Laminin, laminin-entactin and extracellular matrix are equally appropriate adhesive substrates for isolated adult rat cardiomyocyte culture and experimentation

Although techniques for isolating and culturing adult cardiomyocytes were developed four decades ago, it still remains a challenge to isolate high yields of healthy viable cardiomyocytes, to maintain them in culture, and to use them successfully in experiments. This is due to the difficulty in deciding which adhesive substrate and buffer composition to use. Therefore this study aimed to (1) identify a robust experimental buffer to sustain survival of cultured adult rat cardiomyocytes (ARCMs) during control normoxic conditions, and (2) to identify an adhesive substrate that provides optimal cell adherence, not only during normoxia, but especially during simulated ischemia-reperfusion (SIR) experiments. Adhesion and viability of ARCMs were evaluated on laminin, laminin-entactin (LE), and extracellular matrix (ECM) at concentrations between 25–200 ug/ml, in three different normoxic experimental buffers and under SIR conditions. Differences in normoxic buffer composition had no effect on the adherence of ARCMs, but had a significant effect on mitochondrial function and thus cell viability. HEPES buffered PBS supplemented with 10 mM glucose was not sufficient to sustain cell viability unless 2 mM pyruvate was added, yet a cocktail of PBS and M199 provided an even greater viability. Finally, laminin, LE, and ECM retained similar numbers of ARCMs per concentration, but only provided efficient adhesion at concentrations ≥ 100 ug/ml.

尽管成年心肌细胞的分离与培养技术已于四十年前研发成功，但要获取高产量、健康且具有活性的心肌细胞，并在体外培养体系中维持其存活状态，最终成功将其应用于实验研究，仍是一项极具挑战性的工作。这一难题的根源在于，如何筛选适配的黏附底物与缓冲液组分，目前仍缺乏统一的最优方案。因此本研究旨在实现两大研究目标：(1) 筛选出可在常氧对照条件下，维持体外培养的成年大鼠心肌细胞（adult rat cardiomyocytes, ARCMs）存活的稳定实验缓冲液；(2) 筛选出既能在常氧环境下提供良好黏附效果，更可在模拟缺血再灌注（simulated ischemia-reperfusion, SIR）实验中实现最优细胞黏附的黏附底物。本研究在三种不同的常氧实验缓冲液体系及模拟缺血再灌注条件下，针对浓度范围为25~200 μg/ml的层粘连蛋白（laminin）、层粘连蛋白-巢蛋白复合物（laminin-entactin, LE）以及细胞外基质（extracellular matrix, ECM），对成年大鼠心肌细胞的黏附能力与细胞活性进行了系统评估。研究结果表明，常氧缓冲液的组成差异对成年大鼠心肌细胞的黏附能力无显著影响，但会显著影响线粒体功能，进而改变细胞活性。仅添加10 mM葡萄糖的羟乙基哌嗪乙硫磺酸（HEPES）缓冲的磷酸盐缓冲液（phosphate buffered saline, PBS），无法维持细胞活性，除非额外加入2 mM丙酮酸钠；而将PBS与M199培养基混合配制的复合缓冲液，则可进一步提升细胞存活活性。最后，在相同浓度条件下，层粘连蛋白、层粘连蛋白-巢蛋白复合物与细胞外基质所能黏附的成年大鼠心肌细胞数量并无显著差异，但仅当底物浓度≥100 μg/ml时，三者方可实现高效的细胞黏附。