Directed fusion of cardiac spheroids into larger heterocellular microtissues enables investigation of cardiac action potential propagation via cardiac fibroblasts.

Tae Yun Kim,Ulrike Mende,Zhilin Qu,Michelle E King,Alexander R Markes,Amenawon O Okundaye,Bum-Rak Choi,Celinda M Kofron,Alexander V Panfilov

doi:10.1371/journal.pone.0196714

Abstract

Multicellular spheroids generated through cellular self-assembly provide cytoarchitectural complexities of native tissue including three-dimensionality, extensive cell-cell contacts, and appropriate cell-extracellular matrix interactions. They are increasingly suggested as building blocks for larger engineered tissues to achieve shapes, organization, heterogeneity, and other biomimetic complexities. Application of these tissue culture platforms is of particular importance in cardiac research as the myocardium is comprised of distinct but intermingled cell types. Here, we generated scaffold-free 3D cardiac microtissue spheroids comprised of cardiac myocytes (CMs) and/or cardiac fibroblasts (CFs) and used them as building blocks to form larger microtissues with different spatial distributions of CMs and CFs. Characterization of fusing homotypic and heterotypic spheroid pairs revealed an important influence of CFs on fusion kinetics, but most strikingly showed rapid fusion kinetics between heterotypic pairs consisting of one CF and one CM spheroid, indicating that CMs and CFs self-sort in vitro into the intermixed morphology found in the healthy myocardium. We then examined electrophysiological integration of fused homotypic and heterotypic microtissues by mapping action potential propagation. Heterocellular elongated microtissues which recapitulate the disproportionate CF spatial distribution seen in the infarcted myocardium showed that action potentials propagate through CF volumes albeit with significant delay. Complementary computational modeling revealed an important role of CF sodium currents and the spatial distribution of the CM-CF boundary in action potential conduction through CF volumes. Taken together, this study provides useful insights for the development of complex, heterocellular engineered 3D tissue constructs and their engraftment via tissue fusion and has implications for arrhythmogenesis in cardiac disease and repair.

Highlights

Three-dimensional (3D) platforms bridge the gap between two-dimensional (2D) cell culture and intact tissue, since appropriate cell-cell and cell-extracellular matrix (ECM) interactions and architecture in a 3D environment are important determinants of tissue differentiation and function [1,2,3,4]
We developed elongated 3D microtissues comprised of cardiac myocytes (CMs) separated by compact volumes of cardiac fibroblasts (CFs), using individual spheroids comprised of CMs or CFs as building blocks
We developed cardiac 3D elongated microtissues by fusing individual 3D spheroids comprised of CMs and/or CFs

Summary

Introduction

Three-dimensional (3D) platforms bridge the gap between two-dimensional (2D) cell culture and intact tissue, since appropriate cell-cell and cell-extracellular matrix (ECM) interactions and architecture in a 3D environment are important determinants of tissue differentiation and function [1,2,3,4]. The generation of tissue culture platforms with extensive cell-cell contacts is important for a variety of biomedical engineering applications including tissue constructs for in vivo replacement and repair and in vitro models of cellular interactions with other cells, materials, and drugs. When cells are not provided with natural or synthetic surfaces or matrices to attach to, they interact with each other, aggregate, and self-organize into multicellular spheroids in a process known as self-assembly [5,6,7]. Little is known about how multiple cell types self-organize into distinct regions or layers as they fuse within engineered tissue or the functional behavior of cells during fusion

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