Cimetta Elisa, Pizzato Sara, Bollini Sveva, Serena Elena, De Coppi Paolo, Elvassore Nicola
Department of Chemical Engineering, University of Padua, Via Marzolo 9, 35131 Padua, Italy.
Biomed Microdevices. 2009 Apr;11(2):389-400. doi: 10.1007/s10544-008-9245-9.
Micropatterning and microfabrication techniques have been widely used to pattern cells on surfaces and to have a deeper insight into many processes in cell biology such as cell adhesion and interactions with the surrounding environment. The aim of this study was the development of an easy and versatile technique for the in vitro production of arrays of functional cardiac and skeletal muscle myofibers using micropatterning techniques on soft substrates. Cardiomyocytes were used for the production of oriented cardiac myofibers whereas mouse muscle satellite cells for that of differentiated parallel myotubes. We performed micro-contact printing of extracellular matrix proteins on soft polyacrylamide-based hydrogels photopolymerized onto functionalized glass slides. Our methods proved to be simple, repeatable and effective in obtaining an extremely selective adhesion of both cardiomyocytes and satellite cells onto patterned soft hydrogel surfaces. Cardiomyocytes resulted in aligned cardiac myofibers able to exhibit a synchronous contractile activity after 2 days of culture. We demonstrated for the first time that murine satellite cells, cultured on a soft hydrogel substrate, fuse and form aligned myotubes after 7 days of culture. Immunofluorescence analyses confirmed correct expression of cell phenotype, differentiation markers and sarcomeric organization. These results were obtained in myotubes derived from satellite cells from both wild type and MDX mice which are research models for the study of muscle dystrophy. These arrays of both cardiac and skeletal muscle myofibers could be used as in vitro models for pharmacological screening tests or biological studies at the single fiber level.
微图案化和微制造技术已被广泛用于在表面上对细胞进行图案化处理，并更深入地了解细胞生物学中的许多过程，如细胞粘附以及与周围环境的相互作用。本研究的目的是开发一种简便且通用的技术，用于在软质底物上利用微图案化技术体外制备功能性心肌和骨骼肌肌纤维阵列。心肌细胞用于制备定向心肌肌纤维，而小鼠肌肉卫星细胞用于制备分化的平行肌管。我们在光聚合到功能化载玻片上的基于聚丙烯酰胺的软质水凝胶上进行细胞外基质蛋白的微接触印刷。我们的方法在使心肌细胞和卫星细胞在图案化的软水凝胶表面上实现极高的选择性粘附方面被证明是简单、可重复且有效的。心肌细胞形成排列整齐的心肌肌纤维，在培养2天后能够表现出同步收缩活性。我们首次证明，在软水凝胶底物上培养的小鼠卫星细胞在培养7天后融合并形成排列整齐的肌管。免疫荧光分析证实了细胞表型、分化标志物和肌节组织的正确表达。这些结果是在源自野生型和MDX小鼠（肌肉营养不良研究模型）卫星细胞的肌管中获得的。这些心肌和骨骼肌肌纤维阵列可作为体外模型用于药理学筛选试验或单纤维水平的生物学研究。
