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是完全兼容的，并且不会随时间降解。 <ol start="2"><li>改变容器（2）乙醇溶液（3）每次使用，以避免交叉污染整个实验和最大限度地减少不必要的颗粒，可能会导致堵塞的面前。 </li></ol> 2。实验准备<ol><li>这里的容器（2）和（3）在超声浴中FO每次使用前ř5-10分钟。这有助于从以前的实验中去除任何污染颗粒。 </li><li>干净的工作空间在生物安全柜中，用70％乙醇溶液。 </li><li>将它们放置在生物安全柜内进行喷涂之前的所有材料（3容器和镊子），用70％乙醇溶液。 </li></ol> 3。装配<ol><li>在工作​​区设置下2-3低皮棉湿巾。 </li><li>从他们用镊子取出容器塑料水库和设置他们的湿巾，方便的从内表面乙醇溶液蒸发。轻轻敲击表面上的储器或吹空气通过它们，以便除去乙醇溶液中。 </li><li>将O形圈到他们对水库相应的插槽中。 </li><li>从各自的容器中取出固定器和芯片，让乙醇挥发（约1-2分钟）。 </li><li>使用镊子，将所需的芯片面朝上（ 即接入孔向上）的持有人。提高持有人与芯片眼平，以确保芯片平放在支架，必要时调整的镊子。重要提示：如果设备不在其持有人妥适，是有风险的，这将在随后的步骤中突破。 </li><li>接着，轻轻地放在水库在支架上，并与剪辑对齐。要小心，O型圈在此过程中不属于他们的插槽。 </li><li>轻轻向下按压水库，直到它们卡入到位。确保该储层的两侧是安全的，该芯片似乎是在正确的位置。 </li></ol> 4。细胞的制备<ol><li>对于贴壁细胞（初级或已建立的线）：板的细胞在实验前1-2天，使得它们在实验的当天不超过80％汇合。 </li><li>放置在悬浮细胞（在PBS中的或相关的媒体），和目标为1.0×10 6个细胞/ ml至1.0×10 7个CEL的操作浓度LS /毫升。注意：我们不是由于细胞浓度观察到交付绩效显著的变化。 </li><li>混合单元和希望分娩材料在单独的管中，以获得所需的材料的浓度进行实验。重要提示：由于描述的传递方法依赖于扩散，以促进分娩，更高的材料浓度将产生更高的交付。如果可能的话，建议使用用于初始试验的所需材料的1μM的溶液。在今后的实验中根据需要这个浓度可以被滴定下来。与此设备使用的最低报浓度为10纳米21。 </li></ol> 5。作业<ol><li>细胞和交付材料的吸液管混合成一个水库（目前的设计有一个最大150微升的容量）。注：大多数设备的设计是完全可逆的，因此流过通道的方向并不重要。样品可装入任一两个水塘。 </LI&gt; <li>连接高压油管，以填充水库和拧紧螺母，以确保适当的密封（手指紧往往是足够了）。 </li><li>调节压力以在调节器所需的水平。这个控制着细胞通过设备行进的速度。需要注意的是信元流不启动，直到按钮被按下。 </li></ol>注：在先前的工作中，氮气或压缩空气已制作同样作为载气。 <ol start="4"><li>提高设备到眼睛的高度和方向，使在储存器中的液体是很容易看见。注：追踪液柱切断系统的贮存器被排空之前。 </li><li>按下按钮加压水库，并开始信元流。 </li><li>当液面是从贮存器的底部大约为2毫米，迅速转动调节到0 psi至停止流动。重要提示：如果其中一个发生故障停止流动水库被清空之前，一是风险自收藏品弹出样品Ñ​​水库。还要注意的是，如果流体柱不动以可观的速度，或者减慢基本上相对于其初始流速（ 例如，3倍慢）时，车载装置可能是堵塞并需要更换。经营堵塞设备可以带来更高的细胞死亡。 </li><li>从适当的水库收集处理后的细胞，并将其放置在所需收集管/板。重要提示：不要稀释的细胞收集在任何缓冲区在这个阶段作为porated细胞会继续摄取材料长达10分钟20。在此之后的窗口已经过去了，淡化所需的介质/缓冲细胞。 </li><li>根据需要5.7 - 收集更多的处理过的细胞，或尝试不同的实验条件下，重复步骤5.1。回想一下，这些芯片是可逆的，因此，样品可以安装在任一油箱。请务必根据需要，如果他们堵塞交换芯片。丢弃堵塞设备。 </li></ol> 6。拆卸<ol><li&gt;轻轻地推开夹臂断开主座水库。 </li><li>将每个部分在适当的储存容器（第1节详述）。 </li><li>将使用的芯片在处置一个单独的容器。 </li></ol>

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作者阿蒙Sharei，罗伯特·兰格和Klavs F.詹森是SQZ生物技术公司，生产在这篇文章中所使用的微流体装置的股东。

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</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

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

cell-squeezing-as-robust-microfluidic-intracellular-delivery

Research

JoVE Journal

Bioengineering

12.7K 次观看.  Massachusetts Institute of Technology. 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。 

视频: Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

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 ...

科研

生物工程

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

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 high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

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

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

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

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 their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

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

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

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 ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

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

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

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

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

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

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...