Fedorow C, McGann L E, Korbutt G S, Rayat G R, Rajotte R V, Lakey J R
Department of Surgery, University of Alberta, Edmonton, Canada.
Cell Transplant. 2001;10(7):651-9.
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 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees 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 microm/min at 4 degrees C, 10 degrees C, and 22 degrees 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 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees 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 microm/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 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees 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 microm/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 microm/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 microm/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 microm/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.
开发有效的低温保存胰岛方案是胰岛移植治疗I型糖尿病的重要一步。我们一直在探索将新生猪的胰岛作为移植组织的替代来源。目前的冷冻保存方案是根据经验得出的，但可以通过对冷冻保存过程中的渗透反应进行建模来优化。本研究确定了从新生猪胰腺分离的细胞的渗透和冷冻保护剂渗透参数。关键参数包括：细胞体积的非渗透活性部分、水力传导率、不同温度下二甲基亚砜（DMSO）和乙二醇（EG）的渗透系数，以及这些转运过程的活化能。将新生猪胰岛分散成单个细胞，并使用与计算机相连的电子粒子计数器在渗透变化过程中记录动力学和平衡细胞体积。基于 Kedem-Katchalsky 方法，将数据拟合到细胞渗透反应的理论描述中。在不存在冷冻保护剂的情况下，4℃、10℃和22℃时的水力传导率（Lp）分别计算为0.050±0.005、0.071±0.006和0.300±0.016μm/min/atm（平均值±标准误，n = 7、6或9）。将这些值绘制在阿累尼乌斯图中时，得到的活化能值为16.69 kcal/mol。1 M DMSO的溶质渗透率（Ps）值在4℃、10℃和22℃时分别为0.89±0.12、1.86±0.28和5.33±0.26μm/min（n = 11、8或10），活化能为15.98 kcal/mol。暴露于1 M DMSO的细胞的Lp值在4℃、10℃和22℃时分别为0.071±0.006、0.084±0.008和0.185±0.014μm/min/atm。这些值的活化能为8.95 kcal/mol。2 M DMSO在相同温度下的Ps值为1.11±0.13、1.74±0.19和7.68±0.12μm/min，计算得到的活化能为17.89 kcal/mol。在2 M DMSO存在下的Lp值在4℃、10℃和22℃时分别为0.070±0.006、0.085±0.008和0.192±0.009μm/min/atm，活化能为9.40 kcal/mol。1 M EG溶液在三个测试温度下的Ps值为1.01±0.13、1.45±0.25和4.90±0.48μm/min。得到的活化能为14.60 kcal/mol。相应的Lp值为0.071±0.007、0.068±0.006和0.219±0.012μm/min/atm，活化能为10.96 kcal/mol。2 M EG存在下新生猪胰岛细胞的溶质渗透率为1.03±0.15、1.42±0.23和5.56±0.22μm/min；活化能为15.70。在2 M EG存在下细胞的Lp值为0.068±0.008、0.071±0.006和0.225±0.010μm/min/atm；这些值的活化能为11.49 kcal/mol。这些关键的低温生物学参数允许对完整胰岛在冷冻保存过程中的渗透反应进行数学建模，这可能会进一步改善新生猪胰岛的低温保存。
