2020 Progress Report: A Neurovascular Unit on Chip for Reducing Animals in Organophosphate NeurotoxicologyEPA Grant Number: R839504
Title: A Neurovascular Unit on Chip for Reducing Animals in Organophosphate Neurotoxicology
Investigators: Cliffel, David , May, Jody , McLaughlin, BethAnn
Current Investigators: Cliffel, David , May, Jody , McLaughlin, BethAnn , Neely, M Diana
Institution: Vanderbilt University , Vanderbilt University Medical Center
EPA Project Officer: Aja, Hayley
Project Period: August 1, 2019 through July 31, 2020 (Extended to July 31, 2023)
Project Period Covered by this Report: August 1, 2019 through July 31,2020
Project Amount: $850,000
RFA: Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment (2018) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
We are developing an organ on a chip platform entitled the Neurovascular Unit on Chip (NVU) for neuronal development and toxicity studies that faithfully replicates the blood-brain barrier. This NVU is multicell type-based with dynamic metabolic and signaling methods of assessing chemical and drug toxicity without the limitations of animal studies or standard cell death/morphology toxicology assays. Our objective is to demonstrate that the NVU provides equal if not superior information than conventional animal toxicity assays for organophosphate compounds of interest like chlorpyrifos. We have developed the microclinical analyzer and cellular signaling assays to measure in situ both the acute and chronic responses of the multiple cell lines that recreate neuronal tissues to exposures to potential drugs and other chemicals. Our approach has the advantage providing quantitative information regarding a variety of cellular activities, including metabolism, membrane transport, protein translation, and hence provides a comprehensive approach to absorption, distribution, metabolism, excretion (ADME) and toxicological (TOX) profiles.
In this year, we establish human model systems (transwell and microfluidic tissue chips) comprised of neurons and glial cells derived from induced pluripotent stem cells (hiPSCs) to assess the effects of CPF on the human CNS. The Neely laboratory has already developed protocols and routinely differentiates glutamatergic cortical neurons, mesencephalic dopamine neurons and astrocytes from hiPSCs. This first year we have thus focused our efforts on developing a differentiation protocol to derive basal forebrain cholinergic neurons from hiPSC. All forebrain cholinergic neurons share a common developmental origin in the embryonic medial ganglionic eminence (MGE).
Temporal measurement of cellular response is vital to resolving the mechanisms and pathways that are affected by neurotoxins. The 8-channel screen-printed electrodes (SPE) provide a low cost, real-time, and robust monitoring system for a maximum of eight analytes simultaneously.
Each working electrode is separated into a single microfluidic channel and is modified to only detect a single analyte with limited interference. Polymeric and enzymatic films can be deposited onto the working electrodes to make each electrode specific. Electrochemical measurements can be taken and used to determine changes in analyte concentration and cellular response. In this first year we have modified the previous developed electrochemical biosensors to selectively and quantitatively measure four neurotransmitters, including glutamate, acetylcholine, adenosine, and dopamine. This biosensor provides a platform to obtain bioinformation of neurological functions particularly following treatment with chlorpyrifos and other neurological toxicants.
Finally, we have worked to improve on the ex situ measurements of BBB membrane integrity and permeability experiments within the NVU by creating an NVU device composed entirely of human cells and by adding in situ transendothelial electrical resistance (TEER) measurements, in addition to the expanded neurotransmitter sensor array. TEER measurements provide a direct measurement of the resistance of the BBB membrane, and thus can immediately identify any changes to the membrane permeability resulting from chlorpyrifos exposure.
In the next year period, we are bringing the new human-derived cholinergic neurons to the fully-humanized NVU setup along with the dopaminergic and glutaminergic neurons, and we conduct the chlorpyrifos dose responses using the enhanced microclinical analyzer with its four neurotransmitter and TEER sensors.