Figtree Gemma A, Bubb Kristen J, Tang Owen, Kizana Eddy, Gentile Carmine
Sydney Medical School, The University of Sydney, Sydney, NSW, Australia.
Cells Tissues Organs. 2017;204(3-4):191-198. doi: 10.1159/000477436. Epub 2017 Aug 4.
Spheroid cultures are among the most explored cellular biomaterials used in cardiovascular research, due to their improved integration of biochemical and physiological features of the heart in a defined architectural three-dimensional microenvironment when compared to monolayer cultures. To further explore the potential use of spheroid cultures for research, we engineered a novel in vitro model of the heart with vascularized cardiac spheroids (VCSs), by coculturing cardiac myocytes, endothelial cells, and fibroblasts isolated from dissociated rat neonatal hearts (aged 1-3 days) in hanging drop cultures. To evaluate the validity of VCSs in recapitulating pathophysiological processes typical of the in vivo heart, such as cardiac fibrosis, we then treated VCSs with transforming growth factor beta 1 (TGFβ1), a known profibrotic agent. Our mRNA analysis demonstrated that TGFβ1-treated VCSs present elevated levels of expression of connective tissue growth factor, fibronectin, and TGFβ1 when compared to control cultures. We demonstrated a dramatic increase in collagen deposition following TGFβ1 treatment in VCSs in the PicroSirius Red-stained sections. Doxorubicin, a renowned cardiotoxic and profibrotic agent, triggered apoptosis and disrupted vascular networks in VCSs. Taken together, our findings demonstrate that VCSs are a valid model for the study of the mechanisms involved in cardiac fibrosis, with the potential to be used to investigate novel mechanisms and therapeutics for treating and preventing cardiac fibrosis in vitro.
与单层培养相比，球体培养能够在特定的三维微环境中更好地整合心脏的生化和生理特征，因此是心血管研究中探索最多的细胞生物材料之一。为了进一步探索球体培养在研究中的潜在用途，我们构建了一种新型的心脏体外模型——血管化心脏球体（VCSs），方法是将从新生大鼠心脏（1 - 3日龄）分离的心肌细胞、内皮细胞和成纤维细胞在悬滴培养中共同培养。为了评估VCSs在重现体内心脏典型病理生理过程（如心脏纤维化）方面的有效性，我们随后用已知的促纤维化剂转化生长因子β1（TGFβ1）处理VCSs。我们的mRNA分析表明，与对照培养相比，经TGFβ1处理的VCSs中结缔组织生长因子、纤连蛋白和TGFβ1的表达水平升高。在天狼星红染色切片中，我们证明TGFβ1处理后VCSs中的胶原蛋白沉积显著增加。阿霉素是一种著名的心脏毒性和促纤维化剂，可引发VCSs中的细胞凋亡并破坏血管网络。综上所述，我们的研究结果表明，VCSs是研究心脏纤维化相关机制的有效模型，具有在体外研究治疗和预防心脏纤维化新机制及疗法的潜力。
