Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip
Introduction
Engineering cardiac tissues poses a series of critical challenges that need to be addressed in order to translate basic research products from bench to clinical practice [1], [2], [3]. The engineered cardiac organoids coupled with microfluidic bioreactors (e.g. heart-on-chips) have also found increasing applications in functioning as enabling in vitro biomimetic models to study pathology, measure cardiotoxicity, and develop new therapeutics [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. The first challenge in engineering cardiac organoids and their on-chip forms lies in the fact that mature cardiomyocytes exhibit limited self-renewing potential [14]. In this framework, induced pluripotent stem cells (iPSCs) hold great promise, due to their wide availability and the possibility to differentiate into multiple cell lineages including cardiomyocytes [3], [11], [15], [16]. Second, the alignment of cardiomyocytes and their organization into bundles characterized by spontaneous and synchronous contraction further complicate the development of biologically relevant cardiac tissues [1], [2], [3], [17], [18]. Third, the generation of thick (cardiac) tissue constructs requires the introduction of microvascular networks in order to provide oxygen and nutrients, remove waste products, and eventually promote vessel anastomosis with the host vasculature [3], [19], [20].
Several approaches have so far been explored to generate functional tissue constructs including the myocardium [21], [22], [23], [24]. For example, scaffold-free multicellular cardiac spheroids have been developed that could spontaneously and synchronously contract [21], [22]. While the cardiac spheroids have served an important role in drug testing and have been widely used due to the ease of preparation, these constructs lack the directionality characteristic of the physiological myocardium, which is critical to maintain the long-term functionality of the engineered cardiac tissues. On the other side, scaffold-based techniques provide an ideal support for cell adhesion, distribution, and responses [12], [18], [25], [26], [27]. Importantly, the architecture of the scaffolds can be conveniently modulated in order to promote the biological relevance of the engineered tissues by tuning spatial organizations that mimic their in vivo counterparts [25]. In this context, Freed and co-workers demonstrated that anisotropic scaffolds bearing an accordion-like honeycomb structure could induce the generation of highly oriented cardiac fibers [26]. Radisic and colleagues developed a biowire approach to induce the differentiation and alignment of the cardiomyocytes from human pluripotent stem cells [27]. Healy and co-workers recently engineered aligned cardiac tissues by populating microfilament arrays with cardiomyocytes [12]. Our group has also recently developed hydrogel substrates with aligned ridges/grooves via photopatterning to improve the adhesion and alignment of cardiomyocytes [18]. Strategies have further been investigated to integrate blood vessels into engineered tissues including the myocardium [28], [29], [30], [31]. For example, Leong and colleagues have provided a general and versatile strategy by using transwell-mediated layering of endothelial cells and tissue cells for drug testing [30], [31]. However, generating volumetric cardiac tissues containing embedded endothelial networks remains challenging.
Bioprinting has recently emerged as a promising technology to produce geometrically defined structures in three dimensions (3D), significantly improving their physiological relevance through architectural mimicry of native tissues and organs [32], [33]. Particularly, bioprinting overcomes major drawbacks of conventional scaffold-based approaches including limited control over the 3D structures of engineered tissues and thus reduced reproducibility. The bioprinting process is usually biocompatible, allowing for direct encapsulation of bioactive molecules and cells. Furthermore, bioprinting may enable vascularization of the engineered tissue constructs based on sacrificial methods [34], [35], [36] or direct deposition [37], [38], providing additional versatility in producing vascularized cardiac organoids.
In this work we present a novel hybrid strategy based on 3D bioprinting, to engineer endothelialized myocardial tissues (Fig. 1). Based on the microfluidic technology that we developed in our previous work [37], we directly encapsulated endothelial cells within the bioprinted microfibrous lattices to inuce their migration towards the peripheries of the microfibers to form a layer of confluent endothelium. Different from our previous report, however, this 3D bioprinted endothelialized microfibrous scaffold, together with precisely controlled macroscale anisotropic architecture of the microfibers, was then seeded with cardiomyocytes to induce the formation of myocardium with improved alignment capable of spontaneous and synchronous contraction. When further combined with a specially designed microfluidic perfusion bioreactor, the resulting endothelialized-myocardium-on-a-chip platform was adopted to screen pharmaceutical compounds for their cardiovascular toxicity. Finally, we investigated the possibility to translate such a model to endothelialized human myocardium and their on-chip forms that were responsive to drugs using human iPSC (hiPSC)-derived cardiomyocytes.
Section snippets
Cell culture
Human umbilical vein endothelial cells (HUVECs) and green fluorescent protein (GFP)-labeled HUVECs were obtained from Lonza and cultured in endothelial growth medium (EGM, Lonza). Neonatal rate cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats following our established protocol approved by the Institutional Animal Care and Use Committee at the Brigham and Women's Hospital [39]. The cells were then maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 vol%
Bioprinting 3D microfibrous scaffolds
Our novel 3D bioprinting approach allowed us to conveniently generate multilayer hydrogel microfibrous scaffolds using an Organovo Novogen MMX bioprinter (Fig. 2A), which was optimized through the implementation of a custom-designed coaxial nozzle for continuous extrusion of the bioink [37]. The internal needle, having a size of 210 μm (27G), was fed with the bioink composed of a mixture of hydrogel precursors, i.e. alginate, GelMA, and photoinitiator Irgacure 2959; the crosslinking solution, i.
Conclusions and perspectives
In summary, we have presented a novel strategy to construct endothelialized-myocardial-tissues by adopting an innovative bioprinting technology. The endothelial cells, encapsulated inside the microfibers composing the backbone of the scaffolds, gradually migrated towards the peripheries of the microfibers to form a layer of confluent endothelium. The assembly of the endothelial cells within the bioprinted microfibers resembling a blood vessel structure was enabled by the composite bioink
Acknowledgements
The authors gratefully acknowledge funding by the Defense Threat Reduction Agency (DTRA) under Space and Naval Warfare Systems Center Pacific (SSC PACIFIC) Contract No. N66001-13-C-2027. The authors also acknowledge funding from the National Institutes of Health (AR057837, DE021468, D005865, AR068258, AR066193, EB022403, EB021148), the Air Force Office of Scientific Research Award #FA9550-15-1-0273, and the Presidential Early Career Award for Scientists and Engineers (PECASE). Y.S.Z.
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A.A. and S.B. contributed equally to this work.