細胞シグナル伝達

Cell Signaling
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細胞シグナル伝達経路は、細胞の外から来たメッセージを伝達します。細胞外の分子が細胞表面の受容体に結合すると、細胞内のシグナリングが始り、細胞が反応します。これらの微細な変化は急激に起こることが多い一方で、数分から数時間にかけて持続し、身体に重大な影響を及ぼすこともあります。

Axion BioSystems の Maestro では、インピーダンス変化の検出により、細胞の変化をラベルフリーで連続して、測定・解析することが可能です。数日間に渡る連続したデータ測定は、受容体媒介シグナリングなどのカイネティクスに関するより多くの理解が得られます。

 

受容体介在シグナリングの検証

Gタンパク質共益受容体 (G protein-coupled receptor、GPCRs) は、最大の貫通型受容体の1つです。GPCRの結合は構造変化と下流の応答として現れますが、これらの変化をMaestro のインピーダンス測定で検出することが可能です。本事例では、Maestro Zを用いて、Calu-3細胞株の、イソプレテレノール、ヒスタミン、サイトカラシンDへの応答を、数日間に渡り連続して測定しました。各化合物に対する細胞の応答のカイネティクスの差異が顕著に示されました。

Cells grown to confluence on a microelectrode array

 

Calu-3 cells seeded on a Cytoview-Z plate and impedance was continuously monitored on the Maestro Z
Impedance measurement at 20 mins post dose of different concentrations of isoproterenol.
Cell signaling dynamics varied with compound mechanism.

(A) CytoView-Z プレート上にCalu-3細胞を播種し、Maestro Zにてインピーダンスを測定した。βアドレナリン受容体作動薬であるイソプロテレノールを投与したところ、急激なインピーダンスの減少が得られた。
(B) イソプレテレノール投与から20分後(A図点線箇所)のインピーダンス値を示す。高濃度投与においてインピーダンス値が最も低く、また最低濃度投与のインピーダンスは投与前の状態に回復した。
(C) 3種の化合物投与によるインピーダンスの変化を示す。ヒスタミン(オレンジ色、100 μM) 投与においては、インピーダンスが急激に減少した。また、サイトカラシンD(グレー色)投与においては、投与直後に急激に増加した後、緩やかに減少し、アクチン重合阻害と細胞周期停止が示唆された。

Cell Signaling assay protocol

Maestroによるインピーダンスアッセイはとても簡単です。事前コーティングされた CytoView-Zプレート上に細胞を播種します (Hour 0)。Maestroシステムにプレートを搭載すると同時に、温度・CO₂ 濃度制御とインピーダンス測定が開始されます。細胞の増殖・電極への接着に伴いインピーダンスが上昇します (Hour 0 ~ 24-72)。

細胞がプレート上でコンフル状態なった後、化合物などを投与し、以降数日間に渡って細胞シグナル伝達をラベルフリーで測定します。

 

 

Maestro Z user

 

Maestro Z/ ZHT, Pro/EdgeによるGPCR細胞シグナル伝達アッセイ : 特徴

  • 経時的観察 - 96或いは384 well 同時に測定します。GPCRなど細胞シグナル伝達を連続して測定し、専用アプリで、実験室の外からでもライブデータの確認が可能です。

  • ラベルフリー  - 平面電極によるインピーダンス測定は、染色・試薬などを必要としません。ラベルフリー測定で数日間に渡る測定・観察が可能です。

  • インキュベータ不要  - Maestroには温度・CO2濃度コントローラが内蔵されています。インキュベータ等の周辺装置は不要。安定した環境下で数日間に渡る連続測定が可能です。

  • 細胞可視 - CytoView-Z 96 well プレート底面中央部は透明になっています。必要に応じて、細胞の観察が可能です。 

  • 培養から測定まで同一プレート使用 - アッセイの全行程を同一プレートで行います。他のハイスループット・プラットフォーム(例:フローサイトメータ)のような容器の入れ替えなどは不要。細胞への負担を最小限に抑えることができます。

  • スマートフォン・アプリ - 専用のスマートフォンアプリに対応しています。数日間に渡るシグナル伝達変化の様子を、実験室の外からでも、リアルタイムに観察して頂けます。

  • 簡単 - セミ・オートメーションシステムです。ハードウエアの操作はボタン1つ。専用のソフトは、インピーダンスの変化をリアルタイムで表示します。解析結果のエクスポートも容易です。

Impedance Technology

Impedance - General

 

Impedance: For real-time cell analysis

Impedance-based cell analysis is a well-established technique for monitoring the presence, morphology, and behavior of cells in culture. Impedance describes the obstruction to alternating current flow. To measure impedance, small electrical currents are delivered to electrodes embedded in a cell culture substrate. The opposition to current flow from one electrode to another defines the impedance of the cell-electrode interface. When cells are present and attached to the substrate, they block these electrical currents and are detected as an increase in impedance.

Impedance is sensitive to many aspects of cell behavior: attachment, spreading, shape,  cell-cell connections (e.g. tight junctions), and death. Even small transient changes, such as swelling or signaling, are detectable by impedance. Because impedance is noninvasive and label free, the dynamics of these changes can be monitored in real time over minutes, hours, or even days without disturbing the biology.

Interdigitated electrodes embedded in the cell culture substrate at the bottom of each well detect small changes in the impedance of current flow caused by cell presence, attachment, and behavior.

Interdigitated electrodes embedded in the cell culture substrate at the bottom of each well detect small changes in the impedance of current flow caused by cell presence, attachment, and behavior.

In the example below, the electrodes are initially uncovered before cells are added. The electrical current passes easily and the impedance is low. When cells begin to attach and cover the electrodes, less electrical current passes and the impedance is high. After dosing with a cytotoxic agent, cells die or detach, and the impedance decreases back towards baseline.

Cells on electrode
Dosing cells and recording impedance

Impedance measures how much electrical signal (orange arrows) is blocked by the cell-electrode interface. Impedance increases as cells cover the electrode and decreases back to baseline due to cell death.

 

Continuous cell monitoring

Many cell-based assays are endpoint assays, limited to a single snapshot in time. Repeating these assays at multiple time points can be labor intensive, time consuming, and costly. Key time points can be easily missed. Impedance-based cell analysis is nondestructive and label free, meaning that cellular dynamics can be monitored continuously.

The impedance assay can be used to characterize dynamic cell profiles, revealing how cells grow, attach, and interact over time. Each cell type exhibits a different cell profile, or “fingerprint”, of dynamic cell behavior. These profiles are sensitive to cell type, density, purity, and environmental factors. In this example, the Maestro Z impedance assay readily distinguished cell profiles across different cell densities and cell types.

HeLa cells were seeded on a CytoView-Z plate at varying densities and the impedance was continuously monitored by the Maestro Z
Impedance scaled proportionally with cell density and readily distinguished different densities of the same cell type.
Maestro monitored the growth of three cell types, HeLa, A549, and Calu-3, and readily distinguishes their distinct cell profiles over time.

(A, B) HeLa cells were seeded on a CytoView-Z plate at varying densities and the impedance was continuously monitored by the Maestro Z. Impedance scaled proportionally with cell density and readily distinguished different densities of the same cell type. (C) Maestro monitored the growth of three cell types, HeLa, A549, and Calu-3, and readily distinguishes their distinct cell profiles over time.

 

 

The Maestro Z impedance assay can also be used to capture the kinetics of cell responses to drugs or immune cell therapies. The kinetics, which cannot be captured by endpoint assays, often provide key insights into the efficacy of novel therapies. In the example below, the Maestro Z impedance assay was used to quantify the kinetics of cytotoxicity of chemotherapy agents.

A549 were dosed with Doxorubicin, vehicle (DMSO), or Tergazyme. Wells dosed with Tergazyme showed an immediate decrease in impedance, reflecting complete cell death.
Cells dosed with 1 uM doxorubicin reached 50% cytolysis at 31 hrs.
Higher doses of Doxorubicin resulted in a slower decrease in impedance and cell death

A549 cells were dosed with dox, vehicle (DMSO), or tergazyme. Wells dosed with tergazyme showed an immediate decrease in impedance, reflecting complete cell death. Higher doses of dox resulted in a slower decrease in impedance and cell death. Cells dosed with 1 μM dox reached 50% cytolysis at 31 hrs.

 

Different frequencies reveal cell properties

Impedance varies with frequency, such that different frequencies reveal different aspects of cell biology. The small currents used to measure impedance will always take the path of least resistance. At low frequencies, such as 1 kHz, the impedance of the cell membrane is relatively high, forcing the current to flow under and between the cells. Low frequencies provide details about barrier integrity, the presence of gap junctions, and transepithelial or transendothelial resistance (TEER).

At high frequencies, such as 41.5 kHz, the impedance (and capacitive reactance) of the cell membrane is relatively low. Thus, most of the current couples capacitively through the cell membranes, providing information about the cell layer such as confluency and coverage.

In other words, low frequencies are sensitive to “what” cells are there, whereas high frequencies are sensitive to “how many” cells are there. The Maestro Z impedance assay uses multiple frequencies to provide the most information about the cells, simultaneously, continuously, and in real time.

Multiple frequencies were used to simultaneously and continuously monitory the coverage and barrier function
TEER, measured at 1 kHz, reveals that only Calu-3 cells form a strong barrier, as they express tight junctions to block flow between neighboring cells.

Multiple frequencies were used to simultaneously and continuously monitor the coverage and barrier function (TEER) of Calu-3 and A549 cells. Coverage, measured as resistance at 41.5 kHz, increases over time for both cell types. TEER, measured at 1 kHz, reveals that only Calu-3 cells form a strong barrier, as they express tight junctions to block flow between neighboring cells.