Name: a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex
Uploaded: 2017-04-11T12:30:00
Description: Watch this Scientific Journal Video about A Protocol for Using FÃ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

由托马斯·F.和凯特·米勒·杰弗里斯MEMORIA信托（以DEC）和美国国立卫生研究院授予R35GM119617（以DEC）这项工作是支持的。共焦显微镜成像在所述纳米材料VCU表征核心（NCC）设施执行。

1.获取Nesprin-2G传感器DNA和其它质粒DNA <ol><li>从商业来源获得Nesprin-2G TS（张力传感器），Nesprin-2G HL（无头）控制，mTFP1，金星和TSmod。传播的所有DNA的质粒，并使用标准的大肠杆菌菌株，如DH5-α，净化他们如先前12,13说明。 </li></ol> 2.转染细胞与Nesprin-2G和其它质粒DNA <ol><li>生长NIH 3T3成纤维细胞的细胞至70-90％铺满在6孔细胞培养...

<ol>
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一种方法和跨Nesprin-2G，在核LINC复合体的蛋白质的机械张力的活细胞成像演示，在上面概述的。在此之前的工作，各种技术，如微吸管，磁珠术和显微激光烧蚀，已经被用于对细胞核申请应变，并测量它的体积的材料特性16，17，18。 然而，直到我们最近的工作，还没有研究已经直接表明拉力在活细胞中2</...

力敏感，遗传编码的FRET传感器最近出现的用于在活细胞中测量基于拉伸力，提供深入了解的机械力跨蛋白1，2，3，4施加的重要工具。有了这些工具，研究人员能够以非侵入性图像内的力量在使用传统的荧光显微镜的活细胞。这些传感器由通过弹性肽3分离的FRET对（供体和受体荧光蛋白，最频繁蓝色供体和黄色受体）的。在对比C-或N-末端标记，该传感器被插入到测量跨蛋白质传递的机械力的蛋白质的内部位点，表现为一个分子应变计。增加跨越传感器的结果的机械张力在FRET-P之间的距离增加空气，导致降低FRET 3。其结果是，该FRET是负相关的拉伸力。 这些基于荧光的传感器已被用于粘着斑蛋白（纽蛋白3和踝蛋白4），细胞骨架蛋白（α辅肌动蛋白5），和细胞-细胞连接蛋白显影（E-钙粘蛋白6，7，VE-钙粘蛋白8，和PECAM 8）。在这些生物传感器中最常用的和充分表征的弹性连接体被称为TSmod和由40个氨基酸，（GPGGA）8，其从所述蜘蛛丝蛋白衍生鞭的重复序列组成。 TSmod已经显示出表现为线性弹性纳米弹簧，与FRET响应要求1至拉伸力3 5对-N。鞭状的不同长度可以被用于改变所述动态řTSmod FRET力灵敏度9的安格。除了鞭状，血影蛋白重复5和绒毛头戴受话器肽（称为HP35）4已被用作在类似力的生物传感器4 FRET对之间的弹性的肽。最后，最近的一份报告显示，TSmod也可用于检测压缩力10。 我们最近开发了用于细胞核-细胞骨架（LINC）复合蛋白Nesprin2G的接头的力传感器，通过使用TSmod插入称为迷你Nesprin2G（ 图2C），其行为类似于内源性一个先前开发的截短的蛋白Nesprin2G Nesprin-2G 11。在LINC复杂包含了从外面导致细胞核内，连接细胞质细胞骨架的核纤层的多种蛋白质。 Nesprin-2G是结构蛋白结合到两个肌动蛋白骨架在细胞质和核周空间SUN蛋白质。使用我们的生物传感器，我们能够证明Nesprin-2G是受制于NIH3T3成纤维细胞肌动球蛋白2依赖紧张。这是第一次力在核LINC复杂的跨蛋白直接测量，它很可能成为了解力在力学生物学的核心作用的重要工具。 下面的协议提供的如何使用Nesprin-2G力传感器，包括在哺乳动物细胞中Nesprin张力传感器（Nesprin-TS）的表达Nesprin-细胞的FRET图像的表达，以及采集和分析了详细的方法TS。使用配备有光谱检测器的倒共焦显微镜，如何测量敏化发射的描述使用FRET光谱分离，并设置比例FRET成像。输出比率的图像可以被用来制造相对QUAntitative力比较。虽然该协议的重点是Nesprin-TS的成纤维细胞中的表达，这是很容易适应其它哺乳动物细胞，包括细胞系和原代细胞。此外，该协议，因为它涉及到图像采集和分析FRET可以容易地适用于已经被用于其它蛋白开发了其他基于FRET的力生物传感器。

<table border="0" cellpadding="0" cellspacing="0" width="1183">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-TS DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68127</td>
			<td style="width:475px;">Retrieve from https://www.addgene.org/68127/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-HL DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68128</td>
			<td>Retrieve from https://www.addgene.org/68128/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mTFP1 DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">54613</td>
			<td>Retrieve from https://www.addgene.org/54613/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mVenus DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">27793</td>
			<td>Retrieve from https://www.addgene.org/27793/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">TSmod DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">26021</td>
			<td>Retrieve from https://www.addgene.org/26021/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Competent Cells</td>
			<td style="width:108px;">Bioline</td>
			<td style="width:119px;">BIO-85026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Liquid LB Media</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10855001</td>
			<td>https://www.thermofisher.com/order/catalog/product/10855001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Solid LB Bacterial Culture Plates</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">L5667</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/l5667?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Ampicillin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Spectrophotometer</td>
			<td style="width:108px;">Biorad</td>
			<td style="width:119px;">273 BR 07335</td>
			<td>SmartSpec Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">quartz cuvette</td>
			<td style="width:108px;">Biorad</td>
			<td align="right" style="width:119px;">1702504</td>
			<td>Cuvette for SmartSpec Plus</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">&#160;DNA isolation kit</td>
			<td style="width:108px;">Macherey-Nagel</td>
			<td align="right" style="width:119px;">740412.5</td>
			<td>NucleoBond Xtra Midi Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">6-well cell culture dish</td>
			<td style="width:108px;">Falcon-Corning</td>
			<td align="right" style="width:119px;">353046</td>
			<td>Multiwell 6-well Polystrene Culture Dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Dulbecco&#39;s Modified Eagle Medium, (DMEM) cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">11995-065</td>
			<td>DMEM(1x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16170-078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">reduced serum cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">31985-070</td>
			<td>Reduced Serum Medium, &#34;optimem&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Lipid Carrier Solution</td>
			<td style="width:108px;">invitrogen</td>
			<td style="width:119px;">11668-019</td>
			<td>Lipid Reagent, &#34;Lipofectamine 2000&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">1.5mL sterile plastic tube</td>
			<td style="width:108px;">Denville</td>
			<td style="width:119px;">c2170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Trypsin</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>0.25% Trypsin-EDTA (1x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">glass-bottom microscope viewing dish</td>
			<td style="width:108px;">In Vitro Scientific</td>
			<td style="width:119px;">D35-20-1.5-N</td>
			<td>35mm Dish with 20mm Bottom Well #1.5 glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Fibronectin</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">33016015</td>
			<td>fibronectin human protein,plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">14190-144</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">15mL sterile centrifuge tube</td>
			<td style="width:108px;">Greiner bio-one</td>
			<td align="right" style="width:119px;">188261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">swinging rotor centrifuge&#160;</td>
			<td style="width:108px;">Thermo electron</td>
			<td style="width:119px;">Centra CL2</td>
			<td>Swinging rotor thermo electron 236</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">cell culture biosafety hood</td>
			<td style="width:108px;">Forma Scientific</td>
			<td align="right" style="width:119px;">1284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;climate controlled cell culture incubator</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">3596</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">inverted LED widefield fluorescent microscope</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">EVOS FL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Clear HEPES buffered imaging media</td>
			<td style="width:108px;">Molecular Probes</td>
			<td style="width:119px;">A14291DJ</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Fetal bovine Serum</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:483px;">Temperature Controlled-Inverted confocal w/458 and 515nm laser sources&#160;</td>
			<td style="width:108px;">Zeiss</td>
			<td style="width:119px;">&#160;LSM 710-w/spectral META detector</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Outgrowth Media</td>
			<td style="width:108px;">Newengland Biolabs</td>
			<td style="width:119px;">B9020s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">NIH 3T3 Fibroblasts</td>
			<td style="width:108px;">ATCC</td>
			<td style="width:119px;">CRL-1658</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

按照上述协议，质粒DNA从DNA库中获取并转化入大肠杆菌细胞中。表达该传感器DNA 的大肠杆菌从LB /氨苄青霉素平板上选择并在液体LB肉汤扩增。以下载体的扩增，DNA质粒使用标准，市售的DNA提取试剂盒纯化为TRIS-EDTA缓冲液中。使用分光光度计，纯化的DNA被量化为微克/毫升（ 图1A，1-2天）的标准浓度。 ?...

Watch this Scientific Journal Video about A Protocol for Using FÃ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

一种利用共振能量转移（FRET）的生物传感器-force议定书衡量所有核LINC复杂的机械力

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

The overall goal of this procedure is to determine forces on the linker of the nucleoskeleton cytoskeleton, or LINC complex, through FRET microscopy in living cells via a biosensor designed for the LINC complex protein, nesprin-2G. This method can help answer keys questions in the field of mechanotransduction, such as the forces placed on proteins in the nucleus of living cells, and how these forces change under variant conditions. The main advantage of this technique is its ability to measure very small levels of force on proteins and living cells.Generally, individuals new to this method will struggle with capturing images. Optimizing the microscope laser power and gain to receive a good signal between all experimental groups is difficult. To begin, grow NIH 3T3 fiber blast cells to between 70%and 90%confluence in a six-well cell culture dish in a standard cell culture incubator with temperature and CO2 regulation.In a cell culture hood, remove the medium and rinse each well with approximately 1 milliliter of a reduced serum cell medium. Add 800 microliters of reduced serum cell medium to each well, and place the six-well chamber in the incubator. Then, pipette 700 microliters of reduced serum cell medium into a 1.5 milliliter tube labeled L with 35 microliters of lipid carrier solution to form the lipo mix.Mix the suspension by pipetting. Next, pipette 100 microliters of the reduced serum cell medium into each of six 1.5 milliliter tubes labeled one through six. Pipette the plasma DNA for nesprin tension sensor, nesprin headless zero force control, mTFP1, and venus into tubes one through six, as described in the text protocol.Do not reuse pipette tips when pipetting different types of DNA. Then, pipette 100 microliters of the lipo mix from the L tube into each labeled tube, and mix by repeated pipetting, using a clean pipette for each tube. Incubate the suspension for 10 to 20 minutes.Next, add 200 microliters from each labeled tube to a well of the prepared six-well chamber containing cells. Label the top of each well with the corresponding DNA added. Then, place the six-well chamber in an incubator for four to six hours.Following incubation, aspirate the medium, and add one to two milliliters of reduced serum cell medium to rinse. Aspirate the reduced serum cell medium, and add two milliliters of trypsin to each well before placing the six-well dish in the incubator for five to 15 minutes. While the cells detach in the incubator, coat six glass-bottom viewing dishes with a layer of fibronectin at a concentration of 20 micrograms per milliliter dissolved in phosphate-buffered saline.Allow the dishes to coat the surface in the cell culture hood. Neutralize the trypsin by adding two milliliters of DMEM once the cells are detached. Then, transfer the contents of each well to a labeled 15 milliliter centrifuge tube and spin down at 90 times g for five minutes in a swinging rotor centrifuge.Aspirate the supernatant and re-suspend each cell pellet in 1, 000 microliters of DMEM using a pipette. Aspirate the fibronectin mixture from the glass dishes and pipette 1, 000 microliters of each cell suspension onto the dishes. After the cells settle to the bottom of the glass dishes, add another one milliliter of media to each well.Allow the cells to attach and express sensor in the cell incubator for at least 18 to 24 hours. To verify the transfection efficiency, use a fluorescent microscope equipped with an excitation frequency near 462 nanometers for mTFP1, or 525 nanometers for venus. Use an emission filter centered near 492 nanometers for mTFP1, or 525 nanometers for venus.Roughly 18 to 24 hours after completing the transfection, use an inverted fluorescent microscope to examine the efficiency of transfection by comparing the number of fluorescent cells to the total number of cells in view. If live cells cannot be imaged within 48 hours after transfection, fix the cells in 4%paraformaldehyde for five minutes. Allow cells to express sensor for 24 to 48 hours before fixation.Store the cells in PBS and view after 48 hours. In a cell culture hood, replace the cell medium with imaging medium supplemented with 10%fetal bovine serum. Place the viewing dishes in a temperature controlled confocal microscope stage.Place the glass viewing dish with mTFP1 transfected cells over the oil objective at 60X magnification with a numerical aperture of 1.4. Locate mTFP1-expressing cells in widefield fluorescent mode. With a fluorescent cell in the field of view, select the spectral detection mode and capture the spectral image.Include all frequencies beyond 458 nanometers using 10 nanometer increments. Select a bright fluorescent region on the cell. Add the fluorescent ROI mean as the normalized intensity to the spectral database by clicking Save Spectra to Database.Optimize the laser power and gain such that a good signal to noise ratio is achieved. Settings will vary for different equipment. Repeat the process for venus-expressing cells and remain in spectral mode.Use an excitation frequency of 488 nanometers instead of 458 nanometers. Adjust the power settings as needed, but do not change the emission frequencies. Select a bright fluorescent ROI of around a 20-pixel radius.Add the fluorescent ROI mean as the normalized intensity to the spectral database, by clicking Save Spectra to Database. To capture the unmixed images, first switch the capturing mode to spectral unmixing. Add the spectral fingerprints of venus and mTFP1 to the unmixing channels.Set the excitation laser back to a 458 nanometer argon source. Place the nesprin tension sensor viewing dish above the 60X oil objective. After focusing on a fluorescent cell with sufficiently bright expression, adjust the gain and laser power to optimize the signal to noise ratio.Capture a minimum of 15 to 20 images of nesprin tension sensor cells with relatively similar brightness and avoid excessive pixel saturation. Repeat the image capturing process with nesprin headless cells using identical imaging parameters. A representative 10X magnified image of 3T3 fibroblasts transfected with nesprin tension sensor using a lipid mediated carrier is shown here.Typically, only a fraction of cells are transfected in a given field of view. Shown here is a representative pair of two 3T3 nuclei expressing nesprin-TS in the spectrally unmixed venus and mTFP channels. A merged color image of the two channels is shown in the far right panel.Here is a representative sampling of nesprin-TS masked nuclei from stable expressing MDCK cells on patterned and unpatterned substrates. Ratio images are defined as the ratio of the venus channel divided by the mTFP channel and can be used to make relative quantitative force comparisons. Once mastered, this technique can be done in about eight hours if it's performed properly.While attempting this procedure, it's important to remember to carefully label DNA tubes during the transfection, and keep the imaging parameters constant while capturing spectrally result images. After watching this video, you should have a good understanding of how to transfect cells with the nesprin tension sensor and capture spectrally resolved images to computer FRET index that correlates with force on the tension sensor. Though in this case, a FRET sensor is used for nesprin, it can also be applied to other proteins in the cell where measuring force is desired.

Research

JoVE Journal

Bioengineering

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

将生物探针使用硅烷偶联剂的硅光学生物传感器

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

科研

生物工程

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

小型和广角X-射线散射研究生物大分子的解决方案

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

定量荧光共振能量转移（福斯特共振能量转移）SENP1蛋白酶的动力学测定分析

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

成比例的生物传感器测量活酵母细胞中的线粒体氧化还原状态和ATP

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

从体素知识：实用指南复电镜三维数据的分割

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...