This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.
Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection.
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.
1. Preparing the biosafety cabinet for the EpiAirway tissues
2. Unpacking the EpiAirway tissues
3. Maintenance of EpiAirway tissues
4. Inoculation of EpiAirway tissues
5. Quantification of the NTHi inoculum
6. Long-term co-culture with NTHi
7. Harvesting of EpiAirway tissues
8. Representative results:
Scanning electron micrographs of (A.) uninfected EpiAirway tissue and (B.) tissue after a 5-day co-culture with NTHi are shown in Figure 1. Long-term co-culture with NTHi does not result in significant damage to the apical tissues, underscoring the utility of the EpiAirway model. A graph depicting the number of internalized bacteria quantified over time inside tissues is shown in Figure 2. Both results are quite reproducible, making the EpiAirway tissues a consistent and biologically relevant in vitro model of the human upper airway in which to study NTHi host-pathogen interactions.
Figure 1. Scanning electron microscopy of EpiAirway tissues. A. Uninfected control tissue. B. Tissue after five days of co-culture with NTHi. No significant damage to the apical surface is observed in the infected tissues.
Figure 2. Number of NTHi internalized over time during EpiAirway co-culture. Strain R2866 was inoculated at approximately 1.0 x 107 CFU/insert (Day 0), then harvested for internalized bacteria at each indicated time point. Bars represent n=3 replicates in at least duplicate. Error bars are SD.
This method allows the investigation of long-term host-pathogen interactions in a biologically relevant background of primary human respiratory tissues at the air-liquid interface. Here we have used NTHi as the infecting organism, but the interaction of any bacterium that does not introduce unacceptable cytotoxicity over time can be quantified with this method. The EpiAirway model can also be used for the study of viruses, drugs, or chemicals that impact the human upper airway5; 6; 7; 8. We have maintained uninfected tissues for more than 40 days, and tissues infected with NTHi for at least 10 days. We expect that infected tissues could be maintained for significantly longer, if desired.
The limitations of this method are similar to that of any in vitro model, including the inability to reconstitute a competent immune system in these tissues. Although daily washes of the tissues are performed to mimic normal mucociliary clearance, establishing a natural outlet for the mucin produced in these tissues would be more desirable9. Further, contact with NTHi is known to induce mucin expression in human respiratory epithelial cells, exacerbating the problem10. On the other hand, the daily EpiAirway washes can be saved and assayed for proteins or enzymatic activities of interest, adding an important dimension to the method and increasing its usefulness.
One key step in the successful performance of this method is the mechanical disruption of the tissues prior to drop-plating the NTHi (step 7.10). Because the EpiAirways are highly differentiated and composed of multiple cell types, the tissues are not as easy to disintegrate as a cell monolayer, even after saponin treatment. In addition, NTHi are notoriously sensitive to compounds that aid in the disaggregation of tissues. Therefore, we have found that passing the cell suspension through a 26 gauge needle greatly improves our ability to generate consistent and repeatable quantification of internalized or cell-associated bacteria.
Once this technique is mastered, it can also be utilized to investigate the relative ability of mutant bacterial strains to survive as compared to their wild-type parents, allowing the investigator to characterize the effects of specific genetic mutations.
The authors have nothing to disclose.
We would like to thank Patrick Hayden (MatTek) for helpful discussions, and Robert Smith and Libby Perry of Georgia Health Sciences University for their EM skills. This study was funded by NIDCD grant DC010187 to D.A.D.
Name of reagent | Company | Catalogue# | Comments |
---|---|---|---|
Saponin | Calbiochem | 558255-25GM | 1% in D-PBS without calcium or magnesium, filter sterilize |
1 X Dulbecco’s phosphate-buffered saline with calcium and magnesium | Lonza | 17-513Q | |
1 X Dulbecco’s phosphate-buffered saline without calcium or magnesium | Lonza | 17-515Q | |
EpiAirway antibiotic-free tissues | MatTek | AIR-100-ABF | |
EpiAirway antibiotic-free maintenance media | MatTek | AIR-100-MM-ABF | Supplied with kit |
10X phosphate-buffered saline solution | EMD | 6506 | Dilute to 1X before use |
Gelatin | JT Baker | 2124-01 | Add to a final concentration of 0.1% in 1 X PBS and autoclave |
Difco GC Medium Base (chocolate agar) | VWR | 90002-016 | Autoclave 36 g in 500 ml ddH2O and cool to 60°C |
BBL Hemoglobin (chocolate agar) | VWR | 90000-662 | Autoclave 10 g in 500 ml ddH2O, cool to 60°C and mix with the GC medium base above |
BD BBL IsoVitaleX enrichment (chocolate agar) | VWR | 90000-414 | Cool the mixture of GC medium base and hemoglobin to 55°C and add 10 ml of rehydrated IsoVitaleX, pour chocolate agar plates |
Dissecting forceps, fine tip, curved | VWR | 82027-406 | |
Self-sealing sterilization pouches | VWR | 89140-802 | |
Gentamicin sulfate, 10 mg/ml | Lonza | 17-519Z | Add 10 microliters/ml to EpiAirway MM for the gentamicin kill |