We have previously described and published our protocol for 3D bioprinting of monolayer cardiac patches of size ~ 3.6 × 3.6 mm using only cardiospheres, without the use of biomaterials(Ong, Fukunishi, Zhang, et al. 2017 (link)). The thickness of the mono layer patch in this study was about 350–400 um. In brief, a 3D bioprinter (Regenova, Cyfuse Biomedical K.K., Tokyo, Japan) was used to create biomaterial-free cardiac patches. The predetermined 3D geometry (one single layer of spheroids) was designed using the software of the 3D bioprinter. In a sterile environment, the 3D bioprinter identified the locations of the spheroids within the wells of the ultra-low adhesion 96-well tissue culture plate by automated software detection, a robotic arm then used vacuum suction to pick up, transfer and load the cell aggregates individually onto a needle array in exact spatial coordinates according to the 3D design. After bioprinting, the cardiac patch was allowed to mature for 3 days (72 hours) culture within the needle array, while placed on a shaker (Compact Digital Microplate Shaker, Thermo Scientific, Waltham, MA) at 150 rounds per minute (rpm) in the incubator. During this time, the spheroids fuse to form a cardiac patch with synchronous beating function. The needle array was removed (decannulation) and the cardiac patches were cultured for 7–10 days before implantation surgery.