Name: measurement of the compressibility of cell and nucleus based on acoustofluidic microdevice
Uploaded: 2022-07-14T15:00:00
Description: Watch this Scientific Journal Video about Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice at JoVE.com

本研究得到了中国国家自然科学基金（批准号12075330和U1932165）和中国广东省自然科学基金（批准号2020A1515010270）的支持。

1. 声流体微器件的制造与组装
<ol><li>微流控芯片的制造。<ol><li>设计一个只有一个入口和出口的单通道芯片，如图 1所示。对于测量单元，将微通道的矩形横截面保持在 740 μm 宽和 100 μm 深。为了测量细胞核，将微通道的宽度和深度分别更改为250μm和100μm。</li>
		<li>通过反应离子蚀刻 在 硅晶片上制备微通道。通过阳极粘合15用一块透明?...

<ol>
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常用的细胞力学测量方法有AFM、微量移液器抽吸、微流控方法、平行板技术、光学镊子、光学拉伸器和声学方法20。微流体方法可以使用三种方法：微收缩，拉伸流和剪切流。其中，光学担架、光镊、声学方法、拉伸流和剪切流方法都是非接触式测量。与接触式测量相比，非接触式测量可以有效避免接触或局部变形引起的电池损坏问题。光镊对测量环境非常敏感21</s...

细胞力学特性在肿瘤转移、细胞恶性转化和放射敏感性中起重要作用1，2。为了深入了解细胞力学性能在上述过程中的作用，准确测量细胞力学至关重要，并且测量不应对细胞造成损害，以便进行后续培养和分析。测量过程应尽可能快，否则如果将细胞长时间从培养环境中取出，可能会影响细胞活力。
现有的电池力学测量方法面临一些局限性。一些方法，如磁扭曲细胞术、磁镊和粒子跟踪微流变学，由于将颗粒引入细胞3，4，5 而引起细胞损伤。通过与细胞接触进行测量的方法，如原子力显微镜（AFM），微量移液器抽吸，微收缩和平行板技术，也容易发生细胞损伤，并且通量难以提高6，7，8。此外，电离辐射会使细胞变平并增加其附着力9;因此，有必要测量悬浮液中的全细胞力学。
针对上述挑战，研制了一种基于声流体方法10、11、12、13、14的细胞力学测量系统。通道宽度与声学半波长相匹配，从而在微通道的中线处创建一个驻波节点。在声辐射力的作用下，细胞或标准珠可以移动到声压节点。由于标准磁珠的物理性质（尺寸、密度和可压缩性）是已知的，因此可以确定声能密度。然后，通过记录细胞在声场中的运动轨迹，可以获得细胞的可压缩性。可以实现悬浮状态细胞的无损高通量测量。本文将介绍微流控芯片的设计、系统的建立和测量步骤。已经对各种类型的肿瘤细胞进行了测量，以验证该方法的准确性。通过调节压电陶瓷的共振频率和微通道的宽度，该方法的应用范围已扩展到亚细胞结构（如细胞核）。此外，还研究了不同剂量药物诱导的EMT或X射线照射后细胞压缩性的变化。结果表明，该方法作为研究生化变化与细胞力学性能之间相关性的有力工具具有广泛的适用性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin(1x)</td>
			<td>GIBCO</td>
			<td>15050-065</td>
			<td></td>
		</tr>
		<tr>
			<td>502 glue</td>
			<td>Evo-bond</td>
			<td></td>
			<td>cyanoacrylate glue</td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td>lung adenocarcinoma</td>
		</tr>
		<tr>
			<td>Cytonucleoprotein and cytoplasmic protein extraction kit</td>
			<td>Beyotime</td>
			<td>P0027</td>
			<td>Contains cytoplasmic protein extraction reagents A and B</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium (DMEM)&#160;</td>
			<td>corning</td>
			<td>10-013-CVRC</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Srum(FBS)</td>
			<td>AUSGENEX</td>
			<td>FBS500-S</td>
			<td></td>
		</tr>
		<tr>
			<td>HCT116</td>
			<td>ATCC</td>
			<td>CCL247</td>
			<td>colorectal carcinoma</td>
		</tr>
		<tr>
			<td>Heat-resistant glass</td>
			<td>Pyrex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s L-15 medium&#160;</td>
			<td>GIBCO</td>
			<td>11415-064</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF-7</td>
			<td>ATCC</td>
			<td>HTB-22&#160;</td>
			<td>breast Adenocarcinoma</td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>HTB-26&#160;</td>
			<td>breast Adenocarcinoma</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM)</td>
			<td>corning</td>
			<td>10-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td>corning</td>
			<td>21-040-cvc</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Beyotime</td>
			<td>ST506</td>
			<td>100mM</td>
		</tr>
		<tr>
			<td>Polybead Polystyrene Red Dyed Microsphere&#160;</td>
			<td>polysciences</td>
			<td>15714</td>
			<td>The diameter of microshpere is 6.00&#181;m</td>
		</tr>
		<tr>
			<td>propidium iodide(PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184Silicone ELASTOMER</td>
			<td>Dow-Corning</td>
			<td>1673921</td>
			<td>Contains prepolymers and curing agents</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Beyotime</td>
			<td>C0011</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of the compressibility of cell and nucleus based on acoustofluidic microdevice

细胞力学在肿瘤转移、细胞恶性转化和放射敏感性中起重要作用。在这些过程中，研究细胞的机械性能通常具有挑战性。传统的基于接触的测量方法，如压缩或拉伸，容易造成细胞损伤，影响测量精度和后续细胞培养。贴壁状态下的测量也会影响准确性，尤其是在照射后，因为电离辐射会使细胞变平并增强粘附。在这里，已经开发出一种基于声流体方法的细胞力学测量系统。通过记录细胞在声力作用下的运动轨迹可以获得细胞压缩性，可以实现悬浮状态下的快速无损测量。本文详细介绍了芯片设计、样品制备、轨迹记录、参数提取和分析的协议。基于该方法测量了不同类型肿瘤细胞的压缩性。通过调整压电陶瓷的谐振频率和微通道的宽度，也实现了原子核压缩性的测量。结合免疫荧光实验的分子水平验证，比较药物诱导的上皮到间充质转化（EMT）前后的细胞压缩性。此外，揭示了不同剂量X射线照射后细胞压缩性的变化。本文提出的细胞力学测量方法具有通用性和灵活性，在科学研究和临床实践中具有广阔的应用前景。

本文提出了一种基于声流体微器件的快速无损细胞可压缩性测量系统的构建协议，并展示了其在不同情况下测量细胞和细胞核的优势。 图1 显示了微流体通道的示意图。声流体微器件的组件和组件如图 2所示。 图3 显示了测量系统的设置。在声场力的作用下，细胞和粒子将向微通道的中线移动，如图 4A所示?...

Watch this Scientific Journal Video about Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice at JoVE.com

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice

这里提出了一个协议，用于构建一个基于声流体微器件的快速且无损的系统来测量细胞或细胞核的可压缩性。考察了上皮-间充质转化或电离辐射后肿瘤细胞力学性能的变化，证明了该方法在科学研究和临床上的应用前景。

基于声流体微器件的细胞和细胞核可压缩性测量

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the ...

细胞力学在肿瘤转移、细胞恶性转化和放射敏感性中起重要作用。声流体法可以在悬浮状态下实现快速无损的测量。该技术可用于反映上皮向间充质转化过程中自压缩性的变化和分离循环肿瘤细胞，显示出其在癌症诊断中的应用前景。首先，用直径为一毫米的空心针在入口和出口的PDMS块上打孔，将PDMS块与芯片绑定。然后将PDMS块和芯片背面朝上放入等离子清洁器中一分钟。将PDMS模块上的孔与芯片入口和出口对齐后，将PDMS模块轻轻按压芯片15秒，导致PDMS模块与芯片表面之间发生粘合。要将PTFE导管连接到芯片，请切割两根内径为0.8毫米，长度为10厘米的导管。将内径为0.7毫米，长度为1.5厘米乘90度的不锈钢针弯曲成L形，并将其连接到导管的一端。将不锈钢针插入PDMS块的孔中。对于入口，将 19 号分配针连接到导管的另一端，作为注射器的连接器。之后，注入去离子水以测试整个通道的密封性。对于压电陶瓷组件，请选择宽度为 5 毫米的预切压电陶瓷片。然后在压电陶瓷的一端焊接焊丝。通过在压电陶瓷上滴一滴氰基丙烯酸酯胶水，将压电陶瓷粘在芯片背面的中间。用牙签擦平胶水并去除多余的胶水。然后快速将其按在芯片上并继续按压约一分钟。要安装微型设备，请切割一块PDMS作为微型设备的底座，并使用双面胶带将底座的一侧粘在入口和出口PDMS块上，另一侧粘在透明载玻片上。将整个微器件固定在显微镜载物台上，以将芯片保持在一个焦平面上。为了制备聚苯乙烯标准颗粒溶液，将0.05毫升聚苯乙烯颗粒溶液加入到10毫升PBS中并充分混合。然后通过用PBS以90%汇合度洗涤贴壁细胞来制备细胞悬液。加入500微升0.25胰蛋白酶在室温下一至两分钟。去除胰蛋白酶后，加入一毫升完全培养基并通过移液形成细胞悬液。接下来将细胞悬液以100G离心五分钟。除去上清液并重悬于0.5至1毫升PBS中以获得细胞悬液。然后用血细胞计数器计数细胞，浓度约为每毫升300至500，000个细胞。如前所述制备细胞核悬液后，除去上清液，每20微升细胞调色板加入200微升细胞质蛋白提取试剂A并充分混合。然后将上述混合物以220G涡旋5秒钟，然后置于冰浴中10分钟。接下来，在孵育后的溶液中加入10微升细胞质蛋白提取试剂B。涡旋后，置于冰浴上一分钟，然后再次以 220 G 涡旋五秒钟。然后，最后在 1000 G 下在 4 摄氏度下离心五分钟。除去上清液并将调色板重悬于一毫升PBS中。然后在 1000 G 的温度下以 4 摄氏度离心四分钟。除去上清液后，重悬于100微升PBS中作为细胞核悬浮液。将台盼蓝加入上述细胞核悬浮液中，并在室温下染色四分钟。接下来，用10倍物镜计算倒置显微镜下的细胞核数量。用PBS缓冲液稀释上述细胞核悬浮液至每毫升200至300， 000个细胞核的浓度。然后通过70微米筛过滤细胞核悬浮液。要设置测量系统，请打开显微镜的光源并打开相机软件。使用4x物镜找到微通道的中间位置，即压电陶瓷的位置。连接导线后，分别将它们焊接到压电陶瓷信号发生器输出的正极和负极。然后将注射器放在显微注射泵上并将其连接到入口导管。要从微通道收集液体，请在出口导管的末端放置一个小容器。要确定测量参数，请用注射器吸出聚苯乙烯颗粒溶液。然后将溶液注入芯片微通道，同时避免气泡和芯片微通道，以确保准确测量。将信号发生器的输出设置为频率为 1 兆赫兹、峰峰值电压为 10 伏的符号信号，直到观察到粒子向微通道的中线移动，并在到达中线后沿中线保持向前运动。将一毫升细胞或细胞核悬浮液与标准颗粒溶液以一比一的比例混合后，用注射器将其注入微通道以测量细胞和细胞核。当细胞或细胞核进入视野并打开信号发生器时，开始使用CCD相机进行记录。然后依次用去离子水、75%酒精和去离子水冲洗微通道，以备后用。在不同时间间隔的声场力作用下，观察细胞和颗粒向微通道中线移动。对能量、密度和单元压缩性进行了计算，并从最佳拟合结果中获得了粒子的声能密度和实测运动轨迹。从细胞中分离出高纯度和完整的细胞核，并通过获取细胞核的运动轨迹进行细胞核压缩性测量。测量并比较了不同类型癌细胞和不同剂量药物诱导的EMT或X射线照射后的细胞的压缩性。收集测量的细胞，发现培养48小时后与未处理组相比，细胞增殖和活力没有显着差异。表明压缩率测量对细胞增殖和存活没有影响。最重要的是微调信号的频率，直到观察到粒子向微通道的中线移动。

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

该<em&gt;果蝇</em&gt;成虫盘瘤模型：基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

细胞侵袭和迁移过程的评价: 视频 Microscope-based 刮伤法与博伊登室法的比较

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

黑色素瘤细胞衍生因子对骨髓脂肪分化的双重影响

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

pT3N0M0 胃癌组织石蜡嵌入幻灯片上的弹性染色

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

用水凝胶微室阵列 (HMCA) 板进行肿瘤细胞侵袭和抗转移药物筛选的分析

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

Cell Cycle-specific Measurement of ÃƒÅ½Ã‚Â³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

细胞周期特异性测量βH2AX和细胞凋亡后热源毒性应力通过流细胞测定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...