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Tissue Engineering

Tissue Engineering - Poster Session C

Poster L4 - Influence of Microstructure Design on Cardiac Tissue Morphogenesis and Stability

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  The field of tissue engineering relies on combining cells and other materials in an optimal way to accurately mimic physiological microenvironments, allowing the generation and study of tissue functions in vitro. For cardiac tissue engineering applications, various platforms have been developed for drug testing, disease modeling, and regenerative medicine. Several platform designs are currently used to model three-dimensional (3D) environments of cardiomyocytes in vitro; however, these tissues fail over time due to lack of adhesion, high tension, or contractility.1 Given that the micromechanical characteristics of these platforms are finely tuned to adapt and influence the cardiac tissues’ morphology and function, modifying microstructure design can be a feasible strategy to stabilize and improve 3D-engineered tissues.
To investigate the effect of platform microstructures on cardiac tissue morphogenesis, stability, and function, we used two-photon direct laser writing (TPDLW) and polydimethylsiloxane (PDMS) casting techniques to generate 3D cardiac tissue culture platforms with varying stiffnesses and shapes. Two designs were tested, one with cantilever-like rectangular posts and another with cylindrical brush-like posts. Each design was then fabricated with PDMS using two different crosslinker ratios, 1:10 (E ~1 MPa) and 1:20 (E~2 MPa). Microtissues composed of human-induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts were generated on platforms with different elastic moduli and anchorages while morphological changes, structure, and function were monitored.

Materials and

Methods:  The molds were designed using computer-aided design (SolidWorks) and printed on silicon wafers using TPDLW (Photonic Professional GT+, Nanoscribe) and the commercially available photoresist IP-S (Nanoscribe). To remove the excess unpolymerized resist, the 3D-printed molds were submerged in poly ethyl glycol mono ether acetate for 1 hour. The PDMS devices were made using PDMS molding. Two groups of devices were fabricated by filling the printed mold with PDMS made with 1:10 and 1:20 ratios of cross-linker and prepolymer (SYLGARD 184, Dow). All the devices were left in a vacuum desiccator for at least an hour to ensure the removal of any air bubbles in the wells. The molds were then left in an oven at 60° C overnight until the PDMS was completely polymerized. Before cell seeding, the PDMS templates were sterilized in 70% ethanol followed by UV irradiation for 15 min and treated with 2% Pluronic F127 (BASF) to reduce cell adhesion. The hiPSCs featured in this study were created from the PGP1 donor from the Personal Genome Project. hiPSCs were maintained in Matrigel-coated until they reached sufficient (80 to 100%) confluency to initiate differentiation, following the procedure shown in Ref. 2. Microtissues were generated using the protocols described before.2,3 Briefly, cell suspensions (90% hiPSC-CMs and 10% fibroblasts) in collagen or fibrin-based hydrogels were placed in designed seeding wells. Morphologies of the microtissues compacting between the posts were characterized using optical microscopy (Nikon Eclipse Ti).

Results, Conclusions, and Discussions: Results and

Discussion: Here, we simultaneously varied the anchorage stiffness and post geometry of platforms designed for making cardiac microtissues. The device with lower modulus (1 MPa) showed increased bending of the micropillars before tissue culture compared to the device with higher modulus (Fig. 1b). Front views of these posts, and cardiac microtissues formed between them are shown in Fig. 1c. Preliminary findings indicate that the tissues remained attached only to devices with thinner, brush-like posts after two weeks, suggesting these structures provide optimal conditions for tissue stability. Fig. 1d illustrates the spontaneous contractions recorded from the top and bottom of cardiac tissue, with the peak displacement of the top and the bottom layer being approximately 32 um and 15 um, respectively. This difference in contractility possibly occurs due to the stiffer environment at the base and the softer environment at the top, demonstrating that anchorage geometries lead to morphology and contractility changes even within one tissue.

Conclusion: The study demonstrates that varying the mechanical and geometric properties of 3D culture platforms significantly affects cardiac tissue stability and morphology. Devices with thinner, brush-like structures support better tissue attachment, suggesting that optimizing these properties can enhance tissue culture outcomes. These insights are crucial not only for cardiac tissue engineering but also have broader implications in other areas of biomedical research, such as regenerative medicine, where the mechanical environment plays a key role in cell behavior and tissue formation.

Acknowledgements (Optional):  This work was supported by NSF CELL-MET ERC award no. EEC-1647837. Researchers on this project received support from the Biological Design Center of Boston University and STEM Pathways of Boston University (Department of Defense DoD STEM FY20 Award (HQ00342110008).

References:
(1) H. Wang et al., “Necking and failure of constrained 3D microtissues induced by cellular tension,” Proceedings of the National Academy of Sciences, vol. 110, no. 52, pp. 20923–20928, Dec. 2013, doi: https://doi.org/10.1073/pnas.1313662110.
(2) R. K. Jayne et al., “Direct laser writing for cardiac tissue engineering: a microfluidic heart on a chip with integrated transducers,” Lab on a Chip, vol. 21, no. 9, pp. 1724–1737, 2021, doi: https://doi.org/10.1039/d0lc01078b.
(3) M. Ma et al., "High throughput screening system for engineered cardiac tissues." Frontiers in Bioengineering and Biotechnology 11 (2023): 1177688.