Osmotic and cryoprotectant permeation characteristics of islet cells isolated from the newborn pig pancreas

The development of effective protocols for the low-temperature banking of p ancreatic islets is an important step in islet transplantation for the trea tment of type I diabetes mellitus. We have been exploring the use of islets from the newborn pig as an alternative source of tissue for transplantatio n. Current cryopreservation protocols are empirically derived, but may be o ptimized by modeling osmotic responses during the cryopreservation process. This study determined the osmotic and cryoprotectant permeability paramete rs 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 gly col (EG) at varying temperatures, and the activation energies of these tran sport processes. Newborn pig islets were dispersed into single cells and ki netic and equilibrium cell volumes were recorded during osmotic excursions using an electronic particle counter interfaced to a computer. Data were fi tted to theoretical descriptions of the osmotic responses of cells, based o n the Kedem-Katchalsky approach. The hydraulic conductivity (L,) in the abs ence of cryoprotectant was calculated as 0.050 +/- 0.005, 0.071 +/- 0.006, and 0.300 +/- 0.016 mum/min/atm at 4 degreesC, 10 degreesC, and 22 degreesC , respectively (mean +/- SEM, n = 7, 6, or 9). These values give an activat ion energy value of 16.69 kcal/mol when put into an Arrhenius plot. The sol ute permeability (P-s) values for 1 M DMSO were 0.89 +/- 0.12, 1.86 +/- 0.2 8, and 5.33 +/- 0.26 mum/min at 4 degreesC, 10 degreesC, and 22 degreesC, r espectively (n = 11, 8, or 10) giving an activation energy of 15.98 kcal/mo l. The L-p values for cells exposed to 1 M DMSO were 0.071 +/- 0.006, 0.084 +/- 0.008, and 0.185 +/- 0.014 mum/min/atm at 4 degreesC, 10 degreesC, and 22 degreesC, respectively. The activation energy for these values was 8.95 kcal/mol. The P-s values for 2 M DMSO were 1.11 +/- 0.13, 1.74 +/- 0.19, a nd 7.68 +/- 0.12 mum/min for the same temperatures, with a calculated activ ation energy of 17.89 kcal/mol. The L-p values in the presence of 2 M DMSO were 0.070 +/- 0.006, 0.085 +/- 0.008, and 0.192 +/- 0.009 mum/min/atm at 4 degreesC, 10 degreesC, and 22 degreesC, respectively, with an activation e nergy of 9.40 kcal/mol. Solutions of 1 M EG gave P-s values of 1.01 +/- 0.1 3, 1.45 +/- 0.25, and 4.90 +/- 0.48 mum/min at the three test temperatures. The resulting activation energy was 14.60 kcal/mol. The corresponding L-p values were 0.071 +/- 0.007, 0.068 +/- 0.006, and 0.219 +/- 0.012 mum/min/a tm with an activation energy of 10.96 kcal/mol. The solute permeabilities i n 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 mum/min; the activation energy was 15.70. Th e L-p values for cells in the presence of 2 M EG were 0.068 +/- 0.008, 0.07 1 +/- 0.006, and 0.225 +/- 0.010 mum/min/atm; the activation energy for the se values was 11.49 kcal/mol. These key cryobiological parameters permit th e mathematical modeling of osmotic responses of intact islets during the cr yopreservation process, which may lead to further improvements in the low t emperature storage of islets from newborn pigs.