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低コスト・ハイスループットな
平面パッチクランプ装置の開発
Development of high throughput planar patch clamp system
at low cost
高橋賢、深澤太郎、国安築、成瀬恵治
岡山大学大学院・医歯薬学総合研究科
Ken Takahashi, Taro Fukasawa, Kizuku Kuniyasu, Keiji Naruse
Department of Physiology, Graduate School of Medicine Dentistry
and Pharmaceutical Sciences, Okayama University
パッチクランプ Patch clamp
• 細胞内外に流れる電流を記録
• 1991年にノーベル生理学・医学賞を受賞
• 創薬:てんかん、不整脈、糖尿病
Ion
Glass
pipette
Ion channel
A
• Record electric current via ion channels
• Awarded Novel prize in 1991
• Drug discovery: epilepsy, arrhythmia and diabetes
従来法の問題 Problems
• 高価 expensive
• 大きくて複雑 huge and complicated
• 職人芸が必要 requires difficult technique
• 低スループット low throughput
目 標 Goal
• 安価 inexpensive
• 小さくてシンプル small and simple
• 簡単操作 easy to use
• 高スループット high throughput
従来法との比較 Advantages
従来法 traditional 平面法 planar
Requirements
平面電極の作成 Planar electrode
特許番号(patent No):4899051
PDMS: Polydimethylsiloxiane
Excimer light
Pore diameter: 1 – 4 um
Thickness: 100 um
実験方法 Experiment
Suction
Electrode
Cell
A
電極の性能 Performance of electrode
ΩΩ
電極抵抗
Electrode resistance
シール抵抗
Seal resistance
Important for
good recording
シール抵抗 Seal resistance
1
10
100
1000
10000
0 2 4
Resistance(MΩ)
Aperture size (um)
Maximum resistance
22%39%
Rate of successful GΩ seal
(Xiaohui et al., 2006)
Seal resistance %
> 5.0 GΩ 6.9
1.0 ~ 5.0 GΩ 16.8
100 ~ 999 MΩ 5.3
< 99 MΩ 45.8
Bad partitions
(hole blocked etc.)
25.2
24%
Our system Other system
細 胞 Cell
• HEK293 細 胞 の 強 制 発 現 系 (HEK293 ex-
pression system)
限界希釈法 limited dilution Blue: DAPI, Green: anti-V5
電流の記録 Current recording
A
Voltage
command
pulse
Current
recording
BK channel transfected
HEK293 cell
K: 140 mM
Slope conductance:
273.74±2.61pS
40 mV
80 mV
O1
O2
C
O1
O2
C
50ms
10pA
電流の記録 Current recording
+80 mV +70 mV
+60 mV +50 mV
pA
0
1
2
sec
単一チャネル記録 Single channel recording
1
10
100
1000
-35 -30 -25 -20 -15 -10 -5 0
頻度
電流値(pA)
2
1
0
データの正確性 Precision
-30
-20
-10
0
10
20
30
-80 -40 0 40 80
I (pA)
V (mV)
285.4 pS 273.7±2.6 pS
(Tang et al., 2003)
Our data Known data
その他の細胞 Other cells
60 mV
80 mV
50 ms
2 pA
H9c2: Rat ventricular
cardiomyocyte cell line
価格(参考) Price
Our productN company’s
10,000,000
($127,800)
2,500,000
($32,000)
まとめ Summary
• 安価、小型・シンプル、簡単操作のパッ
チクランプ装置を開発した。
• We developed an inexpensive, small and
easy-to-use patch clamp system.
展 望 Perspective
• 適用可能細胞の拡充
• 機器の完成度を上げて製品化
• ハイスループット化
• Expand applicable cell lines
• Develop product
• Develop high throughput system

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Development of high throughput planar patch clamp system at low cost