Development of a three-dimensional pre-vascularized scaffold-free contractile cardiac patch for treating heart disease

Noguchi, Ryo; Nakayama, Koichi; Itoh, Masatoshi; Kamohara, Keiji; Furukawa, K; Oyama, Jun‐ichi; Node, Koichi; Morita, Shigeki

doi:10.1016/j.healun.2015.06.001

Cited by 100 publications

(83 citation statements)

References 25 publications

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“…A fixed endothelial cell component of 15% was chosen based on a previous report demonstrating prevascularization of cardiac patches using endothelial cells 21 . Cardiospheres did not form when only hiPSCs or hiPSC-CMs were used; (Figs S2 and 3A,B).…”

Section: Resultsmentioning

confidence: 99%

Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Ong

Fukunishi

Zhang

et al. 2017

Sci Rep

216

169

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We have developed a novel method to deliver stem cells using 3D bioprinted cardiac patches, free of biomaterials. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), fibroblasts (FB) and endothelial cells (EC) were aggregated to create mixed cell spheroids. Cardiac patches were created from spheroids (CM:FB:EC = 70:15:15, 70:0:30, 45:40:15) using a 3D bioprinter. Cardiac patches were analyzed with light and video microscopy, immunohistochemistry, immunofluorescence, cell viability assays and optical electrical mapping. Cardiac tissue patches of all cell ratios beat spontaneously after 3D bioprinting. Patches exhibited ventricular-like action potential waveforms and uniform electrical conduction throughout the patch. Conduction velocities were higher and action potential durations were significantly longer in patches containing a lower percentage of FBs. Immunohistochemistry revealed staining for CM, FB and EC markers, with rudimentary CD31+ blood vessel formation. Immunofluorescence revealed the presence of Cx43, the main cardiac gap junction protein, localized to cell-cell borders. In vivo implantation suggests vascularization of 3D bioprinted cardiac patches with engraftment into native rat myocardium. This constitutes a significant step towards a new generation of stem cell-based treatment for heart failure.

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Section: Resultsmentioning

confidence: 99%

Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Ong

Fukunishi

Zhang

et al. 2017

Sci Rep

216

169

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“…In our laboratories, we previously generated spheroid cultures of vascular cells with biochemical, morphological, and physiological properties typical of in vivo blood vessels [Gentile et al, 2008;Fleming et al, 2010]. 3D in vitro cocultures of isolated CMs, ECs, and CFs from different origin have been explored, using either scaffolds [Caspi et al, 2007, Tulloch et al, 2011 or a scaffold-free technique, such as spheroid cultures for regenerative medicine purposes [Stevens et al, 2009, Noguchi et al, 2016 or drug testing [Ravenscroft et al, 2016]. From the molecular point of view, TGFβ1 signaling plays a major role in dilated or hypertrophic cardiomyopathy, with the consequent regulation of cardiac fibrotic and hypertrophic remodeling [Dobaczewski et al, .…”

Section: Discussionmentioning

confidence: 99%

Vascularized Cardiac Spheroids as Novel 3D in vitro Models to Study Cardiac Fibrosis

Figtree

Bubb

Tang

et al. 2017

Cells Tissues Organs

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Spheroid cultures are among the most explored cellular biomaterials used in cardiovascular research, due to their improved integration of biochemical and physiological features of the heart in a defined architectural three-dimensional microenvironment when compared to monolayer cultures. To further explore the potential use of spheroid cultures for research, we engineered a novel in vitro model of the heart with vascularized cardiac spheroids (VCSs), by coculturing cardiac myocytes, endothelial cells, and fibroblasts isolated from dissociated rat neonatal hearts (aged 1-3 days) in hanging drop cultures. To evaluate the validity of VCSs in recapitulating pathophysiological processes typical of the in vivo heart, such as cardiac fibrosis, we then treated VCSs with transforming growth factor beta 1 (TGFβ1), a known profibrotic agent. Our mRNA analysis demonstrated that TGFβ1-treated VCSs present elevated levels of expression of connective tissue growth factor, fibronectin, and TGFβ1 when compared to control cultures. We demonstrated a dramatic increase in collagen deposition following TGFβ1 treatment in VCSs in the PicroSirius Red-stained sections. Doxorubicin, a renowned cardiotoxic and profibrotic agent, triggered apoptosis and disrupted vascular networks in VCSs. Taken together, our findings demonstrate that VCSs are a valid model for the study of the mechanisms involved in cardiac fibrosis, with the potential to be used to investigate novel mechanisms and therapeutics for treating and preventing cardiac fibrosis in vitro.

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“…invented the micro-needle based method 8,39,40 . It was called 'Kenzan' method, after the traditional art Ikebana, where for floral arrangements the stalks are impaled in a dome-shaped metal needle array called 'Kenzan' (in Japanese ken=sword; zan=mountain).…”

Section: The 'Kenzan' Methods Of Bioprintingmentioning

confidence: 99%

“…In another example, a planar construct was made from pulsating spheroids, prepared from three human cell types: cardiomyocytes, endothelial cells and fibroblasts 40 . This construct was surgically applied in vivo atop of beating rat hearts.…”

Section: Cell Spheroids As Building Blocks For Bioprintingmentioning

confidence: 99%

Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting

Moldovan

Hibino

Nakayama

2017

Tissue Engineering Part B: Reviews

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264

204

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Abstract.Bioprinting is a technology with the prospect to change the way many diseases are treated, by replacing the damaged tissues with live, de novo created bio-similar constructs. However, after more than a decade of incubation and many proofs-of-concept, the field is still in its infancy. The current stagnation is the consequence of its early success: the first bioprinters, and most of those which followed, were modified versions of the 3D printers used in additive manufacturing, redesigned for layer-by-layer dispersion of biomaterials. In all variants (inkjet, micro-extrusion or laser-assisted), this approach is material-('scaffold'-) dependent and energy-intensive, making it hardly compatible with some of the intended biological applications. Instead, the future of bioprinting may benefit from the use of gentler, scaffoldfree bio-assembling methods. A substantial body of evidence has accumulated indicating this is possible by use of preformed cell spheroids, which have been assembled in cartilage, bone and cardiac muscle-like constructs. However, a commercial instrument capable to directly and precisely 'print' spheroids has not been available until the invention of the microneedles-based ('Kenzan') spheroid assembling, and the launching in Japan of a bioprinter based on this method. This robotic platform laces spheroids into predesigned contiguous structures with micron-level precision, using stainless steel micro-needles ("kenzans') as temporary support. These constructs are further cultivated until the spheroids fuse into cellular aggregates and synthesize their own extracellular matrix, thus attaining the needed structural organization and robustness. This novel technology opens wide opportunities for bio-engineering of tissues and organs.

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Development of a three-dimensional pre-vascularized scaffold-free contractile cardiac patch for treating heart disease

Abstract: We consider the application of our scaffold-free 3-dimensional tissue engineering technology to cardiac regeneration therapy is feasible and expect that this technology will become a promising tool for the treatment of end-stage heart failure.

Cited by 100 publications

References 25 publications

Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Vascularized Cardiac Spheroids as Novel 3D in vitro Models to Study Cardiac Fibrosis

Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting

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