Division of Orthopaedic Surgery, Department of Surgery, University of Alberta, Edmonton, AB, Canada T6G 2B7.
Cryobiology. 2012 Jun;64(3):185-91. doi: 10.1016/j.cryobiol.2012.01.006. Epub 2012 Jan 18.
Vitrification is a method of cryopreservation by which cells and tissues can be preserved at low temperatures using cryoprotective agents (CPAs) at high concentrations (typically ≥6.0 M) to limit the harmful effects of ice crystals that can form during cooling processes. However, at these concentrations CPAs are significantly cytotoxic and an understanding of their toxicity characteristics and interactions is important. Therefore, single-CPA and multiple-CPA solutions were evaluated for their direct and indirect toxicities on chondrocytes.
Chondrocytes were isolated from human articular cartilage samples and exposed to various single-CPA and multiple-CPA solutions of five common CPAs (dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), glycerol (Gy) and formamide (Fm)) at both 6.0 and 8.1 M concentrations at 0 °C for 30 min. Chondrocyte survival was determined using a fluorescent cell membrane integrity assay. The data obtained was statistically analyzed and regression coefficients were used to represent the indirect toxicity effect which a specific combination of CPAs exerted on the final solution's toxicity.
Multiple-CPA solutions were significantly less toxic than single-CPA solutions (P<0.01). The indirect toxicity effects between CPAs were quantifiable using regression analysis. Cell survival rates of approximately 40% were obtained with the four-CPA combination solution DMSO-EG-Gy-Fm. In the multiple-CPA combinations, PG demonstrated the greatest degree of toxicity and its presence within a combination solution negated any benefits of using multiple lower concentration CPAs.
Multiple-CPA solutions are less cytotoxic than single-CPA solutions of the same total concentration. PG was the most toxic CPA when used in combinations. The highest chondrocyte survival rates were obtained with the 6.0 M DMSO-EG-Gy-Fm combination solution.
玻璃化是一种通过使用高浓度（通常≥6.0M）的细胞保护剂（CPAs）来保存细胞和组织的低温保存方法，以限制冰晶在冷却过程中形成所带来的有害影响。然而，在这些浓度下，CPAs 具有明显的细胞毒性，因此了解其毒性特征和相互作用非常重要。因此，评估了单-CPA 和多-CPA 溶液对软骨细胞的直接和间接毒性。
从人关节软骨样本中分离软骨细胞，并在 0°C 下将其暴露于五种常见 CPAs（二甲亚砜（DMSO）、乙二醇（EG）、丙二醇（PG）、甘油（Gy）和甲酰胺（Fm））的各种单-CPA 和多-CPA 溶液中，浓度分别为 6.0 和 8.1M，持续 30 分钟。通过荧光细胞膜完整性测定法确定软骨细胞存活率。对获得的数据进行统计学分析，并使用回归系数表示特定 CPAs 组合对最终溶液毒性的间接毒性效应。
多-CPA 溶液的毒性明显低于单-CPA 溶液（P<0.01）。可以使用回归分析量化 CPAs 之间的间接毒性效应。DMSO-EG-Gy-Fm 四-CPA 组合溶液的细胞存活率约为 40%。在多-CPA 组合中，PG 的毒性最大，其存在于组合溶液中否定了使用多种低浓度 CPAs 的任何好处。
多-CPA 溶液比相同总浓度的单-CPA 溶液的细胞毒性更小。PG 是组合使用时毒性最大的 CPA。DMSO-EG-Gy-Fm 6.0M 组合溶液获得的软骨细胞存活率最高。
