Multiwell Optogenetic Stimulation For The Precise Control Of In Vitro Cellular Network Activity - Neural And Cardiac Applications
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1
Nanion Technologies, Germany
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2
Axion BioSystems, Inc., United States
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3
Axion BioSystems, Inc.
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4
Axion BioSystems, Inc.
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5
Nanion Technologies, Germany
Motivation: Microelectrode array (MEA) technology platforms offer direct connection of key biological variables, such as gene expression or ion channels, to measures of cellular and network function. While MEA electrodes can deliver electrical stimuli, optogenetic stimulation can provide advantages such as targeting of specific cell types, the ability to suppress activity, minimal stimulus artifact, and uniform stimulus delivery across a culture. This motivated the need for the development and validation of a multiwell optical stimulation system for high throughput experimentation suitable for drug screening and phenotypic disease modeling.
Material and Methods: Data were obtained using the Maestro® platform (Axion BioSystems) and LumosTM (Axion BioSystems), a multiwell optogenetic stimulation device capable of illuminating each well of a standard 48-well microplate with 192 independently addressable LEDs (475nm, 530nm, 612nm, 655nm). We show data obtained from cryopreserved primary and human iPSC-derived neurons and cardiomyocytes from diverse providers, transfected with channelrhodopsin-2 (ChR2) or archaerhodopsin (ArchT). Optogenetic experiments were conducted at least 14d post transduction to allow full expression of the opsin. Data was analyzed using Axion’s AxIS analysis software.
Results: Here, we describe the validation and application of a commercial optical stimulation system for independent control of light delivery to each well of a multiwell MEA (Figure 1) using neuronal and cardiomyocyte cultures (1). In rodent cortical cultures, we were able to obtain data by using optogenetic stimuli to induce or suppress specific activity phenotypes. Functional expression was evaluated by detecting extracellular action potential firing in response to blue light pulses for ChR2 expressing wells (a single action potential elicited by each pulse of light, Figure 2A) and extended pulses of green light for ArchT-expressing wells (suppressed action potential firing during green light delivery, Figure 2B) (1). For the ChR2 well, each pulse of blue light initiated a burst of network activity that was consistent across repetitions. Induction of seizure-like activity may be used to screen for proconvulsant liability or the efficacy of anti-epileptic drugs, while suppression of seizure-like activity may be used to tune activity states in “disease-in- a-dish” models of epilepsy. We show that optogenetic stimulation by LumosTM enables tuning of network states across cultures to improve assay sensitivity (1). To prove this, we characterized evoked response metrics as a function of light intensity before and after dosing (Figure 3). Evoked metrics were sensitive to the addition of neuro-active compounds, such as picrotoxin and carbamazepine. Each pulse of blue light elicited a burst of activity, which increased in magnitude and duration in response to picrotoxin addition (1).
Additionally, our results with human iPSC-derived cardiomyocytes expressing ChR2 show that optogenetic stimulation can also be used to control the beating frequency (1). This optical pacing resulted in increased reliability and sensitivity of the repolarization measurement (Figure 4A). Arrhythmic indicators, like EADs, are also sensitive to the beating frequency of cardiomyocyte networks (Figure 4B). We have shown optogenetic stimulation to be a useful tool to control for emergent arrhythmic events or more precisely quantify cardiac arrhythmias (1). Addition of sotalol (10µM) produced EADs on every beat at the spontaneous beat period (top). The EADs progressively disappeared as the pacing rate was increased (middle), with no EADs present at a paced beat period of one second (1). Thus, optogenetic stimulation can be used to control for emergent arrhythmic events, or more precisely quantify cardiac arrhythmias.
Discussion: The Maestro multiwell MEA platform connects key biological variables to cellular and network function by extracting information from complex biological systems in vitro. Optogenetics enables cells to be controlled by light, offering the opportunity to precisely and selectively control or manipulate complex in vitro cell models. Here, we have successfully integrated optical stimulation capabilities within a multiwell MEA system. This advancement enhances control over cultured network activity, enabling advance in vitro modeling and modulation of electrical phenotypes for diseases such as epilepsy, autism, Alzheimer’s and ALS as well as cardiac arrhythmias.
Conclusion: Our findings demonstrate the potential of optically-integrated multiwell MEA systems to enable high-throughput drug screening and phenotypic modeling of diseases. The device was shown to independently deliver precisely controlled levels of robust optical stimulation at selectable wavelengths to 48 MEA wells. Future device work will expand to higher well counts (e.g. 96 wells) and development of enhanced control and analysis methodologies. Together, the Maestro® and LumosTM improve the reliability and sensitivity of existing assay screens, while simultaneously enabling new directions in high throughput network electrophysiology.
References:
(1) Isaac P. Clements I.P, et al. 2016, Proc. of SPIE Vol. 9690 96902C-1; Optogenetic stimulation of multiwell MEA plates for neural and cardiac applications.
Figure Legend:
Figure1. A) Array of 48 LED banks and metallic reflectors. B) The light delivery device mates to the lid of a 48-well microplate, forming a seal around a distributed gas delivery system for environmental control.
Figure 2. Response of individual neurons to pulses of A) blue light (475nm) for ChR2 + and B) green light (530nm) ArchT+ cells.
Figure 3. Quantification of optically-evoked neural activity for screening applications. ChR2-expressing neural cultures dosed with picrotoxin exhibited prolonged network bursts following stimulation with blue light.
Figure 4. A) Example cardiac field potential in response to optical pacing of the culture beat rate. B) Arrhythmic events, like EADs (arrows), were also systematically modulated by pacing culture beat rate in the presence of sotalol (10µM).
Keywords:
Neurons,
MEA,
cardiomyocytes,
optogenetics,
channelrhodopsin,
HTS,
iPSCs,
Multiwell
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
oral
Topic:
MEA Meeting 2016
Citation:
Dragicevic
E,
Clements
IP,
Millard
D,
Nicolini
A,
Chvatal
S,
George
M,
Ross
J and
Fertig
N
(2016). Multiwell Optogenetic Stimulation For The Precise Control Of In Vitro Cellular Network Activity - Neural And Cardiac Applications.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00030
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Elena Dragicevic, Nanion Technologies, München, Germany, elena.dragicevic@nanion.de