CONSTRUCTION AND ANALYSIS OF AN ARTIFICIAL

NEURONAL NETWORK USING A NEURON-COLLECTING,

MICRO-PATTERNING METHOD BASED ON

A MULTI-ELECTRODE ARRAY SYSTEM

Hideyuki Terazono

, Hyonchol Kim

, Masahito Hayashi

, Akihiro Hattori

Hiroyuki Takei

1,3

and Kenji Yasuda

1,2

Yasuda"On-chip Molecular Cell" Project, Kanagawa Academy of Science and Technology, Kanagawa, Japan

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan

Department of Life Sciences, Faculty of Life Sciences, Toyo University, Tokyo, Japan

Keywords: Artificial neuronal networks, Hippocampal neurons, Micro-chip, Agarose, Alginate, Micro-patterning,

Multi-electrode.

Abstract: We developed three techniques to make artificial neuronal networks constructed from rat hippocampal

neurons. 1) a method of non-invasively collecting primary cultured neurons and their deposition, 2) a

technique for microprocessing agarose for the purpose of assembling artificial neuronal networks, 3) a

multi-electrode array system for measurement of the multi-point extracellular potential of neurons. The

three techniques allow us to assemble and evaluate artificial neuronal networks constructed from particular

cells. We can manipulate neuro-transmission pathways and investigate roles played by the innate period or

stability information for each individual cell in the framework of physiological mechanism. It is thus

possible to construct and demonstrated the actual neuronal networks simulated by the computed neural

networks.

1 INTRODUCTION

Neuronal networks in the brain form acceptable

patterns for external information such as long term

potentiation (LTP) or long term depression (LTD)

(

Pelletier JG, 2008). Therefore, it is thought that cells

in the neuronal networks form acceptable or

resistible spatial patterns for external information.

So far, neuronal networks have been analysed

both in vivo and in vitro, but it was very difficult to

analyse informational hysteresis because neurons in

the brain and a culture dish make a self-formation of

synapse that we can’t manipulate. If an artificial

neuronal network can be constructed with desired

neuron types and synapse direction, the

informational hysteresis of neurons in neuronal

networks can be analysed very easily.

With this aim, we developed three techniques for

making artificial neuronal networks, 1) a technique

for picking cells from a group of primary cultured

neurons in a non-invasive fashion using a digestible

thin sheet and depositing the selected cells in a

micropattern, 2) a technique for microprocessing

agarose for assembling artificial neuronal networks

through manipulation of the direction of

neurotransmission on a culture dish, 3) a technique

for measuring the multi-point extracellular potential

of neurons with a multi-electrode array system, these

cells can be also stimulated during measuring if

necessary.

The three techniques allow us to assemble and

evaluate artificial neuronal networks constructed

from particular cells. We can manipulate neuro-

transmission and investigate the physiological

mechanism such as the innate period or stability

information for each individual cell.

2 METHOD

2.1 Neuron Preparation and

Cultivation

Dispersed cultures of hippocampal cells were

304

Terazono H., Kim H., Hayashi M., Hattori A., Takei H. and Yasuda K..

CONSTRUCTION AND ANALYSIS OF AN ARTIFICIAL NEURONAL NETWORK USING A NEURON-COLLECTING, MICRO-PATTERNING METHOD

BASED ON A MULTI-ELECTRODE ARRAY SYSTEM.

DOI: 10.5220/0003641203040307

In Proceedings of the International Conference on Neural Computation Theory and Applications (NCTA-2011), pages 304-307

ISBN: 978-989-8425-84-3

 2011 SCITEPRESS (Science and Technology Publications, Lda.)

prepared from E18 of Wistar rats according to the

National Institutes of Health guidelines for

laboratory animal care and safety. The hippocampal

formation was dissected out from anesthetized

animals in ice-cold Hanks balanced salt solution.

Then hippocampal formation was treated with

0.25% trypsin (Wako) and 0.01% DNase I (Sigma)

at 37°C for 30 min. After adding Fatal bovine serum,

cells were centrifuged at 1,000 rpm for 5 min. The

remaining cells were dispersed in 2 mL Neurobasal

(Invitrogen Neurobasal medium) supplemented with

2% B-27 (Invitrogen) and 1% penicillin-

streptomysin at 37°C. For primary cultures and

recultures, neurons and glias were plated onto a 35-

mm culture dish coated with poly-L-lysine (Iwaki) at

a cell density of 1.0 × 10

cells / cm

at 37°C in a

humidified 5% CO

and 95% air atmosphere.

2.2 Formation of a Cell Collection Dish

for Primary Neurons and Neurons

Network

80 µL of 1.5% sodium alginate was put on a 35-mm

culture dish and sodium alginate was spin-coated at

3,000 rpm for 10 sec, and dried. Subsequently, the

sodium alginate was gelled by applying 1.5% CaCl

and a calcium alginate thin layer was made on the

culture dish. Next, 200mM poly-l-lysine hydro-

borate (PLL) and 4% polyethyleneimine（PEI）was

coated.

2.3 Detaching and Transferring the

Cells

For detaching and transferring cells, a glass capillary

whose internal diameter was 0.6 mm was heated,

pulled, and fire polished to make the internal

diameter around 80 µm using a puller (Narishige)

and a micro forge (Narishige). The micro-capillary

was fire polished and siliconized by sigmacote

(Sigma). To detach the cells, the capillary was filled

with a culture medium with 5mM EDTA ･ 2Na

(Dojindo Laboratories)

2.4 Re-culture of the Collected Cells

Releasing of the medium containing EDTA from the

microcapillary and retrieving cells was controlled by

adjusting the air pressure in the microcapillary using

a pneumatic manual micro-injector (Eppendorf). The

retrieved cells were put onto another culture dish

and cultivated. A second cultivation dish for neuron

was coated with PLL.

Figure 1: The procedure of collecting a cultured single

neuron.

2.5 Agarose Microprocessing System

for the Single Cultivation and

Regulating the Direction of Neurite

Microstructures (microchamber and microchannel)

to ﬁx cell positions, guide neurites and form the

network patterns were created using a photothermal

etching method. A 1480-nm infrared laser beam was

focused on the agar thin layer through the objective

lens of the microscope on the culture dish, causing

the agar at the focal point to melt (Fig.2).

Figure 2: Device configuration of an agarose

microprocessing system.

2.6 An agarose Microprocessing and

Multi-electrode Array System for

the Measurement of an Action

Potential of an Artificial Neuronal

Network

A multi-electrode array (MEA) chip was formed on

a glass slide consisting of either a 8×8 or 16×4

CONSTRUCTION AND ANALYSIS OF AN ARTIFICIAL NEURONAL NETWORK USING A

NEURON-COLLECTING, MICRO-PATTERNING METHOD BASED ON A MULTI-ELECTRODE ARRAY SYSTEM

305

electrode array. This system enables measurement of

extracellular action potentials of single neurons and

a sampling rate of 100 kHz per channel can be used.

The MEA chip was made of indium tin oxide (ITO)

whose transparency facilitate subsequent

microfabrication of microchamber and

microchannel, so that this system allow us to

fabricate agarose microchambers and microchannels

on the MEAs chip. To measure action potential of

artificial neuronal network of neurons, agarose

microstructures was made on the MEAs chip.

3 RESULTS

3.1 Preparation of a Collection Dish for

Primary Neurons

Sodium alginate was put on a culture dish, and

coated by spin-coater, and then dried in air.

Subsequently, sodium alginate was gelled by

applying CaCl

solution. A thin calcium alginate

sheet was dried and washed. Subsequently, PLL and

PEI were coated.

Primary neurons adhered onto the PLL-micro-

contact-printed alginate dish and extended the

neurites and axons very well.

3.2 Collection of Cultured Primary

Hippocampal Neurons and

Re-cultivation

Primary hippocampal neurons were initially cultured

on the detaching-culture dish for neurons. After a

few minutes, neurons started to adhere on the

detaching-culture dish. After 1 day, hippocampal

neurons extended their neurites. After confirming

that neurites of cells were extended, the medium

containing EDTA was loaded by a micro-capillary.

Then calcium alginate around the target cell was

immediately solated, and target cells were released

from the layer. The released neurons were collected

with a pipette very easily. They were cultured on

another dish coated with PLL. All steps from cell

collecting to re-culturing required less than 2 min.

The collected neuron retained their shapes and did

not shrink, and re-cultivated neurons are extended

their neurites immediately (Fig3).

Figure 3: Micrographs of each step for collecting cultured

neuron.

3.3 Neuron Cultivation on the

Agarose-micropatterned Chamber

Primary neurons dissected from hippocampal

formation were initially cultured in an agarose

microchamber. Neurons transferred by a micro-

pipette adhered onto the microchamber. After 1 day,

neurite of neurons extended along the

microchamber. Neuron didn’t adhere onto the

bottom of the small chamber whose diameter was

20µm. Furthermore, neurons didn’t extend neurites

along the microchannel whose width was less than

7µm.

3.4 Measurement of the Extracellular

Action Potential of Micropatterned

Single Neurons

A microchamber was made on top of each electrode

and these microchambers were interconnected via

micro-channels. The Primary hippocampal neurons

were initially cultured on the detaching culture dish

for neuron. After a few minutes, neurons started to

adhere onto the detaching culture dish. After 1 day,

hippocampal neurons extended their neurites. After

7days, the extracellular action potential was

recorded (Fig.4).

Figure 4: An extracellular action potential recording from

single neuron on the micropatterned structure.

２0µV

0.1 sec

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4 DISCUSSION

Alginic acid is a viscous gum derived from algae

and is composed of β-D-mannuronate and α-L-

guluronate. Calcium alginate, which is a salt of

alginic acid, is harmless to cells and used as a

scaffold in tissue transplantation (Heise, 2005).

However, cells cannot adhere to intact calcium

alginate. In this study, alginate sheet that have both

properties that transform sol/gel state and

adhesiveness of cells could be made. Using this

sheet, a specific cell can be collected without

exfoliating surrounding cells.

Recently, Okano et al. have developed

techniques, which allow us to detach cells from

culture dishes without using digestive reagents9.

Temperature dependent polymer, poly (N-

isopropylacrylamide) (PIPAAm) changes the

hydrophilic/ hydrophobic property in a temperature-

dependent manner (Masuda, 2008). PIPAAm is

hydrophobic at 37°C and hydrophilic at 20°C, so

that cells on the PIPAAm coated culture dish can be

detached from the culture dish without perturbing

the extracellular matrix and intercellular connection

such as tight junctions. Such a method gives us cell

sheets that retain intercellular connections. Using

this technique, the stick cardiac tissue stacked mono-

layered cardiac cell sheet can be made.

However, individual cells that have specific

property cannot be collected with this method

because temperature cannot be controlled on the

scale of micrometer. In fact, dispersed cultured cells

have heterogeneous properties while if averaging the

physiologically property, dispersed culture cells are

apparently homogeneous. So that, to align the

physiological properties homogeneously, it must be

necessary to develop a method to collect each single

cell from culture dishes non-invasively.

On the other hand, our method is suitable for

collecting single cells or small clusters of cells.

Therefore, for example, if there are several types of

differentiated or undifferentiated cells derived from

ES or iPS cells in the culture dish, our method can

allow us to collect only targeted cells.

Moreover, primary neurons were cultured on the

agarose-micropatterned chamber. The cultured

neurons extended neurite along the microchannel.

Furthermore, the extracellular action potential of

single neuron can be measured by an agarose-

micropatterned multielectrode array.

The results of three techniques; the noninvasive

collection method of neuron, agarose

microproceesing method and multielectrode array,

allow us to make artificial neuronal networks using

neurons regulating direction of neurotransmission,

and to measure the activity of artificial neuronal

networks. The next stage of the study is to construct

basic components working in the actual brain.

5 CONCLUSIONS

We developed three techniques 1) a non-invasive

neuron collection method, 2) an agarose micro-

processing technique, 3) a multielectrode array

system. These techniques allow us to construct and

demonstrated the actual neuronal networks

simulated by the computed neural networks.

ACKNOWLEDGEMENTS

We greatly thank Ms. Misa Sasajima, Ms. Hiromi

Mikami, and Ms. Tamae Takato for their technical

assistance. This work was financially supported by

the Kanagawa Academy of Science of Technology.

REFERENCES

Pelletier J. G., Lacaille J. C., 2008, O. Prog Brain Res.

169, 241-250.

Heise, M., Koepsel, R., Russell, A. J. & McGee, E. A.

2005, Reprod Biol Endocrinol 3, 47.

Masuda, S., Shimizu, T., Yamato, M. & Okano, T. 2008,

Adv. Drug Del. Rev. 60, 277-285.

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NEURON-COLLECTING, MICRO-PATTERNING METHOD BASED ON A MULTI-ELECTRODE ARRAY SYSTEM

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