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The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology. (Olle Korsgren)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Clinical Immunology. (Olle Korsgren)
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2009 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 87, no 3, 370-5 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The isolation of islets from the human pancreas critically depends on an efficient enzyme blend. Previous studies have solely focused on the presence of collagenase and neutral protease/thermolysin. Despite improved characterization of these components, the lot-related variability in efficacy still persists suggesting that additional so far disregarded enzymes are required for efficient islet cleavage. METHODS: Varying activities of a tryptic-like enzyme were identified within collagenase NB1 lots, which were selected according to a matched ratio between tryptic-like and collagenase activity (TLA-ratio). Rat and human pancreata were processed with current standard procedures. RESULTS: Increasing the TLA-ratio from 1.3% to 10% reduced pancreas dissociation time in rats by 50% without affecting islet yield, viability, or posttransplant function in diabetic nude mice. Enhancing the TLA-ratio from 1.3% to 12.6% for human pancreas processing resulted in a significant reduction of recirculation time and increased incrementally human islet yield without affecting purity, in vitro function or recovery after culture. Optimized pancreas digestion correlated with a higher percentage of islet preparations fulfilling quality criteria for clinical transplantation. CONCLUSIONS: We conclude that TLA is an effective component that should be included in moderate amounts in enzyme blends for human islet isolation to optimize the efficiency and minimize the lot-related variability.

Place, publisher, year, edition, pages
2009. Vol. 87, no 3, 370-5 p.
Keyword [en]
human islet transplantation, human islet isolation, collagenase, enzymes
National Category
Research subject
Medical Science
URN: urn:nbn:se:uu:diva-103604DOI: 10.1097/TP.0b013e31819499f0ISI: 000263397400010PubMedID: 19202441OAI: oai:DiVA.org:uu-103604DiVA: diva2:218536
Available from: 2009-05-20 Created: 2009-05-20 Last updated: 2015-06-15Bibliographically approved
In thesis
1. Standardization of Islet Isolation and Transplantation Variables
Open this publication in new window or tab >>Standardization of Islet Isolation and Transplantation Variables
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Currently, the transplantation of islets of Langerhans is a viable means to maintain control of blood sugar levels and reduce the risk of hypoglycemia in defined populations with brittle type I diabetes mellitus or those requiring pancreatectomy. However, the process of islet isolation is highly variable and not all isolations result in islet numbers or quality suitable for transplantation.

This thesis aimed to improve transplantation success through optimization and standardization of the isolation process and to identify pretransplant variables associated with early islet engraftment.

A previously disregarded enzyme activity, tryptic-like activity (TLA), has been identified to influence pancreas digestion efficiency and islet isolation success in both the preclinical and clinical situations. For human pancreases, islet isolation success rates improved from 0% in the lowest TLA group to over 50% in the highest TLA groups without affecting islet quality. These findings should help standardize evaluation of enzymes for clinical islet isolation.

A closed, automated, pump-made gradient system was compared to the open, manual method for islet separation. No differences were observed in expected gradient volumes, islet yields or total purities between the two methods. The pump-made gradient system successfully removed manual influences on density gradient production while fulfilling regulatory requirements for closed system processing.

Islet quantification was evaluated with computer-assisted digital imaging analysis (DIA) and a semi-closed assessment system. By using the DIA system method, which measures islet purity and pellet volume instead of manual counting methods, variation in islet counts and purity reduced by almost half.

By using a transplant outcome measurement of C-peptide adjusted by blood glucose and creatinine, we identified four pretransplant factors that affect early transplant outcome. Of the four factors, one was related to the organ transport time, one to function of the islets, and two to the transplanted tissue volume. When these four factors were put into a predictive model, it accounted for about 40% of the transplant outcome.

The work contained in this thesis identifies and optimizes a number of critical elements related to islet isolation and transplantation protocols.


Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 76 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 669
Islet isolation, standardization, enzyme, gradient separation, digital imaging analysis, DIA, transplantation outcome, islet transplantation, prediction
National Category
Biomedical Laboratory Science/Technology
Research subject
Medical Cell Biology; Computerized Image Processing
urn:nbn:se:uu:diva-150247 (URN)978-91-554-8066-0 (ISBN)
Public defence
2011-05-23, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Available from: 2011-05-02 Created: 2011-03-28 Last updated: 2011-07-01Bibliographically approved

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