Name: determining ciliary function and membrane impermeability of the pseudostratified lung airway epithelium
Uploaded: 2025-02-21T12:00:00.000+00:00
Duration: 7 min 40 s
Description: Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified ...

This work&#160;was funded by NIH P20GM113123 and UND SMHS pilot grant.

The protocol should be performed in a sterile condition under a biosafety level 2 laminar flow hood (biosafety cabinet). All the medium and solutions should be thawed at 37 &#176;C prior to use. Centrifugation should be performed at 4 &#176;C. All the materials used in the experiment must be sterile. The primary cells (deidentified donor) were obtained in collaboration with Dr. Kristina Bailey at the University of Nebraska Medical Center (UNMC), Omaha, NE. The primary cells are from deidentified donors, and the cells do not fall under the NIH Human Subject. The cells were obtained under an approved Material Transfer Agreement (MTA) between the University of North Dakota, Grand Forks, ND, and UNMC, Omaha, NE. Here, we used passaged 4 NHBE cells. Although higher passage NHBE cells (up to passage 8) were also shown to differentiate into airway epithelium, we routinely use passage four NHBE cells because there is no obvious difference in whole genome transcription profiles among passages 1 to 46,14.
1. Primary cell culture
NOTE: Careful handling of the primary cells is crucial. The whole protocol should avoid harsh treatment and excessive pipetting of the primary cells.
<ol>
	<li>Using a sterile serological pipette add 5 mL of collagen to a 100 mm tissue culture (TC) dish to pre-coat the dish. Incubate the TC dish with collagen at room temperature (RT) for 20 min.</li>
	<li>Using a sterile serological pipette, aspirate the collagen from the TC dish and replace it with 8 mL of pre-thawed complete airway epithelial cell (AEC) medium supplemented with 2% (v/v) penicillin-streptomycin and 1% (v/v) amphotericin B (Table 1).</li>
	<li>Thaw one vial (containing 5 x 105 cells) of primary normal human bronchial epithelial (NHBE) cells in a water bath at 37 &#176;C.</li>
	<li>With a 5 mL sterile serological pipette, add 1 mL of pre-thawed complete AEC medium to the cells dropwise to acclimatize the NHBE cells, and collect all the cells in a sterile 15 mL conical tube.</li>
	<li>Pellet the cells by centrifugation at 212 x g for 5 min at 4 &#176;C. Gently discard the medium from the tube and resuspend the cells in the leftover medium drops in the tube by gentle tapping. 
	NOTE: Excessive and harsh tapping should be avoided.</li>
	<li>Using a 5 mL sterile serological pipette, slowly add 2 mL of complete AEC medium to the resuspended cells and collect all the cells.</li>
	<li>Add the cells to the plate pre-added with 8 mL of complete AEC medium. Move the TC dish sideways to distribute the cells in the dish uniformly.</li>
	<li>Incubate the cells at 37 &#176;C in an 85%-95% humid CO2 cell culture incubator. After 24 h, observe the NHBE cells under the microscope at 10x or 20x for their attachment to the TC dish and growth. If the cells are suspended in the medium, this shows dead cells.</li>
	<li>After 48 h, aspirate the old medium from the TC dish with a sterile serological pipette.</li>
	<li>Using a sterile serological pipette, add 10 mL of pre-warmed, complete AEC medium to the cells. Add the media slowly to one corner; avoid direct pouring over the top of the cells.</li>
	<li>Change the complete AEC media every 48 h until the cells reach 95%-100% confluency.</li>
</ol>
2. Air-Liquid interface culture
<ol>
	<li>Using a P200 pipette, add 100 &#181;L of collagen to a pre-coated 6.5 mm transwell insert with 0.4 &#956;m-pore polyester&#160;membrane (from here on referred as inserts) and incubate at RT for 20 min.</li>
	<li>With a sterile serological pipette, aspirate the medium from the confluent NHBE cells.</li>
	<li>Add 5 mL of 1x Dulbecco&#39;s Phosphate Buffer Saline (DPBS) with a sterile serological pipette to one corner of the TC dish.</li>
	<li>Gently swirl the plate to wash the cells and aspirate the 1x DPBS with a sterile serological pipette. Ensure cells do not dry out at any step during the protocol.</li>
	<li>Using a serological pipette, add 5 mL of TrypsinLE to the cells and incubate at 37 &#176;C in a humid CO2 cell culture incubator until all the cells are dissociated from the bottom surface of the plate.</li>
	<li>Using a sterile serological pipette, collect all the cells in a 15 mL conical tube. With a sterile serological pipette, add 3 mL of trypsin to the TC dish and collect any residual cells.</li>
	<li>Pellet the cells by centrifugation at 212 x g for 5 min at 4 &#176;C. Discard the medium and gently tap the tube to resuspend the cells in the leftover trypsin drops in the tube.</li>
	<li>Using a serological pipette add 2 mL of complete AEC medium to the resuspended cells.</li>
	<li>Count the cells using a hemacytometer or automated cell counter.</li>
	<li>Dilute the cells with complete AEC media, with each insert receiving 5 x 104 cells in a volume of 200 &#181;L.</li>
	<li>With sterile forceps, transfer the inserts to an empty well in a 24-well culture dish.</li>
	<li>Using a P1000 pipette, add 500 &#181;L of complete AEC media to the basal well of the plate.</li>
	<li>Using sterile forceps, transfer the inserts back to the basal well with added complete AEC media.</li>
	<li>With a P200 pipette, add 5 x 104 resuspended NHBE cells in 200 &#181;L of the complete AEC medium to the apical side of the insert. Incubate the cells at 37 &#176;C in an 85% - 95% humid CO2 cell culture incubator.</li>
	<li>After 24 h, observe the cells under the microscope to see if they have formed a confluent layer in the well. 
	NOTE: If the cells do not form a confluent layer, change the basal AEC medium every 48 h with fresh, complete AEC medium till the cells form a confluent monolayer. While changing basal media, transfer the inserts to the empty well with sterile forceps. Aspirate the old medium with a P1000 pipette, add fresh medium, and place the inserts back in fresh, complete AEC medium.</li>
	<li>Once the cells are confluent, transfer the inserts to an empty well with sterile forceps. Using a P1000 pipette, aspirate all the basal AEC medium.</li>
	<li>With a P1000 pipette, add 500 &#181;L of fresh complete air-liquid interface differentiation (ALI) medium supplemented with 2% (v/v) penicillin-streptomycin and 1% (v/v) amphotericin B to the basal well.&#160;</li>
	<li>Transfer the inserts to the basal well with added complete ALI medium using sterile forceps. Using a P200 pipette, gently aspirate the apical AEC medium from the well. 
	NOTE: The apical side should not be added with the complete ALI medium. Aspirate the AEC medium carefully from the apical side to avoid any cell detachment.</li>
	<li>Incubate the cells at 37 &#176;C in a humid CO2 cell culture incubator. After 48 h, transfer the inserts to an empty well with sterile forceps.</li>
	<li>Using a P1000 pipette, aspirate the old medium and add fresh 500 &#181;L of complete ALI medium.</li>
	<li>With sterile forceps, place the inserts back in the well with fresh medium. Keep changing the complete ALI medium every 48 h till day 28 (four weeks).</li>
	<li>On day 28, using a P200 pipette, add 100 &#181;L of 1x DPBS to the apical side of the inserts and incubate at 37 &#176;C in a humid CO2 cell culture incubator for 30 min. Aspirate the 1x DPBS with a P200 pipette. 
	NOTE: DPBS should be added by&#160;pouring&#160;on the wall&#160;instead of directly to the cells. While aspirating DPBS, avoid touching the epithelium and vigorous pipetting because any damage to the epithelium can compromise the epithelium (tissue-like) integrity.</li>
	<li>From the 2nd week of differentiation, evaluate mucus production by observing a possible darker layer due to mucus on the epithelium under the microscope. The excessive mucus can be removed by DPBS washing. Briefly, add 200 &#181;L of 1x DPBS on the apical side of the inserts on a 24-well plate and incubate the plate at 37 &#176;C in a humid CO2 cell culture incubator for 30 min. Remove DPBS and keep the plate at 37 &#176;C in a humid CO2 cell culture incubator.</li>
</ol>
3. Determination of ciliary beat frequency
NOTE: For determination of CBF at 37 &#176;C (if necessary), the microscope needs to have an environment chamber incubator providing 5% CO2 at 37 &#176;C and humidity.
<ol>
	<li>Turn on the microscope with its attached computer and the environment chamber. Open the door of the chamber to place the plate containing the inserts on the microscope stage.</li>
	<li>Once the plate is placed on the stage, close the chamber. Using the microscope stage operating knob, move the plate to bring the inserts into view.</li>
	<li>Select the 20x magnification lens in the phase-contrast mode. Observe the plate through the eyepiece of the microscope and focus the cell using the tuning knob.</li>
	<li>On the computer screen, click on SAVA system icon. SAVA program should be installed on the computer connected to the microscope.</li>
	<li>On the software interface, click on Configure Experiment. On the next tab, click on Create Experiment.</li>
	<li>A new tab will appear, give details of the directory where the data will be stored in the Enter Experiment Directory Name and experiment details in Enter Experiment Description, then click OK.</li>
	<li>Click The Experiment that was created and is shown in the Directory under the Experiments.</li>
	<li>Click on Modify and provide sample details in the Sample Description. Click on Add then on Exit to exit from this tab.</li>
	<li>On the main software interface on the experiment slot, select the experiment created from the dropdown menu on the main interface.</li>
	<li>Click on Record Video and monitor the ciliary beat frequency (CBF) on the computer screen. The screen will show focused ciliary movement.</li>
	<li>For each insert, record 6 different random fields for 2.1 s at 120 frames per second. Click on Save Video to monitor the CBF data.</li>
	<li>To analyze data, click on Analyse Video on the software interface. On the next tab, click on OK.</li>
	<li>On the following tab, click on Analyse All. Download the spreadsheet generated for every experiment in the computer SAVA data acquisition folder in the computer.</li>
	<li>Take Gaussian mean frequency for data presentation.</li>
</ol>
4. Quantification of transepithelial electrical resistance
NOTE: TEER quantification needs liquid at the apical side of the airway epithelium. To establish this protocol, add 100 &#181;L of 1x DPBS at the apical side of the airway epithelium in the inserts that already contain the basal medium (experimental) or 500 &#181;L of 1x DPBS.
<ol>
	<li>Turn on the EVOM2 volt ohmmeter using the power switch. Connect the Test Resistor to the EVOM2 at the INPUT slot.</li>
	<li>Monitor the reading on the EVOM2 screen. If it is above or below 1000, then it needs calibration. Using forceps, turn the ADJ switch at the EVOM2 volt ohmmeter till it shows a uniform 1000 reading.</li>
	<li>Take an empty insert for experimental control reading. Using a P200 pipette add 100 &#181;L of 1x DPBS at the apical side of the empty insert, while 500 &#181;L of 1x DPBS is added at the basal side with a P1000 pipette.</li>
	<li>Disconnect the Test Resistor and connect the STX2 electrode at the INPUT slot on EVOM2. Gently clean the STX2 electrode with 70% ethanol.</li>
	<li>Insert the STX2 electrode in the emptyinsert with 1x DPBS, keeping the shorter electrode leg on the apical part of each insert and the longer electrode leg on the basal part.</li>
	<li>Record the stable reading on the EVOM2 screen, as the reading can fluctuate due to movement or other undetermined reasons.</li>
	<li>Insert the STX2 electrode in the insert with 28-day differentiated epithelium, keeping the shorter electrode leg on the apical part of each insert and the longer electrode leg on the basal part in the medium.</li>
	<li>Record the stable reading on the EVOM2 screen. Take several independent readings to calculate a reliable average. Subtract the background (empty insert reading) from the experimental insert reading for calculation.</li>
</ol>

Assessing Biophysical Properties of Differentiated Airway Epithelium 

<ol>
	<li>GBDCRD Collaborators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+chronic+respiratory+diseases+and+risk+factors,+1990-2019:+an+update+from+the+Global+Burden+of+Disease+Study+2019.">Global burden of chronic respiratory diseases and risk factors, 1990-2019: an update from the Global Burden of Disease Study 2019.</a> E Clin Med. 59, 101936 (2023).</li><li>Fendrick, A. M., Monto, A. S., Nightengale, B., Sarnes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+economic+burden+of+non-influenza-related+viral+respiratory+tract+infection+in+the+United+States.">The economic burden of non-influenza-related viral respiratory tract infection in the United States.</a> Arch Intern Med. 163 (4), 487-494 (2003).</li><li>LeMessurier, K. S., Tiwary, M., Morin, N. P., Samarasinghe, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+barrier+as+a+safeguard+and+regulator+of+defense+against+Influenza+A+virus+and+Streptococcus+pneumoniae.">Respiratory barrier as a safeguard and regulator of defense against Influenza A virus and Streptococcus pneumoniae.</a> Front Immunol. 11, 3 (2020).</li><li>Plasschaert, L. W., 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=A+single-cell+atlas+of+the+airway+epithelium+reveals+the+CFTR-rich+pulmonary+ionocyte.">A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.</a> Nature. 560 (7718), 377-381 (2018).</li><li>Calven, J., Ax, E., Radinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+airway+epithelium-A+central+player+in+Asthma+pathogenesis.">The airway epithelium-A central player in Asthma pathogenesis.</a> Int J Mol Sci. 21 (23), 8907 (2020).</li><li>Talukdar, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RSV-induced+expanded+ciliated+cells+contribute+to+bronchial+wall+thickening.">RSV-induced expanded ciliated cells contribute to bronchial wall thickening.</a> Virus Res. 327, 199060 (2023).</li><li>Osan, J., 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=Goblet+cell+hyperplasia+increases+SARS-CoV-2+infection+in+chronic+obstructive+pulmonary+disease.">Goblet cell hyperplasia increases SARS-CoV-2 infection in chronic obstructive pulmonary disease.</a> Microbiol Spectr. 10 (4), e0045922 (2022).</li><li>Srinivasan, B., 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=TEER+measurement+techniques+for+in+vitro+barrier+model+systems.">TEER measurement techniques for in vitro barrier model systems.</a> J Lab Autom. 20 (2), 107-126 (2015).</li><li>Bustamante-Marin, X. M., Ostrowski, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia+and+mucociliary+clearance.">Cilia and mucociliary clearance.</a> Cold Spring Harb Perspect Biol. 9 (4), a028241 (2017).</li><li>Rhee, C. S., 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=Ciliary+beat+frequency+in+cultured+human+nasal+epithelial+cells.">Ciliary beat frequency in cultured human nasal epithelial cells.</a> Ann Otol Rhinol Laryngol. 110 (11), 1011-1016 (2001).</li><li>Scopulovic, L., Francis, D., Pandzic, E., Francis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+cilia+beat+frequency+using+high-speed+video+microscopy:+Assessing+frame+rate+requirements+when+imaging+different+ciliated+tissues.">Quantifying cilia beat frequency using high-speed video microscopy: Assessing frame rate requirements when imaging different ciliated tissues.</a> Physiol Rep. 10 (11), e15349 (2022).</li><li>Jing, J. C., Chen, J. J., Chou, L., Wong, B. J. F., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+detection+of+ciliary+beating+pattern+and+frequency+in+the+upper+airway+using+phase+resolved+Doppler+optical+coherence+tomography.">Visualization and detection of ciliary beating pattern and frequency in the upper airway using phase resolved Doppler optical coherence tomography.</a> Sci Rep. 7 (1), 8522 (2017).</li><li>Yuksel, H., Turkeli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+epithelial+barrier+dysfunction+in+the+pathogenesis+and+prognosis+of+respiratory+tract+diseases+in+childhood+and+adulthood.">Airway epithelial barrier dysfunction in the pathogenesis and prognosis of respiratory tract diseases in childhood and adulthood.</a> Tissue Barriers. 5 (4), e1367458 (2017).</li><li>Rayner, R. E., Makena, P., Prasad, G. L., Cormet-Boyaka, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Normal+Human+Bronchial+Epithelial+(NHBE)+Cell+3D+Cultures+for+in+vitro+Lung+Model+Studies.">Optimization of Normal Human Bronchial Epithelial (NHBE) Cell 3D Cultures for in vitro Lung Model Studies.</a> Sci Rep. 9 (1), 500 (2019).</li><li>Osan, J. K., DeMontigny, B. A., Mehedi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+for+protein+detection+in+PFA-fixed+paraffin-embedded+SARS-CoV-2-infected+COPD+airway+epithelium.">Immunohistochemistry for protein detection in PFA-fixed paraffin-embedded SARS-CoV-2-infected COPD airway epithelium.</a> STAR Protoc. 2 (3), 100663 (2021).</li><li>Talukdar, S. N., McGregor, B., Osan, J. K., Hur, J., Mehedi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+Syncytial+Virus+Infection+Does+Not+Induce+Epithelial-Mesenchymal+Transition.">Respiratory Syncytial Virus Infection Does Not Induce Epithelial-Mesenchymal Transition.</a> J Virol. 97 (7), e0039423 (2023).</li><li>Christopher, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+temperature+and+anesthetic+agents+on+ciliary+function+in+murine+respiratory+epithelia.">The effects of temperature and anesthetic agents on ciliary function in murine respiratory epithelia.</a> Front Pediatr. 2, 111 (2014).</li><li>Smith, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ciliaFA:+a+research+tool+for+automated,+high-throughput+measurement+of+ciliary+beat+frequency+using+freely+available+software.">ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software.</a> Cilia. 1, 14 (2012).</li></ol>

Authors have no conflict of interest.

Human primary airway epithelium (ALI culture) is increasingly used as an in vitro lung model for biological assessments to investigate function and mechanisms and to reduce animal use14. ALI has several advantages over any monolayer submerged culture. For example, it provides a tissue-like pseudostratified airway epithelium that mimics the apical side of the lung airway6,7,14. However, the quality and integrity of the differentiated pseudostratified airway epithelium should be evaluated to enhance the reliability and reproducibility of the experiment. Two crucial biophysical metrics that provide information on the quality of highly differentiated apical airway epithelium are ciliary function and membrane impermeability. While these properties can be evaluated at the early stage of the primary epithelial&#160;cell differentiation at an ALI, a 3- or 4-week differentiation has been used reliably to produce a pseudostratified airway epithelium in infectious disease research6,15,16. Differences in the ability to grow and differentiate into the airway epithelium were observed in different donors14. Thus, donor-to-donor variation more likely contributes to different CBF and TEER values. Additionally, temperature may also impact CBF reading. A microscope with an environment chamber that provides temperature, CO2, and humidity, similar to a cell culture incubator, can be used. We successfully use this protocol to determine CBF and TEER of the airway epithelium before studying different viral infections6,7. Here, we also determined both CBF and TEER of the airway epithelium obtained from either healthy adult or adult with COPD. We found similar CBF between healthy and COPD donors; in contrast, COPD has higher TEER value than that of healthy. Whether chronic condition plays an important role in the increased membrane integrity is yet to be determined.
CBF can be determined as soon as ciliary movement is visible under a microscope, and we determined the CBF as early as 2 weeks of differentiation. Thus, the quality of the epithelium can be monitored over time by determining CBF. However, CBF can more or less be influenced by temperature and other common environmental factor17, thus an environment chamber providing a cell culture environment (e.g., 37 &#176;C and 5% CO2) can be attached to the microscope6. However, we did not find a substantial difference in CBF measured with or without an environment chamber. CBF may vary within different fields for the same insert plate. We recommend taking at least 6 different field readings for eachinsert. CBF of the respiratory ciliated epithelial cells was reported between 3 to 18 Hz18. However, patients with a rare genetic disorder, primary ciliary dyskinesia, have different ciliary beat patterns and frequency13. The percentage of area for ciliary function in the same insert can also be determined. We observed a substantially higher mucus production at the apical side of the 3- or 4-week differentiation airway epithelium. Thus, a washing step with 1x DPBS is recommended to overcome low CBF reading due to the excessive mucus present at the apical side of the epithelium. For ciliary function determination, we quantified CBF using the SAVA system. However, there is an open source-based CBF quantifying method, CiliaFA, available as an alternative method to the SAVA system18. To determine ciliary function at 37 &#176;C, if necessary, an environment chamber can be attached to the microscope to provide 5% CO2 and control the temperature at 37 &#176;C.
For membrane integrity determination of the airway epithelium, we quantified TEER using an EVOM2 ohm meter. We have not compared different vendors&#39; TEER determination machines to find an alternative to the EVOM2 ohm meter. Like CBF, TEER can be determined from 2 weeks of differentiation. A TEER greater than 500 ohm/cm2 is considered good for a healthy epithelium with tight junctions6,7. At least three TEER readings from the same insert should be taken for rigor and reproducibility. Care should be taken while taking the readings, and the epithelium should not be poked, which can damage it.
ALI is becoming a gold standard for studying respiratory infectious diseases. ALI-developed tissue-like pseudostratified airway epithelium enables us to create studies that examine cellular processes, disease patterns, infection research, and therapeutic development using primary epithelial cells derived from many donors with varying genetic backgrounds.

Chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis are some of the major health concerns worldwide1. The prevalence of respiratory diseases caused by viruses such as human influenza A virus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also cause economic and public health burden2. Therefore, there is an immense need to develop treatment regimens for respiratory illnesses that lead to irreversible respiratory tissue damage. The respiratory epithelial tissue itself is not only involved in oxygen uptake but also provides a barrier to protect the body from invading pathogens and hazardous chemicals3. The respiratory epithelium has a complex cellular organization composed of three major cell types: ciliated cells, goblet cells, and basal cells. Recently, there has been a report on the presence of novel but rare ionocyte cells in the airway epithelium4. The complex tissue functions in a coordinated fashion to provide innate immune responses such as secretion of antimicrobial peptides, cytokines release to activate an adaptive immune response, and mucociliary clearance5. The lack of a suitable airway model is one of the obstacles to the study of respiratory infections and the development of treatments.
The air-liquid interface (ALI) model is becoming a key tool for research on respiratory diseases6. It is an effective in vitro lung airway model as the primary lung airway cells differentiate into a pseudostratified airway epithelium composed of at least three types of cells that generally reside at the apical side of the airway. First, ciliated cells cover the majority of the apical side of the airway and contribute to mucociliary clearance. Second, goblet cells produce mucus and are the largest cells that co-reside with ciliary cells at the apical side of the airway. Third, basal cells reside at the basal layer of the airway and are the progenitor cells that differentiate into different epithelial cells5,7. Although ionocytes and tuft cells are rare in the airway epithelium, they may also play a role in ciliary function and membrane permeability4. This technique differs from the traditional submerged cell culture, which does not mimic the in vivo lung environment. Since ALI is produced from primary epithelial cells derived from healthy people, patients with asthma, and COPD, it offers a more diverse platform for studying responses to infection and disease pathophysiology.
The health assessment of the ALI-developed cultures is an essential aspect to monitor, ensuring the cultured cells&#39; viability, functionality, and physiological relevance. It enhances the confidence in the integrity, reliability, and reproducibility of ALI cultures. As previously published, our group has been evaluating the ALI culture integrity by assessing two biophysical parameters: ciliary function by quantifying ciliary beat frequency (CBF) and epithelial barrier integrity by determining transepithelial electrical resistance (TEER)6,7,8. Mucociliary clearance (MCC) is one of the important features of the airway epithelium carried by ciliated cells. Specialized organelles called cilia on the surface of ciliated cells beat in metachronal waves to clear the airways of infections and inhaled particles stuck in the mucous layer of the airway epithelium. Effective MCC is totally dependent upon proper ciliary activity9. A good CBF is indicative of a healthy, intact airway epithelium; hence, monitoring of the CBF for ALI cultures provides valuable insight into the epithelium&#39;s integrity10. Although there are multiple ways to quantify CBF, for example, high-speed video microscopy11 and phase-resolved Doppler optical coherence tomography12, each method needs technical expertise and specialized equipment. In this protocol, we provide an easier and less -technical way of quantifying CBF of the differentiated airway epithelium. We also described a method for quantifying TEER of the same epithelium, which indicates epithelial tissues&#39; integrity and barrier function13.

<table><tbody><tr><td>1x DPBS</td><td>Thermo Fisher Scientific, USA</td><td>14190-144</td><td></td></tr><tr><td>Airway Epithelial Cell Growth Medium</td><td>PromoCell, Germany</td><td>C-21060</td><td></td></tr><tr><td>Amphotericin B</td><td>Thermo Fisher Scientific, USA</td><td>15290-026</td><td></td></tr><tr><td>EVOM2 volt Ohmmeter</td><td>World Precision Instruments, USA</td><td>Not applicable</td><td></td></tr><tr><td>Heparin Solution</td><td>STEMCELL technologies, USA</td><td>07980</td><td></td></tr><tr><td>Hydrocortisone stock</td><td>STEMCELL technologies, USA</td><td>07926</td><td></td></tr><tr><td>Microscope: Leica DMI8 (Leica Microscope) with 120f/sec high-speed camera (Basler) associated with the microscope. Microsope also fixed with stage chamber incubator i8.</td><td>Leica, USA</td><td>Not applicable</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Corning, USA</td><td>30-002-CI</td><td></td></tr><tr><td>PneumaCult-ALI Basal Medium&amp;nbsp;</td><td>STEMCELL technologies, USA</td><td>05002</td><td></td></tr><tr><td>PureCol</td><td>Advanced BioMatrix, USA</td><td>5005</td><td></td></tr><tr><td>Sisson-Ammons video analysis (SAVA) system. Software V.2.1.15&amp;nbsp;</td><td>Amson Engineering, USA</td><td>Not applicable</td><td></td></tr></tbody></table>

determining ciliary function and membrane impermeability of the pseudostratified lung airway epithelium

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified airway epithelium that mimics the apical side of the lung airway. While the differentiation of primary lung airway cells is expected, the assessment of biophysical properties like ciliary function and membrane impermeability provides a quality assessment of the airway epithelium and ensures the reliability of the experiment. Here, we describe a straightforward protocol for the development of multi-cellular pseudostratified airway epithelium in ALI culture and assess two important biophysical properties: ciliary function and membrane impermeability. To determine ciliary function, we captured the ciliary movement of a 4-week differentiated airway epithelium using a high-speed camera attached to an inverted microscope, followed by quantifying ciliary beat frequency (CBF) using the Simon Amon Video Analysis (SAVA) system. We also measured transepithelial electrical resistance (TEER) using a volt-ohm meter to determine the epithelial barrier integrity of the airway epithelium.

We evaluated both ciliary function and membrane impermeability of the airway epithelium obtained from differentiating primary NHBE cells from two independent deidentified donors (two nonsmoking, healthy adults or adults with COPD, above 50-year-old females). For determining ciliary function, we determined the CBF of the 28-day differentiated airway epithelium. We observed CBF reached at least 3 Hz for both airway epithelium, which was comparable to the previously published results (Figure 1)6,7,14. We also evaluated membrane impermeability on the same airway epithelium by determining TEER. We found TEER around 800 ohm/cm2 for both airway epithelium, similar to previously published data (Figure 2)6,7,14. Thus, our study highlights the processes for evaluating optimal phenotypic and functional endpoints of the airway epithelium when developing an in vitro lung model.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67094/67094fig01.jpg" /> 
Figure 1: CBF of 4-week differentiated airway epithelium either from healthy adults or adults with COPD primary NHBE cells. The data were obtained by combining six independent readings (randomly). The error bar represents the standard error of the mean. The data represent at least two independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67094/67094fig01large.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/67094/67094fig02.jpg" /> 
Figure 2: TEER of 4-week differentiated airway epithelium either from healthy adults or adults with COPD primary NHBE cells. The background signal (obtained by using an empty insert with medium) was subtracted from all readings. The signal was then normalized for the insert&#39;s cell area, which is 0.33 cm2. The data were obtained by combining three independent readings (randomly). The error bar represents the standard error of the mean. The data represent at least two independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67094/67094fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Complete airway epithelial cell (cAEC) medium for cell growth</td>
		</tr>
		<tr>
			<td>Reagent&#160;</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Airway epithelial cell (AEC) growth Medium</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Growth medium supplement mix</td>
			<td>1 ready mix (12 mL)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>10 mL (2 % V/V)</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>5 ml (1 % V/V)</td>
		</tr>
		<tr>
			<td colspan="2">Complete PneumaCult-ALI basal medium for cell differentiation</td>
		</tr>
		<tr>
			<td>Reagent&#160;</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>PneumaCult ALI medium</td>
			<td>450 mL</td>
		</tr>
		<tr>
			<td>PneumaCult 10x medium supplement&#160;</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>PneumaCult-ALI maintenance supplement</td>
			<td>5 x 1 mL</td>
		</tr>
		<tr>
			<td>Heparin Solution</td>
			<td>1 mL&#160;</td>
		</tr>
		<tr>
			<td>Hydrocortisone Stock Solution</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>10 mL (2 % V/V)</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>5 mL (1 % V/V)</td>
		</tr>
	</tbody>
</table>
Table 1: Cell growth and differentiation media.

Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

Air-liquid interface culture is commonly used to develop pseudostratified airway epithelium by differentiating primary normal human bronchial epithelial cells that mimic the apical side of the lung airway. Here, we describe an easy protocol for determining its quality by monitoring its biophysical properties, such as ciliary function and membrane integrity.

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified ...

determining-ciliary-function-membrane-impermeability-pseudostratified

Nanyang Technological University

Research

JoVE Journal

Biology

431 Views.  University of North Dakota. Ons onderzoek heeft tot doel twee kritieke biofysische eigenschappen van pseudogestratificeerd luchtwegepitheel te meten, die kunnen worden verkregen door normale menselijke bronchiale epitheelcellen te differentiëren in een lucht-vloeistofinterface. We identificeerden verschillende kenmerken van verschillende respiratoire virussen door gebruik te maken van pseudogestratificeerd luchtwegepitheel. Bijvoorbeeld respiratoir syncytieel virusgedreven cytoskeletal...

Video: Bepaling van de ciliaire functie en membraanondoorlaatbaarheid van het pseudogestratificeerde longluchtwegepitheel

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Immunology and Infection

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Neuroscience

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (&gt;21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

We describe a protocol to establish an air-liquid interface (ALI) culture model utilizing neonatal tracheal airway epithelial cells (nTAEC) and perform physiologically relevant hyperoxia exposure to study the effect of atmospheric-induced oxidative stress on cells derived from the developing neonatal airway surface epithelium.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes ...

Developmental Biology

Airway Model for Studying Lung Barrier Integrity, Disease, and Drug Discovery

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

Author Spotlight: Modeling Human Airway Remodeling and Viral Responses Using Isogenic Epithelial, Endothelial, and Immune Cells

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...

Medicine

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

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