Developing High Density Mechanobiology Assay Platforms for Stretch and Contractility Compatible with Laboratory Automation

Abstract: Complex in vitro models are increasingly utilized in target identification and screening efforts. However, in the mechanobiology space, these models are often developed as individual chips or in lower throughput formats which significantly limits their implementation in larger automated workflows. To address this, our team has developed consumables and instrumentation that can both accurately model the physiological processes of stretch and contractility and do so while maintaining 192 and 96 well footprints compatible with laboratory automation.

Here we will first describe DEFLCT (Devices Enabling Functional Linear Contractility of Tissues), a cantilever deflection assay platform that can be easily deployed in commercially available tissue culture plates. To demonstrate this tool in a drug discovery workflow, we have used the system to culture human primary bronchial smooth muscle cells in microscale tissue models simulating the muscle lining of upper airway. Using these 3D constructs, we profiled the impact of several inflammatory cytokines present in asthmatic airway on tissue contractility, tissue remodeling, and downstream gene expression. Among these, we identified TGF-β1 and IL-13 specifically as significant contractile agonists. To further explore potential mechanisms for this cytokine induced behavior, we conducted a pilot kinase inhibitor screen of 78 small molecules with known targets to identify key pathway intermediaries regulating tissue hypercontractility. Inhibition of PKC, AKT, and mTOR signaling pathways were all found to prevent contractility despite the presence of TGF-β1, while myosin kinase inhibitors did not significantly reduce tissue contraction. Collectively these data demonstrate the utility of the DEFLCT system in generating dynamic tissue models within standard assay plates that can be leveraged in compound screening experiments.

Additionally, we will briefly highlight another, more recent effort to develop a linear stretch assay also compatible with automation systems. To do this, we have designed both a custom stretching device and an entirely flexible 192 well assay plate that can be used in existing laboratory instrumentation. Extensive characterization of material deformation and failure modes was carried out to ensure device integrity over stretch protocols longer than 24 hours. Viability, gene expression, and morphology of seeded cells pre and post stretch indicate a pronounced phenotypic shift following mechanical loading over short and long timescales.

Together, we believe these platforms take important steps to enable higher throughput approaches for mechanobiology modeling. Their development will hopefully encourage increased complex screening efforts and aid in the uncovering of new targets and therapeutics that cannot be easily identified in static monolayer-based experiments.