Biomedical research has relied on animal studies and conventional cell cultures for decades. Recently, microphysiological systems (MPS), also known as organs-on-chips, that recapitulate the structure and function of native tissues in vitro, have emerged as a promising alternative1. However, current MPS typically lack integrated sensors and their fabrication requires multi-step lithographic processes2. Here, we introduce a facile route for fabricating a new class of instrumented cardiac microphysiological devices via multi-material 3D printing. Specifically, we designed six functional inks, based on piezo-resistive, high conductance, and biocompatible soft materials that enable integration of soft strain gauge sensors within micro-architectures that guide the self-assembly of physio-mimetic laminar cardiac tissues. We validated that these embedded sensors provide non-invasive, electronic readout of tissue contractile stresses, inside cell incubator environments. We further applied these devices to study drug responses, as well as the contractile development of human stem cell derived laminar cardiac tissues over four weeks.

Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches.

Defining the chronic cardiotoxic effects of drugs during preclinical screening is hindered by the relatively short lifetime of functional cardiac tissues in vitro, which are traditionally cultured on synthetic materials that do not recapitulate the cardiac microenvironment. Because collagen is the primary extracellular matrix protein in the heart, we hypothesized that micromolded gelatin hydrogel substrates tuned to mimic the elastic modulus of the heart would extend the lifetime of engineered cardiac tissues by better matching the native chemical and mechanical microenvironment. To measure tissue stress, we used tape casting, micromolding, and laser engraving to fabricate gelatin hydrogel muscular thin film cantilevers. Neonatal rat cardiac myocytes adhered to gelatin hydrogels and formed aligned tissues as defined by the microgrooves. Cardiac tissues could be cultured for over three weeks without declines in contractile stress. Myocytes on gelatin had higher spare respiratory capacity compared to those on fibronectin-coated PDMS, suggesting that improved metabolic function could be contributing to extended culture lifetime. Lastly, human induced pluripotent stem cell-derived cardiac myocytes adhered to micromolded gelatin surfaces and formed aligned tissues that remained functional for four weeks, highlighting their potential for human-relevant chronic studies.

The physiologic role of smooth muscle structure in defining arterial function is poorly understood. We aimed to elucidate the relationship between vascular smooth muscle architecture and functional contractile output. Using microcontact printing and muscular thin film technology, we engineered in vitro vascular tissues with strictly defined geometries and tested their contractile function. In all tissues, vascular smooth muscle cells (VSMCs) were highly aligned with in vivo-like spindle architecture, and contracted physiologically in response to stimulation with endothelin-1. However, tissues wherein the VSMCs were forced into exaggerated spindle elongation exerted significantly greater contraction force per unit cross-sectional area than those with smaller aspect ratios. Moreover, this increased contraction did not occur in conjunction with an increase in traditionally measured contractile phenotype markers. These results suggest that cellular architecture within vascular tissues plays a significant role in conferring tissue function and that, in some systems, traditional phenotype characterization is not sufficient to define a functionally contractile population of VSMCs.

Introduction To evaluate the viability of a muscle tissue, it is essential to measure the tissue’s contractile performance as well as to control its structure. Accurate contractility data can aid in development of more effective and safer drugs. This can be accomplished with a robust in vitro contractility assay applicable to various types of muscle tissue. Methods The devices developed in this work were based on the muscular thin film (MTF) technology, in which an elastic film is manufactured with a 2D engineered muscle tissue on one side. The tissue template is made by patterning extracellular matrix with microcontact printing. When muscle cells are seeded on the film, they self-organize with respect to the geometric cues in the matrix to form a tissue. Results Several assays based on the “MTF on a chip” technology are demonstrated. One such assay incorporates the contractility assay with striated muscle into a fluidic channel. Another assay platform incorporates the MTFs in a multi-well plate, which is compatible with automated data collection and analysis. Finally, we demonstrate the possibility of analyzing contractility of both striated and smooth muscle simultaneously on the same chip. Discussion In this work, we assembled an ensemble of contractility assays for striated and smooth muscle based on muscular thin films. Our results suggest an improvement over current methods and an alternative to isolated tissue preparations. Our technology is amenable to both primary harvests cells and cell lines, as well as both human and animal tissues.

Soft hydrogels such as alginate are ideal substrates for building muscle in vitro because they have structural and mechanical properties close to the in vivo extracellular matrix (ECM) network. However, hydrogels are generally not amenable to protein adhesion and patterning. Moreover, muscle structures and their underlying ECM are highly anisotropic, and it is imperative that in vitro models recapitulate the structural anisotropy in reconstructed tissues for in vivo relevance due to the tight coupling between sturcture and function in these systems. We present two techniques to create chemical and structural heterogeneities within soft alginate substrates and employ them to engineer anisotropic muscle monolayers: (i) microcontact printing lines of extracellular matrix proteins on flat alginate substrates to guide cellular processes with chemical cues, and (ii) micromolding of alginate surface into grooves and ridges to guide cellular processes with topographical cues. Neonatal rat ventricular myocytes as well as human umbilical artery vascular smooth muscle cells successfully attach to both these micropatterned substrates leading to subsequent formation of anisotropic striated and smooth muscle tissues. Muscular thin film cantilevers cut from these constructs are then employed for functional characterization of engineered muscular tissues. Thus, micropatterned alginate is an ideal substrate for in vitro models of muscle tissue because it facilitates recapitulation of the anisotropic architecture of muscle, mimics the mechanical properties of the ECM microenvironment, and is amenable to evaluation of functional contractile properties.

Many potential new asthma therapies that show promise in the pre-clinical stage of drug development do not demonstrate efficacy during clinical trials. One factor contributing to this problem is the lack of human-relevant models of the airway that recapitulate the tissue-level structural and functional phenotypes of asthma. Hence, we sought to build a model of a human airway musculature on a chip that simulates healthy and asthmatic bronchoconstriction and bronchodilation in vitro by engineering anisotropic, laminar bronchial smooth muscle tissue on elastomeric thin films. In response to a cholinergic agonist, the muscle layer contracts and induces thin film bending, which serves as an in vitro analogue for bronchoconstriction. To mimic asthmatic inflammation, we exposed the engineered tissues to interleukin-13, which resulted in hypercontractility and altered relaxation in response to cholinergic challenge, similar to responses observed clinically in asthmatic patients as well as in studies with animal tissue. Moreover, we reversed asthmatic hypercontraction using a muscarinic antagonist and a β-agonist which are used clinically to relax constricted airways. Importantly, we demonstrated that targeting RhoA-mediated contraction using HA1077 decreased basal tone, prevented hypercontraction, and improved relaxation of the engineered tissues exposed to IL-13. These data suggest that we can recapitulate the structural and functional hallmarks of human asthmatic musculature on a chip, including responses to drug treatments for evaluation of safety and efficacy of new drugs. Further, our airway musculature on a chip provides an important tool for enabling mechanism-based search for new therapeutic targets through the ability to evaluate engineered muscle at the levels of protein expression, tissue structure, and tissue function.

Using a tongue-inspired in vitro platform, Nesmith et al. demonstrate that DMD myoblasts fail to align and polarize with respect to extracellular matrix cues in the same manner as healthy myoblasts, resulting in diminished myotube formation and weaker contractile strength.