Fabrication and Characterization of 3D Printed, 3D Microelectrode Arrays for Interfacing with a Peripheral Nerve-on-a-Chip.

We present a nontraditional fabrication technique for the realization of three-dimensional (3D) microelectrode arrays (MEAs) capable of interfacing with 3D cellular networks in vitro. The technology uses cost-effective makerspace microfabrication techniques to fabricate the 3D MEAs with 3D printed base structures with the metallization of the microtowers and conductive traces being performed by stencil mask evaporation techniques. A biocompatible lamination layer insulates the traces for realization of 3D microtower MEAs (250 µm base diameter, 400 µm height). The process has additionally been extended to realize smaller electrodes (30 µm × 30 µm) at a height of 400 µm atop the 3D microtower using laser micromachining of an additional silicon dioxide (SiO2) insulation layer. A 3D microengineered, nerve-on-a-chip in vitro model for recording and stimulating electrical activity of dorsal root ganglion (DRG) cells has further been integrated with the 3D MEA. We have characterized the 3D electrodes for electrical, chemical, electrochemical, biological, and chip hydration stability performance metrics. A decrease in impedance from 1.8 kΩ to 670 Ω for the microtower electrodes and 55 to 39 kΩ for the 30 µm × 30 µm microelectrodes can be observed for an electrophysiologically relevant frequency of 1 kHz upon platinum electroless plating. Biocompatibility assays on the components of the system resulted in a large range (∼3%-70% live cells), depending on the components. Fourier-transform infrared (FTIR) spectra of the resin material start to reveal possible compositional clues for the resin, and the hydration stability is demonstrated in in-vitro-like conditions for 30 days. The fabricated 3D MEAs are rapidly produced with minimal usage of a cleanroom and are fully functional for electrical interrogation of the 3D organ-on-a-chip models for high-throughput of pharmaceutical screening and toxicity testing of compounds in vitro.

Modeling chemotherapy-induced peripheral neuropathy using a Nerve-on-a-chip microphysiological system.

Organ-on-a-chip devices that mimic in vivo physiology have the potential to identify effects of chemical and drug exposure in early preclinical stages of drug development while relying less heavily on animal models. We have designed a hydrogel rat nerve-on-a-chip (RNoaC) construct that promotes axon growth analogous to mature nerve anatomy and is the first 3D in vitro model to collect electrophysiological and histomorphic metrics that are used to assess in vivo pathophysiology. Here we culture embryonic rat dorsal root ganglia (DRG) in the construct to demonstrate its potential as a preclinical assay for screening implications of nerve dysfunction in chemotherapy-induced peripheral neuropathy (CIPN). RNoaC constructs containing DRG explants from E15 rat pups were exposed to common chemotherapeutics: bortezomib, oxaliplatin, paclitaxel, or vincristine. After 7 days of treatment, axons were electrically stimulated to collect nerve conduction velocity (NCV) and the peak amplitude (AMP), which are two clinical electrophysiological metrics indicative of healthy or diseased populations. We observed decreased NCV and AMP in a dose-dependent manner across all drugs. At high drug concentrations, NCV and AMP were lower than control values by 10-60%. Histopathological analysis revealed that RNoaC exhibit hallmarks of peripheral neuropathy. IC50 values calculated from dose-response curves indicate significant decrease in function occurs before decrease in viability. Our data suggest electrophysiology recordings collected from our RNoaC platform can closely track subtle pathological changes in nerve function. The ability to collect clinically relevant data from RNoaCs suggests it can be an effective tool for in vitro preclinical screening of peripheral neuropathy.

Engineering a 3D functional human peripheral nerve in vitro using the Nerve-on-a-Chip platform.

Development of "organ-on-a-chip" systems for neuroscience applications are lagging due in part to the structural complexity of the nervous system and limited access of human neuronal & glial cells. In addition, rates for animal models in translating to human success are significantly lower for neurodegenerative diseases. Thus, a preclinical in vitro human cell-based model capable of providing critical clinical metrics such as nerve conduction velocity and histomorphometry are necessary to improve prediction and translation of in vitro data to successful clinical trials. To answer this challenge, we present an in vitro biomimetic model of all-human peripheral nerve tissue capable of showing robust neurite outgrowth (~5 mm), myelination of hNs by primary human Schwann cells (~5%), and evaluation of nerve conduction velocity (0.13-0.28 m/sec), previously unrealized for any human cell-based in vitro system. To the best of our knowledge, this Human Nerve-on-a-chip (HNoaC) system is the first biomimetic microphysiological system of myelinated human peripheral nerve which can be used for evaluating electrophysiological and histological metrics, the gold-standard assessment techniques previously only possible with in vivo studies.