(K-413) Identifying Cell Number Ratios For Producing Cardiac Tissue With Favorable Contractile Function And Vascularization

Introduction:: Cardiovascular disease is the leading global cause of mortality. Patients with heart disease suffer from the loss or damage of heart tissue, resulting in significant impairment of cardiac functionality (Lam & Wu., 2012). The current demand for heart transplants is high and continues to increase every year, whereas the number of donor hearts stays relatively the same per year. This critical disparity between demand and supply underscores the need for alternative solutions. Myocardial tissue engineering, utilizing hiPSC-derived cardiomyocytes, has emerged as a promising approach to produce bioartificial cardiac tissues (BCTs) that closely resemble human heart tissue. BCTs offer potential regenerative or replacement options for diseased cardiac tissue. However, achieving functional and structurally similar BCTs requires consideration of various cell types found in cardiac tissue, including endothelial cells, cardiac fibroblasts, and pericytes. To attain favorable contractile function and efficient vascularization, it is crucial to determine the optimal cell number ratios for producing BCTs. Additionally, the establishment of a well developed endothelial network within the tissues is vital for proper nutrient diffusion. In this context, this study aims to explore suitable cell number ratios and endothelial network development in BCTs. By understanding these factors, we can pave the way for more effective tissue engineering strategies and address the urgent need for alternative treatments for heart disease patients.

Materials and Methods:: Prior to BCT production, we calculated the cell number ratio in Table1 for each experimental group where the four groups contain varying cell compositions with or without pericytes. To start BCT production we prepared a falcon of each combination of hiPSC-derived cells and combined these with a human collagen and Geltrex matrix. This solution was then dispensed in 6.0mm silicone molds between two removable titanium rods. As the tissues form and solidify, they will adhere to the titanium rods, allowing for the transportation and stretching of the tissues over the next twenty-one days. To promote maturation of the BCTs, they are stretched in increments of 0.4mm on day seven, eleven, fifteen, and nineteen. To assess the endothelial network formation and the remodeling of the BCTs, we capture bright field and fluorescent images on each of the stretching days. On day twenty-one, the tissues will undergo a force measurement to quantify the contractile forces for comparison between each group. 

Results, Conclusions, and Discussions:: Suitable cell number ratios are determined by the tissues’ contractile forces and their ability to form and maintain an endothelial network. In Figure 1, the active forces of test 1, test 2, and the control group were relatively similar, while the fetal heart group exhibited higher contraction forces when subjected to electrical stimuli. Furthermore, as shown in Figure 2, the fetal heart group expressed forces of 3.4 mN/mm^2, whereas the control group produced only around half that amount, with forces of approximately 1.7 mN/mm^2. This difference between the experimental groups is also evident in Figure 3, where the fetal heart group presented a higher range of stiffness compared to the other groups. The clear demarcation between the experimental groups, especially the fetal heart group in comparison to the other compositions, reinforces these findings. Importantly, an ANOVA test confirmed the statistical significance of these differences. Figure 4 illustrates the development of an endothelial network on day eleven in all four groups, but this network significantly disappears on day twenty-one for the test groups, whereas some of the network remains intact in the control and fetal heart group. Based on the force measurement results and the fluorescent pictures on day twenty-one, we can conclude that the fetal heart group is a suitable ratio for tissue production involving this combination of cell types. The potential applications of this research include the production of bioartificial cardiac tissues with favorable contractile forces and an endothelial network.

Acknowledgements (Optional): : This research was made possible by the National Science Foundation's (NSF) International Research Experience for Students (IRES) Track I: Process Development for Cell and Tissue Biomanufacturing (Award Number: 1952614) in collaboration with Dr. Elizabeth Lipke and Dr.Robert Zweigerdt at Auburn University and Medizinische Hochschule Hannover. I would also like to thank my mentor Rajesh Vivekanandan and my Primary Investigator Dr. Ina Gruh.

References (Optional): : Lam, M. T., & Wu, J. C. (2012). Biomaterial applications in cardiovascular tissue repair and regeneration. Expert review of cardiovascular therapy, 10(8), 1039–1049. https://doi.org/10.1586/erc.12.99