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Islet Transplantation a Technical Challenge: Studies on Human Pancreas Preservation and Enzymatic Digestion
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Islet transplantation has found its niche in diabetes treatment. It has contributed to a better quality of life and better glycemic control of patients with diabetes suffering from severe hypoglycemia that are not eligible for vascularized pancreas transplantation. Islet isolation is a technically challenging procedure. The different studies within this doctoral thesis aim to improve and standardize different steps in the isolation procedure. They are in particular looking to improve human pancreas preservation during cold storage, to optimize islet release from the exocrine tissue and to assess whether the isolated islet yield can be predicted from a biopsy.

We found that pancreas preservation with pre-oxygenated perfluorodecalin (two-layer method) did not improve the ischemic tolerance of the human pancreas as compared to cold storage with the University of Wisconsin (UW) solution. Furthermore, in pancreas with long cold ischemia time (CIT) (>10 hours), Histidine-Tryptophan-Ketoglutarate (HTK) had a limited preservation capacity as compared with the UW solution with respect to isolation outcome. We also found that during enzymatic pancreas digestion, Vitacyte HA was able to provide a similar islet yield and quality as Serva NB1 with less collagenase activity and shorter digestion time. We further describe the first experience with a new GMP manufactured enzyme called Liberase MTF-S for successful human islet isolation. Finally, we found that the isolated islet yield could not be predicted from a biopsy taken from the head of the pancreas concerning solely morphological parameters of the islets tissue.

The improvement of pancreas preservation will allow for marginal organs with prolonged cold ischemia time to expand the donor pool. Better knowledge of how the pancreatic extracellular matrix is digested by collagenase will lead to a fast and predictable islet release from the exocrine tissue. By standardizing the isolation procedure and improving organ selection we will increase the success rate in human islet isolation, thereby making islet transplantation available for more patients.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 692
Keyword [en]
Islet transplantation, Human islet isolation, Pancreas preservation, Two-layer method, Perfluorocarbon, Cold storage, Cold ischemia, University of Wisconsin solution, Histidine-tryptophan-ketoglutarate, HTK, Pancreas preservation, Prediction, Collagenase
Keyword [es]
Trasplante de islotes, Aislamiento de islotes, Preservación del pancreas, Método de las dos capas, Colagenasa, Solución de Wisconsin, HTK, Histidina-triptófano-ketoglutarato, Predicción
National Category
Endocrinology and Diabetes Surgery
Research subject
Endocrinology and Diabetology; Cell Research; Clinical Immunology
Identifiers
URN: urn:nbn:se:uu:diva-156893ISBN: 978-91-554-8126-1 (print)OAI: oai:DiVA.org:uu-156893DiVA: diva2:435005
Public defence
2011-09-23, Rudbecksalen, Rudbecklaboratoriet. Dag Hammarskjölds väg 20. SE-751 85, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-09-02 Created: 2011-08-10 Last updated: 2011-11-03Bibliographically approved
List of papers
1. No beneficial effect of two-layer storage compared with UW-storage on human islet isolation and transplantation
Open this publication in new window or tab >>No beneficial effect of two-layer storage compared with UW-storage on human islet isolation and transplantation
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2007 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 84, no 7, 864-869 p.Article in journal (Refereed) Published
Abstract [en]

Background. Shipment of pancreata between distant centers is frequently associated with prolonged cold ischemia time (CIT) that leads to poorer outcomes for islet transplantation. Clinical pilot trials have indicated that oxygenation of explanted human pancreata utilizing the two-layer method (TLM) allows the use of marginal donor pancreata for islet transplantation. The present study aimed to clarify whether TLM enhances the ischemic tolerance of human pancreata. Methods. We analyzed retrospectively the outcome of 200 human islet isolations performed after TLM preservation or storage in University of Wisconsin solution (UWS). Results. Donor characteristics and digestion parameters did not vary significantly between TLM-preserved and UWS-stored pancreata. No differences were observed between experimental groups with regard to islet yield, purity, or dynamic glucose stimulation index after either short or prolonged CIT. However, CIT and stimulation index were negatively correlated in each experimental group. The isolation outcome in donors aged ≥60 years was not increased after TLM preservation when compared to UWS storage. No effect was observed regarding islet posttransplant function in recipients with established kidney grafts. Conclusions. The present study suggests that the ischemic tolerance of human pancreata cannot be extended by TLM preservation. In addition, TLM does not seem to improve the isolation outcome for pancreata from elderly donors.

Keyword
Clinical islet transplantation, Cold storage, Marginal donors, Two-layer method
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-16849 (URN)10.1097/01.tp.0000284584.60600.ab (DOI)000250232600010 ()17984839 (PubMedID)
Available from: 2008-06-05 Created: 2008-06-05 Last updated: 2017-12-08Bibliographically approved
2. Using HTK for Prolonged Pancreas Preservation Prior to Human Islet Isolation
Open this publication in new window or tab >>Using HTK for Prolonged Pancreas Preservation Prior to Human Islet Isolation
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2012 (English)In: Journal of Surgical Research, ISSN 0022-4804, E-ISSN 1095-8673, Vol. 175, no 1, 163-168 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Histidine-tryptophan-ketoglutarate (HTK) has been established as an alternative to University-of-Wisconsin solution (UWS) for abdominal organ preservation, but data about HTK efficiency to preserve pancreata during prolonged cold ischemia time (CIT) are conflicting. In human islet transplantation, HTK provided similar isolation outcomes after short CIT. The present study aimed to investigate whether islets can be successfully isolated from HTK-preserved pancreata after prolonged CIT compared with UWS.

MATERIALS AND METHODS: Sixty-four human pancreata retrieved from donors meeting criteria for kidney donation were perfused utilizing either HTK or UWS and preserved for more or less than 10 h prior to islet isolation. Along with parameters related to isolation and islet quality assessment, the dry-to-wet weight ratio was evaluated.

RESULTS: Donor- and procurement-related factors did not vary between HTK- and UWS-perfused pancreata. The dry-to-wet weight ratio was lower in HTK-preserved pancreata indicated tissue edema (21.0% ± 3.5% versus 24.8% ± 2.0%, P = 0.007). Isolation-related variables differed between experimental groups after prolonged CIT with respect to purified packed tissue volume (9.1 ± 5.0 versus 17.2 ± 8.1 μL/g, P = 0.004) and islet yield (1910 ± 980 versus 3150 ± 1420 IE/g, P = 0.012). Islet purity and survival after culture were similar after HTK or UWS perfusion. The preservation solution did not affect in vitro function and transplantability of isolated islets.

CONCLUSIONS: Compared with UWS, HTK has similar efficiency to preserve human pancreata for subsequent islet isolation during <10 h CIT but seems to be limited for prolonged cold storage.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-156890 (URN)10.1016/j.jss.2011.03.012 (DOI)000303472500031 ()21550052 (PubMedID)
Available from: 2011-08-10 Created: 2011-08-10 Last updated: 2017-12-08Bibliographically approved
3. Vitacyte Collagenase HA: A Novel Enzyme Blend for Efficient Human Islet Isolation
Open this publication in new window or tab >>Vitacyte Collagenase HA: A Novel Enzyme Blend for Efficient Human Islet Isolation
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2009 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 88, no 12, 1400-1402 p.Article in journal, Letter (Refereed) Published
National Category
Surgery Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-111858 (URN)10.1097/TP.0b013e3181bd1441 (DOI)000273200000013 ()20029339 (PubMedID)
Note

Letter. Authors were listed in the wrong order in the original publication (erratum in Transplantation, 2010, vol. 89, issue 7, p. 907)

Available from: 2009-12-28 Created: 2009-12-28 Last updated: 2017-12-12Bibliographically approved
4. Mammalian Tissue-Free Liberase: A New GMP-Graded Enzyme Blend for Human Islet Isolation
Open this publication in new window or tab >>Mammalian Tissue-Free Liberase: A New GMP-Graded Enzyme Blend for Human Islet Isolation
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2010 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 90, no 3, 332-333 p.Article in journal, Letter (Refereed) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-135881 (URN)10.1097/TP.0b013e3181e117e3 (DOI)000280581200020 ()
Available from: 2010-12-13 Created: 2010-12-09 Last updated: 2017-12-11Bibliographically approved
5. Predicting the outcome of human islet isolation
Open this publication in new window or tab >>Predicting the outcome of human islet isolation
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background. Islet transplantation is currently being explored as a possible treatment to diabetes mellitus. Islet isolation from the human pancreas is a technically demanding process and the success rate even in the most experienced GMP facilities is only about 50%. The aim of this study was to investigate whether isolation outcome can be predicted from a pancreas biopsy taken during organ procurement.

Methods. The outcome of 29 human islet isolations was retrospectively studied. Biopsies from the pancreatic head were immunostained for insulin to study islet morphology and size distribution utilizing a digital analysis system. Isolations were categorized as successful if they yielded more than 2000 IE/g. 

Results. Pellet volume after collagenase digestion and islet purity was higher in the successful group. None of the morphology variables, i.e. islet number (IN/mm2), islet equivalent number (IE/IN) and percentage of insulin positive area in the biopsy, differed significantly between the study groups.

Conclusions. No single morphological feature observed in a biopsy taken from the head of pancreas can predict the outcome of islet isolation from the human pancreas, even if using the same enzyme blend in standardized human islet isolation procedure.

Identifiers
urn:nbn:se:uu:diva-156891 (URN)
Available from: 2011-08-10 Created: 2011-08-10 Last updated: 2011-11-03

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