Kumar Naresh, Sridharan Divya, Palaniappan Arunkumar, Dougherty Julie A, Czirok Andras, Isai Dona Greta, Mergaye Muhamad, Angelos Mark G, Powell Heather M, Khan Mahmood
Department of Emergency Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, United States.
Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH, United States.
Front Bioeng Biotechnol. 2020 Sep 16;8:567842. doi: 10.3389/fbioe.2020.567842. eCollection 2020.
Recent advances in cardiac tissue engineering have shown that human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a three-dimensional (3D) micro-environment exhibit superior physiological characteristics compared with their two-dimensional (2D) counterparts. These 3D cultured hiPSC-CMs have been used for drug testing as well as cardiac repair applications. However, the fabrication of a cardiac scaffold with optimal biomechanical properties and high biocompatibility remains a challenge. In our study, we fabricated an aligned polycaprolactone (PCL)-Gelatin coaxial nanofiber patch using electrospinning. The structural, chemical, and mechanical properties of the patch were assessed by scanning electron microscopy (SEM), immunocytochemistry (ICC), Fourier-transform infrared spectroscopy (FTIR)-spectroscopy, and tensile testing. hiPSC-CMs were cultured on the aligned coaxial patch for 2 weeks and their viability [lactate dehydrogenase (LDH assay)], morphology (SEM, ICC), and functionality [calcium cycling, multielectrode array (MEA)] were assessed. Furthermore, particle image velocimetry (PIV) and MEA were used to evaluate the cardiotoxicity and physiological functionality of the cells in response to cardiac drugs. Nanofibers patches were comprised of highly aligned core-shell fibers with an average diameter of 578 ± 184 nm. Acellular coaxial patches were significantly stiffer than gelatin alone with an ultimate tensile strength of 0.780 ± 0.098 MPa, but exhibited gelatin-like biocompatibility. Furthermore, hiPSC-CMs cultured on the surface of these aligned coaxial patches (3D cultures) were elongated and rod-shaped with well-organized sarcomeres, as observed by the expression of cardiac troponin-T and α-sarcomeric actinin. Additionally, hiPSC-CMs cultured on these coaxial patches formed a functional syncytium evidenced by the expression of connexin-43 (Cx-43) and synchronous calcium transients. Moreover, MEA analysis showed that the hiPSC-CMs cultured on aligned patches showed an improved response to cardiac drugs like Isoproterenol (ISO), Verapamil (VER), and E4031, compared to the corresponding 2D cultures. Overall, our results demonstrated that an aligned, coaxial 3D cardiac patch can be used for culturing of hiPSC-CMs. These biomimetic cardiac patches could further be used as a potential 3D model for "clinical trials in a dish" and for cardiac repair applications for treating myocardial infarction.
心脏组织工程学的最新进展表明，在三维（3D）微环境中培养的人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）与其二维（2D）对应物相比，具有更优异的生理特性。这些3D培养的hiPSC-CMs已被用于药物测试以及心脏修复应用。然而，制造具有最佳生物力学性能和高生物相容性的心脏支架仍然是一项挑战。在我们的研究中，我们使用静电纺丝技术制备了一种排列整齐的聚己内酯（PCL）-明胶同轴纳米纤维贴片。通过扫描电子显微镜（SEM）、免疫细胞化学（ICC）、傅里叶变换红外光谱（FTIR）和拉伸试验对贴片的结构、化学和力学性能进行了评估。将hiPSC-CMs在排列整齐的同轴贴片上培养2周，并评估其活力[乳酸脱氢酶（LDH测定）]、形态（SEM、ICC）和功能[钙循环、多电极阵列（MEA）]。此外，使用粒子图像测速技术（PIV）和MEA来评估细胞对心脏药物的心脏毒性和生理功能。纳米纤维贴片由高度排列的核壳纤维组成，平均直径为578±184 nm。无细胞同轴贴片明显比单独的明胶更硬，极限拉伸强度为0.780±0.098 MPa，但表现出类似明胶的生物相容性。此外，在这些排列整齐的同轴贴片表面（3D培养）上培养的hiPSC-CMs呈细长的杆状，肌节排列有序，这可通过心肌肌钙蛋白-T和α-肌动蛋白的表达观察到。此外，在这些同轴贴片上培养的hiPSC-CMs形成了功能性合胞体，这可通过连接蛋白-43（Cx-43）的表达和同步钙瞬变得到证明。此外，MEA分析表明，与相应的2D培养相比，在排列整齐的贴片上培养的hiPSC-CMs对异丙肾上腺素（ISO）、维拉帕米（VER）和E4031等心脏药物表现出更好的反应。总体而言，我们的结果表明，排列整齐的同轴3D心脏贴片可用于hiPSC-CMs的培养。这些仿生心脏贴片可进一步用作“培养皿中的临床试验”的潜在3D模型，以及用于治疗心肌梗死的心脏修复应用。
