免疫細胞キリング

Immuno-oncology applications
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がん免疫療法の分野では、免疫システムを利用してがん治療を施す様々な研究に力が注がれています。免疫細胞によるがん細胞の殺傷(キリング)は有望な治療法の1つとされており、ナチュラルキラー (NK) 細胞、細胞傷害性T細胞、B細胞(Bリンパ球)など、様々な適応(獲得)及び自然免疫システムを有する細胞が使用されています。特に、キメラ抗原受容体T(CAR-T)細胞療法は注目を集め、研究が盛んに行われています。

Axion BioSystemsのMaestroシリーズは、インピーダンスの変化検出により、細胞の生死を経時的にラベルフリーで測定します。長期間に渡る継続的な測定は、免疫細胞によるがん細胞への攻撃を終始追跡し、重要なイベントを見逃しません。また、エンドポイントアッセイのような、アッセイの繰り返しも不要です。

エフェクター細胞によるがん細胞傷害を継続的に観察

 

膠芽腫(グリオブラストーマ)細胞は転移の早い脳腫瘍の1つで、有効な治療薬も開発されていません。免疫システムを有するエフェクターT細胞は、その特異性、また生来の免疫システムを用いることから、有望な治療法の1つとされています。Maestroシリーズによるインピーダンスアッセイでは、がん細胞の増殖から免疫細胞による傷害を、時間の経過と共にリアルタイムで測定・観察します。

 

Continuous monitoring of impedance over 24 hours.  The addition of activated T-cells reduced the impedance measurement
The kill curve for different densities of activated T-cells
The kill time (50%) at different doses of activated T-cells.

(A) U87膠芽腫細胞を、3種の異なる密度(n=12)でCytoView-Zプレートに播種し、Maestro Zでインピーダンスの変化を連続して測定した。24時間後、活性化されたヒトT細胞を10:1の割合で添加(各密度毎にn=4ウェル)した。T細胞添加のウェルでは、インピーダンスが減少し、T細胞によるU87細胞への傷害が示唆された。一方、T細胞非添加のウェルでは、インピーダンスが連続して上昇した(水色:U87 25k)。(B) T細胞(100k, 250k, 500kの3密度)によるCytolysisを示す。(C) T細胞添加密度毎のKill Time 50% (50% Cytolysis到達までの時間)を示す。高濃度添加時ほど、速いスピードで到達した。

Immuno-oncology assay protocol steps

Maestroによる、インピーダンスアッセイはとても簡単です。ターゲット細胞をマルチウェルフォーマットのCytoView-Zプレート上に播種し、プレートをMaestroシステムに装着します (Day 0) 。ターゲット細胞のプレートへの接着・増殖に伴い、インピーダンスが上昇します。

1-2日後、エフェクター細胞(CAR-T細胞など)をCytoView-Zプレートに添加し、ターゲット細胞の反応を数日間に渡り記録します。アッセイの全行程はラベルフリーで行い、専用ソフトウエアでリアルタイムに表示・解析が可能です。

 

 

Maestro Z user

 

Maestroシリーズによる免疫細胞キリングアッセイの特徴

  • 経時的観察 : ターゲット細胞の増殖からエフェクター細胞によるキリングの全過程をリアルタイムで測定します。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ウェル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)のように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。数日間に渡る免疫細胞のキリングの様子を、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.