Name: preparation and structural evaluation of epithelial cell monolayers in a physiologically sized microfluidic culture device
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Description: Watch this Scientific Journal Video about Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device at JoVE.com

The authors acknowledge Alan Shepardson for designing the cutting pattern for the 3M adhesive and mylar sheet used in microfluidic channel construction and for testing the cell culture media flow rate and syringe pump programming. Funding was supplied by NIH R01 HL0142702, NSF CBET 1706801, and the Newcomb-Tulane College Dean&#39;s Grant.

The NCI-H441 human epithelial lung cell line was used for the present study (see Table of Materials).
1. Cell culture in the microfluidic channel
<ol>
	<li>Fabricate the microfluidic channel and perform the pre-treatment following the steps below.
	<ol>
		<li>Obtain a single-channel flow array (see Table of Materials) and separate the upper portion from the polycarbonate base plate.</li>
		<li>Obtain a #1.5 rectangular coverglass (thickness ...

<ol>
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S., Katara, G., Hemvani, N., Chitnis, S., Chitnis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+disinfection+by+exposure+to+germicidal+UV+light.">Surface disinfection by exposure to germicidal UV light.</a> Indian Journal of Medical Microbiology. 26 (3), 241 (2008).</li><li>Sandell, L., Sakai, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+culture.">Mammalian cell culture.</a> Current Protocols Essential Laboratory Techniques. 5 (1), 4 (2011).</li><li>New Era Pump Systems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Phaser+Programmable+Syringe+Pump:+NE-1000+Series+User+Manual.">Multi-Phaser Programmable Syringe Pump: NE-1000 Series User Manual.</a> New Era Pump Systems. , (2014).</li><li>Thermo Fisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+Data+Sheet:+Image-iT+Fixative+Solution+(4%+formaldehyde,+methanol-free).">Safety Data Sheet: Image-iT Fixative Solution (4% formaldehyde, methanol-free).</a> Thermo Fisher Scientific. , (2018).</li><li>Thavarajah, R., Mudimbaimannar, V. 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C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+cell+membranes.">Permeabilization of cell membranes.</a> Immunocytochemical Methods and Protocols. 588, 63-66 (2009).</li><li>Thermo Fisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ActinGreen+488+ReadyProbes+Reagent+Protocol.">ActinGreen 488 ReadyProbes Reagent Protocol.</a> Thermo Fisher Scientific. , (2022).</li><li>Slowfade Glass soft-set Antifade Mountant. 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The presented protocol describes the culture, crosslinking fixation, staining, permeabilization, and confocal microscopic visualization of NCI-H441 human lung epithelial cells in the dynamic environment of a single-channel microfluidic flow array, as well as in the static environment of a traditional eight-well chambered coverglass. With any microfluidic cell culture protocol, the flow conditions of the cell culture media are of paramount importance, as the high-rate flow has the potential to wash away the cells or inter...

Acute respiratory distress syndrome (ARDS) is an acute condition arising from insult to and propagation of injury in the lung parenchyma, resulting in pulmonary edema of the alveoli, inadequate gas exchange, and subsequent hypoxemia1. This initiates a cycle of pro-inflammatory cytokine release, neutrophil recruitment, toxic mediator release, and tissue damage, which itself incurs a further inflammatory response2. Additionally, pulmonary surfactant, which stabilizes the airways and prevents damage caused by repetitive recruitment/derecruitment (R/D), may be inactivated or otherwise rendered dysfunctional by the chemical processes occurring during ARDS, resulting in further stress and injury to the surrounding parenchyma3. If sufficient damage is sustained, mechanical ventilation may be necessary to ensure adequate systemic oxygenation4. However, mechanical ventilation imposes its own challenges and traumas, including the possibility of ventilator-induced lung injury (VILI), characterized as injury to the lung parenchyma caused by the mechanical stresses imposed during overinflation (volutrauma) and/or the R/D of the air-liquid interface in the fluid-occluded airway (atelectrauma)5. The pressure gradient experienced by epithelial cells exposed to an air-liquid interface (as in a fluid-occluded bronchiole) in the atelectrauma model can result in a permeability-originated obstructive response (POOR), leading to a POOR-get-POORer virtuous cycle of injury6,7,8.
In vitro experimentation can provide micro-scale insights into these phenomena, but current studies in microfluidic channel environments with physiologically relevant dimensions face several challenges9. For one, optimizing cell culture conditions poses a significant barrier to entry for cell culture research in microfluidic environments, as there exists a narrow intersection within which media flow parameters, culture duration, and other culture conditions permit optimal cell layer formation. This includes the diffusion limitations imposed by the oxygen-impermeable nature of the microfluidic culture channel enclosure. This necessitates careful consideration of media flow parameters, as low flow rates can deprive cells of oxygen, especially those farthest from the inlet; on the other hand, high flow rates can push cells out of the culture channel or result in improper or uneven layer development. Diffusion limitations may be addressed by using oxygen-permeable materials such as polydimethylsiloxane (PDMS) in an air-liquid interface (ALI) culture apparatus; however, many conventional microfluidic culture channels, such as those of the electric cell-substrate impedance sensing (ECIS) system, are inherently oxygen-impermeable, given the nature of the manufactured enclosure10. This protocol aims to provide a technique for analyzing cell layers cultured in an oxygen-impermeable enclosure.
When comparing the viability of culture conditions, observations of specific layer characteristics, such as the presence of a monolayer, surface topology, confluency, and layer-thickness uniformity, are necessary to determine whether the cell layer produced by a particular set of culture conditions meets the desired specifications and are indeed relevant to the experimental design. A limited evaluation may be performed by methods such as ECIS, which utilizes measurements of electric potential (voltage) created by resistance to high-frequency alternating current (AC) (impedance) imposed by electrically-insulating membranes of cells cultured on gold electrodes within the flow array. By modulating the frequency of AC applied to cells, specific frequency-dependent cellular properties of the cells and cell layers such as surface adherence strength, tight-junction formation, and cell proliferation or confluency may be targeted and examined11. However, these indirect forms of measurements are somewhat difficult to interpret at the onset of an experiment, and may not quantify all relevant aspects of the cell layer. Simply observing the cell layer under a phase-contrast microscope may reveal the nature of certain qualities such as confluency; however, many relevant characteristics such as the presence of a monolayer and layer-thickness uniformity require a three-dimensional (3D) evaluation that is not possible with brightfield, phase-contrast, or fluorescent microscopic imaging12.
The objective of this study was to develop a filamentous-actin staining technique to allow for imaging-based verification of a monolayer and the evaluation of cell layer uniformity using confocal laser scanning microscopy (CLSM). Filamentous-actin (F-actin) was deemed an appropriate target for the fluorophore conjugate, due in part to the way that F-actin tightly follows the cell membrane, allowing for a visual approximation of the entire cell volume13. Another important benefit of targeting F-actin is the manner in which staining of F-actin visually elucidates cytoskeletal disruptions or alterations imposed by the stresses and strains experienced by the cells. Crosslinking fixation with methanol-free formaldehyde was used to preserve the morphology of the cells and the cell layer, as dehydrating fixatives such as methanol tend to flatten cells, grossly distorting the cell layer and altering its properties14,15.
To determine the ability of the layer evaluation technique to mitigate these challenges, cells were cultured in traditional eight-well culture chambers as well as in microfluidic channels to evaluate the differences, if any, in the cell layers that were produced. For fixed culture wells, eight-well chambered coverglass units were used. For microfluidic culture, flow arrays (channel length 50 mm, width 5 mm, depth 0.6 mm) were optimized to culture immortalized human lung epithelial (NCI-H441) cells in an environment with dimensions physiologically relevant to the terminal bronchioles present in the respiratory zone of the human lung16. While this protocol was developed with the culture environment of ECIS flow arrays in mind, it may apply to any oxygen-impermeable dynamic-culture environment for which evaluation of cultured cell layer characteristics or culture conditions is necessary.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A1R HD25 Confocal Microscope System</td>
			<td>Nikon</td>
			<td>A1R HD25</td>
			<td>https://www.microscope.healthcare.nikon. 
			com/products/confocal-microscopes/a1hd25-a1rhd25/specifications</td>
		</tr>
		<tr>
			<td>ActinGreen 488 ReadyProbes Reagent (AlexaFluor 488 phalloidin)</td>
			<td>Invitrogen</td>
			<td>R37110</td>
			<td>https://www.thermofisher.com/order/catalog/product/R37110</td>
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			<td>Adhesive Transfer Tape Double Linered</td>
			<td>3M</td>
			<td>468MP</td>
			<td>https://gizmodorks.com/3m-468mp-adhesive-transfer-tape-sheet-5-pack/</td>
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			<td>Air-Tite&#160;HSW Soft-Ject Disposable Syringes</td>
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			<td>BAISDY 4 mil (0.1 mm) Thick Mylar Sheet</td>
			<td>BAISDY</td>
			<td>AS022</td>
			<td>https://www.amazon.ca/Stencil-Perfect-Silhouette-Machines-BAISDY/dp/B07RJJ9BNC</td>
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			<td>Branson Ultrasonics&#160;M Series Ultrasonic Cleaning Bath</td>
			<td>Branson Ultrasonics</td>
			<td>15-336-100</td>
			<td>https://www.fishersci.com/shop/products/m-series-ultrasonic-cleaning-bath/15336100</td>
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			<td>Corning&#160;Fibronectin, Human</td>
			<td>Fisher Scientific</td>
			<td>CB-40008</td>
			<td>https://www.fishersci.com/shop/products/corning-fibronectin-human-3/CB40008?keyword=true</td>
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			<td>DPBS, calcium, magnesium</td>
			<td>Gibco</td>
			<td>14040133</td>
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			<td>ECIS Cultureware Disposable Electrode Arrays 8 x 10 ECIS Flow Array</td>
			<td>Applied BioPhysics</td>
			<td>1F8x10E PC</td>
			<td>https://www.biophysics.com/cultureware.php#1F8x10E</td>
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			<td>Enterprise Technology Solutions UV Sterilizer Cabinet, White</td>
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			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td>https://www.thermofisher.com/order/catalog/product/26140079</td>
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			<td>Finnpipette F2 Variable Volume Pipettes</td>
			<td>Thermo Scientific</td>
			<td>4642090</td>
			<td>https://www.thermofisher.com/order/catalog/product/4642090</td>
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			<td>Fisherbrand 50mL Easy Reader Plastic Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
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			<td>Fisherbrand&#160;Cover Glasses: Rectangles (#1.5)</td>
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			<td>Fisherbrand&#160;Sterile Syringes for Single Use</td>
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			<td>https://www.fishersci.com/shop/products/sterile-syringes-single-use-12/14955458</td>
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			<td>Gibco RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>https://www.thermofisher.com/order/catalog/product/11875093</td>
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			<td>Image-iT Fixative Solution (4% formaldehyde, methanol-free)</td>
			<td>Invitrogen</td>
			<td>FB002</td>
			<td>https://www.thermofisher.com/order/catalog/product/FB002</td>
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			<td>ImageJ Fiji</td>
			<td>ImageJ</td>
			<td>ImageJ Fiji</td>
			<td>https://imagej.net/downloads</td>
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		<tr>
			<td>Immersion Oil F 30 cc</td>
			<td>Nikon</td>
			<td>MXA22168</td>
			<td>https://www.microscope.healthcare.nikon. 
			com/products/accessories/immersion-oil/specifications</td>
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			<td>Large-Capacity Reach-In CO2&#160;Incubator, 821 L, Polished Stainless Steel</td>
			<td>Thermo Scientific</td>
			<td>3950</td>
			<td>https://www.thermofisher.com/order/catalog/product/3950</td>
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			<td>Laxco LMC-3000 Series Brightfield Compound Microscope System</td>
			<td>Laxco</td>
			<td>LMC3BF1</td>
			<td>https://www.fishersci.com/shop/products/lmc-3000-series-brightfield-compound-microscope-system-8/LMC3BF1</td>
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			<td>Masterflex Fitting, Nylon, Straight, Male Luer Lock to Hose Barb Adapters, 1/16&#34; ID; 25/PK</td>
			<td>Masterflex</td>
			<td>ZY-45505-31</td>
			<td>https://www.masterflex.com/i/masterflex-fitting-nylon-straight-male-luer-lock-to-hose-barb-adapters-1-16-id-25-pk/4550531?PubID=ZY&#38;persist=true&#38;ip=no&#38; 
			gclid=Cj0KCQiA3rKQBhCNARIsAC 
			UEW_Zb5yXy1em6bGs0a9KFOk5k 
			pdlkHCvAEslHumdqcnlwSN0MdR0 
			udmwaAuDHEALw_wcB</td>
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			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>0016</td>
			<td>https://www.microsoft.com/en-us/download/details.aspx?id=56547</td>
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			<td>National Target All-Plastic Disposable Syringes</td>
			<td>Thermo Scientific</td>
			<td>03-377-24</td>
			<td>https://www.fishersci.com/shop/products/national-target-all-plastic-disposable-syringes/0337724#tab8</td>
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			<td>NCI-H441 Human Epithelial Lung Cells</td>
			<td>American Type Culture Collection (ATCC)</td>
			<td>HTB-174</td>
			<td>https://www.atcc.org/products/htb-174</td>
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			<td>NE-1600 Six Channel Programmable Syringe Pump</td>
			<td>New Era Pump Systems</td>
			<td>NE-1600</td>
			<td>https://www.syringepump.com/NE-16001800.php</td>
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			<td>NIS Elements AR</td>
			<td>Nikon</td>
			<td>NIS Elements AR</td>
			<td>https://www.microscope.healthcare.nikon. 
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			<td>Invitrogen</td>
			<td>R37605</td>
			<td>https://www.thermofisher.com/order/catalog/product/R37605?SID=srch-srp-R37605</td>
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			<td>Thermo Scientific</td>
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			<td>https://www.thermofisher.com/order/catalog/product/155361</td>
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			<td>Fisher Scientific</td>
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			<td>https://www.fishersci.com/shop/products/parafilm-m-wrapping-film-3/S37441</td>
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			<td>PendoTech</td>
			<td>ZY-19406-49</td>
			<td>https://www.masterflex.com/i/pendotech-3-way-stopcock-polysulfone-male-female-luer-inlet-x-female-luer-branch/1940649</td>
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			<td>Phosphate Buffered Solution (PBS), pH 7.4</td>
			<td>Gibco</td>
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			<td>https://www.thermofisher.com/order/catalog/product/10010023</td>
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			<td>https://www.thermofisher.com/order/catalog/product/A3890401#/A3890401</td>
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			<td>https://www.sigmaaldrich.com/US/en/product/sigma/47036</td>
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			<td>13-100-752PM</td>
			<td>https://www.fishersci.com/shop/products/1300-series-class-ii-type-a2-biological-safety-cabinet-package-promo/p-9049003#?keyword=biosafety%20hood</td>
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			<td>Cole-Parmer</td>
			<td>EW-95702-01</td>
			<td>https://www.coleparmer.com/i/tygon-transfer-tubing-biopharm-platinum-cured-silicone-1-16-id-x-1-8-od-50-ft/9570201?searchterm=95702-01</td>
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preparation and structural evaluation of epithelial cell monolayers in a physiologically sized microfluidic culture device

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.

The presented method allows for the visualization of epithelial cell layers cultured in microfluidic culture channels and uses a demonstration in traditional fixed-well cell culture environments as validation. Images acquired will exist on a spectrum of quality, signal intensity, and cellular target specificity. Successful images will demonstrate high contrast, allowing for image analysis and quantification of data for subsequent statistical evaluation. Unsuccessful images will be dim, fuzzy, blurry, or otherwise unusabl...

Watch this Scientific Journal Video about Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device at JoVE.com

Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

The presented protocol describes the development and use of a phalloidin-based filamentous-actin staining technique with confocal laser scanning microscopy (CLSM) to visualize adherent cell layer structure in microfluidic dynamic-culture channels and traditional fixed-well static-culture chambers. This approach aids in evaluating cell layer confluency, monolayer formation, and layer-thickness uniformity.

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as ...

This protocol serves to lower a barrier to entry for cell culture experimentation and enables researchers to evaluate and characterize adherent cell monolayers cultured in dynamic microfluidic environments. The primary advantage of this technique is that it enables both qualitative and quantitative evaluation of cell monolayer characteristics to serve as a basis for further monolayer-dependent experimentation. This method enables the recapitulation of an alveolar epithelium in vitro, permitting exploration of the dynamic responses during acute respiratory distress syndrome, ARDS, as well as ventilator-induced lung injury, VILI.To begin, obtain a single channel flow array. Clean the cover glass surface in an ultrasonic bath. Immerse the cover glass in poly-d-lysine at room temperature for five minutes.Then dry it at 60 degrees Celsius for 30 minutes. Next, affix double-sided adhesives in Mylar spacers. Laser cut as described in the manuscript to the cover glass and ensure that the channel cutouts are precisely aligned.Affix a rectangular cover glass to the bottommost adhesive strip. Use the peeled adhesive papers to cover the portions of adhesive still exposed. After assembly is complete, apply firm and equal pressure to the construction.Using a syringe filled with deionized water, rinse the channel. Sterilize the channel enclosure in an ultraviolet sterilizer for 30 minutes. Using a sterile technique, treat the channel with human fibronectin solution prepared in PBS and incubate for at least 30 minutes at 37 degrees Celsius.In the sterile laminar flow hood, prepare NCI H441 cell suspension using RPMI 1640 medium with 10%FBS. Use 0.25 milliliters of this cell suspension to fill the channel and a portion of the ports. Using brightfield microscopy, verify that the cells have been distributed evenly within the channels.Add the media reservoir and stopcock to the channel. Begin culturing the channel at 37 degrees Celsius with 5%carbon dioxide. Use a programmable syringe pump to draw spent media out from the channel and fresh media into the channel from a sterile media reservoir attached to the channel inlet.In a chemical fume hood, dilute a 4%formaldehyde solution into DPBS to create a 1%solution and store in an appropriately labeled tube. Prepare the 2%formaldehyde solution in a similar fashion. Transfer the formaldehyde solutions to appropriately labeled five milliliter syringes.Draw up 20 milliliters of DPBS into a separate 20 milliliter syringe which will be used for the washing steps. Next, remove the microfluidic channel from the culture apparatus and secure it in the chemical fume hood. To assemble the fixation and staining apparatus, attach a segment of transfer tubing to the side port of a three-way stopcock via male Luer lock to hose barb adapter.Then connect the stopcock to the inlet port of the flow array. Attach another segment of transfer tubing to the outlet port of the existing stopcock using the same type of hose barb adapter. Finally, secure the free ends of both transfer tubes now designated as waste lines into a chemical and biohazard appropriate waste container.Ensure the stopcock is blocking off the flow array inlet port and flush the waste line with DPBS. Then turn the stopcock to block off the waste line and slowly wash the cells with two milliliters of DPBS. Slowly push two milliliters of 1%fixative through the channel.Let it sit for five minutes and repeat with 2%fixative. Then wash the cells three times with fresh DPBS as demonstrated previously. Prepare the 0.1%saponin solution as described in the manuscript.Draw up additional DPBS into a 20 milliliter syringe for use in the washing steps. Cover the tube with aluminum foil. Add the filamentous actin staining phalloidin reagent and a nucleus staining Hoechst reagent to the 0.1%saponin solution.Then transfer the solution to a foil covered syringe. Flush the waste line with a small amount of permeabilizing and staining solution. Then introduce two milliliters of the solution to the microfluidic channel.Cover the channel with aluminum foil to block the light. After 30 minutes, flush out the permeabilizing solution with two milliliters of DPBS twice for five minutes each before gently drying the channel. Using a micropipette, introduce a minimal amount of a soft set anti-fade mountant to the microfluidic channel ensuring to cover the bottom surface completely.Then seal the end to the channel. Using a brightfield microscope, quickly verify the integrity of the cell layer before storing the channel in a container to protect it from light. Test imaging locations by taking reference scans and z-stacks until desired image parameters and conditions have been met.Using the flow array base plate as a reference, construct z-stacks at various locations along the length of the channel. Finally, analyze cross-sectional data, depth map data, and any other relevant characteristics to evaluate cell layer properties. The successful use of the technique is demonstrated in the microfluidic dynamic culture environment through image acquisition at least one centimeter away from the inlet and the outlet.In a representative culture duration experiment, successful monolayer production was observed when the cells were cultured for 24 hours. However, when cultured for 48 hours, undesirable multilayer formation was seen. The data collected from each of the five microfluidic channel imaging locations also reveals the relationship between increased culture duration and increased cross-sectional area, suggesting uneven layer formation or overgrowth occurs within 48 hours of culture.The depth map of a central location within the microfluidic channel cultured for 24 hours was obtained, which is useful for the qualitative evaluation of layer characteristics. This protocol's three most crucial aspects are proper channel construction, accurate culture and media flow conditions, and careful use of the fixation and staining apparatus. Following this procedure, other methods may be used in parallel to validate the experimental viability of the produced cell monolayers such as electric cell-substrate impedance sensing, ECIS, or transepithelial electrical resistance, TEER.

Research

JoVE Journal

Bioengineering

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