While dermal fibroblasts adhere within 4 hours, NCP took at least 3 days to adhere and to spread on the substrate. For this reason, the spinner culture was carried by using a cycle of 10 minutes at rest and 40 minutes at 30 rpm for up to 3 days of culture. After this time, the dynamic culture switched to continuous stirring at the velocity of 30 rpm up to 9 days of culture. During the culture time, NCP was able to

adhere to and colonize the microbeads, recreating a structure quite similar to that of native muscle, composed by cardiomyocytes and fibroblast embedded in a collagen network as shown in Figure 3A. CME showed uniform cell distribution and important Inhibitors,research,lifescience,medical biological structure such as sarcomere actin filaments; connexin-43 junctions were also present. One of the most important features of CME is the self-beating capability generated Inhibitors,research,lifescience,medical by the spontaneous

beat of the cardiomyocytes fraction present in the tissue equivalent. After 4 days of culture, cardiac microtissues were collected from the spinner culture, and their spontaneous contraction was monitored by video microscopy. The tissue Inhibitors,research,lifescience,medical organization continued till the end of the culture, and at each time point, beating and contracting microcardiac muscle was observed with a beating rate of 45 bpm (Figure 3B). Interestingly, when placed in close proximity, two micro CMEs having different beating frequencies were able to synchronize after 40 minutes. After 1 day, the CMEs underwent a fusion process, and the resulting tissue was made by a microtissue with a single beating frequency. Figure 3 Masson trichrome of micro CME; (B) beating properties Inhibitors,research,lifescience,medical of the micro CME; the peak represents a single beat. The translation of a microscaffold-based bottom-up strategy from dermis to cardiac muscle tissue fabrication resulted in a promising strategy for heart tissue engineering. Despite other Inhibitors,research,lifescience,medical techniques such as cell sheet or cellular find more spheroids, the presence of the microscaffold aids tissue reorganization and ECM synthesis, simultaneously representing a structure that guarantees mechanical support. The presence of the scaffold does not hinder the electromechanical properties of the CME, which showed spontaneous beating

and synchronization properties. This indicates that the CME can be used as an implantable living microtissue for infracted zone regeneration. Conclusion below The strategy presented in this work highlights the production of functional tissue in vitro, made up of endogenous ECM and tunable in size and shape. In light of this, the tissues created could be useful in regenerative medicine as they could be injected as living microtissues in damaged sites or, if assembled in large 3D tissues, could be used as a patch for extended damages. The versatility of this technology paves the way for in vitro biofabrication of several kinds of tissues, leading to an increased availability of “living” tissue or organ substitutes. Funding Statement Funding/Support: The authors have no funding disclosures.