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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>0016</td>
			<td>https://www.microsoft.com/en-us/download/details.aspx?id=56547</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>NIS Elements AR</td>
			<td>Nikon</td>
			<td>NIS Elements AR</td>
			<td>https://www.microscope.healthcare.nikon. 
			com/products/software/nis-elements/nis-elements-advanced-research</td>
		</tr>
		<tr>
			<td>NucBlue Live ReadyProbes Reagent (Hoechst 33342)</td>
			<td>Invitrogen</td>
			<td>R37605</td>
			<td>https://www.thermofisher.com/order/catalog/product/R37605?SID=srch-srp-R37605</td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek Chambered Coverglass</td>
			<td>Thermo Scientific</td>
			<td>155411</td>
			<td>https://www.thermofisher.com/order/catalog/product/155361</td>
		</tr>
		<tr>
			<td>Parafilm M Wrapping Film</td>
			<td>Fisher Scientific</td>
			<td>S37441</td>
			<td>https://www.fishersci.com/shop/products/parafilm-m-wrapping-film-3/S37441</td>
		</tr>
		<tr>
			<td>PendoTech 3-Way Stopcock, Polysulfone, Male/Female Luer Inlet x Female Luer Branch</td>
			<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>
		</tr>
		<tr>
			<td>Phosphate Buffered Solution (PBS), pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>https://www.thermofisher.com/order/catalog/product/10010023</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Gibco</td>
			<td>A3890401</td>
			<td>https://www.thermofisher.com/order/catalog/product/A3890401#/A3890401</td>
		</tr>
		<tr>
			<td>Reynolds Aluminum Wrap Foil</td>
			<td>Reynolds</td>
			<td>458742928317</td>
			<td>https://www.amazon.com/Reynolds-Wrap-Aluminum-Foil-Square/dp/B00UNT0Y2M</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Millipore Sigma (Sigma Aldrich)</td>
			<td>47036</td>
			<td>https://www.sigmaaldrich.com/US/en/product/sigma/47036</td>
		</tr>
		<tr>
			<td>SlowFade Glass Soft-set Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>S36917-5X2ML</td>
			<td>https://www.thermofisher.com/order/catalog/product/S36917-5X2ML</td>
		</tr>
		<tr>
			<td>Thermo Scientific 1300 Series Class II, Type A2 Biological Safety Cabinet Package</td>
			<td>Thermo Scientific</td>
			<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>
		</tr>
		<tr>
			<td>Tygon Transfer Tubing, BioPharm Platinum-Cured Silicone, 1/16&#34; ID x 1/8&#34; OD; 50 Ft</td>
			<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>
		</tr>
	</tbody>
</table>

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.

preparation-structural-evaluation-epithelial-cell-monolayers

Research

JoVE Journal

Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...