Poster U7 - Cell Spheroid-Loaded Cardiac Extracellular Matrix Patch for Heart Repair

Introduction: Decellularized porcine myocardium slices (dPMS) have demonstrated potential as a cardiac extracellular matrix (cECM) patch for myocardial infarction treatment. cECM can provide the chemical and physical environment in native cardiac tissue. Human mesenchymal stem cell (MSC) based therapies have shown great promise as potential treatments for cardiac therapy. MSCs can differentiate into multiple lineages and have been shown to exert therapeutic effects through their paracrine actions. However, traditional cell suspension scaffold seeding techniques have limited cell retention in porous scaffolds such as dPMS, which impacts effective recellularization and cell delivery efficiency. Conversely, spheroids are larger units with more uniformly organized adhesion molecules, which may improve contact and adherence to scaffold surfaces. Additionally, the optimized intracellular signaling and three-dimensional architecture found in cell spheroids have been shown to enhance cell organization and cell-cell interactions, resulting in increased paracrine functionality, suggesting greater immunomodulatory and regenerative therapeutic potential. This study investigates the feasibility of using human mesenchymal stem cell spheroids in conjunction with dPMS to fabricate an MSC spheroid loaded dPMS in vitro for applications in cardiac cell therapy. To achieve our goal, we sliced decellularized porcine myocardial tissue and seeded hMSC spheroids on dPMS. The effects of various seeding parameters (e.g., spheroid size, dPMS thickness) on cell viability, cell retention, cell morphology, and infiltration were analyzed.

Materials and

Methods: Decellularized porcine myocardial slices (dPMS) were produced following a previously published protocol. dPMS of 300 μm thickness were obtained via cryosectioning. hMSC Spheroids were fabricated with GFP transfected hMSCs (Cyagen, Passage 6) using 12-well Aggrewell™ 800 culture plates (STEMCELL Technologies). For spheroid morphological analysis, spheroids were fabricated at 500, 1000, 2000, 3000, and 5000 cells per spheroid and seeded on dPMS. Three days following seeding, cells were fluorescently imaged, and dPMS fiber orientation, spheroid morphology, and shape descriptors were analyzed using FIJI. Spheroids were fabricated at 1000, 3000, and 5000 cells per spheroid and seeded on dPMS for spheroid migration analysis. Cells were imaged using fluorescent tile scan microscopy on days 5, 10, and 15, and cellular migration was analyzed using FIJI.

Results, Conclusions, and Discussions: Extensive spheroid sprouting with precise alignment was observed in all spheroid size groups as early as day 3, as shown in Fig. 1A. Cells appeared more organized and show greater motility in the 1000, 2000, 3000, and 5000 cells/spheroid groups. Spheroids composed of 1000, 2000, 3000, and 5000 hMSCs showed a significantly higher degree of alignment than the group consisting of 500-cell spheroids (Fig. 1B). We were able to visualize proliferation, migration, and distinct morphological changes for seeded spheroids across the entire cultured area through day 15 (Fig. 2A). On days 10 and 15, seeded spheroids could migrate to inhabit a significantly greater area than on day 5 (Fig. 2B).
In this study, we have successfully developed a dPMS cell delivery and conditioning platform that can harness the benefits of hMSC-derived spheroids in cardiac cell therapy. So far, results indicate that hMSC spheroids firmly attach to the dPMS scaffold, align with its fibrous structure, and continue to migrate and proliferate throughout the scaffold for up to 15 days. We are currently investigating the further effects of spheroidal loading with various spheroid sizes and different thicknesses of dPMS. Additionally, we want to characterize and quantify the paracrine activity of the hMSC spheroids loaded onto the dPMS. The information obtained from our study will provide novel insights needed to improve decellularized cardiac tissue as a potential cardiac cell therapy.

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