Cell Survival in Different 3D-Printed Geometric Forms of Human iPSC-Derived Engineered Heart Tissue

Background: Engineered Heart Tissue (EHT) is a promising technique to repair heart muscle defects in-vivo, and can additionally be used for pharmacological drug testing in-vitro. Due to the absence of an in-vitro vascularization, the geometry of EHTs crucially impacts nutrient and oxygen supply by diffusion capacity. We analyzed the surface-area-to-volume-ratio (AS/V) of different geometric forms to determine the ideal EHT shape in terms of in-vitro cell survival.

Methods: Different geometries with varying mathematically derived AS/V ratios were calculated (structure A (ring)AS/V=40 mm²/440 µL3, structure B (figure of eight) 26 mm²/440 µL3). Custom-made casting molds were 3D printed. Cardiomyocytes were differentiated from human induced pluripotent stem cells (hiPSC) by modulating Wnt/β-Catenin pathways. For EHT generation, 4*106 cardiomyocytes were combined with a fibrin/thrombin mixture to create three-dimensional constructs. Cell viability was evaluated during a longitudinal cultivation process of one week. EHTs were analyzed by histology, quantitative Real Time-PCR assays and cytometric studies. Cell survival was additionally visualized using bioluminescence imaging (BLI).

Results: Using 3D-printed casting molds, spontaneously beating EHTs can be generated in various geometric shapes. Histological experiments showed a higher cell survival for structure A compared with structure B. In both shapes, a dense network of Troponin-T positive cells was observed near the tissue surface while there was a lower cell number at the core of the EHT. The quantitative RT-PCR analyses (n = 8) showed a higher Troponin-T gene expression in shape A, (Ct Mean = 25.85 ± 1.37) compared with shape B (Ct Mean = 28.41 ± 2.34). In cytometric studies (n = 6) we evaluated 15% more Troponin-T cells in structure A with 73% (±12%), compared with B with 58% (±11%), p = 0.04. BLI (n = 8) demonstrated significantly higher cell survival in structure B (ROI= A:1.14*106p/s and B:8.47*105 p/s, p < 0.001) compared with structure A.

Conclusion: 3D-printing allows to create EHTs in all desired geometric shapes. Due to the absence of an in-vitro vascularization, surface-area-to-volume-ratio crucially impacts cell survival. Optimizing the geometry with a better surface-area-to-volume-ratio in structure A demonstrated a significantly higher cell survival measured by RT-PCR, Bioluminescence imaging and cytometric studies.