INTEGRATION OF CO-CULTURE CONDITIONS AND 3D GELATIN METHACRYLOYL HYDROGELS TO IMPROVE HUMAN-INDUCED PLURIPOTENT STEM CELLS-DERIVED CARDIOMYOCYTES MATURATION

Ilaria Gisone¹Monica Boffito²Elisa Persiani¹Roberta Pappalardo²Elisa Ceccherini¹Andrea Alliaud²Manuela Cabiati¹Rossella Laurano²Letizia Guiducci¹Chiara Caselli¹Rosetta Ragusa¹Claudio Cassino³Chiara Ippolito¹Silvia Del Ry¹Susanna Sartori²Antonella Cecchettini¹Salvador Fernández Arroyo⁴Gianluca Ciardelli²Federico Vozzi^1*

• ¹Pisa Research Area, National Research Council (CNR), Pisa, Tuscany, Italy
• ²Polytechnic University of Turin, Turin, Italy
• ³Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale, Alessandria, Piedmont, Italy
• ⁴Eurecat (Spain), Barcelona, Catalonia, Spain

Introduction: Human- induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an excellent alternative to animals for in vitro cardiac studies. However, their immature fetal phenotype represents an important limit to consider. Approaches proposed to overcome this issue are based on better reproducing the in vivo native CMs microenvironment. In the present work, a biomimetic environment to enhance hiPSC-CMs maturation was developed by combining a 14-day co-culture of hiPSC-CMs and Human Coronary Artery Endothelial cells (HCAECs) in a 3D Gelatin Methacryloyl (GelMA) hydrogel system. GelMA was successfully synthesized with two different DoMs (i.e., 30-40 % and 96-97%) and used to prepare hydrogels at 5, 7.5 and 10% w/v concentrations. Both GelMA DoM and hydrogel concentration appeared as tuning parameters of gel mechanical properties, with Young’s Modulus of photo-cured gels ranging between ca. 4 and 55 kPa. Within this plethora, photo-cured gels prepared from GelMA with ca. 96% DoM solubilized at 5% w/v concentration showed E value (8.70 ± 0.12 kPa) similar to the native cardiac tissue and were thus selected to design bioengineered cardiac tissue models upon hiPSC-CMs and HCAECs loading. A direct comparison with the classical 2D monoculture of hiPSC-CMs highlighted the improved maturation profile achieved by hiPSC-CMs in the 3D GelMA system, as demonstrated by the higher expression of cardiac maturation markers (TNNT2, ACTN2, Myl2, MYH 7, CX43 and PPAR-), in association with proteomics and transcriptomics data, that showed the modulation of specific biological pathways related to cardiac differentiation and contraction processes in the 3D system. A more in-depth investigation of cell health and function also suggested a higher viability and less suffering condition for cells co-cultured in the 3D hydrogel. Conclusion: Our results demonstrated that the 3D bioengineered model proposed here represents a good replica of the native cardiac tissue environment, improving the hiPSC-CMs maturation profile, thus opening the opportunity for its application in disease modeling and toxicological screening studies.