Department of Anesthesiology, Vanderbilt University Medical Center.
Department of Anesthesiology, Vanderbilt University Medical Center; Anesthesiology, TVHS VA Medical Center; Department of Pharmacology, Vanderbilt University;
J Vis Exp. 2021 Oct 13(176). doi: 10.3791/62913.
Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells (ECs) can protect cardiomyocytes (CMs) from reperfusion injury through cell-cell interactions. Co-cultures can help investigate the role of cell-cell interactions. A mixed co-culture is the simplest approach but is limited as isolated treatments and downstream analyses of single cell types are not feasible. To investigate whether ECs can dose-dependently attenuate CM cell damage and whether this protection can be further optimized by varying the contact distance between the two cell lines, we used Mouse Primary Coronary Artery Endothelial Cells and Adult Mouse Cardiomyocytes to test three types of cell culture inserts which varied in their inter-cell layer distance at 0.5, 1.0, and 2.0 mm, respectively. In CMs-only, cellular injury as assessed by lactate dehydrogenase (LDH) release increased significantly during hypoxia and further upon reoxygenation when the distance was 2.0 mm compared to 0.5 and 1.0 mm. When ECs and CMs were in nearly direct contact (0.5 mm), there was only a mild attenuation of the reoxygenation injury of CMs following hypoxia. This attenuation was significantly increased when the spatial distance was 1.0 mm. With 2.0 mm distance, ECs attenuated CM injury during both hypoxia and hypoxia/reoxygenation, indicating that sufficient culture distancing is necessary for ECs to crosstalk with CMs, so that secreted signal molecules can circulate and fully stimulate protective pathways. Our findings suggest, for the first time, that optimizing the EC/CM co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of ECs in CM-protection against simulated ischemia/reperfusion injury. The goal of this report is to provide a step-by-step approach for investigators to use this important model to their advantage.
缺血性心脏病是全球范围内导致死亡和残疾的主要原因。再灌注会导致除缺血以外的额外损伤。内皮细胞 (ECs) 可以通过细胞间相互作用保护心肌细胞 (CMs) 免受再灌注损伤。共培养可以帮助研究细胞间相互作用的作用。混合共培养是最简单的方法，但受到限制，因为无法对单一细胞类型进行孤立处理和下游分析。为了研究 ECs 是否可以剂量依赖性地减轻 CM 细胞损伤，以及通过改变两种细胞系之间的接触距离是否可以进一步优化这种保护作用，我们使用小鼠原代冠状动脉内皮细胞和成年小鼠心肌细胞来测试三种类型的细胞培养插入物，它们在细胞间层距离上分别为 0.5、1.0 和 2.0 毫米。在仅 CMs 中，用乳酸脱氢酶 (LDH) 释放评估细胞损伤，当距离为 2.0 毫米时，与 0.5 和 1.0 毫米相比，缺氧期间和再氧合后细胞损伤显著增加。当 ECs 和 CMs 几乎直接接触 (0.5 毫米) 时，缺氧后 CMs 的再氧合损伤仅轻度减轻。当空间距离为 1.0 毫米时，这种衰减显著增加。当距离为 2.0 毫米时，ECs 在缺氧和缺氧/再氧合期间减轻了 CM 损伤，这表明需要足够的培养间距以使 ECs 与 CMs 进行细胞间通讯，以便循环并充分刺激保护途径的信号分子。我们的研究结果首次表明，优化 EC/CM 共培养的空间环境对于提供一个有利的体外模型来测试 ECs 在保护 CM 免受模拟缺血/再灌注损伤中的作用是必要的。本报告的目的是为研究人员提供一个分步方法，以便他们利用这一重要模型发挥优势。
