笔者想感谢以下资金来源:健康研究加拿大学院（CIHR），自然科学和工程研究理事会（NSERC）。

1。 LabVIEW程序的开发<ol><li>质量流量控制器（MKS M100B海量弗洛控制器）连接到氦气线，以确保精确的气体流量。这个单元由一个输入电压控制的，介于0和5 V.在LabView的程序的控制回路确定所需的电压在任何给定的时间范围内。计算机之间的接口连接是通过一个数据采集设备（NI USB-6009）进行的质量流量控制器，和一个12伏电源提供电源的质量流量控制器。 </li><li>两个定位线性滑动是用来控制在6孔板中，每一个方向的移动。每个组件包括一个MA15系列Velmex Unislide装配加上一个步进电机，而这两个组件是由一个单一Velmex VXM步进电机控制器来控制。该控制器允许为每个电机的手动慢跑或接受外部串行命令的字符串，它的组合允许针对特定的移动patterns。几个模式可以被编程到LabVIEW程序，要求用户提供唯一所需的焊炬速度以及该路径的尺寸。 </li></ol> 2。设备的研制<ol><li>打开氦气瓶和调节器两个。 </li><li>转动电机控制器上。 </li><li>打开LabVIEW程序，并确认所有条件。 </li><li>请确保该平台为中心。要检查，看看每个电机阶段和验证每个车厢不靠近任何阶段结束。要么有程序中心​​的车厢或者通过使用对电机控制器的前慢跑控制手动调整。 </li><li>如果这是当天的第一次运行时，建议以无细胞的运行程序一次，以确保安装工作正常。 </li></ol> 3。气体射流毛细管喷嘴高度的制备<ol><li>从支架上取下毛细管和零高度拨号。 </li><li>将6孔板Øn个平台。 </li><li>替换和降低气体喷射毛细​​管到井的底部，直至它碰到盖玻片。固定在支架中的毛细管。 </li><li>使用高度表盘，提高毛细血管3毫米。标记在毛细管基地这个高度。 </li><li>提高毛细管到安全高度，然后取出6孔板。 </li></ol> 4。细胞渗透协议<ol><li>培养贴壁细胞系生长至汇合的玻璃盖玻片在使用标准培养技术的6孔板中。对于HeLa细胞，种子的6孔板中，每孔2×10 5个细胞，并孵育在治疗前48小时。 </li><li>准备包含所需向量解适当的音量。为hrGFPII-1，50纳克/微升在1x PBS中的浓度提供了在HeLa细胞中的转染后24小时的强荧光信号。对于葡聚糖的研究中，80微克/毫升的10 kDa的绿色荧光葡聚糖推荐。此后，该解决方案将被称为一个的"向量解决方案"。 </li><li>从井除去细胞培养基和冲洗三次，用1×PBS。 </li><li>吸取1,370微升的载体溶液制备步骤4.2为好。这将导致约1.3mm的液下深度。 </li><li>将包含在所述平台的中心的矢量溶液阱。降低气体喷嘴向先前标记级别指示为3毫米的幻灯片上述喷嘴的高度。 </li><li>在LabVIEW程序设定氦的流量，并选择要使用的所需的运动模式。选择"运行"准备开始通透通讯协定时。会有在此期间，在质量流量控制器正在准备提供必要的流量的初始滞后期。一旦适当的流量已经达到时，LabVIEW程序将启动步进马达来移动以及在期望的通透模式。一旦该平台已停止，等待大约30秒，取出向量的解决方案。注意:最优透附近发生的100到200帕的动态压力在喷嘴出口处，取决于毛细管直径。 </li><li>用1×PBS洗好三倍。 </li><li>用新鲜的培养基填充好。 </li><li>对于实验所需数量4.6 - 重复步骤4.3。注意:一定要包括控制样本，包括油井被充满向量解决方案，而该气体射流被辗过他们。离开溶液，对照孔约1分钟，然后进行步骤4.5和4.6。 </li><li>如果运行的hrGFP的转染实验中，返回6孔板到培养箱24小时，以允许转染材料由细胞中转录。如果运行葡聚糖通透性实验，返回6孔板到孵化器15分钟。 </li></ol> 5。细胞成像<ol><li>如果需要的话，马上前成像，染液适当的活/死染色评估细胞死亡。 </li><li>与approp细胞图像riate显微镜研究转/通透效果。 </li></ol>

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作者宣称他们没有竞争的经济利益。

惰性气体喷射通透是贴壁细胞转染的有效技术。它提供了传送生物分子进入细胞的使用的机械力，从而省去了潜在的有害化学物质或病毒载体的能力。该技术可以潜在地给研究者和临床医生的效率和相对简单的方法来精确地转染的细胞。透化的选择性也是唯一允许研究人员只是将某些细胞中的单个菌落这可能是在多种应用中是有用的。 一些参数可以为个别的实验，如毛细管?...

随着生物医学的发展和细胞力学的理解，传递生物分子进入细胞已成为许多研究领域和药物治疗是至关重要的。不同的技术已被开发，通过细胞膜，进入细胞胞质溶胶中引入外来分子。这些通常可分类为:病毒，化学的或机械的技术。病毒的技术能够转染遗传物质，例如使用病毒载体1的RNA和DNA。病毒的技术往往具有高的效率在某些细胞系中，但是它们有用于免疫和炎症反应2的潜力。常见的化学转染方法包括沉淀磷酸钙3，细菌外毒素4和脂质体转染5。这些技术已被证明是有效的，然而，某些问题时，如细胞毒性和非特异性。此外，如钙phosphat技术ë沉淀已发现具有转染效率可达70％，但仅在某些细胞系中，很少在原代细胞6。这是值得注意的是越来越多的研究工作正在投入的原代细胞，特别是在临床使用和DNA功能的研究设计各种处理的情况下。 通过机械刺激细胞膜瞬时中断是用于将外来分子进入细胞的一种替代方法。技术包括:使用超声波来破坏细胞膜10-12，粒子轰击就证明了"基因枪"，其拍摄粒子结合有显微注射分子7，利用电场来破坏膜8,9电穿孔，声孔效应基因进入细胞13，以及最近的，已被证明在时间上通透哺乳动物细胞14,15的流体剪切应力的应用。虽然技工人的方法避免上述的一些问题，它们通常伴随着较低的效率和非常复杂的和专门的设置。大气压辉光放电火炬（APGD-T）于麦吉尔开发的功能化表面和体外 16分离贴壁细胞的初始目标。偶然，人们发现，一活/死染色被用来在某种程度上被取入细胞而不被加入渗透剂。此外，这似乎只发生在其中发生在火炬路径匹配井的禁区。调查的APGD-叔的通透能力，以及继续在对照研究中，我们发现，如果没有激发等离子体的载体惰性气体射流也能够透化细胞。这违背了最初的假设，即由等离子体射流产生的活性物种暂时受损的细胞膜功能，并建议只是式机械密封卡尔力量足以引起细胞通透性。从这里，研究持续在我们的实验室，试图量化和表征惰性气体射流的通透性效率以及看看它的转染能力16。 通过这些研究，已经发现，微孔做实际上形式在质膜，并且这些孔隙倾向于在大约5秒15重新密封。我们已经证明了该技术的效用在体外和体内在鸡胚绒毛尿囊膜上。

<table border="1">
	<tbody>
		<tr>
			<td>Vitality hrGFPII-1</td>
			<td>Agilent Technologies Canada</td>
			<td>240143-57</td>
			<td>Green fluorescent protein for transfection experiments on HeLa cells</td>
		</tr>
		<tr>
			<td>Dextran, AlexaFluor 488; 10,000 MW, Anionic, Fixable</td>
			<td>Life Technologies Inc.</td>
			<td>D22910</td>
			<td>dextran for permeabilization experiments</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Life Technologies Inc.</td>
			<td>L3224</td>
			<td>for counterstaining of mammalian cells</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>for mounting slides when imaging</td>
		</tr>
		<tr>
			<td>Ultra High Purity Helium</td>
			<td>Praxair Canada Inc.</td>
			<td>HE 5.0UH-T</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone DMEM/ High Glucose Media - 500 ml</td>
			<td>Thermo Scientific</td>
			<td>SH30022.01</td>
			<td>for HeLa cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>For DMEM media</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) 1x</td>
			<td></td>
			<td></td>
			<td>Produced in lab</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. List of required reagents</td>
		</tr>
		<tr>
			<td>6-Well Clear TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>#1 22 x 22 Coverslips</td>
			<td>Fisher</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td>WPI Sizing Information</td>
		</tr>
		<tr>
			<td>ID (mm): 1, 0.86, 0.68, 0.5 Order #: 1B200, 1B150, 1B120, 1B100</td>
		</tr>
		<tr>
			<td>Mass-Flo Controller</td>
			<td>MKS</td>
			<td>M100B01314CS1BV</td>
			<td>Mass flow controller, Series M100B, requires an external 12 V power supply</td>
		</tr>
		<tr>
			<td>USB-6009 DAQ Device</td>
			<td>National Instruments</td>
			<td>779026-01</td>
			<td></td>
		</tr>
		<tr>
			<td>UniSlide Assembly with stepping motor and controller</td>
			<td>Velmex</td>
			<td></td>
			<td>Two series MA15 UniSlide Assemblies, fitted with Vexta 1.8&#176; stepping motors provided by Velmex, and controlled by a VXM Stepping Motor Controller</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. List of required equipment</td>
		</tr>
	</tbody>
</table>

permeabilization of adhered cells using an inert gas jet

各种细胞的转染技术存在并且这些可以细分到三个大类:病毒，化学和机械。这个协议描述了一种机械方法，用惰性气体喷射，可以方便的通常不可渗透大分子转移到细胞中，以时间上通透贴壁细胞。我们相信这种技术的工作原理是赋予对贴壁细胞的质膜的剪切力，从而在临时形成微孔。一旦创建了这些孔，然后将细胞可渗透的遗传物质和其它生物分子。所涉及的机械力，还是从他们的衬底运行永久损坏或脱落细胞的风险。有，因此，一个狭窄的范围内的惰性气体动力学，其中该技术是有效的。惰性气体喷流在不同的透贴壁细胞系包括HeLa细胞，HEK293和人的腹主动脉内皮细胞被证明有效。这个协议是适当的在p在体外和，贴壁细胞ermeabilization因为我们已经证明， 在体内 ，显示出它可以在今后的临床应用中用于研究和潜在的。它也有一个空间限制的方式透化细胞的优势，这可能被证明是一个有价值的研究工具。

HeLa细胞以利用氦的气体喷射用0.86毫米内径毛细管10 kDa的绿色荧光葡聚糖透示于图1。细胞进行复通透与2μl/ ml的ETHD-1溶液（LIVE / DEAD活力/细胞毒性试剂盒）进行可视化细胞死亡之后。紧随复染，盖玻片封片和成像。该图中示出了在相对于对照样品三个不同的出口压力运行氦气喷射的效果。注意的是，透磁迹宽度和效率增加具有更高的动态压力和细胞死亡仅表现出轻微增加（A</str...

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Permeabilization of Adhered Cells Using an Inert Gas Jet

这个协议描述了用于贴壁细胞用惰性气体喷射的暂时通透性的方法。这种技术有利于遗传物质和生物分子转移到贴壁哺乳动物细胞中通过机械力的利用来破坏细胞膜。

粘附细胞用惰性气体喷射的透

Various cell transfection techniques exist and these can be broken down to three broad categories: viral, chemical and mechanical. This protocol describes ...

permeabilization-of-adhered-cells-using-an-inert-gas-jet

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.6K 次观看.  McGill University. 这个协议描述了用于贴壁细胞用惰性气体喷射的暂时通透性的方法。这种技术有利于遗传物质和生物分子转移到贴壁哺乳动物细胞中通过机械力的利用来破坏细胞膜。 

视频: Permeabilization of Adhered Cells Using an Inert Gas Jet

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

科研

生物工程

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

从藻类水热液化使用二维气相色谱时间飞行质谱法水提取部分的定性鉴定

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the light distortion during the propagation within the fiber. Our methodology is based on digital optical phase conjugation employing only a single spatial light modulator, where the optical wavefront is individually modulated at different regions of the modulator, one region per light signal. Digital optical phase conjugation approaches are considered to be faster than other wavefront shaping approaches, where (for example) a complete determination of the wave propagation behavior of the fiber is performed. In contrast, the presented approach is time-efficient since it only requires one calibration per light signal. The proposed method is potentially appropriate for spatial division multiplexing in communications engineering. Further application fields are endoscopic light delivery in biophotonics, especially in optogenetics, where single cells in biological tissue have to be selectively illuminated with high spatial and temporal resolution.

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

通过光纤使用波前整形多个信号的传输

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

用数字全息显微镜 (DHM) 定量化低浓度微生物

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

采用两种 Electrohydrodynamic 射流的高分辨率模式: 需求下降和近场静电纺丝

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

使用噬菌体显示肽库筛选和识别针对纤维细胞生长因子受体2的小肽

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

使用DNA对细胞进行简单、经济、模块化的图案化

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

具有透化检测功能的连续流动微电穿孔系统的制造和操作

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...