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Standardization of Islet Isolation and Transplantation Variables
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. (forskargrup Korsgren)
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 669
Keyword [en]
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
Identifiers
URN: urn:nbn:se:uu:diva-150247ISBN: 978-91-554-8066-0OAI: oai:DiVA.org:uu-150247DiVA: diva2:406870
Public defence
2011-05-23, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-05-02 Created: 2011-03-28 Last updated: 2011-07-01Bibliographically approved
List of papers
1. The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation
Open this publication in new window or tab >>The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation
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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.

Keyword
human islet transplantation, human islet isolation, collagenase, enzymes
National Category
Surgery
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-103604 (URN)10.1097/TP.0b013e31819499f0 (DOI)000263397400010 ()19202441 (PubMedID)
Available from: 2009-05-20 Created: 2009-05-20 Last updated: 2015-06-15Bibliographically approved
2. Human islet separation utilizing a closed automated purification system
Open this publication in new window or tab >>Human islet separation utilizing a closed automated purification system
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2008 (English)In: Cell Transplantation, ISSN 0963-6897, E-ISSN 1555-3892, Vol. 17, no 12, 1305-1313 p.Article in journal (Refereed) Published
Abstract [en]

A central step within the human islet isolation process is the separation of islets from contaminating exocrine tissue utilizing linear, continuous density gradients manufactured by means of manually controlled standard gradient makers (SGM). The present study was performed to develop a closed, automated purification system (APS) that customizes density gradient profiles aiming to standardize and optimize human islet purification. Digested human pancreata were pooled, split evenly, and incubated in UW solution according to our standard protocol (n = 11). Continuous density gradient centrifugation was performed in parallel in two refrigerated COBE 2991 cell separators loaded with light (1.076 g/ml) and heavy (1.097 g/ml) Ficoll utilizing either an SGM or two computer-controlled pumps connected to Ficoll-containing bags. Quality control included islet equivalent (IE) yield, purity, in vitro function, and islet cytokine expression. Gradient profiles demonstrated that the APS readily customizes linear and nonlinear gradients. In comparison to the SGM, the APS recovered a higher percentage of the expected volume of continuous gradients (90.0 +/- 1.1% vs. 98.2 +/- 2.0%, p < 0.05). Islet yield (120,468 +/- 15,970 vs. 114,570 +/- 15,313 IE, NS) and purity (51.7 +/- 4.8% vs. 54.4 +/- 4.9%, NS) were nearly identical utilizing the SGM or APS. Decreased MCP-1, IL-6, and IL-8 expression indicated that APS-purified islets were possibly exposed to less proinflammatory stress. Compared to standard procedures, similar success and gentle continuous density gradient separation of human islets is feasible utilizing the APS. The APS facilitates the standardization of this complex procedure according to cGMP standards.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-105000 (URN)10.3727/096368908787648100 (DOI)000264577900003 ()19364068 (PubMedID)
Available from: 2009-05-31 Created: 2009-05-31 Last updated: 2011-07-01Bibliographically approved
3. Quantification of the Islet Product: Presentation of a Standardized Current Good Manufacturing Practices Compliant System With Minimal Variability
Open this publication in new window or tab >>Quantification of the Islet Product: Presentation of a Standardized Current Good Manufacturing Practices Compliant System With Minimal Variability
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2011 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 91, no 6, 677-683 p.Article in journal (Refereed) Published
Abstract [en]

Background. Accurate islet quantification has proven difficult to standardize in a good manufacturing practices (GMP) approved manner. Methods. The influence of assessment variables from both manual and computer-assisted digital image analysis (DIA) methods were compared using calibrated, standardized microspheres or islets alone. Additionally, a mixture of microspheres and exocrine tissue was used to evaluate the variability of both the current, internationally recognized, manual method and a novel GMP-friendly purity-and volume-based method (PV) evaluated by DIA in a semiclosed, culture bag system. Results. Computer-assisted DIA recorded known microsphere size distribution and quantities accurately. By using DIA to evaluate islets, the interindividual manually evaluated percent coefficients of variation (CV%; n = 14) were reduced by almost half for both islet equivalents (IEs; 31% vs. 17%, P = 0.002) and purity (20% vs. 13%, P = 0.033). The microsphere pool mixed with exocrine tissue did not differ from expected IE with either method. However, manual IE resulted in a total CV% of 44.3% and a range spanning 258 kIE, whereas PV resulted in CV% of 10.7% and range of 60 k IE. Purity CV% for each method were similar approximating 10.5% and differed from expected by +7% for the manual method and +3% for PV. Conclusion. The variability of standard counting methods for islet samples and clinical quantities of microspheres mixed with exocrine tissue were reduced with DIA. They were reduced even further by use of a semiclosed bag system compared with standard manual counting, thereby facilitating the standardization of islet evaluation according to GMP standards.

Keyword
Islet evaluation, Digital imaging analysis, Purity, Volume, GMP
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-149562 (URN)10.1097/TP.0b013e31820ae48e (DOI)000288115800023 ()21248660 (PubMedID)
Available from: 2011-03-22 Created: 2011-03-21 Last updated: 2011-07-01Bibliographically approved
4. Transplantable functional islet mass – predictive biomarkers of graft function in islet after kidney transplanted patients
Open this publication in new window or tab >>Transplantable functional islet mass – predictive biomarkers of graft function in islet after kidney transplanted patients
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The ability to predict clinical function of a specific islet batch released for clinical transplantation using standardized variables remains an elusive goal. Analysis of donor, islet isolation, quality control and recipient variables was undertaken in 110 islet after kidney (IAK) transplants and correlated to the pre- to 28-day posttransplant change in C-peptide to glucose and creatinine ratio (ΔCP/GCr). Using backward multiple regression the variables positively associated to ΔCP/GCr were islet volume transplanted (p<0.001) and glucose stimulated insulin secretion (SI) (p=0.009). Factors negatively associated to ΔCP/GCr were cold ischemia time (CIT) (p=0.002) and total tissue volume (p=0.009). Donor age, donor body mass index, number of retrieved organs from the donor, preservation solution, islet insulin content, body weight of the recipient of the islets had no influence on transplant function. The transplantable functional islet mass (TFIM), accounting for islet volume transplanted, SI, CIT, and total tissue volume explained 39% of the variance of the clinical outcome in the IAK data set. Therefore, the TFIM provides a straightforward and potent tool to guide the decision to utilize a specific islet preparation for clinical transplantation.

Keyword
Islet transplantation, kidney function, predict outcome, transplantable islet mass
National Category
Surgery
Research subject
Medicine
Identifiers
urn:nbn:se:uu:diva-150243 (URN)
Available from: 2011-03-28 Created: 2011-03-28 Last updated: 2015-06-16

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