An Ionically Conductive Hydrogel Amplifies Cardiomyocyte Signaling in Vitro

An Ionically Conductive Hydrogel Amplifies Cardiomyocyte Signaling In Vitro

Authors: Dominic Pizzarella^*1, Katelyn Neuman¹, Anthony Zappala¹, Ryan Koppes, PhD¹

¹Northeastern University Department of Chemical Engineering, Boston MA

Introduction: According to the World Heart Federation (WHF), cardiovascular diseases are the world’s leading cause of death, with 20.5 million deaths in 2021 alone [1]. The lack of treatment options is partly due to limitations in translatability of conventional screening platforms, 2D monolayer cultures and animal models, to human physiology [2]. Considering these limitations, hydrogels have shown promise as an alternative screening platform. Hydrogels are 3D crosslinked networks of hydrophilic, insoluble polymers [3]. These characteristics allow emulation of the native Extracellular Matrix (ECM) which results in a biocompatible material with tunable properties [3]. Despite these advances, it remains difficult to characterize electrophysiology in the hydrogel environment because of the intrinsic insulation properties of conventional hydrogel polymers [4]. This project investigates an ionically conductive hydrogel’s ability to amplify action potentials (AP) from a culture of neonatal rat Cardiomyocyte (CM) cells. This work seeks to establish a biocompatible, ionically conductive, tunable scaffold that could create a physiologically accurate platform to screen for cardiovascular disease medications.

Materials and Methods: Ionic Liquid (IL) and Gelatin Methacrylate (GelMA) were synthesized based on previous methods [5,6]. Proton Nuclear Magnetic Resonance (¹H NMR) was conducted after synthesis to confirm the chemical structure. The hydrogels were fabricated with a photo-initiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), using blue light (405 nm). To assess biocompatibility, a Live/Dead Viability assay and Immunocytochemistry were performed [6]. The elastic modulus was investigated following prior protocols [6] CM signaling was assessed by recording the AP and field potential with a microelectrode array (MEA). Before primary cell isolation, a chip was assembled onto a MEA (Figure 1A). Cells were encapsulated into a Gel-Amin (8% Gelatin Methacryloyl + 3.5% Choline Acrylate) or 9% GelMA control (n=2) in a cell density of 1e5 cells/µl. CM AP activity (i.e., Spikes) was monitored on day 7 post-seeding with a spike detection threshold of 20 µV. Spikes were analyzed using the MEAnalyzer tool [7].

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Results: There were no significant differences in cell viability between the Gel-Amin and GelMA hydrogels (Figure 1 B). MEA testing revealed that incorporation of an IL significantly amplified action potentials (AP). This resulted in an interconnected, synchronous CM culture. The raster plot (Figure 1 C) shows a time stamp whenever a spike was recorded by the MEA. Based on the spike threshold, the Gel-Amin hydrogel was able to amplify CM signaling. This property allowed for a synchronous CM culture that was efficiently monitored by a MEA. Conversely, the GelMA hydrogel showed little activity which highlights the scaffolds limitations for excitable tissue studies.

Conclusion: The ionically conductive, Gel-Amin hydrogel overcame the insulating nature of the GelMA polymer. This allowed for amplification of APs and an interconnected CM culture in vitro. Future work will be done to apply the Gel-Amin hydrogel to the development of a physiologically relevant heart-on-a-chip system.

References: [1] Deaths from Cardiovascular Disease Surged 60% Globally over the Last 30 years: Report. World Heart Federation. https://world-heart-federation.org/news/deaths-from-cardiovascular-disea.... [2] Marei I, et al. 3D Tissue-Engineered Vascular Drug Screening Platforms: Promise and Considerations. Frontiers in Cardiovascular Medicine 2022, 9, 847554. https://doi.org/10.3389/fcvm.2022.847554. [3] Sánchez-Cid, Pablo et al. “Novel Trends in Hydrogel Development for Biomedical Applications: A Review.” Polymers vol. 14,15 3023. 26 Jul. 2022, doi:10.3390/ [4] Del Valle, L et al. (2017). Hydrogels for Biomedical Applications: Cellulose, Chitosan, and Protein/Peptide Derivatives. Gels (Basel, Switzerland), 3(3), 27. https://doi.org/10.3390/gels3030027 [5] I. Noshadi Sci. Rep., (2017) 4345 [6] J. Soucy. Tissue Eng. Part A., (2018) 1393-1405. [7] Dastgheyb, Raha M et al. “MEAnalyzer - a Spike Train Analysis Tool for Multi Electrode Arrays Neuroinformatics vol. 18,1 (2020): 163-179. doi:10.1007/s12021-019-09431-0