Poster U6 - Cardiac ECM-derived microgels for hiPSC-CMs culture

Introduction: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) present unprecedented opportunities in disease modeling, drug discovery, and regenerative medicine [1]. However, conventional culture methods inadequately recapitulate the intricate three-dimensional (3D) microenvironment of native cardiac tissue, resulting in suboptimal cell viability, function, and maturation [2, 3]. To overcome these challenges, there is a growing interest in the development of biomimetic scaffolds that can provide the necessary cues to support hiPSC-CMs growth and maturation in vitro. Recent studies highlight the significance of cardiac-derived extracellular matrix (ECM) in providing structural and biochemical cues for left ventricular remodeling [4]. While ECM-based hydrogels have been extensively applied for heart disease studies [5], traditional bulk hydrogels exhibit significant limitations in promoting cell and vessel invasion due to their nano-porous structure [6]. Microgels have emerged as a promising platform for cell culture, offering a 3D microenvironment conducive to cell growth and function. In this study, we aim to combine the advantages of microgels and ECM to investigate the feasibility of cardiac-derived ECM microgels as a scaffold for the culture of hiPSC-CMs and to assess their impact on CMs viability, function, and maturation.

Materials and

Methods: Porcine myocardium-derived ECM was prepared according to a published protocol [7]. Cardiac-ECM microgels were fabricated by electrospray. Briefly, the neutralized cardiac ECM solution (12.5 mg/mL) was mixed with sodium alginate (SA) to obtain a final concentration of ECM (4, 6, and 8 mg/mL) and a constant concentration of 2% SA. ECM/SA solution was loaded into a syringe with a 26G blunt needle for electrospray (Voltage: 17.5kV), and 3% CaCl2 solution was used as a collection container. The obtained ECM/SA microgels were crosslinked with 0.5% glutaraldehyde, followed by treated with 5% sodium citrate to remove SA. The morphology of ECM microgels was observed using SEM. The swelling ratio and water content of ECM microgels were measured. H9C2 cells were used to evaluate the biocompatibility of ECM microgels. hiPSC-CMs maturation on ECM microgels was assessed by immunostaining, calcium transient, and gene expression.

Results, Conclusions, and Discussions: Three different concentrations of ECM microgels were fabricated with diameters of around ~300 µm, and the morphology of ECM microgels is spheric with interconnected pores and fibrous structure. All microgels had a similar swelling ratio and water content. The relative cell growth rate (compared to day 1) of H9C2 cells on ECM microgels was significantly higher than the TCP group. The differentiated H9C2 cells exhibited greater muscle bundle-like structures and higher α-actinin expression on ECM microgels compared with the TCP group on day 7. For hiPSC-CM maturation, the beating frequency of CMs on ECM microgels was faster than that of the TCP group. The time to peak time and mean amplitude on ECM microgels were greater than the TCP group on day 14. The α-actinin, connexin-43, and Troponin T expression of CMs on ECM microgels were significantly upregulated compared to the TCP group on day 14. The therapeutic effectiveness of ECM microgels is currently being evaluated in a rat acute MI model.

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