Liu C, Benson C T, Gao D, Haag B W, McGann L E, Critser J K
Cryobiology Research Institute, Methodist Hospital of Indiana, Inc., Indianapolis 46202, USA.
Cryobiology. 1995 Oct;32(5):493-502. doi: 10.1006/cryo.1995.1049.
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.
随着临床胰岛移植的迅速发展，对活胰岛冷冻保存的需求日益增加。基础低温生物学需要确定几个低温生物物理参数，以预测最佳冷冻保存程序。这些参数包括水渗透率或水力传导率（Lp）及其活化能（Ea）、细胞质膜对一种或多种冷冻保护剂的渗透率（Ps）及其Ea、细胞体积的非渗透活性部分（Vb）以及细胞内冰晶形成温度。对于胰岛细胞，这些参数以前尚未见报道。在本研究中，测定了分离的单个金黄仓鼠胰岛细胞的Lp、其Ea和Vb。还测量了相应外分泌细胞的Lp和Vb参数。当通过玻意耳-范特霍夫关系进行检查时（线性回归，两种细胞类型的r均为0.99），在实验范围内胰岛细胞和外分泌细胞似乎都是理想的渗透计。这些图的外推得出胰岛细胞的Vb值为0.40，胰腺外分泌细胞的Vb值为0.45。为了确定Lp，使用电子粒子计数器测量了在不等渗条件下（范围从145 mOsm/kg至1.35 Osm/kg）细胞体积随时间的动力学变化（dv/dt）。通过将实验数据拟合到一个描述渗透水跨质膜移动的微分方程，以最小二乘法曲线拟合来生成Lp值。对于胰腺胰岛细胞，在22℃时Lp测定为0.25±0.03微米/分钟/大气压（平均值±标准差，n = 14），在37℃、8℃和0℃时分别为0.54±0.07（n = 10）、0.06±0.008（n = 9）和0.01±0.001（n = 9）。基于四个不同温度下的平均Lp值，从阿伦尼乌斯图的斜率计算出Lp的Ea。在0至37℃之间Ea为16.21千卡/摩尔。基于这些值，预测冷冻保存胰腺胰岛细胞的最佳冷却速率约为0.5℃/分钟。单个外分泌细胞在22℃时的Lp测定为3.73±1.75微米/分钟/大气压（n = 13），约为相应胰岛细胞Lp值的10倍。
