The protocols described here address key questions such as replication kinetics, host cell tropism, innate immune induction, and cytotoxicity induction in the nasal epithelium during infections with human coronaviruses. This protocol enables the study of human coronavirus host interactions in an organoid microenvironment that closely recapitulates features of the in vivo nasal epithelium, including its heterogeneous cellular population and mucociliary functions. This technique can be applied to other ALI culture systems such as those derived from the lower airway.
Additionally, the system can be used to mimic clinical disease states like asthmatic or cystic fibrosis airways. Grow and differentiate the nasal air-liquid interface, or ALI cultures, in transwells having a basal and an apical chamber. Expand the dissociated human sinonasal cells under submerged condition, following which, remove the media from the apical chamber to allow differentiation of the cultures in an air-liquid interface.
On the day prior to infection, wash the ALI cultures apically with phosphate-buffered saline, or PBS. To do so, add 200 microliters of warmed PBS into the apical compartment of each transwell and incubate the plate at 37 degrees Celsius for five minutes. Aspirate the PBS and repeat the washing process twice.
Then, replace the basal medium with 500 microliters of fresh medium per well and equilibrate the culture overnight at the intended infection temperature. On the next day, dilute the virus in serum-free DMEM to achieve the desired multiplicity of infection in a total inoculum volume of 50 microliters. Add the viral inoculum apically to the culture and place it back in the incubator for one hour.
Every 15 minutes during incubation, gently rock the plate forward and backward as well as side to side by holding it firmly with both hands to ensure a uniform adsorption of the viral inoculum. After incubation, aspirate the viral inoculum and ensure its complete removal by washing each infected culture three times with PBS as described previously. If desired, collect the third PBS wash to confirm adequate removal of the input virus.
Replace the basal medium on the infected ALIs every 72 hours. At predetermined time points after the infection, add 200 microliters of PBS to the apical chamber of each infected transwell. Pipette the PBS up and down five times to ensure maximum apically-shed virus collection, and transfer the entire volume into a microcentrifuge tube to collect the apical surface liquid, or ASL sample.
Next, serially dilute the sample to quantify the concentration of viral particles in the ASL using a standard viral plaque assay. As outlined on the screen, select the cell type depending on the human coronavirus, or HCoV, being titrated. If desired, confirm the absence of basally-released virus by plaque assay of undiluted basal medium collected at various time points post-infection.
To measure TEER on nasal ALI cultures, clean, equilibrate, and blank the EVOM instrument per the manufacturer's instructions. Use an empty transwell with no nasal cells for blanking and record the blank measurement. To wash residual basal medium, transfer each infected transwell to a pre-labeled, clean, 24-well plate containing 500 microliters of PBS with calcium and magnesium in each well.
Then, add 200 microliters of PBS containing calcium and magnesium to the apical compartment of each transwell. Add one milliliter of PBS containing calcium and magnesium to the Endohm-6 measurement chamber. Place each transwell into the Endohm-6 measurement chamber using forceps and close the chamber by replacing the lid.
Ensure that the apical electrode is submerged in the 200 microliters of PBS in the apical compartment. The basal electrode remains at the bottom of the chamber. Once the EVOM reading stabilizes, record the raw TEER measurement.
If titering is desired, collect the ASL sample after TEER measurement. Convert the raw TEER readings to final measurements in ohms per centimeter squared using the equation shown on the screen. For human coronaviruses, or HCoVs, assess the TEER at 24-or 48-hour intervals post-infection.
Ensure to include a negative control at each time point. For the medium or background control, use PBS with the same composition as the one used for collecting the ASL sample. For the positive control, treat the control ALA cultures apically with 200 microliters of 2%Triton X-100.
After incubating for 10 to 15 minutes, collect the entire volume as a Triton sealing sample, following the plate layout consisting of Triton-positive control samples, mock background control samples, PBS control samples, and experimental samples. Load the lactate dehydrogenase, or LDH plate, in triplicate. Finally, calculate the percentage cytotoxicity relative to the Triton sealing value using the displayed equation, The average apically-shed viral titers from nasal ALI cultures infected with four HCoVs revealed that each of the viruses replicated productively, though SARS-CoV-2 and HCoV-229E replicated most efficiently.
MERS-CoV intracellular and apically-shed viral titers were approximately the same at 48 hours post-infection, or HPI. Co-staining of the infected cultures with antibodies specific to viral antigens and markers for ciliated or goblet cells showed that SARS-CoV-2 and HCoV-NL63 primarily infected ciliated cells, but MERS-CoV predominantly infected non-ciliated goblet cells. The difference in TEER from baseline to a given post-infection time point or delta-TEER for both SARS-CoV-2 and HCoV-NL63 infections were negative at 192 HPI, unlike MERS-CoV infection, which resulted in no significant change in TEER.
Negative delta-TEER values indicate damage to epithelial barrier integrity. HCoV-229E caused epithelial barrier dysfunction at an earlier time point of 96 HPI, but recovered to mock levels later in the infection. TEER traces depicting raw TEER data for each infected transwell over time allowed for the visualization of TEER trends over the course of infection.
Quantification of apically-released LDH during infection revealed that SARS-CoV-2, HCoV-NL63, and HCoV-229E caused significant cytotoxicity in nasal cultures, while MERS-CoV did not. It's crucial to understand viral replication kinetics in nasal cultures, as these determine the optimal time points for other downstream analysis such as cytotoxicity and innate immune induction. TEER measurements are most informative if taken sequentially on the same cultures, both prior to infection and then at various times post-infection in order to monitor changes in TEER over the course of infection.
Our current research applies RNA sequencing approaches to the described system to understand transcriptional changes during human coronavirus infections, as well as ELISA techniques to measure cytokine and other protein production.
