Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method.
Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method.
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DOI：
10.1016/j.cryobiol.2020.10.017
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发表时间：
2021-03
期刊：
影响因子：
2.7
通讯作者：
Higgins AZ
中科院分区：
文献类型：
--
作者：
Warner RM;Ampo E;Nelson D;Benson JD;Eroglu A;Higgins AZ
玻璃化状态的冷冻保存对于长期保存可能因结冰而受损的组织和器官具有巨大的潜力。然而，玻璃化这些标本所需的高浓度冷冻保护剂 (CPA) 所带来的毒性仍然是一个障碍。为了应对这一挑战，我们之前开发了一种数学方法，基于毒性成本函数的最小化来设计毒性较小的 CPA 平衡方法。该方法用于设计牛肺动脉内皮细胞 (BPAEC) 与甘油平衡的改进方法。为了充分利用毒性成本函数方法，描述其他 CPA 的毒性动力学至关重要，包括常用于玻璃化的多 CPA 混合物。在这项工作中，我们使用自动液体处理来表征五种最常见的 CPA（甘油、二甲基亚砜 (DMSO)、丙二醇、乙二醇和甲酰胺）及其 BPAEC 的二元和三元混合物的毒性动力学。在此过程中，我们开发了可用于更快、更准确地确定毒性动力学的实验方法。我们的结果强调了一些常见的 CPA 毒性趋势，包括乙二醇的毒性相对较低，以及随着 CPA 浓度的增加，毒性普遍增加。我们的结果还表明了潜在的降低毒性的新方法，包括甘油对甲酰胺的令人惊讶的毒性中和作用。将来，该数据集将作为扩展我们的 CPA 毒性模型的基础，从而能够将毒性成本函数方法应用于包含多个 CPA 的玻璃化解决方案。
Cryopreservation in a vitrified state has vast potential for long-term storage of tissues and organs that may be damaged by ice formation. However, the toxicity imparted by the high concentration of cryoprotectants (CPAs) required to vitrify these specimens remains a hurdle. To address this challenge, we previously developed a mathematical approach to design less toxic CPA equilibration methods based on the minimization of a toxicity cost function. This approach was used to design improved methods for equilibration of bovine pulmonary artery endothelial cells (BPAEC) with glycerol. To fully capitalize on the toxicity cost function approach, it is critical to describe the toxicity kinetics of additional CPAs, including multi-CPA mixtures that are commonly used for vitrification. In this work, we used automated liquid handling to characterize the toxicity kinetics of five of the most common CPAs (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide), along with their binary and ternary mixtures for BPAEC. In doing so, we developed experimental methods that can be used to determine toxicity kinetics more quickly and accurately. Our results highlight some common CPA toxicity trends, including the relatively low toxicity of ethylene glycol and a general increase in toxicity as the CPA concentration increases. Our results also suggest potential new approaches to reduce toxicity, including a surprising toxicity neutralization effect of glycerol on formamide. In the future, this dataset will serve as the basis to expand our CPA toxicity model, enabling application of the toxicity cost function approach to vitrification solutions containing multiple CPAs.
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