Human Bronchial Epithelial Cell Culture Models for Cigarette Smoke and Vaping Studies

Abstract

Despite the continuing public health efforts to stop or reduce smoking, cigarette smoke use remains popular in the youth and adult population. Recent surge in the use of electronic cigarette and vaping products has created another major health challenge in public health. There is an urgent need to use physiologically relevant models to study the health effect of smoking or vaping in human subjects. Airway diseases such as bronchitis [1-3] have been described in people who smoke, vape or both. Here, we will describe methods to collect, expand and culture human airway epithelial cells from endobronchial brushings, and expose these cells cultured at air-liquid interface to cigarette smoke or electronic cigarette vapor.

1. Introduction

The use of cigarette (tobacco) smoke and particularly electronic cigarette smoke is still on the rise. The impact of firsthand and secondhand exposures to smoke on public health is highly concerning. Not only the lung, but also other organs such as the cardiovascular system and brain are affected by smoke exposures [4-6]. It has been well established that cigarette smoke exposure is associated with the development of chronic pulmonary obstructive disease (COPD), exacerbations of asthma [7] and lung cancer. Vaping is also associated with asthma [8]. Recently, electronic cigarette (e-cig) or vaping product use-associated lung injury (EVALI) has been reported in the youth population. Clinically, EVALI manifests as an acute lung disease resembling viral illness. The lung pathological and radiological studies in EVALI patients show various lung lesions, including organizing pneumonia and diffuse alveolar damage[9]. The etiological studies of EVALI are still in its infancy. Although vaping of tetrahydrocannabinol (THC) and cannabidiol (CBD) mixture with nicotine has been considered as the major contributor to EVALI, recent discovery of vitamin E acetate (Vit-E Ac) in bronchoalveolar lavage (BAL) fluid of EVALI patients[10] suggests a new or an additional player in EVALI. This is not only because EVALI is relatively new to the scientific community, but also due to the lack of a model system that allows us to directly and robustly test the health effect of Vit-E Ac/THC containing vaping product on human lungs. Human airway epithelium represents the first line of host defense, and is the initial site exposed to cigarette smoke or e-cig. Airway epithelial cells consist of several major types of cells including ciliated cells, mucous goblet cells, secretory cells and basal cells. These cells use both mechanical (e.g. mucociliary escalator) and biochemical (e.g. antimicrobial substances) mechanisms to exert host defense functions. However, impaired immunity and excessive pro-inflammatory responses resulting from exposures to smoke in the context of respiratory bacterial or viral infections have been reported by using various cigarette smoke or e-cig exposure systems in human airway epithelial cells [11-15]. So far, there is no standardized or unified protocol for exposing cultured human airway epithelial cells to cigarette smoke or e-cig to study the detrimental health and biological effects. Here, we provide detailed protocols that have been briefly described in our publications [11-14] with some modifications.

2. Materials

2.1. Processing of airway epithelial cells from endobronchial brushings

2.1.1. Collection of Bronchial Epithelial Cells

1. Epithelial cell bronchial cytology brush for adults (Medical Engineering Laboratory)

2. Single-sheathed nylon cytology brush (Olympus, Tokyo, Japan)

3. 2 pairs of sterile forceps

4. Wire cutter

5. Phosphate buffered saline (PBS)

6. 15 ml conical centrifuge tubes

2.1.2. Processing bronchial epithelial cells

1. 100mm tissue culture dishes

2. PBS

3. 15 ml conical centrifuge tubes

4. Sterile transfer pipettes

5. Hemocytometer

6. Microcentrifuge tubes

7. 4°C centrifuge

8. Trypan blue

9. BronchiaLife medium: BronchiaLife™ Epithelial Airway Medium (Lifeline Cell Technology, Carlsbad, CA)

2.2. Culture and expansion of isolated brushed bronchial epithelial cells under submerged condition

1. 60 mm Corning® BioCoat™ Collagen I-coated culture dishes (Corning Life Sciences)

2. BronchiaLife medium: BronchiaLife™ Epithelial Airway Medium (Lifeline Cell Technology, Carlsbad, CA)

3. Trypsin-EDTA solution (Millipore Sigma)

4. Trypsin neutralizing solution (Millipore Sigma)

5. Sterile transfer pipettes

2.3. Air-liquid interface (ALI) culture of expanded brushed bronchial epithelial cells

1. Transwell Plates (Corning, 12 inserts, clear transwell membrane, 0.4μm pores, 1.12 cm² growth area)

2. Transwell Plates (Corning, 24 inserts, clear transwell membrane, 0.4μm pores, 0.33 cm² growth area)

3. Bovine collagen: PurCol Bovine Type I Collagen (3mg/ml, Inamed)

4. 1N NaOH

5. 12 well culture plates (Thermo Fisher Scientific)

6. 24 well culture plates (Thermo Fisher Scientific)

7. PneumaCult™-Ex Medium (STEMCELL Technologies)

8. PneumaCult™-ALI complete base medium (STEMCELL Technologies): Add 50 ml PneumaCult™-ALI 10X supplement to 450 ml PneumaCult™-ALI base medium.

9. PneumaCult™-ALI maintenance medium (STEMCELL Technologies): Combine 98.3 ml PneumaCult™-ALI complete base medium with 1 ml PneumaCult™-ALI maintenance supplement, 0.2 ml 0.2% heparin solution and 0.5 ml 0.2 mM hydrocortisone stock solution. All solutions are from STEMCELL Technologies.

2.4. Cigarette smoking exposure

1. MasterFlex L/S Economy Variable Speed Drive (Cole-Parmer)

2. Smoking chamber: British American Tobacco (BAT) exposure chamber (Curbridge Engineering, Hampshire, UK)

3. Fisherbrand™ Variable-Flow Peristaltic Pumps (Thermo Fisher Scientific)

4. Dulbecco's Modified Eagle Medium (DMEM)

5. Research cigarette 2R4F (University of Kentucky, Lexington, KY, USA)

6. Masterflex L/S® Precision pump tubing (Cole-Parmer)

2.5. Vaping protocol

1. MasterFlex L/S Economy Variable Speed Drive (Cole-Parmer)

2. British American Tobacco (BAT) exposure chamber

3. Fisherbrand™ Variable-Flow Peristaltic Pumps (Thermo Fisher Scientific)

4. E-liquid pods: JUUL Labs (www.juul.com) Virginia tobacco flavor at 3% (35 mg/ml) nicotine strength

5. JUUL USB charger: JUUL Labs (www.juul.com)

6. JUUL battery: JUUL Labs (www.juul.com)

7. Masterflex L/S® Precision pump tubing (Cole-Parmer)

2.6. Collection of airway epithelial cells at the end of the cell culture experiment

1. PBS

2. Microcentrifuge tubes

3. Forceps

4. Buffer RLT (Qiagen) for processing RNA

5. Radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) for processing protein

6. 10% formalin solution (Thermo Fisher Scientific)

3. Methods

The methods described below are based on our publications [11-14,16] and our recent optimization of the vaping protocol in human airway epithelial cells.

3.1. Isolation of human airway epithelial cells from endobronchial brushings

1. Endobronchial brushings (Note 1) are performed during bronchoscopy on the subsegmental bronchi of human subjects with the approval of Institutional Review Board (IRB).

2. Cut the brushes from the handle using a wire cutter and place the brushes (n = 1 to 6) into ice-cold PBS (10 ml) in a 50 ml conical microcentrifuge tube until time of processing.

3. Transfer the brushes, along with the PBS, in the 50 ml tube to a sterile 100 mm culture dish.

4. Using a sterile transfer pipet, gently rinse over the brush with 3 ml of PBS five to six times and transfer the washed content into the same 50 ml conical centrifuge tube as above.

5. Rinse the dish with 10 ml of fresh PBS and repeat an additional wash of the brush. Transfer the rinsed content into the same 50 ml conical centrifuge tube as above.

6. Centrifuge the cell collection tube at 590 x g for 10 minutes at 4°C.

7. Discard the supernatant and re-suspend the cell pellet gently in 2 ml BronchiaLife medium with a serological pipet.

8. Take 10 μl of the cell suspension and mix it with 10 μl of Trypan blue to determine total live cell count using a hemacytometer.

3.2. Expansion of human brushed bronchial epithelial cells

1. Seed 10⁵ live bronchial epithelial cells in 3 ml of the BronchiaLife medium per 60 mm collagen coated dish to expand the cells.

2. On day 3 of the culture, change the cell culture medium. Change the medium every other day or every 3 days if it falls into the weekend.

3. Expand the cells for up to 10 days or until 80% confluent, whichever comes first. Do not let the cells overgrow.

3.3. Air-liquid interface (ALI) culture of human bronchial epithelial cells

3.3.1. Trypsinization of cultured human bronchial epithelial cells

Once cells reach 80 to 90% confluency, it is time to pass the cells for ALI culture or for further expansion by trypsinization. Delay of passing the cells may result in cell senescence.

1. Pre-warm the frozen trypsin solution at 37°C for 30 minutes.

2. Remove the culture medium from the 60 mm culture dish.

3. Wash cells with pre-warmed (37°C) PBS once.

4. Remove the PBS and add 3 ml of trypsin to each 60 mm culture dish.

5. Incubate at 37°C for 2 to 5 minutes.

6. Monitor the detachment of cells every 30 seconds under an inverted microscope (Note 2).

7. Shake the cell culture dish and determine if about 80% of cells are detached.

8. Stop the trypsinization by adding 3 ml of trypsin neutralizing solution per dish.

9. Gently blow the remaining cells that are not detached with a transfer pipet.

10. Collect the detached cells in a 15 or 50 ml sterile conical centrifuge tube and spin at 230 x g for 10 minutes at 4°C.

11. After the centrifugation, discard supernatant and add 3 ml of ice cold BronchiaLife medium to resuspend the cell pellet.

12. Perform cell count using a hemacytometer to determine the total live cell count.

3.3.2. Collagen coating of transwell inserts

Please try to use freshly prepared transwell inserts for ALI culture, and do not use them if they were prepared more than 7 days ago. You can choose the 12-well or 24-well transwell inserts for your experiment depending on your cell need and budget.

1. Add 0.5 ml of sterile 10x PBS and 30 μl of 1N NaOH to 4 ml of bovine collagen (3 mg/ml) in a sterile 15 ml conical centrifuge tube on ice.

2. Gently mix with a serological pipette to avoid any bubbles.

3. Add a drop of the collagen mixture onto pH paper. The collagen mixture should be slightly acidic with pH around 6-7. If needed, adjust the pH by adding 1N HCL or 1N NaOH.

4. Add 160 μl of the collagen mixture to the center of each 12-well transwell insert or add 50 μl to the 24-well transwell insert.

5. In a small circular motion, shake the plate for ~10 seconds to spread the collagen evenly over the membrane.

6. Let the collagen soak into the transwell membrane for about 10 seconds with the plate cover off.

7. Tilt the plate and remove the floating collagen by pipetting.

8. With the plate lid off, let the collagen dry for 1 hour at room temperature under sterile cell culture hood.

9. Cover the plate after 1 hour and keep the transwells under hood until use.

3.3.3. ALI culture of human bronchial epithelial cells on collagen-coated transwell inserts

1. Warm refrigerated PneumaCult™-Ex Medium in a water bath at 37°C for 30 minutes before use.

2. Dilute the cell suspension from trypsinization with the appropriate volume of the PneumaCult™-Ex Medium to yield the final concentration of cells at 4x10⁴ in 300 μl per 12-well transwell insert or at 1.3x10⁴ in 100 μl per 24-well transwell insert.

3. Add 300 μl or 100 μl of cells to the apical side of each 12-well or 24-well transwell insert. Add 1.2 ml or 0.6 ml of the PneumaCult™-Ex Medium to the basolateral side of each 12-well or 24-well transwell.

4. Place the plates in an incubator set at 37°C, 5% CO₂.

5. Change medium at both apical and basolateral sides every other day or every 3 days (if it falls into the weekend) until the cells reach 100% confluence.

6. Use the PneumaCult™-ALI maintenance medium to start air-liquid interface culture by reducing the culture medium on the apical side of the transwell inserts to 50 μl (12-well plate) or 17 μl (24-well plate), while keeping the same volume (1.2 ml or 0.6 ml) on the basolateral side.

7. Change medium every other day or every 3 days (if it falls into the weekend), and remove the mucus after 1 week of culture (Note 3).

8. Let the cells culture at air-liquid interface for at least 21 days to induce full mucociliary differentiation (Figure 1).

Figure 1.

Human bronchial epithelial cells grown at the air-liquid interface culture for 21 days show mucociliary differentiation. H&E staining, magnification, X200.

3.4. Exposure of fully differentiated human bronchial epithelial cells to cigarette smoke

In the literature, both cigarette smoke extract (CSE) and whole cigarette smoke (WCS) have been used to study the health effect of cigarette smoke. Since WCS contains all phases (e.g. particulates, gas, and vapor) of cigarette smoke that smokers inhale, it is more physiologically relevant than CSE in the study of cigarette smoke health effects. Nonetheless, we provide methods here for both CSE and WCS preparation.

3.4.1. Preparation of cigarette smoke extract (CSE)

1. Cut a fresh piece of a large tube and a fresh piece of a small tube (MasterFlex L/S Precision Pump Tubing) for each experiment.

   1. Cut enough length for each tube to run through the clamps on the MasterFlex L/S Economy Variable Speed Drive and to have enough for the cigarette and media.
   2. Cut the end (about 5 mm) of a 1000 μl pipette tip
   3. Use the cut 1000 μl pipette tip to connect the small and the large tubes.
   4. Ensure that there is no leak of tube and pipette tip connections by sealing the connecting part using parafilm.

2. Insert one research cigarette 2R4F to the large tube.

3. In the fume hood, set up the system to generate CSE (Figure 2)

   1. Place the tubing under the clamp of the MasterFlex L/S Economy Variable Speed Drive.
   2. Push clamp down and turn knob to speed 2.
   3. Make sure that the small tube is all the way to the bottom of the 50 ml conical tube filled with 25 ml of warmed cell culture BronchiaLife medium and capped with parafilm.
   4. Make sure to use the air to test if media is bubbling before cigarette is lit.
   5. Turn machine on and light cigarette. Use a metal tweezer to pick up cigarette ash during the CSE preparation.

4. Once cigarette has burned out, use the tweezer to drop it into the 50 ml conical centrifuge tube with water.

5. Dispose the used tubes in a hazard bag in the hood and clean the pump with 70% ethanol.

6. Recap the smoked media, which are defined as 100% CSE.

7. Under the sterile cell culture hood, use a 5 ml syringe to pass the CSE solution through a 0.22μm filter to sterilize CSE for cell culture.

Figure 2.

Generation of cigarette smoke extract using the Master Flex L/S Economy Drive.

3.4.2. Exposure of human bronchial epithelial cells cultured at air-liquid interface to cigarette smoke extract

1. Use the PneumaCult-ALI maintenance medium to dilute 100% sterile CSE into desired concentrations (e.g. 1%, 5% and 10%).

2. After 21 days of ALI culture, wash the apical surface of epithelial cells with 300 μl (12-well transwell) or 100 μl (24-well transwell) of warm PBS twice to remove any mucus.

3. Add 100 μl (12-well transwell) or 50 μl (24-well transwell) of the CSE solution at desired concentration(s) to the apical surface of epithelial cells. Incubate cells with CSE solution for the duration of your study.

4. Refresh CSE treatment if the duration of the experiment is more than 48 hours.

3.4.3. Preparation of whole cigarette smoke

1. To prepare whole cigarette smoke, use the similar MasterFlex L/S Economy Variable Speed Drive set up as described for CSE preparation.

2. Connect the tube directly to a 1000 ml flask (Figure 3), and clamp the outlet tube.

3. Turn machine on and light cigarette until it burns out.

4. Clamp the entry of the tube into the flask so the smoke will be kept in the flask.

Figure 3.

Whole cigarette smoke exposure system for human bronchial epithelial cells grown at the air-liquid interface culture.

3.4.4. Exposure of human bronchial epithelial cells cultures at air-liquid interface to whole cigarette smoke

1. After 21 days of ALI culture wash the apical surface of epithelial cells with 300 μl (12-well transwell) or 100 μl (24-well transwell) of warm PBS twice to remove any mucus.

2. Add 50 μl (12-well plate) or 17 μl (24-well plate) of the PneumaCult-ALI maintenance medium to the apical surface of epithelial cells. Using forceps, place transwell inserts into a normal 12-well or 24-well plate with no media. The transwells will be subsequently transferred to the smoking chamber (British American Tobacco exposure chamber) – see Step 5.

3. Expose transwells to whole cigarette smoke or air as described in Steps 4 to 10 below. Treat “Air” samples first and then move to the whole cigarette smoke to avoid smoke contamination in the “Air” samples.

4. In the fume hood, set up the whole cigarette smoke exposure system (Figure 3).

   1. Fisherbrand Variable-Flow Peristaltic Pump is for circulating the media to reduce the accumulation of toxic substances (e.g. tar) at the bottom of transwells during smoke exposure.
   2. Set up media tubing with “in” tube on the bottom of the smoking chamber and “out” tube at the top off-set spot.
   3. Pour 50ml of warmed DMEM into the bottom of the smoking chamber.
   4. Make sure that the small hole where the media pump pokes down is lined up correctly with the marked spots (it will ruin a transwell if in the wrong spot).
   5. Thread the tubing through the opening making sure that both ends are within the black clamp holes at the bottom and close the lever on top.
   6. The right side goes into the center of the chamber set up, and the left side is left open for airflow and e-cig.

5. Place the transwells into the smoking chamber filled with 50 ml DMEM (Figure 4).

6. Close the top of the chamber tight.

7. Set the Fisherbrand Variable-Flow Peristaltic Pump to “prime” speed and “forward” to get the media into the tubing, then switch speed to “fast” when system is primed and leave on “forward” setting for the remainder of the treatment time.

8. For “Air” treatment, use the tubing designated for “Air Only” in the Masterflex L/S, and set the speed to 2 and expose the cells to air for 10 minutes.

9. To exposure the cells to whole cigarette smoke, remove the clamps from the tubes connecting the 1000 ml flask at the entry and the outlet.

10. Turn on the MasterFlex L/S Economy Variable Speed Drive, selecting speed 2 for 10 minutes to allow all the smoke stored in the flask to flow into the smoking chamber (Note 4).

11. After 10 minutes of smoke exposure, wash the apical surface with 150 μl (12-well transwell) or 50 μl (24-well transwell) warm PBS twice.

12. Add 50 μl (12-well plate) or 17 μl (24-well plate) of the PneumaCult-ALI maintenance medium back to the apical surface.

13. Move the transwell plates back to the incubator set at 37°C, 5% CO₂ to continue the air-liquid interface culture for the duration of your experiment.

14. To clean the equipment, push 100% ethanol through the media tubing and air pump tubing with the squirt bottle over the sink, then hang the tubing in the hood to dry overnight.

15. Remove the DMEM medium from the smoking chamber, and rinse it with diH₂O, spray with 100% ethanol and wipe down.

Figure 4.

Transwells with human bronchial epithelial cells grown at the air-liquid interface culture in the smoking chamber.

3.5. Exposure of human bronchial epithelial cells cultured at ALI to electronic cigarette (e-cig) vape

We adopted the whole cigarette smoke protocol to expose well-differentiated human bronchial epithelial cells to e-cig vape (Figure 5).

Figure 5.

Electronic cigarette (e-cig) exposure system for human bronchial epithelial cells grown at the air-liquid interface culture.

1. After 21 days of ALI culture, wash the apical surface of epithelial cells with 300 μl (12-well transwell) or 100 μl (24-well transwell) of warm PBS twice to remove any mucus.

2. Using forceps, place transwell inserts into a normal 12-well or 24-well plate with no media.

3. Expose transwells to e-cig vapor or air as described in Steps 4-9 below. Treat “Air” samples first and then move to the e-cig treatments to avoid e-cig contamination in the “Air” samples.

4. In the fume hood, set up the vaping system (Figure 5).

   1. Fisherbrand Variable-Flow Peristaltic Pump is for circulating the media in the smoking chamber to reduce the accumulation of any potential toxic substances at the bottom of the transwells.
   2. Set up media tubing with “in” tube on the bottom of the smoking chamber and “out” tube at the top off-set spot.
   3. Pour 50ml of warmed DMEM into the bottom of the smoking chamber.
   4. Make sure the small hole where the media pump pokes down is lined up correctly with the marked spots (it will ruin a transwell if in the wrong spot).
   5. Thread the tubing through the opening making sure that both ends are within the black clamp holes at the bottom and close the lever on top.
   6. The right side goes into the center of the chamber set up, and the left side is left open for airflow and e-cig.

5. Using forceps, place the transwells into the smoking chamber filled with 50 ml DMEM.

6. Close the top of the chamber tight.

7. Set the Fisherbrand Variable-Flow Peristaltic Pump to “prime” speed and “forward” to get the media into the tubing, then switch speed to “fast” when system is primed and leave on “forward” setting for the remainder of the treatment time.

8. For “Air” treatment, use the tubing designated for “Air Only” in the Masterflex L/S, and set the speed to 10 and expose the cells to air for 16 minutes.

9. For vaping (e-cig treatment), use the tubing designated for “E-vape Only” in the Masterflex L/S, insert the e-cig (e.g. JUUL battery plus e-liquid pod) into the tube, and set the speed to 10.

   1. Expose the cells to e-vape for 15 times with 4 seconds puffs at a flow rate of ~8.75 ml/second with 56 sec rest (Note 5 and Note 6).

10. When done with treatments, turn off the pumps and remove transwells and place them back into the 12-well or 24-well transfer plate using forceps.

11. Move transwell inserts back into the ALI culture media in a 12- or 24-well plate.

12. Add 50 μl (12-well plate) or 17 μl (24-well plate) of the PneumaCult-ALI maintenance medium back to the apical surface.

13. Move the transwell plates back to the incubator set at 37°C, 5% CO₂ to continue the ALI culture for the duration of your experiment.

14. To clean the equipment, push 100% ethanol through the media tubing and air pump tubing with the squirt bottle over the sink, then hang the tubing in the hood to dry overnight.

15. Remove the DMEM medium from the smoking chamber, and rinse it with diH₂O, spray with 100% ethanol and wipe down.

3.6. Collection of human bronchial epithelial cells cultured at ALI for outcome analysis

1. To collect apical surface components, add 300 μl (12-well plate) or 100 μl (24-well plate) of warmed 1xPBS to the apical surface, and incubate at 37°C for 10 minutes.

2. Rinse the apical surface thoroughly to remove any components including mucus, place the supernatants in a 1.5 ml tube and store the tube at −80°C.

3. Collect the basolateral supernatants in a 1.5 ml tube and store the tube at −80°C.

4. Use a surgical scalpel to remove the transwell membrane that contains cells.

5. Cells for RNA and western blot work:

   1. Cut the transwell membrane into two halves.
   2. One half will be placed into a 1.5 ml tube filled with buffer RLT for extracting RNA.
   3. The other half of the transwell membrane will be placed into a tube filled with RIPA buffer for processing the protein for western blot.
6. Cells for histology or immunofluorescent staining:

   1. Use the entire or half of the transwell membrane depending on your experimental design.
   2. Place the transwell membrane into a 12-well or 24-well plate filled with 10% formalin to fix the cells.
   3. Perform the immunofluorescent staining using the transwell membrane or send it to your histology lab to perform tissue embedding in paraffin for further tissue cutting and staining.

4. Notes

1. Cell yield from a brush varies. Generally, up to 10⁵ of cells can be obtained using a small brush (e.g. single-sheathed nylon cytology brush. Cell yield is higher (up to 5x10⁵ cells/brush) if the big brush (e.g. the one from Medical Engineering Laboratory) is used.

2. Occasionally agitate or turning the dish will help identify detaching cells.

3. To remove mucus from the apical surface of airway epithelial cells, add 300 μl (12-well plate) or 100 μl (24-well plate) of warmed PBS to the apical surface. Incubate at 37°C, 5% CO₂ for 10 minutes to help dissolve the mucus. Gently wash the apical surface with the PBS to remove excess mucus, after which continue with the media change as stated.

4. We measured the nicotine concentration in the apical surface of airway epithelial cells using the gas chromatography-mass spectrometry. Our whole cigarette smoke system for cell cultures mimics human smokers' airway milieu as nicotine levels (12.5±0.4 μg/ml) in apical supernatants are similar to those in epithelial lining fluid (6 - 52 μg/ml) of human smokers.

5. Based on ISO standard for conventional cigarette smoking which is a 35ml puff volume, 1x 2 sec puff every 60sec, and a flow rate of 17.5 ml/sec. Our pump won’t do a 17.5 ml/sec puff, so we opted for half the flow rate for twice the time. Our flow rate was estimated by the upside-down cylinder in bucket of water displacement method.

6. Nicotine concentrations in the apical media of e-vapor exposed airway epithelial cells were measured in a range of 0.86-1.79 μg/ml 24 hours after one exposure to e-vapor. This concentration is relevant to human vapers based on the serum nicotine concentrations, which are about 1000 times lower than those in the airway epithelial lining fluid.