Engineering viscoelastic hydrogels for cardiomyocyte culture

Introduction: : Heart disease is the leading cause of death; however, research progress of cardiomyopathies has been limited by the lack of accurate models of human disease. Viscoelasticity in particular is important to model as it is exhibited by all tissues in the body, can influence several cell functions, and has been understudied with respect to the heart. Ultimately, we aim to measure the viscoelasticity of the heart to provide concrete measurements to incorporate into our engineered alginate biomaterials, to better understand how human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) are influenced by viscoelasticity.

Materials and

Methods: : The viscoelasticity of human tissue was evaluated by stress relaxation testing on a custom-built microindenter. We characterized the stress relaxation by quantifying the τ1/2, or the time a sample takes to reach half of its maximum value. To determine how viscoelasticity was influenced by fibrosis we performed picrosirius red staining to see the collagen fiber size, alignment and compaction. We engineered alginate hydrogels with stress relaxation properties similar to that of healthy cardiac tissue through PEG conjugation to enhance the stress relaxation. Grooves were imprinted onto the surface of the hydrogel using a PDMS mold. Hydrogels were then coated with Matrigel and Oregon Green for visualization of hydrogel surface. We evaluated how viscoelasticity influenced iPSC-CMs compared to elastic materials through confocal imaging of contracting cells.

Results, Conclusions, and Discussions:: We found that healthy human tissue becomes slower during Ischemic heart failure and correlates with fibrosis (Fig.1A-B). We then engineered alginate hydrogels with either viscoelastic ionic crosslinks or elastic covalent crosslinks. We added PEG spacers to the ionic gels to make the stress relaxation faster (Fig.1C). We found the alginate with PEG had the fastest relaxation time that was not significantly different from our healthy human tissue (Fig.1D). We engineered our material to exhibit grooves to facilitate iPSC-CM alignment (Fig. E-F). Cells on viscoelastic gels exhibited a more cylindrical morphology, reminiscent of cardiomyocytes in the heart (Fig. 1G,H). Additionally, the cells on viscoelastic gels beat at higher speed, as seen by the speed and displacement of the fluorescent beads in the gel (Fig. 1I). Our results indicate that the heart is fast relaxing, and this property can be mimicked in an alginate hydrogel system through PEG modification. We further showed that when seeding iPSC-CMs on these hydrogels, viscoelastic substrates exhibited a more cylindrical morphology and had faster and increased displacement during contraction. These data suggest that cardiomyocytes on viscoelastic substrates exhibit increased maturity, both morphologically and functionally. Future work includes looking at other maturity markers, such as calcium handling, cytoskeletal maturity, and differential gene expression as well as investigating how iPSC-CMs react to slower relaxing materials.

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