Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Summary

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

Abstract

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.

Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

Introduction

Inhalation of toxic airborne particles is a public health concern, leading to a multitude of health risks worldwide and many millions of deaths annually^1,2. Climate change, the ongoing industrial development and the rising demand for energy, agricultural and consumer products have contributed to the increase of pulmonary diseases over the last years^3,4,5,6. Knowledge and evaluation of inhalable substances regarding their acute inhalation toxicity provide the basis for hazard assessment and risk management, but this information is still lacking for a wide range of these substances^7,8. Since 2006, the EU chemical legislation REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) requires that already existing and newly introduced products undergo a toxicological characterization including the inhalation route before being placed on the market. Therefore, REACH focuses on alternative and animal-free methods, the implementation of the "3R" principle (Replacement, Refinement, and Reduction of animal experiments) and the use of appropriate in vitro models⁹. In recent years, many different and adequate non-animal inhalation toxicity testing models (e.g., in vitro cell cultures, lung-on-a-chip models, precision cut lung slices (PCLS)) have been developed in order to assess the acute inhalation toxicity of airborne particles^5,7,10,11. In terms of in vitro cell culture models, cultivated cells can be exposed under submerged conditions or at the ALI (Figure 1). However, the validity of submerged exposure studies is limited with regard to the evaluation of the toxicity of airborne compounds especially particles. Submerged exposure techniques do not correspond to the human in vivo situation; the cell culture medium covering the cells may affect the physico-chemical properties and thus, the toxic properties of a test substance^12,13. ALI in vitro inhalation models allow the direct exposure of cells to the test substances without interference of the cell culture medium with test particles, thus, mimicking human exposure with higher physiological and biological similarity than submerged exposures^12,14.

For regulatory processes such as REACH, however, only animal models are available in the field of acute inhalation toxicology, as no alternative in vitro methods have been sufficiently validated and officially accepted so far¹⁴. For this purpose, test models have to be validated according to the requirements of the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM) principles on test validity¹⁵.

A former pre-validation study and a recently completed validation study successfully demonstrated the application area of the CULTEX RFS exposure system and its transferability, stability, and reproducibility¹³. This exposure system is an in vitro cell-based exposure system that enables homogenous exposure of cells to gases, particles or complex atmospheres (e.g., cigarette smoke) at the ALI due to its radial aerosol distribution concept and the conduction of the test aerosol in a continuous flow over the cells¹⁶. The basic module of this radial flow system consists of the inlet adapter, the aerosol guiding module with a radial aerosol distribution, the sampling and socket module, and a locking module with a hand wheel (Figure 2). The generated particles reach the cells via the inlet adapter and the aerosol guiding module and are deposited on the cell culture inserts, which are located in the three radially arranged exposure chambers of the sampling module. The aerosol guiding module as well as the sampling module can be heated by connecting to an external water bath¹⁷.

Within the framework of both studies, A549 cells were used for all exposure experiments. The cell line A549 is a human immortalized epithelial cell line that is very well-characterized and has been used as an in vitro model for type II alveolar epithelial cells in numerous toxicological studies. The cells are characterized by lamellar bodies, the production of surfactant and a number of inflammation-relevant factors¹⁸. They also show properties of bronchial epithelial cells due to their mucus production¹⁹. Moreover, they can be cultured at the ALI. Although this cell line is deficient in building cell-cell contacts, the cultivation of these cells is much more convenient, less cost expensive and results derived thereof are donor-independent compared to primary cells²⁰.

A549 cells were seeded in 6-well cell culture inserts (PET membrane, 4.67 cm², pore size 0.4 mm) with a density of 3.0 x 10⁵ cells per insert and cultivated for 24 h under submerged conditions. Cells were then exposed in three independent laboratories to clean air and three different exposure doses (25, 50, and 100 µg/cm²) of 20 test substances at the ALI. The exposure dose is correlated to the deposition time resulting in a constant particle rate of 25 µg/cm², 50 µg/cm² and 100 µg/cm² onto the cells after 15, 30 or 60 min, respectively. The deposited particles, however, were not washed off after deposition, but remained on the cells for 24 h. The deposition times of the particles were therefore 15, 30 and 60 min, but the exposure of the cells lasted for 24 h in total. The deposition rate of the test substances was determined in preliminary experiments according to previous methods¹⁷.

Cell viability as an indicator of toxicity was assessed 24 h after particle deposition using a cell viability assay. Special focus was set on the quality of clean air controls, the optimization and refinement of the exposure protocol, the intra- and inter-laboratory reproducibility and the establishment of a prediction model (PM). Substances that led to a decrease of cell viability below 50% (PM 50%) or 75% (PM 75%) at any of the three exposure doses were considered to exert an acute inhalation hazard. Results were then compared to existing in vivo data (based on at least one reliable study according to OECD test guideline (TG) 403 or TG 436^21,22), leading to an overall concordance of 85%, with a specificity of 83% and a sensitivity of 88%²³.

Besides the measurement of cell viability, other endpoints such as cytokine release, examination of the cell lysate or membrane integrity via LDH assay can be assessed but were not required for the validation study. Thus, the exposure system (e.g., CULTEX RFS) was proven as a predictive screening system for the qualitative assessment of the acute inhalation toxicity of the tested airborne particles, representing a promising alternative method to animal testing. The following protocol is recommended for exposure experiments to airborne particles using this exposure system.

Protocol

NOTE: The protocol of one exposure experiment covers a period of three days.

Day 1

1. General preparations and cultivation of cells

NOTE: The human lung adenocarcinoma epithelial cell line A549 was used for exposure experiments. Cells must be handled under sterile conditions. Other cell lines that are suitable for cultivation at the ALI can be used.

1. Prepare the growth medium (Dulbecco's Minimum Essential Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 5 µg/mL gentamycin) and the exposure medium (DMEM, supplemented with 5 µg/mL gentamycin and a final HEPES concentration of 100 mM).
2. Culture A549 cells in growth medium at 37 °C in a humidified atmosphere containing 5% CO₂.
3. Cultivate cells in cell culture flask of 75 cm² (T-75) in 14 mL of growth medium until a confluence of 80-90% before splitting (every 2-3 days) and a passage of 35.
4. Calculate the suspension volume and the required number of cell culture inserts (three cell culture inserts as incubator controls and cell culture inserts for clean air and particle exposure) and cell culture plates.
5. Before trypsinization and seeding of cells, add 2.5 mL of tempered growth medium to each well of a 6-well plate. Place the cell culture inserts without cells carefully inside the wells and add 1 mL growth medium to every cell culture insert. Incubate the 6-well plates for at least 30 min in the incubator (37 °C, 5% CO₂).

2. Trypsinization of cells

1. Temper phosphate buffered saline (PBS) and growth medium at 37 °C and the trypsin/EDTA (0.05%/ 0.02%) solution at room temperature.
2. Aspirate the cell culture medium from the cell culture flask and wash the cells carefully with 8 mL of pre-heated PBS.
3. Remove the PBS, add 2 mL of the trypsin/EDTA (0.05%/ 0.02%) solution to the cells and incubate for maximal 6 min. in the incubator at 37 °C. Control the detachment process under the microscope.
4. Neutralize the trypsin activity by adding 8 mL pre-heated growth medium, detach the cells by gently tapping sideways on the flask and resuspend the cells by repeated pipetting up and down.
5. Transfer the cell suspension to a 50 mL tube. Determine the cell number for further procedure (e.g., seeding of cells, passage of cells).

3. Determination of cell number

NOTE: Cell concentration was determined using a cell counter or counting chambers.

1. Dilute 100 µL of the cell culture suspension in a cup filled with 10 mL of isotonic solution. Tilt the cup slowly without shaking.
2. Determine number of viable cells/mL and cell viability based on the cell-specific measurement parameters of A549 cells.

4. Seeding of cells onto microporous membranes in cell culture inserts

NOTE: The exposure system is equipped with special adapters to enable the use of commercial inserts from different suppliers and of different sizes. For these exposure experiments, 6-well plates and the corresponding cell culture inserts were used. All working steps have to be done under sterile conditions.

1. Provide pre-heated growth medium under sterile cell culture conditions (laminar flow).
2. Prepare a sufficiently high volume of the cell suspension with a cell concentration of 3.0 x 10⁵ cells/mL.
3. After tempering the plates for 30 min, aspirate the medium within the cell culture inserts and seed 1 mL of A549 cells with a density of 3.0 x 10⁵ cells/mL in each cell culture insert. Distribute the cell suspension by gentle rocking.
4. Incubate the cell culture inserts with the cell suspension for 24 h (37 °C and 5% CO₂).

5. Pressing of test substances

NOTE: Test substances were pressed into powder cakes using a fully controllable hydraulic press. The press package can apply a maximum force of 18 kN, which is displayed as the current oil pressure (in bar) of the press package. Press conditions (pressing pressure, time of pressing) of unknown test substances have to be established and characterized in preliminary tests. Depending on the press properties of a substance, different pressing parameters and kinds of pressing plunger can be used.

CAUTION: Wear protective equipment when pressing toxic or dangerous substances.

1. Set the pressing time via the time control on the front side of the press.
2. Open the compressed air supply at the compressed air valve. Set the compressed air pressure to approximately 2 bar (indicated by a pressure gauge on the front side) using the pressure regulator on the front side of the press or on the compressed air valve of the compressed air supply. Pull out the drawer, press the Press button and read the pressing pressure on the digital pressure switch.
   1. Read just the pressure at the pressure regulator if the pressure is too high or too low.
3. Assembly the substance container and ensure that the glass cylinder is correctly centered (Supplementary Figure 1). Fill the substance container with a small amount of the test substance. Insert the plunger into the substance container and turn it slightly back and forth to evenly distribute the powder in the container.
4. Place the substance container with the plunger in the drawer and press the Press button. The hydraulic piston of the press moves onto the plunger and exerts a pressure on the test substance for the set pressing time. Open the drawer and remove the plunger.
5. Repeat steps 5.3 and 5.4 until the substance container is at least half full.
6. After completion of the pressing work, remove the substance container from the drawer and turn it upside down to remove loose and deposited particles.
7. If the substance container is not needed at the same day, close the substance container with parafilm in order to prevent the test substance from drying out or absorbing moisture.

Day 2

6. Assembly of the exposure system and connecting the peripheral equipment

NOTE: A more detailed view is provided in Figure 3, Supplementary Figure 2 and Supplementary Figure 3. Assemble both modules and the aerosol generator according to the manufacturer's instructions.

1. Place the exposure system on a solid and even surface, with the water supply facing forward. Connect the mass flow controllers with the aerosol generator and a three-necked bottle connected to the module for clean air exposure.
2. Connect the flow controller and the vacuum pump. Connect the tubes from the flow controllers with the tube connector on the attachments of the aerosol guiding module. Connect the tubes on the other side of the flow controllers with the vacuum pump. Make sure that the flow is going from the module through the flow controllers to the vacuum pump.
3. Connect the water bath with the heating water supply. The water supply is going from the water bath to the water inlet on the aerosol guiding module. Connect the water outlet of the aerosol guiding module with the water inlet of the sampling module. Close the circle with a connection from the water outlet of the sampling module to the water bath.
4. Place the aerosol generator including the elutriator close to the exposure module and connect the excess lines of the elutriator and the exposure and clean air module with large micro filters, and the suction of the exposure chambers with small micro filters (e.g., disposable filters). The elutriator serves as a reservoir for the generated particulate atmosphere and retains particles bigger than approx. 7 µm, whereas smaller particles are transported to the exposure module.
5. Connect the computer used to control the aerosol generation to the USB port of the aerosol generator top via a USB cable and the power supply to the power supply port. Connect the AC power plug of the power supply unit to a socket (220-240 V).
6. Connect the pipes for the medium supply and removal with two pumps. Instead of using a pump for the medium supply, the medium can also be filled manually.

7. Preparation for clean air and particle exposure

1. Turn on the vacuum pump, the flow controllers and the water bath (37 °C) for a warm-up period of at least 30 min.
2. Open the compressed air supply. Set the mass flow controllers to 8 L/min for the supply line to the aerosol generator and to 3 L/min for the supply line to the three-necked bottle. Close the tabs of the mass flow controllers.
   NOTE: These values may vary depending on the characteristics of the test substance.
3. Adjust the flow controllers via the computer to regulate the module flow (1.5 L/min) and the chamber suction (30 mL/min).

8. Leakage test of the radial flow system

NOTE: The leakage check must be performed under vacuum and for both modules (exposure and clean air module) in order to ensure that the module has been reassembled properly.

1. Remove the inlet adapter and the condensate reflector from the aerosol guiding module. Close the three aerosol feeding bores in the aerosol guiding module with plugs and the medium supply connections at the sampling module with dummy flaps.
2. Connect the vacuum lines without the filter with the tube connector of the aerosol guiding module. Close the module by using the hand wheel and measure the value of the flow controllers. The values should decrease some minutes after closing below 5 mL/min.
3. After the impermeability check, remove all plugs and dummy flaps, insert the inlet adapter and the condensate reflector into the aerosol guiding module and connect the pipes for medium supply and removal.

9. Aerosol generation

1. Start the computer and the software (Supplementary Figure 4). Start the aerosol generator software by double clicking on the aerosol generator start button on the desktop of the computer. A message window appears and asks if the settings should be reset or not. Click Yes if the software is started for the first time that day. Set the values for Slide Position and Scraper Position to the default values. Click No to keep the values for Slide Position and Scraper Position or the slide is not in the starting position.
2. Screw a substance scraper into the pipe, which is located in the central opening of the aerosol generator top.
   NOTE: Depending on the press characteristics, distinct types of substance scraper can be used.
3. Use the button Homing Mode if the substance scraper is not in the lowest position.
4. Place the substance container with the pressed test material upside down over the substance scraper. Ensure that the glass of the substance container faces the front. Make sure that the two holes in the substance container fit onto the two pins of the aerosol generator top. Place the locking plate in the slot over the substance container and tighten the black screw.
5. Change the values for Feed (0.24 to 20 mm/h) and Rotation (1 to 800 revs/h) to the desired settings. The particle concentration can be modified by increasing or decreasing the Feed value or the carrier gas flow rate.
6. Use the downward arrows to push down the slide with the substance container until the substance scraper is near the pressed substance.
7. Open the compressed air supply to the aerosol generator with a tap of the mass flow controller and start the aerosol generation by clicking on the Start button. Set the Feed rate to 15-20 mm/h to avoid long waiting times.
8. Control the correct particle generation by observing the fine dust cloud with a small flashlight (positioned from below behind the glass tube of the Elutriator). Change the value for Feed back to the desired settings when the first aerosol vapor reaches continuously the Elutriator and click on the Stop button.

10. Exposure experiments

1. Start the medium supply with pre-heated exposure medium and fill the sampling modules until the downpipes are covered while the module is open. Use the medium pump or fill the medium manually (25 mL per individual exposure chamber).
2. Insert blind cell culture inserts (inserts without cells) into the exposure module. Pump the exposure medium down until the downpipes are covered with medium and the lower side of the inserts are in contact with medium.
3. Start the aerosol generator, close the exposure module and connect the exposure module to the exposure module outlet of the aerosol generator. Give the aerosol generator a lead time of at least 20-30 min before exposures are started in order to enable a stable generation of particles.
4. Prepare the post-incubation plates for the incubator controls and the exposed cell culture inserts during the lead time. Add 1.5 mL of growth medium per well and incubate the plates in the incubator (37 °C, 5% CO₂).
5. After the lead time, seal the exposure module outlet of the Elutriator with a rubber plug and remove the blind inserts. Refill the exposure medium (using the pump or manually) until the downpipes are covered with medium. Now, open also the compressed air supply to the clean air module with the tap of the mass flow controller (3 L/min)
6. Remove the cell culture inserts from the 6-well plates with the help of a tweezer. Pour the growth medium carefully from the cell culture inserts off by toppling the inserts and aspirate and discard the residual liquid using a pipette. Place the inserts in the exposure chambers of both modules, the exposure and clean air module.
7. Close the modules and start the exposure experiments by connecting the exposure module to the exposure module outlet of the aerosol generator and the clean air module to the carrier gas supply simultaneously.
   NOTE:The particle concentration can be modified by increasing/decreasing the Feed value, the carrier gas flow rate or the time of exposure.
8. Disconnect the exposure and clean air modules after completion of the experiment and seal the exposure module outlet.
9. Stop the compressed air supply and the aerosol generator by clicking on the Stop button.
10. Open the exposure and clean air module and transfer the cell culture inserts to the prepared post-incubation plates using a tweezer. Incubate the 6-well plates for 24 h (37 °C, 5% CO₂) at the ALI.
    NOTE: Repeat steps 10.5 -10.10 if further exposure experiments are planned.
11. Lift the cell culture inserts, that are used as incubator controls to the ALI under the same conditions as the exposed cell culture inserts and incubate them for 24 h (37 °C, 5% CO₂) at the ALI.
12. Use the button Homing Mode to remove the substance container. Close the aerosol generator software by clicking on the X in the upper right-hand corner and turn off the computer.
13. After completion of all exposure experiments, clean the aerosol generator and both exposure modules. Close the substance container with parafilm if the test substance will be further used within the next days.

Day 3

11. Cell viability

NOTE: Cell viability was determined 24 h after particle deposition by measuring the mitochondrial activity using the WST-1 assay. The assay was performed according to the manufacturer's protocol. Cell viability can also be determined by using other cell viability tests (e.g., XTT).

1. Temper growth medium at 37 °C and thaw the WST-1 solution protected from light. Prepare an appropriate number of new 6-well plates with 2.5 mL growth medium per well and incubate the plates in the incubator.
2. Prepare the WST-1 dilution by diluting a sufficient amount of WST-1 1:7 in growth medium
3. Insert the cell culture inserts 24 h after exposure in the new prepared 6-well plates. Add 1 mL of the fresh-prepared WST-1 solution to each cell culture insert. Rock the plates carefully in order to distribute the solution homogenously on the cells. Incubate the 6-well plates with the cell culture inserts for 1 h (37 °C, 5% CO₂).
4. Transfer 100 µL of the supernatant in triplicates from each 6-well to a 96-well plate. Measure the absorbance at 450 nm with a reference wavelength of 650 nm using a microplate reader.

12. Statistics

1. Normalize the cell viability of the individual incubator controls to 100%.
2. Express the viability of the exposed cells in relation to the individual incubator controls. Cytotoxicity of test substances was compared to the respective incubator controls and used as an indicator of toxicity.

Representative Results

The CULTEX RFS is a specially designed modular in vitro exposure system that enables the direct and homogenous exposure of cells at the ALI. Within a former pre-validation study, the general applicability of this exposure system and its transferability, stability and reproducibility were successfully demonstrated. In a recent research project funded by the German Federal Ministry of Education and Research, the exposure system was successfully validated and established as a prediction model (PM) for acute inhalation hazards of the tested compounds. As the quality of the clean air controls turned out to be a critical parameter during the pre-validation study, several protocol and method optimizations (e.g., change of cell culture inserts, stabilization of the pH of the exposure medium by increasing the HEPES concentration to 100 mM) were implemented at the beginning of the validation study, leading to highly stable and reproducible results and a substantial improvement of clean air viability data across all three laboratories (Figure 4). A549 cells were then exposed at the ALI to three different exposure doses (25, 50 and 100 µg/cm²) of 20 pre-selected and coded test substances in the three independent laboratories, and cytotoxicity (used as an indicator of toxicity) was compared to the respective incubator controls. Thirteen coded substances were thereby tested in triplicates, seven coded substances as single experiments. Test substances were considered to exert an acute inhalation hazard when cell viability decreased below 50% (PM 50%) or 75% (PM 75%).

As shown exemplarily in Figure 5, exposure of A549 cells to different test substances exhibited no, medium or a strong toxicity. As all experiments were conducted independently in three laboratories, data were analyzed regarding the reproducibility within and between the laboratories and the predictivity of the exposure system. Depending on the applied PM (PM 50% or PM 75%), the within-laboratory and the between-laboratory reproducibility ranged from 90-100%, demonstrating the robustness and transferability of this method. As all tested substances had relevant available in vivo reference data (based on at least one reliable study according to OECD TG 403 or TG 436 using a traditional LC₅₀ protocol and a concentration x time (C x t) protocol), comparison of the in vivo and in vitro data revealed an overall concordance of 85% (17/20) with a specificity of 83% (10/12) and a sensitivity of 85% (7/8) (Table 1). Only two substances were classified as falsely positive and one as falsely negative.

In summary, our results of the validation study present a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of the selected airborne particles.

Figure 1: Exposure of cells at the ALI or under submerged conditions. A549 cells can be either exposed with a test substance (blue arrows and dots) at the ALI (left) through an inlet of the exposure system or with the test substance diluted in exposure medium (blue dots) creating submerse conditions (right). Red dotted lines represent the fill levels of exposure medium (bright red) in the respective experimental setup. This figure has been modified from Tsoutsoulopoulos et al.²⁴. Please click here to view a larger version of this figure.

Figure 2: The exposure module. Schematic overview of the basic module of the radial flow system consisting of the inlet adapter, the aerosol guiding module, the sampling and socket module and a locking module with a hand wheel. This figure has been modified from Aufderheide et al.¹⁷. Please click here to view a larger version of this figure.

Figure 3: Overview of the exposure system. The components are the two exposure modules for clean air and particle exposure, the aerosol generator including the elutriator and corresponding control unit, and the medium pumps. This figure has been modified from Aufderheide et al.¹⁷. Please click here to view a larger version of this figure.

Figure 4: Clean air viability data after optimization of the test method. Exposure of cells to clean air led to no decrease of cell viability over time, leading to a substantial improvement of clean air viability data compared to the pre-validation study. All clean air controls were pooled for each laboratory (Lab 1-3) and exposure time (n = 46 per laboratory and point in time). Data are displayed as boxplots with a median line and the range indicated by whiskers. This figure has been modified from Tsoutsoulopoulos et al.²³. Please click here to view a larger version of this figure.

Figure 5: Exposure of A549 cells to different test substances. (A) Exposure of A549 cells to tungsten(IV) carbide showed no decrease of cell viability for the three different exposure doses and appeared to be non-toxic (n = 3 per laboratory and exposure dose). (B) Exposure of cells to tetrabromophthalic anhydride resulted for all laboratories in a moderate toxicity presenting a good dose response curve (n = 1 per laboratory and exposure dose). (C) Zinc dimethyldithiocarbamate exhibited a strong toxicity, leading to a decreased cell viability already after a deposited dose of 25 µg/cm² (n = 3 per laboratory and exposure dose). Error bars represent standard deviations. This figure has been modified from Tsoutsoulopoulos et al.²³. Please click here to view a larger version of this figure.

		Reference results in vivo		Results in vitro
Substance	OECD TG	CLP regulation	Toxicity	PM50%	PM75%	Accordance in vivo / in vitro
Tungsten(IV) carbide	403	n. c	0	0	0	Yes
Tungsten(IV) carbide nano	-	n. c.	0	0	0	Yes
N,N'-ethylenebis(N-acetylacetamide)	EPA OPPTS 870.1300	n. c.	0	0	0	Yes
Silicon dioxide	403	n. c.	0	0	0	Yes
Diammonium hydrogenortho-phosphate	403	n. c.	0	0	0	Yes
Disodium fluorophosphate	403	n. c.	0	0	0	Yes
Neodymium oxide	436	n. c.	0	0	0	Yes
Potassium hydrogen monopersulfate	403	n. c.	0	0	0	Yes
Cyclohepta-pentylose	403	n. c.	0	0	0(2x) / 1(1x)	(Yes)
Vanadium(III) oxide	403	n. c.	0	0	0(2x) / 1(1x)	(Yes)
Tetrapotassium pyrophosphate	403	n. c.	0	1	1	No
Tetra-bromophthalic anhydride	403 (similar)	n. c.	0	1	1	No
Cetylpyridinium chloride	403	Acute Tox. 2	1	1	1	Yes
N-Lauroylsarcosine sodium salt	403	Acute Tox. 2	1	1	1	Yes
Zinc dimethyldithio-carbamate	403	Acute Tox. 2	1	1	1	Yes
Copper(II) hydroxide	403	Acute Tox. 2	1	1	1	Yes
Zinc selenite	436	Acute Tox. 3	1	1	1	Yes
Sodium metavanadate	403	Acute Tox. 4	1	1	1	Yes
Divanadium pentaoxide	403	Acute Tox. 4	1	1	1	Yes
Cadmium telluride	403	Acute Tox. 4 (n. c.)	1	0	0	No
n. c. = not classified; 0 = non-toxic; 1 = toxic; CLP = classification, labelling and packaging

Table 1: Accordance between in vivo and in vitro results. Out of 20 substances, 10 substances were classified as correctly negative, and seven substances correctly as positive, leading to a concordance of 85% (17/20). This table has been modified from Tsoutsoulopoulos et al.²³. (Test No. 403: Acute Inhalation Toxicity, Test No. 436: Acute Inhalation Toxicity - Acute Toxic Class Method; Acute Tox. 2 = fatal, Acute Tox. 3 = toxic, Acute Tox. 4 = harmful).

Supplementary Figure 1: Assembly of the substance container. Picture taken from CULTEX DG (Dust Generator) User Manual. Please click here to view a larger version of this figure.

Supplementary Figure 2: Aerosol guiding module of the exposure system. A) Top view and B) bottom view of the aerosol guiding module. Picture taken from CULTEX RFS (Radial Flow System) User Manual. Please click here to view a larger version of this figure.

Supplementary Figure 3: The aerosol generator. A) Schematic overview of the aerosol generator, consisting of the aerosol generator top and the Elutriator. Detailed view of B) the aerosol generator top and C) the Elutriator. Picture taken from CULTEX DG (Dust Generator) User Manual. Please click here to view a larger version of this figure.

Supplementary Figure 4: The aerosol generator control software. Picture taken from CULTEX DG (Dust Generator) User Manual. Please click here to view a larger version of this figure.

Discussion

Many non-animal inhalation toxicity testing models have been developed in recent years in order to gain information about the acute inhalation hazard of inhalable particles and to reduce and replace animal experiments according to the 3R principle²⁵.

In terms of cell culture models, exposure of cells can be done under submerged conditions or at the ALI. Exposing cells under submerged conditions may affect the physico-chemical properties and thus, the toxic properties of a test substance¹². In vitro ALI inhalation models, however, mimic the human exposure situation with higher biological and physiological similarity than submerged exposure and are therefore better suited for analyzing the acute inhalation toxicity of airborne particles. The significance of the CULTEX RFS with respect to other existing exposure modules is not only the exposure of cells under ALI conditions but also the very homogenous distribution and deposition of particles. In contrast to sequential exposure models with linear aerosol guidance, the modular design of this exposure method enables a radial supply line leading to a very homogenous deposition of particles on the cells¹⁷.

The most important point for successful exposure experiments is the stable and congruent quality of the clean air controls. Special attention must be paid that the viability of the clean air controls is not affected over time and as close as possible at 100% compared to the corresponding incubator controls. Factors that play an important role regarding clean air viability are the choice of suitable cell culture inserts, the pH value of the exposure medium, and the composition of the clean air. In terms of cell culture inserts, a good quality and a high density of pores must be guaranteed. This ensures a better medium supply and a higher relative humidity inside the cell culture inserts, protecting the cells from desiccation²⁶. By using cell culture inserts with side wall openings, special insert sleeves must be used in order to avoid leakage of test particles through the side wall openings which could lead to a possibly contamination of the exposure medium. A shift of the pH value higher than 8 can already have a toxic effect on the cells and therefore leading to an impairment of cell viability²⁷. This occurs especially in prolonged particle deposition times (e.g., 60 min) if the clean air contains less than 5% CO₂ or the HEPES concentration of the exposure medium is too low which has to be avoided.

A critical issue of the protocol is the pressing of the test substances. The test substances have to be sufficiently compressed to a powder cake within the substance container in order to enable a stable particle exposure. Thus, the substances have to be characterized in preliminary experiments regarding their press properties and in order to obtain information about which press plunger, type of scraping blade or feed rates have to be used.

The maximum pressing pressure of the hydraulic press, however, is 10 kN, which represents at the same time the load limit for the glass cylinder and thus, a limitation of the pressing process. The substance container cannot withstand higher pressing forces than 10 kN. A higher pressing force might offer the pressing of crystalline substances and thus, extend the applicability of this press but would require more robust substance containers.

Moreover, this exposure system which is primarily designed for the investigation of airborne particle exposures can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules. Exchanging the aerosol generator with a membrane nebulizer and using a stainless-steel exposure module enabled the exposure of cells to highly toxic liquid aerosols²⁴.

A further critical issue is the overload effect. Cell viability can be affected not only by toxicological properties of a test substance but also by the amount of a substance deposited on the cells. This exposure system shows indeed substantial similarity to the physiological conditions in the human alveolar region but does not contain any clearance mechanisms for removing particles. It is therefore very important that the cell viability is not impaired due to a too large number of particles.

The protocol herein describes the homogenous exposure of cultivated human lung cells at the ALI to airborne particles. The reproducibility, its robustness and transferability make the current exposure system applicable as an in vitro screening method for the qualitative assessment of inhalable particles regarding their acute inhalation toxicity. Because no alternative in vitro methods have been sufficiently validated so far, acute pulmonary toxicity is still being assessed by exposing animals (e.g., whole-body chambers, nose or mouth-only methods). All commonly accepted OECD test guidelines for the acute inhalation toxicity (e.g., TG 403, TG 433 and TG 436) are based on animal models at present^21,22,28,29. One future direction will be therefore to apply at the Organization for Economic Cooperation and Development (OECD) for the acceptance as an in vitro test guideline for the acute inhalation toxicity.

Materials

Name	Company	Catalog Number	Comments
Cells
A549	ATCC	CCL-185
Cell culture medium and supplies
DMEM	Biochrom, Berlin, Germany	FG 0415	used as growth medium
DMEM	Gibco-Invitrogen, Darmstadt, Germany	22320	used as exposure medium
FBS superior	Biochrom, Berlin, Germany	S 0615
Gentamycin (10mg/mL)	Biochrom, Berlin, Germany	A 2710
HEPES 1M	Th. Geyer, Renningen, Germany	L 0180
PBS	Biochrom, Berlin, Germany	L 1825
Trypsin/EDTA (0.05%/0.02%)	Biochrom, Berlin, Germany	L 2143
Cell culture material
CASY Cups	Roche Diagnostic GmbH, Mannheim, Germany	REF 05651794
Cell culture plates	Corning, Wiesbaden, Germany	3516	6­-well plates
Corning Transwell cell culture inserts	Corning, Wiesbaden, Germany	3450	24mm inserts; 6-­well plates; 0.4 µm
Chemicals
CASYton	Roche Diagnostic GmbH, Mannheim, Germany	REF 05651808001
Compressed Air (DIN EN 12021)	Linde Gas Therapeutics GmbH, Oberschleißheim, Germany	2290152
WST-1	Abcam, Cambridge, United Kingdom	ab155902
Instruments + equipment
CASY Cell Counter	Schärfe System GmbH, Reutlingen, Germany
Circulation thermostat	LAUDA, Lauda-Königshofen, Germany	Ecoline RE 100
CULTEX HyP - Hydraulic Press	Cultex® Technology GmbH, Hannover, Gemany
CULTEX insert sleeve	Cultex® Technology GmbH, Hannover, Gemany
CULTEX RFS - Radial Flow System Type 2 (module for particle exposure)	Cultex® Technology GmbH, Hannover, Gemany
CULTEX RFS - Radial Flow System Type 2 (module for clean air exposure)	Cultex® Technology GmbH, Hannover, Gemany
CULTEX supply
Flow controller 0-30 ml/min (IQ-Flow)	Bronkhorst Deutschland Nord GmbH
Flow controller 0-1,5 l/min (EL-Flow)	Bronkhorst Deutschland Nord GmbH
Filters (large)	Munktell & Filtrak GmbH, Sachsen, Germany	LP-050	Munktell Sterile Filter; Particle retention efficiency > 99,999%
Filters (small)	Parker Hannifin Corporation, Mainz, Germany	9933-05-DQ	Balston disposable filter
Medium pump	Cole-Parmer GmbH, Wertheim, Germany	Ismatec IPC High Precision Multichannel Dispenser	digital peristaltic pump
Microplate Reader Infinite M200 Pro	Tecan Deutschland GmbH, Crailsheim, Germany
Vakuum pump	KNF, Freiburg, Germany	N86 KT.18
Vögtlin mass flow controller 0,2-10 l/min	TrigasFI GmbH	Vögtlin red-y compact regulator, Typ-Nr.: GCR-C3SA-BA20
Water Bath	LAUDA, Lauda-Königshofen, Germany	Ecoline Staredition RE 104