Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions

The use of nasal specimens was approved by the University of Pennsylvania Institutional Review Board (protocol # 800614) and the Philadelphia VA Institutional Review Board (protocol # 00781).

1. Infection of nasal ALI cultures

NOTE: Acquisition of clinical specimens, as well as growth and differentiation of nasal ALI cultures, is outside the scope of this paper. Specific methods for culturing primary nasal epithelial cells can be found in recently published works utilizing these cultures^18,22,23. The below protocols can additionally be applied to commercially available nasal epithelial ALI cultures if desired. Protocols and volumes detailed below are applicable to 24-well plate transwell inserts (6.5 mm diameter, 0.33 cm² membrane surface area). If using ALI cultures grown on larger transwells (i.e., 12-well plates, 12 mm diameter, 1.12 cm² surface area), adjust the volumes proportionally to reflect the transwell size.

1. Day before infection:
   1. Wash ALI cultures 3x with phosphate-buffered saline (PBS) apically (add ~200 μL of warmed PBS, place in 37 °C incubator for 5 min, aspirate PBS, and repeat).
   2. Replace basal medium (500 μL).
   3. Allow cultures to equilibrate at the temperature at which infections will be conducted overnight (i.e., if infecting at 33 °C, place cultures in a 33 °C incubator after PBS washes).
      NOTE: HCoVs associated with the common cold such as HCoV-229E and HCoV-NL63 are reported to replicate more efficiently at 33 °C. Additionally, the temperature of the in vivo nasal epithelium is 33 °C (this differs from the temperature of the lung, which is 37 °C).
2. Dilute virus as needed in serum-free Dulbecco's Modified Eagle Medium (DMEM) to achieve the desired multiplicity of infection (MOI) in a total inoculum volume of 50 μL.
   NOTE: Infections have typically been conducted at MOI = 5 (high MOI); however, infections at MOI = 0.5 (low MOI) have also been used for SARS-CoV-2 and common cold-associated HCoVs, and these result in comparable peak viral titers, but with variable kinetics (either MOI is acceptable).
3. Add inoculum apically, and place cultures back into the incubator for 1 h.
4. Rock plates gently every 15 min during infection (holding plate firmly in both hands, rock forward and backward and side to side to ensure uniform adsorption of viral inoculum).
5. After 1 h of incubation, aspirate viral inoculum, and wash each infected culture 3x with PBS to ensure removal of viral inoculum (for each wash, add 200 μL of PBS, incubate for 5 min, and aspirate or remove with a pipette).
6. If desired, collect the third PBS wash to confirm adequate removal of input virus.
7. Replace the basal medium with fresh medium on infected ALIs every 72 h during infection.

2. Collection of apical surface liquid (ASL) and titration of shed virus

1. At predetermined time points following infection, add 200 μL of PBS to apical chamber of each infected transwell.
   NOTE: Relevant time points vary depending on HCoV of interest and range from 24 h to 192 h post infection (see the representative results section for viral replication data for various HCoVs).
2. Pipette PBS up and down 5x to ensure maximal collection of apically shed virus, and collect entire volume into a microcentrifuge tube (this is the ASL sample).
   NOTE: ASL includes shed viral particles in addition to mucus and other products secreted apically from ALI cultures.
3. Quantify infectious virus in ASL via standard viral plaque assay (serially dilute virus-containing samples in order to quantify the concentration of viral particles).
   NOTE: Cell types and incubation period used for plaque assay will depend on the virus used: SARS-CoV-2 (VeroE6 cells); MERS-CoV (VeroCCL81 cells); HCoV-NL63 (LLC-MK2 cells); HCoV-229E (Huh7 cells). Specifics on how to conduct viral plaque assays are beyond the scope of this manuscript but have been detailed previously in a Journal of Visualized Experiments (JoVE) publication²⁴.
4. If desired, collect the basal medium at various times post-infection to confirm the absence of basally released virus. HCoVs are typically released apically from nasal epithelial cells, but confirm this via plaque assay of undiluted basal medium.
5. Store ASL samples at −80 °C if quantification by plaque assay will not occur on the day of collection.

3. Quantification of intracellular virus

1. After collecting ASL sample, move each transwell into a clean 24-well plate preloaded with 500 μL of DMEM containing 2% fetal bovine serum (FBS) basally.
   NOTE: DMEM with 2% FBS is used to stabilize the virus during subsequent freeze-thaw cycles.
2. Wash each transwell 3x apically with PBS to ensure complete removal of apically shed virus.
3. After aspirating to remove the final PBS wash, add 100 μL of DMEM with 2% FBS to the apical compartment.
4. Move the plate containing transwells with both apical and basal media into a −80 °C freezer, and complete three consecutive freeze-thaw cycles to lyse the cells.
5. After the final freeze-thaw cycle, pool apical (100 μL) and basal (500 μL) media into a clean tube.
6. Centrifuge at 500 × g for 10 min at 4 °C to pellet any cellular debris.
7. Collect the supernatant. This is the intracellular virus sample for titration via standard plaque assay.
   NOTE: Three-fold dilution occurs during the collection process relative to ASL collection; ASL samples are collected in 200 μL of PBS, while intracellular virus sample is collected into a total volume of 600 μL.

4. Transepithelial electrical resistance (TEER) measurement

NOTE: For TEER measurement, PBS supplemented with calcium and magnesium (PBS + Ca²⁺/Mg²⁺) should be used. An epithelial volt/ohmmeter set to read in ohms is used (see the Table of Materials).

1. Clean, equilibrate, and blank the EVOM instrument according to the manufacturer's instructions; use an "empty" transwell with no nasal cells added for blanking. Record blank TEER measurement.
   NOTE: If using multiple viruses, the EVOM instrument must be cleaned stringently between conditions to avoid cross-contamination (washes with 70% ethanol followed by deionized water are sufficient).
2. Move each infected transwell into a prelabeled, clean 24-well plate with 500 μL of PBS + Ca²⁺/Mg²⁺ basally to wash residual basal medium from the transwells.
3. Add 200 μL of PBS + Ca²⁺/Mg²⁺ to the apical compartment of each transwell.
4. Add 1 mL of PBS + Ca²⁺/Mg²⁺ to the Endohm-6 measurement chamber.
5. Move each transwell into the Endohm-6 measurement chamber, and replace the lid of the chamber so that the apical electrode rests in the 200 μL of PBS in the apical compartment; the basal electrode is built into the bottom of the Endohm-6 chamber.
6. Allow the EVOM reading to stabilize, and record raw TEER measurement.
7. Collect ASL sample as described above after taking TEER measurement if titering is desired (collect in the 200 μL of PBS + Ca²⁺/Mg²⁺ that was added for TEER measurement).
   NOTE: ASL sample collection can induce micro-tears in the epithelial barrier that may confound TEER readings, so ASL must be collected after TEER measurement. Further, basal medium must not be changed immediately before TEER readings as this may also impact TEER values.
8. To convert raw TEER readings to final measurements in Ohms/cm², subtract blank TEER value, and multiply this value by the surface area of the transwell membrane using equation (1):
   TEER = [TEER reading – blank TEER value] × (surface area of transwell)    (1)
9. For HCoVs, evaluate TEER every 24 h or 48 h following infection; the kinetics of TEER changes often vary among viruses and necessitate troubleshooting at various time points.
10. When measuring TEER, always include mock-infected cultures and evaluate at each time point (negative control).
    NOTE: For mock cultures, TEER measurements should remain stable or may slightly increase from baseline after differentiation of the cultures is completed. Surface area of membrane supports will vary depending on size and manufacturer of transwells; 24-well transwells typically have a surface area of 0.33 cm².

5. Measurement of cytotoxicity during infection via lactate dehydrogenase (LDH) assay

NOTE: In this work, LDH content in ASL samples was quantified using a commercially available cytotoxicity detection kit. LDH signal in basal medium was often below the limit of detection and often less reproducible than LDH quantified in ASL samples from HCoV-infected cultures.

1. Prepare additional controls necessary for LDH assay.
   1. Background control: use PBS (use PBS containing calcium and magnesium if ASL samples were collected in this way).
   2. Positive control (ceiling value): treat ALIs apically with Triton X-100. Collect Triton control wells (typically use three ALI cultures for this at each time point).
      1. Add 200 μL of 2% Triton X-100 in PBS directly to the apical compartment of the transwell.
      2. Incubate for 10-15 min to allow cells to completely lyse.
      3. Collect the entire volume as a Triton ceiling sample.
   3. Negative control (low control/baseline LDH release): collect ASL from mock-infected ALI cultures derived from the same donor as infected cultures.
      1. Collect negative control mock ASL following ASL collection procedure above.
         NOTE: The same transwells can be used for this negative control for all time points, whereas fresh transwells will be necessary for each time point for the Triton control.
2. To allow for quantification of shed virus in addition to LDH readings from each ASL sample, load an optically clear flat-bottom black 96-well plate as follows:
   1. Dilute all samples in PBS, using 45 μL of sample and 55 μL of PBS (100 μL total volume).
   2. Treat Triton positive control and mock background control samples in the same manner.
      NOTE: This dilution allows for triplicate loading of each experimental sample (45 μL x 3) and enough residual ASL volume for titering via plaque assay.
   3. Load LDH plate in triplicate: Triton ceiling, mock background control, PBS control, and experimental samples.
   4. Prepare the reaction mixture as indicated by the manufacturer (dye solution + catalyst).
      1. Add 100 μL of reaction mixture to each well, and incubate the plate at room temperature for 20 min protected from light.
   5. After 20 min, measure absorbance at 492 nm.
   6. Calculate percentage cytotoxicity relative to the Triton ceiling value using equation (2).
      Cytotoxicity (%)      (2)

6. Preparation of nasal ALI cultures for immunofluorescence (IF) imaging

1. Move the transwell into a fresh 24-well plate, and wash 3x apically with PBS (collect the first PBS wash as ASL if titering; then, perform two additional PBS washes to remove excess shed virus that might hinder IF quality)
2. After the final PBS wash, cover the transwell apically and basally in 4% PFA.
3. Incubate for 30 min to fix in 4% PFA, and then remove and wash 3x with PBS.
4. Excise the transwell support containing the cells using a sharpened razor blade or scissors.
5. To increase the number of IF targets for each transwell, cut each membrane in half, and stain each half with different antibody combinations.
6. Permeabilize with 0.2% Triton X-100 in PBS for 10 min.
7. Block with 10% normal donkey serum and 1% BSA in PBST (PBS + 0.2% Triton X-100) for 60 min at room temperature.
   NOTE: Protect samples from light exposure from this step forward.
8. Incubate in primary antibody solution overnight at 4 °C. Dilute all antibodies 1:1,000 in blocking buffer.
   NOTE: Representative antibodies for HCoV nucleocapsid staining, as well as epithelial cell type markers and cytoskeletal stains, are listed below (see the Table of Materials for manufacturer information and catalog numbers).
   1. For HCoV antigen staining, use antibodies targeting SARS-CoV-2 nucleocapsid, MERS-CoV nucleocapsid, and HCoV-NL63 nucleocapsid.
   2. To identify epithelial cell types, use antibodies targeting goblet cell marker MUC5AC and ciliated cell marker type IV β-tubulin.
   3. For cytoskeletal markers, use antibodies directed against phalloidin (binds F-actin) and epithelial cell adhesion marker: EpCAM (CD326).
9. Incubate in secondary antibody solution for 60 min at room temperature; use secondary antibody dyes diluted 1:1,000 in blocking buffer.
10. After staining is complete, transfer the membrane onto a glass slide with a spatula, orient the transwell with the apical side toward the slide, and add mounting solution. Allow to settle for 15-30 min prior to applying clear nail polish around edges.
11. Acquire images using a confocal microscope (Z-axis step: 0.5 μm; sequential scanning)^1,16,17.
    ​NOTE: After fixation of cultures in 4% PFA and washing with PBS, fixed cultures can be stored at 4 °C for weeks to months prior to staining and preparation for imaging. After staining and mounting of membranes, samples can be stored long-term (>2 years) at 4 °C in the dark.

7. Collection of intracellular protein for western immunoblotting or RNA for RT-qPCR analysis

1. Collect ASL as described above if quantifying viral titers (protocol section 2).
2. Move the transwell to a clean 24-well plate, as scraping the membrane can lead to breaking the insert.
3. For western blot analysis, collect total protein lysate in 125 μL of RIPA buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 1% NP40) supplemented with protease inhibitors and phosphatase inhibitors.
4. Add 125 μL of RIPA buffer to the apical compartment, and incubate for 5-10 min.
5. Scrape the membrane using a P200 pipette tip to remove any remaining attached cells, and collect the entire volume (scrape vigorously across the entire surface of the membrane, and then pipette up and down multiple times to collect the entire sample).
6. Incubate protein lysate samples on ice for 10 min, and then centrifuge at maximum speed (20,000 × g) for 10 min at 4 °C.
7. Mix the supernatant with 4x Laemmli sample buffer with β-mercaptoethanol (reducing agent) according to manufacturer protocols.
8. Boil protein samples at 95 °C for 5 min, and then run using traditional western blotting protocols^1,16,17.
9. For collection of total RNA, use a commercially available RNA extraction kit of choice.
   NOTE: The apical compartment of 24-well transwell inserts has a maximal volume of ~200 μL; thus, we perform two sequential washes on each culture to reach total recommended volume. Details below correspond to recommended collection of RNA samples in a 350 μL total volume.
10. Add 200 μL of lysis buffer to the apical compartment of the infected transwell.
11. Let sit for 5-10 min, scrape any remaining cells from the membrane using a pipette tip, and collect the entire volume into a labeled microcentrifuge tube.
12. Add 150 μL of additional lysis buffer to the apical compartment of the transwell, and pipette up and down before collecting into same microcentrifuge tube.
13. Extract RNA according to the manufacturer's protocol.