P22 - Development of a modular microphysiological system to innervate 3D tissues for disease modeling and toxicity testing

Introduction: : More than 90% of drug candidates fail in clinical trial stages and cannot gain Food and Drug Administration (FDA) approval, with 30% of these failures attributed to unmanageable toxicity, mostly due to the inadequacy of existing disease and screening models [1, 2] . The function of most organs in the body is affected by the autonomic nervous system (ANS). For example, glandular organs, including salivary glands (SGs), are highly innervated, which is central to physiological function, organogenesis, and tissue repair [3]. However, the physiologic importance of the ANS has been largely ignored in tissue models for drug/toxicity screening [4]. A wide range of commonly prescribed drugs, including chemotherapeutic agents, antimicrobials, cardiovascular medications, psychotropics, and anticonvulsants, can induce peripheral nervous system (PNS) disorders by directly causing neuropathy, disrupting neurotransmission, or interacting with receptors expressed in target tissues [5, 6]. ANS dysfunction can lead to many disorders, including Parkinson’s disease, Alzheimer’s disease, hypertension, diabetic autonomic neuropathy, spinal cord injury, and xerostomia [7]. The current gold standard animal models for preclinical testing are impractical for drug screening as they are costly, low-throughput, and prompt ethical concerns [8-10]. Hence, there is a critical unmet need for innovative, reliable in vitro disease modeling and drug efficacy and toxicity screening. The first-ever functional human and mouse SG tissue chips (non-innervated) in an array-based platform for high-throughput radioprotective drug discovery were recently reported [11]. In this work, we have developed a modular multi-organ-on-a-chip platform for tissue innervation and toxicity screening using the SG as the target tissue.

Materials and

Methods: : Combining expertise in 3D tissue culture platform (microbubble (MB) array), hydrogel-based extracellular matrix (ECM), and silicon nitride (SiN) nanomembrane microfluidic technologies, a functional hybrid microphysiological system (MPS) for innervating spheroids/organoids, was developed using a salivary gland tissue mimetic (SGm) model (Fig. 1). A double-sided open MB array is bonded to a SiN microporous nanomembrane (~400 nm thick, 2 micron pore size) with pressure sensitive adhesive (PSA) and integrated into a microfluidic device called the µSiM (microphysiological system enabled by a silicon membrane). The nanomembrane separates the nerve compartment from the SGm cultured in the MBs. The gap between the two compartments, is filled with a hydrogel ECM engineered to support robust neurite extension and tissue innervation. PC-12 cells differentiated into sympathetic neurons (a division of the ANS) are used as the nerve model. The ability of the system to innervate SGm is studied in the hybrid MPS, where the nerve tissue is cultured on the underside of the nanomembrane in a perfusable basal compartment, and SGm are generated in the double-sided open MB array in the apical compartment of the device in an arrayed format (~40 microtissues/spheroids per device). Upon demonstration of the platform as a functional system, the device will be employed for toxicity testing using agents with potential xerostomia (dry mouth) effects.

Results, Conclusions, and Discussions:: A practical MPS device was developed for multi-organ culture and high-throughput screening (U.S. Patent No 63/560, 129 patent pending). The ECM hydrogel composition, stiffness, and swelling ratio are engineered to support both SGm growth and neurite/axon vertical extension. The preliminary results have shown that a combination of collagen type 1 and Matrigel provides a proper base for SGm formation and enables vertical 3D neurite extension of more than 300 µm. In a co-culture of SGm and sympathetic neurons, SGm produced growth factors, including nerve growth factor (NGF) intensified the neurite extension for up to 600 µm, implying the importance of multi-organ culture (U.S. Patent No 63/778, 553 patent pending). I will demonstrate that the model is functional (gene expression study, protein analysis, and [Ca2+] signaling measurement) and qualify the platform as a drug development tool (DDT) for evaluating drug-induced xerostomia (dry mouth) effect. A third organ, like vasculature can also be integrated into the hydrogel for neurovascularized tissue development, where vessels can additionally infiltrate the target tissue. The novel platform can enable microfluidics, disease modeling through multi-organ development, high-throughput drug discovery, and toxicity testing in a single device. A lot of organs, including SGs, lungs, kidneys, heart, and the GI tract system, that are affected by the ANS can leverage this novel technology. The device will have a significant potential for progression to qualification through the FDA as a drug development tool to enhance the predictivity of non-clinical studies, and reduce, refine, and replace the reliance on animal testing in alignment with the FDA Modernization Act 2.0 and Roadmap to Reducing Animal Testing in Preclinical Safety Studies.

Acknowledgements and/or References (Optional):: This project acknowledges financial support from the Dermatology Department of the University of Rochester Medical Center and a pilot award funded by the Clinical and Translational Science Institute (CTSI) at the University of Rochester. We acknowledge Professor Benjamin Miller for sharing his resources and advice.