MEA assay with human iPSC-derived neurons
generated by a rapid differentiation method

021

Tomoaki Nakayamaa, Manabu Seob, Tomoyuki Aratania, Toshihiko Hosoyaa(e-mail; tomoaki.nakayama@jp.ricoh.com)
a. Drug Discovery Business Department, Healthcare Business Group, RICOH COMPANY, Ltd., Japanb. Elixirgen Scientific, United States

Introduction
Neurotoxicity is a major cause of failures in drug development. Animal models have been insufficient for precise prediction of neurotoxicity because of the species difference.
To overcome this difficulty human neurons generated from induced pluripotent stem cell (iPSC) have been extensively tested. A major assay system for this purpose isneuronal networks cultured on microelectrode arrays (MEAs). In this paper we investigated whether Quick-NeuronTM Excitatory (EX-SeV-CW50065) generated with therecently developed quick differentiation method (Quick-TissueTM technology ) are suited to neurotoxicological assays. In addition, we optimized the fabrication and culturing toenable transportation of live-cell MEAs.

ResultsTM
1. Reliable pharmacological responses of Quick-NeuronExcitatory(a)Negative
control
Acetaminophen

Neurotransmitter
Glutamate(AMPA/NMDAR agonist)

GABA(GABAR agonist)

Dopamine(DopamineR agonist)

CNQX(AMPA/KinateRantagonist)*: p<0.05*: p<0.05*: p<0.05

Antiepileptic
drug

Receptor antagonist
Gabazine(GABA(A)R antagonist)

Chlorpromazine(Dopamine D2R antagonist)

Carbamazepine(Na+ channel blocker)

Amoxicillin(Beta-lactam antibotic)

Strychnine(Glycine R, Acetylcholine Rantagonist)*: p<0.05*: p<0.05*: p<0.05*: p<0.05

ConvulsantControl

Control

Control

Control

Control

Control

ControlControl

Control

Control

100 μM

10 μM

10 μM

10 μM

5 μM

0.3 μM

0.1 μM

100 μM

300 μM

0.3 μM(b)
Changes of spike
Testing density
number1-100 μM
No change
1-100 μM↗↘
0.1-10 μM↘

Mode

Receptors / Ion channels

Chemicals

Negative control

Glutamate receptors
GABA receptors

Acetaminophen
Glutamate
GABA

Dopamine receptors

Dopamine

0.1-10 μM↘↗

Muscarine receptors

Pilocarpine(HCl)0.1-10 μM↗

Histamine receptors
AMPA, Kainate receptors
GABA(A) receptors
Dopamine D2 receptors
NMDA receptors
GABA(A) receptors
Histamine H1 receptors

Histamine
CNQX
Gabazine
Chlorpromazine
D-AP5
Picrotoxin
Ketotifen

0.3-30 μM
0.5-50 μM
0.3-30 μM
0.1-10 μM
0.5-50 μM
0.1-10 μM
0.1-10 μM↗
↘
↗
↗↘
No change↗
↗↘

2.3 μM
2.8 μM
0.122 μM（D1)
2.76 μM (D2)
1.66 μM (D5)18 μM(M1,3)
4.5 μM(M2)24 μM
0.92/6.1μM
0.2 μM
0.363 μM
4.1 μM
2.4 μM-Antiepileptic
Drug

Sodium channels

Carbamazepine

3-300 μM↘

131 μM

Amoxicillin

1-100 μM↗

100 μM- 10 mM※

Convulsantbeta-lactam antibotic
Glycine receptors,
Acetylcholine receptors
Potassium channels

Strychnine

0.3-30 μM↗↘

17-40 nM4-AP

0.3-30 μM↗

147/117 μM

NeuroTransmitter

Receptor
Antagonist

Ref: IC50MEA(microelectrodearray)

Device: Maestro Cytoview 48 well plate, Axion Biosystems
Neurons : Quick-NeuronTM Excitatory, Elixirgen ScientificAstrocytes : Human astrocytes, ThermoFisher
Medium: Neurobasal plus based medium
Culture: DIV51
Evaluation : Relative change of number of spikes

Human iPSC-derived neuron generated with transcription-factor-induced differentiation of iPSCs were cocultured with human astrocytes. The cells were plated onto MEA plates (48 wells, 16 electrodes/well,Axion BioSystems). (a) (Top) The spike numbers generated in response to the indicated compounds(three concentrations) were divided by that generated in response to the control compound (0.1% DMSO).
(Middle and bottom) Raster plots. (b) Summary of the results.

Quick Tissue Neurons show expected responses to neurotransmitters, receptor
antagonists, channel blockers, antiepileptic drugs, and convulsant.

※Since the IC50 value of amoxicillin is unknown, the IC50 value for other GABA receptors of β-lactams is described.

3. Overseas transportation of live-cell MEAs

2. Optimization of culture stability

Neurons : Quick-NeuronTM Excitatory, Elixirgen Scientific
Astrocytes : Human astrocytes, ThermoFisher
Culture: DIV49-52
Medium: Neurobasal plus based medium

Shinnosuke Koshizuka, Momoko Shionoiri (RICOH).

Neurons: Quick-NeuronTM Excitatory, Elixirgen ScientificMedium : Brainphys based medium

From Tokyo to Baltimore, 33 hours(e)(c)
Coating method A

Coating method B32 hour(d)Medium change Proportion
Low

Medium change
frequency
HighLowHigh(f)(g)

Active electrodes

Number of active electrode(N=8 wells)
20

Drug response after transportation

Control (DMSO)

4AP (3 μM)

4AP (30 μM)

15
10
50
before transport after transport(c) Top: Immunostaining of laminin on the surface of MEAs. Bottom: Photographs ofneuron cultures. Coating method B improved laminin adhesion and suppressed cellaggregation. (d) The effect of medium change on cell aggregation.

(e) The course of the transportation. (f) The number of active electrodes of MEAs before
and after the transportation. (g) Response to 4-Aminopyridine (4-AP), a potassiumchannel blocker, after the transportation.

Conclusions and future works
Conclusions✓ Quick-tissue neurons exhibit excellent pharmacological responses and are suited to neurotoxicological assays.
✓ Optimized culture conditions enable transportation of live-cell MEAs overseas.

Future work
✓ We will evaluate the cells with a larger number of compound.
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