fluorescence micropipette aspiration assay to study live cell rbc mechanosensing

蛍光マイクロピペット吸引を用いたシングルセルメカノバイオロジー

The unfolding discoveries in the world of cellular behaviors have accentuated the role of mechanical stimuli, such as tension, fluid shear stress, compression, and substrate stiffness, in dictating dynamic cellular activities such as adhesion, migration, and differentiation. These mechanobiological aspects are of paramount importance in elucidating how cells interact with and respond to their physiological environments, impacting various biological processes1,2.
Over the past decade, micropipette-based aspiration assays have stood out as a versatile tool in studying diverse cellular responses to mechanical stimuli. This technique offers valuable insights into the intrinsic mechanical properties of living cells at the single-cell level, including cellular elastic modulus, stiffness, and cortical&#160;tension. These assays enable the measurement of various mechanical parameters, such as cell membrane&#160;tension, pressure exerted on the cell membrane, and cortical tension (summarized in Table 1). Studying the aspirational forces has enriched our understanding of how they influence cellular functions and processes, particularly in the realm of membrane dynamics, including fragmentation, elongation, and budding3,4.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mechanical Parameter</td>
			<td>Description</td>
			<td>Seminal Approaches</td>
		</tr>
		<tr>
			<td>Cell Stiffness</td>
			<td>Measurement of a cell&#39;s mechanical rigidity and elasticity.</td>
			<td>Aspiration of the cell membrane and analysis of deformation response to the negative pressure20,21.</td>
		</tr>
		<tr>
			<td>Adhesion Strength</td>
			<td>Evaluation of how strongly cells adhere to surfaces.</td>
			<td>Application of controlled suction to detach adhered cells from a substrate2,22.</td>
		</tr>
		<tr>
			<td>Membrane Tension</td>
			<td>Assessment of the tension or stress within cell membranes.</td>
			<td>Measurement of the membrane deformation in response to applied pressure23,24.</td>
		</tr>
		<tr>
			<td>Viscoelastic Properties</td>
			<td>Characterization of a cell&#39;s combined viscous and elastic behavior.</td>
			<td>Analysis of the time-dependent deformation response to aspiration23,25.</td>
		</tr>
		<tr>
			<td>Deformability</td>
			<td>Determination of how easily a cell can change shape.</td>
			<td>Evaluation of the extent of deformation under controlled suction20,24.</td>
		</tr>
		<tr>
			<td>Surface Tension</td>
			<td>Measurement of the tension at the cell&#39;s surface.</td>
			<td>Assessment of the pressure required to form a micropipette membrane protrusion26.</td>
		</tr>
		<tr>
			<td>Cell-Material Interaction</td>
			<td>Study of interactions between cells and materials or substrates.</td>
			<td>Aspiration of cells in contact with different materials and observation of interactions2,24.</td>
		</tr>
		<tr>
			<td>Cell-Cell Interaction</td>
			<td>Examination of interactions between neighboring cells.</td>
			<td>Aspiration of a group of cells and analysis of their intercellular forces27.</td>
		</tr>
	</tbody>
</table>
Table 1: Mechanical parameters characterized by the micropipette aspiration assay.
The micropipette-based aspiration technique has been widely used to study red blood cells (RBCs), assessing the deformability and various mechanical characteristics of RBCs, which is essential in understanding their function in the circulatory system. RBCs exhibit remarkable adaptability, preserving their mechanical versatility against deformation when navigating through the intricate capillary network and inter-endothelial clefts5,6. During this journey, RBCs must traverse through passages as narrow as 0.5-1.0 &#181;m, subjecting themselves to a multitude of mechanical forces, including tension and compression7,8,9. They also have high sensitivity to the shear stress generated by blood flow during circulation10. These processes promote the activation of regulatory mechanisms involving calcium influx, a crucial signaling event with well-established roles in cellular responses to mechanical stimuli11,12. The complex mechanisms governing the calcium-mediated mechanosensing remain compelling subjects of ongoing investigation.
In this context, the fMPA stands as an effective approach to reveal the extent of calcium mobilization under precisely controlled mechanical forces, allowing for the simultaneous application of mechanical modulation (using the micropipette aspiration system) and visualization of calcium intensity (using fluorescent indicators). It particularly mimics the physiological scenario when the RBC travels through narrowing blood vessels. It is worth noting that the fMPA system we developed can generate pressure with a resolution of 1 mmHg. The implemented high-speed camera can achieve a temporal resolution of 100 ms and a spatial resolution at the submicron-meter level. These configurations ensure the precise application of mechanical forces to live cells and simultaneously capture the resulting cellular signaling. Moreover, due to the integrative engineered nature of this setup, the micropipette aspiration assay can be readily adapted to complement other equipment or techniques, enabling further exploration of the intricacies of cell mechanics. This versatility stands as an additional advantage of this approach.

著者スポットライト:蛍光微小吸引による生細胞メカノバイオロジーの進歩

赤血球メカノセンシングを調査するための蛍光マイクロピペット吸引アッセイ

The scope of our research is directed towards lifestyle mechanobiology, specifically the behavior of cells where mechanical stress is applied. We are investigating the intracellular signaling in single cells. The proposed FMPA system utilizes fluorescence imaging combined with mechanical stimuli.That is, the aspiration pressure. There are currently further advancements that have attempted to combine more than two techniques with the microaspiration setup like microfluidics or image analysis software. Current experimental challenges lie with the fact that the setup is quite labor intensive and is operator dependent.As the system is manually operated, there are inconsistencies that arise in specific steps. For example, filament preheating. The findings of research demonstrated there was a corresponding increase in the influx of calcium ions into the RBCs when the pressure was incrementally increased between negative 10 millimeter mercury to negative 40 millimeter mercury.This suggests that RBCs poses the ability to sense changes in their mechanical environment and respond by rapid calcium-related channel activities. This study places particular emphasis on the application of FMPA as a crucial tool for unveiling the nuanced mechanosensitive responses showcased by RBCs under varying stimuli.

生細胞赤血球メカノセンシングを研究するための蛍光マイクロピペット吸引アッセイ

まず、ダイヤモンドペンシルを使用して、標準的なガラスカバースリップを3等分します。真空グリースを使用して、カバースリップストリップの一部を自家製のチャンバーホルダーの底に貼り付けます。同様に、カバースリップストリップの2番目のピースをチャンバーホルダーの上部に取り付けます。次に、ピペットガンを使用して、200マイクロリットルのラベル付きRBC懸濁液を2つのカバースリップの間に注入します。マイクロピペット吸引装置を組み立てるには、細胞チャンバーを顕微鏡プラットフォームのホルダーステージに取り付けます。チャンバーの位置を対物レンズの真下になるまで調整します。次に、接続されている貯水槽の液面を下回るまでホルダーを下げます。脱塩した水をマイクロピペットに注入し、34ゲージの針を備えたシリンジを使用して気泡を取り除きます。マイクロピペットホルダーの端を半分ほど緩め、ホルダーから数秒間水が滴り落ちるのを待ちます。次に、懸濁液を充填したマイクロピペットをホルダーチップに挿入し、ホルダーネジを締めます。マイクロピペットをセルチャンバーに入れます。次に、ピペットと赤血球を顕微鏡で見つけます。ピペットチップを下げて、配置されたRBCと水平になっていることを確認します。貯水池の高さを調整して、先端の油圧をゼロにします。次に、貯水池を少し上げて、微妙な陽圧を発生させます。488ナノメートルの蛍光励起光源をONにします。蛍光灯カメラと透過カメラの電源を入れます。対応するソフトウェアで、両方のカメラの露光時間、関心領域、およびビニングサイズを入力します。次に、[Multi-Dimensional Acquisition]パネルで取得番号を2000に設定します。必要な保存ディレクトリを選択します。次に、マイクロマニピュレーターの助けを借りてマイクロピペットを見つけます。空気圧クランプをオンにして、コントロールボックスが外部モードになっていることを確認します。ノブをゆっくりと回して、システム内のオフセット圧力を補正します。空圧クランプを制御するソフトウェアを起動します。電気制御パネルを使用して、20ミリボルト/ミリメートルの水銀変換係数で圧力を制御し続けます。次に、システム内の圧力をゼロにし、マイクロピペットを赤血球の近くに慎重に移動させ、貯水槽の位置を調整して、マイクロピペットの先端に微妙な陽圧を発生させます。カメラソフトウェアで撮影を開始し、蛍光シャッターをオンにします。計算された電圧の大きさをコントロールパネルに入力して、目的の圧力に到達し、RBCを吸引します。あらかじめ設定した期間圧力を保持してから、解放します。同様に、さらに分析する前に、次の赤血球で実験を繰り返します。圧力が徐々に上昇すると、赤血球へのカルシウムイオンの動員が増加しました。

Research

JoVE Journal

Biology

Fluorescence Micropipette Aspiration Assay to Study Live Cell RBC Mechanosensing

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to ...

生物学

Fabrication of Micropipettes for Fluorescence Aspiration Assay to Study Live Cell RBC Mechanosensing

生細胞赤血球メカノセンシング研究のための蛍光吸引アッセイ用マイクロピペットの作製