Collagenase does Not Persist in Human Islets following Isolation

2012 ◽  
Vol 21 (11) ◽  
pp. 2531-2535 ◽  
Author(s):  
Sarah E. Cross ◽  
Stephen J. Hughes ◽  
Anne Clark ◽  
Derek W. R. Gray ◽  
Paul R. V. Johnson
Keyword(s):  
Human Islets ◽  
Isolation Procedure ◽  
Human Islet ◽  
The Other ◽  
Islet Isolation ◽  
Time Points ◽  
Bacterial Collagenase ◽  
University Of Wisconsin ◽  
Collection Phase ◽  
Wisconsin Solution

Optimal human islet isolation requires the delivery of bacterial collagenase to the pancreatic islet–exocrine interface. However, we have previously demonstrated the presence of collagenase within human islets immediately following intraductal collagenase administration. This potentially has significant implications for patient safety. The present study aimed to determine if collagenase becomes internalized into islets during the isolation procedure and if it remains within the islet postisolation. Islet samples were taken at various stages throughout 14 clinical human islet isolations: during digest collection, following University of Wisconsin solution incubation, immediately postisolation, and after 24 h of culture. Samples were embedded in agar, cryosectioned, and then assessed by immunolabeling for collagenase and insulin. Immunoreactivity for collagenase was not observed in isolated islets in any preparation. Collagenase labeling was detected in one sample taken at the digest collection phase in one islet preparation only. No collagenase-specific labeling was seen in islets sampled at any of the other time points in any of the 14 islet preparations. Collagenase that enters islets during intraductal administration is washed out of the islets during the collection phase of the isolation process and thus does not remain in islets after isolation. This observation alleviates some of the important safety concerns that collagenase remains within islet grafts.

Human Islet Isolation Outcomes From Pancreata Preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin Solution

2006 ◽  
Vol 82 (7) ◽  
pp. 983-985 ◽  
Author(s):  
Payam Salehi ◽  
Michael A. Hansen ◽  
Jose G. Avila ◽  
Barbara Barbaro ◽  
Antonio Gangemi ◽  
...  
Keyword(s):  
Human Islet ◽  
Islet Isolation ◽  
University Of Wisconsin Solution ◽  
University Of Wisconsin ◽  
Wisconsin Solution

Excellence of the two-layer method (University of Wisconsin solution/perfluorochemical) in pancreas preservation before islet isolation

Surgery ◽  
1997 ◽  
Vol 122 (2) ◽  
pp. 435-442 ◽  
Author(s):  
Yasuki Tanioka ◽  
David E.R Sutherland ◽  
Yoshikazu Kuroda ◽  
Thomas R Gilmore ◽  
Tor C Asaheim ◽  
...  
Keyword(s):  
Islet Isolation ◽  
University Of Wisconsin Solution ◽  
University Of Wisconsin ◽  
Layer Method ◽  
Pancreas Preservation ◽  
Wisconsin Solution

scholarly journals Research-Focused Isolation of Human Islets From Donors With and Without Diabetes at the Alberta Diabetes Institute IsletCore

Endocrinology ◽  
2015 ◽  
Vol 157 (2) ◽  
pp. 560-569 ◽  
Author(s):  
James Lyon ◽  
Jocelyn E. Manning Fox ◽  
Aliya F. Spigelman ◽  
Ryekjang Kim ◽  
Nancy Smith ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Glycated Hemoglobin ◽  
Organ Procurement ◽  
Human Islets ◽  
Human Islet ◽  
Islet Function ◽  
Small Contribution ◽  
Islet Isolation ◽  
Diabetes Status

Abstract Recent years have seen an increased focus on human islet biology, and exciting findings in the stem cell and genomic arenas highlight the need to define the key features of mature human islets and β-cells. Donor and organ procurement parameters impact human islet yield, although for research purposes islet yield may be secondary in importance to islet function. We examined the feasibility of a research-only human islet isolation, distribution, and biobanking program and whether key criteria such as cold ischemia time (CIT) and metabolic status may be relaxed and still allow successful research-focused isolations, including from donors with type 1 diabetes and type 2 diabetes. Through 142 isolations over approximately 5 years, we confirm that CIT and glycated hemoglobin each have a weak negative impacts on isolation purity and yield, and extending CIT beyond the typical clinical isolation cutoff of 12 hours (to ≥ 18 h) had only a modest impact on islet function. Age and glycated hemoglobin/type 2 diabetes status negatively impacted secretory function; however, these and other biological (sex, body mass index) and procurement/isolation variables (CIT, time in culture) appear to make only a small contribution to the heterogeneity of human islet function. This work demonstrates the feasibility of extending acceptable CIT for research-focused human islet isolation and highlights the biological variation in function of human islets from donors with and without diabetes.

scholarly journals Iodixanol Density Gradient Preparation in University of Wisconsin Solution for Porcine Islet Purification

2003 ◽  
Vol 3 ◽  
pp. 1154-1159 ◽  
Author(s):  
Michael P.M. Van der Burg ◽  
John M. Graham
Keyword(s):  
Adverse Effects ◽  
Human Islet ◽  
Porcine Pancreas ◽  
Isolated Pancreatic Islets ◽  
University Of Wisconsin Solution ◽  
Porcine Islet ◽  
University Of Wisconsin ◽  
Ficoll Gradient ◽  
The University ◽  
Wisconsin Solution

Previously published as Graham, J.M. (2002) Purification of Islets of Langerhans from porcine pancreas. TheScientificWorldJOURNAL 2, 1657–1661. ISSN 1537-744X; DOI 10.1100/tsw.2002.847.Generally, prior to the purification of isolated pancreatic islets, the collagenase-digested tissue is incubated in the University of Wisconsin solution (UWS; ~320 mOsm) for osmotic stabilization to preserve or improve the density differences between islets and acinar fragments. The adverse effects arising from the subsequent pelleting and resuspension of the islets in a second, different (often highly hyperosmotic) purification solution are avoided in the protocol described here; preparation of the purification medium is simply achieved by mixing the UWS preincubated islets with a second UWS containing the inert impermeant iodixanol. Flotation of the islets isolated from juvenile porcine pancreases through this mildly hypertonic (~380 mOsm) gradient of iodixanol-UWS achieves a much higher recovery of islets of an improved viability than the customary method using a Ficoll gradient. The method has been extended to human islet purification.

scholarly journals Comparison of Purification Solutions with Different Osmolality for Porcine Islet Purification

Cell Medicine ◽  
2017 ◽  
Vol 9 (1-2) ◽  
pp. 53-59 ◽  
Author(s):  
Chika Miyagi-Shiohira ◽  
Naoya Kobayashi ◽  
Issei Saitoh ◽  
Masami Watanabe ◽  
Yasufumi Noguchi ◽  
...  
Keyword(s):  
Salt Solution ◽  
Stimulation Index ◽  
Human Islets ◽  
Human Islet ◽  
Low Density ◽  
Islet Isolation ◽  
Continuous Density ◽  
Yield Rate ◽  
Purification Efficiency ◽  
Porcine Islet

The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank's balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.

Cell Preservation in University of Wisconsin Solution during Isolation of Canine Islets of Langerhans

1994 ◽  
Vol 3 (4) ◽  
pp. 315-324 ◽  
Author(s):  
Michael P.M. Van Der Burg ◽  
Onno R. Guicherit ◽  
Marijke Frölich ◽  
Frans A. Prins ◽  
Jan Anthonie Bruijn ◽  
...  
Keyword(s):  
Density Gradient ◽  
Acinar Cells ◽  
Cell Swelling ◽  
Type I ◽  
Islet Isolation ◽  
University Of Wisconsin Solution ◽  
Isolation And Purification ◽  
Density Separation ◽  
University Of Wisconsin ◽  
Wisconsin Solution

Allogeneic islet transplantation in Type I diabetic patients is considerably hampered by the variable outcome of islet isolation and purification. After collagenase digestion of the pancreas, islet isolation is traditionally performed under hypothermic conditions in physiological solutions such as Hanks and RPMI. The University of Wisconsin solution (UWS) has been shown superior for hypothermic preservation of the pancreas. We, therefore, compared the UWS and RPMI for canine islet isolation and subsequent purification in either a conventional hyperosmotic density gradient of dextran in Hanks, or a novel normosmotic density gradient of Percoll in UWS. The isolation solution did not affect islet yield before purification (51% of the native islet mass). Loss of amylase (30%) and swelling of the acinar cells were observed in RPMI. In contrast, no loss of amylase and slight shrinkage of the acinar cells were observed in the UWS. Cell swelling affected the density separation and viability of the cells. Dextran density separation resulted in a 15% purity and 41% recovery of the islets isolated in RPMI, as compared to a 93% purity and 52% recovery of islets isolated in UWS. Percoll density separation improved the purity (99%) and recovery (74%) of islets isolated in UWS. Islets isolated in UWS demonstrated a superior basal and glucose stimulated insulin release during perifusion. Electron microscopy demonstrated a well-preserved islet ultrastructure after isolation in both solutions — except for slightly swollen mitochondria after isolation in RPMI. Autotransplantation of islets in pancreatectomised dogs was successful both after isolation in UWS and RPMI. We conclude that prevention of cell swelling during isolation and purification in the UWS resulted in an improved yield of viable and consistent virtually pure islets. Prevention of cell swelling during islet isolation should facilitate the analysis and control of other factors affecting outcome in man.

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