Walcerz D B, Diller K R
MRC Medical Cryobiology Group, University Department of Surgery, Cambridge, U.K.
J Microsc. 1991 Feb;161(Pt 2):297-311. doi: 10.1111/j.1365-2818.1991.tb03091.x.
The rational design of cryopreservation protocols for living tissues demands an understanding of the mechanisms of mass transport between cells and their environment throughout the entire process. We have developed a new microscope stage to enable a specimen to be viewed continuously during a preservation protocol, including the addition and removal of cryoprotective additives and freezing and thawing. The specimen is contained in a sealed chamber having inlet and outlet ports for admitting and collecting perfusate solution, the entire volume of which may be exchanged with a time constant of 1-5 s, depending on the solution viscosity. The temperature of the active area of the stage is regulated by the standard techniques of convection cryomicroscopy over a range in excess of 50 to -100 degrees C. A series of experiments has been performed on this system to measure the osmotic behaviour of rat pancreas islets during the addition and removal of dimethyl sulphoxide at temperatures between 25 and -10 degrees C. The technique involves mounting a single islet onto the low-temperature stage so that it is constrained from lateral movement by a specially sized mesh. Both the system temperature and chemical composition are monitored and controlled simultaneously and independently; as a consequence, virtually any defined cryopreservation protocol may be imposed on the specimen. For making permeability measurements, the bathing medium of the specimen may be changed very rapidly to produce a defined osmotic stress. Alternatively, the specimen may be subcooled to a specific and fixed subzero temperature, at which point ice is nucleated in the extracellular medium, creating a near instantaneous change in composition. The temporal alteration in specimen size is monitored by video microscopy and quantified by computer vision analysis methods. One of several mass transfer models is fitted to the data to estimate the membrane permeability based on the assumption of either transport dominated by the movement of water or simultaneous coupled flows of water and cryoprotective agent.
活组织冷冻保存方案的合理设计需要了解整个过程中细胞与其环境之间的质量传输机制。我们开发了一种新的显微镜载物台，以便在保存方案过程中能够连续观察标本，包括添加和去除冷冻保护添加剂以及冷冻和解冻过程。标本置于一个密封腔室中，该腔室有用于灌注液进出的端口，根据溶液粘度，灌注液的总体积可在1至5秒的时间常数内进行交换。载物台活性区域的温度通过对流低温显微镜的标准技术在超过50至 -100摄氏度的范围内进行调节。已经在该系统上进行了一系列实验，以测量大鼠胰岛在25至 -10摄氏度之间添加和去除二甲基亚砜期间的渗透行为。该技术包括将单个胰岛安装在低温载物台上，使其通过特殊尺寸的网格限制横向移动。系统温度和化学成分同时且独立地进行监测和控制；因此，几乎任何定义的冷冻保存方案都可以施加于标本。为了进行渗透率测量，标本的浴液可以非常迅速地更换以产生确定的渗透应力。或者，标本可以过冷到特定的固定零下温度，此时细胞外介质中会形成冰核，从而导致成分几乎瞬间发生变化。通过视频显微镜监测标本大小的时间变化，并通过计算机视觉分析方法进行量化。基于水的移动主导传输或水和冷冻保护剂同时耦合流动的假设，将几种传质模型之一拟合到数据中以估计膜渗透率。
