<meta charset="utf8"></head><body>与真菌伴侣共培养蔗糖分泌蓝细菌的方案

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >10x Ph1 objective</td>
			<td >Carl Zeiss AG</td>
			<td >46 04 01-9904</td>
			<td ></td>
		</tr>
		<tr >
			<td >525/40 nm Bandpass Filter</td>
			<td >Beckman Coulter, Inc.</td>
			<td >A01-1-0051</td>
			<td>FITC filter (in 488 nm laser)</td>
		</tr>
		<tr >
			<td >660/10 nm Bandpass Filter</td>
			<td >Beckman Coulter, Inc.</td>
			<td>A01-1-0055</td>
			<td>APC channel (in 638 nm laser)</td>
		</tr>
		<tr >
			<td >710/50 nm Bandpass Filter</td>
			<td >Beckman Coulter, Inc.</td>
			<td>B71092</td>
			<td>PC5.5 channel (in 561 nm laser)</td>
		</tr>
		<tr >
			<td >Accuvettes for Multisizer 4e</td>
			<td>Beckman Coulter, Inc.</td>
			<td>A35473</td>
			<td>Sample cups with lids</td>
		</tr>
		<tr >
			<td >Aperture tube 30 &#956;M for Multisizer 4e</td>
			<td>Beckman Coulter, Inc.</td>
			<td>C92760</td>
			<td>Capillary</td>
		</tr>
		<tr >
			<td >Autoclave</td>
			<td >Lab Associates</td>
			<td >EQM-ARBP</td>
			<td>for sterilisation of material</td>
		</tr>
		<tr >
			<td >Baffled flasks ROTILABO&#174;, Straight neck, 250 ml</td>
			<td >Carl Roth GmbH + Co. KG</td>
			<td >LY95.1</td>
			<td></td>
		</tr>
		<tr >
			<td >BG11 / CoYBG11 components</td>
			<td>different suppliers</td>
			<td ></td>
			<td >see Table 1&#160;</td>
		</tr>
		<tr >
			<td >Bottle top sterile filter</td>
			<td >Sigmaaldrich</td>
			<td >Z222593</td>
			<td>to sterile media if you dont want to use autoclave</td>
		</tr>
		<tr >
			<td >Capillary 45 &#181;m</td>
			<td >Omni Life Science</td>
			<td>OLS 5651738</td>
			<td></td>
		</tr>
		<tr >
			<td >CASY Cell Counter Model TTC</td>
			<td>Schaerfe Systems</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >CASY cups</td>
			<td >Omni Life Science</td>
			<td>OLS 5651794</td>
			<td></td>
		</tr>
		<tr >
			<td >CASYclean</td>
			<td >Omni Life Science</td>
			<td>OLS 5651786</td>
			<td>Cleaning solution</td>
		</tr>
		<tr >
			<td >CASYton solution</td>
			<td >Omni Life Science</td>
			<td>OLS 5651808</td>
			<td>Isotonic solution&#160;</td>
		</tr>
		<tr >
			<td >Centrifuge tubes (50 mL)</td>
			<td >Sigmaaldrich</td>
			<td >CLS430828</td>
			<td></td>
		</tr>
		<tr >
			<td >CO2</td>
			<td >BOC</td>
			<td >270182-L</td>
			<td>to supply cyanobacteria axenic cultures and co-cultures</td>
		</tr>
		<tr >
			<td >CONTRAD&#174; 70</td>
			<td >Beckman Coulter, Inc.</td>
			<td>81911</td>
			<td>Deep clean solution</td>
		</tr>
		<tr >
			<td >Cover glass 20 &#215; 26 x 0.4 mm</td>
			<td >VWR International GmbH</td>
			<td>631-1190</td>
			<td>Cover glass for counting chamber</td>
		</tr>
		<tr >
			<td >Cuvettes</td>
			<td>Sarstedt&#160;</td>
			<td>67.742</td>
			<td></td>
		</tr>
		<tr >
			<td >Cyanobacteria BG-11 Freshwater Solution</td>
			<td >Merck KGaA</td>
			<td>C3061</td>
			<td></td>
		</tr>
		<tr >
			<td >CytExpert Software v. 2.6</td>
			<td >Beckman Coulter, Inc.</td>
			<td ></td>
			<td>Cytometer application software</td>
		</tr>
		<tr >
			<td >CytoFLEX Sheath Fluid</td>
			<td >Beckman Coulter, Inc.</td>
			<td >B51503</td>
			<td>Sheat fluid</td>
		</tr>
		<tr >
			<td >Cytometer CytoFLEX S</td>
			<td >Beckman Coulter, Inc.</td>
			<td >BE51180</td>
			<td></td>
		</tr>
		<tr >
			<td >DxH Cleaner for Multisizer 4e</td>
			<td>Beckman Coulter, Inc.</td>
			<td>628022</td>
			<td>Cleaning solution</td>
		</tr>
		<tr >
			<td >Eppendorf tube centrifuge (small)</td>
			<td >Labstac</td>
			<td >CEN16-15</td>
			<td>to centrifuge less than 2mL of culture</td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td >Carl Roth GmbH + Co. KG</td>
			<td >K928.1</td>
			<td></td>
		</tr>
		<tr >
			<td >FACS tubes</td>
			<td >Beckman Coulter, Inc.</td>
			<td >2523749</td>
			<td>tubes for semi-automatic mode</td>
		</tr>
		<tr >
			<td >FlowClean Cleaning Agent, 500ml</td>
			<td >Beckman Coulter, Inc.</td>
			<td>C48093</td>
			<td>Daily clean solution</td>
		</tr>
		<tr >
			<td >Graduated cylinders</td>
			<td >Sigmaaldrich</td>
			<td >Z131121</td>
			<td>for preparation of media</td>
		</tr>
		<tr >
			<td >HEPPSO</td>
			<td >Merck KGaA</td>
			<td>R426725</td>
			<td></td>
		</tr>
		<tr >
			<td >Infors MultiTron photoincubator</td>
			<td >Infors AG</td>
			<td ></td>
			<td>white LED light</td>
		</tr>
		<tr >
			<td >IPTG</td>
			<td >Merck KGaA</td>
			<td >I6758</td>
			<td>Prepare aliquots, store at -20&#176;C, thaw and keep on ice&#160;</td>
		</tr>
		<tr >
			<td >Isoton II for Multisizer 4e</td>
			<td>Beckman Coulter, Inc.</td>
			<td>8448011</td>
			<td>Isotonic solution&#160;</td>
		</tr>
		<tr >
			<td >Kaluza v. 2.2</td>
			<td >Beckman Coulter, Inc.</td>
			<td >B16406</td>
			<td>Cytometry data analysis software</td>
		</tr>
		<tr >
			<td >KimWipes</td>
			<td >VWR International GmbH</td>
			<td >115-2221</td>
			<td>Lint-free tissues</td>
		</tr>
		<tr >
			<td >KOH</td>
			<td >Merck KGaA</td>
			<td >221473</td>
			<td></td>
		</tr>
		<tr >
			<td >KPO3</td>
			<td >Noah Chemicals</td>
			<td>7790-53-6</td>
			<td></td>
		</tr>
		<tr >
			<td >Measuring beakers</td>
			<td >VWR</td>
			<td >213-3747</td>
			<td></td>
		</tr>
		<tr >
			<td >Micro test plate, 96 well, slip-on lid, flat base, PS, transparent</td>
			<td >SARSTEDT AG &#38; Co. KG</td>
			<td >82.1581.001</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope base</td>
			<td >Carl Zeiss AG</td>
			<td >47 09 18-9902/16</td>
			<td></td>
		</tr>
		<tr >
			<td >Microsope head</td>
			<td >Carl Zeiss AG</td>
			<td>47 30 14</td>
			<td></td>
		</tr>
		<tr >
			<td >Milli-Q water purification system</td>
			<td >Merck Millipore</td>
			<td>C85358</td>
			<td></td>
		</tr>
		<tr >
			<td >Mulitisizer 4e particle counter</td>
			<td>Beckman Coulter, Inc.</td>
			<td>B23005</td>
			<td>Alternative to the CASY Cell Counter</td>
		</tr>
		<tr >
			<td >Neubauer improved, depth 0.02 mm</td>
			<td>Assistent Germany</td>
			<td></td>
			<td>Counting chamber</td>
		</tr>
		<tr >
			<td >Neubauer improved, depth 0.1 mm</td>
			<td >Paul Marienfeld GmbH &#38; Co. KG</td>
			<td >640010</td>
			<td>Counting chamber</td>
		</tr>
		<tr >
			<td >Ready to Use Daily QC Fluorospheres</td>
			<td >Beckman Coulter, Inc.</td>
			<td >C65719</td>
			<td>Reference for QC</td>
		</tr>
		<tr >
			<td >SCHOTT bottles</td>
			<td >Dutscher</td>
			<td >90347</td>
			<td>for storage</td>
		</tr>
		<tr >
			<td >Specord&#174;200 Plus Spectrophotometer</td>
			<td>Analytic Jena GmbH</td>
			<td ></td>
			<td>OD600/750</td>
		</tr>
		<tr >
			<td >Sucrose</td>
			<td >Merck KGaA</td>
			<td>84100</td>
			<td></td>
		</tr>
		<tr >
			<td >Table top centrifuge (big)</td>
			<td >Labstac</td>
			<td >CEN18-06R</td>
			<td>collect culture biomass</td>
		</tr>
		<tr >
			<td >Yeast Nitrogen Base, without amino acids, without ammonium sulfate</td>
			<td >Thermo Fisher Scientific Inc.</td>
			<td >11743014</td>
			<td></td>
		</tr>
	</tbody>
</table>

assembly and quantification of co-cultures combining heterotrophic yeast with phototrophic sugar-secreting cyanobacteria

<meta charset="utf8"></head><body>异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...

assembly-quantification-co-cultures-combining-heterotrophic-yeast

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

540 次观看.  Heinrich Heine University Düsseldorf. 该协议为共培养物的设置和定量监测提供了全面的指南，包括光合自养分泌糖的蓝细菌和异养酵母。 

视频: 异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

科研

生物工程

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

细胞外基质蛋白纳米纤维和纳米结构工程采用表面引发大会

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

生物膜的微阵列用刮墨刀型组合方式形成

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

人卵巢组织与工程化内皮细胞联合移植: 一种以细胞为基础的快速灌注与直接分泌的融合策略

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

离子通道掺杂脂质膜组成的生物分子记忆器的组装与表征

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...