3D biofabrication of microtissues with human induced pluripotent stem cell derived cardiomyocates and human foetal cardiac fibroblasts in CELLINK hydrogels
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.
To assess the application of CELLINK GelMA, GelXA, and GelXA LAMININK 521 (in graphs shortened GelLAM) in 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 foetal cardiac fibroblasts (hfCF) in the hydrogels. These were used to fabricate 3D microtissues to establish a coculture model.
BACKGROUND AND RESULTS
iPSC-CM in coculture 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, displayed in the adjacent figure.
In addition, the beating rate of the cells in the constructs was quantified on multiple days to assess the functionality of the constructs. GelMA 30s demonstrated the highest beating rates, matching human resting heart rates of 40-60 bpm displayed in the adjacent figure. Interestingly, the GelXA LAMININK 521 60s constructs reached a beating rate of 40 bpm on day five and remained at that rate, indicating stability in the constructs. For GelMA 60s a reduction in beating rate on days seven and 10 was observed; to understand this behavior, the experiment should be repeated with a larger database of measurements to increase robustness. Variation of the beating rate could be due to the timing of the experiments in relation to recent media changes.
Interestingly, the GelXA LAMININK 521 and GelMA groups displayed uniform, synchronized contractions without electric stimulation resembling functionality in the native myocardial tissue. 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 30s and 60s were asynchronous between multiple clusters of contracting cells. This indicates that the GelXA gels were not sufficiently rearranged by the hfCFs to allow coupling of cardiomyocytes.
Finally, constructs were stained with Hoechst nuclear stain and Vimentin antibody. The adjacent figure visualizes iPS-CMs and hfCFs with Hoechst nuclear stain (blue) and vimentin (red) in hfCFs in A) GelMA 30s, B) GelXA 30s, C) GelLAM 30s, D) GelMA 60s, E) GelXA 60s, F) GelLAM 60s. Vimentin is an intermediate filament protein present in hfCFs, enabling visualization of cell orientation in the construct. Zuppinger et al. identifies Vimentin in human iPSCs; however, co-expression of Vimentin with cardiac marker c-Troponin has not been observed in iPSC-CMs in our lab. In all constructs, parallel alignment of cells is observed. To distinguish between iPSC-CMs and hfCFs, repetition with the addition of cardiomyocyte specific markers such as alpha-actinin or c-Troponin would be advised.
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 coculture model that can be used as a foundation for tissue engineering applications and for developing a high-throughput model for drug testing.
Zuppinger, C. Gibbons, G. Dutta-Passecker, P. Segiser, A. Most, H. Suter, T.M. Characterization of cytoskeleton features and maturation status of cultured human iPSC-derived cardiomyocytes. Eur J Histochem. 2017. 61. 2763.