PhD Scientific Days 2025

Pharmaceutical Sciences and Health Technologies III.

Development of a 3D HL-1 Cardiomyocyte Model System Using Self-Assembling Peptide

Name of the presenter

Szász Zsófia

Institute/workplace of the presenter

Department of Genetics, Cell- and Immunobiology, Semmelweis University

Authors

Zsófia Szász¹, Petra Pőcze¹, Kata Nóra Enyedi², Dorina Lenzinger¹, Angéla Takács¹, László Kőhidai¹, Eszter Lajkó¹

1: Department of Genetics, Cell- and Immunobiology, Semmelweis University
2: Institute of Chemistry, Eötvös Loránd University

Text of the abstract

Introduction: Drug development begins in the preclinical phase with target identification and in vitro testing. There has been a growing focus on developing advanced 3D cell culture models that better replicate the complexity of in vivo tissues compared to traditional 2D monolayer cultures. These models not only allow for a more accurate understanding of drug mechanisms but also offer improved prediction of potential side effects (e.g. cardiotoxicity) before in vivo experiments.
Aim: The objective of this study was to develop a hydrogel-based 3D cardiomyocyte model using a self-assembling peptide (EAK-16-II). We also aimed to investigate (i) the effects of extracellular matrix (ECM) components - specifically fibronectin and laminin - (ii) and drug (daunorubicin – Dau) targeting peptide conjugates on spheroid size and cell viability.
Methods: HL-1 mouse cardiomyocytes were cultured in a self-assembling peptide hydrogel matrix for 3 or 6 days, with or without fibronectin or laminin. Spheroid size was measured using ImageJ from ZEISS Celldiscoverer7 images. Cardiomyocyte markers were analyzed via indirect immunocytochemistry using a Leica SP8 confocal microscope. To evaluate the model, 3-day spheroids were treated with Dau-peptide conjugates for 3 days, and viability was assessed with the CellTiter-Glo 3D assay.
Results: After 3 days, HL-1 cells formed spheroids within the hydrogel matrix with a mean diameter of 33±9.1 μm, increasing to 38±11.1 μm by day 6. The spheroids were cultured for up to a period of 6 days, and there was no decrease in cell viability. Adding fibronectin or laminin did not significantly alter spheroid size or viability. The HL-1 cells retained their cardiomyocyte markers, including α-actinin and caveolin, within the spheroids. Upon treatment with the Dau-based peptide conjugates, the 3D model exhibited greater sensitivity compared to 2D cultures, as evidenced by cell viability remaining above 50% in the 3D model, while viability in the 2D cultures stayed above 75%.
Conclusion: We successfully developed a 3D cardiomyocyte model using the self-assembling EAK-16-II peptide hydrogel. Although the resulting spheroids were relatively small, this platform shows promise for future development as a preclinical tool to evaluate cardiotoxic side effects of drug candidates prior to in vivo testing.
Funding: RRF-2.3.1-21–2022-00003, EKOP-2024-120