The authors wish to thank Elisabeth Blanchard for her technical assistance. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

1. Culturing Calu-3 Cells for Use in Transwell Cultures
Safety Measures: Perform all procedures in a biosafety cabinet using sterile culture technique.
<ol>
 <li>To thaw cells from frozen storage:
 <ol>
 <li>Prepare Eagle's Minimum Essential Medium containing 20% heat-inactivated fetal bovine serum (FBS), 0.1 mM non-essential amino acids, 2 mM L-glutamine, and10 mM HEPES pH 7.4 (EMEM-20%+S). Sterile-filter the prepared medium using a 0.2 &mu;m pore-size filter, and warm in a water bath to 37 &deg;C. To be more reflective of the in situ airway epithelium environment, medium is not supplemented with antibiotics or antimycotics.</li>
 <li>Thaw a frozen cryovial of Calu-3 cells rapidly (&lt; 1 min) in a 37 &deg;C water bath.</li>
 <li>Transfer the thawed cells to a 50 ml sterile conical tube and add 30 ml of warmed EMEM-20%+S.</li>
 <li>Pellet the cells by centrifugation for 5 min at 1,000-1,200 &times; g, without refrigeration, and with low brake speed.</li>
 <li>Decant the supernatant gently and resuspend the cells in 5 ml of warmed EMEM-20%+S by gently pipetting up and down. Seed 2 to 5 &times; 106 cells per well into a 6-well tissue culture plate and incubate at 37 &deg;C, 7% CO2 in air atmosphere until the cells are 75% - 80% confluent. Check daily; up to 7 days may be required. Every 3-4 days aspirate the medium from the cells and replace it with fresh EMEM-20%+S.</li>
 </ol>
 </li>
 <li>To subculture cells:
 <ol>
 <li>Warm 0.05% trypsin - 0.02% EDTA in a 37 &deg;C water bath. Aspirate the medium from the cells, and wash cells once with warmed 0.05% trypsin - 0.02% EDTA to remove excess medium and serum. Remove the wash and add 1 ml of warmed trypsin per well to the cells.</li>
 <li>Incubate at 37 &deg;C and 7% CO2 and monitor under a microscope every 5 min until at least 75% of cells detach from the well(s) of the tissue culture plate. This may take between 5 and 30 min.</li>
 <li>Wash cells in EMEM-20%+S to remove excess trypsin. Transfer the trypsinized cells to a 50 ml sterile conical tube. Multiple wells of cells from one 6-well culture plate may be pooled into one 50 ml sterile conical tube. Add 30 ml of EMEM-20%+S and pellet the cells by centrifugation at 1,000-1,200 &times; g, without refrigeration, and with low brake speed.</li>
 <li>Decant the supernatant gently and resuspend the cells in fresh EMEM-20%+S by gently pipetting up and down. Using steps 1.2.1 through 1.2.3 above to trypsinize cells, continue to subculture cells into tissue culture dishes as described in steps 1.2.5 through 1.2.7 when they are between 75 - 80% confluent.</li>
 <li>Subculture from one well of a 6-well plate up to one T-25 cm2 flask in a final volume of 5 ml of EMEM-20%+S, seeding between 0.5 to 1 &times; 106 cells/flask.</li>
 <li>Using 5 ml warmed trypsin per T-25 cm2 flask to detach cells, subculture from one T-25 cm2 flask up to one T-75 cm2 flask in a final volume of 10 ml of EMEM-20%+S, seeding between 1 to 5 &times; 106 cells/flask.</li>
 <li>Using 8 ml warmed trypsin per T-75 cm2 flask to detach cells, subculture from one T-75 cm2 flask into 2 T-75 cm2 flasks. For optimal cell growth, do not subculture cells into flasks larger than T-75 cm2 or subculture at a ratio greater than 1 parent flask:3 new flasks.</li>
 </ol>
 </li>
 <li>Once cells have been subcultured, completely remove and replace medium every 2 to 3 days until they reach 75-90% confluence. Cells may take up to 3 weeks to reach 75-90% confluence. For optimal generation of polarized cultures, subculture cells before they grow beyond 90% confluence as a monolayer.</li>
 <li>Continue to subculture cells until enough cells have grown to seed the desired number of Transwell inserts. Each Transwell insert requires 2 &times; 105 cells. For optimal performance generating polarized cultures, use cells that have undergone no more than 10 subcultures.</li>
</ol>
2. Growing Polarized Calu-3 Liquid-covered Cultures (LCC)
Safety Measures: Perform all procedures in a biosafety cabinet using sterile culture technique.
<ol>
 <li>Prepare EMEM containing 10% FBS (EMEM-10%) and warm in a water bath to 37 &deg;C.</li>
 <li>Prepare a 24-well Transwell plate for seeding. Using sterile forceps, move Transwell inserts from the interior to the exterior rows of the plate without touching the insert membrane. Cells should only be subcultured into wells on the exterior rows of the plate. To prevent the introduction of air bubbles into the basolateral compartments, add 600 &mu;l of EMEM-10% to each one by angling the pipette against the wall of each compartment and slowly releasing the medium into the well.</li>
 <li>Detach cells from T-75 cm2 tissue. Subculture flasks using warmed trypsin, and wash in 30 ml of EMEM-20%+S per every 2 flasks of trypsinized cells, as described in step 1.2.</li>
 <li>Thoroughly resuspend the washed cells in 5 ml of EMEM-10% by gently pipetting up and down.</li>
 <li>Determine viable and total cell counts by the trypan blue exclusion method. Proceed only if 80% - 90% are viable.</li>
 <li>In a 50 ml sterile conical tube, dilute cells to a concentration of 2 &times; 106 viable cells/ml with EMEM-10%.</li>
 <li>Add cells to the apical compartment of each Transwell. To wells A1 and D1, which will be cell-free control wells, that is, blanks, add 100 &mu;l of EMEM-10% without cells. To each of the remaining wells, add 100 &mu;l of resuspended Calu-3 cells, gently angling the pipette against the interior guide channel of the insert wall and slowly releasing the cells. Do not touch the pipette to the insert membrane. To maintain a homogeneous suspension of Calu-3 cells, gently agitate the cell suspension during this seeding step.</li>
 <li>Incubate Transwell plate at 37 &deg;C and 7% CO2 in air atmosphere. Place plate in an incubator where physical disturbance will be minimal. To improve the efficiency of polarization, do not stack plates on top of each other.</li>
 <li>To maintain cultures until polarized and ready to use in experiments, completely replace medium in Transwells, as described in steps 2.10 through 2.13 below. Medium should be completely replaced three days after being subcultured into Transwells, and then on a cycle alternating between the fourth and then third day after the previous feeding, until cells are fully polarized.</li>
 <li>Warm EMEM-10% in a water bath to 37 &deg;C.</li>
 <li>Gently aspirate medium from Transwell inserts. With a capillary pipette attached to a vacuum trap with a gentle vacuum, or with a standard 1 ml pipette, aspirate medium from cell-free wells A1 and D1, then from seeded wells, first removing apical medium from all wells, and then removing basolateral medium from all wells. Do not touch insert membranes while aspirating medium.</li>
 <li>Add 200 &mu;l of EMEM-10% to the apical compartment of cell-free wells A1 and D1, then to the seeded wells, directing the medium into the apical compartment using the side of the insert to guide the pipette tip. Do not add medium directly onto cells, and do not touch the insert membrane.</li>
 <li>Add 600 &mu;l of EMEM-10% to the basolateral compartments. To prevent the introduction of air bubbles into the basolateral compartments, add medium to each one by angling the pipette against the wall of each compartment and slowly releasing the medium into the well.</li>
</ol>
3. Evaluating Resistance Development of Calu-3 LCC
Safety Measures: Perform all procedures in a biosafety cabinet using sterile culture technique.
<ol>
 <li>Evaluate trans-epithelial electrical resistance (TEER) of Calu-3 LCC 30 min after medium changes; the evaluation can be performed after each medium change if desired. Perform measurements under sterile conditions. Begin measurements with the cell-free control wells A1 and D1 (the blanks) to obtain baseline measurements, and continue with measurements for each well.
 <ol>
 <li>Under sterile conditions, transfer the STX2 electrode to a 50 ml centrifuge tube containing 70% ethanol in sterile water, and sterilize 15 min.</li>
 <li>Calibrate and test the voltohmmeter for use according to manufacturer's directions.</li>
 <li>Remove the electrode from the ethanol, air dry 5-10 sec, and rinse the electrode with sterile EMEM-10%.</li>
 <li>Set the mode switch of the voltohmmeter to the RESISTANCE setting, and turn power ON.</li>
 <li>Gently place the electrode into one of the 3 ports that allow access into the basolateral compartment of one Transwell culture. Place electrode so that the longer lead just lightly touches the bottom of the outer well and remains vertical, and the shorter lead is in the tissue culture medium of the apical compartment, without touching the insert membrane.</li>
 <li>Push "Measure R" button, and wait for the reading to stabilize. Repeat for the other 2 ports for each well and record the measurements from all three ports, for a total of three measurements per well. Continue to measure the resistance for all wells of cells.</li>
 <li>Clean the electrode by soaking 5-10 min in 70% ethanol, rinsing in sterile dH2O, and drying thoroughly. Store in the original container.</li>
 <li>Calculate the resistance of each well, using Equation 1. 
 &Omega; actual = &Omega; sample - &Omega; blank , Equation 1 
 where &Omega; sample is the average measurement from a seeded well and &Omega; blank is the average measurement from the 2 wells, A1 and D1, containing inserts and medium, but no cells.</li>
 <li>Calculate the unit area resistance, using Equation 2. 
 &Omega; actual&times; effective membrane area = &Omega; &times; cm2, Equation 2 
 where the effective membrane area is 0.33 cm2 for 24-well Transwell inserts.</li>
 </ol>
 </li>
 <li>Verify that polarization is complete by performing a secondary assay, measuring passive sodium fluorescein diffusion between the apical and basolateral compartments, on one to three Transwell cultures. The wells used for the sodium fluorescein assay should be discarded immediately after completion of the assay.
 <ol>
 <li>Prepare non-fluorescent buffer (118 mM NaCl; 4.75 mM KCl; 2.53 mM CaCl2.2H2O; 2.44 mM MgSO4; 1.19 mM KH2PO4; 25 mM NaHCO3 in sterile water; sterile filter with 0.45 &mu;m filtration device). Prepare sodium fluorescein at 1 mg/ml in non-fluorescent buffer, sterile filter, wrap in aluminum foil, and store at 4 &deg;C, protected from light. Warm to room temperature before use.</li>
 <li>After completing resistance measurements, carefully remove medium from both the basolateral and apical compartments of one to three individual Transwell cultures.</li>
 <li>Rinse Transwell cultures. Gently add 600 &mu;l room-temperature sterile Dulbecco's PBS (D-PBS) to the basolateral compartments and 100 &mu;l room-temperature sterile D-PBS to the apical compartments.</li>
 <li>Gently remove D-PBS from wells. Add 600 &mu;l sterile non-fluorescent buffer to the basolateral compartments. Add 100 &mu;l sterile 1 mg/ml sodium fluorescein to the apical compartments.</li>
 <li>Incubate the plate at 37 &deg;C for 1 hr. CO2 is not required during this incubation.</li>
 <li>During incubation, prepare a sodium fluorescein standard curve ranging between 20 &mu;g/ml and 0 &mu;g/ml, using non-fluorescent buffer to dilute sodium fluorescein. Prepare at least 8 different concentrations of sodium fluorescein, each in a final volume of at least 600 &mu;l. Keep standard curve dilutions protected from light until ready to measure absorbance.</li>
 <li>Transfer the non-fluorescent buffer sample from the basolateral compartment of each test Transwell to a separate clean tube for analysis. Remove the test Transwells used to examine passive sodium fluorescein diffusion from the plate and discard. Return the plate with remaining Transwells to the incubator.</li>
 <li>Place 100 &mu;l of each standard solution in triplicate into a 96-well flat-bottomed plate.</li>
 <li>Prepare three dilutions of each basolateral sample (1:2, 1:20, and 1:50) in non-fluorescent buffer, and add 100 &mu;l of each undiluted sample and of each dilution, in duplicate, into a 96-well flat-bottom plate.</li>
 <li>Measure sample absorbance on an ELISA plate reader at 486 nm or 490 nm.</li>
 <li>Determine the concentration of sodium fluorescein in the basolateral compartment by comparing the absorbance of samples against the absorbance values for the sodium fluorescein standard curve.</li>
 </ol>
 </li>
</ol>
4. Infecting Polarized Calu-3 LCC with Respiratory Virus
Safety Measures: Perform all procedures in a biosafety cabinet using sterile culture technique, at a biosafety level appropriate for the virus being used.
<ol>
 <li>Dilute virus in serum-free EMEM so that desired inocula would be in 100 &mu;l.</li>
 <li>Wash cells in serum-free EMEM. Gently aspirate medium from all wells, removing the apical medium then the basolateral medium. With a capillary pipette attached to a vacuum trap with a gentle vacuum, or with a standard 1 ml pipette, aspirate medium from cell-free wells A1 and D1, then from seeded wells, first removing the apical medium from all wells, and then removing the basolateral medium from all wells. Do not touch insert membranes while aspirating medium.
 <ol>
 <li>Add 100 &mu;l of serum-free EMEM to the apical compartment of cell-free wells A1 and D1, and then to the seeded wells, directing medium into the apical compartment using the side of the insert to guide the pipette tip. Do not add medium directly on cells, and do not touch the insert membrane.</li>
 <li>Add 600 &mu;l of serum-free EMEM to the basolateral compartments. Add medium to each Transwell by angling the pipette against the wall of the compartment and slowly releasing the medium into the well.</li>
 <li>Gently aspirate serum-free medium from wells.</li>
 </ol>
 </li>
 <li>Beginning with uninfected or mock-infected wells, add appropriate virus dilutions to the apical compartment of Transwells. For uninfected wells, add 100 &mu;l of serum-free EMEM to the apical compartment of appropriate Transwells, for mock-infected wells, add 100 &mu;l of virus-free preparation diluted in serum-free EMEM to the apical compartment of appropriate Transwells, and for virus infection, dilute virus in serum-free EMEM and add 100 &mu;l to the apical compartments of appropriate Transwells.</li>
 <li>Add 600 &mu;l of serum-free EMEM to all basolateral compartments.</li>
 <li>Incubate at 37 &deg;C and 7% CO2 for 2 hr.</li>
 <li>Aspirate medium from wells, first from uninfected and mock-infected wells, then from infected wells. Remove apical supernatants first, followed by basolateral supernatants.</li>
 <li>Replace medium with 200 &mu;l of EMEM-10% in the apical compartments and 600 &mu;l of EMEM-10% in the basolateral compartments.</li>
</ol>

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No conflicts of interest declared.

When establishing Calu-3 LCC in Transwell inserts, cells may not polarize at all, or may not fully polarize, as defined by a TEER &ge; 1,000 &Omega;&times;cm2 and &le; 1% sodium fluorescein dye equilibration between the apical and basolateral compartments. In addition, Calu-3 cells in LCC may fully polarize, but TEER may be inconsistent between measurements. Although fluctuations in TEER measurements of Calu-3 LCC are normal from day to day, once fully polarized, dramatic swings in TEER are not expected until the culture naturally declines with age, which may be as little as 5 weeks or as long as 12 weeks after seeding.
The ability of Calu-3 LCC to polarize depends in part on how cells are maintained and subcultured before use in the Transwell system. Cells that have grown beyond 90% confluence as a monolayer during subculturing, that have been subcultured more than 10 times from frozen stock, or that have not been supplied with fresh medium on a regular schedule are less likely to fully polarize, and any polarization is likely to decline rapidly. Incomplete or total lack of polarization may also be attributed to variation in the material and pore size of Transwells used for Calu-3 LCC, and lot-to-lot variation in Transwells of similar composition and pore size may also affect polarization. Larger pore sizes can allow Calu-3 to grow through the Transwell membrane into the basolateral compartment, preventing the culture from polarizing. An absence of polarization may also be due to bacterial growth, indicated by clouded culture medium, which leads to subsequent breakdown of the tight junctions between Calu-3 cells.
Variable TEER measurements of Calu-3 LCC may be caused by mechanical disruptions of the Calu-3 LCC cell monolayers, the inserts, or the plates themselves. Medium changes and TEER measurements should be performed without pipette tips or electrode leads touching the cells. While performing these operations, care should be taken to avoid introducing air bubbles into the apical and basolateral compartments, which will disrupt the ability of the voltohmmeter to detect resistance. The ability of the voltohmmeter to detect resistance in a culture that is actually polarized may also limited by protein buildup on the electrode leads. This build-up may be removed with gentle sanding, or may be corrected by replacing the electrode.
Once Calu-3 LCC completely polarize and the TEER is no longer increasing, Calu-3 LCC are ready for use as an in vitro model for characterizing host lung epithelial cell responses to respiratory infection. This system permits better characterization of directional responses to pathogens compared to monolayer-cultured lung cell lines traditionally used to study respiratory pathogens, such as A549 and HEp-2 cells, with the additional advantages of Calu-3 LCC being more rapid to develop, more easily obtained, and less expensive to generate than primary, polarized, differentiated NHBE. Similar to NHBE, polarized Calu-3 demonstrate tight junction formation, and produce mucins. However, unlike NHBE, polarized Calu-3 cells do not differentiate into layers of basal cells and ciliated columnar epithelial cells, and few polarized Calu-3 cells develop cilia-like projections19. Thus, although useful for examining polarized responses of airway epithelial cells to respiratory insult, polarized Calu-3 LCC are not an ideal model to examine airway development or remodeling in response to respiratory insult or injury. Mucus production by cultured cells in vitro may impact cellular infectivity, as well as release of infectious virus and virus spread in a polarized model, and a direct comparison of the mucus production between polarized Calu-3 LCC and polarized, differentiated NHBE has not been reported. A549 and HEp-2 cells are easier to culture than Calu-3 cells, however, unlike Calu-3 LCC, they do not form polarized cultures when grown on Transwell inserts, and are thus not ideal models for examining in vitro the responses of polarized epithelial cells to respiratory virus infection.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Calu-3</td>
 <td>ATCC</td>
 <td>HTB-55</td>
 <td></td>
 </tr>
 <tr>
 <td>0.05% Trypsin - 0.02% EDTA</td>
 <td>Gibco/Invitrogen</td>
 <td>25300 </td>
 <td></td>
 </tr>
 <tr>
 <td>Eagle's Minimum Essential Medium (EMEM)</td>
 <td>Gibco/Invitrogen</td>
 <td>07-00100DK </td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal bovine serum (FBS)</td>
 <td>HyClone</td>
 <td>SH30070.03 </td>
 <td>Heat-inactivate, 56 &deg;C, 30 mins</td>
 </tr>
 <tr>
 <td>Non-essential amino acids 100X (10 mM)</td>
 <td>Gibco/Invitrogen</td>
 <td>11140 </td>
 <td>Store at 4 &deg;C in the dark</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco/Invitrogen</td>
 <td>25030 </td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Gibco/Invitrogen</td>
 <td>15630 </td>
 <td></td>
 </tr>
 <tr>
 <td>EMEM-10% FBS (EMEM-10%)</td>
 <td></td>
 <td></td>
 <td>Supplement EMEM with heat-inactivated FBS to 10% serum, sterile-filter</td>
 </tr>
 <tr>
 <td>EMEM-20% FBS + supplements (EMEM-20%+S)</td>
 <td></td>
 <td></td>
 <td>Supplement EMEM to final concentrations: heat-inactivated FBS, 20%; 1X amino acids; 2 mM L-glutamine; 10 mM HEPES; sterile-filter </td>
 </tr>
 <tr>
 <td>24-well Transwell plates</td>
 <td>Corning Costar</td>
 <td>3472 </td>
 <td>3 &mu;m pore size, polyester</td>
 </tr>
 <tr>
 <td>Trypan blue</td>
 <td>Gibco/Invitrogen</td>
 <td>15250 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma</td>
 <td>E7023 </td>
 <td>Prepare to 70% using sterile dH2O</td>
 </tr>
 <tr>
 <td>Dulbecco's PBS (D-PBS)</td>
 <td>Invitrogen</td>
 <td>14040 </td>
 <td></td>
 </tr>
 <tr>
 <td>Non-fluorescent buffer</td>
 <td></td>
 <td></td>
 <td>118 mM NaCl; 4.75 mM KCl; 2.53 mM CaCl2&times;H2O; 2.44 mM MgSO4; 1.19 mM KH2PO4; 25 mM NaHCO3 in sterile water; sterile-filter</td>
 </tr>
 <tr>
 <td>Sodium fluorescein</td>
 <td>Sigma</td>
 <td>6377 </td>
 <td>1 mg/ml in sterile non-fluorescent buffer; sterile-filter, protect from light; store at 4 &deg;C up to 6 months</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT </td>
 </tr>
 <tr>
 <td>Voltohmmeter</td>
 <td>World Precision Instruments</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>STX2 electrode</td>
 <td>World Precision Instruments</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA plate reader</td>
 <td></td>
 <td></td>
 <td>Capable of measuring A486 or A490</td>
 </tr>
 </tbody>
</table>

establishing a liquid-covered culture of polarized human airway epithelial calu-3 cells to study host cell response to respiratory pathogens in vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

When grown as liquid-covered cultures (LCC) in Transwell culture systems, as illustrated in Figure 1, Calu-3 cells polarize, developing distinct apical and basolateral surfaces. Following the method described here, the trans-epithelial electrical resistance (TEER) of Calu-3 LCC reaches a plateau at or above 1,000 &Omega;&times;cm2 within 3 weeks after seeding, an example of which is shown in Figure 2. The tight junctions formed between polarized cells prevent passive equilibration of small molecules between the apical and basolateral compartments. Thus, a modified sodium fluorescein equilibration assay is used to confirm polarization of Calu-3 LCC 15,17,18 . As the TEER of Calu-3 cell monolayers in LCC increases, the amount of fluorescein that passively equilibrates into the basolateral compartment decreases. Once the TEER is 1,000 &Omega;&times;cm2, the amount of fluorescein that equilibrates into the basolateral compartment is &le; 1%, as shown in Figure 3; therefore, Calu-3 LCC are considered to be fully polarized when the TEER is &ge; 1,000 &Omega;&times;cm2. The peak TEER measurement of Calu-3 LCC may vary from experiment to experiment. However, once TEER values plateau for any given experiment, a fully polarized, uninfected Calu-3 LCC may be stable for 5 through 12 weeks post-seeding. Absence of resistance development in Transwell-cultured Calu-3 may be caused by several factors as outlined in Table 1. Once the TEER of Calu-3 LCC plateaus at or above 1,000 &Omega;&times;cm2, the model is ready to be used to examine airway epithelial cell responses to respiratory pathogens, including respiratory syncytial virus (RSV). Exposure to RSV results in a more rapid decline in polarized culture integrity compared to a mock-infection of cells (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/50157/50157fig1.jpg" /> 
 Figure 1. Cross-section representation of Transwell-cultured cells. Cells are grown on the apical surface of the insert membrane.
<img alt="Figure 2" fo:content-width="4in" fo:src="/files/ftp_upload/50157/50157fig2highres.jpg" src="/files/ftp_upload/50157/50157fig2.jpg" /> 
 Figure 2. Development of trans-epithelial electrical resistance (TEER) and polarization of Calu-3 cells after seeding in Transwell inserts. At each time point, TEER is presented as median &Omega; x cm2 &plusmn; SEM of 32 independent wells from one representative experiment.
<img alt="Figure 3" fo:content-width="4in" fo:src="/files/ftp_upload/50157/50157fig3highres.jpg" src="/files/ftp_upload/50157/50157fig3.jpg" /> 
 Figure 3. Passive equilibration of sodium fluorescein into the basolateral compartment of Calu-3 cell monolayers is inhibited as cells become polarized. Cumulative data from four independent experiments is presented. Each data point represents an individual measurement.
<img alt="Figure 4" fo:content-width="4in" fo:src="/files/ftp_upload/50157/50157fig4highres.jpg" src="/files/ftp_upload/50157/50157fig4.jpg" /> 
 Figure 4. RSV infection of polarized Calu-3 LCC accelerates a decline in monolayer integrity. Polarized Calu-3 cells were infected with RSV-A2 at an MOI=1 on day 0, and polarity was monitored for 8 weeks post-infection. One representative experiment is shown. Data are presented as median resistance of 8 independent wells per infection &plusmn; SEM per time point. *p&lt;0.05 between mock- and RSV-A2-infected cultures, as determined by Student's t-test.
<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Issue</td>
 <td>Resolution(s)</td>
 </tr>
 <tr>
 <td>Resistance is not measurable</td>
 <td>
 <ul>
 <li>Remove any air bubbles in the wells</li>
 <li>Use Calu-3 cells that have been subcultured from frozen stock less than 10 times</li>
 <li>Do not allow Calu-3 cells to reach 100% confluence in monolayer subculture</li>
 <li>Check that cells are not growing on the plastic well below the insert (indicating that cells may have grown through the pores of the insert)</li>
 <li>Allow 2-3 weeks post-seeding for full resistance development</li>
 <li>Check the composition and pore size of inserts used, change if necessary (recommended polyester insert, 0.3 &mu;m pore size, no coatings, see materials for details)</li>
 <li>Check the quality of the electrode, sanding gently to remove accumulated proteins from the tip, and replacing if necessary</li>
 <li>Check medium for bacterial growth</li>
 </ul>
 </td>
 </tr>
 <tr>
 <td>Resistance develops, but full polarization (&ge;1,000 &Omega;&times;cm2) is not attained</td>
 <td>
 <ul>
 <li>Allow additional time for polarization to develop (may occasionally take up to 4 weeks)</li>
 <li>Use Calu-3 cells that have been subcultured from frozen stock less than 10 times</li>
 <li>Do not allow Calu-3 cells to reach 100% confluence in monolayer subculture</li>
 <li>Consistently replace medium in Transwell cultures, alternating between the third and then fourth day after previous feeding</li>
 <li>Only culture cells on inserts that are placed in the exterior rows of plates</li>
 <li>Use a different lot of Transwell inserts</li>
 </ul>
 </td>
 </tr>
 <tr>
 <td>Cells fully polarize, but resistance is not consistent from one reading to the next</td>
 <td>
 <ul>
 <li>Do not disrupt or stack plates in the incubator</li>
 <li>Do not cause vibration in the biosafety cabinet while Transwell plates are being handled</li>
 <li>Remove any air bubbles in the wells</li>
 <li>Do not disturb the cell layer with pipette tips when changing media or performing TEER readings</li>
 <li>Use 200 &mu;l medium in the apical compartment; lower volume may result in unstable TEER readings</li>
 <li>Check the quality of the electrode, sanding gently to remove accumulated proteins from the tip, and replace if necessary</li>
 </ul>
 </td>
 </tr>
 </tbody>
</table>
Table 1. Trouble-shooting problems that may arise when culturing Calu-3 cells in Transwells. Multiple factors contribute to full polarization of Calu-3 cells in liquid-covered cultures, the most likely of which are highlighted in this table.

Watch this Scientific Journal Video about Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro at JoVE.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

establishing-liquid-covered-culture-polarized-human-airway-epithelial

Research

JoVE Journal

Immunology and Infection

22.3K Views.  Centers for Disease Control and Prevention (CDC). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Video: Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...