Dual Dependence of Cryobiogical Properties of Sf21 Cell Membrane on the Temperature and the Concentration of the Cryoprotectant

The Sf21 cell line is extensively used for virus research and producing heterologous recombinant proteins. To develop optimal strategies for minimizing cell injury due to intracellular ice formation and excessive volume shrinkage during cryopreservation, the fundamental transport properties including the osmotic inactive volume (Vb), the hydraulic conductivity (Lp), and the glycerol permeability (Ps) of Sf21 cell membrane at 25, 15, 5 and −2°C were characterized using a micro-perfusion chamber. The effects of temperature on the hydraulic conductivity and the glycerol permeability of Sf21 cell membrane, reflected by the activation energies, were quantitatively investigated. It was found that the hydraulic conductivity decreases along with the increase of the final CPA concentration at a given temperature, and quantitative analysis indicates that the hydraulic conductivity has a significant linear attenuation along with the increase of the concentration of glycerol. Therefore, we incorporate the concentration dependence of the hydraulic conductivity into the classic Arrhenius relationship by replacing the constant reference value of the hydraulic conductivity at the reference temperature with a function that is linearly dependent on the CPA concentration. Consequently, the prediction of the Arrhenius relationship is improved, and the novel Arrhenius relationship could be very important to the development of optimal strategies for cell cryopreservation.Sf21细胞系（Sf21 cell line）被广泛应用于病毒学研究与异源重组蛋白的生产。为开发可最小化低温保存过程中因细胞内冰形成与过度体积收缩所致细胞损伤的最优策略，本研究采用微灌注腔，对Sf21细胞膜在25℃、15℃、5℃及-2℃下的基本转运特性进行了表征，所表征的特性包括渗透非活动体积（osmotic inactive volume, Vb）、水导率（hydraulic conductivity, Lp）以及甘油通透率（glycerol permeability, Ps）。本研究以活化能为量化指标，考察了温度对Sf21细胞膜水导率与甘油通透率的影响。研究发现，在给定温度下，水导率随最终低温保护剂（cryoprotective agent, CPA）浓度的升高而降低；定量分析结果表明，水导率随甘油浓度的升高呈现显著的线性衰减趋势。据此，本研究将水导率的浓度依赖性纳入经典阿伦尼乌斯关系中，将参考温度下水导率的恒定参考值替换为与低温保护剂浓度呈线性相关的函数。由此，阿伦尼乌斯关系的预测精度得到显著提升，该新型阿伦尼乌斯关系对于细胞低温保存最优策略的开发具有重要指导意义。