Mechanical Stimulation Enhances Development of Scaffold‐Free, 3D‐Printed, Heart Tissue Grafts

Mechanical Stimulation Enhances Development of Scaffold‐Free, 3D‐Printed, Engineered Heart Tissue Grafts

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 their 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,

they 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, their 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. Their work highlights the critical role of mechanical conditioning as an important engineering strategy toward developing clinically applicable, scaffold-free human cardiac tissue grafts.

The adult human heart is unable to naturally recover from severe traumatic, ischemic, or chronic damage, due to its limited regenerative potential. Although heart transplantation can address end-stage heart failure, organ shortages limit therapeutic availability. Therefore, human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have been used to develop engineered heart tissues, with the ultimate goal of using in vitro grown tissues to surgically repair injured human hearts. Recreating the material and cellular properties of adult cardiac tissue has proven to be a complex engineering challenge. Many approaches to date focus on crafting a scaffolding material on to which cardiac myocytes are embedded and grown. For example, cardiomyocytes from neonatal rats can be mixed with collagen-I and extracellular matrix factors, then mechanically conditioned, after which they adopt an adult-like phenotype. Cardiomyocytes for human technology would ideally be derived from hESCs or hiPSCs. Despite initial successes, multi-factorial interactions emerged between the embedded cells and their surroundings that highlight unsolved difficulties in tuning engineered scaffolds. In a simple example, an excessive added extracellular matrix yields a stiff microenvironment, which can result in overexertion and subsequent failure of the embedded cardiomyocytes. Conversely, insufficient extracellular matrix material yields a tissue that is too weak to be mechanically stressed, in turn causing cardiomyocyte differentiation. Various spatiotemporal feedback effects present in the developmental context which affects cell identity must be accounted for, including but not limited to biochemical, electrical, mechanical, and shear stress necessary for proper function. Although some groups have achieved success in guiding hiPSC-derived CMs grown in

scaffolding toward in vitro adult physiologic functionality, alternatively called a matured state, the

scaffolding material itself may still be immunogenic, result in toxic degradation products, interfere with cell-to-cell connections, promote fibrous tissue formation during degradation, or be a mechanical mismatch with the recipient tissue microenvironment.

Mechanical stimulation of EHT grafts results in maintenance of gross tissue morphology and sarcomere elongation as early as one 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 four weeks of culture. Minor scale marks

= 1 millimeter. (D) Immunohistochemical analysis of unstretched and stretched tissues at one and four weeks of culture (green = troponin T and blue = DAPI. Scale bar = 10 µm). Quantification of the average sarcomere length at one week (E) and four weeks (F) of culture; bars represent mean ± standard deviation, n=3. *p<0.05, **p<0.001.

Their 3D-printing approach to growing engineered heart tissue, as described here, is designed to circumvent these complexities of a typical strategy that would otherwise use externally supplied scaffold or extracellular matrix as a major tissue-structuring substrate. Instead, they use a scaffold-free 3D-printing process to position and grow hiPSC-derived cardiomyocytes, then rely on the formation of biologically-deposited and regulated extracellular matrix supplied by human fibroblasts intercalated within the engineered graft. They find that the application of static stress to their 3D-printed engineered heart tissue grafts promotes their maturation by regulating the formation of extracellular matrix, improving their super-cellular structuring and mechanical capabilities, and upregulating expected cardiac cell fate markers1.

Engineered heart tissue (EHT) grafts are created using a scaffold-free 3D-printing method, then stretched after mounting onto polydimethylsiloxane molds.

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.

  1. Lui, C., Chin, A. F., Park, S., Yeung, E., Kwon, C., Tomaselli, G., Chen, Y., & Hibino, N. (2021). Mechanical Stimulation Enhances Development of Scaffold-Free, 3D-Printed, Engineered Heart Tissue Grafts. Journal of tissue engineering and regenerative medicine, 10.1002/term.3188. Advance online publication.

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