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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/64148/64148fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 interfere with the normal assembly of the cell monolayer. Meanwhile, low-rate flow has the potential to "starve" or "suffocate" (insufficient nutrient and oxygen availability, respectively) the cell layer due to the diffusion limitations imposed by the impermeable nature of the microfluidic channel as well as the flow characteristics30. By beginning with a 10 min waiting period (during which cells remain suspended in the same media that originally filled the channel), then advancing to a flow rate of 0.2 &#181;L/min, before finally ramping up to 10 &#181;L/min over the course of 4 h, these competing needs are balanced to promote both cell adherence and survival.
Another critical step in the protocol is the proper preparation of the microfluidic channel, including construction and pre-treatment. Care must be taken to ensure that the coverglass is mounted properly to the chamber construct to avoid leakage and potential contamination. Proper attachment of the coverglass is also necessary to ensure that the channel height is consistent and accurately reproduced between successive trials using different constructed units. Minor variability may be introduced by the tolerance in thickness of the adhesive strips used as well as the relative compressibility. Minimizing any variability will help with the consistency of experimental results. Proper pre-treatment of the constructed channel is important to ensure that the cells are allowed to adhere to and initiate proliferation on the glass surface. Ultrasonic cleaning of the glass improves Poly-D-Lysine adherence, promoting adherence to fibronectin. Adding fibronectin, a glycoprotein naturally synthesized and secreted by human epithelial cells, further promotes the adherence of the NCI-H441 cells to the coverglass surface, allowing for normal cell monolayer development31. 
Further, one of the most important aspects of this protocol is the imaging location. Despite carefully controlled preparation, due to the columnar volume available in the regions of the two channel openings, more cells will be present on the coverglass surface in the area directly underneath as well as adjacent to these openings, even when adhering to proper cell culture techniques. For this reason, regions near the openings should not be considered as part of the "normal" cultured cell layer, as there will be significant multilayering/cell-stacking occurring there. Instead, the central region of the microfluidic channel is an appropriate area to image, as this location is undisturbed by the error imposed by channel height variation near the openings. The electrode locations on the flow array can be used to guide imaging locations, and as a rule of thumb, all scans intended to capture layers truly representative of the microfluidic channel conditions should be taken at analogous locations within the bounds of the two farthest-apart electrodes on the flow array. Additionally, in the protocol described, the 40x magnification objective is specified for use; however, this may be modified to suit the user's needs. 40x magnification was chosen as the optimal magnification because of its ability to offer high-resolution, high-magnification imaging while still retaining a sufficient number of cells to serve as a statistically valid sample.
One modification that can be made to the microfluidic channel includes altering the channel height, which can be achieved by varying the number of spacers and adhesives used to construct the microfluidic channel. Alteration of the channel height will likely necessitate adjusting the flow parameters to ensure that the shear stress does not remove cells during the feeding. As such, any variation in channel height will impact the ideal initial rate, rate of ramp-up, and final rate of media flow. The described protocol in this article may serve as a means by which suitable flow rates may be experimentally derived, in conjunction with indirect methods such as ECIS. Additionally, flow rates may be modified to suit the needs of different cell lines, which may require adaptation to establish optimal conditions. Another modification that may be made, with some additions to the steps in this protocol, is to expand the use-case of the described technique beyond the relative comparison of the cross-sectional area of cell layers. With appropriate calibration using microspheres or microbeads of known dimensions, accurate volumetric, topologic, and absolute cross-sectional area measurements may be taken. Calibration using a reference object is necessary in this instance due to the confocal microscope's limited Z-resolution and the potential for over- or under-sampling in the Z-dimension due to the manual control over Z-stack step size that confocal scanning systems offer32. 
One limitation of this protocol is the lack of true volumetric measurement due to the lack of vertical calibration steps. The calibration mentioned above and the verification technique utilizing fluorescent microbeads of known dimensions may be used for true-to-life scaling. Additionally, the present protocol involves the use of pre-prepared, ready-to-use stains. Immunofluorescent staining using primary and secondary antibodies often requires a multi-day protocol that is more involved than the one this article discusses. However, it is likely possible to utilize immunofluorescent staining in this manner, though some experimentation may be necessary to optimize the workflow. Given the additional steps involved in immunofluorescent staining protocols, care must be taken to avoid shearing of the cultured cell layer during the numerous wash steps, as this may alter the properties of the cell layer ultimately evaluated and therefore invalidate any conclusions that would follow. Further developments of this technique may include automation of the image and data analyses, including region-of-interest (ROI) detection and quantification for the cross-sectional area determination. Additionally, expanding this technique to include different intra- and extra-cellular target structures (perhaps staining for targets such as tight-junction proteins) may broaden the use-case of this method to 3D co-localization within microfluidic cell culture devices. 
To produce the most physiologically representative cell layers, lung epithelial cells must be cultured in microfluidic channels with an air-liquid interface (ALI) at the culture surface, which is typically accomplished using polydimethylsiloxane (PDMS), a flexible material made permeable by the introduction of &#181;m-scale pores. ALI culture conditions allow for the formation of cell layers that most closely resemble the physiological features of the in vivo lung epithelium33. While technically possible, using ECIS in an ALI culture environment with a PDMS culture surface in a dynamic microfluidic environment is impractical and inaccessible for most researchers, as there are no commercially available apparatuses for such a complex cell culture assembly34. One reason for this difficulty may be the paradoxical need for a permeable culture surface that is still capable of acting as a solid electrode for the purpose of ECIS measurements. As such, there currently exists the need for a method to visually verify the characteristics and physiologic relevance of cell layers cultured in oxygen-impermeable culture environments such as those present in ECIS flow arrays. 
This method expands on the traditional method of ensuring proper confluency using two-dimensional microscopy by allowing for 3D visualization, which greatly broadens the number of cell layer characteristics that can be observed, quantified, and analyzed. Additionally, this permits a subjective determination of the presence of a monolayer, layer uniformity, and other properties deemed relevant for experimental purposes. This protocol is important because it serves as a means for the visual verification and evaluation of cell layers being produced in many oxygen-impermeable environments, such as in ECIS experiments evaluating barrier function properties of epithelial cells exposed to atelectrauma. This also serves as a method that can be used in future work to determine whether a set of dynamic microfluidic culture conditions produces cell layers that are relevant and appropriate for further experimentation.

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><tbody><tr><td>A1R HD25 Confocal Microscope System</td><td>Nikon</td><td>A1R HD25</td><td>https://www.microscope.healthcare.nikon.&lt;br/&gt; 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></tr><tr><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></tr><tr><td>Air-Tite&amp;nbsp;HSW Soft-Ject Disposable Syringes</td><td>Air-Tite RL5</td><td>14-817-53</td><td>https://www.fishersci.com/shop/products/air-tite-hsw-soft-ject-disposable-syringes-6/1481753#?keyword=syringe%20leur%20locking%205ml</td></tr><tr><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></tr><tr><td>Branson Ultrasonics&amp;nbsp;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></tr><tr><td>Corning&amp;nbsp;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></tr><tr><td>DPBS, calcium, magnesium</td><td>Gibco</td><td>14040133</td><td>https://www.thermofisher.com/order/catalog/product/14040133?SID=srch-srp-14040133</td></tr><tr><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></tr><tr><td>Enterprise Technology Solutions UV Sterilizer Cabinet, White</td><td>Enterprise Technology Solutions</td><td>50-211-1163</td><td>https://www.fishersci.com/shop/products/uv-sterilizer-cabinet-white/502111163</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26140079</td><td>https://www.thermofisher.com/order/catalog/product/26140079</td></tr><tr><td>Finnpipette F2 Variable Volume Pipettes</td><td>Thermo Scientific</td><td>4642090</td><td>https://www.thermofisher.com/order/catalog/product/4642090</td></tr><tr><td>Fisherbrand 50mL Easy Reader Plastic Centrifuge Tubes</td><td>Fisher Scientific</td><td>06-443-21</td><td>https://www.fishersci.com/shop/products/fisherbrand-higher-speed-easy-reader-plastic-centrifuge-tubes-8/p-193269</td></tr><tr><td>Fisherbrand&amp;nbsp;Cover Glasses: Rectangles (#1.5)</td><td>Fisher Scientific</td><td>12-544-GP</td><td>https://www.fishersci.com/shop/products/cover-glasses-rectangles-promo-22/12544GP#coverglass</td></tr><tr><td>Fisherbrand&amp;nbsp;Sterile Syringes for Single Use</td><td>Fisher Scientific</td><td>14-955-458</td><td>https://www.fishersci.com/shop/products/sterile-syringes-single-use-12/14955458</td></tr><tr><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.&lt;br/&gt; com/products/accessories/immersion-oil/specifications</td></tr><tr><td>Large-Capacity Reach-In CO&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;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&quot; 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&amp;amp;persist=true&amp;amp;ip=no&amp;amp;&lt;br/&gt; gclid=Cj0KCQiA3rKQBhCNARIsAC&lt;br/&gt; UEW_Zb5yXy1em6bGs0a9KFOk5k&lt;br/&gt; pdlkHCvAEslHumdqcnlwSN0MdR0&lt;br/&gt; 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.&lt;br/&gt; 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&quot; ID x 1/8&quot; 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 unusable for subjective or quantitative evaluation. Some images may be suitable for subjective layer characteristic determination while still being of insufficient quality for quantitative analysis. Thus, the degree and care to which the protocol is followed will directly impact the suitability of a given image to serve as a data source for statistical analysis.
Figure 4 represents the successful use of the technique in the context of a microfluidic dynamic-culture environment and shows image acquisition at the inlet and outlet of two microfluidic devices. Figure 4A,B provides examples of layers cultured for 24 h, and Figure 4C,D presents examples of layers cultured for 48 h. Figure 5 illustrates the associated data collected from the microfluidic channel experiment, incorporating three samples from each of the five microfluidic channel imaging locations indicated in Figure 2 from each culture duration. Error bars signify standard deviations.
Figure 6 depicts a 2D image of the XY plane in the center of the microfluidic channel, and Figure 7 depicts a 3D view of the same location with depth color-coding. Figure 8 represents successful image acquisition within a density-duration experiment analyzing the effects of initial cell seeding density and culture duration on NCI-H441 cell layer formation in the eight-well chambered coverglass environment. Figure 8A-C indicates culture durations of 24 h, 48 h, and 96 h, respectively. Figure 8X-Z indicates initial cell seeding densities of 180,000, 90,000, and 45,000 cells/cm2, respectively. Figure 9 presents quantitative data collected from the eight-well chambered coverglass experiment, including samples from three locations in each density-duration match-up. Error bars represent standard deviations and p-value tests were performed to determine statistically significant differences between apparently close values.
These two representative experiments within the dynamic microfluidic and static eight-well environments are distinct use-case examples that demonstrate how the technique may contextualize experimental findings by visually confirming the presence or absence of physiologically relevant monolayers.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64148/64148fig04.jpg" /> 
Figure 4: NCI-H441 cell layers cultured in the microfluidic channel for 24 and 48 h, imaged at the inlet-side and outlet-side locations as depicted in Figure 2. (A) 24 h, inlet-side. (B) 24 h, outlet-side. (C) 48 h, inlet-side. (D) 48 h, outlet-side. Blue represents the nuclei staining and green represents the staining of filamentous-actin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64148/64148fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64148/64148fig05.jpg" /> 
Figure 5: Graphical representation of the data collected during the 24-48 h microfluidic channel imaging experiment. This includes six cross-sectional area samples from each of five locations along the relevant length of the microfluidic channel (as depicted in Figure 2). Error bars represent standard deviations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64148/64148fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64148/64148fig06.jpg" /> 
Figure 6: 2D image taken in the XY plane in a central location within the microfluidic channel. Blue represents the nuclei staining and green represents the staining of filamentous-actin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64148/64148fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64148/64148fig07.jpg" /> 
Figure 7: 3D model of the same microfluidic channel location as Figure 6. The color-coding depicts depth data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64148/64148fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64148/64148fig08.jpg" /> 
Figure 8: NCI-H441 cell layers cultured in the eight-well chambered coverglass. For 24 h (A), 48 h (B), and 96 h (C) at initial seeding densities of 180,000 (X), 90,000 (Y), and 45,000 (Z) cells/cm2. Blue represents the nuclei staining and green represents the staining of filamentous-actin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64148/64148fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/64148/64148fig09.jpg" /> 
Figure 9: Graphical representation of the data collected during the density-duration eight-well culture experiment. This includes six cross-sectional area samples from each density-duration match-up. Error bars represent standard deviations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64148/64148fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Universidad San Sebastian

Research

JoVE Journal

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Biology

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

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

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

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

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

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.
Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues 1-6. The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties.
The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type1, 7-13. Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

A rotating cell culture system that allows epithelial cells to grow under physiological conditions resulting in 3-D cellular aggregate formation is described. The aggregates generated display in vivo-like characteristics not observed in conventional culture models and serve as a more accurate organotypic model system for a multitude of scientific investigations.

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

Analysis of Blood-Brain Barrier Models via Immunofluorescence Staining

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

Author Spotlight: Advancing the Use of Tissue Chip Technology for Studying Human Tissues 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...

A Reconfigurable Barrier Tissue Platform with Open-Well and Microfluidic Modes

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems.
Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.

Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue

This protocol describes a reconfigurable membrane-based cell culture platform that integrates the open-well format with fluid flow capabilities. This platform is compatible with standard protocols and allows for reversible transitions between open-well and microfluidic culture modes, accommodating the needs of both engineering and bioscience laboratories.

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. ...

Advanced Two-Layered 3D Co-Culture System for Epithelial and Fibroblast Cells

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

Author Spotlight: Advancing 3D Coculture Systems with PVA-PCL Nanofibrous Membranes 

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...