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Long-term cryopreservation of islets of Langerhans would be advantageous to a clinical islet transplantation program. Fundamental cryobiology utilizes knowledge of basic biophysical characteristics to increase the understanding of the preservation process and possibly increase survival rate. In this study several of these previously unreported characteristics have been determined for individual islet cells isolated from Golden hamster islets. Using an electronic particle counting device and a temperature control apparatus, dynamic volumetric response of individual islet cells to anisosmotic challenges of 1.5 M dimethyl sulfoxide (DMSO) and 1.5 M ethylene glycol (EG) were recorded at four temperatures (8, 22, 28, and 37 degreesC). The resulting curves were fitted using Kedem and Katchalsky equations which describe water flux and cryoprotectant agent (CPA) flux based on hydraulic conductivity (Lp), CPA permeability (Ps), and reflection coefficient (final sigma) for the membrane. For Golden hamster islet cells, Lp, Ps, and final sigma for DMSO at 22 degreesC were found to be 0.23 +/- 0.06 microm/min/atm, 0.79 +/- 0.32 x 10(-3) cm/min, and 0.55 +/- 0.37 (n = 11) (mean +/- SD), respectively. For EG at 22 degreesC, Lp equaled 0.23 +/- 0.06 microm/min/atm, Ps equaled 0.63 +/- 0.20 x 10(-3) cm/min, and final sigma was 0.75 +/- 0.17 (n = 9). Arrhenius plots (ln Lp or ln Ps versus 1/temperature (K)) were created by adding the data from the other three temperatures and the resulting linear regression yielded correlation coefficients (r) of 0.99 for all four plots (Lp and Ps for both CPAs). Activation energies (Ea) of Lp and Ps were calculated from the slopes of the regressions. The values for DMSO were found to be 12.43 and 18.34 kcal/mol for Lp and Ps (four temperatures, total n = 52), respectively. For EG, Ea of Lp was 11.69 kcal/mol and Ea of Ps was 20.35 kcal/mol (four temperatures, total n = 58)., (Copyright 1998 Academic Press.)

Coupled with the rapid development of clinical pancreatic islet transplantation, there is an increasing requirement for cryopreservation of viable islets. Fundamental cryobiology requires determination of several cryobiophysical parameters to predict optimal cryopreservation procedures. These include water permeability or hydraulic conductivity (Lp) and its activation energy (Ea), the permeability of the cell plasma membrane to a cryoprotectant(s) (Ps) and its Ea, the osmotically inactive fraction of cell volume (Vb), and the intracellular ice formation temperature. For islet cells, these parameters have not previously been reported. In the present studies, the Lp, its Ea, and Vb were determined for isolated individual golden hamster pancreatic islet cells. The Lp and Vb parameters were also measured for corresponding exocrine cells. Both islet and the exocrine cells appeared to be ideal osmometers over the experimental range when examined by the Boyle Van't-Hoff relationship (linear regression, r = 0.99 for both types of cells). Extrapolation of these plots generated Vb values of 0.40 for the islet cells and 0.45 for the pancreatic exocrine cells. To determine the Lp, kinetic changes of cell volume over time (dv/dt) in response to anisoosmotic conditions (ranging from 145 mOsm/kg to 1.35 Osm/kg) were measured using an electronic particle counter. The experimental data were fitted to generate the Lp values by least-squares curve fitting to a differential equation describing osmotic water movement across the plasma membrane. For pancreatic islet cells, the Lp was determined to be 0.25 +/- 0.03 microns/min/atm (mean +/- SD, n = 14) at 22 degrees C, 0.54 +/- 0.07 (n = 10), 0.06 +/- 0.008 (n = 9), and 0.01 +/- 0.001 (n = 9) at 37, 8 and 0 degrees C, respectively. The Ea for Lp was calculated from the slope of the Arrhenius plot based upon the mean Lp values at the four different temperatures. The Ea was 16.21 Kcal/mol between 0 and 37 degrees C. Based upon these values, an optimal cooling rate for cryopreserving pancreatic islet cells is predicted to be approximately 0.5 degrees C min. The Lp for the individual exocrine cells was determined to be 3.73 +/- 1.75 microns/min/atm (n = 13) at 22 degrees C, which was approximately 10 times the Lp value of the corresponding islet cells.

Oocytes provide an excellent model cell type for the exploration of the hypothesis that there is a genetic influence to inter-animal variability in plasma membrane water permeability (Lp) and its activation energy (Ea). Although variability among oocytes pooled across animals has been previously published, variability of oocyte Lp and its Ea among individual animals has yet to be determined. Using a microdiffusion chamber, individual oocytes from five Golden hamsters and six ICR mice were exposed to a 900-mOsm NaCl solution at 37, 22, or 3 degrees C. The resulting change in cell volume over time (dv/dt) was analyzed and hydraulic conductivity (Lp) estimated. The Arrhenius plots (lnLp vs (1/K) x 1000) of the oocytes from each animal were determined and the Ea's were found not to be different among individual Golden hamsters (P > 0.05) with a mean of 7.96 +/- 0.96 Kcal/mol (mean +/- SD). However, the Ea's of the oocytes among individual ICR mice were shown to be significantly different (P < 0.05), with a mean of 11.38 +/- 2.2 Kcal/mol (mean +/- SD). Furthermore, an analysis of the within-mouse and among-mouse variability of Lp was undertaken. The results demonstrated that there was significant variability among ICR mouse oocyte Lp values at all temperatures; however, there was no significant variability within animals. In summary, these data support the hypothesis that significant variability exists in the Lp of ova among mice of the outbred ICR strain. In contrast, the data from the inbred Golden hamster oocyte donors do not show significant variability.