Kim Haeng-Hoon, Lee Sheong-Chun
Department of Well-being Resources, Sunchon National University, Korea.
Cryo Letters. 2012 Jul-Aug;33(4):271-9.
Although an appropriate cryopreservation protocol is a prerequisite for basic studies and large-scale implementation as well as further cryopreservation studies, the process relies on trial and error. Among the vitrification-based cryopreservation techniques, droplet-vitrification produces high post-cryopreservation recovery. However, the protocol itself cannot solve the problems engaged in plant cryopreservation, prominently due to dehydration with cytotoxic vitrification solutions. This paper proposes a set of treatments to develop droplet-vitrification using a standard procedure associated with additional treatments and alternative vitrification solutions. The proposed standard protocol consists of a progressive preculture with 0.3 M sucrose for 31 h and with 0.7 M for 17 h, loading with vitrification solution C4-35% (17.5 percent glycerol + 17.5 percent sucrose, w/v) for 20 to 40 min, dehydration with vitrification solutions A3-90 percent (37.5 percent glycerol + 15% DMSO + 15 percent EG + 22.5 percent sucrose) for 10 to 30 min or B1-100 percent (PVS3) for 40 to 120 min at room temperature, cooling the samples using aluminum foil strips, rewarming by plunging into pre-heated (40 degree C) unloading solution (0.8 M sucrose) and further unloading for 20 to 60 min, depending on size and permeability of the materials. Using this systematic approach we can identify whether the material is tolerant or sensitive to chemical toxicity and to the osmotic stress of dehydration with vitrification solutions, thus revealing which is the main barrier in solution-based vitrification methods. Based on the sensitivity of samples we can design a droplet-vitrification procedure, i.e. preculture, loading, dehydration with vitrification solutions, cooling and rewarming. Using this approach, the development of appropriate droplet-vitrification protocol is facilitated.
尽管合适的冷冻保存方案是基础研究、大规模实施以及进一步冷冻保存研究的先决条件，但该过程依赖反复试验。在基于玻璃化的冷冻保存技术中，液滴玻璃化法能产生较高的冷冻保存后复苏率。然而，该方案本身无法解决植物冷冻保存中存在的问题，主要是因为细胞毒性玻璃化溶液会导致脱水。本文提出了一套处理方法，通过与额外处理及替代玻璃化溶液相关的标准程序来改进液滴玻璃化法。所提出的标准方案包括：用0.3 M蔗糖进行31小时的逐步预培养，再用0.7 M蔗糖进行17小时的预培养；用玻璃化溶液C4-35%（17.5%甘油 + 17.5%蔗糖，w/v）加载20至40分钟；在室温下用玻璃化溶液A3-90%（37.5%甘油 + 15%二甲基亚砜 + 15%乙二醇 + 22.5%蔗糖）脱水10至30分钟或用B1-100%（PVS3）脱水40至120分钟；使用铝箔条冷却样品；将样品投入预热至40摄氏度的卸载溶液（0.8 M蔗糖）中复温，并根据材料的大小和渗透性进一步卸载20至60分钟。通过这种系统方法，我们可以确定材料对化学毒性以及玻璃化溶液脱水造成的渗透胁迫是耐受还是敏感，从而揭示基于溶液的玻璃化方法中的主要障碍。基于样品的敏感性，我们可以设计液滴玻璃化程序，即预培养、加载、用玻璃化溶液脱水、冷却和复温。使用这种方法，有助于开发合适的液滴玻璃化方案。
