2D and 3D-Organized Cardiac Cells Shows Differences in Cellular Morphology, Adhesion Junctions, Presence of Myofibrils and Protein Expression

Cardiac cells are organized in vivo in a complex tridimensional structural organization that is crucial for heart function. While in vitro studies can reveal details about cardiac cell biology, usually cells are grown on simplified two-dimensional (2D) environments. To address these differences, we established a cardiac cell culture composed of both 2D and three-dimensional (3D)-organized cells. Our results shows significant differences between the two culture contexts in relation to the overall morphology of the cells, contraction ability, proliferation rate, presence of intercellular adhesion structures, organization of myofibrils, mitochondria morphology, endoplasmic reticulum contents, cytoskeletal filaments and extracellular matrix distribution, and expression of markers of cardiac differentiation. Cardiac cells grown in 2D-context displayed a flattened and well spread shape, were mostly isolated and their cytoplasm was filled with a large network of microfilaments and microtubules. In contrast, 3D-cells were smaller in size, were always in close contact with each other with several cellular junctions, and displayed a less conspicuous cytoskeletal network. 3D-cells had more mitochondria and myofibrils and these cells contract spontaneously more often than 2D-cells. On the other hand, endoplasmic reticulum membranes were present in higher amounts in 2D-cells when compared to 3D-cells. The expression of desmin, cadherin and alpha-actinin was higher in 3D-aggregates compared to 2D-spread cells. These findings indicate that the tridimensional environment in which the cardiac cells are grown influence several aspects of cardiac differentiation, including cell adhesion, cell shape, myofibril assembly, mitochondria contents and protein expression. We suggest that the use of this cardiac culture model, with 2D and 3D-context cells, could be useful for studies on the effects of different drugs, or growth factors, giving valuable information on the biological response of cells grown in different spatial organizations.体内的心脏细胞以复杂的三维结构组织形式排布，这对心脏功能的维持至关重要。尽管体外研究能够揭示心脏细胞生物学的诸多细节，但常规实验中细胞通常被培养于简化的二维（2D）环境中。为了弥补这一差异，我们构建了同时包含二维（2D）与三维（3D）结构化细胞的心脏细胞培养模型。本研究结果显示，两种培养环境下的细胞在多项特征上存在显著差异，涵盖细胞整体形态、收缩能力、增殖速率、细胞间黏附结构的存在情况、肌原纤维组织形式、线粒体形态、内质网含量、细胞骨架丝与细胞外基质分布，以及心脏分化标志物的表达水平。在二维环境中培养的心脏细胞呈现扁平舒展的形态，大多呈孤立状态，其细胞质内充满了密集的微丝与微管网络。与之相反，三维培养的细胞体积更小，始终彼此紧密接触并形成多种细胞连接，且细胞骨架网络的辨识度较低。三维培养细胞拥有更多的线粒体与肌原纤维，且其自发收缩频率高于二维培养细胞。另一方面，与三维培养细胞相比，二维培养细胞的内质网膜含量更高。结蛋白（desmin）、钙粘蛋白（cadherin）与α-辅肌动蛋白（alpha-actinin）的表达水平在三维细胞聚集体中显著高于二维铺展细胞。上述研究结果表明，心脏细胞所处的三维培养环境会影响心脏分化的多个方面，包括细胞黏附、细胞形态、肌原纤维组装、线粒体含量以及蛋白质表达。我们认为，这种同时包含二维与三维培养细胞的心脏模型，可用于探究不同药物或生长因子的作用效果，为揭示不同空间排布下细胞的生物学应答提供极具价值的研究工具。