This work was supported in part by the National Science Foundation (CBET 0846453) and the National Institutes of Health (1R01GM089999).

1. Fabrication of silicon masters using standard SU-8 photolithography1 (not shown in this video).
<ol>
 <li> Use standard SU-8 photolithography methods to create a SU-8 &quot;masters&quot; (SU-8 2050, MicroChem, Newton, MA) for fabricating the different components (PDMS membranes of different thickness, co-culture chamber) in a cleanroom. Such master molds can be fabricated at any microfabrication facility (e.g., Stanford Microfluidics Foundry; <a href="http://thebigone.stanford.edu/foundry/">http://thebigone.stanford.edu/foundry/</a>). </li>
 <li> Prior to PDMS replication, expose the SU-8 masters to fluorosilane vapor in a desiccator attached to a vacuum source to facilitate easy release of PDMS from the master. Add a drop of fluorosilane to a paper towel and apply vacuum for one min. Remove the vacuum and allow 30 min for deposition of fluorosilane. Keep the SU-8 master in a closed container for future use. </li>
</ol>
2. Replica molding of PDMS from SU-8 master2
<ol>
 <li> The fluidic layer and the control layer are made by replica molding of PDMS from SU-8 master.</li>
 <li> Mix the PDMS pre-polymer and cross-linker at a 10:1 wt. ratio and degas in a desiccator for 1hour (or until air bubbles are removed from the PDMS mixture).</li>
 <li> Place the SU-8 master in a petri dish and carefully pour the PDMS mixture on top of the SU-8 master. Degas the PDMS again.</li>
 <li> Spin the mold at 1200 rpm for one minute to get a uniform PDMS thickness of around 100&mu;m on the mold.</li>
 <li> Heat the petri dish containing the PDMS and the SU-8 master mold at 80&deg;C for one hour to cure the PDMS. </li>
 <li> Remove the cured PDMS-covered SU-8 master from the hotplate.</li>
 <li> Peel the PDMS mold from the SU-8 master.</li>
 <li> Drill four holes in the PDMS mold using a 20 gauge blunt-end needle for two bacteria inlets, one outlet and one pneumatic port.</li>
</ol>
3. Bonding of Multilayer PDMS devices: This device is assembled with three layers -a top layer which contains the main channel, a middle pneumatic layer, and a bottom bacterial layer that contains the channels for bacteria. The three layers are assembled as described below.
<ol>
 <li> Wet the PDMS membranes with methanol and carefully peel them from their SU-8 master mold using a clean forceps. Place membrane on a teflon sheet.</li>
 <li> Place the pneumatic membrane and the fluidic layer on an oxygen plasma chamber. Turn on plasma for 40 sec (oxygen pressure 10 sccm, RF power 100W). </li>
 <li> After exposure to plasma, align (within 10 minutes) the membrane on the bottom PDMS channel using forceps. Adding methanol on the membrane and the PDMS layers helps free movement of the layers, and makes alignment easier. In this case, the device is placed on a piece of gauze to allow the methanol vapors to escape.</li>
 <li> Cure the aligned PDMS layers on a hotplate at 80&deg;C for 8 h.</li>
 <li> After bonding the pneumatic membrane onto the bottom layer, repeat steps 3.2 - 3.4 to bond the top channel onto the assembled composite PDMS device.</li>
</ol> 
4. Assembling the PDMS device3,4
<ol>
	<li> Drill holes in the inlet and outlet ports used for seeding eukaryotic cells and bacteria.</li>
 <li> Connect Tygon tubing (0.01 inch O.D.) to the ports and operate the pneumatic layer by pulling with a 20mL syringe.</li>
 <li> Place the PDMS model and a pre-cleaned glass slide into an oxygen plasma chamber. Turn on plasma for 20 sec (oxygen pressure 20 sccm, RF power 300W) without applying the vacuum. </li>
 <li> In order to prevent contact between the island wall (PDMS) bonding to the glass slide, immediately connect a syringe to the device and apply vacuum by pulling the syringe and holding it in place.</li>
 <li> Gently press the PDMS model onto the glass (within 2 minutes).</li>
 <li> Cure the PDMS device at 80&deg;C for 10 minutes.</li>
</ol>
5. Treating the glass surface with fibronectin
<ol>
 <li> Sterilize device under UV for 15 minutes after connecting Tygon tubing to every port.</li>
 <li> Fill and flow the sterilized PBS into the channel to remove any debris in the channel. Apply flow for 10 minutes to remove air bubbles. The air bubbles can easily escape the PDMS because of its gas permeability. </li>
 <li> Fill a color dye solution into the pneumatic channel to prevent formation of air bubbles in the channel.</li>
 <li> To facilitate eukaryotic cell seeding on the glass, introduce a 1 &mu;g/mL solution of fibronectin into the device and incubate for 40 min at 37&deg;C. </li>
 <li> Wash the excess fibronectin with 1 mL of growth media.</li>
</ol>
6. Formation and measurement of commensal E. coli biofilm in islands
<ol>
 <li> Dilute the commensal bacteria (e.g., E. coli BW25113/pCM18) in M9 minimal media to OD600~1 for forming a commensal biofilm in the closed chambers/islands. </li>
 <li> Close the island wall by applying positive-liquid pressure. A homemade air-to-liquid pressure converter is used to prevent air bubble formation in the channel. </li>
 <li> Introduce the commensal E. coli into the islands with the valve closed, and allow bacteria to attach for 1 hour at 32&deg;C.</li>
 <li> Wash the unattached bacteria and perfuse a dilute bacterial suspension (OD600~0.05) at 0.25mL/hr for 12 hours.</li>
 <li> Rinse the biofilm with fresh DMEM growth media to remove loosely-attached bacteria and prepare for eukaryotic cell attachment.</li>
 <li> Image the biofilm using a Leica TCS SP5 Confocal Laser Scanning Microscope (Bannockburn, IL), and visualize the biofilm architecture using the IMARIS software5.</li>
</ol>
 7. Seeding eukaryotic cells around bacterial islands6-8
<ol>
 <li> Trypsinize HeLa cells from tissue culture flask and resuspend in DMEM media for a seeding density of 5 x 105 cells/mL. </li>
 <li> Introduce HeLa cells into the device at a flow rate of 2 mL/min using a Picoplus syringe pump (Harvard Apparattus, MA). Ensure that the island wall is lowered so that the bacterial biofilm is separate from the HeLa cell region. This step should be performed on the stage of a light microscope so that the uniformity of seeding can be determined.</li>
 <li> Clamp the cell outlet to reduce the movement of the cell suspension in the channel and incubate for 6 h at 37&deg;C in a 5% CO2 incubator.</li>
 <li> Perfuse the device with growth medium at 0.5 mL/hr to remove unattached cells. </li>
 <li> Refresh the DMEM media for HeLa and commensal E. coli every 6 hours. </li>
</ol>
8. Introduction of pathogenic bacteria into the island and exposure to eukaryotic cells
<ol>
 <li> Prepare E. coli O157:H7 (EHEC) to infect HeLa cells at a multiplicity of infection (m.o.i) of 100:1 200:1 (number of EHEC per HeLa cell).</li>
 <li> Rinse and wash out loosely-attached commensal bacteria. </li>
 <li> Introduce a dilute EHEC suspension (OD600 ~ 0.1) into the islands.</li>
 <li> Incubate device at 37&deg;C in a 5% CO2 incubator for 6 hours without flow to allow EHEC to be exposed to signals present in the commensal biofilm.</li>
 <li> Lift the PDMS wall to expose EHEC in the commensal islands to HeLa cells for 6 hours.</li>
 <li> Rinse the channel with PBS and stain for live and dead cells using the Live/Dead kit (L3224, Invitrogen, CA).</li>
 <li> Image multiple locations on a Zeiss Axiovert 200M fluorescence microscope and estimate the number of live and dead cells.</li>
</ol>
9. Representative Results 9
The sequence of events in GI tract infections was mimicked by developing a monolayer of HeLa cells and a commensal E. coli biofilm and exposing EHEC to the commensal biofilm prior to infection of HeLa cells. The steps involved in fabrication of the microfluidic co-culture model are shown in Figure 1. Figure 2A shows a 3-dimensional rendering of the co-culture device. The two regions in the co-culture device the eukaryotic cell culture chamber and the bacterial island are shown in Figure 2B with different color dyes (purple for the eukaryotic regions and green for the bacterial island). The feasibility of isolating the bacterial culture regions from the surrounding eukaryotic region is shown in Figure 2C using color dyes. Figure 3 shows representative results from co-culture of epithelial cells and bacteria. Figure 3A shows colonization of the commensal biofilm (green) island by EHEC (red). Figure 3B shows the juxtaposition of epithelial cells and commensal E. coli with EHEC in the bacterial island. The RFP expressing EHEC and GFP expressing commensal bacteria are localized in the island when the PDMS wall is lowered, illustrating the feasibility of isolating the bacterial island from epithelial cells. 
<img src="/files/ftp_upload/1749/1749fig1.jpg" alt="Figure 1" /> Figure 1: Cell seeding scheme in the co-culture model. (1) The PDMS wall is lowered to form an island, commensal bacteria are introduced into the island, and fibronectin is flowed around the island. (2) HeLa cells are seeded in the regions surrounding the island. (3) After HeLa cells reach confluence and the commensal biofilm has developed, EHEC is introduced into the island. (4) The PDMS wall is lifted up to expose HeLa cells surrounding the island to EHEC. Inset shows details of valve operation.
<img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="Figure 2a" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="Figure 2bc" /> Figure 2: Microfluidic model for co-culture of epithelial cells and bacteria. (A) Three-dimensional rendition of the co-culture device showing pneumatically-actuated trapping regions for forming bacterial islands among epithelial cells. Each bacterial island (1200 &mu;m diameter and 1000 &mu;m apart) has a separate inlet and outlet for providing growth media and removing waste from the island. (B) Micrograph of the co-culture device with color dyes showing the different regions (epithelial cell zone, bacterial islands). (C) The fidelity of the pneumatic trapping system is shown by lowering the PDMS wall (left panel) using a pneumatically-activated channel (blue), introducing purple dye into the closed island islands, and flowing yellow dye around it for 48 h. When the PDMS wall is raised (right panel), the island region is exposed to the surrounding yellow dye. Scale bar represents 500 &mu;m.
<img src="/files/ftp_upload/1749/1749fig3.jpg" alt="Figure 3" /> Figure 3: Co-culture of HeLa cells and bacteria. (A) Fluorescence image of RFP-expressing EHEC and GFP-expressing E. coli BW25113 in island. (B) Overlay of transmitted, green, and red fluorescence images in the device. Scale bar represents 200 &mu;m.

<ol>
	<li>McDonald, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prototyping+of+microfluidic+devices+in+poly(dimethylsiloxane)+using+solid-object+printing.">Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.</a> Anal Chem. 74, 1537-1545 (2002).</li><li>Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+integrated+passive+valves+and+pumps+for+flexible+polymer+3-dimensional+microfluidic+systems.">Design and fabrication of integrated passive valves and pumps for flexible polymer 3-dimensional microfluidic systems.</a> Biomed Microdevices. 4, 117-121 (2002).</li><li>Baek, J. Y., Park, J. Y., Ju, J. I., Lee, T. S., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatically+controllable+flexible+and+polymeric+microfluidic+valve+fabricated+via+in+situ+development.">A pneumatically controllable flexible and polymeric microfluidic valve fabricated via in situ development.</a> J Micromech Microeng. 15, 1015-1020 (2005).</li><li>Grover, W. H., Ivester, R. H., Jensen, E. C., Mathies, R. A., A, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+multiplexed+control+of+latching+pneumatic+valves+using+microfluidic+logical+structures.">Development and multiplexed control of latching pneumatic valves using microfluidic logical structures.</a> Lab Chip. 6, 623-631 (2006).</li><li>Lee, J., Jayaraman, A., Wood, T. K., K, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indole+is+an+inter-species+biofilm+signal+mediated+by+SdiA.">Indole is an inter-species biofilm signal mediated by SdiA.</a> BMC Microbiol. 7, 42-42 (2007).</li><li>Hsu, C. H., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+with+tunable+topographies.">Microfluidic devices with tunable topographies.</a> Appl Phys Lett. 86, 023508-023508 (2005).</li><li>Hui, E. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell-cell+interactions.">Micromechanical control of cell-cell interactions.</a> Proc Natl Acad Sci USA. 104, 5722-5726 (2007).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+sensing+hydrogel+microstructures+into+micropatterned+hepatocellular+cocultures.">Integrating sensing hydrogel microstructures into micropatterned hepatocellular cocultures.</a> Langmuir. 25, 3880-3886 (2009).</li><li>Kim, J., Hegde, M., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+epithelial+cells+and+bacteria+for+investigating+host-pathogen+interactions.">Co-culture of epithelial cells and bacteria for investigating host-pathogen interactions.</a> Lab-on-Chip. , (2009).</li></ol>

Conventional assays for pathogen attachment and colonization utilize a monolayer of eukaryotic cells in tissue culture plates into which pathogens are added. These models are not physiologically relevant as they do not incorporate a commensal bacterial biofilm developed on eukaryotic cells. Simple addition of a pre-grown bacterial culture to eukaryotic cells is unlikely to lead to this conformation as biofilms are highly organized structures that develop over time, and it is extremely difficult, if not virtually impossib...

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		<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>SU-8 2050</td>
<td>&nbsp;</td>
<td>Microchem Corp, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>high-resolution (16,256 dpi) photolithography mask</td>
<td>&nbsp;</td>
<td>Fineline-Imaging Inc, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>77279</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning, WI</td>
<td>184 SIL ELAST KIT 0.5KG</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH30002.02</td>
<td>&nbsp;</td>
<tr>
<td>Programmable spin coater</td>
<td>&nbsp;</td>
<td>Laurell Tech Corp</td>
<td>WS0650S</td>
<td>&nbsp;</td>
<tr>
<td>Mask aligner</td>
<td>&nbsp;</td>
<td>Neutronix-Quintel, PA</td>
<td>Q4000</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen plasma etcher</td>
<td>&nbsp;</td>
<td>March Plasma System, CA</td>
<td>CS-1701</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Live/Dead Viability/Cytotoxicity Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>L-3224</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

Watch this Scientific Journal Video about Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions at JoVE.com

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

microfluidic-co-culture-epithelial-cells-bacteria-for-investigating

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JoVE Journal

Biology

14.0K Views.  Texas A&M University. Этот протокол описывает микрожидкостных совместно культуры модель для одновременного и локализованные культуры эпителиальных клеток и бактерий. Эта модель может быть использована для исследования роли различных растворимых молекулярные сигналы на патоге...

Video: Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...