title: Analysis of Calcium Transients and Uniaxial Contraction Force in Single Human Embryonic Stem Cell-Derived Cardiomyocytes on Microstructured Elastic Substrate with Spatially Controlled Surface Chemistries.
abstract: The mechanical activity of cardiomyocytes is the result of a process called excitation-contraction coupling (ECC). A membrane depolarization wave induces a transient cytosolic calcium concentration increase that triggers activation of calcium-sensitive contractile proteins, leading to cell contraction and force generation. An experimental setup capable of acquiring simultaneously all ECC features would have an enormous impact on cardiac drug development and disease study. In this work, we develop a microengineered elastomeric substrate with tailor-made surface chemistry to measure simultaneously the uniaxial contraction force and the calcium transients generated by single human cardiomyocytes in vitro. Microreplication followed by photocuring is used to generate an array consisting of elastomeric micropillars. A second photochemical process is employed to spatially control the surface chemistry of the elastomeric pillar. As result, human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be confined in rectangular cell-adhesive areas, which induce cell elongation and promote suspended cell anchoring between two adjacent micropillars. In this end-to-end conformation, confocal fluorescence microscopy allows simultaneous detection of calcium transients and micropillar deflection induced by a single-cell uniaxial contraction force. Computational finite elements modeling (FEM) and 3D reconstruction of the cell-pillar interface allow force quantification. The platform is used to follow calcium dynamics and contraction force evolution in hESC-CMs cultures over the course of several weeks. Our results show how a biomaterial-based platform can be a versatile tool for in vitro assaying of cardiac functional properties of single-cell human cardiomyocytes, with applications in both in vitro developmental studies and drug screening on cardiac cultures.
authors.affiliation: CNR Institute of Neuroscience , Corso Stati Uniti 4, 35127 Padova, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Grespan
authors.affiliation: Department of Industrial Engineering, University of Padova , Via Marzolo 9, 35131 Padova, Italy.|Venetian Institute of Molecular Medicine , Via Orus 2, 35129 Padua, Italy.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Martewicz
authors.affiliation: Department of Industrial Engineering, University of Padova , Via Marzolo 9, 35131 Padova, Italy.|Venetian Institute of Molecular Medicine , Via Orus 2, 35129 Padua, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Serena
authors.affiliation: Institute Charles Sadron, University of Strasbourg , 23 rue du Loess, 84047 Strasbourg, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Le Houerou
authors.affiliation: Department for Microsystems Engineering, University of Freiburg , Georges-Köhler Allee 103, 79110 Freiburg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Rühe
authors.affiliation: Department of Industrial Engineering, University of Padova , Via Marzolo 9, 35131 Padova, Italy.|Venetian Institute of Molecular Medicine , Via Orus 2, 35129 Padua, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Elvassore
mesh_terms: D002118:Calcium / Q000737:chemistry*; D002454:Cell Differentiation; D002478:Cells, Cultured; D020724:Elastomers; D000066449:Human Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D055595:Mechanical Phenomena; D055616:Microtechnology; D032383:Myocytes, Cardiac / Q000166:cytology*
chemical_list: D020724:Elastomers; D002118:Calcium