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Development Of A Novel in Vitro Method to Study Effects of Volatile Compounds on Neural Activity
Citation:
Rice, O., Q. Krantz, E. Puckett, P. Evansky, K. Wallace, T. Freudenrich, M. Higuchi, AND T. Shafer. Development Of A Novel in Vitro Method to Study Effects of Volatile Compounds on Neural Activity. Society of Toxicology (SOT) 63rd Annual Meeting and ToxExpo, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25407025
Impact/Purpose:
Poster presented to the Society of Toxicology (SOT) 63rd Annual Meeting and ToxExpo March 2024
Description:
Background and Purpose: Current in vitro toxicology methods typically rely on exposure of cells to compounds that were added to the culture media. For neurotoxicity, in vitro exposure of rat cortical neural networks plated on multi-well microelectrode array plates (MEAs) have been used to evaluate >1000 compounds for their effects on electrical activity. This exposure method works well for compounds that are easily soluble in aqueous solution; however, volatile compounds that are not easily solubilized in media are problematic to test in this manner. Many volatiles are known to be neurotoxic; as such, the current inability to test volatiles in vitro creates a gap in the ability of New Approach Methods to evaluate neurotoxicity for uncharacterized volatile compounds. To address this, a novel manifold has been developed that allows for in vitro exposure of cells to volatile compounds in the gas phase. Herein, preliminary experiments are presented to optimize conditions for the use of this manifold on MEA plates, including optimization of the volume of media, density of cells, and gas flow rates. Methods: A manifold was designed to deliver volatile compounds in the gas phase to each well of 48-well MEA plates. Above each column of wells, two parallel tubes are drilled into the manifold from opposite sides; one for intake and the other for exhaust. Above each well of the MEA plate, holes drilled into the manifold connect to the inlet and exhaust tubes. These are used to create a continuous flow of a known gas concentration over each well. Before passing through the manifold, the gas is heated to 37 ¿C and humidified to 85%. To allow for fast equilibration between gas and liquid (media) phases, we sought to minimize the media volume without impacting cellular electrical activity........ Results: Initial testing of multiple media volumes shows that cells are viable in 100-500μL media without a statistically significant difference in their activity. Additional results demonstrate that a density of 150K cells/well produces consistent activity with most tested media volumes. Specifically, media volumes of 100μL, while not equal in mean firing rate and synchrony to the standard 500μL volume, have similar trends across the 2 hr reduced media trial, indicating that 100μL can be used in compound experiments if no electrical artefacts due to turbulence are found. Results from turbulence testing indicate no significant difference in activity between media volumes, but a significant difference between wells exposed to 5% CO2 and wells without. The number of active electrodes were constant for all conditions studied, whereas the mean firing rate and synchrony were higher for the wells exposed to CO2 regardless of media volume. Because all endpoints evaluated are comparable between the volumes of 100 and 500μL artefacts caused by turbulence are negligible in the endpoints that are used. Well by well analysis additionally shows no large differences due to well placement in the plate. Conclusions: These experiments demonstrate the optimal cell density and volume for use in the manifold is 150,000 cells per well with 100μL of media during exposures. The manifold has also been shown to transport gas through the wells without causing any change in neural activity at the flow rates we evaluated. Next, to test whether volatile gases will equilibrate with the media and impact neuronal activity, toluene will be used as an initial test agent. Toluene is known to impact the function of voltage-gated calcium channels, as well as NMDA, GABAA, and nicotinic receptors. As such, it is expected to alter electrical activity measured by the MEA which would confirm successful function of the manifold. (This abstract does not reflect US EPA policy.)
URLs/Downloads:
DOI: Development Of A Novel in Vitro Method to Study Effects of Volatile Compounds on Neural Activity
RICE_SOT POSTER V7.PDF (PDF, NA pp, 812.307 KB, about PDF)