Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Summary

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.

Abstract

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.

Protocol

1. Preparing the biosafety cabinet for the EpiAirway tissues

1. Wearing a dedicated lab coat, with hair tied back and gloves on, start the laminar flow. After 5 minutes, move any tools in the cabinet (pipettors, tips, centrifuge tubes, etc.) to one side, spray the biosafety cabinet interior and sash with 70% ethanol and wipe down with clean paper towels. Spray the cleaned interior again with 70% ethanol and leave to dry. Move the tools to the clean side and repeat the ethanol procedure.
2. Use aerosol barrier pipette tips and dedicated pipettors in the cabinet. To use individually-wrapped sterile serological pipettes, pass the plugged end through the....

Discussion

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 airway^{5; 6; 7; 8}. We have maintained uni.......

Materials

List of materials used in this article

Name	Company	Catalog Number	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 Inc.	17-513Q
1 X Dulbecco’s phosphate-buffered saline without calcium or magnesium	Lonza Inc.	17-515Q
EpiAirway antibiotic-free tissues	MatTek Corp.	AIR-100-ABF
EpiAirway antibiotic-free maintenance media	MatTek Corp.	AIR-100-MM-ABF	Supplied with kit
10X phosphate-buffered saline solution	EMD Millipore	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 international	90002-016	Autoclave 36 g in 500 ml ddH2O and cool to 60°C
BBL Hemoglobin (chocolate agar)	VWR international	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 international	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 international	82027-406
Self-sealing sterilization pouches	VWR international	89140-802
Gentamicin sulfate, 10 mg/ml	Lonza Inc.	17-519Z	Add 10 microliters/ml to EpiAirway MM for the gentamicin kill