Micropatterning Alginate Substrates for in vitro Cardiovascular Muscle on a Chip.

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.

Human airway musculature on a chip: an in vitro model of allergic asthmatic bronchoconstriction and bronchodilation.

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 ß-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.

Microfluidic heart on a chip for higher throughput pharmacological studies.

We present the design of a higher throughput "heart on a chip" which utilizes a semi-automated fabrication technique to process sub millimeter sized thin film cantilevers of soft elastomers. Anisotropic cardiac microtissues which recapitulate the laminar architecture of the heart ventricle are engineered on these cantilevers. Deflection of these cantilevers, termed Muscular Thin Films (MTFs), during muscle contraction allows calculation of diastolic and systolic stresses generated by the engineered tissues. We also present the design of a reusable one channel fluidic microdevice completely built out of autoclavable materials which incorporates various features required for an optical cardiac contractility assay: metallic base which fits on a heating element for temperature control, transparent top for recording cantilever deformation and embedded electrodes for electrical field stimulation of the tissue. We employ the microdevice to test the positive inotropic effect of isoproterenol on cardiac contractility at dosages ranging from 1 nM to 100 µM. The higher throughput fluidic heart on a chip has applications in testing of cardiac tissues built from rare or expensive cell sources and for integration with other organ mimics. These advances will help alleviate translational barriers for commercial adoption of these technologies by improving the throughput and reproducibility of readout, standardization of the platform and scalability of manufacture.