The Effect of UW Solution and Its Components on the Collagenase Digestion of Human and Porcine Pancreas

University of Wisconsin (UW) solution is used extensively as a cold storage solution during the procurement and transport of the pancreas prior to islet isolation. However, it has been observed that UW inhibits the collagenase digestion phase of human but not porcine islet isolation, resulting in poor islet yields and islets of poor viability. The aim of this study was, therefore, to confirm this species difference and to determine which components of UW are responsible for the inhibition in the human. In the initial experiment, blocks of human and porcine pancreas (n = 7) were incubated in test tubes containing collagenase at a concentration of 4 mg/mL at 37°C dissolved in 4 mL of either Hanks' solution or UW. Every 5 min the tubes were manually shaken and the degree of tissue dissociation scored on a scale of + and +++. Our results confirm the inhibition of collagenase digestion in the human but not the pig. Using the same methodology, we then investigated the components of UW that were causing the observed inhibition in the human pancreas (n = 7). This time the collagenase was dissolved in individual or combinations of UW components. Using Hank's as a control, the results were then expressed as a median ratio. The components found to be most inhibitory were magnesium, the Na+/K+ ratio, hydroxyethyl starch (HES), and adenosine. Allopurinol in combination with either lactobionate or glutathione was markedly inhibitory (i.e., median ratio 1.8 and 1.9, respectively). The most inhibitory solution tested was a combination of the three components raffinose, glutathione, and lactobionate (median ratio 2.1). This combination was almost as inhibitory as UW itself (median ratio 2.7). These findings are essential for the development of effective cold-storage solutions for the human pancreas that do not inhibit the subsequent collagenase digestion phase of islet isolation.

Download Full-text

Effective Islet Isolation Method with Extremely High Islet Yields from Adult Pigs

Cell Transplantation ◽

10.3727/000000005783982512 ◽

2005 ◽

Vol 14 (10) ◽

pp. 757-762 ◽

Cited By ~ 36

Author(s):

Yukihide Yonekawa ◽

Shinichi Matsumoto ◽

Teru Okitsu ◽

Takashi Arata ◽

Yasuhiro Iwanaga ◽

...

Keyword(s):

Large Scale ◽

New Method ◽

Human Pancreas ◽

Islet Isolation ◽

Isolation Method ◽

Porcine Pancreas ◽

Porcine Islet ◽

Single Donor

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase™ HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 ± 213,486 IE) (mean ± SE) before purification and 800,000 IE (879,815 ± 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 ± 11,532 IE, p < 0.01) and the Ricordi method (317,073 ± 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.

Download Full-text

Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution

Journal of Clinical Medicine ◽

10.3390/jcm8101561 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1561 ◽

Cited By ~ 3

Author(s):

Kazuho Kuwae ◽

Chika Miyagi-Shiohira ◽

Eri Hamada ◽

Yoshihito Tamaki ◽

Kai Nishime ◽

...

Keyword(s):

Stimulation Index ◽

Islet Isolation ◽

Preservation Solution ◽

Porcine Pancreas ◽

Porcine Islet ◽

University Of Wisconsin ◽

Pancreas Preservation ◽

Purification Methods ◽

Pancreas Weight ◽

Wisconsin Solution

Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.

Download Full-text

Preservation of the human pancreas before islet isolation using a two-layer (UW solution???perfluorochemical) cold storage method1

Transplantation Journal ◽

10.1097/00007890-200212270-00031 ◽

2002 ◽

pp. 1809-1811 ◽

Cited By ~ 49

Author(s):

Jonathan R. T. Lakey ◽

Toshiaki Tsujimura ◽

A. M. James Shapiro ◽

Yoshikazu Kuroda

Keyword(s):

Cold Storage ◽

Human Pancreas ◽

Islet Isolation ◽

Uw Solution

Download Full-text

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

The Scientific World JOURNAL ◽

10.1100/tsw.2003.107 ◽

2003 ◽

Vol 3 ◽

pp. 1154-1159 ◽

Cited By ~ 14

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.

Download Full-text

PROLONGED PRESERVATION OF HUMAN PANCREAS BEFORE ISLET ISOLATION WITH THE TWO-LAYER COLD STORAGE METHOD

Transplantation Journal ◽

10.1097/00007890-199905150-00432 ◽

1999 ◽

Vol 67 (9) ◽

pp. S645 ◽

Cited By ~ 1

Author(s):

Y. Tanioka ◽

Y. Suzuki ◽

T. Deai ◽

K. Suzuki ◽

I. Matsumoto ◽

...

Keyword(s):

Cold Storage ◽

Human Pancreas ◽

Islet Isolation ◽

Storage Method

Download Full-text

Notice of forthcoming erratum

The Scientific World JOURNAL ◽

10.1100/tsw.2003.73 ◽

2003 ◽

Vol 3 ◽

pp. 826-826

Keyword(s):

Density Gradient ◽

Islets Of Langerhans ◽

Porcine Pancreas ◽

University Of Wisconsin Solution ◽

Porcine Islet ◽

University Of Wisconsin ◽

Published Paper ◽

Wisconsin Solution

The recently published peer-reviewed protocol article entitled Purification of Islets of Langerhans from Porcine Pancreas by John M. Graham (TheScientificWorldJOURNAL (2002) 2, 1657�1661, ISSN 1537-744X; DOI 10.1100/tsw.2002.847) is being revised. TheScientificWorldJOURNAL will shortly publish the article as an erratum entitled Iodixanol Density Gradient Preparation in University of Wisconsin Solution for Porcine Islet Purification. Links between the published paper and erratum will be made to facilitate cross-referencing.22 August 2003TheScientificWorldJOURNAL

Download Full-text

The effect of UW solution and its components on the collagenase digestion of human and porcine pancreas

Cell Transplantation ◽

10.1016/0963-6897(95)00039-z ◽

1995 ◽

Vol 4 (6) ◽

pp. 615-619 ◽

Cited By ~ 18

Author(s):

H Contractor

Keyword(s):

Porcine Pancreas ◽

Uw Solution ◽

Collagenase Digestion

Download Full-text

A Comparison of Pancreatic Islet Purification using Iodixanol with University of Wisconsin Solution and with Na-Lactobionate and Histidine Solution

Cell Medicine ◽

10.1177/2155179018775071 ◽

2018 ◽

Vol 10 ◽

pp. 215517901877507 ◽

Cited By ~ 1

Author(s):

Yoshiki Nakashima ◽

Chika Miyagi-Shiohira ◽

Nana Ebi ◽

Eri Hamada ◽

Yoshihito Tamaki ◽

...

Keyword(s):

Survival Rate ◽

Pancreatic Islets ◽

Stimulation Index ◽

Islet Isolation ◽

University Of Wisconsin Solution ◽

Porcine Islet ◽

University Of Wisconsin ◽

Purification Methods ◽

Isolation Performance ◽

Wisconsin Solution

Purification of pancreatic islets is an important step in islet isolation for islet transplantation. In this study, to investigate how a solution composed mainly of Na-lactobionate and histidine (HL) influences the purification of islets, iodixanol was added to a purified solution for porcine islet isolation. A solution (IU) made by adding iodixanol to University of Wisconsin solution and a solution (IHL) made by adding iodixanol to HL solution were used to evaluate the islet isolation performance. We noted no significant differences between the two purification methods with regard to the islet yield, survival rate or purity, score, or stimulation index. These results show that IHL solution is as useful as IU solution for islet purification.

Download Full-text