We thank Deborah Studer for the help with the bioprinter.
The work was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n&deg;NMP4-SL-2009-229292.

1. Preparation of the Poloxamer 407 Solution
If available, perform the preparation of the poloxamer solution in a cold room (4 &deg;C). If not available, place a glass bottle in a beaker filled with ice-cold water. At higher temperatures the poloxamer will be above the gel point and will not dissolve properly.
<ol>
	<li>Add 60 ml of ice cold PBS solution into a glass bottle and stir vigorously using a magnetic stirrer.</li>
	<li>Weigh 24.5 grams of poloxamer and add it in small amounts to the cold PBS. Wait until the poloxamer has partially dissolved before adding more.</li>
	<li>Stir the solution until all poloxamer has dissolved.</li>
	<li>Add cold PBS until a final volume of 100 ml is reached. The final concentration will be 24.5% w/v.</li>
	<li>Stop stirring the solution and let it rest at 4 &deg;C until bubbles and foam in the solution have disappeared. Bubbles that are trapped within the gel will be transferred to the printer cartridge and will lead to defects in the printed sacrificial molds.</li>
	<li>Filter (0.22 &mu;m filter) the solution directly into the printing cartridge to remove any unwanted particles that could clog the needle. The filtering step should be performed in a cold room (or if not available with cooled tips, filter etc.) to avoid gelling of the poloxamer in the filter. Keep the loaded cartridge at 4 &deg;C until 30 min before the experiment.</li>
</ol>
2. Preparation of the 3D Printer
The 3D printer used in this work was the "BioFactory" from regenHU. The extrusion part of the system consists of several parts. A cartridge under pressure at the top is attached to a connector via a luer-lock adapter. The connector bridges the spaces between the outlet of the cartridge and the inlet of a solenoid valve. At the outlet of the solenoid valve, needles with different diameters can be used. The material is extruded onto a substrate that is held to a moving stage by vacuum. The major parts of the system are depicted in Figure 1. Other extrusion based systems can be used for the printing process, and the optimization process needs to be done for each system.
<ol>
	<li>Place the solenoid valve (nozzle diameter 0.3 mm) and the needle (inner diameter 0.15 mm) in separate 1.5 ml test tubes filled with ultrapure water and place them in a heated ultrasonic bath to clean for 30 min. Rinse the cleaned valves with ethanol and dry them with a nitrogen gun.</li>
	<li>Install the valve and needle in the printer as well as an empty, clean cartridge.</li>
	<li>Apply 3 bar pressure to the system and blow out any residual liquids from the installed valve and needle with compressed air. For small needle diameters, it is recommended to have a filter (common syringe filter, 0.45 &mu;m pore size) installed at the exit of the compressed air to avoid entry of small particles that could clog the needle.</li>
	<li>Turn the pressure off and install the cartridge loaded with the poloxamer. The cartridge should be taken out of the refrigerator approximately 30 min before mounting the cartridge so the poloxamer can reach room temperature and gel.</li>
	<li>Apply 3 bar pressure to the system and dispense poloxamer until it reaches the needle tip and is extruded in a continuous strand.</li>
</ol>
3. Optimization of the Printing Parameters
To create accurate 3D structures, the printing process has to be optimized for the chosen material and concentration. Depending on the viscosity and the 3D printing system each material will yield a specific dispensing volume and line thickness for a fixed set of parameters.
<ol>
	<li>With a suitable CAD software (able to create ISO files from the drawings), draw a single line about the same length as the structure that you intend to print.</li>
	<li>Place a microscope glass slide 25 mm x 75 mm x 1 mm or any other substrate in the printer and secure it by turning on the vacuum.</li>
	<li>In the printer software, set the solenoid valve to a high frequency of 50 Hz and set a high pressure of 3 bar.</li>
	<li>Print one layer of a single line with a stage speed of 300 mm/min.</li>
	<li>Reduce the pressure until the desired line width is reached. You can also control the volume that is extruded via the opening time of the valve.</li>
	<li>Reduce the frequency of the valve until no continuous line can be printed anymore. Choose a frequency above this value.</li>
</ol>
Note: Once the desired line width and continuous lines are achieved, determine the optimal stage speed and layer thickness i.e. the lift of the needle after one printed layer.
<ol start="7">
	<li>Print several layers on top of each other and see if the needle is in the right position above the previous layer after several printed layers. Adjust the layer thickness (needle lift) so that each layer is printed on top of the next one (Figure 3).</li>
	<li>Decrease the stage speed of the stage from 300 mm/min step-wise so that extruded layers start and end at the same positions as the previous ones (Figure 4). Too high stage speeds cause the stage to be moving before the extruded material has touched the previous layer.</li>
	<li>For printing the pillar structures follow steps 3.1.-3.8., but instead of drawing a single line draw a single point. The parameters to focus on when printing the pillars are the pressure (regulates layer thickness and pillar diameter of poloxamer), the opening time of the valve (extruded volume) and the residence time of the print head at the position where the pillar should be deposited.</li>
	<li>When the parameters are optimized, printing several layers of a line should result in a solid wall, or in case of the points, a pillar. Save the parameters for later use.</li>
</ol>
4. Printing and Elution of the Reverse Mold
Use the parameters found during the optimization procedure from this point on.
<ol>
	<li>Print the inner structure (here it is a pillar array) on a glass microscope slide and let it dry overnight. This a) reduces the size and thickness of the structures and b) provides better adhesion between the structure and the substrate, so lift-off during the backfilling can be avoided.</li>
	<li>With the CAD software, draw a structure that consists of an outer wall surrounding the structure you intend to have eluted away and filled. Print the structure with poloxamer. The printing of the wall will take 6 min.</li>
</ol>
Attention: The wall has to be printed at least 3.5 mm away from the inner structure because of the dimensions of the needle. Otherwise the printing of the outer wall will destroy the inner structure
<ol start="3">
	<li>Prepare the solution you want to backfill your sacrificial mold with (here 1% agarose in deionized water). The agarose solution should have a temperature between 35 &deg;C and 45 &deg;C. Beneath this temperature, the agarose will solidify too quickly; above this temperature, it might destroy the printed pillars because the poloxamer structure will soften.</li>
	<li>Slowly fill the sacrificial mold with the backfilling solution using a pipette. This should be done slowly to avoid destruction of the structure inside the wall.</li>
	<li>Let the backfilled solution gel or crosslink it depending on the polymer used. In the case of agarose the solidification took place at 4 &deg;C for 10 min.</li>
	<li>Place the backfilled sacrificial mold in an ice bath for 10 min to elute the poloxamer structure.</li>
	<li>Blot the backfilled structure with a paper tissue and place it on a new glass microscope slide. Press the structure carefully onto the glass microscope slide to avoid leakage of the third hydrogel from the void into the space between the backfilled structure and the glass microscope slide.</li>
</ol>
5. Filling of the Voids
<ol>
	<li>To fill the voids left by the eluted poloxamer, fill the intended polymer solution into a syringe equipped with a 30 G needle. In this example, we used a 1% alginate methacrylate in 0.15 M NaCl solution with the addition of 0.05% w/v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 2.5% v/v of Alexa-488 conjugated fibrinogen. The Alexa-488 conjugated fibrinogen was added for visualization purposes.</li>
	<li>Photopolymerize the polymer with a high intensity UV lamp (100 Watt, 365 nm, distance from substrate was 3.5 cm) for 5 min and image the construct using an epi-fluorescence or confocal microscope.</li>
</ol>

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The authors have nothing to declare.

Here we present, for the first time, the use of a thermoresponsive polymer for a sacrificial mold that can be quickly eluted in cold water due to the gel-sol transition of poloxamer of ~20 &deg;C. The speed of the entire process makes poloxamer interesting for the rapid creation of biopolymer structures which cannot be printed with adequate resolution. The technique described here can be used for patterning one hydrogel within another hydrogel or for the creation of microfluidic channels as has been previously reported f...

Tissue engineering approaches have made much progress over the last years with respect to regeneration of human tissues and organs19,20. However, until now, the focus of tissue engineering has been often limited to tissues that have a simple structure or small dimensions such as the bladder21,22 or the skin23-25. The human body, however, contains many complex three-dimensional tissues where cells and extracellular matrix are arranged in a spatially defined manner. To manufacture these tissues, a technique is required that can place cells and extracellular matrix scaffolding within a three-dimensional construct at specified positions. Bioprinting has the potential to be such a technique where the vision of manufacturing complex three-dimensional tissues can be realized10,11,26-28.
Bioprinting is defined as "the use of material transfer processes for patterning and assembling biologically relevant materials - molecules, cells, tissues, and biodegradable biomaterials - with a prescribed organization to accomplish one or more biological functions"4. It encompasses several different techniques that work at different resolutions and length scales, ranging from the sub-micron resolution of two-photon polymerization29 to a resolution of 150 &mu;m to 420 &mu;m for extrusion printing1,12,30. Not a single material or material combination will satisfy the requirements of each method31. For extrusion printing, the key parameters are viscosity and gelation time32, where high viscosity and rapid gelation are desirable.
3D printing is a technique which allows the easy creation of sacrificial molds for creating complex geometries30,33,34. This process is based on the construction of a mold using a rapid prototyping technique such as an extrusion bioprinter. The created sacrificial mold is used to form complex structures from materials which are difficult to print due to their low viscosity and slow gelation time. The method presented here involves the creation of a sacrificial mold consisting of a material that dissolves quickly at low temperature and can be extruded accurately. The block copolymer poly(ethylene glycol)99-poly(propylene glycol)67-poly(ethylene glycol)99 (also known as Pluronic F127 or poloxamer 407) fulfills these requirements. It has already been used in a modified version in extrusion printing1 but, to our knowledge, has never been used for printing in its unmodified version due to its instability in liquid environments. Poloxamer 407 also shows an inverse thermal responsive behavior18 i.e. it changes from a gel to a sol upon cooling. Most importantly, it can be printed into complex arbitrarily curved structures with very high fidelity. This allows the creation of a structured hydrogel from a low viscosity material, in this case slow gelling agarose, by pipetting the solution into the printed sacrificial mold. The combination of printing the sacrificial mold with high fidelity and its quick elution from the casted structured hydrogel makes it a fast and flexible method to create molds with different geometries without the use of a mask or a stamp as it is often required in lithographic methods. The casted structured hydrogel can be further filled with another material that is not suitable for extrusion printing due to its low viscosity. This is in our case a low viscosity alginate methacrylate solution. Here we present the method of thermoresponsive reverse sacrificial molds for hydrogel patterning using the example of a pillar array.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Poloxamer (Pluronic F127)</td>
 <td>Sigma</td>
 <td>P2443</td>
 <td ></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=10010015" target="_blank">10010-015</a></td>
 <td ></td>
 </tr>
 <tr>
 <td>CAD software</td>
 <td>regenHU</td>
 <td>BioCAD</td>
 <td ></td>
 </tr>
 <tr>
 <td>Alginate methacrylate</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td>
 <td>Invitrogen</td>
 <td>F13191</td>
 <td ></td>
 </tr>
 <tr>
 <td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Lonza</td>
 <td>50004</td>
 <td ></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Bioprinter</td>
 <td>regenHU</td>
 <td>Biofactory</td>
 <td ></td>
 </tr>
 <tr>
 <td>Valve</td>
 <td>regenHU</td>
 <td>300 &mu;m Nozzel Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>regenHU</td>
 <td>150 &mu;m Inner Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Zeiss Axioobserver with ApoTome</td>
 <td>Zeiss</td>
 <td></td>
 <td ></td>
 </tr>
 <tr>
 <td>UV Light Source</td>
 <td>UVP</td>
 <td>Blak-Ray B-100AP High Intensity UV Lamp</td>
 <td>100 W</td>
 </tr>
 </tbody>
</table>

printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3d cell culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

The representative results show that the reverse mold technique (depicted in Figure 2) will create a structured gel that can be filled with a second material. At the beginning of every printing process the printing parameters are first optimized. Step-wise adjustments of the parameters will result in printed multilayered constructs depicted in Figure 3 and Figure 4 when single lines are printed. If the layer thickness (the needle lift after one printed layer) is too low,...

Watch this Scientific Journal Video about Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture at JoVE.com

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

printing-thermoresponsive-reverse-molds-for-creation-patterned-two

Research

JoVE Journal

Immunology and Infection

15.0K Views.  Cartilage Engineering & Regeneration. A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Video: Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.