The silicone tube carried a suspension of fibrin gel and rat CMs. practices for post-fabrication conditioning of 3D engineered constructs for effective tissue development and stability, then concludes with prospective points of interest for engineering cardiac tissues in vitro. Cardiovascular three-dimensional bioprinting has the potential to be translated into the clinical setting and can further serve to model and understand biological principles that are at the root of cardiovascular disease in the laboratory. 1.?Introduction Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in most developed countries such as the United States and is a broad term given to a set of pathologies that affect the myocardium, heart valves, or vasculature in the body [1, 2]. The progression of CVD usually leads to the deterioration of one or more of the structures and cells of the heart and will, at their end-stage, need to replacement in order to improve the prognosis of patients affected. Current medical practices usually involve grafting tissues from the patients own body, donors, animals, or synthetically made constructs. Autografts, such as coronary artery bypass grafts, are performed by harvesting part of the patients own saphenous vein or other Edicotinib vessel to treat ischemia . End stage heart failure is usually treated by allografting a heart from a donor , while some valve replacement surgeries normally involve xenografting bovine or porcine heart valves . Synthetic valves  and vascular grafts  can also be implanted to treat CVD. Although each type of graft holds promise in treating one of the pathologies associated with CVD, each has their set of disadvantages that include, but are not limited to, a shortage of donor organs that are readily available , anticoagulation therapy , immune rejection , and limited sturdiness . As such, other avenues for readily available and compatible treatments are needed. Cardiovascular tissue engineering endeavors to repair damaged or ineffective blood vessels, heart valves, and cardiac muscle . Current strategies to accomplish such a feat include the differentiation of stem cells into mature and functional tissues on biomaterials that support the tissues growth and development. The biomaterials of choice usually involve either natural or synthetic hydrogels, or decellularized matrices as they provide a porous, interconnected polymeric network that allow cells to migrate, proliferate, and receive the nutrients that are essential to their survival . Moreover, because of their potential to reduce the immune rejection of grafts, decrease thrombogenic effects, and prospectively have tissues available on demand, the use of autologous and allogenic stem cells are a warm topic in cardiac tissue engineering [13, 14]. Generation of cardiac structures requires the integration of cardiac fibroblasts, cardiomyocytes, and endothelial cells, derived from multiple stem cell sources (Physique 1). Open in a separate window Physique 1: Cells for cardiac tissue engineering. These cells can be derived from multiple stem cell sources as shown in the physique. Reproduced with permission from . Three-dimensional bioprinting technology, an additive manufacturing technique that employs a layer-by-layer approach, has been implemented to develop the next generation of cardiac patches. Numerous efforts have been made to 3D bioprint functional cardiac tissues-on-a-chip using biomaterials such as scaffolds or bioinks that could restore the functions of the damaged myocardium. Some of the biomaterials that were utilized to 3D bioprint myocardial tissue include alginate , collagen [17, 18], gelatin [19, 20], hyaluronic acid , and decellularized extracellular matrix scaffolds, among others Edicotinib . Despite the many successes of 3D printed scaffold based cardiac patches, they are not without imperfections. Scaffolds have a Edicotinib high probability of rapid degeneration, eventually resulting in reduced physical or mechanical stability [23, 24]. However, 3D bioprinting of scaffold-free cardiac patches have yielded satisfactory outcomes. Atmanli et al. exhibited the fabrication of functional cardiac patches using microcontact 3D bioprinting of double transgenic murine committed ventricular progenitors (CVPs) . These patches were found to maintain the unique architecture of the native myocardial tissue . A separate study conducted by Ong et al. fabricated spontaneously beating biomaterial-free cardiac patches by 3D bioprinting mixed cell spheroids made up of aggregates of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), fibroblasts, and ECs . Bioprinted cardiac patches fabricated from gelatin methacrylate-cardiac extracellular matrix (GelMA-cECM) hydrogel based bioinks laden with human cardiac progenitor cells were found to increase the angiogenic potential and showed vascularization IL8 when applied on rat hearts . Other research groups have also met with success in constructing scaffold-free 3D bioprinted cardiac patches exhibiting viability, vascularization, and engraftment of cells following implantation [26, 27]. Motivated by these outcomes, we added fibrin to our previously optimized photopolymerizable gelatin-based bioink to fabricate cardiac cell-laden constructs with hiPS-CMs or CM cell lines and cardiac fibroblasts (CFs). The cell-laden bioprinted constructs were crosslinked via a two-step procedure including the visible-light crosslinking of furfuryl-gelatin followed by the chemical crosslinking.