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).
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.
Inhalation of toxic airborne particles is a public health concern, leading to a multitude of health risks worldwide and many millions of deaths annually1,2. 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 years3,4,5,6. 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 substances7,8. 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 particles5,7,10,11. 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 substance12,13. 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 exposures12,14.
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 43621,22), 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.
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.
2. Trypsinization of cells
3. Determination of cell number
NOTE: Cell concentration was determined using a cell counter or counting chambers.
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.
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.
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.
7. Preparation for clean air and particle exposure
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.
9. Aerosol generation
10. Exposure experiments
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).
12. Statistics
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.
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 present21,22,28,29. 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.
The authors have nothing to disclose.
This work was supported by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF, Germany (Grant 031A581, sub-project A-D)) and by the German Research Foundation (Deutsche Forschungsgesellschaft, DFG, Research Training Group GRK 2338).
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 |