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 0.17 mm) with dimensions of 60 mm x 22 mm. Clean surfaces of the coverglass in an ultrasonic bath and treat one side with a 0.1 mg/mL solution of Poly-D-Lysine at room temperature for 5 min before drying at 60 &#176;C for 30 min.</li>
		<li>Affix a 0.13 mm thick double-sided adhesive (see Table of Materials), laser-cut to accommodate the dimensions of the flow array top and the flow channel (50 mm length, 5 mm width), to the flow array top, taking care to precisely align the channel cut-outs17.</li>
		<li>Affix a 0.1 mm thick mylar spacer (see Table of Materials), laser-cut to accommodate the dimensions of the flow array top and the flow channel, to the adhesive strip, taking care to precisely align the channel cut-outs.</li>
		<li>Repeat steps 1.1.3 and 1.1.4 until desired channel height is achieved (for example, for a channel height of 0.6 mm, use two spacers and three adhesive strips).</li>
		<li>Affix a rectangular coverglass to the bottom-most adhesive strip with the Poly-D-Lysine-treated side facing the adhesive. After assembly is complete, as indicated in Figure 1, apply firm and equal pressure to the top and bottom of construction and hold for 1 min. 
		NOTE: Construction of the channel enclosure, including coverglass, adhesives, spacers, and flow array top, is now complete.</li>
		<li>Rinse the channel with de-ionized water using a syringe, simultaneously checking for leaks.</li>
		<li>Sterilize channel enclosure in an ultraviolet (UV) sterilizer for 30 min18.</li>
		<li>Using sterile technique, treat the channel with 2.0 &#181;g/mL human fibronectin (see Table of Materials) in phosphate buffered saline (PBS) and incubate for at least 30 min at 37 &#176;C19.</li>
	</ol>
	</li>
	<li>Perform cell culture in the microfluidic channel following the steps below.
	<ol>
		<li>In a sterile laminar flow hood, use a micropipette to transfer an even suspension of NCI-H441 cells in RPMI 1640 medium with 10% fetal bovine serum (FBS) (see Table of Materials) to seed two microfluidic channels, each with cells at a surface density of 150,000 cells/cm2.
		<ol>
			<li>For 50 mm x 5 mm x 0.6 mm channels, use 0.25 mL of a 2.5 x 106 cells/mL suspension to fill each channel, as well as a portion of the ports. Verify that cells have been distributed evenly within the channels using a brightfield microscope.</li>
		</ol>
		</li>
		<li>Culture the two channels for 24 h and 48 h respectively at 37 &#176;C with 5% CO2 using a programmable syringe pump (see Table of Materials), drawing spent media out from the channel and fresh media into the channel from a sterile media reservoir attached to channel inlet comprised of a cut-open 20 mL syringe covered with paraffin film.
		<ol>
			<li>After a 10 min waiting period following cell seeding, introduce and pump fresh media from the reservoir through the channel at a variable flow rate beginning at 0.2 &#181;L/min and ramping up to 10 &#181;L/min for 4 h, maintaining at that rate thereafter20. 
			NOTE: This variable flow rate provides culture conditions that allow cells to (1) gravitationally settle to the culture surface, (2) adhere to the culture surface, and (3) form a confluent monolayer.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform cell fixation inside the microfluidic channels using formaldehyde solution. 
	CAUTION: Formaldehyde is toxic and must be handled in an appropriate chemical fume hood21.
	<ol>
		<li>In a chemical fume hood, prepare formaldehyde solutions by using 4% formaldehyde in PBS (methanol-free) (see Table of Materials) to create two 4 mL portions, diluting the first to a concentration of 1% formaldehyde and the second to 2% formaldehyde using Dulbecco&#39;s phosphate buffered saline (DPBS; with Ca2+ and Mg2+) as the diluent. Transfer formaldehyde solutions to separate 5 mL syringes and label accordingly. Draw up 20 mL of DPBS into a separate 20 mL syringe.</li>
		<li>Remove microfluidic channels from the culture apparatus and place them into the chemical fume hood.</li>
		<li>Assemble the fixation and staining apparatus.
		<ol>
			<li>Attach a 10 cm segment of transfer tubing to the side port of a three-way stopcock via a male Luer lock to hose barb adapter (see Table of Materials), then connect the stopcock to the inlet port of the flow array.</li>
			<li>Next, attach another 10 cm segment of transfer tubing to the outlet port of the flow array using the same type of hose barb adapter.</li>
			<li>Finally, secure the free ends of both transfer tubes in a chemical and biohazard-appropriate waste container, such as a labeled empty 50 mL conical centrifuge tube.</li>
		</ol>
		</li>
		<li>Turn the stopcock to block 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 cells with 2 mL of DPBS. Repeat the flushing step using the new solution every time. A new solution (or concentration of solution) is introduced to the channel.</li>
		<li>Slowly push 2 mL of 1% fixative solution through the channel and then allow to sit for 5 min22.</li>
		<li>Slowly push 2 mL of 2% fixative solution through the channel and then allow to sit for 15 min.</li>
		<li>Wash cells by slowly introducing 2 mL of fresh DPBS to the channel in three separate instances (5 min each).</li>
		<li>Complete steps 1.3.3-1.3.7 for both microfluidic channels in parallel.</li>
	</ol>
	</li>
	<li>Stain, permeabilize, and add mounting media to the cells in the microfluidic channel.
	<ol>
		<li>Prepare 0.1% saponin solution by adding 1 mg of saponin (see Table of Materials) per mL of DPBS to produce 4 mL of solution and gently vortex to mix23. Draw up 8 mL of DPBS into a 20 mL syringe.</li>
		<li>Add an F-actin-staining phalloidin reagent and a nucleus-staining Hoechst reagent (see Table of Materials) to the 0.1% saponin solution, at two drops (0.1 mL) of each reagent per mL of saponin solution. Keep prepared staining/permeabilizing solution away from light by covering with aluminum foil24.</li>
		<li>Flush the line with a small amount of staining/permeabilizing solution (as described in step 1.3.4), then introduce 2 mL of the solution to the microfluidic channel and cover the channel with aluminum foil before allowing it to sit at room temperature for 30 min.</li>
		<li>Flush out the staining/permeabilizing solution twice with 2 mL of DPBS for 5 mins per flush.</li>
		<li>For better image quality, add a suitable mounting media (with an index of refraction closely matching the microscope objective oil and cover glass, see Table of Materials) into the channel.
		<ol>
			<li>Using a micropipette, introduce a minimal amount of a soft-set antifade mountant to each port of the microfluidic channel, ensuring that the bottom surface is completely covered and no bubbles are trapped within the desired imaging area25. Seal the ends of the channel and verify cell layer integrity by observing under a brightfield microscope.</li>
		</ol>
		</li>
		<li>Complete steps 1.4.3-1.4.5 for both microfluidic channels in parallel. 
		NOTE: Image cells as soon as possible after staining for maximum image quality. If photobleaching occurs or long-term storage is desired, other mounting media with antifade or sample-preserving properties may be used. Note that hard-curing mounting media will distort the 3D structure of the cells and, by extension, the cell layer; for this reason, soft-setting mounting media is preferable26.</li>
	</ol>
	</li>
	<li>Image cells in the microfluidic channel following the steps below.
	<ol>
		<li>Adjust the confocal microscope (see Table of Materials) settings, including laser power, gain, offset, and scan parameters such as scan speed, scan area, scan format, resolution, and pinhole diameter27.</li>
		<li>Test imaging location by taking reference scans as well as Z-stacks until desired image parameters and conditions have been met. Dial-in parameters at sequentially higher-magnification objectives until the 40x oil-immersion objective has been reached and optimized28.</li>
		<li>Using the flow array base plate as a reference, construct Z-stacks at five locations, at the would-be location of the first electrode on the inlet side, halfway between the center and the previous location, the center, halfway between the center and the location of the last electrode (on the outlet side), and at the last electrode, as indicated in Figure 2.</li>
	</ol>
	</li>
	<li>Perform image processing and data analysis.
	<ol>
		<li>Export XZ and YZ cross-sections using the confocal microscope software package (see Table of Materials).</li>
		<li>Using an image-processing software (see Table of Materials) with edge-detection capabilities (thresholding value 15.0), measure the total image area in pixels by using the Magic Wand tool outside of the image, then the area excluding the total cross-sectional area of the cell layer in pixels by using the Magic Wand tool in the portion of the image exterior to the cell layer29.</li>
		<li>Using data-processing software (see Table of Materials), subtract the exterior area pixel value from the total area pixel value to find the cross-sectional area pixel value.</li>
		<li>Convert cross-sectional area pixel values to &#181;m2 values by multiplying pixel values by the square of the &#181;m/pixel value, as indicated in the microscope software for the particular image (0.31 &#181;m/pixel for Z-stacks taken in 1024p resolution with no zoom on a 40x oil-immersion lens).</li>
		<li>Calculate means and standard deviations of data and graph results.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64148/64148fig01.jpg" /> 
Figure 1: Exploded-view schematic of the microfluidic channel construction. The top element is the top portion of the flow array, thin grey elements are adhesive strips, thin blue elements are mylar spacers, and the bottom element is the rectangular coverglass. <a href="https://www.jove.com/files/ftp_upload/64148/64148fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64148/64148fig02.jpg" /> 
Figure 2: Five imaging locations along the consistently layer-producing region of the microfluidic culture channel. Imaging locations are as follows: inlet-side, near where the first electrode would be on the intact flow array; halfway between inlet-side location and the center of the channel; center of the channel; halfway between the center and the outlet-side location, and outlet-side, near where the last electrode would be on the intact flow array. <a href="https://www.jove.com/files/ftp_upload/64148/64148fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Cell culture in the eight-well chambered coverglass
<ol>
	<li>Perform pre-treatment of the eight-well chambered coverglass.
	<ol>
		<li>Obtain a sterile eight-well chambered coverglass manufactured with a #1.5 coverglass and cell adherence-increasing surface treatment (Figure 3, see Table of Materials).</li>
		<li>Using sterile technique, treat the surface of culture wells with 2.0 &#181;g/mL human fibronectin in PBS and incubate for at least 30 min at 37 &#176;C19.</li>
	</ol>
	</li>
	<li>Perform cell culture in the chambered coverglass following the steps below.
	<ol>
		<li>In a sterile laminar flow hood, transfer 0.5 mL portions of solutions of NCI-H441 cells evenly suspended in RPMI 1640 medium with 10% FBS at volumetric densities of 81,000, 162,000, and 324,000 cells/mL to seed the culture wells at surface densities of 45,000, 90,000, and 180,000 cells/cm2, respectively. Verify that cells have been evenly distributed within the wells using a brightfield microscope.</li>
		<li>Culture cells for 24 h, 48 h, and 96 h at 37 &#176;C with 5% CO2, replacing media daily.</li>
	</ol>
	</li>
	<li>Perform formaldehyde fixation in the eight-well chambered coverglass. 
	CAUTION: Formaldehyde is toxic and must be handled in an appropriate chemical fume hood21.
	<ol>
		<li>In a chemical fume hood, prepare formaldehyde solutions by making two portions of 4% formaldehyde in PBS (methanol-free), diluting the first to a concentration of 1% formaldehyde and the other to 2% formaldehyde using Dulbecco&#39;s phosphate buffered saline (DPBS; with Ca2+ and Mg2+) as the diluent.</li>
		<li>Remove the eight-well culture chambered coverglass from the incubator and place it into the chemical fume hood.</li>
		<li>Gently wash cells with 0.5 mL of DPBS using a micropipette by slowly introducing liquid along the upper portion of the corner of each well.</li>
		<li>Remove existing liquid in each well by slowly extracting it from the corner of the wells using a micropipette. Using the liquid-introduction method (step 2.3.3), introduce 0.5 mL of 1% fixative solution into each well and allow it to sit for 5 min22.</li>
		<li>Remove existing liquid in each well using the liquid-extraction method mentioned in step 2.3.4. Using the liquid-introduction method mentioned in step 2.3.3, introduce 0.5 mL of 2% fixative solution into each well and allow it to sit for 15 min.</li>
		<li>Using the liquid introduction and extraction methods (steps 2.3.3 and 2.3.4), wash cells by introducing and removing 0.5 mL of fresh DPBS in each well in three separate instances for 5 min each.</li>
	</ol>
	</li>
	<li>Perform staining, permeabilization, and mounting media addition in the eight-well chambered coverglass.
	<ol>
		<li>Prepare 0.1% saponin solution by adding 1 mg of saponin per mL of DPBS and gently vortex to mix23.</li>
		<li>To the 0.1% saponin solution, add two drops (0.1 mL) each of an F-actin-staining phalloidin reagent and a nucleus-staining Hoechst reagent per mL of saponin solution. Keep the prepared solution away from light by covering with aluminum foil24.</li>
		<li>Introduce 0.2 mL of staining/permeabilizing solution to each well and cover the chambered coverglass with aluminum foil before allowing it to sit at room temperature for 30 min.</li>
		<li>Flush out staining/permeabilizing solution twice with 0.5 mL of DPBS.</li>
		<li>For better image quality, add a suitable mounting media (with an index of refraction closely matching the microscope objective oil and cover glass) into the wells.
		<ol>
			<li>Using a micropipette, introduce a minimal amount of a soft-set antifade mountant to each well, ensuring that the bottom surface is completely covered and no bubbles are trapped within the desired imaging area25. Verify cell layer integrity by observing under a brightfield microscope. 
			NOTE: Image cells as soon as possible after staining for maximum image quality. If photobleaching occurs or long-term storage is desired, other mounting media with antifade or sample-preserving properties may be used. Note that hard-curing mounting media will distort the 3D structure of the cells and, by extension, the cell layer, so soft-setting mounting media is preferable26.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform imaging in the eight-well chambered coverglass.
	<ol>
		<li>Adjust the confocal microscope settings, including laser power, gain, offset, and scan parameters such as scan speed, scan area, scan format, resolution, and pinhole diameter27.</li>
		<li>Test imaging location by taking reference scans as well as Z-stacks until desired image parameters and conditions have been met. Dial-in parameters at sequentially higher-magnification objectives until the 40x oil-immersion objective has been reached and optimized28.</li>
		<li>Construct Z-stacks of three random locations in each seeding density/culture duration match-up.</li>
	</ol>
	</li>
	<li>Perform image processing and data analysis.
	<ol>
		<li>Export XZ and YZ cross sections using the confocal microscope software package.</li>
		<li>Using an image-processing software with edge-detection capabilities (thresholding value 15.0), measure the total image area in pixels by using the Magic Wand tool outside of the image, then the area excluding the total cross-sectional area of the cell layer in pixels by using the Magic Wand tool in the portion of the image exterior to the cell layer29.</li>
		<li>Using a data-processing software, subtract the exterior area pixel value from the total area pixel value to find the cross-sectional area pixel value.</li>
		<li>Convert cross-sectional area pixel values to &#181;m2 values by multiplying pixel values by the square of the &#181;m/pixel value as indicated in the microscope software for the particular image (0.31 &#181;m/pixel for Z-stacks taken in 1024p resolution with no zoom on a 40x oil-immersion lens).</li>
		<li>Calculate means and standard deviations and graph results.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64148/64148fig03.jpg" /> 
Figure 3: Diagram of the eight-well chambered coverglass used for the fixed-well culture, staining, and imaging experiment comparing the effects of initial cell seeding density and culture duration on the formation of cell layers. <a href="https://www.jove.com/files/ftp_upload/64148/64148fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Matthay, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome.">Acute respiratory distress syndrome.</a> Nature Reviews Disease Primers. 5, 18 (2019).</li><li>Rawal, G., Yadav, S., Kumar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome:+An+update+and+Review.">Acute respiratory distress syndrome: An update and Review.</a> Journal of Translational Internal Medicine. 6 (2), 74-77 (2018).</li><li>Bilek, A. M., Dee, K. C., Gaver, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+surface-tension-induced+epithelial+cell+damage+in+a+model+of+pulmonary+airway+reopening.">Mechanisms of surface-tension-induced epithelial cell damage in a model of pulmonary airway reopening.</a> Journal of Applied Physiology. 94 (2), 770-783 (2003).</li><li>Modrykamien, A. M., Gupta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> Baylor University Medical Center Proceedings. 28 (2), 163-171 (2017).</li><li>Jacob, A. -. M., Gaver, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atelectrauma+disrupts+pulmonary+epithelial+barrier+integrity+and+alters+the+distribution+of+tight+junction+proteins+ZO-1+and+Claudin+4.">Atelectrauma disrupts pulmonary epithelial barrier integrity and alters the distribution of tight junction proteins ZO-1 and Claudin 4.</a> Journal of Applied Physiology. 113 (9), 1377-1387 (2012).</li><li>Kay, S. S., Bilek, A. M., Dee, K. C., Gaver, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+gradient,+not+exposure+duration,+determines+the+extent+of+epithelial+cell+damage+in+a+model+of+pulmonary+airway+reopening.">Pressure gradient, not exposure duration, determines the extent of epithelial cell damage in a model of pulmonary airway reopening.</a> Journal of Applied Physiology. 97 (1), 269-276 (2004).</li><li>Jacob, A. M., Gaver, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+the+influence+of+cell+topography+on+epithelial+mechanical+stresses+during+pulmonary+airway+reopening.">An investigation of the influence of cell topography on epithelial mechanical stresses during pulmonary airway reopening.</a> Physics of Fluids. 17 (3), 031502 (1994).</li><li>Gaver, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+POOR+get+POORer:+A+hypothesis+for+the+pathogenesis+of+ventilator-induced+lung+injury.">The POOR get POORer: A hypothesis for the pathogenesis of ventilator-induced lung injury.</a> American Journal of Respiratory and Critical Care Medicine. 202 (8), 1081-1087 (2020).</li><li>Jain, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+ultra-thin+alveolar-capillary+basement+membrane+mimics.">Reconstruction of ultra-thin alveolar-capillary basement membrane mimics.</a> Advanced Biology. 5 (8), 2000427 (2021).</li><li>Byrne, M. B., Leslie, M. T., Gaskins, H. R., Kenis, P. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+study+the+tumor+microenvironment+under+controlled+oxygen+conditions.">Methods to study the tumor microenvironment under controlled oxygen conditions.</a> Trends in Biotechnology. 32 (11), 556-563 (2014).</li><li>Szulcek, R., Bogaard, H. J., van Nieuw Amerongen, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+cell-substrate+impedance+sensing+for+the+quantification+of+endothelial+proliferation,+barrier+function,+and+motility.">Electric cell-substrate impedance sensing for the quantification of endothelial proliferation, barrier function, and motility.</a> Journal of Visualized Experiments. (85), e51300 (2014).</li><li>Jaccard, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+method+for+the+rapid+and+precise+estimation+of+adherent+cell+culture+characteristics+from+phase+contrast+microscopy+images.">Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images.</a> Biotechnology and Bioengineering. 111 (3), 504-517 (2013).</li><li>Hagiyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modest+static+pressure+suppresses+columnar+epithelial+cell+growth+in+association+with+cell+shape+and+cytoskeletal+modifications.">Modest static pressure suppresses columnar epithelial cell growth in association with cell shape and cytoskeletal modifications.</a> Frontiers in Physiology. 8, 00997 (2017).</li><li>Srinivasan, M., Sedmak, D., Jewell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fixatives+and+tissue+processing+on+the+content+and+integrity+of+Nucleic+Acids.">Effect of fixatives and tissue processing on the content and integrity of Nucleic Acids.</a> The American Journal of Pathology. 161 (6), 1961-1971 (2002).</li><li>Zhu, L., Rajendram, M., Huang, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+fixation+on+bacterial+cellular+dimensions+and+integrity.">Effects of fixation on bacterial cellular dimensions and integrity.</a> Iscience. 24 (4), 102348 (2021).</li><li>Lust, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Pulmonary System. XPharm: The Comprehensive Pharmacology Reference. , 1-6 (2007).</li><li>EpilogueLaser. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FusionSeries:+Pro+&amp;+Edge+Laser+System+Manual+and+Original+Instructions.">FusionSeries: Pro &amp; Edge Laser System Manual and Original Instructions.</a> EpilogueLaser. , (2022).</li><li>Chitnis, D. 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. K., Rao, U. K., Ranganathan, K., Elizabeth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+basics+of+routine+formaldehyde+fixation.">Chemical and physical basics of routine formaldehyde fixation.</a> Journal of Oral and Maxillofacial Pathology. 16 (3), 400-405 (2012).</li><li>Jamur, M. 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. Thermo Fisher Scientific Available from: <a target="_blank" href="https://www.thermofisher.com/order/catalog/product/S36917-5X2ML?SID=srch-hj-S36917-5X2ML">https://www.thermofisher.com/order/catalog/product/S36917-5X2ML?SID=srch-hj-S36917-5X2ML</a> (2022)</li><li>Ravikumar, S., Surekha, R., Thavarajah, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mounting+media:+An+overview.">Mounting media: An overview.</a> Journal of Dr. NTR University of Health Sciences. 3 (5), 1-8 (2014).</li><li>Shihan, M. H., Novo, S. G., Le Marchand, S. J., Wang, Y., Duncan, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+quantitating+confocal+fluorescent+images.">A simple method for quantitating confocal fluorescent images.</a> Biochemistry and Biophysics Reports. 25, 100916 (2021).</li><li>North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing?+A+beginners'+guide+to+practical+pitfalls+in+image+acquisition.">Seeing is believing? A beginners' guide to practical pitfalls in image acquisition.</a> Journal of Cell Biology. 172 (1), 9-18 (2006).</li><li>Ferriera, F., Rasband, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+User+Guide.">ImageJ User Guide.</a> National Institutes of Health. , (2012).</li><li>Halldorsson, S., Lucumi, E., G&#243;mez-Sj&#246;berg, R., Fleming, R. M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+challenges+of+microfluidic+cell+culture+in+polydimethylsiloxane+devices.">Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.</a> Biosensors and Bioelectronics. 63, 218-231 (2015).</li><li>Smith, H. S., Riggs, J. L., Mosesson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+fibronectin+by+human+epithelial+cells+in+culture.">Production of fibronectin by human epithelial cells in culture.</a> American Association for Cancer Research. 39 (10), 4138-4144 (1979).</li><li>Sieck, G. C., Mantilla, C. B., Prakash, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+measurements+in+confocal+microscopy.">Volume measurements in confocal microscopy.</a> Methods in Enzymology. 307, 296-315 (1999).</li><li>Heijink, I. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+cell+adhesion+in+airway+epithelial+cell+types+using+electric+cell-substrate+impedance+sensing.">Characterisation of cell adhesion in airway epithelial cell types using electric cell-substrate impedance sensing.</a> European Respiratory Journal. 35 (4), 894-903 (2009).</li><li>Zhang, X., Wang, W., Li, F., Voiculescu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretchable+impedance+sensor+for+mammalian+cell+proliferation+measurements.">Stretchable impedance sensor for mammalian cell proliferation measurements.</a> Lab on a Chip. 17 (12), 2054-2066 (2017).</li></ol>

The authors declare no conflicts of interest.

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>
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			<td>https://www.microscope.healthcare.nikon. 
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		<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>
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			<td>Branson Ultrasonics&#160;M Series Ultrasonic Cleaning Bath</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>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>26140079</td>
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			<td>Finnpipette F2 Variable Volume Pipettes</td>
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			<td>4642090</td>
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			<td>Fisherbrand 50mL Easy Reader Plastic Centrifuge Tubes</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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>ImageJ Fiji</td>
			<td>ImageJ</td>
			<td>ImageJ Fiji</td>
			<td>https://imagej.net/downloads</td>
		</tr>
		<tr>
			<td>Immersion Oil F 30 cc</td>
			<td>Nikon</td>
			<td>MXA22168</td>
			<td>https://www.microscope.healthcare.nikon. 
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		</tr>
		<tr>
			<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>
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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; 
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		</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>
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		<tr>
			<td>National Target All-Plastic Disposable Syringes</td>
			<td>Thermo Scientific</td>
			<td>03-377-24</td>
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		</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>
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		<tr>
			<td>NIS Elements AR</td>
			<td>Nikon</td>
			<td>NIS Elements AR</td>
			<td>https://www.microscope.healthcare.nikon. 
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		</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>
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		<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>
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		<tr>
			<td>Parafilm M Wrapping Film</td>
			<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>Phosphate Buffered Solution (PBS), pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>https://www.thermofisher.com/order/catalog/product/10010023</td>
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		<tr>
			<td>Poly-D-Lysine</td>
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			<td>Millipore Sigma (Sigma Aldrich)</td>
			<td>47036</td>
			<td>https://www.sigmaaldrich.com/US/en/product/sigma/47036</td>
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		<tr>
			<td>SlowFade Glass Soft-set Antifade Mountant</td>
			<td>Invitrogen</td>
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			<td>https://www.thermofisher.com/order/catalog/product/S36917-5X2ML</td>
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			<td>Thermo Scientific 1300 Series Class II, Type A2 Biological Safety Cabinet Package</td>
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			<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 ...

preparation-structural-evaluation-epithelial-cell-monolayers

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Bioengineering

1.2K Views.  Tulane University. 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.

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

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

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 ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

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.