A strategy for fabrication of a three-dimensional tissue construct containing uniformly distributed embryoid...

Growing three-dimensional (3D) scaffolds that contain more than a few layers of seeded cells in vitro is crucial for the creation of thick and viable cardiac tissues in vivo. Embryonic stem cells (ESCs) have been used as an alternative cell source for cardiac repair; however, dissociated ESCs show poor viability in the scaffold and do not form the embryoid body (EB)-like structures. In this study, a strategy intended for cultivating EB-derived cells (EBDCs) uniformly in a porous 3D tissue scaffold was developed. This strategy employed techniques of formation of spherically symmetric EBs in a thermo-responsive hydrogel system, production of cell sheets of EBDCs in a similar hydrogel system coated with collagen and fabrication of sliced porous tissue scaffolds. The prepared EBs were collected and plated evenly in the cell-sheet culture system. After 8 days in culture, a continuous sheet of EBDCs with cell beating was obtained; our qPCR and flow cytometric analyses showed that the collagen-coated on the cell-sheet culture system can significantly enhance the population of cardiac-lineage cells. The produced EBDC sheets were then sandwiched into the sliced porous tissue scaffold. After reculture, the seeded EBDCs were redistributed uniformly throughout the scaffold, with a significant increase in mechanical strength. Cardiac-specific myosin heavy chain and alpha-actinin were expressed for some cells grown in the scaffold, while connexin 43 was clearly expressed at the cell borders. Additional studies such as employing purification techniques to enrich the population of cardiomyocytes are needed to further improve the developed tissue constructs as a bioengineered cardiac patch.