PDF(Pubmed)
Abstract：
H9c2 myoblasts are a cell line derived from embryonic rat heart tissue and demonstrate the ability to differentiate to cardiac myotubes upon reduction of the serum concentration (from 10% to 1%) and addition of all-trans retinoic acid in the growth medium. H9c2 cells are increasingly being used as an easy-to-culture proxy for some functions of cardiomyocytes. The cryobiology of cardiac cells including H9c2 myoblasts has not been studied as extensively as that of some cell types. Consequently, it is important to characterize the cryobiological response and systematically develop well-optimized cryopreservation protocols for H9c2 cells to have optimal and consistent viability and functionality after thaw for high quality studies with this cell type. In this work, an interrupted slow cooling protocol (graded freezing) was applied to characterize H9c2 response throughout the cooling profile. Important factors that affect the cell response were examined, and final protocols that provided the highest post-thaw viability are reported. One protocol uses the common cryoprotectant dimethyl sulfoxide combined with hydroxyethyl starch, which will be suitable for applications in which the presence of dimethyl sulfoxide is not an issue; and the other protocol uses glycerol as a substitute when there is a desire to avoid dimethyl sulfoxide. Both protocols achieved comparable post-thaw viabilities (higher than 80%) based on SYTO 13/GelRed flow cytometry results. H9c2 cells cryopreserved by either protocol showed ability to differentiate to cardiac myotubes comparable to fresh (unfrozen) H9c2 cells, and their differentiation to cardiac myotubes was confirmed with i) change in cell morphology, ii) expression of cardiac marker troponin I, and iii) increase in mitochondrial mass.
摘要：
H9c2成肌细胞是源自胚胎大鼠心脏组织的细胞系,并且在血清浓度降低(从10%至1%)和在生长培养基中添加全反式视黄酸时,证明分化为心脏肌管的能力。H9c2细胞越来越多地被用作心肌细胞某些功能的易于培养的代表。包括H9c2成肌细胞在内的心肌细胞的冷冻生物学尚未像某些细胞类型那样广泛地研究。因此,重要的是表征冷冻生物学反应，并系统地开发优化的冷冻保存方案，使H9c2细胞在解冻后具有最佳和一致的活力和功能，以进行这种细胞类型的高质量研究。在这项工作中,在整个冷却曲线中应用中断缓慢冷却方案(分级冷冻)来表征H9c2响应。检查了影响细胞反应的重要因素，报告了提供最高解冻后活力的最终方案。一种方案使用普通的冷冻保护剂二甲基亚砜与羟乙基淀粉结合，这将适用于二甲基亚砜的存在不是问题的应用;当需要避免二甲基亚砜时，其他方案使用甘油作为替代品。基于SYTO13/GelRed流式细胞术结果,两种方案实现了相当的解冻后活力(高于80%)。通过任一方案冷冻保存的H9c2细胞显示与新鲜（未冷冻）H9c2细胞相当的分化为心脏肌管的能力，并通过i）细胞形态的变化证实了它们向心肌管的分化，ii)心脏标志物肌钙蛋白I的表达，和iii)线粒体质量的增加。
