Cell Damage due to Osmotic Stress and Microscale Mass Transfer relative to Cryopreservation

在本研究中，我们通过实验研究了高渗电解质溶液在非冷冻条件下对细胞的损伤和死亡，并探讨了细胞冷冻损伤的潜在原因。其次，提出并建立了细胞损伤与死亡的反应动力学数学模型。1)将人前列腺癌细胞系PC-3细胞从生理盐水等渗溶液中暴露于高渗电解质溶液（NaCl浓度C_m，暴露时间τ）中，在恒温T℃条件下恢复到等渗溶液中。然后在25℃生理盐水中测定细胞膜完整性，测定细胞活力。T在4 ~ 25℃范围内设3个值，C_m在1.8 ~ 4.0M范围内设4个值，τ在2 ~ 170min范围内设17个值。2)从实验结果中得到细胞的存活曲线与细胞死亡的概率分布和概率密度函数τ的函数关系，细胞死亡的特征，更多的暴露时间等，以阐明它们对τ， C_m和T的依赖关系。浓度越高、温度越高，活性降低越明显。当细胞活力为0.5时，暴露时间τ_<0.5>为细胞活力下降的特征暴露时间时，细胞活力与无因次暴露时间τ/τ_<0.5>的分布相似，与浓度和温度无关。这意味着细胞损伤和死亡的过程是动态的和相似的，这表明其机制也可能是相似的。4)在细胞活力实验结果的基础上，建立了细胞损伤与死亡过程的两步损伤动力学模型。这里考虑了细胞的三种状态：旺盛、受损但存活（中等状态）和死亡。提出并研究了四种模型。通过反问题分析确定了模型中的反应速率常数，并确定了四种模型中的最佳模型。用该模型的解析解成功地模拟了细胞活力。得到了细胞死亡的概率分布和概率密度函数，以及反应动力学性质。反应动力学常数随C_m和t的增大而增大

In this study, cell damage and death due to the hypertonic electrolyte solution under the unfrozen condition were investigated experimentally in relation to the potential causes of freezing damage of cells. Next, a mathematical model with a form of reaction kinetics was proposed and developed for the cell damage and death.1) The cells from a human prostatic adenocarcinoma cell line (PC-3) were exposed from an isotonic solution (physiological saline) to hypertonic electrolyte solution (NaCl concentration C_m and exposure time τ) back to the isotonic solution under a constant temperature, T℃. Then, the cell membrane integrity was assessed in physiological saline at 25℃ to measure the cell viability. T was set at three values between 4 and 25℃, C_m at four values between 1.8 and 4.0M, and τ at seventeen values between 2 and 170min.2) From the experimental results, survival curve of cell as a function of τ, probability distribution and probability density function of cell death, characteri … More stic exposure times of cell death, etc. were obtained to clarify the dependency of them on τ, C_m, and T.3) The cell viability decreased with increasing exposure time. The decrease in viability was steeper at a higher concentration and higher temperature. When a characteristic exposure time for the decrease in cell viability was defined as exposure time τ_<0.5> at 0.5 in cell viability, the viability versus nondimensional exposure time τ/τ_<0.5> had a similar distribution independently of concentration and temperature. This means that the cell damage and death proceed kinetically and similarly, suggesting that the mechanism may be also similar.4) The cell damage and death process was formulated with a kinetic model of two-step damage on the basis of experimental results of the cell viability. Here, three states of cells : vigorous, damaged but living (medium state), and dead were considered. Four kinds of model were proposed and investigated. A reaction rate constant in the model was determined by the inverse problem analysis and the best model of four was determined. The cell viability was simulated successfully by the analytical solution from the present model. Also, the probability distribution and probability density function of cell death, and reaction kinetic properties were obtained. The reaction kinetic constant increased with an increase in C_m and T. Less