Geometrically optimized cardiac microtissues attenuate inflammation and promote vascularization following omental implantation

Introduction: The fabrication of highly vascularized myocardial tissues with physiologically relevant cell densities and an endothelial network fully integrated within the parenchymal space poses a significant challenge in cardiac tissue engineering. The simple co-culture of cardiomyocytes (CMs) with endothelial cells (ECs) and stromal cells only results in premature and segmented lumen structures without sufficient perfusion. Although biomaterial-based and 3D-printed vascular networks provide full control over topology and confer immediate functionality, they inhibit dynamic crosstalk between CMs and ECs and have limited remodeling ability. Moreover, the incorporation of a functional vasculature is critical for achieving cardiac grafts large enough to support clinically meaningful functions. Although human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) serve as a promising cell source for these grafts, their poor post-transplantation survival secondary to ischemia and the host immune response limits their therapeutic potential. To overcome these limitations, we have developed a method of generating microsized uniaxial cardiac tissues, referred to as cardiac μtissues, using hiPSC-CMs and cardiac fibroblasts (cFBs) for stromal support. These tissues maintain their architectural integrity and cellular alignment and yet remain within the oxygen diffusion limit due to their small size. We show that when co-cultured with ECs and stromal cells, these µtissues induce the formation of continuous, interconnected vascular networks.

Materials and

Methods: Five pillar-based platforms were designed with different geometries and pillar numbers, then fabricated using PDMS molds with soft lithography to investigate the effect of pillar arrangement on cellular alignment. Vascular µtissues consisting of GFP-HUVECs and dental pulp stem cells (DPSCs) were initially used to identify patterns with highly reproducible tissue compaction around mold features. Given the oxygen diffusion limit, tissue width and depth did not exceed 400 µm. Live/dead staining was performed to evaluate cell damage following µtissues pipette harvesting. Cardiac μtissues were then fabricated using hiPSC-CMs and cFBs seeded at ratio of 10:1 on three 2-pillar designs, with cardiac Troponin T (cTnT) immunostaining used to evaluate sarcomere alignment. Coherency quantification and traces of pillar displacement were used to identify the design with the highest contractile forces. Dispersed hiPSC-CMs or cardiac μtissues were subsequently co-cultured with ECs and DPSCs (ratio of 2:1:1 and 4:1:1, respectively) in fibrin hydrogels to evaluate vessel network formation by fluorescence microscopy and VE-cadherin immunostaining over 14 days of culture. Culture supernatant for both groups was collected on 10 day for LDH assay, quantification of cell-free mitochondrial DNA (mtDNA), cytokine profiling, and proteomics analysis. Finally, dispersed hiPSC-CMs or cardiac µtissues were embedded in fibrin hydrogels with or without the addition of GFP-HUVECs and DPSCs and transplanted onto the omentum of 7 week-old athymic nude rats. One week following implantation, omental samples were removed for contractility assessment (excitation threshold (ET) and maximum capture rate (MCR)) and histological/immunohistochemical studies (H&E, cTNT, CD68, CD206, and CD31).

Results, Conclusions, and Discussions: Vascular µtissues cultured on PDMS molds with the 2-pillar design demonstrated more consistent tissue formation and improved cellular alignment; consequently, designs with 3 or 4 pillars were eliminated from further experiments. Live/dead staining demonstrated minimal cellular damage from pipette harvesting. Following cardiac µtissue fabrication and immunostaining with cTNT, coherency quantifications revealed that tissues from both the “eight” and “candy” shaped wells exhibited higher sarcomere alignment and had consistently higher success rates of tissue formation. By tracing pillar displacement, we found that tissues formed in the “candy” shaped wells had the highest contractile forces and shortest contractile durations. At day 7 and day 14 of CM/EC/DPSC and μtissue/EC/DPSC co-culture within fibrin hydrogels, vascularization of the CM/EC/DPSC group was poor; in contrast, the μtissue/EC/DPSC group demonstrated formation of continuous and interconnected vessels as quantified by increased vessel junction density, increased average vessel length, and reduced lacunarity. LDH assay and quantification of cell-free mtDNA performed on the culture supernatant were indicative of significantly lower cell damage and cytotoxicity in the μtissue/EC/DPSC group. Cytokine profile analysis and proteomic analysis revealed upregulation of pro-angiogenic and cardioprotective cytokines and downregulation of proteins associated with immune system activation. Importantly, cardiac µtissues showed unprecedented survival and engraftment one week following omental implantation in a rodent model, and retained their contractility as quantified by ET/MCR measurements similar to pre-implantation values. This was unlike omental tissues containing dispersed hiPSC-CMs, the vast majority of which were removed by the host inflammatory response. Correspondingly, omental samples containing µtissues also had a significant decrease in the number of CD68+ macrophages and a larger proportion of CD206+ macrophages, indicative of a pro-healing phenotype. Moreover, despite the loss of GFP-HUVECs following transplantation, cardiac µtissues nevertheless showed increased vessel density and graft perfusion via vascular ingrowth from the host omental vasculature. These findings show that cardiac µtissues promote host endothelial ingrowth to generate functional, vascularized grafts with excellent cellular retention, preserved architecture, and stable contractile properties, without the need to incorporate pre-established luminal networks. Such µtissues thus offer a novel approach to improving cell-based therapies directed at cardiac remuscularization and functional recovery.

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