<meta charset="utf8"></head><body>使用 Vessels-on-a-Plate 快速制造血管网络的 3D 生物打印

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological Pipette</td>
			<td>SPL</td>
			<td>SPL 91010</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe&#160;</td>
			<td>Shinchang Medical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>3D Bioprinter&#160;</td>
			<td>T&#38;R Biofab</td>
			<td>3DX-Printer</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate&#160;</td>
			<td>SPL</td>
			<td>37206</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Safety Cabinets</td>
			<td>CHC LAB</td>
			<td>PCHC-777A2-04,&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Brightfield Inverted Microscopes</td>
			<td>Leica</td>
			<td>DMi1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit (CCK8)</td>
			<td>GlpBio</td>
			<td>GK10001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit (CCK8)</td>
			<td>GlpBio</td>
			<td>GK10001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flask 75T</td>
			<td>SPL</td>
			<td>70075</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, 10 mL</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO, Cell Culture Grade</td>
			<td>Sigma aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow-Corning, PDMS-Sylgard 184a Kit</td>
			<td>DOW</td>
			<td>DC-184</td>
			<td></td>
		</tr>
		<tr>
			<td>DOWSIL SE 1700 Clear W/C 1.1 KG Kit&#160;</td>
			<td>DOW</td>
			<td>2924404</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS - 1x</td>
			<td>Welgene</td>
			<td>LB001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium MV 2 (Ready to use)</td>
			<td>Promocell</td>
			<td>C-22022</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Micro pipette(1000,200,100,20,10)</td>
			<td>eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol 99.9%</td>
			<td>Duksan</td>
			<td>D5</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>F8630-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Dextran 70 kDa</td>
			<td>Sigma Aldrich</td>
			<td>46945-100MG-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent beads (1.0 &#956;m, green)</td>
			<td>Sigma Aldrich</td>
			<td>L1030-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>GelMA-powder (Gelatin methacrylate) 50 g</td>
			<td>3D Materials&#160;</td>
			<td>20JT29</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco, Recovery Cell Culture Freezing Medium, 50 mL</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HUVECs (Human Umbillical Vein Endothelial Cells)</td>
			<td>Promocell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo SCIENTIFIC</td>
			<td>Forma STERI-CYCLE i160 CO2 Incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen, Live/dead viability/cytotoxicity Kit (for mammalian cells)</td>
			<td>Thermo Fisher</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphate powder&#160;</td>
			<td>Tokoyo Chemical Industry CO.</td>
			<td>85073-19-4&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Marienfeld Superior, Counting chamber cover</td>
			<td>Marienfeld Superior</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Marienfeld Superior, Hemocytometer, cell counting chamber</td>
			<td>Marienfeld Superior</td>
			<td>HSU-0650030</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>eppendorf</td>
			<td>Centrifuge 5920 R</td>
			<td></td>
		</tr>
		<tr>
			<td>NCViewer.com</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen tank</td>
			<td>WORTHINGTON INDUSTRIES</td>
			<td>LS750</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnicure UV Laser</td>
			<td>EXCELITAS</td>
			<td>SERIES 1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>amcor</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution (100x)</td>
			<td>GenDEPOT</td>
			<td>CA005-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary Mixer</td>
			<td>THINKY CORPORATION, japan</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma treatment machine</td>
			<td>FEMTO SCIENCE</td>
			<td>CUTE-1MPR</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma aldrich</td>
			<td>P2443-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-made buffer, (P2007-1) 10x PBS</td>
			<td>Biosesang</td>
			<td>PR4007-100-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent storage cabinet</td>
			<td>ZIO FILTER TECH</td>
			<td>SC2-30F-1306D1-BC</td>
			<td></td>
		</tr>
		<tr>
			<td>Real time Live cell Imaging Microscope</td>
			<td>Carl ZEISS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>SAMSUNG</td>
			<td>RT50K6035SL</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCKER 2D digital</td>
			<td>IKA</td>
			<td>4003000</td>
			<td></td>
		</tr>
		<tr>
			<td>Scoop-Spatula</td>
			<td>CacheBy</td>
			<td>SL-SCO7001-EA</td>
			<td></td>
		</tr>
		<tr>
			<td>sigma,Trypsin-EDTA solition, 0.25%</td>
			<td>Sigma aldrich</td>
			<td>T4049-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Thermo Fisher scientific</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Barrel Tip Cap</td>
			<td>FISNAR</td>
			<td>3051806</td>
			<td></td>
		</tr>
		<tr>
			<td>Tally counter</td>
			<td>Control Company</td>
			<td>&#160;C23-147-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapered Nozzle (18 G)</td>
			<td>Mushashi</td>
			<td>TPND-18G-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapered Nozzle (22 G)</td>
			<td>Mushashi</td>
			<td>TPND-22G-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapered nozzle 20 G</td>
			<td>Musashi</td>
			<td>TPND-20G-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>T7326-1KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer, 4-channel</td>
			<td>ETL</td>
			<td>SL.Tim3005</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution</td>
			<td>Promocell</td>
			<td>C-41120</td>
			<td></td>
		</tr>
		<tr>
			<td>UG 24 mL UG ointment jar</td>
			<td>Yamayu</td>
			<td>No. 3-53</td>
			<td></td>
		</tr>
		<tr>
			<td>UG 58 mL UG ointment jar</td>
			<td>Yamayu</td>
			<td>No. 3-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>DAIHAN Scientific</td>
			<td>WB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Weight machine</td>
			<td>Sartorius</td>
			<td>bce2241-1skr</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput bioprinting method for modeling vascular permeability in standard six-well plates with size and pattern flexibility

<meta charset="utf8"></head><body>作者聚焦：用于高通量血管模型制造的自动化生物打印

高通量生物打印方法，用于模拟具有尺寸和图案灵活性的标准六孔板中的血管通透性

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

high-throughput-bioprinting-method-for-modeling-vascular-permeability

Research

JoVE Journal

Bioengineering

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

816 次观看.  Chonnam National University. 开发针对血管通透性（动脉粥样硬化等常见疾病的前兆）的新型疗法需要严格的临床前测试。然而，复杂的制造方法通常会阻碍体外模型的可重复性和高通量生产，而这是药理学测试的先决条件。在这里，我们利用多材料生物打印技术的自动化来解决这个问题。通过将传统的孔板系统与计算机控制的 3D 打印相结合，我们简化了制造过程，简化?...

视频: 作者聚焦：用于高通量血管模型制造的自动化生物打印

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

生产尺寸控制肿瘤细胞球体采用微孔板大数

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

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 Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

多井板膜插入系统中3D 组织模型渗透扩散的测定与模拟方法

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...