Abstract Body: Introduction: Human pluripotent stem cells (hPSC)-derived cardiac organoids (COs) have gained increasing interest in biotechnology due to their characteristics like diverse cardiovascular cell types, 3D complex structure, and function that recapitulates the human heart in early development. However, current methods for vascularizing COs are not systematic and robust.

Hypothesis: We hypothesize that culturing hPSC-derived COs dynamically will further enhance vascularization in COs.

Materials and Methods: Two conditions were created to compare vascularization in COs: 1) dynamic culturing (Dyn) and 2) static culturing (St). Firstly, hPSCs were cultured for two days in ultra-low attachment plates to develop spheroids. The spheroids were then transferred to pillar plates followed by direct cardiac differentiation the next day. Live-cell imaging was used to monitor the process of vascularization for up to 16 days and the corresponding fluorescence intensity of each cell type from its reporter expression was analyzed. The cardiac function was measured by video-based contractility analysis. Data were presented as mean ± standard error of the mean (SEM). The student’s t-test was used for the statistical analysis.

Results: Cardiomyocytes (CMs) and endothelial cells (ECs) were imaged on pillars under the two conditions as shown in Fig. 1A. Successful cardiac differentiation was present, displaying hPSC-CM contraction on day 12 and onwards. Higher intensity values of hPSC-CMs under Dyn were observed in all four days compared to St as shown in Figs. 1B&C. The hPSC-ECs intensity displayed greater intensity values compared to St, demonstrating enhanced vascularization in VCOs due to the active Dyn as shown in Figs. 1D&E. The functionality was improved in Dyn as indicated with higher contraction velocity as shown in Fig. 1F.

Conclusion: This study demonstrates the effects of incorporating dynamic culture for enhancing cardiogenesis and vasculogenesis in hPSC-derived COs. The enhanced hPSC-VCOs hold promise for studying early cardiovascular development, congenital heart disease modeling, and drug screening and discovery.