FIELD OF THE INVENTION

The present invention pertains generally to additive manufacturing systems and methods. More specifically, the present invention can pertain to systems and methods for additive manufacturing of cardiac tissues and organ models. The present invention can be particularly, but not exclusively, useful as systems and methods for additive manufacturing of cardiac tissues and organ models that can seed a three dimensional endothelial bed with cardiomyocytes using a controlled anisotropy, in order to generate aligned myocardium capable of spontaneous and synchronous contraction.

BACKGROUND OF THE INVENTION

Engineering cardiac tissues poses a series of critical challenges that need to be addressed in order to translate basic research products from bench to clinical practice. The engineered cardiac organoids coupled with microfluidic bioreactors (e.g. heart-on-chips) have also found increasing applications in functioning as enabling in vitro biomimetic models to study pathology, measure cardiotoxicity, and develop new therapeutics. One of the first challenges in engineering cardiac organoids and their on-chip forms in the prior art can be the fact that mature cardiomyocytes exhibit limited self-renewing potential. In this framework, induced pluripotent stem cells (iPSCs) hold great promise, due to their wide availability and the possibility to differentiate into multiple cell lineages including cardiomyocytes. Second, the alignment of cardiomyocytes and their organization into bundles characterized by spontaneous and synchronous contraction further complicate the development of biologically relevant cardiac tissues. Third, the generation of thick (cardiac) tissue constructs requires the introduction of microvascular networks in order to provide oxygen and nutrients, remove waste products, and eventually promote vessel anastomosis with the host vasculature.

Several approaches have so far been explored in the prior art to generate functional tissue constructs including the myocardium. For example, scaffold-free multicellular cardiac spheroids have been developed that could spontaneously and synchronously contract. While the cardiac spheroids have served an important role in drug testing and have been widely used due to the ease of preparation, these constructs can lack the directionality characterized by the physiological myocardium, which can be critical to maintain the long-term functionality of the engineered cardiac tissues.

On the other hand, prior art scaffold-based techniques can provide an ideal support for cell adhesion, distribution, and responses. Importantly, the architecture of the scaffolds can be conveniently modulated in order to promote the biological relevance of the engineered tissues by tuning spatial organizations that mimic in vivo counterparts. For example, it has been demonstrated that anisotropic scaffolds bearing an accordion-like honeycomb structure could induce the generation of highly oriented cardiac fibers. Biowire approaches to induce the differentiation and alignment of the cardiomyocytes from human pluripotent stem cells have also been discussed in the literature. Still further, it has been shown that cardiac tissues can be populated with microfilament arrays seeded with cardiomyocytes to engineer aligned cardiac tissues; hydrogel substrates with aligned ridges/grooves via photopatterning to improve the adhesion and alignment of cardiomyocytes have recently been developed. Other strategies have further been investigated to integrate blood vessels into engineered tissues including the myocardium. However, generating volumetric cardiac tissues containing embedded endothelial networks remains challenging.

Bioprinting has recently emerged as a promising technology to produce geometrically defined structures in three dimensions (3D), significantly improving their physiological relevance through architectural mimicry of native tissues and organs. Particularly, bioprinting overcomes major drawbacks of conventional scaffold-based approaches including limited control over the 3D structures of engineered tissues and thus reduced reproducibility. Additionally, the bioprinting process is usually biocompatible, allowing for direct encapsulation of bioactive molecules and cells. Still further, bioprinting may enable vascularization of the engineered tissue constructs based on sacrificial methods or direct deposition, providing additional versatility in producing vascularized cardiac organoids.

In view of the above, it can be an object of the present invention to provide systems and methods for additive manufacturing of myocardial tissue that can yield functional cardiac tissue that is capable of contraction. Still another object of the present invention can be to provide systems and methods for additive manufacturing of myocardial tissue that can yield functional cardiac tissue for use in regenerative medicine, drug screening, and potentially disease modeling application. Yet another object of the present invention can be to provide systems and methods for additive manufacturing of myocardial tissue that can yield functional tissue, which can further yield the perfusion of such an endothelialized network in the future upon usage of sacrificial bioinks that can be removed, to enhance the biomimetic properties of produced vascularized organoids. Another object of the present invention can be to provide systems and methods for additive manufacturing of myocardial tissue that can eventually be somewhat easy to use in a cost-efficient manner.

SUMMARY OF THE INVENTION

A bioreactor and methods for using said bioreactor for drug screening, can include a microfibrous hydrogel scaffold, that can be made of a composite alginate-gelatin methacryloyl (GeIMA) bioink, and that can have endothelial cells directly embedded within the scaffold. The scaffold can further be seeded with cardiomyocytes so that said bioreactor has a controlled anisotropy, and the scaffold can be placed in a chamber defined by a PDMS half pieces, that compress the scaffold slightly when the PDMS half pieces are fastened to each other. The chamber could certainly be made of other materials as chosen by the end user, including but not limited to, thermoplastics, glass, etc.

The hydrogel scaffold can be composed of a plurality of serpentine layers, with each serpentine layer having a primary axis defined by the serpentine layer tines. Successive serpentine layers can be placed on each other in a cross-hatch configuration, so that the primary axes of successive layers are perpendicular. This configuration can establish a plurality of rectangular holes that have an aspect ratio when viewed in plan view. The aspect ratio can vary from 2×2, 2×3, 2×4, and 2×5. The successive layers that have a primary axis in the same direction can be placed in the scaffold so that they are offset from each other. The microfibrous hydrogel scaffold can be manufactured using additive manufacturing techniques.

In some embodiments, the cardiomyocytes can be neonatal rat cardiomyocytes. In still other embodiments, cardiomyocytes can be human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, and in particular hiPSC-derived cardiomyocytes from the candidate patient for whom the drug screening process is being accomplished.

