A Novel Microelectrode Array System for High-Throughput Neurotoxic Screening
Axion Biosystems, Atlanta, GA J. Ross, S. Rajaraman, E. Brown, M. McClain, E. McConnell, and T. O’Brien
761.03
1. ABSTRACT Microelectrode arrays (MEAs) are an important tool for scien<fic discovery and medical advancement. Because MEAs can ac<vely manipulate and monitor cellular ac<vity at both the single-‐cell and network level, these tools provide insight into complex cellular interac<ons. MEAs, however, possess two constraints that prevent their use as a high-‐throughput instrument: (1) extracellular s<mula<on temporarily induces a s<mula<on ar<fact that disrupts the monitoring of cellular ac<vity, and (2) MEA systems typically limit sample processing to a few samples at a <me. In order to address these constraints, we developed novel charge-‐control circuitry and cost-‐effec<ve MEMS processes to remove the s<mula<on ar<fact and implement a scalable, high-‐throughput MEA system. MEAs have a wide range of applica<ons and are increasingly finding a role in evalua<ng compounds for safety pharmacology or environmental monitoring. Our objec<ve is to increase the throughput for iden<fying neuroac<ve proper<es in uncharacterized compounds. To this end, in conjunc<on with the Environmental Protec<on Agency, we are tes<ng the performance of the mul<well MEA system on a number of chemicals, including established environmental contaminants and a set of control compounds with iden<fied neuroac<ve mechanisms. In this study, we present preliminary spontaneous ac<vity, characterizing ini<al results for correctly discrimina<ng known neuroac<ve compounds from inert controls.
2. CONCEPTUAL FRAMEWORK
3. DEVELOPING ANSI/SBS COMPLIANT MEA SENSOR PLATES
5. SYSTEM INTEGRATION
4. SCALABLE SIMULTANEOUS STIMULATION & RECORDING ELECTRONICS
6. SCREENING GOALS & COMPOUND SELECTION
7. SCREENING APPROACH & PRELIMINARY RESULTS
8. CONCLUSIONS & FUTURE STUDIES
A! B!
(1) Use the 768 channel, mul<well MEA system with 12-‐well MEAs.
(2) Assess the ability of a set of 27 chemicals to alter spontaneous network ac<vity in primary cultures of cor<cal neurons.
(3) At 14 to 21 DIV, record 30 min. of baseline ac<vity, then 30 min. of ac<vity in presence of 50 µM (or highest soluble concentra<on) of chemical.
By defini)on, unintended altera)ons in neural structure or func)on are neurotoxic: Following acute or chronic exposures, at concentra<ons that do not affect general viability, an altera<on in the structure or func<on in any part of the CNS or PNS is considered neurotoxic (Costa, 1998; Defranchi 2011)
Mean Firing Rate (MFR) is a simple and effec)ve MEA metric to iden)fy neurotoxicity: In vitro neural cultures form spontaneously ac<ve func<onal neuronal networks that retain the basic processes underlying in vivo physiological behavior (Novellino 2011). Mean firing rate (ac<on poten<als per second) is a sensi<ve measure of neurotoxic effects (Defranchi 2011; Novellino 2011; Johnstone 2010). However, processing many samples simultaneously remains a challenge.
Altera)on in neural func)on (neurotoxicity) is “holis)cally” captured by MEAs: Neural structure or func<on may be altered by many different mechanisms (receptor modula<on, metabolic disruptors, etc.). Independent of the mechanism, these alterna<ons induce a func<onal change that is recorded by the MEA (Johnstone 2010).
Microelectrode Arrays (MEAs): A grid of microelectrodes monitors and controls electroac<ve <ssues and cultures. Each electrode or channel simultaneously records extracellular voltages, detec<ng both unit-‐level ac<on poten<als and field poten<als.
(1) Primary rat cor<cal neurons are cultured on the MEA-‐
(2) Spontaneous extracellular ac<vity is recorded on each electrode
(3) Unit level ac<on poten<als on each channel are detected and quan<fied.
Microfabrica)on across large surface-‐areas presents conflic)ng requirements: Increasing the throughput of MEA experiments requires distribu<ng microscale electrodes across mul<ple, widely-‐distributed wells. In order to address this complex problem, large area fabrica<on processes (e.g., PCB, Pick-‐and-‐Place) were combined with small area fabrica<on (e.g., MEMS, Nanotechnology).
An<cipated performance benefits using a 48-‐well MEA plate to capture neuroac<vity dose-‐response curves for 12 compounds
(1) Die Fabrica)on: MEA sensors are microfabricated on 300µm thick glass substrates u<lizing a two-‐layer surface micromachining process.
(2) Packaging: Glass die are assembled on a PCB with through-‐holes to enable inverted microscopy. The MEA die is then wirebonded to the PCB.
(3) Post Processing: A low impedance surface coa<ng of nano-‐porous pla<num is electrodeposited.
Mul)well electronics must impart mul)ple func)ons to each electrode: Engaging 768 electrodes to perform simultaneous s<mula<on and recording requires sophis<cated, miniaturized electronics. Therefore, our goal was to capture mul<ple electrophysiological func<ons inside an integrated circuit (IC); including amplifica<on, s<mula<on, ar<fact elimina<on, and basic signal processing.
Neurotoxicity Screening Objec)ves: (1) Assess the ability the mul<well MEA system to efficiently screen
environmental compounds (2) Compare results to data collected using single well MEAs (3) Establish appropriate approaches and parameters
Compounds selected for screening:
20 Test Compounds: Chemicals known to alter neuronal ac<vity, with a preference for compounds for which previous MEA data exists.
• 3 Glutamatergic compounds
• 4 GABAA antagonists • 2 GABAA modulators • 2 Nico<nic agonists
7 Nega7ve controls: Chemicals that are not neuroac<ve or neurotoxic under normal exposure condi<on, including acetaminophen, saccharin, and sorbitol)
Acknowledgements: Mul<well MEA development work is supported by a phase II NIH SBIR Grant (R44 NS062477). Neurotoxicity screening research is supported by a Coopera<ve Research and Development Agreement (CRADA) between Axion Biosystems and the Environmental Protec<on Agency. The authors wish to thank Dr. Timothy Shafer for the preliminary screening results and for selec<on of the test compounds.
High-‐level schema<c of a single channel within the 64-‐channel IC Neural recordings on the s<m-‐ula<on channel with (b) and without (a) ar<fact elimina<on
The system must manage large data volumes and coordinate mul)ple func)ons simultaneously: The following systems components were developed to allow con<nuous applica<on of ‘down-‐stream’ s<mulus commands during uninterrupted ‘up-‐stream’ data sampling: (1) A mechanical interface, to physically interface the electrodes; (2) an analog front-‐end, to monitor and manipulate cellular ac<vity; (3) a data acquisi<on subsystem, to manage analog-‐to-‐digital conversion; and (4) a digital control system, to provide system communica<ons and controls.
Mechanics, Analog front-‐end, & ADC
Processing Plahorm
Soiware control & analysis
64 Ch. S<m & Record IC
Cell Cell
• 2 Acetylcholinesterase Inhibitors • 4 Sodium Channel Modulators • 3 Neurotoxic Heavy Metals
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Spike-‐rate ac<vity maps, illustra<ng contras<ng neuro ac<vity pajerns. The ac<vity map color codes and interpolates ac<vity for all 64 channels within a culture well. The average, well-‐wide spike rate is used to iden<fy neuroac<vity changes aier dosing (below).
Packaged Plate
Single-well MEA system
(1 well/plate)
Maestro Multiwell MEA system
(48-wells/plate)
MEA Plates Required (1) 48 12
Compounds evaluated/day 1/6 6
Days to complete Experiment (2) 72 2
(1) Assumes single-well plates are cleaned and reused 12 times. Assumes 48-well plates are discarded after each experiment.
(1) Assumes single-well plates are cleaned and reused 12 times. Assumes 48-well plates are discarded after each experiment.
(1) Assumes single-well plates are cleaned and reused 12 times. Assumes 48-well plates are discarded after each experiment.
(2) Single-well MEA System: Eight recordings completed per day using eight single-well plates. Multiwell MEA System: 288 recordings completed per day using six 48-well plates.
(2) Single-well MEA System: Eight recordings completed per day using eight single-well plates. Multiwell MEA System: 288 recordings completed per day using six 48-well plates.
(2) Single-well MEA System: Eight recordings completed per day using eight single-well plates. Multiwell MEA System: 288 recordings completed per day using six 48-well plates.
In this study, a 768-‐channel, simultaneous s<mula<on and recording mul<well MEA system was developed. As a first applica<on, the system was used to inves<gate the rela<ve change in spike rate for 5 compounds and 42 cultures. The results of the remaining compounds (panel 6) will be reported by year-‐end. Ongoing work will op<mize the approach and parameters used to iden<fy neuroac<vity changes. Future studies will determine the minimum channels required for a viable neurotoxicity assay and will inves<gate the use of evoked ac<vity to increase sensi<vity.
In development: 48-‐well, SBS Compliant MEA plate.
Pt electrodes Pt Ground Impedance Live/Dead Stain
