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Osmotic and Cryoprotectant Permeation Characteristics of Islet Cells Isolated From the Newborn Pig Pancreas

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Abstract:



The development of effective protocols for the low-temperature banking of pancreatic islets is an important step in islet transplantation for the treatment of type I diabetes mellitus. We have been exploring the use of islets from the newborn pig as an alternative source of tissue for transplantation. Current cryopreservation protocols are empirically derived, but may be optimized by modeling osmotic responses during the cryopreservation process. This study determined the osmotic and cryoprotectant permeability parameters of cells isolated from the pancreas of newborn pigs. Key parameters are: the osmotically inactive fraction of cell volume, hydraulic conductivity, the permeability coefficients of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) at varying temperatures, and the activation energies of these transport processes. Newborn pig islets were dispersed into single cells and kinetic and equilibrium cell volumes were recorded during osmotic excursions using an electronic particle counter interfaced to a computer. Data were fitted to theoretical descriptions of the osmotic responses of cells, based on the Kedem-Katchalsky approach. The hydraulic conductivity (Lp) in the absence of cryoprotectant was calculated as 0.050 ± 0.005, 0.071 ± 0.006, and 0.300 ± 0.016 μm/min/atm at 4°C, 10°C, and 22°C, respectively (mean ± SEM, n = 7, 6, or 9). These values give an activation energy value of 16.69 kcal/mol when put into an Arrhenius plot. The solute permeability (Ps) values for 1 M DMSO were 0.89 ± 0.12, 1.86 ± 0.28, and 5.33 ± 0.26 μm/min at 4°C, 10°C, and 22°C, respectively (n = 11, 8, or 10) giving an activation energy of 15.98 kcal/mol. The Lp values for cells exposed to 1 M DMSO were 0.071 ± 0.006, 0.084 ± 0.008, and 0.185 ± 0.014 μm/min/atm at 4°C, 10°C, and 22°C, respectively. The activation energy for these values was 8.95 kcal/mol. The Ps values for 2 M DMSO were 1.11 ± 0.13, 1.74 ± 0.19, and 7.68 ± 0.12 μm/min for the same temperatures, with a calculated activation energy of 17.89 kcal/mol. The Lp values in the presence of 2 M DMSO were 0.070 ± 0.006, 0.085 ± 0.008, and 0.192 ± 0.009 μm/min/atm at 4°C, 10°C, and 22°C, respectively, with an activation energy of 9.40 kcal/mol. Solutions of 1 M EG gave Ps values of 1.01 ± 0.13, 1.45 ± 0.25, and 4.90 ± 0.48 μm/min at the three test temperatures. The resulting activation energy was 14.60 kcal/mol. The corresponding Lp values were 0.071 ± 0.007, 0.068 ± 0.006, and 0.219 ± 0.012 μm/min/atm with an activation energy of 10.96 kcal/mol. The solute permeabilities in the presence of 2 M EG for newborn pig islet cells were 1.03 ± 0.15, 1.42 ± 0.23, and 5.56 ± 0.22 μm/min; the activation energy was 15.70. The Lp values for cells in the presence of 2 M EG were 0.068 ± 0.008, 0.071 ± 0.006, and 0.225 ± 0.010 μm/min/atm; the activation energy for these values was 11.49 kcal/mol. These key cryobiological parameters permit the mathematical modeling of osmotic responses of intact islets during the cryopreservation process, which may lead to further improvements in the low temperature storage of islets from newborn pigs.

Keywords: Key words: Islet cell; Cryopreservation; Basic cry

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/000000001783986323

Affiliations: 1: *Department of Surgery, University of Alberta, Edmonton, Canada T6G 2N8 2: †Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada T6G 2N8 3: ‡Surgical-Medical Research Institute, University of Alberta, Edmonton, Canada T6G 2N8 4: §Barbara Davis Center for Childhood Diabetes, Denver, CO

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/ct249
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