Single cell determination of cardiac microtissue structure and function using light sheet microscopy

Abstract

Native cardiac tissue is comprised of heterogeneous cell populations that work cooperatively for proper tissue function; thus, engineered tissue models have moved toward incorporating multiple cardiac cell types in an effort to recapitulate native multicellular composition and organization.Cardiac tissue models comprised of stem cell-derived cardiomyocytes require inclusion of nonmyocytes to promote stable tissue formation, yet the specific contributions of the supporting nonmyocyte population on the parenchymal cardiomyocytes and cardiac microtissues have yet to be fully dissected. This gap can be partly attributed to limitations in technologies able to accurately study the individual cellular structure and function that comprise intact 3D tissues. The ability to interrogate the cell-cell interactions in 3D tissue constructs has been restricted by conventional optical imaging techniques that fail to adequately penetrate multicellular microtissues with sufficient spatial resolution. Light sheet fluorescence microscopy overcomes these constraints to enable single cell-resolution structural and functional imaging of intact cardiac microtissues.Multicellular spatial distribution analysis of heterotypic cardiac cell populations revealed that cardiomyocytes and cardiac fibroblasts were randomly distributed throughout 3D microtissues.Furthermore, calcium imaging of live cardiac microtissues enabled single-cell detection of cardiomyocyte calcium activity, which showed that functional heterogeneity correlated with spatial location within the tissues. This study demonstrates that light sheet fluorescence microscopy can be utilized to determine single-cell spatial and functional interactions of multiple cell types within intact 3D engineered microtissues, thereby facilitating the determination of structure-function relationships at both tissue-level and single-cell resolution. Impact StatementThe ability to achieve single-cell resolution by advanced 3D light imaging techniques enables exquisite new investigation of multicellular analyses in native and engineered tissues. In this study, light sheet fluorescence microscopy was used to define structure-function relationships of distinct cell types in engineered cardiac microtissues by determining heterotypic cell distributions and interactions throughout the tissues as well as by assessing regional differences in calcium handing functional properties at the individual cardiomyocyte level.