Engineering a 3D human myocardial tissue model

Casper van der Ven and Nino Chirico are PhD students in the Regenerative Medicine Programme at Utrecht University, the Netherlands. They research cardiac tissue engineering and regeneration under supervision of Professor. J.P.G. Sluijter and Dr. A. van Mil in the Experimental Cardiology department of the University Medical Center Utrecht.

Aim

To assess the application of CELLINK GelMA, GelXA, and GelXA LAMININK 521 (in graphs shortened GelLAM) a 3D human in vitro model of myocardial tissue was engineered. Induced pluripotent stem cells (iPSC) were differentiated into cardiomyocytes (CMs) and combined with human fetal cardiac fibroblasts (hfCF) in the hydrogels. These were used to fabricate 3D microtissues to establish a co-culture model.

The G-code to this three-layered grid structure can be found on Bioverse!

Increasing cell viability

iPSC-CM in co-culture with hfCFs in a near-native concentration were mixed into GelMA, GelXA, or GelXA LAMININK 521 and cultured for 11 days at 37°C and 5% CO2. Two different photocrosslinking times were evaluated; 30 and 60 seconds with a 405 nm module.

An Alamar Blue assay was employed to assess cell viability and an increase in cell viability seven days compared to three days after bioprinting was observed across all conditions, displayed in the adjacent figure.

Constructs match human beating rates

In addition, the beating rate of the cells in the constructs was quantified on multiple days to assess the functionality of the constructs. GelMA 30 s demonstrated the highest beating rates, matching human resting heart rates of 40-60 bpm displayed in the adjacent figure. Interestingly, the GelXA LAMININK 521 60 s constructs reached a beating rate of 40 bpm on day five and remained at that rate, indicating stability in the constructs. For GelMA 60 s a reduction in beating rate on day seven and ten was observed; to understand this behavior, the experiment should be repeated with a larger database of measurements to increase robustness.

Synchronized contractions

Interestingly, the GelXA LAMININK 521 and GelMA groups displayed spontaneous uniform, synchronised contractions, resembling functionality in the native myocardial tissue. The contraction of the GelXA LAMININK 521 at day seven can be seen in the adjacent video. Synchronous contractions are indicative of cardiomyocyte networking, enabling intracellular communication and transfer of action potentials to neighboring cells. The GelXA groups did not display these kinds of contractions during the experiment. The contractions observed in the GelXA 30 s and 60 s were asynchronous between multiple clusters of contracting cells.  This indicates that the GelXA gels were not sufficiently re-arranged by the hfCFs to allow coupling of cardiomyocytes.

Parallell cell alignment

Finally, constructs were stained with Hoechst nuclear stain and a Vimentin antibody. The adjacent figure visualizes iPS-CMs and hfCFs with Hoechst nuclear stain (blue) and vimentin (red) in hfCFs in A) GelMA 30 s, B) GelXA 30 s, C) GelLAM 30 s, D) GelMA 60 s, E) GelXA 60 s, F) GelLAM 60 s, scale bars 50 µm. In all constructs, parallel alignment of cells is observed.

Conclusion

This preliminary data show that both GelMA and GelXA LAMININK 521 are promising candidates in establishing a 3D human myocardial tissue model. Synchronous, uniform contractions at rates resembling the resting heart rate in humans were observed in these groups. Further fine-tuning of printing parameters and cell culture conditions will hopefully lead to a reproducible co-culture model that can be used as a foundation for tissue engineering applications and for developing a high-throughput model for drug testing.

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