Wusteman Monica C, Simmonds Joanne, Vaughan David, Pegg David E
Medical Cryobiology Unit, Biology Department, University of York, York YO10 5YW, UK.
Cryobiology. 2008 Feb;56(1):62-71. doi: 10.1016/j.cryobiol.2007.10.177. Epub 2007 Nov 17.
A previous study had suggested the use of a mixture of propanediol and trehalose for the preservation of tissues by vitrification. In this paper, we describe experiments in which stepwise procedures were developed for adding these cryoprotectants to high final concentrations in two rabbit tissues-carotid artery and cornea. The tissue concentration of the additives was measured at the end of each step so that the temperature of the next step could be chosen to reduce toxicity but avoid freezing. This process was arrested when a concentration had been reached that should permit vitrification if the tissues were cooled rapidly to -175 degrees C. They were stored at that temperature; warmed rapidly by conduction; the cryoprotectants removed by stepwise dilution; and appropriate active functions measured. These were contraction and relaxation for arteries and endothelial integrity and ability to control stromal swelling for the corneas. In control experiments the exposure and functional assays were carried out without vitrification. It was shown that the tissue concentration of propanediol was 33%w/w in artery and 30% in cornea. These permitted cooling to -175 degrees C without freezing but devitrification occurred during the warming of the arteries, though not of the corneas, despite the lower tissue concentration reached in the cornea. The function of the vitrified arteries was severely reduced but the endothelium of the corneas was substantially intact although we were unable to demonstrate any ability to control stromal swelling during normothermic perfusion. It appears that concentrations of cryoprotectants sufficient to prevent freezing in these tissues during cooling were well tolerated so long as appropriate stepwise means of addition and removal were used. Devitrification during warming remained a major problem with arteries, but not with corneas. We suggest that the composition of the aqueous phase in the tissue with respect to components other than the vitrifying agents may be crucial here and that the search for agents that will suppress devitrification is an important avenue for further study.
先前的一项研究曾建议使用丙二醇和海藻糖的混合物通过玻璃化来保存组织。在本文中，我们描述了一些实验，其中开发了逐步程序，以便在两种兔组织（颈动脉和角膜）中将这些冷冻保护剂添加到高终浓度。在每个步骤结束时测量添加剂的组织浓度，以便选择下一步的温度以降低毒性但避免冷冻。当达到如果将组织快速冷却至-175℃应允许玻璃化的浓度时，该过程停止。将它们储存在该温度下；通过传导快速升温；通过逐步稀释去除冷冻保护剂；并测量适当的活性功能。对于动脉，这些功能是收缩和舒张；对于角膜，这些功能是内皮完整性和控制基质肿胀的能力。在对照实验中，在没有玻璃化的情况下进行暴露和功能测定。结果表明，丙二醇在动脉中的组织浓度为33%w/w，在角膜中为30%。这些浓度允许冷却至-175℃而不冻结，但在动脉升温过程中发生了脱玻璃化，尽管角膜中达到的组织浓度较低，但角膜未发生脱玻璃化。玻璃化动脉的功能严重降低，但角膜内皮基本完整，尽管我们无法证明在常温灌注期间有任何控制基质肿胀的能力。似乎只要使用适当的逐步添加和去除方法，足以在冷却期间防止这些组织冻结的冷冻保护剂浓度是可以耐受的。动脉升温过程中的脱玻璃化仍然是一个主要问题，但角膜不是。我们认为，组织中水相相对于玻璃化剂以外的成分的组成在此可能至关重要，并且寻找能够抑制脱玻璃化的试剂是进一步研究的重要途径。
