Di Felice Valentina, Cappello Francesco, Montalbano Antonella, Ardizzone Nella Maria, De Luca Angela, Macaluso Filippo, Amelio Daniela, Cerra Maria Carmela, Zummo Giovanni
Human Anatomy Section E. Luna, Department of Experimental Medicine, University of Palermo, Via del Vespro 129, 90127 Palermo, Italy.
Biol Cell. 2007 Dec;99(12):689-99. doi: 10.1042/BC20070043.
Cultivation techniques promoting three-dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel-based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co-cultures to evaluate, on both two- and three-dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat-shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase).
Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co-culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel-cultured cardiomyocytes, areas of high co-localization between HSP90 and eNOS and co-localization coefficients, which indicated the highest correlation with respect to the other substrates.
Our three-dimensional adult cardiomyocyte cultures are suitable for both analysing cell-ECM interactions at electron and confocal microscopy levels and monitoring micro-environment impact on cardiomyocyte phenotype.
促进哺乳动物细胞三维组织的培养技术越来越受到关注，因为它们赋予了天然细胞外基质（ECM）的关键功能，具有再生医学应用的潜力。由于ECM的顺应性会影响许多细胞功能，基于基质胶的凝胶已成为有吸引力的工具，因为其机械性能易于控制。在本研究中，我们利用常用细胞培养底物的化学和机械可调性以及共培养，在二维和三维培养的成年大鼠心肌细胞上评估ECM化学和力学对两种相互作用信号蛋白细胞定位的影响：HSP90（90 kDa热休克蛋白）和eNOS（内皮型一氧化氮合酶）。
将新鲜分离的大鼠心肌细胞培养在纤连蛋白、基质胶凝胶或层粘连蛋白上，或与心脏成纤维细胞共培养，并测试其完整性和活力。作为验证标准，通过透射电子显微镜评估质膜和线粒体的完整性。通过免疫荧光和共聚焦显微镜监测细胞对微环境刺激的敏感性。我们发现HSP90和eNOS的表达及定位受ECM组成变化的影响。对图像的分析显示，在基质胶培养的心肌细胞上，HSP90和eNOS之间存在高共定位区域以及共定位系数，这表明相对于其他底物具有最高的相关性。
我们的三维成年心肌细胞培养适用于在电子显微镜和共聚焦显微镜水平分析细胞 - ECM相互作用以及监测微环境对心肌细胞表型的影响。
