Name: closed system cell culture protocol using hyperstack vessels with gas permeable material technology
Uploaded: 2010-11-29T17:45:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology at JoVE.com

The authors would like to thank Lonza Walkersville for their assistance with closed system knowledge, bag design, loan of bag sealers and welders, as well as creating the bag to HYPERStack schematics.

1. HYPERStack Vessel - Gas Permeable Material Technology Background
<ol>
<li>The HYPERStack Vessel is a multilayered vessel for closed system culturing of cells that relies on gas exchange through a 76.2micron gas permeable polymer film for cellular metabolism.</li>
<li>The HYPERStack vessel differs from more traditional cell culture vessels in that it does not have a "headspace" above the cells inside the vessel. Rather than containing this "headspace" for gas exchange within the vessel, the gas permeable products have air spaces (referred to as "tracheal" spaces) beneath each culture chamber which is open to the atmosphere.</li>
<li>This tracheal space enables the cells growing in each cell culture compartment of the vessel to have equivalent gas exchange.</li>
<li>The gas permeable film allows gas exchange to occur while maintaining a sterile environment. All fluid manipulations for each of the multiple layers occur through a single entry port.</li>
</ol>
2. HYPERStack Vessel Component Summary
<ol>
<li>The Stackette is the individual cell culture compartment that is made up of the top plate and gas permeable film. The cells are cultured within this compartment.</li>
<li>The Liquid Manifold connects each of the 12 stackette layers together within a HYPERStack module. Modules are connected with tubing to form vessels in multiples of 12 layers. The manifold allows the user to make one fluid manipulation to the entire vessel.</li>
<li>The Air Manifold also connects the stackette layers together, but it used to displace air from the vessel when fluid additions occur. It contains a fill line for use during filling.</li>
<li>The Tracheal Space is the open air space between each stackette layer, allowing gas exchange to occur through each layers' gas permeable film.</li>
<li>The Liquid handling tubing is connected to the liquid manifold and is used to make all closed system fluid manipulations. This component can be customized.</li>
<li>The Vent tubing is connected to the air manifold, contains an air filter, and is used to release excess air while maintaining sterility.</li>
<li>The chase tubing has a filter and clamp and are connected to the liquid handling tubing and are used to evacuate the fluid from the liquid handling tubing after filling the vessel.</li>
<li>The Corning Stack Manipulator or CSM is a handling device to assist in placing the vessels in the correct positions during use.</li>
<li>The Filling Wedge is a stainless steel assist used when manually filling the HYPERStack vessels.</li>
</ol>
3. Gas Diffusion Results Through Gas Permeable Film Used in the HYPERStack Vessels
<ol>
<li>In traditional cell growth systems with vented headspace, the oxygen in the media is depleted an average of 50% over 3 days in culture. (Figure 1)</li>
<li>In the same cell growth system, the oxygen gradient in a 3mm height of media is almost 50% greater at the media to headspace junction than it is at the cell layer. (Figure 2)</li>
<li>The diffusion of oxygen through the 76.2micron gas permeable polystyrene film is equal to the diffusion through 2.6mm of media. The gas permeable film used in the HYPERStack vessels allows the gas exchange to occur at the cell layer.</li>
<li>The % of oxygen in the tracheal space of the HYPERStack vessels remains constant as cells grow to confluence. (Figure 3)</li>
</ol>
4. Media Bag Preparation
<ol>
<li>The HYPERStack-12 layer vessel takes 1.3L of media and the HYPERStack-36 layer vessel takes 3.9L of media.</li>
<li>Prior to inoculating your bagged media, if serum is to be used, tube weld the serum bag to the media bag and mix thoroughly.</li>
<li>Clamp off approximately 300 mL of media in the bag using large bag clamps. This will ensure all inoculated cells are used during filling and none are remaining in the media bag. Place media bag on bag stand.</li>
</ol>
5. Inoculating Media
<ol>
<li>Fill a syringe with tubing attached with cell suspension. Tube weld 3/16" tubing from a syringe containing cell suspension to the media bag.</li>
<li>Inject the Cell Suspension into the Media Bag and Mix well.</li>
</ol>
6. Fill Procedure
<ol>
<li>Tube weld the inoculated media bag to the HYPERStack vessel's 3/8" liquid handling tubing. (Using Sterile Connects or Multipurpose connects (MPCs), you can also attach the media bag to the vessel).</li>
<li>Close the clamps on the liquid handling and chase tubes on the HYPERStack.</li>
<li>Place the HYPERStack 36 Layer Vessel into the CSM in the load position; hook the vent filter tube into the holding clamp on the CSM. Tighten the lid and move the CSM to the fill position. By putting the vessel into the correct fill position; the air filter is now in the highest position to prevent wetting during fill operation. The 10&deg; angle permits equilibration of the liquid to the layers during filling. The HYPERStack-12 layer vessel placed on its side on the filling wedge is in the correct position for filling.</li>
<li>Clear air from the fill line by positioning the bag at the same level as the HYPERStack vessel (not higher). Keeping the chase tube clamp closed; open the liquid handling tube clamp and the media bag clamp to allow fluid to enter the vessel.</li>
<li>Using the bag stand, raise the media bag to help the cell suspension flow into the vessel.</li>
<li>Fill the vessel until the all of the inoculated media enters the vessel; the 300 mL of media should still remain in the top portion of the media bag. Remove the clamp from the media bag to continue filling the vessel. As liquid approaches the upper air manifold, slow down the fill rate by lowering the media bag in order to prevent over filling. Slowly bring the liquid level to the fill line and clamp the liquid handling tubing.</li>
</ol>
7. Isolation Procedure to Lock Liquid Into the Layers
<ol>
<li>Bring the HYPERStack vessel in the CSM to the isolation position so both sets of manifolds are in the highest position and lower the media bag below the height of the vessel. The HYPERStack-12 layer vessel can be lifted into the isolation position by placing a hand on the side of the vessel and using the other to lift the filling wedge.</li>
<li>With the chase filter held in an upright position, open the chase tube clamp. This will empty or chase the media in the liquid handling tube back into the media bag. Once the tubing is empty, close the clamp on the media bag tubing.</li>
<li>Keeping the chase tube filter in an upright position, open the clamp on the liquid handling tube on the HYPERStack to allow any remaining liquid to enter and equilibrate in the vessel. Wait 1-2 minutes for this to occur.</li>
<li>Turn the HYPERStack vessel in the CSM so that the manifolds are on the left. Lower the vessel to the load position on the CSM. Rocking the HYPEStack-12 layer vessel slightly to the left will permit the removal of the filling wedge. Close the clamps on both the chase and liquid handling tubing.</li>
<li>The media bag may now be removed from the vessel or it may remain attached for use later in the harvest procedure. To store the attached media bag, roll the empty bag and any remaining media in the bag, and place under the retaining bands in the storage tray of the HYPERStack vessel.</li>
<li>Move the HYPERStack vessel to the incubator. When carrying the vessel care should be taken to keep liquid from entering the air vent filter. This is accomplished by tipping the manifold end of the HYPERStack slightly upward.</li>
</ol>
8. Harvest Protocol
<ol>
<li>To begin the harvest procedure, tube weld the cell dissociation solution and quench bags together to form the harvesting bag assembly. Ensure all tubing clamps are closed. (Use cartoon to demonstrate)</li>
<li>Remove HYPERStack vessel from the incubator, release the media bag from under the retaining bands, and hang on the bag stand. Place the vessel in the CSM in the load position and tighten the lid to secure the vessel. Hook the vent tubing into the holding clamp and move the CSM to the fill position.</li>
<li>Making sure the media bag on the bag stand is hanging lower than the HYPERStack; open the clamp on the media bag tubing and the vessel's liquid handling tubing to allow media to flow into the attached bag.</li>
<li>Once the vessel is about &frac34; empty, change the CSM settings to the final empty position. When the HYPERStack vessel and liquid handling tubing are empty, lift the tubing to chase the media past the clamp on the spent media bag tubing, then close both the clamp on the liquid handling tubing and the clamp on the spent media bag tubing.</li>
<li>Replace the spent media bag with the harvesting bag assembly by tube welding and raise the height of the cell dissociation bag assembly above the height of the HYPERStack using the bag stand.</li>
<li>Place the CSM in the equilibration position. Open the cell dissociation solution tube clamp and transfer the solution into the HYPERStack. When the transfer is complete, close the clamps on the liquid handling tubing and the cell dissociation solution tubing.</li>
<li>Bring CSM to the load position. By holding the horizontal positioning lever open, gently rock the vessel side to side using the wheel, distributing the solution over the cell layers. The vessel may occasionally be returned to the equilibration position during rocking to maintain equal distribution of the solution. It may not be visually apparent that the layers are completely coated, but the dissociation solution will be equally distributed across the surface of the cells. Once the solution has been adequately distributed, release the lever on the CSM to lock the HYPERStack in the load position. Leave the HYPERStack in this position during the required dissociation time for your cells.</li>
<li>Once the cells have detached; using turbidity as a guide, move the CSM to the equilibration position. Care should be taken not to over digest the cells. Open the quench bag clamp and the HYPERStack liquid handling tubing clamp to allow the quench media to enter the vessel. Close the clamp on the HYPERStack liquid handling tubing once the transfer is complete.</li>
<li>Return the CSM to the load position and hold the horizontal positioning lever to rock the vessel side to side.</li>
<li>Using the bag stand, lower the position of the harvest bag assembly below the level of the HYPERStack. Adjust the CSM to the empty position, open the vessel liquid handling tube clamp and transfer the cell solution back into the quench bag. Return the CSM to the load position and disconnect the harvest bag assembly from the vessel using a tube sealer. The cell solution is ready for processing.</li>
</ol>
9. Representative Results
<ul>
<li>Harvesting time needs to be optimized when converting from a standard polystyrene surface to the gas permeable polystyrene surface. Cells tend to release faster on the HYPERStack product.</li>
<li>To view cells under a microscope the vent and liquid handling tubing needs to be clamped off. Place the HYPERStack vessel upside down, with the manifolds pointing down, on the microscope stage.</li>
<li>Prevention steps can be taken to avoid wetting the filters during use which will cause them to stop functioning. By not over filling the HYPERStack vessel and keeping the vent and chase tubing elevated when the clamps are open, no wetting should occur.</li>
<li>The pressure of the closed clamps on the tubing causes it to remain closed after the clamps are opened. Pinching the tubing in the opposite direction will open it.</li>
<li>Media flow time entering and exiting the HYPERStack vessel via gravity feed is based on the media height difference between the top level of media in the HYPERStack vessel and the top level of media in the media container. The greater distance, the faster the flow. The maximum flow is limited by the rating of the filters to 1.5L/min.</li>
<li>Maintaining proper fill volume in the HYPERStack vessel will prevent air bubble formation in the cell growth chambers. Air bubbles can occur due to sampling or incubating in an unhumidified environment due to evaporative water loss. The sampling amount and frequency as well as incubation conditions will determine if additional fluid should be added by the end user.</li>
</ul>
<img src="/files/ftp_upload/2499/2499fig1.jpg" alt="Figure 1" /> 
Figure 1. Oxygen depletion of media in a standard cell culture vessel. The figure shows the decrease in mg/L of oxygen at the level of the cells over 3 days.
<img src="/files/ftp_upload/2499/2499fig2.jpg" alt="Figure 2" /> 
Figure 2. Oxygen gradient in media in a standard cell culture vessel. The figure shows the mg/L of Oxygen is greatest at the media to headspace intersection than at the cell layer.
<img src="/files/ftp_upload/2499/2499fig3.jpg" alt="Figure 3" /> 
Figure 3. Percentage of Oxygen in the tracheal space of the HYPERStack during cell growth. The figure shows that over 96 hours, the percentage of oxygen between the layers, which supplies each gas permeable film bottom, remains constant. This demonstrates the ability of the cells to access oxygen during growth to confluency.

The HYPERStack vessel gas permeable technology is covered under US Patent #: 7745209. 
The authors are employees of Corning Life Sciences, Inc that produces the instrument used in this article.

HYPERStack vessel has successfully grown engineered, primary and stem cells. Fluid additions into the vessel have been conducted via aseptic bag connections by gravity feed and also by pumping from a sterile media container. The vessel provides a greater than 2.5X increase in growth area vs. a stacked plate product of the same volumetric footprint. The HYPERStack-12 has 6,000cm2 growth area, the HYPERStack-36 has 18,000cm2 growth area, and the HYPERStack-120 has 60,000cm2 of growth area. By increasing the surface area, users are able to produce more cells from the same cell population or lot and reduce variability. They are also able to save space and/or labor by using fewer vessels to achieve the same cell output, or increase cell output without adding people, cleanroom suites, or incubators. The system is produced with low particulate assembly methods and without the use of adhesives. It is a closed system which is preassembled with tubing sets and filters, triple bagged and provided sterile.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1.5L Bagged Media</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>TBD</td>
<td>1350ml media + 150ml serum (if required)</td>
</tr>
<tr>
<td>4.0L Bagged Media</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>TBD</td>
<td>3600ml media + 400ml serum (if required)</td>
</tr>
<tr>
<td>HYPERStack Vessel-12</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>10012</td>
<td>&nbsp;</td>
<tr>
<td>HYPERStack Vessel-36</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>10036</td>
<td>&nbsp;</td>
<tr>
<td>Filling Wedge</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>10040</td>
<td>&nbsp;</td>
<tr>
<td>Stack Manipulator</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>TBD</td>
<td>&nbsp;</td>
<tr>
<td>HyQtainer Sub-Assembly Sampling Device</td>
<td>&nbsp;</td>
<td>HyClone/LonzA</td>
<td>SH3B0324.01</td>
<td>&nbsp;</td>
<tr>
<td>600ml Bagged Media Quencher (10% FBS IMDM)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>TBD</td>
<td>&nbsp;</td>
<tr>
<td>600ml Cell Dissociation Solution</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>TBD</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

closed system cell culture protocol using hyperstack vessels with gas permeable material technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

Watch this Scientific Journal Video about Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology at JoVE.com

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

closed-system-cell-culture-protocol-using-hyperstack-vessels-with-gas

Research

JoVE Journal

Biology

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...