Improved quality and yield of islets isolated from human pancreata using a two-step digestion method

Takashi Kenmochi, Masaaki Miyamoto, Satoshi Une, Yoshihiko Nakagawa, Stefan Moldovan, R. Antonio Navarro, Pierre Y. Benhamou, F. Charles Brunicardi, Yoko Mullen

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

A new approach, involving a two-step digestion process and Los Angeles preservation solution 1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty- six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 ± 36,244 islets equivalent to 150 μm (IEQ) and 6,233 ± 681 IEQ/g of pancreas with 83 ± 2.5% purity, whereas the old method yielded a total of 195,587 ± 25,242 IEQ and 3,763 ± 5,509 IEQ/g with 69.2 ± 4.7% purity. Isolated islets in group I maintained a good three- dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.

Original languageEnglish
Pages (from-to)184-190
Number of pages7
JournalPancreas
Volume20
Issue number2
DOIs
Publication statusPublished - 01-03-2000
Externally publishedYes

Fingerprint

Pancreas
Digestion
Los Angeles
Collagenases
Cold Ischemia
Pancreas Transplantation
Islets of Langerhans Transplantation
Ficoll
Streptozocin
Nude Mice
Tail
Transplantation
Insulin
Weights and Measures
Glucose
Enzymes

All Science Journal Classification (ASJC) codes

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism
  • Hepatology
  • Endocrinology

Cite this

Kenmochi, T., Miyamoto, M., Une, S., Nakagawa, Y., Moldovan, S., Navarro, R. A., ... Mullen, Y. (2000). Improved quality and yield of islets isolated from human pancreata using a two-step digestion method. Pancreas, 20(2), 184-190. https://doi.org/10.1097/00006676-200003000-00012
Kenmochi, Takashi ; Miyamoto, Masaaki ; Une, Satoshi ; Nakagawa, Yoshihiko ; Moldovan, Stefan ; Navarro, R. Antonio ; Benhamou, Pierre Y. ; Brunicardi, F. Charles ; Mullen, Yoko. / Improved quality and yield of islets isolated from human pancreata using a two-step digestion method. In: Pancreas. 2000 ; Vol. 20, No. 2. pp. 184-190.
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abstract = "A new approach, involving a two-step digestion process and Los Angeles preservation solution 1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty- six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5{\%} (43 of 46 cases) in group 1, and 56.5{\%} (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 ± 36,244 islets equivalent to 150 μm (IEQ) and 6,233 ± 681 IEQ/g of pancreas with 83 ± 2.5{\%} purity, whereas the old method yielded a total of 195,587 ± 25,242 IEQ and 3,763 ± 5,509 IEQ/g with 69.2 ± 4.7{\%} purity. Isolated islets in group I maintained a good three- dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.",
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Kenmochi, T, Miyamoto, M, Une, S, Nakagawa, Y, Moldovan, S, Navarro, RA, Benhamou, PY, Brunicardi, FC & Mullen, Y 2000, 'Improved quality and yield of islets isolated from human pancreata using a two-step digestion method', Pancreas, vol. 20, no. 2, pp. 184-190. https://doi.org/10.1097/00006676-200003000-00012

Improved quality and yield of islets isolated from human pancreata using a two-step digestion method. / Kenmochi, Takashi; Miyamoto, Masaaki; Une, Satoshi; Nakagawa, Yoshihiko; Moldovan, Stefan; Navarro, R. Antonio; Benhamou, Pierre Y.; Brunicardi, F. Charles; Mullen, Yoko.

In: Pancreas, Vol. 20, No. 2, 01.03.2000, p. 184-190.

Research output: Contribution to journalArticle

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T1 - Improved quality and yield of islets isolated from human pancreata using a two-step digestion method

AU - Kenmochi, Takashi

AU - Miyamoto, Masaaki

AU - Une, Satoshi

AU - Nakagawa, Yoshihiko

AU - Moldovan, Stefan

AU - Navarro, R. Antonio

AU - Benhamou, Pierre Y.

AU - Brunicardi, F. Charles

AU - Mullen, Yoko

PY - 2000/3/1

Y1 - 2000/3/1

N2 - A new approach, involving a two-step digestion process and Los Angeles preservation solution 1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty- six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 ± 36,244 islets equivalent to 150 μm (IEQ) and 6,233 ± 681 IEQ/g of pancreas with 83 ± 2.5% purity, whereas the old method yielded a total of 195,587 ± 25,242 IEQ and 3,763 ± 5,509 IEQ/g with 69.2 ± 4.7% purity. Isolated islets in group I maintained a good three- dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.

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