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Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (Olle Korsgren)ORCID iD: 0000-0003-4511-6149#sthash.NKvqlgKU.dpuf
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2015 (English)In: Transplantation Direct, ISSN 2373-8731, Vol. 1, no 5, 1-6 p.Article in journal (Refereed) Published
Abstract [en]

Background: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential as they synergize with collagenase for effective pancreas digestion. The presence of tryptic-like activity has been implicated in efficient enzyme blends and the present study aimed to evaluate if addition of clostripain, an enzyme with tryptic-like activity, could improve efficacy of the islet isolation procedure.

Methods: Clostripain was added to the enzyme blend just before pancreas perfusion. Islets were isolated per standard method and numerous isolation parameters, islet quality control, and the number of isolations fulfilling standard transplantation criteria were evaluated. Two control organs per clostripain organ were chosen by blindly matching against body mass index, cold ischemia time, hemoglobin A1c, donor sex, and donor age.

Results: There were no differences in pancreas weight, dissection time, digestion time, harvest time, percent digested pancreas, or total pellet volume before islet purification between control or clostripain pancreases. Glucose-stimulated insulin release results were similar between groups. Total isolation islet equivalents, purified tissue volume and islet equivalents/g pancreas as well as fulfillment of transplantation criteria favored clostripain processed pancreases.

Conclusions: The addition of clostripain to the enzyme blend soundly improved islet yields and transplantation rates. It gently aided pancreas digestion and maintained proper islet functionality. The addition of clostripain to the enzyme blend has now been implemented into standard isolation protocols at the isolation centers in Uppsala and in Oslo.

Place, publisher, year, edition, pages
2015. Vol. 1, no 5, 1-6 p.
National Category
Endocrinology and Diabetes
URN: urn:nbn:se:uu:diva-254354DOI: 10.1097/TXD.0000000000000528OAI: oai:DiVA.org:uu-254354DiVA: diva2:843638
Available from: 2015-07-30 Created: 2015-06-08 Last updated: 2015-08-28Bibliographically approved
In thesis
1. Technical challenges in human islet isolation
Open this publication in new window or tab >>Technical challenges in human islet isolation
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transplantation of islets of Langerhans is an effective treatment option for patients with brittle type 1 diabetes mellitus. This treatment restores glucose control and also reduces hypoglycemia. Unfortunately, the outcome from islet isolations is variable, and many preparations do not yield sufficient islet number or islet quality.

The aim of this thesis was to improve the isolation procedure, thereby making more preparations available for clinical transplantation.

A well-established method for pathogen inactivation was applied to human serum used in the islet isolation process. Evaluation of isolated islets stored in medium supplemented with pathogen-inactivated serum showed that pathogen inactivation did not have negative effects. These findings will enable the use of human serum in clinical cell transplantation programs, while simultaneously increasing patient safety.

Pre-incubation of islets prior to gradient separation is an established standard in the field of islet isolation. Through a reduction in the pre-incubation step, isolation time could be reduced by almost an hour without affecting the isolation outcome.

A commercially available protease enzyme, clostripain, was added to the enzyme blend used in islet isolation. Addition of clostripain was found to increase the number of islets isolated as well as the purified tissue volume and fulfillment of transplant criteria. Use of clostripain should help to increase the number of successful isolations.

A newly developed pancreas-specific preservation solution, I-Let protect, was evaluated. As compared to standard preservation solutions, it can be used in situations of prolonged cold ischemic time without affecting the isolation outcome or islet functionality. I-Let protect can also be used in establishing a protocol that would eliminate the need for night- time isolations.

Through the work in this thesis, several key elements in human islet isolation have been optimized, and further knowledge has been gained.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 57 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1119
National Category
Endocrinology and Diabetes
urn:nbn:se:uu:diva-259358 (URN)978-91-554-9283-0 (ISBN)
Public defence
2015-09-18, Auditorium Minus, Gustavianum, Akademigatan 3, Uppsala, 09:15 (English)
Available from: 2015-08-27 Created: 2015-07-31 Last updated: 2015-10-01

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Ståhle, MagnusTufveson, GunnarKorsgren, Olle
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Clinical ImmunologyTransplantation SurgeryDepartment of Immunology, Genetics and Pathology
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