25B05
Extracellular matrix (ECM) patterning by atmospheric-pressure plasmas forarranging neuronal cells on silicon substrates
シリコン基板上への神経細胞配列のための大気圧プラズマによる細胞外マトリックス（ECM)パターニング
Ayumi Ando1, Hidetaka Uno2, Katsuhisa Kitano1, Tsuneo Urisu2 and Satoshi Hamaguchi1
安藤あゆみ1, 宇野秀隆2, 北野 勝久1, 宇理須 恒雄2, 浜口智志1

1

Center for Atomic and Molecular Technologies,Graduate School of Engineering, Osaka University
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
2
FIRST Research Center for Innovative Nanobiodevice, Nagoya University,Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan1
大阪大学 大学院工学研究科 原子分子イオン制御理工学センター 〒565-0871 大阪府吹田市山田丘2-1
2
名古屋大学 革新ナノバイオデバイス研究センター 〒464-8603 名古屋市千種区不老町
A new extracellular matrix (ECM) patterning technique has been developed with using the atmosphericpressure plasmas (APP) application. Arrangement of neuron cells in a desired pattern on a silicon (Si) chipis an important step for the development of neuron cell chips. In this study, ECM layers deposited on a Si
substrate were patterned in 100~200 m sizes by the APP etching and neuronal model cells were shown togrow on the patterned ECM. The results indicate that the process based on APP application for the ECM
provides a simple mean of arranging neuron cells on a relatively large area of a Si surface.

1. Introduction
Control of cell adhesion to and proliferation on a
specific surface connected with electrical circuits is
a technology required for the development of
next-generation biosensors/biochips. For example,
the formation of a large-area neuron network on asilicon (Si) surface is considered to be a crucial stepfor the fabrication of cell chips for diagnosis ofbrain diseases such as Alzheimer’s. Theextracellular matrix (ECM) that consists of avarious types of proteins, polysaccharides, andother polymers serves as a scaffold for cells. For thearrangement of living cells on a semiconductor
substrate, therefore, the formation of patterned
ECM layers on the semiconductor surface is animportant first step. The goal of this study is todemonstrate that the atmospheric pressure plasma(APP) etching method for ECM layers can beapplied to the arrangement of neuron cells on a Sichip. We have confirmed that Specific HEK293cells (Human Embryo Kidney cells) proliferate only
on specific fibronectins patterned by low-frequency
APP jets (LF plasma jets) [1]. In this study, we shall
examine the APP etching techniques for the growthof neuron cells on a Si chip, using PC12 cell (Ratadrenal pheochromocytoma cell line) as a neuronalmodel and Poly-L-Lysine (PLL) as their ECM.

in air by pulsed high voltages applied to anelectrode that surrounds a glass tube inside which ahelium (He) gas flows [1, 2]. In our experiments,
the pulse frequency of power supply was 10 kHz,the inner diameter of the glass tube was 4 mm, andthe distance between the sample substrate and theend of glass tube was 50 mm. The peak-to-peakvoltage applied to the electrode, the flow rate of Hegas, and plasma irradiation time were varied in theprocess of searching optimal conditions for ECM
patterning.
Two different types of stencil masks were used inour experiments; one had three different sets of slits
in area of 1 cm2, as shown in Fig. 2 (a); (1) ten 100
m-wide slits with a slit interval (i.e., the distance
between two successive slits) of 100 m, (2) ten
100 m-wide slits with a slit interval of 200 m,and (3) ten 200 m,-wide slits with a slit interval of100 m, while the other had holes about 100 m, indiameter with an interval of about 100 m, in areaof 1 cm2, as shown in Fig. 2 (b).
He gas

LF highVoltage supply

Glass tube
Electrode

2. Experimental set up
LF plasma jets were applied to a 1515 mm2 Si
substrate coated with ECM and a stainless-steel
stencil mask was directly placed on the ECM layer,
as shown in Fig. 1. LF plasma jets were discharged

Stencil mask
ECM

Plasma jet

50 mm
Si substrate

Fig. 1. Experimental set up for ECM patterning byLF plasma jets.

(a)(a)(b)(b)(1) (2) (3)

10 mm

10 mm
100 mFig. 2. Stainless-steel masks used in this study; (a) has
three different sets of slits, (b) is the micrograph ofmask which has holes.

3. Experimental results
3.1 Fluorescently-labeled Poly-L-Lysine patterning
Si substrate which had been coated with afluorescently-labeled PLL (FITC-PLL, SIGMAP3545, 500 g/ml) layer was patterned by LF
plasma jets. The substrate surfaces after patterning
were observed by fluorescence microscopy. Figure
3 is a fluorescence micrographs of a Si surface. PLL
remaining on the substrate is indicated in white inthese micrographs. Figure 3 (a) shows the PLL
patterns located in the area (3) of Fig. 2 (a) when
LF plasma jets were applied for 15 s uniformly tothe substrate with a peak-to-peak voltage of 7 kV
and a gas flow rate of 4 L/min. The dashed whitelines of Fig. 3 (a) delineate the original slit patternsof the stencil mask. Figure 3 (b) shows afluorescence micrograph of a Si surface patternedwith a mask of holes shown in Fig. 2 (b) after LF
plasma jets were applied for 20 s uniformly to thesubstrate with a peak-to-peak voltage of 7 kV and agas flow rate of 4.5 L/min. It has been observed that,
if LF plasma jets were applied under rightconditions for each mask, PLL deposited on thesubstrate was patterned more or less uniformly
following the mask patterns.
3.2 Cell culture on a patterned PLL
PC12 cells were cultured on a PLL (SIGMAP4707, 0.01 % solution) layer patterned by LF
plasma jets for the cellular adherence examination.
The micrograph of area (3) in Fig. 2 (a) taken 24
hours after the cell seeding is shown in Fig. 4.
Figure 4 shows that cells adhered to andproliferated along the patterned PLL strips althougha small number of cells ran off the edge of thepatterned PLL strips. Similar cell adhesion was also
observed in the areas (1) and (2) of Fig. 2 (a), andon the patterned PLL with a mask of holes shown inFig. 2(b) (data not shown).

100m

100mFig. 3. Fluorescence micrographs of PLL patterns; (a)patterns in the area (3) of the mask shown in Fig. 2 (b), (b)patterns by using the mask shown in Fig.2 (b).

100 mFig. 4. A micrograph of cell arrangement patterns in thearea (3) of Fig. 2 (a) on the Si substrate. The dashed blacklines indicate the original mask slit patterns.

4. Conclusions
It has been demonstrated that PC12 cells can bearranged and grown in a pattern of 100200 msize on a Si substrate by means of an APP etchingfor the ECM layer deposited on the substrate. The
process based on APP etching for the ECM
proposed in our study offers a cost effective meansfor neuron cell proliferation in a desired pattern ona relatively large area of a Si chip. Formation ofneural networks by the growth of axons of neurons
over the patterned ECM is the subject of a future
study.
Acknowledgments
The authors are grateful to Dr. Toshifumi Asanoof Tohoku University for his support in theexperiments. This research has been funded in partby the Grants-in-Aid for Scientific Research from
the Ministry of education, culture, sports, scienceand technology (MEXT) of Japan and This work
was supported by the Joint Studies Program (2010 ~2011) of the Institute for Molecular Science.
References[1] A. Ando, M. A. Sayed, T. Asano, R. Tero, K. Kitano,T. Urisu and S. Hamaguchi, J. Phys.: Conf. Series
232 012019 (2010).
[2] S. Ikawa, K. Kitano, and S. Hamaguchi: Plasma
Process. Polym. 7 (2010) 33.