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Islet Cryopreservation Using Intracellular Preservation Solutions

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Abstract:



Cryopreservation of islets adds great flexibility to clinical islet transplant programs. Methods of islet cryopreservation have traditionally utilized permeating cryoprotectants contained within isotonic solutions without specifically addressing issues of ionic balances, buffering capacity, or oxygen free radicals that occur during hypothermic stresses. These factors may become significant issues during low-temperature storage and during the freezing and thawing process. Since its development in the early 1980s, the University of Wisconsin (UW) organ preservation solution has become the standard vascular flush and preservation solution. Recently, Hypothermosol preservation solution (HTS) was developed as a hypothermic blood substitute. The unique characteristics and composition of these preservation solutions may be important when developing solutions specific for the cryopreservation of cells and tissues. It was the aim of this study to evaluate these two hypothermic preservation solutions as the media used in cryopreservation of islets. Groups of canine islets [5000 islet equivalents (IE)/group] were cryopreserved using the standard protocol of stepwise addition of dimethyl sulfoxide (DMSO) to 2 M, controlled nucleation, slow cooling (0.25°C/min), and rapid thawing (200°C/min). The cryopreservation solutions were made with 1) UW solution, 2) HTS solution, or 3) Medium 199 solution with 10% fetal calf serum (FCS). Additional control groups included islets cryopreserved using 4) HTS, 5) UW solution, and 6) Medium 199 alone, without DMSO. Recovery of islets immediately following thawing was equivalent between the groups with the exception of the islets cryopreserved without DMSO (groups 4–6, p < 0.05). After 48 h of postcryopreservation tissue culture, islet recovery was highest in the groups frozen with UW and HTS (mean ± SEM) (79.8 ± 1.9% and 82.5 ± 1.5%, p < 0.05 vs. group 3, 69.1 ± 3.3%, p < 0.05, ANOVA). Less than 15% of the islets were recovered when they were cryopreserved without the cryoprotectant DMSO (groups 4–6). Functional viability was assessed by measuring the glucose-stimulated insulin secretion during static incubation after 48-h culture. The stimulation indexes were 4.6 ± 1.0, 4.2 ± 0.8, 3.6 ± 1.2, 0.6 ± 0.5, and 0.4 ± 0.2 for islets in groups 1–5, respectively. This study demonstrates that postcryopreservation survival can be improved using intracellular-based preservation solutions, including UW or HTS, in conjunction with DMSO.

Keywords: Key words: Cryopreservation; Pancreatic islets

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/000000001783986369

Affiliations: 1: †Department of Surgery, University of Alberta, Edmonton, Alberta, Canada 2: ‡Department of Medicine, University of Alberta, Edmonton, Alberta, Canada 3: *Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta, Canada 4: ¶Allegheny University of the Health Sciences, Pittsburgh, PA

Publication date: January 1, 2001

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  • 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.
cog/ct/2001/00000010/00000007/ct250
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