Miyake Yuko, Karanis Panagiotis, Uga Shoji
Department of Medical Technology, Faculty of Health Science, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142, Japan.
Cryobiology. 2004 Feb;48(1):1-7. doi: 10.1016/j.cryobiol.2003.10.004.
Conventional methods for the propagation and preservation of parasites in vivo or in vitro have some limitations, including the need for labor, initial isolation and loss of strains, bacterial, and fungal contamination, and changes in the original biological and metabolic characteristics. All these disadvantages are considerably reduced by cryopreservation. In this study, we examined the effects of various freezing conditions on the survival of several protozoan parasites after cryopreservation. The viability of Entamoeba histolytica was improved by seeding (p < 0.05, chi2 test), while this was not so effective for Trichomonas vaginalis. Of six cryoprotectants examined, dimethyl sulfoxide (Me(2)SO), and glycerol showed the strongest cryoprotective effects. The optimum conditions for using Me(2)SO were a concentration of 10% with no equilibration, and those for glycerol were a concentration of 15% with equilibration for 2h. The optimum cooling rate depended on the parasite species. Trypanosoma brucei gambiense and Leishmania amazonensis were successfully cryopreserved over a wide range of cooling rates, whereas the survival rates of E. histolytica, T. vaginalis, Pentatrichomonas hominis, and Blastocystis hominis were remarkably decreased when frozen at improper rates. Unlike the cooling rate, exposure of the protozoans to a rapid thawing method produced better motility for all parasites.
寄生虫在体内或体外繁殖与保存的传统方法存在一些局限性，包括需要人工操作、初始分离和菌株丢失、细菌和真菌污染以及原始生物学和代谢特征的改变。冷冻保存可大大减少所有这些缺点。在本研究中，我们检测了各种冷冻条件对几种原生动物寄生虫冷冻保存后存活情况的影响。通过接种可提高溶组织内阿米巴的活力（p < 0.05，卡方检验），而这对阴道毛滴虫效果不佳。在所检测的六种冷冻保护剂中，二甲基亚砜（Me(2)SO）和甘油显示出最强的冷冻保护作用。使用Me(2)SO的最佳条件是浓度为10%且无需平衡，而甘油的最佳条件是浓度为15%并平衡2小时。最佳冷却速率取决于寄生虫种类。布氏冈比亚锥虫和亚马逊利什曼原虫在较宽的冷却速率范围内均可成功冷冻保存，而当以不适当的速率冷冻时，溶组织内阿米巴、阴道毛滴虫、人五毛滴虫和人芽囊原虫的存活率显著降低。与冷却速率不同，原生动物采用快速解冻方法可使所有寄生虫的运动能力更好。
