Demonstration of doxorubicin's cardiotoxicity and screening using a 3D bioprinted spheroidal droplet-based system.

Doxorubicin (DOX) is a highly effective anthracycline chemotherapy agent effective in treating a broad range of life-threatening malignancies but it causes cardiotoxicity in many subjects. While the mechanism of its cardiotoxic effects remains elusive, DOX-related cardiotoxicity can lead to heart failure in patients. In this study, we investigated the effects of DOX-induced cardiotoxicity on human cardiomyocytes (CMs) using a three-dimensional (3D) bioprinted cardiac spheroidal droplet based-system in comparison with the traditional two-dimensional cell (2D) culture model. The effects of DOX were alleviated with the addition of N-acetylcysteine (NAC) and Tiron. Caspase-3 activity was quantified, and reactive oxygen species (ROS) production was measured using dihydroethidium (DHE) staining. Application of varying concentrations of DOX (0.4 µM-1 µM) to CMs revealed a dose-specific response, with 1 µM concentration imposing maximum cytotoxicity and 0.22 ± 0.11% of viable cells in 3D samples versus 1.02 ± 0.28% viable cells in 2D cultures, after 5 days of culture. Moreover, a flow cytometric analysis study was conducted to study CMs proliferation in the presence of DOX and antioxidants. Our data support the use of a 3D bioprinted cardiac spheroidal droplet as a robust and high-throughput screening model for drug toxicity. In the future, this 3D spheroidal droplet model can be adopted as a human-derived tissue-engineered equivalent to address challenges in other various aspects of biomedical pre-clinical research.

3D Biofabrication of a Cardiac Tissue Construct for Sustained Longevity and Function.

In this study, we developed three-dimensional (3D) printed annular ring-like scaffolds of hydrogel (gelatin-alginate) constructs encapsulated with a mixture of human cardiac AC16 cardiomyocytes (CMs), fibroblasts (CFs), and microvascular endothelial cells (ECs) as cardiac organoid models in preparation for investigating the role of microgravity in cardiovascular disease initiation and development. We studied the mechanical properties of the acellular scaffolds and confirmed their cell compatibility as well as heterocellular coupling for cardiac tissue engineering. Rheological analysis performed on the acellular scaffolds showed the scaffolds to be elastogenic with elastic modulus within the range of a native in vivo heart tissue. The microstructural and physicochemical properties of the scaffolds analyzed through scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy-attenuated total reflectance (ATR-FTIR) confirmed the mechanical and functional stability of the scaffolds for long-term use in in vitro cell culture studies. HL-1 cardiomyocytes bioprinted in these hydrogel scaffolds exhibited contractile functions over a sustained period of culture. Cell mixtures containing CMs, CFs, and ECs encapsulated within the 3D printed hydrogel scaffolds exhibited a significant increase in viability and proliferation over 21 days, as shown by flow cytometry analysis. Moreover, via the expression of specific cardiac biomarkers, cardiac-specific cell functionality was confirmed. Our study depicted the heterocellular cardiac cell interactions, which is extremely important for the maintenance of normal physiology of the cardiac wall in vivo and significantly increased over a period of 21 days in in vitro. This 3D bioprinted "cardiac organoid" model can be adopted to simulate cardiac environments in which cellular crosstalk in diseased pathologies like cardiac atrophy can be studied in vitro and can further be used for drug cytotoxicity screening or underlying disease mechanisms.

Development and Characterization of Furfuryl-Gelatin Electrospun Scaffolds for Cardiac Tissue Engineering.

In this study, three types of electrospun scaffolds, including furfuryl-gelatin (f-gelatin) alone, f-gelatin with polycaprolactone (PCL) in a 1:1 ratio, and coaxial scaffolds with PCL (core) and f-gelatin (sheath), were developed for tissue engineering applications. Scaffolds were developed through single nozzle electrospinning and coaxial electrospinning, respectively, to serve as scaffolds for cardiac tissue engineering. Uniform fibrous structures were revealed in the scaffolds with significantly varying average fiber diameters of 760 ± 80 nm (f-gelatin), 420 ± 110 nm [f-gelatin and PCL (1:1)], and 810 ± 60 nm (coaxial f-gelatin > PCL) via scanning electron microscopy. The distinction between the core and the sheath of the fibers of the coaxial f-gelatin > PCL electrospun fibrous scaffolds was revealed by transmission electron microscopy. Thermal analysis and Fourier transformed infrared (FTIR) spectroscopy revealed no interactions between the polymers in the blended electrospun scaffolds. The varied blending methods led to significant differences in the elastic moduli of the electrospun scaffolds with the coaxial f-gelatin > PCL revealing the highest elastic modulus of all scaffolds (164 ± 3.85 kPa). All scaffolds exhibited excellent biocompatibility by supporting the adhesion and proliferation of human AC16 cardiomyocytes cells. The biocompatibility of the coaxial f-gelatin > PCL scaffolds with superior elastic modulus was assessed further through adhesion and functionality of human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, thereby demonstrating the potential of the coaxially spun scaffolds as an ideal platform for developing cardiac tissue-on-a-chip models. Our results demonstrate a facile approach to produce visible light cross-linkable, hybrid, biodegradable nanofibrous scaffold biomaterials, which can serve as platforms for cardiac tissue engineered models.

Development of an extrusion-based 3D-printing strategy for clustering of human neural progenitor cells.

3D bioprinting offers a simplified solution for the engineering of complex tissue parts for in-vitro drug discovery or, in-vivo implantation. However, significant amount of challenges exist in 3D bioprinting of neural tissues, as these are sensitive cell types to handle via extrusion bioprinting techniques. We assessed the feasibility of bioprinting human neural progenitor cells (NPCs) in 3D hydrogel lattices using a fibrinogen-alginate-chitosan bioink, previously optimized for neural-cell growth, and subsequently modified for structural support during extrusion printing, in this study. The original bioink used in this study was made by adding optimized amounts of high- and medium-viscosity alginate to the fibrinogen-chitosan-based bioink and making it extrudable under shear pressure. The mechanically robust 3D constructs promoted NPC cluster formation and maintained their morphology and viability during the entire culture period. This strategy may be useful for co-culturing of NPCs along with other cell types such as cardiac, vascular, and other cells during 3D bioprinting.