A tissue-engineered scale model of the heart ventricle.

Laboratory studies of the heart use cell and tissue cultures to dissect heart function yet rely on animal models to measure pressure and volume dynamics. Here, we report tissue-engineered scale models of the human left ventricle, made of nanofibrous scaffolds that promote native-like anisotropic myocardial tissue genesis and chamber-level contractile function. Incorporating neonatal rat ventricular myocytes or cardiomyocytes derived from human induced pluripotent stem cells, the tissue-engineered ventricles have a diastolic chamber volume of ~500 µl (comparable to that of the native rat ventricle and approximately 1/250 the size of the human ventricle), and ejection fractions and contractile work 50-250 times smaller and 104-108 times smaller than the corresponding values for rodent and human ventricles, respectively. We also measured tissue coverage and alignment, calcium-transient propagation and pressure-volume loops in the presence or absence of test compounds. Moreover, we describe an instrumented bioreactor with ventricular-assist capabilities, and provide a proof-of-concept disease model of structural arrhythmia. The model ventricles can be evaluated with the same assays used in animal models and in clinical settings.

Demonstration of multi-analyte patterning using piezoelectric inkjet printing of multiple layers.

Patterning substrates with biological reagents is a critical component of biosensor development. Many applications require multi-analyte patterning capabilities, with a need to deposit several species reproducibly with a high degree of precision. We demonstrate a piezoelectric inkjet printing system that is capable of creating sub-millimeter (down to 150microm) patterns of aqueous and nonaqueous reagents with precise placement for biosensor applications. The size, shape, and density of the patterns may be modified by simple adjustments of the patterning parameters. Using this system, two methods of multi-analyte protein patterning for use in biosensor assays are demonstrated. The first method involves the deposition of multiple proteins directly onto a gold substrate. Specific binding of an antibody to the deposited antigen is demonstrated, although nonspecific adsorption of the antibody may limit the utility of this simple method in quantitative biosensor applications. A second, more sophisticated multi-analyte patterning method involves two sequential patterning steps, consisting of an initial deposition onto gold of a mixed thiol layer to provide oriented binding capabilities in a nonfouling background and a second deposition of multiple biotinylated proteins. Highly specific antibody binding to this patterned multi-analyte surface was demonstrated, with minimal nonspecific adsorption to the surrounding regions. Thus, this method produces high-quality, localized, and customizable sub-millimeter patterns in a nonfouling background for multi-analyte bioassay development.

A nanofabricated planar aperture as a mimic of the nerve-muscle contact during synaptogenesis.

The release of synaptogenic factors by the nerve terminal plays a central role in the aggregation of neurotransmitter receptors at the postsynaptic membrane, a precisely timed and localized process that is essential for the correct formation and functioning of the synapse. This process has been difficult to re-capitulate in cell culture because present cell stimulation methods do not have sufficient spatiotemporal control of the delivery of soluble signals. We cultured myotubes atop nanofabricated planar apertures (2-8 microm diameter) to focally stimulate the muscle cell membrane with neural agrin, a synaptogenic factor released by motor neurons during development. Focal agrin delivery through the apertures after myotube fusion results in local aggregation of acetylcholine receptors (AChRs) in the vicinity of the apertures, a process reminiscent of AChR clustering at innervation sites. Since the apertures are spatially organized in microarrays, multiple experiments can be run in parallel on one device. The technique has wide applicability in cell-cell communication studies and cell-based bioassays.

Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin.

During neuromuscular synaptogenesis, the exchange of spatially localized signals between nerve and muscle initiates the coordinated focal accumulation of the acetylcholine (ACh) release machinery and the ACh receptors (AChRs). One of the key first steps is the release of the proteoglycan agrin focalized at the axon tip, which induces the clustering of AChRs on the postsynaptic membrane at the neuromuscular junction. The lack of a suitable method for focal application of agrin in myotube cultures has limited the majority of in vitro studies to the application of agrin baths. We used a microfluidic device and surface microengineering to focally stimulate muscle cells with agrin at a small portion of their membrane and at a time and position chosen by the user. The device is used to verify the hypothesis that focal application of agrin to the muscle cell membrane induces local aggregation of AChRs in differentiated C2C12 myotubes.