Name: cell squeezing as a robust, microfluidic intracellular delivery platform
Uploaded: 2013-11-07T19:45:00
Description: Watch this Scientific Journal Video about Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform at JoVE.com

我们感谢T. Shatova的实验设计和数据分析的有益讨论。 G.天堂的援助和专门知识，流式细胞仪核心在科赫研究所，并在麻省理工学院微系统技术实验室的工作人员的人员表示感谢。这项工作是由卫生部资助RC1 EB011187-02，DE013023，DE016516，EB000351国家机构的支持，部分由美国国家癌症研究所癌症研究中心支持（核心）补助P30-CA14051和MPP-09Call - 兰格-60。

1。存储<ol><li>存储水库，持有人，O型圈和微流体装置中70％的乙醇。使用容器（ 如瓶子或烧杯中），有一个盖子，以防止蒸发和污染的灰尘或颗粒外。放置设备在一个容器（1），水库和O形环在第二容器（2），和持有者在第三（3）。 </li></ol>注意：对于存储的使用70％乙醇中的是保持无菌。如果溶液的唯一成分是乙醇和水（ 即没有变性剂），所有的系统组件应该...

<ol>
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所描述的实验程序（ 即因子大于芯片的设计和操作速度等）的某些方面，可能需要根据系统被应用到的细胞类型和传递材料进行优化。下面地址的一些设计实验时要考虑的最常见因素的讨论。 改善对荧光标记的化合物的输送信号，需要解决的背景荧光源。表面结合和内吞作用，例如，可以通过分娩后清洗细胞5-10分钟培养或进一步加工之前去除多余的材料解决。如果选...

递送大分子到细胞的细胞质中的治疗和研究应用中的关键步骤。纳米颗粒介导的递送，例如，已经显示出在基因治疗1,2潜力，而蛋白质递送是在临床3和实验4的设置影响细胞功能的一个有希望的途径。其他材料，如小分子药物，量子点，或金的纳米粒子，是在应用范围从癌症治疗5,6至细胞内标记7,8，和单分子跟踪9的兴趣。 细胞膜主要是不可渗透的大分子。许多现有技术使用的聚合物纳米颗粒10,11，脂质体12，或化学修饰13，以促进膜破裂或内吞递送。在这些方法中，输送效率和细胞存活率通常是依赖于次结构Ë目标分子和细胞类型。这些方法可以在结构均匀的材料，例如核酸的递送是有效的，但通常不适合用于多结构不同的材料，如蛋白质14,15和纳米材料7的交付。而且，大多数这些方法依赖于核内体破坏机理是经常效率不高，因而留下被困在囊泡结构16多的材料。最后，往往与建立的细胞系使用而开发的方法并不很好的转化为原代细胞。 膜穿孔的方法，如电穿孔17,18和19声孔效应，在某些应用中有吸引力的选择，但是，它们是已知会引起低细胞存活率，并且可以通过目标传递材料的电荷来限制。 细胞的快速机械变形，微流体方法来交付，最近有demonstrATED其优于目前的技 ​​术在细胞重编程20和纳米材料的输送管21的范围内。这种方法依赖于机械破碎Ø细胞膜，促进细胞内传递的存在于周围的缓冲材料。该系统已经证明使潜在的挑战先前的细胞类型（ 如原发性免疫细胞和干细胞）和材料（ 如抗体和碳纳米管）。本文中，用于细胞内递送的目标大分子的这些设备的一般操作进行说明。该过程推广到大多数细胞类型和交付的材料，但是，它是建议一所进行的条件一个简短的优化，详情载于我们先前报道的设计准则20，对任何以前未报告的应用程序。迄今为止，该系统已被​​成功地用于RNA，DNA，金纳米颗粒，量子点，碳纳米管，蛋白质的递送s和葡聚糖聚合物20,21。

<table border="0" cellpadding="0" cellspacing="0" width="777">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:375px;">Device Holder &#38; Plastic reservoir</td>
			<td style="width:136px;">SQZ Biotechnologies</td>
			<td style="width:149px;">Holder</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LSRFortessa Analyzer</td>
			<td>Becton Dickinson</td>
			<td>N/A</td>
			<td>Flow cytometry machine used at the Koch Institute Core Facilities</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic device</td>
			<td>SQZ Biotechnologies</td>
			<td>Cell Squeeze</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O-Rings</td>
			<td>McMaster</td>
			<td>9452K311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressure system to operate device</td>
			<td>SQZ Biotechnologies</td>
			<td>Pressure System</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasound bath</td>
			<td></td>
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</table>

cell squeezing as a robust, microfluidic intracellular delivery platform

细胞的快速机械变形已成为细胞内输送大分子和纳米材料的一种很有前途的，矢量-free方法。这种技术已经显示潜在解决先前具有挑战性的应用;包括，输送到初级免疫细胞，细胞重编程，碳纳米管，量子点传递。这个矢量的无微流体平台依赖于细胞膜的机械破坏，以促进胞内递送的靶材料制成的。本文中，我们描述了用于这些微流体装置，包括装置组件中，细胞制剂，和系统操作的详细方法。这种交付方式需要的设备类型和操作条件对以前未报告应用程序的简短优化。提供的说明书是一般化到大多数细胞类型和传递材料作为本系统不需要专门的缓冲器或化学修饰/共轭的步骤。这项工作也提供的建议，关于如何提高设备的性能和无故障的拍摄与堵塞，低输送效率和细胞活力的潜在问题。

图1包含微流体输送系统的一个说明性示意图。 图2A-B示出了从在荧光共轭3 kDa的葡聚糖20的存在下处理的HeLa细胞与不同设备设计的典型结果。如果程序正确执行，系统的性能将是设备类型和运行速度敏感。因此，人们必须在更复杂的实验之前，优化这些条件，对于给定的应用程序。在图中所示的操作条件的范围内，细胞活力保持在80％以上，但是，必须注意的是?...

Watch this Scientific Journal Video about Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform at JoVE.com

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

细胞的快速机械变形已成为细胞内输送大分子和纳米材料的一种很有前途的，矢量-free方法。这个协议提供了关于如何使用该系统进行范围广泛的应用中的详细步骤。

电池挤压作为一种强大的，微流控细胞内传送平台

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海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

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A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

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高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

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影像学在两层微流控平台拟南芥幼苗的根头发形态

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

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秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

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The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...