence: 99%

“…Studies have shown that NP is integral to effective pancreas dissociation and islet recovery, but a delicate balance remains between islet mass, function and viability 8 - 10 . A recent study detailed islet isolation experience void of NP altogether 11 . The current alternative to NP, thermolysin, has also reported favorable outcomes 12 .…”

Section: Introductionmentioning

confidence: 99%

Clinical islet isolation outcomes with a highly purified neutral protease for pancreas dissociation

O’Gorman

Kin

Pawlick

et al. 2013

Islets

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Background: Pancreas dissociation is a critical initial component of the islet isolation procedure and introduces high variability based on factors including the enzyme type, specificity and potency. Product refinement and alterations to the application strategies have improved isolation outcomes over time; however, islet utilization from donor organs remains low. In this study we evaluate a low endotoxin-high activity grade neutral protease in clinical islet isolation.Materials and Methods: The use of a non-collagenolytic enzyme, either thermolysin or high active neutral protease, was randomized in clinical islet isolations to evaluate efficacy. Additionally a retrospective comparison to neutral protease NB was conducted.Results:The thermolysin group had lower trapped islet population and increased purity and post-culture islet mass in comparison to high active grade neutral protease. Comparison of neutral protease NB GMP grade to high active neutral protease displayed no measurable difference in islet mass or viability and transplantation outcomes at 1 mo post-transplant were favorable for both groups.Conclusions: High activity neutral protease can generate clinical grade islets and may prove beneficial to islet function and viability based on a reduced endotoxin load but dosing of neutral protease requires ongoing optimization.

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Experience of islet isolation without neutral protease supplementation

Cited by 15 publications

References 27 publications

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

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

The Choice of Enzyme for Human Pancreas Digestion Is a Critical Factor for Increasing the Success of Islet Isolation

Clinical islet isolation outcomes with a highly purified neutral protease for pancreas dissociation

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