Assessing the in vitro toxicity of airborne (nano)particles to the human respiratory system: from basic to advanced models

Figures & data

Figure 1. Respiratory tract: cell types and morphology. This illustration was created including images obtained from Smart Servier Medical Art (www.smart.servier.com) CC by 3.0.

Table 1. Main anatomical and physiological differences between humans’ and rodents’ respiratory system. [Adapted from Harkema et al. (2012); Meyerholz et al. (2018); Rackley and Stripp (2012)].

Features	Humans	Rodents
Nose and sinus	Relatively simple nasal turbinate structure; total surface area: 150–200 cm^2.	Complex nasal turbinate structure; total surface area: 2.9 cm^2 [relative surface area (surface area/volume) 5× greater than humans].
	Accessory olfactory organs are not well developed or functional.	Presence of functional accessory olfactory organs.
	Olfactory epithelium ~3% of the nasal cavity.	Olfactory epithelium ~50% of the nasal cavity (well-developed sense of smell).
Pharynx and larynx	Widely disseminated and well-defined pharyngeal tonsils.	Lack of distinct pharyngeal tonsils; widely dispersed lymphoid aggregates.
	No U-shaped laryngeal cartilage and ventral pouch.	Presence of U-shaped laryngeal cartilage and ventral pouch (larger/more prominent in rats than mice).
	Absence of taste buds in this region.	Taste buds are located within the epiglottis, larynx, and pharynx.
Trachea	Trachea internal diameter of ~12 mm.	Trachea internal diameter of ~1.5 mm (mouse).
	Trachea lined by ciliated cells.	The trachea is mostly lined by non-ciliated epithelial cells.
	The submucosa contains numerous tightly packed seromucinous glands.	Submucosal glands are restricted to the proximal (closest to the larynx) trachea.
	Tracheal cartilaginous rings extend for several bronchial generations into the lung.	Tracheal cartilaginous rings are only present in the extrapulmonary airways.
Lungs	The right lung of humans is divided into three lobes, whereas the left lung has two lobes.	The right lung is divided into four lobes, whereas the left lung has one lobe.
	Dichotomous airway branching.	Monopodial airway branching.
	The respiratory zone includes respiratory bronchioles, alveolar ducts, and alveoli.	Respiratory zone includes diminutive respiratory bronchioles (if present), alveolar ducts, and alveoli.
Bronchi to terminal bronchioles	Lung parenchyma includes both bronchi and bronchioles.	Lack of well-developed respiratory bronchioles (defined by lack of cartilage and submucosal glands).
	Relatively abundant mucin-secreting goblet cells.	Less than 1% mucous goblet cells in the epithelium of extrapulmonary bronchi (particularly in adults mice maintained under lab conditions).
Branching pattern	Symmetric (airflow pattern more susceptible to deposition at its bifurcation points).	Asymmetric or monopodial (relatively unimpeded flow).
Breathing mode	Oronasal breathers (both nasal and oral breathing).	Obligate nose breathers (all inhaled air passes through the nasopharynx on its path into the lungs).
Lung organogenesis	~week 3 during human development (average 40 weeks of gestation) as the primitive lungs bud from the foregut endoderm.	~embryonic day 9 in the mouse (average 19–21 days of gestation).
	Undergo many additional rounds of branching before beginning alveolarization.	Mouse lungs develop quickly and do not begin forming alveoli until after birth.

Figure 2. Inhaled particles: deposition mechanisms along the respiratory tract. This illustration was created including images obtained from SlidesCarnival (https://www.slidescarnival.com) CC by 4.0.

Figure 3. Schematic representation of the in vitro models addressed in the present review: (a) Mono- and co-cultures of respiratory cells under submerged and (b) air-liquid interface culture conditions; (c) Spheroid and organoids; (d) 3D cultures and (e) Microfabricated human breathing-inspired lung-on-a-chip microfluidic device [Adapted from Huh et al. (2010)]. The device simulates physiological breathing movements by applying a vacuum into the side chambers causing a stretch of the membrane. This illustration was created including images obtained from Smart Servier Medical Art (www.smart.servier.com) CC by 3.0. The relative costs of each cell system are indicated using a 1 to 4 level ($-).

Table 2. Human epithelial cell lines commonly used in in vitro (nano)particle pulmonary toxicity studies.

Lung region	Human cell line
	Identification	Origin	Features
Bronchial	16HBE14o	Normal bronchial epithelium; Virus transformed (adenovirus 12-simian virus 40 hybrid virus).	Ability to form tight junctions; Express cilia when grown at the air-liquid interface (ALI); Retain important properties and functions of differentiated airway epithelial cells (e.g. mucus-secreting capacity, apical microvilli, etc.).
	BEAS-2B	Normal bronchial epithelium; Virus transformed (adenovirus 12-simian virus 40 hybrid virus).	Do not form tight junctions; Resemble airway basal epithelial cells, however, do not differentiate; Poor barrier function.
	Calu-3	Lung adenocarcinoma from submucosal gland serous cells.	Ability to form tight junctions; Express mucins and some cilia when grown at ALI; Reasonable barrier function.
Alveoli	A549	Lung adenocarcinoma.	Display some characteristics of alveolar epithelial type II (AEC2) cells; Express metabolizing phase I (cytochrome P450 isoenzymes) and phase II enzymes (transferases); Do not form tight junctions; Ability to produce surfactant when grown at ALI; Poor barrier function.
	NCI-H441	Lung papillary adenocarcinoma.	Resemble characteristics of both type II pneumocytes and clubcells; Poor barrier function.
	hAELVi	Lentivirus immortalized.	Morphologically resemblance with alveolar type I (AEC1) cells; Express high levels of metabolizing enzymes and transporters; Ability to form tight junctions; Ability to produce surfactant.

Table 3. Pulmonary (nano)particle toxicity studies using coculture models mimicking relevant human lung barriers.

Human lung barrier	Coculture model (cell lines used)	(Nano)particles tested	Main findings	References
Alveolar-capillary barrier	NCl-H441 and ISO-HAS-1.	SiO2 NP (0.6–6000 μg/mL; submerged conditions; 4 h exposure +20 h recovery period).	Release of IL-6 and IL-8 (early inflammatory events); upregulation of apoptosis markers.	Kasper et al. (2011)
Air-blood barrier	NCI-H441 and HPMECST1.6 R.	CuO and TiO2 NP (25 μg/mL) and PM 10 (22.5 μg/mL) (submerged conditions; 24 h exposure).	Significant modulation of pro-inflammatory (e.g., IL-6 and IL-8) proteins.	Bengalli et al. (2013)
Alveolar epithelial barrier	A549, THP-1 and HMC-1.	SiO2-Rhodamine NP (10 mg/L; ALI conditions; 24 h exposure).	Lower levels of ROS and IL-8.	Klein et al. (2013)
Alveolar epithelial barrier	A549, MDDC and MDM.	MWCNT (1.15 μg/cm^2; ALI conditions; repeated exposure: 3 days).	No cytotoxicity, alterations in cell morphology, or increase in pro-inflammatory markers.	Chortarea et al. (2015)
Lung – blood barrier	Biculture of 16HBE14o and THP-1; triculture of 16HBE14o, THP-1, and HLMVEC.	TiO2, Ag and SiO2 NP (1–243 μg/mL; submerged conditions; 24 h exposure).	Biculture: TiO2 and Ag NP but not pristine SiO2 NP induced cytotoxic effects at relative high doses (83–243 μg/mL); Triculture: no considerable changes were observed.	Smulders et al. (2015)
Alveolar epithelial barrier	A549 and THP-1.	CeO2 and TiO2 NP (1–20 μg/cm^2; submerged and ALI conditions; 24 h exposure).	Inflammation, decreased cell viability, and oxidative stress were observed at ALI; more predictive of in vivo effects.	Loret et al. (2016); Loret et al. (2018)
Air epithelial barrier	hAELVi and THP-1.	Ag (7.25 μg) and starch (41.25 μg) NP (ALI conditions; 24 h exposure).	Cocultures form functional diffusion barriers under ALI conditions.	Kletting et al. (2018)
Air – blood barrier	Biculture of A549 and EA.hy926; triple coculture of A549, THP-1, and EA.hy926.	Ambient PM 2.5 collected from Shanghai city (China) (20–180 μg/mL; ALI conditions; 24 h exposure).	Stronger inflammatory responses and ICAM-1 and caveolin-1 mRNA expression in triculture than in biculture system.	Wang et al. (2019)
Lung epithelial barrier	hAELVi and huAEC.	CeO2 NP (0.1–200 μg/mL; ALI conditions; 24 h exposure).	CeO2 NP induced no toxicity, while ZnO NP were toxic at concentrations between 10–50 μg/mL.	Leibrock et al. (2019)
Air – blood barrier	A549, THP-1, HMC-1 and EA.hy926.	Ag NM (spherical particles, PVP coated nanowires) (0.05–5 μg/cm^2; ALI conditions; 6 and 24 h exposure).	Increased cytotoxicity; increased mRNA levels of the pro-apoptotic gene CASP7, anti-oxidant enzyme HMOX-1, and pro-inflammatory mediators; induction of the NF-kB nuclear translocation.	Fizesan et al. (2019)
Lung – blood barrier	Calu-3, THP-1, and EA.hy926.	Ag NP (coated with tannic acid) (3–30 μg/cm^2; submerged conditions; 24 h exposure).	NP cellular uptake and translocation of Ag NP through the modeled barrier; mild cytotoxicity and reduced secretion of IL-6, IL-8, and TNF-α.	Zhang et al. (2019)
Air-blood barrier	A549, EA.hy 926 and THP-1.	Citrate-capped Au (50 mg/mL), Ag-SiO2 (0.50 mg/mL), and CuO (1.5 mg/mL) NP (ALI conditions; 4 h exposure).	Induced more oxidative stress and higher IL-8 levels.	Wang et al. (2020)

Table 4. Advanced 3D in vitro models of human respiratory tissues.

Model		Features	Advantages and limitations
MucilAir™ (Epithelix Sàrs)	nasal, tracheal, or bronchial epithelial model.	Isolated from human biopsies of the nasal cavity, trachea, and bronchi; Constituted by basal, goblet, and ciliated cells; Under ALI conditions displays tight junctions, cilia beating and mucus production, cytokines, chemokines, and metalloproteinases secretion, and expression of specific respiratory epithelia cytochromes (e.g., P450);	Effective barrier model for the assessment of the permeability/absorption of several compounds Long shelf-life Option of coculture with fibroblasts available Donor with several pathologies available More expensive than conventional cultures
SmallAir™ (Epithelix Sàrs)	small airway model.	Isolated from the distal lung; Constituted by epithelial cells, a large number of club cells, and fewer goblet and ciliated cells; Presents a much thinner epithelium (compared to MucilAir™).	Long shelf-life Option of coculture with fibroblasts available Donor with several pathologies available There are still a limited number of studies using this model More expensive than conventional cultures
EpiAirway™ (MatTek Corporation)	tracheal/bronchial epithelium model.	Derived from normal tracheal and bronchial epithelial cells; Constituted by mucus-producing goblet cells, ciliated cells with actively beating cilia, basal cells, and club cells (club).	More than 30 donors available More expensive than conventional cultures
EpiAlveolar™ (MatTek Corporation)	lower respiratory tract tissue model.	Constituted by alveolar epithelial cells and monocyte-derived macrophages (apical side) and pulmonary endothelial cells (basolateral side).	Remains stable for studies lasting more than 30 days. Option of coculture with fibroblasts available More expensive than conventional cultures
Micro-Lung™ (Cardiff University)	bronchial epithelium model.	Isolated from surgical patients and postmortem donors; Constituted by normal human bronchial epithelial (NHBE) cells, basal, serous, club, goblet, and ciliated cells.	Not commercially available No studies in the literature regarding (nano)particle toxicity
Metabo-Lung™ (Cardiff University)	lung-liver model.	Coculture of NHBE cells with primary human hepatocytes, which allows the biotransformation of inhaled toxicants in an in vivo-like manner.	Not commercially available No studies in the literature regarding (nano)particle toxicity

Table 5. Overview of the existing pulmonary (nano)particle toxicity studies using advanced 3D in vitro models.

Pulmonary 3D model	(Nano)particles tested	Experimental design	Biological endpoints assessed	Main findings	References
MucilAir™ (Epithelix Sàrs)	CeO2 NP	ALI (droplet) exposure; responses assessed after 3, 24, 48 h.	Cytotoxicity (TEER, LDH release); Inflammation (IL-8, MCP-1, sICAM-1, IL-1α, TNFα); Genotoxicity (comet assay and HO-1 expression).	No major toxic effects were observed.	Frieke Kuper et al. (2015)
	MWCNT	Cultures from healthy and asthmatic donors; ALI exposure (ALICE system); cells exposed 5 weeks/5 days per week; responses assessed at weeks 1, 3, and 5.	Cell morphology Cytotoxicity (LDH release); Inflammation (IL-8, IL-6, IP-10 and TGF-β); Oxidative Stress (HMOX-1 and SOD-2 gene expression).	Increased cilia beating frequency; alterations in the mucociliary clearance; no cytotoxicity or morphological changes; (pro)inflammatory and oxidative stress responses after chronic exposure.	Chortarea et al. (2017)
	CuO NP	ALI exposure (Vitrocell® system); Cells exposed for 2 days (two periods of 1 h/day); responses assessed 24 h after exposure.	Cytotoxicity (LDH release); Gene expression of inflammatory markers (MCP-1, IL-8, and IL-6).	No major cytotoxicity; increased expression of inflammation markers (MCP-1 and IL-6).	Kooter et al. (2017)
	CeO2, Mn2O3, CuO, ZnO, Co3O4 and WO3 NP	ALI (droplet) exposure (MucilAir cultures: 24 h; DC cultures: 48 h); responses assessed after 24 h (MucilAir) and 48 h (DC).	Cytotoxicity (LDH release); Inflammation (MCP-1, IL-6, IL-8); DC maturation surface markers (HLA‐DR, CD80, CD83, and CD86)	MucilAir™ cultures: inflammatory responses (increased levels of IL-6, IL-8, and MCP-1) after CuO NP exposure; DC cultures: Mn2O3 NP upregulated all the assessed maturation biomarkers.	Dankers et al. (2018)
	Milled W NP	ALI exposure; single 24 h exposure; responses assessed following exposure and up to 28 days.	Cell viability (Trypan blue); Metabolic activity (resazurin assay); Inflammation (IL-8).	Decrease in barrier integrity; no effect on metabolic activity or cell viability; transient increase in IL-8 secretion at 24 and 96 h after initial exposure.	George et al. (2019)
	SiO2 NP	ALI (droplet) exposure (Vitrocell® Cloud system); daily exposure for 5 times per week, up to 12 weeks.	Cell viability (Alamar Blue); Barrier integrity (TEER).	No changes in the barrier integrity; no cytotoxic effects.	Di Cristo et al. (2020)
	ATO and ZrO2 NP; PGFP (<2.5 µm) and PGNP (0.200 µm) particle fractions derived from high velocity oxy-fuel spraying.	ALI (droplet) exposure (Vitrocell® Cloud system); responses assessed after 24, 48, and 72 h.	Cytotoxicity (LDH release and WST-1 metabolic activity); Genotoxicity (Comet assay); Inflammation (IL-8 and MCP-1).	Mild cytotoxicity at early time points (24 h), cellular recovery at late time points (72 h), and no major genotoxicity for ATO and ZrO2 NP; PGFP affected cell viability, while PGNP caused increased oxidative DNA damage.	Bessa, Brandão, Fokkens, Cassee et al. (2021)
SmallAir™ (Epithelix Sàrs)	Ag NP	ALI exposure (Cultex® RFS system); 3 exposure times (7, 20, and 60 min); responses assessed at 6 or 24 h.	Cytotoxicity (lactate and LDH release); Alterations in immune-related gene expression (real-time PCR analysis for mRNA gene expression).	Minimal cytotoxicity and significant upregulation in the expression of inflammatory-related genes (e.g. IL1R2).	Guo et al. (2018)
EpiAirway™ (MatTek Corporation)	Airborne particulates collected in woodworking and metalworking industries	Submerged exposure; repeated exposures (4 h, 8 h and 72 h)	Cytotoxicity (MTT assay); Gene expression (GAPDH, Gelsolin, Caspase-3, IL-6)	Marked decrease in tissue viability after 8 h (woodworking particles) and a slight reduction after 72 h exposure (metalworking particles); suppressed expression of gelsolin, caspase-3 and IL-6	Pavlovska et al. (2021)
EpiAlveolar™ (MatTek Corporation)	MWCNT	ALI (droplet) exposure (Vitrocell® Cloud system); repeated exposure (3 weeks).	Cytotoxicity (LDH release); Inflammation (IL-1β, TNF-α and IL-8); Profibrotic response (TGF-β, fibronectin, and COL1 release).	Barrier integrity and release of pro-inflammatory and profibrotic markers.	Barosova et al. (2020)

ALI: Air-liquid interface; ALICE: Air-liquid interface cell exposure; ATO: Antimony-tin oxide; COL1: Collagen 1; DC: Dendritic cells; LDH: Lactate dehydrogenase; IL1R2: Interleukin 1 Receptor Type 2; MWCNT: Multiwalled carbon nanotubes; PCR: Polymerase chain reaction; PGFP: Process-generated fine particles; PGNP: Process-generated nanoparticles; RFS: Radial flow system; TEER: Transepithelial electrical resistance.

Table 6. Aerosol generation and exposure systems for cells cultured under air-liquid interface (ALI) conditions.

Aerosol exposure system	Developer/Manufacturer	Features
Minucell	Tippe, Heinzmann, and Roth (2002)	Uses direct flow allowing a greater particle deposition onto cells.
Electrostatic Aerosol in vitro Exposure system (EAVES)	de Bruijne et al. (2009)	Uses electrostatic precipitation for particle deposition onto cells.
Air-Liquid Interface Cell Exposure (ALICE)	Lenz et al. (2009)	Uses dense cloud of droplets generated and transported through an exposure chamber at a specific flow rate, allowing uniform and efficient depositions of particle aerosols; Considered an optimal system to screen NP’ toxicity, particularly in pharmaceutical industries where suspension-based aerosolized NP are used in drug formulations (Duret et al. 2012).
NAVETTA	Frijns et al. (2017)	Applies an electrostatic field to improve particle deposition efficiency, allowing a much higher deposition rate when compared to other ALI setups.
Nano Aerosol Chamber In vitro Toxicity (NACIVT)	Jeannet et al. (2015)	Uses direct flow in a continuous air stream, enabling a more efficient and uniform deposition of airborne (nano)particles onto cells; Computer-controlled temperature and humidity environment, which allows long-term exposures and helps to prevent cellular stress during exposure; Compact and easy to transport; High throughput system; Commercially available.
MicroSprayer® Aerosolizer	Penn (2021)	Commercially available manual aerosolizer; Model IA-1C: requires a very small number of NP to reach the target dose; Leads to the formation of droplets, affecting the homogeneous deposition of particles onto cells; Requires high flow rates for effective particle deposition, which might damage cells by shear stress.
CULTEX®	Aufderheide and Mohr (1999)	Uses electrostatic precipitation for NP deposition onto cells, ensuring close contact between the tested aerosol and cells without any interference of the culture medium; Available as Radial Flow System (RFS) and RFS Compact versions; Popular system among the commercially available.
Vitrocell®	Aufderheide and Mohr (2004)	Modified CULTEX® system; Three available setups: Cloud® system, powder chamber, and automated exposure station; Offers exposure chambers for 6, 12, and 24 well-plates; Most used among the commercially available.
XposeALI®	Inhalation Sciences (2021)	Models adapted to aerosolize dry powders; Commercially available.

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Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.