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Open Access Islet Isolation From Juvenile Porcine Pancreas After 24-h Hypothermic Machine Perfusion Preservation

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Abstract:

Pancreas procurement for islet isolation and transplantation is limited by concerns for the detrimental effects of postmortem ischemia. Hypothermic machine perfusion (HMP) preservation technology has had a major impact in circumventing ischemic injury in clinical kidney transplantation and is applied here to the preservation and procurement of viable islets after hypothermic perfusion preservation of porcine pancreata because pigs are now considered the donor species of choice for xenogeneic islet transplantation. Pancreases were surgically removed from young (<6 months) domestic Yorkshire pigs (25‐32 kg), either before or after 30 min of warm ischemia time (WIT), and cannulated for perfusion. Each pancreas was assigned to one of six preservation treatment groups: fresh controls—processed immediately (cold ischemia <1 h) (G1, n = 7); static cold storage—flushed with cold UW-Viaspan and stored in UW-Viaspan at 2‐4°C for 24 h with no prior WIT (G2, n = 9); HMP perfused on a LifePort® machine at 4‐6°C and low pressure (10 mmHg) for 24 h with either KPS1 solution (G3, n = 7) or Unisol-UHK (G4, n = 7). Additional treatment groups to evaluate the effects of prior warm ischemia examined islet isolation after 30 min WIT in situ without (G5, n = 6) or with subsequent 24-h HMP with KPS1 (G6, n = 7). The pancreas was intraductally distended with Liberase PI enzyme and normothermically digested. The isolated islets were purified by a continuous density-gradient centrifugation. Perfusion-induced glandular edema was G3 = 138 ± 19%, G4 = 160 ± 16%, and G6 = 127 ± 22%. Islet yield (IEQ/g of pancreas) varied between the groups: G1 = 1,425 ± 610, G2 = 1,002 ± 262, G3 = 2,242 ± 449 (p < 0.05 vs. G2), G4 = 1,901 ± 420 (p < 0.05 vs. G2), G5 = 1,756 ± 329, and G6 = 1,396 ± 243. Islet stimulation indices were equivalent between the groups and similar to controls (G1). Insulin content (ng/IE) was different between the treatment groups with the highest insulin content in islets harvested from HMP pancreata. Dithizone staining for islets consistently showed more uniform digestion of the perfused organs, with greater separation of the tissue, less entrapped islets, and higher islet yield and purity. The salutary effects of HMP for 24 h were also manifest after 30-min prior warm ischemia. We conclude that 24 h of HMP is well tolerated, leading to moderate edema but no loss of function of the harvested islets. The edema appears to aid in enzymatic digestion, producing a greater yield and purity of islets compared with pancreas subjected to 24 h of static cold storage.

Keywords: Cold storage; Hypothermic perfusion preservation; Juvenile porcine pancreas; Pancreas preservation; Porcine islets; Porcine pancreas preservation

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/096368910X486316

Affiliations: Cell and Tissue Systems, N. Charleston, SC, USA; Organ Recovery Systems, Itasca, IL, USA. mtaylor@celltissuesystems.com

Publication date: May 1, 2010

More about this publication?
  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.
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