Name: isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry
Uploaded: 2015-06-04T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

This project was funded by the National Science Foundation ECCS grant 10-02165, and the Interdisciplinary Innovation Initiative Program, University of Illinois grant 12035. M.Y. A. was funded at UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer &#8216;Midwest Cancer Nanotechnology Training Center&#8217; Grant R25 CA154015A. Immnunostaining and confocal microscopy imaging were carried out at the Institute for Genomic Biology (IGB), UIUC. M.Y.A. acknowledges the discussions with B. J. Williams of UIUC regarding the isolation experiments. M.Y.A. acknowledges C. Nemeh and Abdul Bhuiya of UIUC for assistance in schematic and materials list preparation.

下面所描述的协议如下UIUC人类研究伦理委员会的指导方针。 1.收集和外科组织消解<ol><li>收集后右结肠切除术（ 图1A和1B）的肿瘤组织样品。收集来自邻近部位的健康组织为好。 </li><li>马上转移的组织，以15毫升小瓶含12毫升HBSS解决方案。继续冰瓶内的绝缘泡沫箱。 </li><li>传送后45分钟的含有组织小瓶组织培养罩作进一步的处理。保持罩内的冰块上的小瓶。 </li><li...

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蜂窝牵引强调最近成为转移状态13一个潜在的生物物理指标。然而，随着原发肿瘤细胞没有实验数据的牵引文献中存在的日期。此外，还未见报道的是不同的刚度聚丙烯酰胺凝胶直接分离培养原发性结肠癌细胞。因此，我们建立对凝胶和聚苯乙烯（ 图2）优化的原发性结肠癌细胞的培养条件。在软凝胶制剂邻近细胞培养表面的荧光微珠封装允许位移场和由人原代细胞（ ?...

近年来它已成为越来越明显的是，机械微环境中，除了生物化学因素，在调节细胞功能中起重要作用。细胞可以感知并就它们所附着到（如在2D培养）或（在3D培养如）包围3-7基板刚度作出响应。通过这样做，细胞可以调节它们的分化3，形态4，迁移/蠕动5，生物物理性质6，生长7，等工序。 癌细胞也使用粘附受体的协调，分层机械化学组合和相关的信号转导的膜蛋白，细胞骨架结构，以及分子马达1,2的二维和三维矩阵刚度作出响应。例如，乳腺上皮细胞（的MECs）上为150Pa衬底培养时形成正常腺泡实质是类似于刚度健康的乳腺组织。有趣的是，它们表现出的显影肿瘤的标志，其结构和转录，当较硬基板（&gt; 5000帕），模仿一个肿瘤基质8的刚度进行培养。此外，另一实验表明，乳房肿瘤发生是伴随着胶原交联和ECM加强9。最近的实验表明，人结肠癌（HCT-8）细胞显示样表型（MLP）转移时，他们就具有生理学相关刚度（20-47千帕）2D基板培养，但不能在非常僵硬（3.6 GPA）衬底-10- 12。这些细胞的第一种形式的肿瘤样细胞团，然后相互分离，从外围开始。作为这种上皮到圆形形态（E至R转变）发生变化时，它们增殖，减少细胞 - 细胞和细胞-ECM粘附，并成为迁移的。 HCT-8细胞培养上很辛苦聚苯乙烯基板没有表现出这些恶性特征。因此，已经假定HCT-8细胞成为转移性由于其暴露于适当的微环境。值得注意的是，这些实验进行了永生化癌细胞株或鼠衍生的原代细胞，不与原代人癌细胞。 最近的一项研究提出，扩充蜂窝牵引应力可被用作一个潜在的生物物理签名为转移细胞13。研究涉及测量牵引力为在聚丙烯酰胺凝胶不同的人类肿瘤细胞株。据发现，转移性癌细胞可以发挥显著较高牵引应力相比，非转移性的细胞在所有情况下13。然而，这些结果直接矛盾对鼠衍生的乳腺癌细胞系14中的早些时候发表的研究结果。此外，最近的一项研究强调了他们的细胞骨架重构亲永生和初级人体细胞之间的显着差异蛋白分析和细胞存活蛋白表达15。因此，重新许多生物物理测定法包括牵引原发性人类癌细胞的是重要的。这将解决的问题的原代细胞是否概括永生化癌细胞系牵引趋势。 这里所描述的协议被优化用于原代人结肠细胞（包括健康的和癌性的），并为它们对软底物（聚丙烯酰胺凝胶），以及在培养皿中培养的隔离。该协议基于消化外科组织样品成单细胞悬浮液16的结果酶解。据我们所知，这是直接在软水剂基质与牵引嵌入荧光微球培养流式细胞仪分离原发性结肠癌肿瘤和正常细胞的第一个示范。透明凝胶基质也让免疫。这个实验表明在F-肌动蛋白组织和差异粘着斑在原代人结肠癌细胞作为基材硬度的变化。该细胞培养平台开辟了探索原代人细胞的各种生物物理性质，如细胞刚度和牵引作为癌症预后参数的可能性。

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	<tbody>
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr >
			<td >HBSS</td>
			<td>Life technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td>Worthington</td>
			<td>LS003736</td>
			<td></td>
		</tr>
		<tr >
			<td >Collagenese</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr >
			<td >3- Aminopropyltrymethoxysilane (ATS)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td >281778</td>
			<td></td>
		</tr>
		<tr >
			<td >Glutaraldehyde</td>
			<td>Polysciences, Inc.</td>
			<td>01201-5</td>
			<td></td>
		</tr>
		<tr >
			<td >Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A4058</td>
			<td></td>
		</tr>
		<tr >
			<td >N- methylenebisacrylamide (bis)</td>
			<td>Sigma-Aldrich</td>
			<td>M1533</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES buffer solution</td>
			<td>Sigma-Aldrich</td>
			<td >83264</td>
			<td></td>
		</tr>
		<tr >
			<td >Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr >
			<td >N&#714;-tetramethylethylenediamine (TEMED)</td>
			<td>Bio-Rad</td>
			<td>161-0801</td>
			<td></td>
		</tr>
		<tr >
			<td >Human fibronectin</td>
			<td>BD biosciences</td>
			<td >354008</td>
			<td></td>
		</tr>
		<tr >
			<td >Hydrazine hydrate</td>
			<td>Sigma-Aldrich</td>
			<td >18412</td>
			<td>Hazardous</td>
		</tr>
		<tr >
			<td >Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT15710</td>
			<td></td>
		</tr>
		<tr >
			<td >Signal enhancer</td>
			<td>Life technologies</td>
			<td>I36933</td>
			<td></td>
		</tr>
		<tr >
			<td >Monoclonal anti vinculin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>V9131</td>
			<td></td>
		</tr>
		<tr >
			<td >Alexa fluor 488 goat anti-mouse IgG</td>
			<td>Life technologies</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr >
			<td >TRITC phalloidin conjugates&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr >
			<td >0.1 &#181;m fluroscent beads</td>
			<td>Life technologies</td>
			<td>F8801</td>
			<td></td>
		</tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Life technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr >
			<td >12 mm2 glass cover slips</td>
			<td>Corning</td>
			<td>2865-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

癌细胞响应不同癌细胞使用粘附受体组成的协调，分层机械化学系统和相关的信号转导的膜蛋白，细胞骨架结构，以及分子马达1,2与基质的机械刚度以复杂的方式。Mechanosensitivity 体外是与永生化细胞系或鼠衍生的原代细胞，不与原代人肿瘤细胞研究主要。因此，知之甚少初级人结肠癌细胞的体外 mechanosensitivity。这里，一个优化的协议被开发，描述从健康和癌外科人类组织样品的原代人结肠细胞的分离。孤立的结肠细胞，然后软（2千帕刚度）和僵硬（10千帕刚度）聚丙烯酰胺水凝胶和刚性聚苯乙烯（〜3.6 GPA刚度）由细胞外基质官能化基底（纤连蛋白培养成功在这种情况下）。荧光微珠嵌入在细胞培养物表面附近软凝胶，和牵引试验被执行以评估使用免费开放存取软件蜂窝收缩应力。此外，在不同的刚度衬底免疫荧光显微镜提供有关主细胞形态，细胞骨架组织及纽蛋白含有粘着斑作为底刚度的函数的有用信息。

上述协议被成功地用于对来自四个不同患者的多个组织样品（n = 12）下的机构审查委员会的指导方针。 图1A示出了从该组织切片的细胞培养物获得的手术后右一代表结肠直肠肿瘤。在转移到层流罩，以便进一步处理后的HBSS溶液的典型组织截面示于图1B。消化外科组织样品成单细胞悬浮液酶解的示意图示于图2。 成功的组织分离后，分离原?...

Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Research

JoVE Journal

Bioengineering

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

从病人的血液样本可行的循环肿瘤细胞的快速分离

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

科研

生物工程

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

手术取出，分离和<em&gt;在体外</em&gt;扩展人类前十字韧带衍生细胞组织工程的应用

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

人体软组织的生物力学特性采用压痕和拉伸试验

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...