(J-400) Engineering viscoelastic alginate hydrogels for human induced pluripotent stem cell cardiomyocyte culture

Introduction:: Heart disease is the leading cause of death worldwide, however research progress of heritable cardiomyopathies has been limited by the lack of cost effective and accurate human models. Human induced pluripotent stem cells (hiPSCs) are a renewable cell source that can be differentiated into human cardiomyocytes (CMs), presenting a reliable tool for in vitro cellular models. The physiological environmental cues that CMs are exposed to are extraordinarily complex and in vitro systems typically fail to mimic key aspects. Viscoelasticity in particular is important to model as it is exhibited by all tissues in the body and can influence several cell functions including spreading, differentiation and proliferation. However, the majority of experiments with CMs to date have used hydrogels that do not exhibit stress relaxation. We hypothesize that culturing hiPSC-CMs on materials mimicking the viscoelasticity of the heart will increase translatability through promotion of a more mature cellular phenotype compared to elastic hydrogels. Our objective here is to replicate the viscoelastic characteristics of the heart through our alginate hydrogel platform.


Materials and Methods:: The viscoelasticity of porcine left ventricle cardiac tissue was tested through stress relaxation experiments on the rheometer and microindenter at 10% strain. Low molecular weight alginate was conjugated with 5 kDa PEG to modulate stress relaxation and crosslinked with 45mM CaSO₄ to control stiffness allowing us to mimic cardiac properties. Elastic alginate was made using a combination of high and low molecular weight alginate and was crosslinked using adipic acid dihydrazide (AAD). GFP α-actinin tagged hiPSC-CMs were seeded onto the hydrogels and their function and morphology were evaluated through calcium imaging, sarcomere imaging, and hydrogel displacement.

Results, Conclusions, and Discussions:: Through stress relaxation testing, we found that porcine cardiac tissue exhibited a fast relaxation profile, reaching half the maximum stress (τ_1/2) at less than 10 s. To replicate this property, we employed PEG-modified alginate to enable a faster stress relaxation. Using this method, we were able to successfully replicate the stress relaxation (heart = 5.2s alginate =16.2s) and elastic moduli (heart = 7.7kPa, alginate = 5.4kPa) properties of porcine left ventricle cardiac tissue (Figure 1A and B). Additionally, by utilizing alginate’s ability to be crosslinked covalently, we made an alginate hydrogel with the same elastic modulus that was not stress relaxing to use as a control for our cellular studies (Figure 1C and D). We further showed that hiPSC-CMs can be seeded onto our viscoelastic alginate platform. Through confocal imaging, we saw interactions between the GFP labeled α-actinin in the CMs and the red fluorescent beads entrapped in the hydrogel (Figure 1E). Although hiPSC-CMs are a power tool in human cardiac modeling, they exhibit properties closer to that of embryonic CMs than adult CMs. One way to increase CM maturity, is to better replicate microenvironmental cues the CMs are presented with in vivo, including stiffness, adhesion type, and mechanical and electrical stimulation. This work is important because it is the first investigation into replicating viscoelastic cardiac properties for hiPSC-CMs. This work will lead to more accurate cell cardiac models to aid in cardiac therapy development, as well as shed light on important cardiac biological processes. Our results show that our alginate platform is a valuable tool that can model the unique viscoelastic response of the heart. This is significant, as the vast majority of materials used for CM culture are elastic. It will further aid us in answering our ultimate question of how hiPSC-CM behave on physiologically relevant viscoelastic substrates compared to elastic.


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