Method Article

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

DOI:

10.3791/64148

⸱

July 1st, 2022

 ,  ,  , 
1Department of Biomedical Engineering, School of Science and Engineering, Tulane University

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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 p....

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Protocol

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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

  1. Fabricate the microfluidic channel and perform the pre-treatment following the steps below.
    1. Obtain a single-channel flow array (see Table of Materials) and separate the upper portion from the polycarbonate base plate.
    2. 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....

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Results

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

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Discussion

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The presented protocol describes the culture, crosslinking fixation, staining, permeabilization, and confocal microscopic visualization of NCI-H441 human lung epithelial cells in the dynamic environment of a single-channel microfluidic flow array, as well as in the static environment of a traditional eight-well chambered coverglass. With any microfluidic cell culture protocol, the flow conditions of the cell culture media are of paramount importance, as the high-rate flow has the potential to wash away the cells or inter.......

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Disclosures

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The authors declare no conflicts of interest.

Acknowledgements

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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.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
A1R HD25 Confocal Microscope SystemNikonA1R HD25https://www.microscope.healthcare.nikon.
com/products/confocal-microscopes/a1hd25-a1rhd25/specifications
ActinGreen 488 ReadyProbes Reagent (AlexaFluor 488 phalloidin)InvitrogenR37110https://www.thermofisher.com/order/catalog/product/R37110
Adhesive Transfer Tape Double Linered3M468MPhttps://gizmodorks.com/3m-468mp-adhesive-transfer-tape-sheet-5-pack/
Air-Tite HSW Soft-Ject Disposable SyringesAir-Tite RL514-817-53https://www.fishersci.com/shop/products/air-tite-hsw-soft-ject-disposable-syringes-6/1481753#?keyword=syringe%20leur%20locking%205ml
BAISDY 4 mil (0.1 mm) Thick Mylar SheetBAISDYAS022https://www.amazon.ca/Stencil-Perfect-Silhouette-Machines-BAISDY/dp/B07RJJ9BNC
Branson Ultrasonics M Series Ultrasonic Cleaning BathBranson Ultrasonics15-336-100https://www.fishersci.com/shop/products/m-series-ultrasonic-cleaning-bath/15336100
Corning Fibronectin, HumanFisher ScientificCB-40008https://www.fishersci.com/shop/products/corning-fibronectin-human-3/CB40008?keyword=true
DPBS, calcium, magnesiumGibco14040133https://www.thermofisher.com/order/catalog/product/14040133?SID=srch-srp-14040133
ECIS Cultureware Disposable Electrode Arrays 8 x 10 ECIS Flow ArrayApplied BioPhysics1F8x10E PChttps://www.biophysics.com/cultureware.php#1F8x10E
Enterprise Technology Solutions UV Sterilizer Cabinet, WhiteEnterprise Technology Solutions50-211-1163https://www.fishersci.com/shop/products/uv-sterilizer-cabinet-white/502111163
Fetal Bovine Serum (FBS)Gibco26140079https://www.thermofisher.com/order/catalog/product/26140079
Finnpipette F2 Variable Volume PipettesThermo Scientific4642090https://www.thermofisher.com/order/catalog/product/4642090
Fisherbrand 50mL Easy Reader Plastic Centrifuge TubesFisher Scientific06-443-21https://www.fishersci.com/shop/products/fisherbrand-higher-speed-easy-reader-plastic-centrifuge-tubes-8/p-193269
Fisherbrand Cover Glasses: Rectangles (#1.5)Fisher Scientific12-544-GPhttps://www.fishersci.com/shop/products/cover-glasses-rectangles-promo-22/12544GP#coverglass
Fisherbrand Sterile Syringes for Single UseFisher Scientific14-955-458https://www.fishersci.com/shop/products/sterile-syringes-single-use-12/14955458
Gibco RPMI 1640 MediumGibco11875093https://www.thermofisher.com/order/catalog/product/11875093
Image-iT Fixative Solution (4% formaldehyde, methanol-free)InvitrogenFB002https://www.thermofisher.com/order/catalog/product/FB002
ImageJ FijiImageJImageJ Fijihttps://imagej.net/downloads
Immersion Oil F 30 ccNikonMXA22168https://www.microscope.healthcare.nikon.
com/products/accessories/immersion-oil/specifications
Large-Capacity Reach-In CO2 Incubator, 821 L, Polished Stainless SteelThermo Scientific3950https://www.thermofisher.com/order/catalog/product/3950
Laxco LMC-3000 Series Brightfield Compound Microscope SystemLaxcoLMC3BF1https://www.fishersci.com/shop/products/lmc-3000-series-brightfield-compound-microscope-system-8/LMC3BF1
Masterflex Fitting, Nylon, Straight, Male Luer Lock to Hose Barb Adapters, 1/16" ID; 25/PKMasterflexZY-45505-31https://www.masterflex.com/i/masterflex-fitting-nylon-straight-male-luer-lock-to-hose-barb-adapters-1-16-id-25-pk/4550531?PubID=ZY&persist=true&ip=no&
gclid=Cj0KCQiA3rKQBhCNARIsAC
UEW_Zb5yXy1em6bGs0a9KFOk5k
pdlkHCvAEslHumdqcnlwSN0MdR0
udmwaAuDHEALw_wcB
Microsoft ExcelMicrosoft0016https://www.microsoft.com/en-us/download/details.aspx?id=56547
National Target All-Plastic Disposable SyringesThermo Scientific03-377-24https://www.fishersci.com/shop/products/national-target-all-plastic-disposable-syringes/0337724#tab8
NCI-H441 Human Epithelial Lung CellsAmerican Type Culture Collection (ATCC)HTB-174https://www.atcc.org/products/htb-174
NE-1600 Six Channel Programmable Syringe PumpNew Era Pump SystemsNE-1600https://www.syringepump.com/NE-16001800.php
NIS Elements ARNikonNIS Elements ARhttps://www.microscope.healthcare.nikon.
com/products/software/nis-elements/nis-elements-advanced-research
NucBlue Live ReadyProbes Reagent (Hoechst 33342)InvitrogenR37605https://www.thermofisher.com/order/catalog/product/R37605?SID=srch-srp-R37605
Nunc Lab-Tek Chambered CoverglassThermo Scientific155411https://www.thermofisher.com/order/catalog/product/155361
Parafilm M Wrapping FilmFisher ScientificS37441https://www.fishersci.com/shop/products/parafilm-m-wrapping-film-3/S37441
PendoTech 3-Way Stopcock, Polysulfone, Male/Female Luer Inlet x Female Luer BranchPendoTechZY-19406-49https://www.masterflex.com/i/pendotech-3-way-stopcock-polysulfone-male-female-luer-inlet-x-female-luer-branch/1940649
Phosphate Buffered Solution (PBS), pH 7.4Gibco10010023https://www.thermofisher.com/order/catalog/product/10010023
Poly-D-LysineGibcoA3890401https://www.thermofisher.com/order/catalog/product/A3890401#/A3890401
Reynolds Aluminum Wrap FoilReynolds458742928317https://www.amazon.com/Reynolds-Wrap-Aluminum-Foil-Square/dp/B00UNT0Y2M
SaponinMillipore Sigma (Sigma Aldrich)47036https://www.sigmaaldrich.com/US/en/product/sigma/47036
SlowFade Glass Soft-set Antifade MountantInvitrogenS36917-5X2MLhttps://www.thermofisher.com/order/catalog/product/S36917-5X2ML
Thermo Scientific 1300 Series Class II, Type A2 Biological Safety Cabinet PackageThermo Scientific13-100-752PMhttps://www.fishersci.com/shop/products/1300-series-class-ii-type-a2-biological-safety-cabinet-package-promo/p-9049003#?keyword=biosafety%20hood
Tygon Transfer Tubing, BioPharm Platinum-Cured Silicone, 1/16" ID x 1/8" OD; 50 FtCole-ParmerEW-95702-01https://www.coleparmer.com/i/tygon-transfer-tubing-biopharm-platinum-cured-silicone-1-16-id-x-1-8-od-50-ft/9570201?searchterm=95702-01

References

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  1. Matthay, M. A., et al. Acute respiratory distress syndrome. Nature Reviews Disease Primers. 5, 18(2019).
  2. Rawal, G., Yadav, S., Kumar, R. Acute respiratory distress syndrome: An update and Review. Journal of Translational Internal Medicine. 6

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Tags

Epithelial Cell MonolayerMicrofluidic Culture DeviceHuman Lung EpithelialConfocal MicroscopyPhalloidin StainingCell Layer EvaluationMicrofluidic Channel ConstructionCell Culture ConditionsActin Cytoskeleton ImagingTransepithelial Electrical Resistance

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