Xu X, Cui Z F, Wilkins R J, Urban J P G
Department of Engineering Science, Oxford University, Parks Road, OX1 3PJ, Oxford, UK.
Cryobiology. 2003 Apr;46(2):161-73. doi: 10.1016/s0011-2240(03)00022-1.
The addition and removal of a cryoprotective agent (CPA) are necessary steps in the cryopreservation of natural or engineered tissue products. However, the introduction and removal of CPAs induces dramatic chemical changes inside tissues and cells and these could cause irreversible damage. This study examined the effect of CPA loading and removal on the intracellular pH of isolated bovine articular chondrocytes using a fluorimetric technique. Chondrocytes that had been isolated from bovine articular cartilage were loaded with the pH-sensitive fluorophore 2('),7(')-bis(carboxyethyl)-5(6)-carboxyfluorescein. After removal of the extracellular fluorophore, the intensity of fluorescence was used to measure the intracellular pH according to a pre-determined calibration curve. Changes of intracellular pH in chondrocytes were measured following their exposure to dimethyl sulfoxide (Me(2)SO) and glycerol at concentrations of 0.6, 0.9, and 1.2M and later to the isotonic or hypertonic solutions that were used to remove the CPA. The effect of the presence of NaCl on the intracellular pH during CPA removal was also examined. The temperature was maintained at 37 degrees C. Trypan blue exclusion was used to quantify cell membrane integrity after the addition and removal of CPA. It was found that when the cells were exposed to CPA, the intracellular pH decreased quickly and recovered gradually later. During CPA removal, the intracellular pH rose following exposure to isotonic Hepes-buffered medium, but the opposite was observed if the Hepes buffer solution contained no NaCl; this was ascribed to the role of NaCl in cell membrane transport. It was noted that the change in intracellular pH correlated with the cell volume excursion, which could be estimated by the Kedem-Katchalsky model, and was linked to cell survival. The resulting alteration of pH inside the cells might contribute to cell damage and loss of function after cryopreservation.
添加和去除冷冻保护剂（CPA）是天然或工程组织产品冷冻保存过程中的必要步骤。然而，CPA的引入和去除会在组织和细胞内部引发显著的化学变化，这些变化可能会导致不可逆转的损伤。本研究使用荧光技术检测了CPA加载和去除对分离的牛关节软骨细胞细胞内pH值的影响。从牛关节软骨中分离出的软骨细胞加载了对pH敏感的荧光团2('),7(')-双（羧乙基）-5(6)-羧基荧光素。去除细胞外荧光团后，根据预先确定的校准曲线，利用荧光强度测量细胞内pH值。在软骨细胞暴露于浓度为0.6、0.9和1.2M的二甲基亚砜（Me(2)SO）和甘油后，以及随后暴露于用于去除CPA的等渗或高渗溶液后，测量细胞内pH值的变化。还检测了在去除CPA过程中NaCl的存在对细胞内pH值的影响。温度维持在37℃。在添加和去除CPA后，使用台盼蓝排斥法对细胞膜完整性进行定量分析。结果发现，当细胞暴露于CPA时，细胞内pH值迅速下降，随后逐渐恢复。在去除CPA过程中，暴露于等渗的Hepes缓冲培养基后细胞内pH值升高，但如果Hepes缓冲溶液不含NaCl，则观察到相反的情况；这归因于NaCl在细胞膜转运中的作用。值得注意的是，细胞内pH值的变化与细胞体积变化相关，细胞体积变化可通过Kedem-Katchalsky模型估算，并且与细胞存活有关。细胞内pH值的最终改变可能导致冷冻保存后细胞损伤和功能丧失。
