Mechanical stimulation enhances development of scaffold-free, 3D-printed, engineered heart tissue grafts

Abstract

Current efforts to engineer a clinically relevant tissue graft from human-induced pluripotent stem cells (hiPSCs) have relied on the addition or utilization of external scaffolding material. However, any imbalance in the interactions between embedded cells and their surroundings may hinder the success of the resulting tissue graft. Therefore, the goal of our study was to create scaffold-free, 3D-printed cardiac tissue grafts from hiPSC-derived cardiomyocytes (CMs), and to evaluate whether or not mechanical stimulation would result in improved graft maturation. To explore this, we used a 3D bioprinter to produce scaffold-free cardiac tissue grafts from hiPSC-derived CM cell spheroids. Static mechanical stretching of these grafts significantly increased sarcomere length compared to unstimulated free-floating tissues, as determined by immunofluorescent image analysis. Stretched tissue was found to have decreased elastic modulus, increased maximal contractile force, and increased alignment of formed extracellular matrix, as expected in a functionally maturing tissue graft. Additionally, stretched tissues had upregulated expression of cardiac-specific gene transcripts, consistent with increased cardiac-like cellular identity. Finally, analysis of extracellular matrix organization in stretched grafts suggests improved remodeling by embedded cardiac fibroblasts. Taken together, our results suggest that mechanical stretching stimulates hiPSC-derived CMs in a 3D-printed, scaffold-free tissue graft to develop mature cardiac material structuring and cellular fates. Our work highlights the critical role of mechanical conditioning as an important engineering strategy toward developing clinically applicable, scaffold-free human cardiac tissue grafts.

Keywords: engineered heart tissue; human-induced pluripotent stem cells (hiPSCs); maturation; mechanical microenvironment; tissue engineering.

FIGURE 1

Engineered heart tissue grafts are created using a scaffold-free, 3D-printing method, then stretched after mounting onto polydimethylsiloxane molds. (a) Human-induced pluripotent stem cell derived cardiomyocytes, cardiac fibroblasts, and human umbilical vein endothelial cells were combined in a ratio of 70%:15%:15% and incubated for 3 days to form cell spheroids. The spheroids are placed onto a stainless steel needle array using a vacuum operated 3D tissue printer, and allowed to culture for 3 days to allow for tissue fusion prior to removal from the stainless steel needle array. (b) Polydimethylsiloxane is cured around a plastic master to form a mold. After the mold is sterilized, stretched tissues are mounted onto the mold while unstretched tissues are allowed to culture free floating in media [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Mechanical stimulation of engineered heart tissue (EHT) grafts results in maintenance of gross tissue morphology and sarcomere elongation as early as 1 week after culture. (a) Light microscopy of an engineered heart tissue immediately after removal from the stainless steel needle array. The tissue (upper) is composed of individual spheroids (lower) of cells (scale bar = 1000 μm for the tissue and scale bar = 200 μm for the spheroid). Gross images of stretched (b) and unstretched (c) engineered heart tissues after 4 weeks of culture. Minor scale marks = 1 mm. (d) Immunohistochemical analysis of unstretched and stretched tissues at 1 and 4 weeks of culture (green = troponin T and blue = DAPI. Scale bar = 10 μm). Quantification of the average sarcomere length at 1 week (e) and 4 weeks (f) of culture; bars represent mean ± standard deviation, n = 3. *p < 0.05 and **p < 0.001 [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Mechanical conditioning of engineered heart tissue grafts results in directional deposition and organization of extracellular matrix. (a) Immunofluorescent staining for collagen I, collagen III, and vimentin revealed increased directionality of extracellular matrix proteins and intermediate filament proteins in stretched compared to unstretched tissues (red = collagen I, collagen III, or vimentin, blue = DAPI; scale bar = 10 μm). (b–d) Quantification reveals increased area of expression per cell for collagen I in the unstretched compared to the stretched tissues, and no significant difference in area of expression in collagen III and vimentin between the two groups. (e–g) Anisotropy increased for collagen I and collagen III fibers and vimentin intermediate filaments in the stretched compared to the unstretched tissues (bars represent mean ± standard deviation, *p < 0.05 and **p < 0.001, n = 3) [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Mechanical stimulation improves flexibility and increases maximal contractile force in engineered heart tissue grafts. (a) Schematic annotation of the electromagnetic tweezers apparatus used to pull on tissue-bound electromagnetic particles. This setup allows measurement of the elastic modulus of the tissue. (b) Maximal contractile force generated by stretched versus unstretched tissues at 4 weeks of culture. (c) Normalized elastic modulus for stretched and unstretched tissues at 4 weeks of culture (bars represent mean ± standard error, *p < 0.05, n = 4) [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

Mechanical stimulation upregulates cardiac-specific gene expression in engineered heart tissue (EHT) grafts. Statistically increased expression of MYH7, CASQ2, and SERCA2 was found in stretched compared to unstretched EHT grafts, as queried by RT-qPCR (bars represent mean ± 95% confidence interval, n = 6; *p < 0.05)