Department of Surgery, University of Alberta, Edmonton, Canada.
Cryobiology. 2010 Aug;61(1):123-7. doi: 10.1016/j.cryobiol.2010.05.008. Epub 2010 Jun 15.
Long-term biologic storage of articular cartilage has proven elusive due to cellular degradation over time or acute damage during attempts at cryopreservation. Vitrification is one option that may result in successful cryopreservation but difficulty with cryoprotective agent (CPA) toxicity at high concentrations of a single cryoprotectant has hindered development of successful protocols. This study was designed to determine the vitrifiability and glass stability of solutions containing combinations of commonly used CPAs and to document CPA interactions that occur. One hundred and sixty-four multi-CPA combination solutions of 6-9 M were evaluated for vitrifiability and glass stability using direct visualization after immersion in liquid nitrogen for 30 min and upon warming. Binary and ordinal logistic regression analysis was used to statistically analyze each CPA for its ability to vitrify and its effect on glass stability in multi-component CPA solutions. Propylene glycol had the greatest incremental contribution to vitrification while formamide had the least contribution. A threshold was established whereby the ability of a solution to vitrify could be determined by calculation. Glass stability was not as clearly defined due to variability in the results; however, contributions of interactions between CPAs to the glass stability of solutions were determined. This study provided values that predict if a solution will vitrify. Furthermore, the glass stability of solutions containing multiple CPAs do not behave as linear additions of binary solutions and interactions between CPAs have a significant effect on the glass stability of these solutions. These variables should be considered when designing vitrification solutions.
由于细胞降解或在冷冻保存过程中的急性损伤，关节软骨的长期生物学储存一直难以实现。玻璃化是一种可能导致成功冷冻保存的选择，但高浓度单一冷冻保护剂的冷冻保护剂（CPA）毒性的困难阻碍了成功方案的发展。本研究旨在确定含有常用 CPA 的组合溶液的玻璃化能力和玻璃稳定性，并记录发生的 CPA 相互作用。使用浸入液氮 30 分钟后的直接可视化方法和升温后，评估了 164 种 6-9 M 的多-CPA 组合溶液的玻璃化能力和玻璃稳定性。使用二元和有序逻辑回归分析，对每个 CPA 使其能够玻璃化的能力及其在多成分 CPA 溶液中对玻璃稳定性的影响进行了统计分析。丙二醇对玻璃化的贡献最大，而甲酰胺的贡献最小。建立了一个阈值，通过计算可以确定溶液玻璃化的能力。由于结果的可变性，玻璃稳定性没有得到明确的定义；然而，确定了 CPAs 之间相互作用对溶液玻璃稳定性的贡献。本研究提供了可以预测溶液是否可以玻璃化的数值。此外，含有多种 CPAs 的溶液的玻璃稳定性不能作为二元溶液的线性添加，并且 CPAs 之间的相互作用对这些溶液的玻璃稳定性有显著影响。在设计玻璃化溶液时应考虑这些变量。
