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Development of high throughput planar patch clamp system at low cost

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Although patch clamp techniques are utilized in a broad range of fields, there are economic and methodological hurdles to overcome to employ them. Here we developed a small, simple, and easy-to-use patch clamp system. A four-chamber cell stage with fluid exchange channels was fabricated for high-throughput recording.

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Development of high throughput planar patch clamp system at low cost

  1. 1. 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
  2. 2. 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
  3. 3. 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
  4. 4. 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
  5. 5. 従来法との比較 Advantages 従来法 traditional 平面法 planar Requirements
  6. 6. 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
  7. 7. 実験方法 Experiment Suction Electrode Cell A
  8. 8. 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
  9. 9. 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
  10. 10. 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
  11. 11. 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
  12. 12. 電流の記録 Current recording +80 mV +70 mV +60 mV +50 mV pA 0 1 2 sec
  13. 13. 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
  14. 14. 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
  15. 15. その他の細胞 Other cells 60 mV 80 mV 50 ms 2 pA H9c2: Rat ventricular cardiomyocyte cell line
  16. 16. 価格(参考) Price Our productN company’s 10,000,000 ($127,800) 2,500,000 ($32,000)
  17. 17. 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.
  18. 18. 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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