Clinic for Horses - Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Germany.
Cryobiology. 2010 Aug;61(1):115-22. doi: 10.1016/j.cryobiol.2010.06.002. Epub 2010 Jun 8.
Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E(Lp), Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezing-induced shift in wavenumber position of the symmetric CH(2) stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H(2)O-bending and -libration combination band, which yielded higher values for both E(Lp) and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition.
细胞膜是细胞冷冻保存过程中冷冻和解冻损伤的主要部位之一。傅里叶变换红外光谱（FTIR）被用于监测马精子冷冻过程中膜相行为和冰形成。在导致细胞脱水的高过冷冰核温度下，膜经历了向高度有序凝胶相的深刻转变。相比之下，低温过冷核温度可能导致细胞内冰形成，使膜脂处于相对水合的流体状态。发现冷冻诱导的膜脱水程度取决于冰核温度，并表现出阿伦尼乌斯行为。甘油的存在并不能防止冷冻诱导的膜相变，但膜脱水发生得更缓慢，且在更宽的温度范围内发生。我们描述了一种从膜相行为数据确定亚零温度下膜水力渗透参数（E（Lp），Lpg）的方法。为此，假设由脂质酰链引起的对称 CH（2）伸缩带的测量到的冷冻诱导的波数位置偏移与细胞脱水成比例。还通过分析 H（2）O 弯曲和 - 摆动组合带来确定膜渗透率参数，与脂质带分析相比，E（Lp）和 Lpg 的值都更高。这些差异可能反映了自由水和膜结合水的传输之间的差异。FTIR 允许在完整细胞中直接评估亚零温度下的膜性质。推导的生物物理膜参数取决于细胞的固有特性以及冷冻扩展剂的组成。
