A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

The goal of current cardiac tissue engineering is to develop a therapy to replace or repair the structure and function of injured myocardial tissue¹. Methods to create 3-D cardiac tissue models exhibiting the important contractile and electrophysiological properties of native cardiac tissue have been rapidly expanding^2,3. A variety of strategies have been explored and used in studies^4,5. These methods range from the use of specific synthetic and natural bioactive hydrogels, such as gelatin, collagen, fibrin, and peptides⁶, to bio-ink deposition technologies² and bioprinting technologies⁷.

It has been shown that scaffold-free methods can produce comparable tissues as biomaterial-based methods, without the drawbacks of incorporating foreign scaffolding material⁸. Oren Caspi et al. demonstrated that the incorporation of various types of cells enables the generation of highly vascularized human engineered cardiac tissue⁹. Chin et al. developed a 3-D printing method for cardiac patch creation from spheroids. Resulting patches are composed of cardiomyocytes, fibroblasts, and endothelial cells in a 70:15:15 ratio¹⁰. Spheroids have been shown to be effective "building blocks" of scaffold-free cardiac tissue creation, as they are resistant against hypoxia and possess sufficient mechanical integrity for implantation^11,12. Previous studies have demonstrated several fabrication methods for spheroid creation, including the use of the hanging drop method, spinner flasks¹³, microfluidic systems¹⁴, and non-adherent culture surfaces uncoated or coated with agarose micro-molds¹⁵. In this protocol, we use the hanging drop device, which contains micropores for condensing hundreds of spheroids at one time.

This study presents a novel and efficient scaffold-free method for cardiac tissue creation, which includes manually seeding the spheroids into a square mold cavity and incubating the tissue on a shaker for maturation. Under usual static culture conditions, oxygen diffusion is limited to the outer aspects of the tissue construct, resulting in central necrosis. However, with the net mold, all the spheroids seeded into the mold are immersed in media with a constant fluidic motion, allowing for the increased diffusion of nutrients and oxygen. Additionally, this mold-based method allows for the simultaneous creation of different-sized tissue patches with minimal manual effort and the resultant tissue can be easily removed from the mold. This novel method allows for the efficient and reproducible creation of scaffold-free, multilayered cardiac patches.