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

Abstract

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.

Keywords: 3D bioprinting; biofabrication; cardiac tissue-on-a-chip; heterocellular coupling; organoids.

Figure 1.

3D printed cardiac scaffolds were developed, and their properties were studied up to 21 days. (A) Rigid 3D printed structures developed through optimized printing parameters. (B) High-throughput 12-well fabrication of the 3D printed scaffolds and their integration within the CubeLab microfluidic channel-based system. (C) A schematic of the high-throughput assembly of the bioprinted scaffolds in 12-well plates and the workflow.

Figure 2.

Swelling analysis. (A) Lyophilized 3D printed annular ring on day 0 (left) and swollen annular ring on day 12 (right). (B) Non-cross-linked versus cross-linked scaffolds shown during the swelling experiment at varying time points. Scale bar in all images corresponds to 2 mm. (C) Swelling ratio of the 3D printed annular rings at various time points in culture media with 3–4 days, 10–11 days, and 20–21 days highlighted to depict significant differences in swelling behavior in the scaffolds studied. Swelling ratio (mean ± SD) for characteristic alginate–gelatin-annular ring scaffolds was studied for 21 days during which the maximum degree of swelling was attained on day 12. Beyond day 12, until day 15, the swelling apparently reached equilibrium as these values did not seem to bear any statistical differences when analyzed and a significantly lower swelling ratio from day 16 to 21 was observed.

Figure 3.

Physicochemical characterization of the 3D printed scaffolds at various time points. (A) Gross and microstructural analysis (using SEM) of the 3D printed annular ring scaffolds showing average pore diameters at various time points. (B) Average pore diameters of scaffolds at varying time points in the culture. (C) ATR-FTIR of the 3D printed scaffolds on days 3 and 21 showing minimal chemical degradation of the scaffolds.

Figure 4.

Rheometric analysis of 3D bioprinted cellular vs acellular scaffolds. Graphs (A, C, E) show the average storage/loss moduli, average complex viscosity, and average elastic modulus results, respectively, after 4 days of culture, where p-values were found to be statistically significant between the cellular and acellular scaffolds (p < 0.05). Graphs (B, D, F) show the average storage/loss moduli, average complex viscosity, and average elastic modulus results, respectively, after 11 days of culture, where p-values were not found to be statistically significant between the cellular and acellular scaffolds (p > 0.05).

Figure 5.

HL-1 contractile cardiomyocytes cultured within the 3D gels. (A) Image captured using the Fluo-8AM staining in the HL-1 cells in gels detecting their intracellular calcium mobilization. (B) Average cell densities isolated from cultures of HL-1 cells in gels on day 1 and after 21 days. (C, D) HL-1 cells cultured atop 2D controls and within gels up to 21 days are depicted in panels (C) and (D), respectively. Scale bar corresponds to 150 μm in both images. (E, F) Immunohistochemistry of HL-1 contractile cardiomyocytes cultured within the gels (F) or on controls (E). The predominant cardiac markers including CX43 and MyoH expressions were confirmed for the HL-1 cells used in this study, while all cells were counterstained using the nuclear stain DAPI. Scale bar corresponds to 50 μm in both images (E) and (F).

Figure 6.

Viability of the cells and their heterocellular coupling in 3D printed scaffolds. (A) MTS assay of cells in 3D printed annular rings at various time points depicts cell viability and maintenance in long-term sustained cultures at 21 days. (B) Fluorescent microscopy image of CMs (red), CFs (green), and ECs (blue) in the 3D printed scaffolds (scale bar depicts 100 μm). (C) Number of heterocellular couplings at various time points in this study.

Figure 7.

FACS analysis. Cardiomyocytes were stained with CellTrace Violet, cardiac fibroblasts with CellTrace Yellow, and endothelial cells with CellTrace Far Red and mixed in a 2:2:1 ratio with the bioink prior to 3D bioprinting. After 3, 10, and 21 days, cells were extracted from the scaffolds and analyzed using a flow cytometer. Graphs (A, D, G) show the % of CTV dye intensity, (B, E, H) show the % of CTY dye intensity, and (C, F, I) show the % of CTFR dye intensity for cardiomyocytes, cardiac fibroblasts, and endothelial cells, respectively. p-values were found to be all statistically significant between the different time points (p < 0.05).

Figure 8.

Immunohistochemistry was performed to probe the presence and the mechanism of interaction with other cells for the EC in bioprinted gels after 5 days of culture. CM and CF were prestained with PKH26 (red) and PKH67 (green), respectively. The ECs were immunostained using primary followed by secondary antibodies targeted toward CD31 (blue). The magnified figure insets are presented in Supporting Figures S5–S7.