The determination of membrane permeability coefficients of canine pancreatic islet cells and their application to islet cryopreservation

Sufficient numbers of pancreatic islets for successful allotransplantation can be achieved by storing and then pooling islets from several donors. Optimal MHC matching and infectious disease screening also require long-term storage of islets, and cryopreservation is currently the only practical approach. Cryopreservation protocols may be optimized by modeling the changes in cell volume and the associated damage incurred during cryoprotectant addition and dilution and during cooling and warming. The objective of the present work was to determine the following biophysical parameters of canine islet cells: the osmotically inactive cell volume (V-b), hydraulic conductivity (L-p), cryoprotectant permeability coefficient (P-s), and the reflection coefficient sigma. A determination of these parameters allows the simulation of cell responses using computer models. Islets were isolated by collagenase digestion and Euro-Ficoll purification. After 24 h culture, islets were dissociated into single cells using trypsin and 2 mM EGTA. The kinetic change in cell volume as a function of time after exposure to 2 M dimethyl sulfoxide (Me2SO) was measured using an electronic particle counter at 22, 5, and -3 degrees C. At -11 degrees C, cells were preloaded with 1 M Me2SO and exposed to 4 M Me2SO to prevent the formation of ice in the working solution. Kedem-Karchalsky theory was used to describe the cell volume change kinetics, and a three-parameter curve fitting was performed using the Marquardt-Levenberg method to determine L-p, P-s, and sigma values. The L-p was determined to be 0.19 +/- 0.05, 0.037 +/- 0.005, 0.020 +/- 0.003, and 0.013 +/- 0.005 mu m.min(-1).atm(-1) (mean +/- SD) at 22, 5, -3, and -11 degrees C, respectively. The P-s values were 1.05 +/- 0.50, 0.15 +/- 0.04, 0.096 +/- 0.028, and 0.067 +/- 0.029 x 10(-3) cm.min(-1) at 22, 5, -3, and -11 degrees C, respectively. The sigma values were 0.81 +/- 0.16, 0.91 +/- 0.09, 0.80 +/- 0.21, and 0.98 +/- 0.04 at 22, 5, -3, and -11 degrees C, respectively. The temperature dependence or activation energy of L-p and P-s was calculated, using the Arrhenius equation, to be 12.7 and 13.5 kcal.mol(-1), respectively. These permeability parameters were used to calculate cell water loss and the likelihood of lethal intracellular freezing during cooling, as well as both water flux and solute concentration gradients across the cell membrane during warming. (C) 1997 Academic Press.