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Technical challenges in human islet isolation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Olle Korsgren)ORCID iD: 0000-0003-4511-6149#sthash.NKvqlgKU.dpuf
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1119
National Category
Endocrinology and Diabetes
Identifiers
URN: urn:nbn:se:uu:diva-259358ISBN: 978-91-554-9283-0 (print)OAI: oai:DiVA.org:uu-259358DiVA: diva2:843905
Public defence
2015-09-18, Auditorium Minus, Gustavianum, Akademigatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-08-27 Created: 2015-07-31 Last updated: 2015-10-01
List of papers
1. Pathogen Inactivation of Human Serum Facilitates its Clinical Use for Islet Cell Culture and Subsequent Transplantation
Open this publication in new window or tab >>Pathogen Inactivation of Human Serum Facilitates its Clinical Use for Islet Cell Culture and Subsequent Transplantation
2011 (English)In: Cell Transplantation, ISSN 0963-6897, E-ISSN 1555-3892, Vol. 20, no 5, 775-781 p.Article in journal (Refereed) Published
Abstract [en]

Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept (R) technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37 degrees C for 3-4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

Keyword
Cell therapy, Cell transplantation, Cell culture, Serum, Pathogen inactivation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-156689 (URN)10.3727/096368910X539056 (DOI)000292532000015 ()
Available from: 2011-08-08 Created: 2011-08-07 Last updated: 2017-12-08Bibliographically approved
2. Human Islet Isolation Processing Times Shortened By One Hour: Minimized Incubation Time Between Tissue Harvest and Islet Purification
Open this publication in new window or tab >>Human Islet Isolation Processing Times Shortened By One Hour: Minimized Incubation Time Between Tissue Harvest and Islet Purification
Show others...
2013 (English)In: Transplantation, ISSN 0041-1337, E-ISSN 1534-6080, Vol. 96, no 12, E91-E93 p.Article in journal, Letter (Refereed) Published
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-220216 (URN)10.1097/01.tp.0000437562.31212.d5 (DOI)000330439900004 ()24342944 (PubMedID)
Available from: 2014-03-17 Created: 2014-03-12 Last updated: 2017-12-05Bibliographically approved
3. Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation
Open this publication in new window or tab >>Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation
Show others...
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.

National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-254354 (URN)10.1097/TXD.0000000000000528 (DOI)
Available from: 2015-07-30 Created: 2015-06-08 Last updated: 2015-08-28Bibliographically approved
4. Evaluation of the new pancreas preservation solution, I-Let Protect, for clinical islet isolation and transplantation
Open this publication in new window or tab >>Evaluation of the new pancreas preservation solution, I-Let Protect, for clinical islet isolation and transplantation
Show others...
2015 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Background

This prospective study aimed to evaluate polydimethylsiloxane 5 (F6H8S5) preservation of pancreases in a clinical setting compared with standard solutions for 1) cold ischemia-time (CIT) <10 hour and 2) a prolonged CIT > 20 hour.

Methods

Part 1. Procured clinical grade pancreases were shipped in either F6H8S5 or in standard preservation solutions, i.e., University of Wisconsin (UW) or Custodiol. F6H5S5 were pre-oxygenated for at least 15 minutes.

Part 2. Included clinical grade pancreases were procured in UW or Custodiol. Upon arrival at the islet isolation laboratory, duodenum was removed followed by rough trimming while F6H8S5 was oxygenated for 15-20 minutes. Trimmed pancreases were immersed into oxygenated F6H8S5 and stored at 4°C over night followed by subsequent islet isolation.

Results

Pancreas preservation using F6H8S5 proved as effective as UW and Custadiol when used within CIT up to 10 hour, both in terms of isolation outcome and islet functionality. Preservation in F6H8S5 of pancreases with prolonged CIT gave results similar to controls with CIT< 10 hours for both isolated islet functionality and isolation outcome.

Conclusions

This prospective study of clinically obtained pancreases indicate a clear benefit of using F6H8S5 on pancreases with prolonged CIT when compared with other organ preservation solutions. F6H8S5 preserved islet quality and quantity compared with islets isolated from pancreases with CIT of less than 10 hour.

National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-254357 (URN)
Available from: 2015-07-31 Created: 2015-06-08 Last updated: 2015-08-28

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