Production of arrays of cardiac and skeletal muscle myofibers by micropatterning techniques on a soft substrate
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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.
KeywordsMicro-patterning Soft substrate Hydrogel Cardiomyocytes Satellite cells Cell array
This work was supported by MIUR, University of Padua, Regione Veneto (Azione biotech II), Città della Speranza.
- A.J. Engler, M.A. Griffin, S. Sen, C.G. Bönnemann, H. LeeSweeney, D.E. Discher, Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments J. Cell Biol. 166, 877–887 (2004a). doi: 10.1083/jcb.200405004 CrossRefGoogle Scholar
- D. Hern, J.A. Hubbell, Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing J. Biomed. Mater. Res 39, 266–276 (1998). doi: 10.1002/(SICI)1097-4636(199802)39:2<266::AID-JBM14>3.0.CO;2-B CrossRefGoogle Scholar
- J.H. Jang, D.V. Schaffer, Microarraying the cellular microenvironment Mol. Syst. Biol. 2(39) (2006)Google Scholar
- S. Rohr, D.M. Scholly, A.G. Kleber, Patterned growth of neonatal rat heart cells in culture. Morphological and electrophysiological characterization Circ. Res 68, 114–130 (1991)Google Scholar
- Y. Xia, G.M. Whitesides, Soft lithography Angew. Chem. Int. Ed 37, 550–575 (1998). doi: 10.1002/(SICI)1521-3773(19980316)37:5<550::AID-ANIE550>3.0.CO;2-G CrossRefGoogle Scholar