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 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.
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.
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
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
2. Cell culture in the eight-well chambered coverglass
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. Please click here to view a larger version of this figure.
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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 7: 3D model of the same microfluidic channel location as Figure 6. The color-coding depicts depth data. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µL/min, before finally ramping up to 10 µL/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 µm-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.
The authors have nothing to disclose.
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's Grant.
A1R HD25 Confocal Microscope System | Nikon | A1R HD25 | https://www.microscope.healthcare.nikon. com/products/confocal-microscopes/a1hd25-a1rhd25/specifications |
ActinGreen 488 ReadyProbes Reagent (AlexaFluor 488 phalloidin) | Invitrogen | R37110 | https://www.thermofisher.com/order/catalog/product/R37110 |
Adhesive Transfer Tape Double Linered | 3M | 468MP | https://gizmodorks.com/3m-468mp-adhesive-transfer-tape-sheet-5-pack/ |
Air-Tite HSW Soft-Ject Disposable Syringes | Air-Tite RL5 | 14-817-53 | https://www.fishersci.com/shop/products/air-tite-hsw-soft-ject-disposable-syringes-6/1481753#?keyword=syringe%20leur%20locking%205ml |
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Corning Fibronectin, Human | Fisher Scientific | CB-40008 | https://www.fishersci.com/shop/products/corning-fibronectin-human-3/CB40008?keyword=true |
DPBS, calcium, magnesium | Gibco | 14040133 | https://www.thermofisher.com/order/catalog/product/14040133?SID=srch-srp-14040133 |
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Gibco RPMI 1640 Medium | Gibco | 11875093 | https://www.thermofisher.com/order/catalog/product/11875093 |
Image-iT Fixative Solution (4% formaldehyde, methanol-free) | Invitrogen | FB002 | https://www.thermofisher.com/order/catalog/product/FB002 |
ImageJ Fiji | ImageJ | ImageJ Fiji | https://imagej.net/downloads |
Immersion Oil F 30 cc | Nikon | MXA22168 | https://www.microscope.healthcare.nikon. com/products/accessories/immersion-oil/specifications |
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National Target All-Plastic Disposable Syringes | Thermo Scientific | 03-377-24 | https://www.fishersci.com/shop/products/national-target-all-plastic-disposable-syringes/0337724#tab8 |
NCI-H441 Human Epithelial Lung Cells | American Type Culture Collection (ATCC) | HTB-174 | https://www.atcc.org/products/htb-174 |
NE-1600 Six Channel Programmable Syringe Pump | New Era Pump Systems | NE-1600 | https://www.syringepump.com/NE-16001800.php |
NIS Elements AR | Nikon | NIS Elements AR | https://www.microscope.healthcare.nikon. com/products/software/nis-elements/nis-elements-advanced-research |
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