Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, China.
Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, China.
Cryobiology. 2014 Apr;68(2):294-302. doi: 10.1016/j.cryobiol.2014.02.011. Epub 2014 Feb 28.
The objective of this study is to determine the cryobiological characteristics of human embryonic kidney (HEK293T) cells. The cell membrane hydraulic conductivity (L(pg)) and the activation energy of water transport (E(Lp)) were determined in the absence/presence of cryoprotectant agent (CPA), while the nucleation rate kinetic and thermodynamic parameters (Ωo(SCN) and κo(SCN)) were determined in the absence of CPA. Since dehydration and intracellular ice formation (IIF) are two factors that may cause damage to cells during the freezing process, systematical freezing experiments were carried out at different cooling rates (5, 10, 15, 20, 30, and 60°C/min) under the commercial available cryomicroscopy (FDCS 196, Linkham, Waterfield, UK) to further explore the cryoinjury mechanism for HEK293T cells. By simultaneously fitting the water transport equation to the experimentally measured volumetric shrinkage data at 5, 10, and 15°C/min, the "combined best fit" membrane permeability parameters for HEK293T cells in both phosphate buffer saline (PBS) and CPA media (0.75M Me2SO in PBS) are determined. They are L(pg)=2.85×10(-14)m/s/Pa (0.17μm/min/atm), E(Lp)=142.91kJ/mol (34.13kcal/mol) (R(2)=0.990), and L(pg)[cpa]=2.73±0.44×10(-14)m/s/Pa (0.16±0.03μm/min/atm), E(Lp)[cpa]=152.52±27.69kJ/mol (36.42±6.61kcal/mol) (R(2)=0.993), respectively. An optimal cooling rate B(opt) (the highest cooling rate without IIF) was determined to be 14.24°C/min in the absence of CPA. Additionally, the ice nucleation parameters (Ωo(SCN) and κo(SCN)) were averaged to be 1.31±0.11×10(8)m(-2)s(-1) and 7.67±2.55×10(9)K(5) for the cooling rates 20, 30, and 60°C/min.
本研究旨在确定人胚肾（HEK293T）细胞的 cryobiological 特征。在没有/存在 cryoprotectant 剂（CPA）的情况下，确定细胞膜水力传导率（L(pg)）和水传输的激活能（E(Lp)），而在没有 CPA 的情况下，确定成核速率动力学和热力学参数（Ωo(SCN)和 κo(SCN)）。由于脱水和细胞内冰形成（IIF）是冷冻过程中可能导致细胞损伤的两个因素，因此在商业可用的 cryomicroscopy（FDCS 196，Linkham，Waterfield，UK）下，以不同的冷却速率（5、10、15、20、30 和 60°C/min）进行系统的冷冻实验，以进一步探讨 HEK293T 细胞的 cryoinjury 机制。通过同时将水传输方程拟合到在 5、10 和 15°C/min 下测量的体积收缩数据，确定了磷酸盐缓冲盐水（PBS）和 CPA 介质（PBS 中的 0.75M Me2SO）中 HEK293T 细胞的“组合最佳拟合”膜渗透参数。它们是 L(pg)=2.85×10(-14)m/s/Pa（0.17μm/min/atm），E(Lp)=142.91kJ/mol（34.13kcal/mol）（R(2)=0.990），以及 L(pg)[cpa]=2.73±0.44×10(-14)m/s/Pa（0.16±0.03μm/min/atm），E(Lp)[cpa]=152.52±27.69kJ/mol（36.42±6.61kcal/mol）（R(2)=0.993）。在没有 CPA 的情况下，确定了最佳冷却速率 B(opt)（没有 IIF 的最高冷却速率）为 14.24°C/min。此外，将冰成核参数（Ωo(SCN)和 κo(SCN)）平均为 1.31±0.11×10(8)m(-2)s(-1)和 7.67±2.55×10(9)K(5)，用于 20、30 和 60°C/min 的冷却速率。
