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.