心脏球体作为体外生物工程心脏组织用于研究人类心脏病理生理学。
Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology.
机构信息
University of Newcastle; University of Sydney; Kolling Institute of Medical Research, Royal North Shore Hospital; University of Technology, Sydney.
University of Sydney; Kolling Institute of Medical Research, Royal North Shore Hospital; University of Technology, Sydney;
出版信息
J Vis Exp. 2021 Jan 23(167). doi: 10.3791/61962.
Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 µM genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.
尽管心脏组织工程学取得了多项进展，但仍有一个主要挑战需要克服，即生成包含多个层次复杂性的完全功能性血管网络，为生物工程心脏组织提供氧气和营养。我们的实验室开发了一种称为“心脏球体”或“CS”的人类心脏三维体外模型。该模型具有典型的人类心脏的生化、生理和药理学特征，是通过共培养其三种主要细胞类型（如心肌细胞、内皮细胞和成纤维细胞）产生的。人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs 或 iCMs）与人类心脏成纤维细胞（HCFs）和人类冠状动脉内皮细胞（HCAECs）在悬滴培养板中以近似于体内的比例共培养三到四天。用针对心肌肌钙蛋白 T、CD31 和波形蛋白的抗体对 CSs 进行共聚焦分析，这些抗体分别是心肌细胞、内皮细胞和成纤维细胞的标志物，结果表明 CSs 具有复杂的内皮细胞网络，类似于人类心脏中发现的天然网络。通过对这些共聚焦图像的 3D 渲染分析可以证实这一点。CSs 还具有典型的人类心脏的细胞外基质（ECM）蛋白，如 IV 型胶原、层粘连蛋白和纤维连接蛋白。最后，CSs 表现出收缩活性，可测量为与仅含有 iCMs 的 CSs 相比更接近人类心脏典型的合胞收缩性。当用心脏毒性抗癌药物（如阿霉素（DOX）治疗白血病、淋巴瘤和乳腺癌）处理时，10 μM 的 DOX 处理 CSs 的活力明显降低。内皮型一氧化氮合酶的基因和化学抑制是 DOX 在 HCFs 和 HCAECs 中的下游靶点，这降低了其在 CSs 中的毒性。鉴于这些独特的特征，CSs 目前被用作研究心脏生化、病理生理学和药理学的体外模型。
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