Poster U10 - Conductive Microfibers Improve Stem Cell-Derived Cardiac Spheroid Maturation

Introduction: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) serve as potential platforms for disease modeling and high throughput drug screening. While 2D hPSC-CM platforms have been extensively explored, these models inherently lack the structural and functional complexity of 3D constructs, which balance tissue maturity and throughput. Though cardiac spheroids allow for high-throughput assays and improved function over 2D models, spheroids lack the functional maturity of more complex models. We have previously shown that conductive polymer microfibers improve 2D hPSC-CM maturity, hence we hypothesize here that the addition of conductive microfibers to 3D cardiac spheroids will accelerate hPSC-CM maturation.

Materials and

Methods: Poly(vinyl alcohol) (PVA) was blended with DI water or poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) to create non-conductive or conductive PEDOT:PSS/PVA polymer scaffolds via electrospinning. Electrospun scaffolds were frozen in DI water and cryo-sectioned at 5, 10, or 20 µm sections to create a library of microfiber lengths. Resulting microfiber solutions were filtered with a 100 µm strainer, lyophilized, and resuspended at a known concentration. Day 15 hPSC-CMs were dissociated and combined with non-conductive or conductive microfibers at 50,000 cells/well at either varying fiber masses or fiber lengths in 96-well u-bottom plates. Cardiac spheroid formation and contractility parameters were tracked over 15 days.

Results, Conclusions, and Discussions: Electrospun scaffolds were cryo-sectioned to create a library of non-conductive and conductive microfibers with variable lengths (Fig. 1a). With fixed 5 µm sectioned scaffolds, we assessed cardiac spheroid formation with 10, 20, or 40 µg of microfibers per spheroid. By tracking the percentage of beating spheroids over time, we found that spheroids formed with 40 µg of conductive microfibers maintained the highest percentage of beating constructs over time, but experienced poor compaction; therefore, spheroids with 20 µg of microfibers (Fig. 1b) were formed with varying fiber lengths to further probe the effect of microfiber length on cardiac spheroid performance. Beats per minute increased, while peak to peak, time to peak, and peak width decreased in the presence of conductive fibers (Fig. 1c), suggesting an improvement in the contractility of conductive cardiac spheroids compared to control spheroids without microfibers or with non-conductive microfibers. Additionally, we found parameter variance decreased over time in our conductive spheroids and was most evident in spheroids formed with 5 µm sectioned microfibers (Fig. 1d). Microfibers sectioned at 5 µm result in fiber ranges from ~2-20 µm, compared to ~3.6 to 21.9 µm for collagen gel fibers, suggesting that recapitulating the morphology of natural extracellular matrix (ECM) fibers is important but that functionality is further improved in the presence of PEDOT:PSS, likely due to its ability to enable electrical cell-cell communication. Thus, cryo-sectioned electrospun microfibers are a viable source of conductivity to improve 3D cardiac spheroid maturity. By recapitulating the morphology of native ECM fibers while providing a source of conductivity, we can facilitate cell-cell electrical communication in distant hPSC-CMs to improve functionality while maintaining a high throughput cardiac spheroid model.

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