OPTIMISATION OF TRIPLE CELL CULTURE FOR STUDYING INDOOR AIR PARTICULATE MATTER
TOXICITY
Tamara Gajšt MSc Thesis General Toxicology and Environmental Health Risk Assessment University of Eastern Finland, Department of Environmental and Biological Sciences May, 2019
Tamara Gajšt: Optimisation of triple cell culture for studying indoor air particulate matter toxicity
MSc thesis 45 pages, 1 appendix (3 pages)
Supervisors: Kati Huttunen, PhD, Docent, Adjunct Professor, University Researcher, UEF and Maria-Elisa Nordberg, M.Sc., Early Stage Researcher, UEF
May, 2019
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keywords: in vitro, triple culture model, pulmonary toxicity, particulate matter, air-liquid interface (ALI)
ABSTRACT
Exposure to particulate matter (PM) has been associated with different pulmonary and cardiovascular conditions. In order to examine the potential mechanisms of adverse effects of PM, several in vitro models have been proposed using different types of either continuous or primary cell lines. The aim of this work was to develop and optimise a triple-culture model that would best represent the in vivo conditions. It consisted of a human alveolar epithelial cell line (A549) and monocytic human cells (THP-1) seeded apically on a semipermeable cell culture insert with a continuous endothelial cell line (EA.hy926) on the basolateral side. Prior to PM exposure, the air-liquid interface (ALI) was formed by removal of apical growth medium (GM).
Synthetic lung lining fluid (LLF) was used for PM sample dilutions during the exposure study.
Different endpoints were measured after exposure to two indoor PM samples collected using two different methods. Cell count and viability were determined, transepithelial electrical resistance (TEER) measured to determine the barrier integrity and pro-inflammatory chemokine IL-8 quantified to assess the inflammatory response. Additionally, RNA was isolated for the transcriptome analysis and total protein concentration of the apical wash was evaluated. Finally, electron and light microscopy were applied to visualise the cellular layers and determine the cell-particle interaction. Results suggest that LLF was suitable for diluting PM as it did not elicit a toxic response. The model was sensitive enough for the IL-8 measurements with significant differences in controls and some PM dilutions. However, the measured TEER values differed from other studies and the total protein concentration was not adequate to identify the PM effects on the triple-culture model. There was a clear indication of cell-particle interaction established in electron microscopy images and a uniform cellular layer confirmed with light microscopy. RNA quality and amount was not sufficient for transcriptome analysis.
First, I would like to express my gratitude to my two supervisors Kati Huttunen and Maria- Elisa Nordberg for the opportunity to conduct a Master thesis under their supervision. I would especially like to thank them for all their help, guidance, super detailed thesis review and mostly patience when waiting for me to finish my thesis. Maria-Elisa, thank you also for all the long days you spent with me in the laboratory – I could not have wished for a better lab-partner and I surely did not imagine I would acquire such a wonderful friend in the end. Thank you.
Secondly, I would like to thank my family and friends for all the moral support, words of encouragement and occasional babysitting giving me a chance to write the thesis.
Finally, my love, my life companion, my fiancé Tine Bizjak, thank you for being there for me every time I need you and for sharing this life with me and thank you for our wonderful son Emil Olav, our never-ending smiley face and all the motivation we’ll ever need in all our future endeavours!
ALI Air-liquid interface
COPD Chronic obstructive pulmonary disease DMEM Dulbecco’s modified Eagle medium ELISA Enzyme-linked immunosorbent assay FBS Fetal bovine serum
HBSS Hank’s Balanced Salt Solution
IL-8 Interleukin 8 or Chemokine (C-X-C motif) Ligand (CXCL)-8 found in humans LLF Lung-lining fluid
LPS Lipopolysaccharide PM Particulate matter
TEER Transepithelial electrical resistance
ACKNOWLEDGEMENTS ... 3
ABBREVIATIONS AND DEFINITIONS ... 4
1. INTRODUCTION ... 7
2. LITERATURE REVIEW ... 9
2.1 PARTICULATEMATTER ... 9
2.2 HUMANRESPIRATORYTRACT ... 10
2.2.1 Lung lining fluid (LLF) ... 11
2.2.2 In vivo models... 12
2.2.3 In vitro models ... 12
3. AIM OF THE WORK... 17
4. MATERIALS AND METHODS... 18
4.1 PMSAMPLINGANDSAMPLEPREPARATION ... 18
4.2 CELLCULTURES ... 18
4.3 CO-CULTUREOPTIMISATIONANDMODELCOMPARISONS ... 19
4.4 TRIPLE-CULTUREPMEXPOSURESTUDY ... 20
4.5 ANALYSESANDMICROSCOPY ... 22
4.5.1 Viability and cell count ... 22
4.5.2 Transepithelial/transendothelial electrical resistance (TEER)... 22
4.5.3 Bradford (protein) assay ... 23
4.5.4 Chemokine IL-8 ... 23
4.5.5 RNA extraction and quality control ... 23
4.5.6 Electron and Light microscopy ... 24
4.6 STATISTICALANALYSIS ... 24
5. RESULTS ... 26
5.1 CO-CULTUREOPTIMISATIONANDMODELCOMPARISONS ... 26
5.2 TRIPLE-CULTUREPMEXPOSURESTUDY ... 28
7. SUMMARY AND CONCLUSIONS... 37
8. REFERENCES ... 38
APPENDICES ... 46
APPENDIX I–STATISTICAL ANALYSIS DETAILS ... 47
1. INTRODUCTION
Human respiratory tract is a target organ for many potentially harmful inhaled particulates and additionally acts as an entry into systemic circulation, thereby also exposing other tissues to hazardous material. The relationship between outdoor particulate matter (PM) exposure and pulmonary as well as cardiovascular diseases has already been widely established (Cachon et al. 2014). However, as people spend the majority of their time indoors, indoor air pollution has gained increased interest among researchers. Commonly found indoor air pollutants are PM, CO, O3, NO2, SO2, volatile organic compounds (VOCs), passive smoke (Bernstein et al. 2008) as well as moisture-related biological agents which have also been connected to several respiratory conditions (e.g. asthma) (WHO Regional Office for Europe 2009).
Animal models provide a valuable resource to study the possible respiratory conditions induced by air pollution exposure. Nevertheless, in addition to ethical considerations, alternative in vitro models provide a faster, more cost-effective way to assess the toxicity of inhalable particulates.
Furthermore, rodents, most commonly used in in vivo inhalation toxicity studies, have physiologically, anatomically and biochemically different respiratory tract compared to humans (Clippinger et al. 2018). Therefore, it is important to develop and optimise in vitro pulmonary models in order to more accurately evaluate relevant human responses.
The alveolar epithelium, with its large surface area, represents an important target for inhaled foreign material and consequently a valuable research area in inhalation toxicology. Co- culturing allows studying responses of multiple cell types after stimulation to an exposure agent. Cellular response induced by PM exposure is often determined by measuring cytotoxicity and release of pro-inflammatory cytokines (e.g. interleukin-8 (IL-8)) because of their importance in progression of inflammation and neutrophil recruitment (Viau et al. 2010).
It is important to co-culture epithelial and endothelial cell lines in air-liquid interface (ALI), since submerged cultivation does not imitate the real in vivo conditions as the exposure agent remains suspended in medium and therefore does not provoke a realistic cell response (Chary et al. 2018). In addition to ALI conditions, a synthetic lung lining fluid (LLF) can be added to the co-culture model to reflect the in vivo conditions of human airways even better. The advancements in creating a valid complex in vitro pulmonary model for inhalation exposure studies is imperative in order for this type of models to be representative and even accepted for regulatory purposes.
The aim of this study was to optimise the triple-culture model for studying indoor air PM exposure and to determine whether the synthetic LLF is suitable for dilution of the indoor air PM samples. The developed triple-culture model provides opportunities for further studies of the effects of PM exposure in ALI.
2. LITERATURE REVIEW
Adverse health effects caused by exposure to polluted air has become a great public health concern in the modern society. Both indoor and outdoor air pollution are found as one of the leading cause for mortality and morbidity worldwide (Cohen et al. 2017) and are a well-known risk factor for many respiratory diseases (Johannson et al. 2015).
One of the main sources of indoor air pollutants is ambient air (Huttunen 2018), levels of which are dependent on different factors, such as human activity, climate, building ventilation and emission sources (Monn 2001). Other major sources attributable to adverse health outcomes in human population include volatile organic compounds (VOCs), radon, environmental tobacco smoke and biological pollutants such as moulds, fungus, endotoxins, bacteria and viruses.
Products of incomplete combustion are one of the most extensive air pollution sources worldwide and include particulate matter (PM), carbon monoxide (CO), nitrogen oxides (NOx) as well as other gaseous pollutants known to be irritating and even carcinogenic (Perez-Padilla et al. 2010). Indoor air pollution arising from solid fuel combustion was found to be one of the main risk factors for global disease burden (Lim et al. 2012).
People spend over 50-90% of their time indoors (Perez-Padilla et al. 2010, Huttunen 2018), depending on their geographical location, gender and age. Consequently, people are exposed to a variety of indoor air pollutants. Humans inhale about 12,000 litres of air per day, exposing the lung epithelium to 25 million particles per hour (Rogers 2007). Increased pollution in environments where people spend the majority of their time means more inhaled potentially health impairing particles, making the protective mechanisms of our airways even more important.
2.1 PARTICULATE MATTER
PM includes air pollution particles, a composite of various organic and inorganic substances that vary in size, which is closely correlated with the location of their deposition in the respiratory tract and are classified accordingly to coarse (PM10, <10 µm in diameter) and fine (PM2.5, < 2.5 µm in diameter) particles (Mazzarella et al. 2006). With advances in technology and new nanomaterials, exposure assessment of fine and ultra-fine PM (<100 nm in diameter) has also become of concern, especially since they are able to enter the bloodstream (Klein et al.
2013) more easily than larger particles and (Geiser, Kreyling 2010) cause severe inflammation and increased cytokine release (Kim, J. S. et al. 2013). The adverse health effects of PM in
human pulmonary system can be local or systemic, therefore affecting the lungs and the cardiovascular system (Donaldson, Macnee 2001, Alfaro-Moreno et al. 2008).
Most common changes in the gene expression of the respiratory tract epithelium attributed to PM exposure are related to oxidant stress and release of pro-inflammatory cytokines, which can lead to exacerbation of chronic inflammatory conditions such as chronic obstructive pulmonary disease (COPD) and asthma (Proud 2008). It has been estimated that up to 19% of adults over 40 years old are affected by COPD, a chronic disease rapidly spreading worldwide and increasing the susceptibility to ischemic heart disease, stroke, pneumonia and lung cancer (Ling, van Eeden 2009). One of the proposed mechanisms by which the particulates are causing adverse outcomes are oxidative stress, inflammation and genotoxicity (Cho et al. 2018).
Therefore, inflammatory responses are of great interest in air pollution exposure studies (Jalava 2008). As a marker of airway inflammation, IL-8 is usually measured due to its important role in leukocyte recruitment (Kim et al. 2013).
It has been estimated that in populated continental areas, 22% of total air particulates are from biological origin and include pollens, fungal spores, bacteria, viruses as well as plant and animal fragments (Jones, Harrison 2004). The potential adverse health effects of these particulates to human health still needs more research, however it has already been shown that exposure to endotoxins increases the risk of asthma development (Tavernier et al. 2005). Biological agents are also present in indoor environments, however their composition can change in case of moisture problems and insufficient ventilation systems. High humidity creates favourable conditions for growth of fungi, mould and bacteria. Moisture-damaged indoor environments therefore represent exposure to increased concentration of biological contaminants and are connected to upper respiratory tract conditions, cough, wheezing, dyspnoea and asthma (WHO Regional Office for Europe 2009, Bornehag et al. 2001) .
2.2 HUMAN RESPIRATORY TRACT
Human airways are a specialised complex system of more than 40 different cell types (Sorokin 1970) which function against any potentially harmful inhaled toxins. For example, it has been shown that PM exposure of lung macrophages results in release of different cytokines (Becker et al. 2003).
Human respiratory tract consists of the upper respiratory tract which includes the nasal cavity, pharynx and larynx and the lower respiratory tract including the trachea, primary bronchi leading to the bronchioles and finally the alveoli, where the gas exchange takes place (Klein et
al. 2011). The entire respiratory tract is covered in morphologically different epithelial cells depending on the region (Willetts 2012) and consists of different amounts of ciliated, Clara, goblet and undifferentiated basal cells (Srinivasan et al. 2015).
The pseudostratified and columnar epithelium in trachea and bronchi consists of ciliated, basal and secretory goblet cells, leading to a reduced cuboidal ciliated and secretory Clara cells in bronchioles. Finally, the squamous alveolar part contains type I and type II cells (Klein et al.
2011), which form the protective tight junctions important in ALI (Bengalli et al. 2013). The existence of ALI in the respiratory tract is essential to prevent the air bubbles entering the blood as well as the blood and plasma from penetrating into the alveoli (Sakolish et al. 2016). Type I cells cover 90% of the alveoli and have a function of a gas-permeable membrane while type II cells cover the rest 10%, form tight junctions, synthesise and secrete surfactants consisting of a variety of proteins and phospholipids (Chang et al. 2008).
The respiratory epithelium has several protective mechanisms against harmful inhaled pathogens, toxins and particulate matter (Luyts et al. 2015). It forms a tight barrier and removes particulates via mucociliary clearance (Chang et al. 2008). Moreover, several inflammatory cytokines and LLF are released as a response to foreign material, acting together as a protective barrier (Kim et al. 2013). Exposure to particulates can impair ALI and lead to lung inflammation and oxidative stress (Luyts et al. 2015). Furthermore, if the particulates penetrate the ALI, they can interact with immune and endothelial cells, enter the systemic circulation and are transferred into different organs (Wang et al. 2019) where they can cause adverse health effects.
2.2.1 Lung lining fluid (LLF)
When the particulates are inhaled, they initially come in contact with the protective lining fluid covering the entire pulmonary epithelium (Bicer 2015, Balogh Sivars et al. 2018). LLF is a composite of proteins and phospholipids (Rauprich et al. 2000) and a primary defensive barrier against any harmful inhaled particles. The composition of the LLF can differ locally due to different cell type secretions in specific region of the airways, (Bicer 2015). Because of LLF heterogeneity, it has been a challenge to produce a synthetic LLF that would mimic the in vivo conditions as realistically as possible. Nevertheless, the use of LLF in in vitro cell models is highly relevant to increase the accuracy of such models and to evaluate the behaviour of inhaled particles (Bicer 2015).
2.2.2 In vivo models
Presently, PM-induced pulmonary toxicity is still widely studied in vivo, commonly using different rodent species (Willetts 2012). The adverse effects of PM are usually studied using hamsters, mice and rats by intratracheal inhalation or intratracheal instillation, the latter allowing for better regulation of concentration and location of the exposure agent (Cho et al.
2018).
In in vivo conditions, deposited inhaled particles in alveoli interact with different elements in ALI, starting with a thin surfactant layer (Geiser, Kreyling 2010). Thereafter, the particles either interact with the pneumocytes, are eliminated by macrophages or penetrate the ALI and end up in the systemic circulation. Throughout the different interactions, protective responses are triggered, resulting in e.g. impaired cell function and release of inflammatory cytokines (Loret et al. 2016).
Even though in vivo models are a big part of toxicological assays, there have been great efforts worldwide trying to minimise the number of experiments on animals. In some instances, animal tests were banned entirely, as with the 7th amendment of the European cosmetics directive (1976) which prohibits animal use in tests of cosmetic formulations and ingredients as of March 11th, 2009 (Klein et al. 2011). Taking into account the stricter regulations and ethical aspects of animal testing, there has been an increased endeavour among researchers to try to find a more affordable and comparable alternative to animal experiments. In addition, the airway of rodents is both functionally and structurally different to human respiratory tract (Carey et al. 2012).
Compared to humans that are oronasal breathers with simple turbinates, rodents classify as obligate nasal breathers with complex nasal turbinates allowing for better protection of lower airways with finer filtration, absorption and excretion of air particulates (Harkema et al. 2006).
Furthermore, an extrapolation to humans is also challenging in terms of different cytokine expression, for instance, rodents do not express the human IL-8 gene (Tarrant 2010).
2.2.3 In vitro models
Faster and more cost-effective alternatives to in vivo assays are different in vitro models. They represent an advantage in determining interactions among cells and inhaled particles in human airways, which is otherwise not easily achieved in vivo (Kim et al. 2013, Rothen-Rutishauser et al. 2005) and contribute greatly in understanding the alveolar damage (Srinivasan et al. 2015).
Different cell lines are used in in vitro models, namely human primary cells, immortalised human cells or cell lines obtained from animal species (Chary et al. 2018). The continuous cell lines are usually virus-transformed and carcinoma derived (Huttunen, Korkalainen 2017). Co- cultures commonly include different epithelial, macrophage, endothelial and dendritic cell lines (Klein et al. 2011).
Several different attempts have been made to obtain a co-culture model that would imitate the human respiratory system as realistically as possible (Huang et al. 2017, Kim et al. 2017). Very simple pulmonary models consist of a single cell line - a monoculture (Mazzarella et al. 2006, Müller et al. 2010, Huang et al. 2017), double-culture (Mukae et al. 2005, Bisig et al. 2019), triple-culture (Wang et al. 2019) or even a tetra-culture (Kim et al. 2017, Klein et al. 2013).
Some examples of co-culture pulmonary cell models used in different exposure studies are summarised in Table 1.
Table 1: Various pulmonary cell models with seeding densities (if available) for each cell line and the exposure agent tested with the model.
Cell model with seeding densities Exposure agent Reference Monoculture A549
Co-culture A549 and U937 (3:1 ratio)
DMS3 or PEV2 (P.
aeruginosa targeting phages)
(Shiley et al. 2017) Monoculture 16HBE14o-; 40,000 cells by well in
12 well plates
Urban PM, DEP and carbon black particles (CB) at 10, 20 or 30 mg/cm2
(Baulig et al. 2003)
Co-culture, THP-1 and A549 (4:1 ratio) PM - wood smoke particles from different combustion cycles at 40 μg/cm2 for 12 and 40h
(Bølling et al. 2012)
Monocultures A549, THP-1, HMC-1, EAHY926 Co-cultures (A549 + HMC-1 in a 10:1 ratio;
THP-1 + HMC-1 in a 2:1 ratio)
Triple-cultures (A549 + THP-1 + HMC-1 in a 10:2:1 ratio)
Urban PM10 (24 h at 0, 10, 30 or 100 mg/cm2).
(Alfaro-Moreno et al. 2008)
Monoculture BEAS-2B cells, seeded at 300,000 cells/well into collagen-coated 6-well plates
10 μg/cm2 (100 μg/mL) of PM
(Longhin et al. 2018) Triple-culture:
EA.hy926 (CRL-2922) seeded at 2.5×10ˆ4 cells/cm2 (basal)
Calu-3 (HTB-55) seeded at 5.0×10ˆ4 cells/cm2 (apical)
THP-1 (TIB-202) seeded at 5.0×10ˆ4 cells/cm2
Silver nanoparticles (AgNPs) at 3 and 30 mg/L
(Zhang et al. 2019)
Tetra-culture Calu-3, THP-1, HMC-1 seeded simultaneously at 10:2:1; (epithelial cells 1.0 × 10ˆ5 cells/apical; macrophage-like cells 0.2 × 10ˆ5 cells/apical and mast cells 0.1 × 10ˆ5 cells/apical) and EA.hy926 (2.0 × 10ˆ5 cells/basal)
Standard reference material (SRM) 1648a, an urban PM at 50 mg/ml
(Kim et al. 2017)
Cell model with seeding densities Exposure agent Reference Triple-culture:
16HBE14o- seeded at 10ˆ6 cells/six-well insert (=2.4x10ˆ5 cells/cm2)
MDM MDDC
Gasoline direct injection (GDI) exhaust particles (tenfold diluted)
(Bisig et al. 2018)
Co-culture HPF and CALU 3 seeded at 3 × 10ˆ5
cells/SW and 5 × 10ˆ5 cells/SW Whole cigarette smoke (WCS) or E-cigarette vapour (ECV)
(Vasanthi Bathrinarayanan et al. 2018)
Monoculture A549 seeded at 2.5 x 10ˆ5 cells/mL into 4.7 cm2
Cu nanoparticles (Kim et al. 2013) Monocultures 16HBE14o-, A549
Co-cultures of 16HBE14o-, A549, MDMs and MDDCs
Endotoxin
lipopolysaccharide (LPS) at 1 and 10 μg/mL for 4 h and 24 h
(Bisig et al. 2019)
Triple-culture
A549 at 1 × 10ˆ6 cells/insert MDM at 5 × 104 cells/insert MDDC at 25 × 104 cells/insert
Dry respirable volcanic ash (VA) at 0.26 ± 0.09 or 0.89 ± 0.29 μg/cm2 and single or repeated VA and diesel exhaust particles (DEP; 0.02 mg/mL)
(Tomašek et al. 2016)
In order to assess the respiratory toxicity, different parameters are measured in cell models following exposure. Cytotoxicity is usually determined by lactate dehydrogenase (LDH) quantification (Cachon et al. 2014, Boland et al. 1999, Zavala et al. 2017), the integrity of cell junctions evaluated by transepithelial electrical resistance (TEER) (Klein et al. 2013, Srinivasan et al. 2015) and cell viability measured by e.g. Resazurin test (Bengalli et al. 2017, Balogh Sivars et al. 2018). Some studies also assess cell morphology by e.g. laser scanning microscopy (Tomašek et al. 2016) and mucociliary clearance using microbead movement imaging (Huang et al. 2017). Commonly, pro-inflammatory cytokine release (e.g. interleukin 8 (IL-8), interleukin 6 (IL-6)) is determined with an enzyme-linked immunosorbent assay (ELISA) (Balogh Sivars et al. 2018). Furthermore, transmission electron microscopy (TEM) or scanning electron microscopy (SEM) are often applied in order to study the cellular morphology and particle interaction after exposure (Rothen-Rutishauser et al. 2005, Dekali et al. 2014, Loret et al. 2016, Wang et al. 2019).
Regardless of the fact that even the most complex co-culture models developed so far do not entirely mimic the actual in vivo conditions, they still represent a system allowing cell-to-cell communication compared to monoculture models and also aid in estimating the hazard of exposure to a certain pollutant (Klein et al. 2013). Moreover, it has been shown that a co-culture system produces an enhanced response in exposure studies compared to monocultures (Ishii et al. 2004, Wottrich et al. 2004, Loret et al. 2016). Even particle exposures of simple monocultures have proven to induce an inflammatory reaction (Becker et al. 2005) or increase
the number of human lung epithelial cells (Bayram et al. 2006). Similarly, soot particle exposure of a co-culture of peripheral human blood monocytes and bronchial epithelial cells caused an autocrine stimulation releasing inflammatory cytokines (Drumm et al. 2000) and ozone exposure of co-cultured bronchial epithelial cells and endothelial cells increased the IL- 6 and IL-8 release in comparison to monocultures of the two cell types (Mögel et al. 1998).
Comparatively, a co-culture of A549 and differentiated THP-1 produced an increase in release of cytokines compared to monocultures when exposed to ambient PM (Wottrich et al. 2004).
Moreover, it was shown that THP-1 monocultures exposed to combustion PM are not suitable to study toxicity pathways, which depend on physio-chemical properties of PM, when compared to a co-culture of A549 and THP-1 (Kasurinen et al. 2018).
According to Chary et al. (2018), the most representative in vitro alveolar model would consist of an alveolar type II epithelial cells (e.g. A549, NCI-H441), an endothelial cell line (e.g.
EA.hy926) forming the capillary lining, dendritic-like cells (e.g. THP-1) and macrophages (e.g.
differentiated THP-1) which serve as phagocytic defence against a foreign agent (Chary et al.
2018). It has been shown that macrophages as well as human pulmonary epithelial cells can phagocyte diesel exhaust particles leading to an increased release of pro-inflammatory cytokines (Mazzarella et al. 2006).
A549 is a human alveolar epithelial cell line, closely resembling the morphological and biochemical properties of human type II alveolar cells derived from human lung non-small cell adenocarcinoma (Srinivasan et al. 2015, Mazzarella et al. 2006, Cho et al. 2018).
Characteristically, the cell line exhibits similarities with human primary alveolar epithelial cells such as cytokeratin expression (Mazzarella et al. 2006). Examination with electron microscopy revealed cytoplasmic inclusion bodies usually present in lung epithelial type II cells in both early and late subculturing levels (Lieber et al. 1976). THP-1 is a monocytic human cell line derived from an infant with acute monocytic leukaemia (Tsuchiya et al. 1980), a macrophage representation commonly used in in vitro studies (Kasper et al. 2017). It has the cytologic, histochemical and functional characteristics of monocytes and similarly to human monocytes, boosts the cell reaction to LPS (Brand et al. 1991). EA.hy926 is a continuous endothelial cell line derived from a human somatic cell hybrid (Rieber et al. 1993) and exhibits several properties of vascular endothelium as it is able to morphologically re-organize forming an in vitro angiogenesis-like structure (Bauer et al. 1992).
Among different approaches in cell cultivation, the two which are most commonly used include submerged cell cultures in growth medium (GM) or culturing in ALI system, where the cells
are cultivated apically on semipermeable cell culture inserts and have direct contact with air (Klein et al. 2011). Most of the in vitro airway models use the membrane filter support for co- culturing several cell lines to imitate the ALI of the respiratory tract (Blume, Davies 2013). An improved imitation of inhalation exposure, higher reproducibility and no alteration of particle properties are among the advantages of cultivating in ALI, however it is a more complex and expensive approach compared to submerged cultivation systems (Klein et al. 2011). Moreover, ALI cultivation systems enable the direct contact to the entire particle exposure dose, whereas in submerged conditions the particles persist suspended in media, which does not reflect the particle deposition mechanism in human airways (Kim et al. 2013).
The correct evaluation of PM-induced toxicity in in vitro assays can be achieved by the utilisation of reliable controls (Bisig et al. 2019). For the exclusion of false negative findings, choosing the relevant control compounds is important (Chary et al. 2018). Consequently, an endotoxin lipopolysaccharide (LPS) is commonly applied as a positive control to the cell system (Loret et al. 2016), resulting in increased production of pro-inflammatory cytokines.
Corresponding negative controls are just as important – eg. untreated cell culture models (usually supplemented with growth medium only) or in case of PM samples as an exposure agent, procedural blanks are important to take during PM sampling.
3. AIM OF THE WORK
Aim of this study was to optimise the triple-culture model for studying indoor air PM toxicity using three different cell lines: human lung epithelial cells (A549), differentiated human macrophage-like cells (THP-1), and human endothelial cells (EA.hy926). Cellular toxicological response was characterised after exposure to cell culture media, synthetic LLF and two indoor air PM samples collected with different sampling methods. Monoculture models were compared with co-culture and triple-culture models. Furthermore, it was tested if the synthetic LLF is toxic itself and assessed whether the concentration and quality of RNA extracted after the exposure is adequate for transcriptome analysis.
4. MATERIALS AND METHODS
4.1 PM SAMPLING AND SAMPLE PREPARATION
The indoor air PM samples used in triple-culture exposure study were collected from a non- moisture damaged urban residence, using two different sampling methods. NIOSH BC251 two- stage bioaerosol cyclone sampler (National Institute for Occupational Safety and Health, NIOSH, Morgantown, WV, USA) was used to collect an air sample (stage 1) at 10 l/min throughout 7 days in 12-hour cycles with a total sampling time of 84 hours. The collected sample was stored at -20ºC. (Tirkkonen et al. 2016). Similarly, the Inspirotec electrostatic sampler was used to collect a 5-day integrated sample with a total sampling time of 60 hours at 89 l/min (Gordon et al. 2015). In both sampling methods, the procedural blanks were used during sampling time (no air flow), which served as control samples. PM-concentration during the sampling was monitored size-resolved with Lighthouse optical PM 3016-IAQ counter during the sampling (data not shown).
The synthetic LLF was prepared according to previous scientific publications (Rauprich et al.
2000, Slade et al. 1993, Sun et al. 2001) and consisted of 0.2 mg/ml cholesterol, 75 µg/ml palmitic acid, 75 µg/ml glyceryl tripalmitate and 1 μg/ml α–tocopherol which were added to a solution of 8 mg/ml dipalmitoylphosphatidylcholine, 1 mg/ml phosphatidylglycerol and phosphatidylethanolamine (0.2 mg/ml in chloroform and the chloroform was evaporated in nitrogen flow at room temperature). 0.5 mg/ml albumin, 50 μg/ml glutathione, 50 μg/ml ascorbic acid and 25 μg/ml uric acid were dissolved in Hank’s Balanced Salt Solution (HBSS) and added to the solution, stirred until homogenous and the pH adjusted with 0.2 M NaOH and 0.2 M H3PO4.
Prior to exposure, the PM samples were suspended in cell culture growth medium (Dulbecco’s modified Eagle medium (DMEM D5546; Sigma Aldrich) supplemented with 10% heat- inactivated FBS, 1% L-glutamine (Sigma Aldrich) and 1% penicillin-streptomycin (Sigma Aldrich) with vigorous agitation and sonication for 15 minutes, followed by dilution of the PM samples to four different concentrations (1:4, 1:8, 1:16, 1:32) in the synthetic LLF. Each dose was tested in quadruplicates.
4.2 CELL CULTURES
Three different continuous cell lines (ATCC, Rockville, MD, USA) were used in the triple cell culture model including human lung epithelial cells (A549), differentiated human macrophage-
like cells (THP-1) and human endothelial cells (EA.hy926). All cell lines were cultured in 75 cm2 cell culture flasks in 37°C, 5% CO2. EA.hy926 and A549 cells were subcultured twice a week by trypsinisation at confluency of 70-90%, while THP-1 cells were subcultured three times a week.
The EA.hy926 were maintained in ATCC growth medium (ATCC 30-2002) supplemented with 10% heat-inactivated fetal bovine serum, FBS (Sigma Aldrich, Darmstadt, Germany) and 1%
penicillin-streptomycin (Sigma Aldrich). The A549 and THP-1 cells were maintained in Dulbecco’s modified Eagle medium (DMEM D5546, Sigma Aldrich) supplemented with 10%
heat-inactivated FBS, 1% L-glutamine (Sigma Aldrich) and 1% penicillin-streptomycin (Sigma Aldrich). DMEM (D5546) supplemented growth medium as stated above, was also used with co-cultures in all exposure studies.
4.3 CO-CULTURE OPTIMISATION AND MODEL COMPARISONS
The triple-culture model was optimised to obtain the most optimal cell line ratios and exposure doses were tested with a positive control (LPS). Additionally, synthetic LLF toxicity was assessed to see if it was suitable for PM dilutions. Furthermore, the responses of monoculture, double-culture and triple-culture models were compared.
In order to obtain an optimal number of cells per insert for the final triple-culture model, several different seeding densities of each cell line were tested (Table 2). First, the trypsinised A549 were seeded in different densities (n=4) apically in a 24-well plate on semipermeable cell culture inserts (ThinCert™, Greiner Bio-One, 0.4 μm pore size, transparent, surface area of 0.336 cm2). Next, trypsinised EA.hy926 were seeded on the basolateral side of the inserts, testing 4 different densities (n=3) as shown in Table 2. Differentiated THP-1 were added on top of A549 on day 4 of co-culturing using 6 different seeding amounts after which the triple- culture was moved to ALI for 24 hours. On day 6, the co-cultures were checked for cell count and viability after which they were stained (Life Technologies Cell TrackerTM Red CMTPX and SYTO® 13 Green, Thermo Fisher Scientific, USA) and checked with fluorescence microscopy. The most optimal seeding density of each cell line for the triple-culture model was determined based on fluorescence microscopy observations checking the confluences of each cell line and looking for monolayered growth.
Table 2: The approximate seeding densities tested for each cell line in the triple-culture model during the optimisation process.
Cell line Approximate seeding densities tested (cells/cm2)
A549 3 000 6 000 9 000 12 000 15 000 18 000
EA.hy926 6 000 12 000 18 000 24 000
THP-1 6 000 12 000 18 000 24 000 30 000 36 000
A comparison of LPS-induced toxicological responses at ALI was also conducted for a monoculture, double-culture and triple-culture model (Figure 1). Each cell model was exposed to a 50 µl of LPS diluted in LLF in four different concentrations (0.1 µg/ml, 0.2 µg/ml, 0.4 µg/ml, 0.8 µg/ml) while the controls were exposed to LLF for 24 hours. The responses were assessed by measurements of cell counts and viabilities, TEER, total protein concentration and IL-8 release (as described in Chapter 4.5).
Figure 1: Monoculture, double culture and triple culture of human respiratory cells in ALI.
4.4 TRIPLE-CULTURE PM EXPOSURE STUDY
The final PM exposure study timeline and workflow is represented in Figure 2. In the developed triple-culture model, first the EA.hy926 cells were seeded on the basolateral side of the ThinCert inserts by inverting the inserts into the wells and allowing the cells to attach for two hours (37°C, 5% CO2). After EA.hy926 attached, the inserts were carefully oriented with the apical side up in the 24-well plate containing 400 μl of pre-warmed (37°C, 5% CO2) growth medium and the A549 cells were seeded apically by pipetting the cell suspension into the insert and mixed gently to ensure an even cell distribution throughout the insert’s membrane.
Figure 2: Timeline and workflow of the exposure study
After a 3-day incubation period (37°C, 5% CO2), the growth medium was changed for a fresh one on both sides of the inserts (200 μl on apical side and 400 μl on basolateral side of the insert) and the inserts were inspected with light microscopy. On the fourth day, the GM was changed, and the cells were examined with light microscopy. Third, the THP-1 were differentiated in 0.25 l/ml PMA in growth medium without FBS for about 30 minutes. When the cells began to attach to the bottom of the cell culturing flask, the PMA medium was removed, the cells were detached and washed with PBS, centrifuged (1200 rpm, 5 min) and re- suspended to fresh growth medium. Then, the medium from apical side of the inserts was removed and THP-1 seeded on top of the A549 cells. The THP-1 were allowed to adhere for 4 hours (37°C, 5% CO2) after which the growth medium was removed from the apical side of the inserts and the cell model was incubated for 24 h in ALI before exposure (Figure 3).
Figure 3: Triple-culture cell model for PM exposure
On exposure day, first 400 µL of fresh GM was added to the wells, followed by a 50 µL addition of each exposure agent in quadruplicates to the apical side of the inserts. The indoor air PM samples were diluted in synthetic LLF in four different concentrations (1:4, 1:8, 1:16, 1:32).
DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6
EA.hy926 and
A549 seeding Incubation
(37°C, 5% CO2) Incubation (37°C, 5% CO2)
Medium change,
microscopy THP-1 seeding Exposure
• EA.hy926 on basal side at
~6,000 cells/cm2
• A549 on apical side at ~12,000 cells/cm2
• Fresh medium
• Light microscopy to check cell growth
DAY 7
Analyses
• PMA treated THP-1 seeding on apical side at
~24,000 cells/cm2
• Fresh medium
• 4 h incubation èALI conditions
4 h incubation
• TEER
• Fresh medium
• Exposure to indoor air PM samples at 4 dillution ratios (1:4, 1:8, 1:16, 1:32) for 24 h
• Ending the exposure (150 µl of PBS to apical side)
• TEER
• Bradford assay
• IL-8
• TEM, SEM, Light microscopy
• RNA extraction
• Cell count and viability
LPS (0.01 µg/ml) was used as a positive control and growth medium as a negative control. In order to exclude the cell response from ALI conditions or LLF itself, control inserts with LLF and no exposure (incubator controls) were also included. Similarly, a set of controls was also made with exposure to PBS only. The exposure was ended after 24 hours (37°C, 5% CO2) by the addition of 150 µl PBS to the apical sides of the inserts.
4.5 ANALYSES AND MICROSCOPY
After exposure, first the TEER measurements were made, followed by collection of the aspirates that were stored at +4°C for further total protein analyses. The growth medium from the basal side of the inserts was collected and stored at -20°C for IL-8 determination. Selected inserts were fixed for SEM, TEM and light microscopy. In addition, cell count and viability were measured and RNA extracted.
4.5.1 Viability and cell count
Viability and cell count assay was performed for cells from the apical side of the selected inserts, including the apoptotic cells. The cells were detached from the inserts by trypsinisation and resuspension and centrifuged (5 min, 8000 rpm, +4°C). Furthermore, cells were stained with ChemoMetec Solution 13 containing Acridine orange (AO) for cell detection and nucleic acid stain DAPI to detect non-viable cells (ChemoMetec A/S, Allerøld, Denmark). Cell suspension was applied on NC-Slides A8™ and viability and cell count analysed with NucleoCounter® NC-3000™ (ChemoMetec, Denmark). The viability result was given as a percentage of viable cells and the cell count in cells/ml.
4.5.2 Transepithelial/transendothelial electrical resistance (TEER)
In order to measure the integrity of the triple-culture model, TEER was measured before and after exposure, using EndOhm chamber paired with EVOM2TM resistance meter (World Precision Instruments, Sarasota, FL, USA).
150 μl of warm PBS was added to the insert, 1 ml to the EndOhm chamber and the resistance was measured. A stabilised TEER value was determined for each insert. Blank controls consisting only of an empty insert with PBS were measured several times between the measurements.
In order to get the cell-specific resistance (Rcell layer), the average control value (RAVGblank) was substracted from each measurement (Rsample) and the end TEER value (RTEER) was calculated as:
𝑅𝑇𝐸𝐸𝑅 [𝛺. 𝑐𝑚2] = 𝑅𝑐𝑒𝑙𝑙 𝑙𝑎𝑦𝑒𝑟[𝛺] ∗ 𝐼𝑎𝑟𝑒𝑎 [𝑐𝑚2]
where Iarea represents the area of the cell culture insert membrane (0.336 cm2). Based on the above calculation, TEER values were expressed in Ω * cm2.
4.5.3 Bradford (protein) assay
The total protein concentration was analysed from the apical wash after exposure using the Bradford assay. 20 mg/ml BSA stock solution in PBS was used as a standard protein. All samples were diluted in PBS and pipetted into a microtiter 96-well plate (ThermoFisher Scientific, Denmark) in triplicates. After the Bradford reagent (Sigma Aldrich, Darmstadt, Germany) was added to all the wells, the plate was incubated for 5 minutes and the absorbance was measured at 570 nm with PerkinElmer Victor3. The total protein concentration was calculated based on the measured absorbance taking into account the used dilution.
4.5.4 Chemokine IL-8
The pro-inflammatory chemokine IL-8 was analysed by ELISA with a commercially available kit Human CXCL8/IL-8 DuoSet ELISA DY208 (R&D Systems, Abingdon, United Kingdom).
The concentration of IL-8 was quantified according to the manufacturer’s protocol using Nunc MaxiSorp™ 96-well plates (ThermoFisher Scientific, Denmark).
In short, the samples were diluted, applied along the standards to wells in triplicates and the absorbance was measured at 450 nm spectrophotometrically with multilabel reader (PerkinElmer Victor3). The final IL-8 concentration was calculated by comparing the results to the standard curve taking into consideration the corresponding sample dilutions.
4.5.5 RNA extraction and quality control
The RNA was extracted using RNeasy Plus Micro Kit including DNase treatment (QIAGEN, Hilden, Germany) according to manufacturer’s protocol. The cells were first harvested by direct lysis from the apical side of the inserts and the total RNA was eluted in 14 l RNase-free water, stored at -70°C and sent to Sequencing Unit of Finnish Institute for Molecular Medicine, FIMM (Helsinki, Finland) for further RNA quality control. Integrity and quantity of RNA were
verified by Caliper GX RNA LabChip (Perkin Elmer) and by Qubit RNA BR system (Thermo Fischer Scientific).
4.5.6 Electron and Light microscopy
In order to study the cellular layers and cell-particle interaction, selected samples were imaged with scanning electron microscopy (SEM; Zeiss Σigma HD / VP (Cambridge, UK), SmartSEM v. 5.07, Pathfinder 1.1 (ThermoFisher Inc, Madison, Wisconsin, USA) and transmission electron microscopy (TEM; JEOL JEM-2100F HR 200 kV field emission analytical electron microscope, Olympus Quemesa camera, Olympus iTEM software), both conducted by SIB Labs (University of Eastern Finland, Kuopio, Finland) following the protocols described below.
For SEM examination, the samples were fixed with a 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH=7.4) for at least 2 hours, then washed with a buffer solution for 20 minutes and dehydrated gradually with ethanol, dried with hexamethylene disilazane for 10 minutes, attached to sample holders and coated with approximately 50 nm layer of gold.
Inserts selected for TEM imaging were prefixed over night with a 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH = 7.4) and washed with 0.1 M phosphate buffer (pH = 7.4) for 20 minutes. Postfixation was conducted with 1% osmiumtetraoxide in 0.1 M phosphatebuffer (pH=7.4) for 1 hour, washed with 0.1 M phosphatebuffer (pH = 7.4) for 20 minutes, dehydrated with ethanol gradually and infiltrated overnight, embedded to a mould after which the samples were polymerised and cut into ultrathin (60-70 nm) sections and stained with 1% uranyl acetate for 30 minutes and lead citrate for 2 minutes.
Light microscopy (Zeiss AxioImager M2 light microscope, 40x objective, AxioCam MRc (Carl Zeiss) with high-resolution colour camera and Axiovision Rel. 4.8 software) was done in order to see the number of cellular layers and cell shapes in different exposures. The slides for light microscopy were separated from TEM blocks before sectioning step was performed with a thickness of 1 µm and coloured with toluidine blue. Slides were then observed with the light microscope and digital images were taken.
4.6 STATISTICAL ANALYSIS
All data is expressed as mean ± standard deviation (SD). Obtained data was analysed in GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA) and Excel (Microsoft, Redmond, WA, USA). In order to assess the differences between groups, nonparametric t-tests (Mann-Whitney test, Wilcoxon test) were used. A two-way ANOVA followed by a
Bonferroni’s post-hoc test was conducted in order to compare control to treated groups and between culture models. Values of p<0.05 were considered statistically significant.
5. RESULTS
5.1 CO-CULTURE OPTIMISATION AND MODEL COMPARISONS
Results of fluorescence microscopy revealed that the A549 were growing nicely in monolayers even in the highest seeding density tested (18 000 cells/cm2), however densities over 12 000 cells/cm2 appeared to have less confluent THP-1 cells seeded on top. Additionally, it was found that the EA.hy926 were forming clusters when seeded over 6000 cells/cm2. Therefore, the most optimal triple-culture model was obtained by culturing 12 000 A549/cm2 and 24 000 THP-1/cm2 apically and 6000 EA.hy926/cm2 on the basolateral side of the insert.
Interestingly, IL-8 secretion was up to 72-fold higher in double-cultures and up to 83-fold higher in triple-cultures compared to monocultures exposed to LPS (Figure 4). Additionally, there was a non-statistically significant difference in IL-8 concentrations between double- culture and triple-culture at all LPS doses. There was however, a statistically significant difference between monoculture and double-culture (p<0.0001) as well as monoculture and triple-culture (p<0.0001) in IL-8 release after LPS exposure at all four doses (Figure 4).
Figure 4: Differences in IL-8 release between mono, double- and triple-culture following exposure to LPS. There was a statistically significant difference between monoculture and double-culture (*; p<0.0001) as well as monoculture and triple-culture (*; p<0.0001) at all four doses. There was no statistical difference in controls between co-cultures (exposed to LLF only; LPS=0 µg/ml).
Figure 5 shows IL-8 secretion profiles expressed as % of control for each cell model. There was a non-statistically significant difference in monoculture IL-8 release at each dose compared to control while both double-culture and triple-culture models had an extremely statistically significant increase (p<0.0001) in IL-8 secretion at each LPS dose compared to the
*
*
* * Triple culture
Double culture Monoculture
corresponding control with an up to an almost 10-fold increase in IL-8 at some LPS exposure doses.
Figure 5: IL-8 secretion profiles in monoculture (A), double-culture (B) and triple-culture (C) model expressed in
% of control ± SEM, n = 4 (LLF exposure only) and significance levels (****, p<0.0001; (two-way ANOVA followed by Bonferroni post-hoc test comparing control means with each dose mean within each model).
TEER values of monoculture, double-culture and triple-culture models exposed to LPS and compared to controls are presented in figure 6. Resistance of the cell layer was not affected by LLF nor LPS exposure.
Figure 6: TEER profiles expressed as mean ± SD before and after LPS exposure in monoculture (A), double- culture (B) and triple-culture (C). Marked statistically significant differences represent differences in TEER value according to the corresponding LPS exposure dose compared to control value in each individual cell model (two- way ANOVA followed by Bonferroni post-hoc test; p<0.0332 (*), p<0.0021 (**), p<0.0002 (***), p<0.0001 (****)).
Cell layer resistance differed statistically significantly between mono- and triple-cultures and also between double and triple-cultures before and after exposure (Figure 7; Appendix 1).
Monoculture Double culture Triple culture
Monoculture Double culture Triple culture
Figure 7: TEER measurements before (A) and after (B) LPS exposure. There were statistically significant differences between monocultures and triple-cultures as well as between double-culture and triple-culture model in both, before and after exposure (two-way ANOVA followed by Bonferroni post-hoc test; see Appendix I for more details on statistically significant differences)
LPS exposure did not induce protein secretion in any of the cell culture models. Figure 8 shows differences in cell counts and viabilities in different cell models following exposure to different LPS concentrations. Compared to corresponding controls, cell counts decreased in all models in all exposure doses, however a statistically significant decrease in all exposure doses was noted only in triple-culture model. Similarly, a statistically significant decrease in viability was only evident at three LPS exposure doses in triple-culture model.
Figure 8: Cell count (A) and viabilities (B) for each cell model expressed as % of corresponding controls for each cell model tested. Data is shown as mean ± SEM with indicated statistically significant differences compared to controls (two-way ANOVA followed by a Bonferroni’s post-hoc test; p<0.0332 (*), p<0.0021 (**), p<0.0002 (***), p<0.0001 (****))
5.2 TRIPLE-CULTURE PM EXPOSURE STUDY
There was no statistically significant difference in cell counts between GM, LLF, LPS, ALI and PBS controls. A statistically significant decrease in viability was observed only in cells exposed to LPS in comparison to GM (p=0.0322). Because of the small sample size of analysed groups exposed to NIOSH and Inspirotec PM samples (n=1 per exposure dose) the statistical analysis
Monoculture Double culture Triple culture
Monoculture Double culture Triple culture
A B
Monoculture Double culture Triple culture
Monoculture Double culture Triple culture
of differences in cell counts and viabilities was not possible, nevertheless, there were no clear differences.
Differences in mean TEER values in controls are presented in Table 3. A statistically significant difference before and after exposure was observed only in LLF and ALI controls.
Table 3: TEER values of controls (growth medium n=7; LLF n=8, LPS n=4, ALI n=8, PBS n=4) before and after exposure presented as mean ± SD. Differences between time points for each control were analysed by a Wilcoxon test (p<0.0021 (**)).
Control TEERbefore exposure (Ohm*cm2) TEERafter exposure (Ohm*cm2)
Blank, GM 16,0 ± 3,0 20,1 ± 5,5
LLF ** 17,1 ± 2,9 19,3 ± 3,7
LPS 17,1 ± 5,8 17,7 ± 3,7
ALI ** 15,4 ± 3,8 32,1 ± 10,7
PBS 18,3 ± 3,6 23,6 ± 2,7
There was no statistically significant difference in mean TEER values between NIOSH Control before and NIOSH 7-day sample before exposure or between NIOSH control after and NIOSH 7-day sample after exposure. Interestingly, TEER values of NIOSH Control after exposure increased statistically significantly compared to before exposure values at 1:16 and 1:4 (Figure 9). In addition, there was a statistically significant increase in NIOSH 7-day TEER values after exposure at dilution factors 1:32 and 1:16, compared to before exposure values.
Figure 9: TEER values between NIOSH Controls (A) and NIOSH 7-day sample (B) before and after exposure presented as mean ± SD (n=4; two-way ANOVA followed by a Bonferroni's post-hoc test; p<0.0332 (*), p<0.0021(**))
Similar to NIOSH PM exposure samples, there were no statistically significant differences in TEER values between Inspirotec control before and the 5-day sample before exposure or
1:32 1:16 1:8 1:4 0
10 20 30 40
PM sample dillution factor TEER (Ohm*cm2)
NIOSH,Control,BE NIOSH,Control,AE
* **
A
1:32 1:16 1:8 1:4 10
15 20 25 30
PM sample dillution factor TEER (Ohm*cm2)
NIOSH 7D,BE NIOSH 7D,AE
* *
B
between Inspirotec control after and 5 day integrate after. Interestingly, there was a statistically significant increase (p=0.0033) in Inspirotec 5-day sample mean TEER value after exposure compared to before exposure at dilutions 1:16, 1:8 and 1:4 (Figure 10).
Figure 10: TEER values between Inspirotec Controls (A) and Inspirotec 5-day sample (B) before and after exposure presented as mean ± SD (n=4; two-way ANOVA followed by a Bonferroni's post-hoc test; p<0.0332 (*), p<0.0021 (**))
There was a statistically significant difference in protein amounts between growth medium control an all other individual control comparisons (GM vs. LLF p=0.0001; GM vs. LPS p=0.0006; GM vs. ALI p<0.0001 and GM vs. PBS p<0.0001). Additionally, there was a statistically significant difference in protein concentration (n=4) between NIOSH control and the 7-day integrate at dilution factor 1:4 (p<0.0002) as well as between Inspirotec control and the 5-day integrate at the same dilution (p <0.0001).
There was a statistically significant (p<0.0001: LPS vs. GM, LPS vs. LLF, LPS vs. ALI, LPS vs. PBS) increase in IL-8 secretion in triple-culture cell models exposed to LPS compared to other controls (growth medium, LLF, ALI and PBS). IL-8 secretion increased statistically significantly after NIOSH sampled PM exposure in dilutions 1:32 and 1:4. Similarly, Inspirotec sampled PM increased IL-8 secretion statistically significantly in dilution 1:4. (Figure 11).
1:32 1:16 1:8 1:4 10
15 20 25
PM sample dillution factor TEER (Ohm*cm2)
A
Inspirotec, Control, BE Inspirotec, Control, AE
1:32 1:16 1:8 1:4 15
20 25 30
PM sample dillution factor
TEER (Ohm*cm2) ***
B
Inspirotec, 5D, BE Inspirotec, 5D, AE
*
Figure 11: IL-8 release following exposure to different dilutions of NIOSH (A) and Inspirotec (B) PM samples.
Data is expressed as mean ± SD (n=4; two-way ANOVA followed by Bonferroni's post-hoc test p<0.0332 (*), p<0.0001 (****))
Interestingly, the released IL-8 amounts in Inspirotec exposed models were lower compared to NIOSH PM sample exposure. The IL-8 concentrations measured from triple-culture models exposed to NIOSH 7-day sample were statistically significantly higher at dilutions 1:32, 1:16 and 1:4 compared to models exposed to Inspirotec 5-day sample, while there was no statistically significant difference between the controls.
RNA was extracted from PM-exposed cells. However, the amount and quality of RNA was inadequate for RNA sequencing studies.
Light microscopy images revealed that cells in both apical and basolateral side were growing as monolayer except for some individual point overlapping on apical side (Figure 12).
Figure 12: Light microscopy image of A) control and B) PM exposed triple culture stained with toluidine blue on semipermeable cell culture insert.
1:32
1:16 1:8 1:4
0 2000 4000 6000 8000 10000
Inspirotec, control Inspirotec, 5D
The PM sample dillution factor
IL-8 concentration (pg/ml) *
B
SEM images confirmed the uniform cellular layer in the triple-culture model in both sides of the cell culture membrane (Figure 13). An interaction of THP-1 cells with the particulate matter can also be observed in Figure 13 (C, D).
Figure 13: SEM images of A) epithelial cells and macrophages, B) endothelial cells, and C-D) PM-exposed cells.
Tight extracellular space was observed in TEM images of ALI and blank control exposed cells as shown in Figure 14.
Figure 14: TEM images of ALI (A) and blank control (B) exposed cells.
TEM images of sampler controls and PM exposed cells are presented in Figure 15, clearly showing loosened extracellular space after PM exposure.
Figure 15: TEM images of Inspirotec control (A), PM exposed cells (Inspirotec) (B), NIOSH control (C) and PM exposed cells (NIOSH) (D).
6. DISCUSSION
A triple-culture model of human alveolar basal epithelial cell line, a monocytic human cell line and a human endothelial cell line was developed in combination with a synthetic LLF to better imitate the pulmonary air-blood barrier in vivo. Several cellular responses were tested after exposure to two different indoor air PM samples.
Synthetic LLF was not acutely toxic for the pulmonary cells. However, composition of LLF may vary between batches (Figure 16) due to mixing challenges during manufacturing process.
Therefore, it is recommended to test each batch of LLF before applying it to the cells.
Figure 16: Failed triple-culture PM exposure experiment due to LLF inconsistency - example of a triple-culture GM control (left; Zeiss microscope, 4x magnification) and LLF control (right; Zeiss microscope, 4x magnification).
The release of inflammatory agents can indicate a potential toxic effect. It has been shown that exposure of a co-culture model at ALI to metal oxide nanoparticles promotes a statistically significant inflammatory response apically (Bengalli et al. 2013). The co-culture comparison proved that the triple-culture model is far more sensitive compared to monocultures as it elicited a much higher cellular response in the measured inflammatory IL-8 when exposed to different LPS concentrations. As there was no clear dose response, lower LPS concentrations should probably be included. Since the lowest dose (0.1 µg/ml) produced statistically significantly higher IL-8 release compared to control, LPS dose used for positive control in PM exposure study was even lower (0.01 µg/ml), similarly provoking a higher IL-8 release compared to other controls. Results of measurements after LPS exposure therefore indicate that the triple-culture model responded well in all measured endpoints. Interestingly, similar studies using LPS as a
