The heart possesses a complex three-dimensional (3D) laminar myofiber organization; however, because engineering physiologically relevant 3D tissues remains a technical challenge, the effects of cardiomyocyte alignment on excitation-contraction coupling, shortening and force development have not been systematically studied. Cellular shape and orientations in 3D can be controlled by engineering scaffold microstructures and encapsulating cells near these geometric cues. Here, we show that a novel method of cell encapsulation in 3D methacrylated gelatin (GelMA) scaffolds patterned via Microscale Continuous Optical Printing (μCOP) can rapidly micropattern neonatal mouse ventricular cardiomyocytes (NMVCMs) in photocrosslinkable hydrogels. Encapsulated cardiomyocytes preferentially align with the engineered microarchitecture and can display morphology and myofibril alignment phenotypic of myocardium in vivo. Utilizing the μCOP system, an asymmetric, multi-material, cantilever-based scaffold was directly printed, so that the force produced by the microtissue was transmitted onto a single deformable pillar. Aligned 3D encapsulated NMVCM scaffolds produced nearly 2 times the force compared to aligned 2D seeded samples. To further highlight the flexibility of μCOP, NMVCMs were encapsulated in several patterns to compare the effects of varying degrees of alignment on tissue displacement and synchronicity. Well aligned myofiber cultured patterns generated 4–10 times the contractile force of less anisotropically patterned constructs. Finally, normalized fluo-4 fluorescence of NMVCM-encapsulated structures showed characteristic calcium transient waveforms that increased in magnitude and rate of decline during treatment with 100 nM isoproterenol. This novel instrumented 3D cardiac microtissue serves as a physiologically relevant in vitro model system with great potential for use in cardiac disease modeling and drug screening.

心脏具有复杂的三维 (3D) 层状肌纤维组织；然而，由于工程生理相关 3D 组织仍然是一个技术挑战，心肌细胞排列对兴奋收缩耦合、缩短和力发展的影响尚未得到系统研究。3D 中的细胞形状和方向可以通过工程支架微结构和封装这些几何线索附近的细胞来控制。在这里，我们表明，一种新颖的在3D细胞封装的方法的甲基丙烯酸酯化的明胶（GelMA）的支架图案经由微型连续光学打印 (μCOP) 可以在可光交联的水凝胶中快速微图案化新生小鼠心室心肌细胞 (NMVCM)。封装的心肌细胞优先与工程微结构对齐，可以在体内显示心肌的形态和肌原纤维对齐表型. 利用 μCOP 系统，直接打印非对称、多材料、基于悬臂的支架，从而将微组织产生的力传递到单个可变形支柱上。与对齐的 2D 种子样品相比，对齐的 3D 封装 NMVCM 支架产生的力几乎是其 2 倍。为了进一步突出 μCOP 的灵活性，NMVCMs 被封装在几种模式中，以比较不同程度的对齐对组织位移和同步性的影响。排列良好的肌纤维培养模式产生的收缩力是非各向异性模式结构的收缩力的 4-10 倍。最后，NMVCM 封装结构的归一化荧光 4 荧光显示出特征性钙瞬态波形，在用 100 nM 异丙肾上腺素治疗期间，其幅度和下降速率增加。在心脏疾病建模和药物筛选方面具有巨大潜力的体外模型系统。