The Society of Toxicology (SOT) held its 62nd Annual Meeting and ToxExpo from March 19 to 23, 2023, at the Music City Center in Nashville, Tennessee. The event featured over 70 scientific sessions, 2,000 presentations, and 250 exhibitors, attracting more than 5,000 attendees.
J. Lowry Curley1, Megan Terral1, Corey Rountree1, Eva Schmidt1, and Michael J. Moore 1, 2
1 AxoSim Inc., New Orleans, LA, USA
2 Department of Biomedical Engineering, Tulane University, New Orleans, LA, USA
Toxicity is a leading reason drugs are withdrawn from the market, with neurotoxicity responsible for 16%. Peripheral nerves are particularly susceptible to off-target effects resulting in permanent sensory-motor deficits, and chemotherapy-induced peripheral neuropathy (CIPN) occurs with a 68% incidence rate and 30% retaining effects after 6 months. CIPN can also affect clinical outcomes, with 91% of cases leading to dose reduction and a 45% discontinuation rate. Preclinical animal models are historically expensive and low-throughput and have largely failed to deliver results that translate to success in the human system. Peripheral nerves (PNs), in particular, lack predictive human-relevant in vitro drug screening models, with less than 7% of neurological drug candidates reaching the marketplace. Microphysiological systems (MPS), including organs-on-chips, which utilize human induced pluripotent stem cell (iPSC)-derived cells to emulate specific organ systems, have emerged as promising screening platforms to bridge the gap between preclinical and clinical success. These systems are attracting attention from the pharmaceutical industry in the hope they will curb attrition rates, lower costs, and reduce reliance on animal models.
Engineering 3D tissues relevant to the nervous system, particularly PNs, is challenging because of the complex ultrastructure and necessity of functional outputs. AxoSim has developed an all-human NerveSim® MPS platform, with human iPSC-derived sensory neurons and primary human Schwann cells for screening neurotoxic compounds using an embedded electrode array (EEA) to record compound action potentials (CAPs) from peripheral nerve cultures. The efficacy of this system was demonstrated by recording cultures exposed to Paclitaxel (PTX).
The NerveSim® MPS is a custom tissue culture plate with integrated electrodes to allow in situ electrophysiological recordings. Each well contains a single cell-restrictive polymer mold and a cell-permissive inner gel that guides axonal growth across a series of 10 microelectrodes. Coculture spheroids were produced from human sensory neurons and Schwann cells in round-bottom multiwell plates then plated in one end of the inner cell-permissive gel. Spheroids were cultured for 4 weeks in a series of culture medias that promote neurite outgrowth through the gel, Schwann cell migration and alignment with axons, and ultimately, Schwann cell myelination. Robust neurite outgrowth of ~5mm was consistently observed, with Schwann cell migration and alignment along neuronal axons confirmed by S100 immunostaining.
Using this system, multiple NerveSim® EEA cultures were stimulated in parallel at multiple distal sites with a stimulation current ramp while recording the CAPs at the cell body and axons. From these data, we collected the conduction velocity (CV), peak response amplitude (AMP), and threshold stimulus strength (TSS), which are all clinically relevant electrophysiological metrics in response to several known to cause peripheral neuropathy. Additionally, histopathology shows phenotypic responses of peripheral neuropathy including decreased fiber densities and increased degenerated fibers. The ability to collect clinically relevant data is an effective tool for in vitro modeling of CIPN towards screening of therapeutics for neuroprotection and neuroregeneration. Current efforts are focused first on increasing the number of myelinated nerve fibers in the NerveSim® sample, and second, on quantifying the effects of well-known demyelinating compounds, such as Cuprizone, to further establish the efficacy of NerveSim® as a drug screening platform.
Corey Rountree1, Monica Metea2, J. Lowry Curley1, Megan Terral1, Eva Schmidt1, and Michael J. Moore 1, 3, 4
1 AxoSim Inc., New Orleans, LA, USA
2 Preclinical Electrophysiology Consulting LLC, Boston, MA, USA
2 Department of Biomedical Engineering, Tulane University, New Orleans, LA, USA
3 Brain Institute, Tulane University, New Orleans, LA, USA
Microphysiological systems (MPS) are an emerging technology with the potential to close the gap between preclinical and clinical studies for neurotoxicity screening and drug development. These systems are attracting attention from the pharmaceutical industry in the hope they will curb attrition rates, lower costs, and reduce reliance on animal models. AxoSim has developed an innovative MPS, the NerveSim® platform, for screening neurotoxic compounds using a custom embedded electrode array (EEA) to record compound action potentials (CAPs) from peripheral nerve (PN) cultures. Bridging the gap between in vitro and in vivo, the NerveSim® platform measures multiple clinically relevant electrophysiological metrics such as conduction velocity (CV), peak response amplitude (AMP), threshold stimulus strength (TSS), and activity dependent slowing (ADS).
The NerveSim® EEA platform is a novel electrophysiology system specifically designed to allow high-throughput electrophysiological recordings of peripheral nerve cultures. The platform is centered on a custom 24-well tissue culture plate with an integrated circuit board that allows both spontaneous and evoked electrophysiological measurements. Each well contains a cell-restrictive outer layer and a growth-permissive inner gel that together guides axonal growth into a 3D nerve-like bundle. At the bottom of the inner gel are a series of 10 microelectrodes (1 mm spacing) that can be used for both recording and stimulation. Dissociated peripheral neuron spheroids, derived from rodent or human models, are placed into the inner gel and cultured for four weeks to ensure mature axonal growth with myelination before conducting electrophysiological testing.
Using a proprietary electrophysiology system, up to 12 NerveSim® cultures can be tested in parallel with current-based electrical stimulation. A typical recording session involves stimulation at multiple distal sites with a stimulation current ramp (1 to 64 µA) while recording the CAPs at the spheroid body as well as along the axons to fully characterize the sensitivity of the neuronal cultures to electrical stimulation. A stimulation frequency ramp (0.5 to 2 Hz) can also be tested at each site to differentiate the responses of different C fiber subtypes based on the presence of ADS. The assay can be performed at multiple timepoints before, during, and after drug dosing to provide a more complete picture of both the scale and timing of pharmacological effects. At each of these timepoints, a full range of electrophysiological metrics (CV, AMP, TSS, and ADS) is collected for comparison to baseline values. Each of these metrics can also be measured and compared based on the distance separating the recording electrode from the spheroid, allowing quantifiable detection of axonopathy versus neuropathy.
The NerveSim® EEA platform can also be multiplexed with common assays, such as a lactate dehydrogenase (LDH) cytotoxicity assay, to provide a more complete profile of neurotoxic effects on peripheral nerve cultures. Moreover, each NerveSim® culture can be examined via immunohistochemistry and fluorescence microscopy to correlate spatial morphology with the
functional, spatially localized electrophysiological data. The combination of electrophysiology, immunohistochemistry, and molecular assays make AxoSim’s NerveSim® EEA platform a unique, high-throughput preclinical screening model specifically designed for investigating peripheral neuropathy, neuroprotection, and neuroregeneration.
