The Ratio Between Collagenase Class I and Class II Influences the Efficient Islet Release From the Rat Pancreas

Brandhorst, Heide; Raemsch-Guenther, N.; Raemsch, C.; Friedrich, O; Huettler, S.; Kurfuerst, M.; Korsgren, Olle; Brandhorst, Daniel

doi:10.1097/tp.0b013e31816050c8

Cited by 36 publications

(35 citation statements)

References 43 publications

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“…Brandhorst et al suggest that the differences from lot to lot may be due to the lack of proper accounting of tryptic-like activity, even in the most pure mixtures of collagenases, such as Liberase HI (Roche, Indianapolis, IN, USA) and collagenase NB1 (Serva, Heidelberg, Germany) used in isolation of human islets for transplantation [17]. The tryptic-like activity in enzyme blends may work in concert with the other enzymes to increase the activity of the digestion, although there is some debate about the damage tryptic-like activity has on the islets [18]. Enzyme blends with high purity and precise notation of the components are used in human islet isolation to ensure consistent activity and reproducibility [17,19].…”

Section: Procedures Of Islet Isolationmentioning

confidence: 99%

A Practical Guide to Rodent Islet Isolation and Assessment

et al. 2009

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Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.

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Section: Procedures Of Islet Isolationmentioning

confidence: 99%

A Practical Guide to Rodent Islet Isolation and Assessment

et al. 2009

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“…Male Lewis rats (Harlan, Hanover, Germany) weighing 300 to 350 g were used as pancreas donors and processed for islet isolation using standard procedures as described previously in detail (6). Pancreas digestion was performed by means of 20 PZ-U of NB 1 and 0.4 dimethylcasein units (DMC-U) of NP (Serva).…”

Section: Rat Islet Isolation and Characterizationmentioning

confidence: 99%

The Importance of Tryptic-like Activity in Purified Enzyme Blends for Efficient Islet Isolation

Brandhorst¹,

Friberg²,

Andersson³

et al. 2009

Transplantation

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“…It was reported that C2 was more efficient in collagen degradation [10] whereas C1 played only supporting role in islet isolations [11]. Study by Brandhorst et al [12] showed that neither C1 nor C2 alone were able to dissociate pancreatic tissue. Rapid and complete loss of peri-islet ECM depends on both collagenase isoform and is best achieved with assistance of other proteolytic enzymes [10] [12] [13].…”

Section: Discussionmentioning

confidence: 99%

Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by <i>Clostridium histolyticum</i>

Berková¹,

Saudek²,

Leontovyč³

et al. 2018

ABB

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Clostridium histolyticum is used for production of several proteolytic enzymes such as elastase, neutral proteases, clostripain and in particular collagenase. Besides industrial applications, collagenase has been indispensable for medical purposes including isolation of pancreatic islets for diabetes treatment. The aim of this study was to optimize the method for production and partial purification of a new collagenase blend and to test its suitability for successful pancreatic islets isolation in a rat model. Bacterial strain of C. histolyticum was sequenced for presence of the collagenase genes. Different fermentation conditions were tested and the process of collagenase extraction was modified and optimized. Samples of collagenases were taken for western blot detection, activity assessment, and ability for dissociation of pancreatic tissue. Findings indicate that concentrated trypton growth medium with pepton was the most suitable for Clostridium growth and collagenase production.Whole genome sequencing revealed two genes for collagenase and also gene for clostripain. Western blot specific detection helped to select useful production modifications. Following these modifications was also improved the yield, morphology and in vitro function of intact pancreatic islets which were finally comparable or better than those achieved using standard blends of collagenase. The results support the use of the new collagenase blend for islet isolation giving thus the opportunity to choose an alternative product. Our next steps would lead to further enzyme purification through scaling up of the production method for a wider use.

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The Ratio Between Collagenase Class I and Class II Influences the Efficient Islet Release From the Rat Pancreas

Cited by 36 publications

References 43 publications

A Practical Guide to Rodent Islet Isolation and Assessment

A Practical Guide to Rodent Islet Isolation and Assessment

The Importance of Tryptic-like Activity in Purified Enzyme Blends for Efficient Islet Isolation

Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by <i>Clostridium histolyticum</i>

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The Ratio Between Collagenase Class I and Class II Influences the Efficient Islet Release From the Rat Pancreas

Cited by 36 publications

References 43 publications

A Practical Guide to Rodent Islet Isolation and Assessment

A Practical Guide to Rodent Islet Isolation and Assessment

The Importance of Tryptic-like Activity in Purified Enzyme Blends for Efficient Islet Isolation

Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by &lt;i&gt;Clostridium histolyticum&lt;/i&gt;

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Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by <i>Clostridium histolyticum</i>