Three-dimensional in vitro alveolar lung model for determining and / or predicting the sensitizing effects of inhalable anhydrides

Abstract

A three-dimensional in vitro alveolar lung model including essentially the five cells types as follows: alveolar type II epithelial cells able to secrete (lung laying) surfactant, endothelial cells which forms the inner lining of capillaries providing a permeable barrier, alveolar dendritic-like cells able to protect inhalable sensitizers and initiate immune response, plasmacytoid dendritic-like cells linking innate and adaptive immunity and macrophage-like cells, able to participate to defense mechanisms by ingesting foreign materials by phagocytosis. Also, a process for preparing this model, and its use for assessing the irritation potential or toxicity of inhalable products on the alveolar barrier of lungs, and also for determining and / or predicting the sensitizing effects of inhalable products on the alveolar barrier of lungs.

Description

THREE-DIMENSIONAL IN VITRO ALVEOLAR LUNG MODEL FOR DETERMINING AND / OR PREDICTING THE SENSITIZING EFFECTS OF INHALABLE ANHYDRIDESFIELD

[0001] The present invention relates to a three-dimensional in vitro alveolar lung model and the process for preparing said model for assessing the irritation potential, toxicity, or sensitizing effects of inhalable products.BACKGROUND

[0002] The widespread use of synthetic compounds, such as fragrances and industrial solvents, has led to an exponential increase in the number of substances to which we are potentially exposed on a daily basis. This has contributed to the rise in respiratory disorders, such as asthma. While asthma can be triggered by genetic predisposition, environmental factors also play a significant role. Asthma can develop after exposure to protein allergens like animal dander, house dust mites, or pollen, but it can also be induced by chemicals. This is particularly common in the workplace, leading to occupational asthma, but exposure to chemicals in everyday products, such as cosmetics or household items, can also contribute. In response to the increased use of chemical products, legislation has been introduced to protect consumers from the associated risks.

[0003] One of the clinical manifestations of asthma and other diseases resulting from exposure to chemicals, such as allergic rhinitis, rhino-conjunctivitis, and sinusitis, is respiratory sensitization. Respiratory sensitization refers to an immune system reaction that occurs in the lungs following repeated exposure to certain substances, potentially leading to allergic or hypersensitive responses. This process involves the activation of the immune system, which can trigger inflammation and other symptoms upon subsequent exposure to the same substance. Over time, respiratory sensitization contributes to the development of allergies, potentially leading to systemic effects and an increase in morbidity.

[0004] Anhydrides, such as phthalic anhydride, trimellitic anhydride (TMA), and hexahydrophthalic anhydride (HHPA), are a class of chemicals widely used in industrial and commercial applications, including the production of resins, coatings, adhesives, andpharmaceuticals. These compounds, particularly acid anhydrides, are known to be potent respiratory sensitizers. Upon inhalation, these compounds react with proteins in the respiratory tract, forming hapten-protein complexes that are recognized by the immune system. This triggers a cascade of immune responses, leading to inflammation and the development of respiratory sensitization. With repeated exposure, symptoms of asthma, such as wheezing, coughing, and shortness of breath, can develop.

[0005] Regulatory and preventive measures are essential to minimize exposure and protect workers from the long-term health effects associated with these compounds. Recently, testing laboratories and regulatory agencies have collaborated to address these challenges. To better characterize the potential risks of these chemicals, it is crucial to fully understand the mechanisms involved in lung respiratory sensitization.

[0006] In the past, animal studies have provided valuable insights into the mechanisms underlying the development of respiratory allergies. However, our understanding of the cellular effects involved remains limited. Various methods have been proposed to identify and differentiate the functional and structural features of allergens, but conclusive results have not been achieved, largely due to biogenic and environmental confounding factors. This knowledge gap hinders the development of predictive in vitro models, leaving in vivo models as the primary tool for characterizing respiratory allergens. Despite the absence of validated methods for identifying and characterizing respiratory sensitizers, regulations such as REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals) encourage the use of non-animal methods for toxicology assessments.

[0007] Recently, a three-dimensional in vitro alveolar lung model comprising four cell types was developed (WO2018122219). This tetraculture model includes an alveolar type II epithelial cell line (A459), differentiated macrophage-like cells (THP-1 differentiated with PMA), dendritic-like cells (THP-1), and endothelial cells (EA.hy 926). The model is exposed at the airliquid interface (ALI) and mimics the alveolar surface of the lungs. It provides a feasible approach to studying the respiratory sensitization potential of inhalable products, chemicals, and particles.

[0008] While the model described above can be used to study respiratory sensitization induced by substances like Mouse Dust Mite (HDM), phthalic anhydride (PA), trimellitic anhydride (TMA), acrolein (Acr), methyl salicylate (MeSa), and sodium dodecyl sulfate (SDS), it was found to be unsuitable for studying certain challenging compounds, such as some compoundsof the class of anhydrides. In our study, no responses were observed using key markers of respiratory sensitization (CD54 and TSLPr) in tests with hexahydrophthalic anhydride (HHPA) and hexahydro-4-methylphthalic anhydride (MHHPA). One key reason for this is that anhydrides rapidly hydrolyze to their corresponding acids once introduced to the apical side of the model. Same limitation may apply for chemicals from diisocyanate class, known to act as respiratory sensitizers, but which are highly reactive and polymerize fast when placed in contact with water. Currently, there is no validated in vitro method available as an alternative to in vivo testing to identify and characterize the potential of chemicals, particularly anhydrides, as respiratory sensitizers.SUMMARY

[0009] The present invention relates to a three-dimensional in vitro alveolar lung model comprising mainly the five cell types as follows:a) alveolar type II epithelial cells able to secrete (lung laying) surfactant, b) endothelial cells which form the inner lining of capillaries providing a permeable barrier,c) alveolar dendritic-like cells,d) plasmacytoid dendritic-like cells linking innate and adaptive immunity, and e) macrophage-like cells, able to participate to defense mechanisms by ingesting foreign materials by phagocytosis, wherein:said three-dimensional in vitro lung model is in the form of a culture well equipped with a porous membrane separating the well into an apical compartment exposed to an air-liquid interface, and a basolateral compartment submerged in a culture medium, said alveolar type II epithelial cells, said alveolar dendritic-like cells, and said macrophage-like cells being present in the apical compartment, and said endothelial cells and said plasmacytoid dendritic-like cells being present at the basolateral compartment and immerged in the culture medium,said porous membrane has pores from 2 to 10 pm, dimensioned to allow migration of the alveolar dendritic-like cells from the apical compartment to the basolateral compartment.

[0010] By “mainly” is meant here that said alveolar lung model comprises essentially the five cells type above, in particular said alveolar lung model does not include mast cells, which could lead to false results.

[0011] By “essentially”, is in particular meant that said model does not include mast cells, that could lead to false results in the assessment of the toxicity or sensitizing effects of inhalable products.BRIEF DESCRIPTION OF THE FIGURES

[0012] Advantages of embodiments of the present invention will be apparent from the following detailed description of the exemplary embodiments. The following detailed description should be considered in conjunction with the accompanying figures in which:

[0013] Fig. 1 shows a schematic drawing of the three-dimensional in vitro alveolar lung model according to the prior art (A) and the invention (B).

[0014] Fig. 2 shows a schematic drawing of an exemplary embodiment of the invention.

[0015] Fig. 3 shows a schematic representation of the in vivo alveoli.

[0016] Fig. 4 shows the molecular mechanism of respiratory sensitization process.

[0017] Fig. 5 shows a schematic drawing of the three-dimensional in vitro alveolar lung model according to the prior art (A) and the invention (B) including differently stained dendritic cells: red stained for dendritic cells seeded in the apical compartment and violet stained for dendritic cells seeded in the basolateral compartment.

[0018] Fig. 6 shows the FACS images of the apical compartment (figure 6a) and of the basolateral compartment (figure 6b) of the Prior art 3D model prepared in Example 2 in which dendritic cells stained with CellTracker® Violet BMQC were seeded in the basolateral compartment only.

[0019] Fig. 7 shows the FACS images of the apical compartment (figure 7a) and of the basolateral compartment (figure 7b) of the Invention 3D model prepared in Example 2 in which the dendritic cells stained with CellTracker® Violet BMQC were seeded in the basolateral compartment and the dendritic cells stained with CellTracker® Deep Red were seeded in the apical compartment.DETAILED DESCRIPTION

[0020] Aspects of the invention are disclosed in the following description and related drawings directed to specific embodiments of the invention. Alternate embodiments may be devised without departing from the spirit or the scope of the invention. Additionally, well-knownelements of exemplary embodiments of the invention will not be described in detail or will be omitted so as not to obscure the relevant details of the invention. Further, to facilitate an understanding of the description discussion of several terms used herein follows.

[0021] As used herein, the word “exemplary” means “serving as an example, instance or illustration.” The embodiments described herein are not limiting, but rather are exemplary only. It should be understood that the described embodiments are not necessarily to be construed as preferred or advantageous over other embodiments. Moreover, the terms “embodiments of the invention”, “embodiments” or “invention” do not require that all embodiments of the invention include the discussed feature, advantage or mode of operation.

[0022] In one or more exemplary embodiment a three-dimensional in vitro alveolar lung model of the invention may be provided comprises:a) alveolar type II epithelial cells,b) endothelial cells,c) alveolar dendritic-like cells,d) plasmacytoid dendritic-like cells,e) macrophage-like cells,and excludes mast cells,said three-dimensional in vitro lung model is in the form of a culture well equipped with a porous membrane separating the well into an apical compartment exposed to an airliquid interface, and a basolateral compartment submerged in a culture medium, said alveolar type II epithelial cells, said alveolar dendritic-like cells, and said macrophage-like cells being present in the apical compartment, and said endothelial cells and said plasmacytoid dendritic- like cells being present at the basolateral compartment and immerged in the culture medium, said porous membrane has pores from 2 to 10 pm, dimensioned to allow the alveolar dendritic-like cells from the apical compartment to the basolateral compartment.

[0023] Also in one or more exemplary embodiments of the invention, the three-dimensional in vitro alveolar lung model consists exclusively of the following cells:a) alveolar type II epithelial cells,b) endothelial cells,c) alveolar dendritic-like cells,d) plasmacytoid dendritic-like cells, ande) macrophage-like cells.said three-dimensional in vitro lung model is in the form of a culture well equipped with a porous membrane separating the well into an apical compartment exposed to an airliquid interface, and a basolateral compartment submerged in a culture medium, said alveolar type II epithelial cells, said alveolar dendritic-like cells, and said macrophage-like cells being present in the apical compartment, and said endothelial cells and said plasmacytoid dendritic- like cells being present at the basolateral compartment and immerged in the culture medium, said porous membrane has pores from 2 to 10 pm, dimensioned to allow migration of the alveolar dendritic-like cells from the apical compartment to the basolateral compartment.

[0024] In an exemplary embodiment the macrophage-like cells are differentiated THP-1 cells (MQ-THP-1), with PMA (Phorbol-12-myristate- 13 -acetate) or 1,25-dihydroxy vitamin D3, which may be differentiated with PMA. This differentiation may be performed during several days, such as from 3 to 10 days, or 5 days.

[0025] In an exemplary embodiment all cells are immortalized mammalian cell lines. Different from primary cell, continuous cell lines are immortalized, which allows maintaining them in culture for a prolonged time (or in theory, indefinitely). Cell lines are more homogeneous and stable over a long time which enhances the reproducibility of results obtained under standardized in vitro conditions.

[0026] In an exemplary embodiment said alveolar type II epithelial cells are A549 cells.

[0027] In an exemplary embodiment said endothelial cells are EA.hy926 cells.

[0028] In an exemplary embodiment said alveolar dendritic-like cells and said plasmacytoid dendritic-like cells are non-differentiated THP-1 cells, which are well established models for human dendritic cells.

[0029] The cell type that requires special consideration in an in vitro model for respiratory sensitization is the dendritic cell. Lung dendritic cells, which are typically distributed above and below the basal membrane of the epithelium throughout the lung, are the most important antigen-presenting cells (APCs) in the lungs. Fig. 4 schematically shows how dendritic cells are involved in the process of respiratory sensitization. Specifically, dendritic cells sense the localmicroenvironment through a wide range of pattern recognition receptors. They also express cytokine receptors, which can lead to activation upon autocrine or paracrine secretion. In the steady state, dendritic cells are immature and have the potential to recognize, capture, and process antigens. After antigen uptake via phagocytosis, micropinocytosis, or receptor-mediated endocytosis, dendritic cells process and present these antigens on their surface. Additionally, dendritic cells require activation, which occurs either through the direct binding of their pattern recognition receptors or indirectly through the release of damage-associated molecules. This activation leads to dendritic cell maturation and the upregulation of adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1), also known as cluster of differentiation 54 (CD54), and co-stimulatory molecules like CD40, CD80 (B7-1), and CD86 (B7-2). The migration of dendritic cells to the lymph nodes is facilitated by the upregulation of C-C chemokine receptor type 7 (CCR7) during maturation. Signals from antigen presentation, co-stimulatory molecules, and cytokines are required for the activation of naive T-cells and the development of acquired immunity. Therefore, dendritic cells are central to the sensitization process, as they are responsible for recognizing and capturing antigens, which triggers the immune response.

[0030] As dendritic cells are central to lung respiratory sensitization, their inclusion and positioning in in vitro culture models are critical for determining whether a model can accurately replicate in vivo physiology. Compared to the tetraculture model in WO2018122219, the present model incorporates alveolar dendritic-like cells seeded in the apical compartment of the insert, alongside plasmacytoid dendritic-like cells seeded in the basolateral compartment. As is shown in Fig. 3, lung dendritic cells are distributed both above and below the basal membrane of the epithelium in alveoli, this design thus more closely mimics the in vivo environment and provides greater histological relevance. Unlike the plasmacytoid dendritic-like cells in the basolateral compartment, the alveolar dendritic cells are responsible for sensing, recognizing, engulfing, or presenting antigens. This function is essential for priming the sensitization process.

[0031] One of another key advantages of adding alveolar dendritic-like cells to the apical compartment of the culture model is their ability to protect anhydrides from hydrolysis before antigen presentation. The tetraculture model in WO2018122219 does not show sensitization responses to hexahydrophthalic anhydride (HHPA) and hexahydro-4-methylphthalic anhydride (MHHPA), both of which are known to hydrolyze within seconds, forming their corresponding acids. However, in the present model, with alveolar dendritic-like cells seeded in the apicalcompartment, HHPA and / or MHHPA antigens can be presented either by the dendritic-like cells themselves or immediately to macrophages, triggering an innate immune response. Subsequently, plasmacytoid dendritic-like cells travel to the lymphoid organs, where signals from antigen presentation, co-stimulatory molecules, and cytokines are required to activate naive T-cells and promote the development of acquired immunity. This configuration ensures that the anhydrides are effectively involved in the lung sensitization process, providing an optimal model for studying anhydride-induced lung sensitization. This prior art model is shown schematically in Fig. 1(A).

[0032] In an exemplary embodiment, as schematically shown in Fig. 1(B), said porous membrane separating the culture well into the apical and the basolateral compartments may be a porous membrane that is part of a membrane insert. Dendritic cells are pivotal in the induction phase of the sensitization process and in their absence the entire process cannot be studied. The porous membrane is dimensioned to allow migration of the the alveolar dendritic-like cells from the apical compartment to the basolateral compartment but on the contrary, the migration of the plasmacytoid dendritic-like cells from the basolateral compartment to the apical compartment does not occur.

[0033] The insert body is made of transparent plastic. The membrane insert comprising the porous membrane is introduced or inserted into the culture well, or another chamber suitable for cell culture, to split the culture well into the two compartments, and is attached to a cell culture insert body that keeps it in place. The membrane inserts may be designated as having an apical side and a basolateral side, which correspond to the apical and the basolateral compartments upon their use. For example, the membrane inserts may be placed on well plates, where cell culture media is added, such that the membrane insert has a lower face, which is the basolateral side, facing the basolateral compartment of the well and an upper face, which is the apical side, facing the apical compartment of the well. Cells grown on the membrane inserts benefit from in vi vo-like conditions and have access to nutrients or substances from the apical and basolateral side. After use, the membrane insert may be removed.

[0034] In an exemplary embodiment the membrane insert may be a hanging cell culture insert comprising a microporous membrane, as shown in Fig. 2.

[0035] In an exemplary embodiment said pore membrane has pores of from 2 to 10 pm. The porous membrane may also have pores of from 3 to 8 pm or of from 4 to 6 pm.

[0036] The present invention also relates to a process for preparing the above three-dimensional in vitro alveolar lung model, comprising co-culture of cells a)-e) in a membrane insert having a porous membrane with pores from 2 to 10 pm, in the following steps:i) seeding a lower face of the membrane insert with 0.24 / 105endothelial cells / cm2, said lower face corresponding to a basolateral side of the membrane insert,ii) at least four hours later, seeding an upper face of the membrane insert with 0.6 / 105alveolar type II epithelial cells / cm2, said upper face corresponding to an apical side of the membrane insert,iii) about 4 days later, adding a cell suspension containing 0.5 million plasmacytoid dendritic-like cells / mL to the basolateral side of the membrane insert, and theniv) adding 1 million alveolar dendritic-like cells mixed with 0.11 million macrophage-like cells on top of the alveolar type II epithelial cells seed, and finally,v) introducing the membrane insert into a co-culture medium of a culture well in order that said endothelial cells and said plasmacytoid dendritic-like cells are present at a basolateral compartment of said culture well and immerged in the co-culture medium; and said epithelial cells, said alveolar dendritic-like cells and said macrophage-like cells are present in an apical compartment of said culture well, at the air-liquid interface.

[0037] In an exemplary embodiment, said macrophage-like cells are THP-1 cells, differentiated with PMA (Phorbol-12-myristate- 13 -acetate), and / or said alveolar type II epithelial cells are A549 cells, and / or said endothelial cells are EA.hy926 cells, and / or said alveolar dendritic-like cells are non-differentiated THP-1 cells, and / or said plasmacytoid dendritic-like cells are non-differentiated THP-1 cells.

[0038] Compared with the model described in WO2018122219, adding another cell layer, the alveolar dendritic-like cells, in the apical compartment leads to more technical challenges. As alveolar dendritic-like cells co-exist with alveolar type II epithelial cells and macrophage-like cells in the apical compartment, it is essential to determine the time and amount of seeding, thus making sure that each cell type maintains its morphology and function. The optimization of standard operating procedure (SOP) includes:a. Apical seeding volume (100-500 pL);b. Co-seeding of alveolar dendritic-like cells with alveolar type II epithelial cells (A549) on Day 0;c. Induce pre-maturation of alveolar dendritic-like cells by cytokines (e.g. IL-4 and GM-CSF) prior seeding.

[0039] The present invention also relates to an in vitro process for determining and / or predicting the sensitizing effects, and / or the irritation potential or toxicity of an inhalable product on the alveolar barrier of lungs, comprising:A) a step of exposing by aerosolization and nebulizing the inhalable product over the apical compartment of a three-dimensional in vitro alveolar lung model = in the form of a culture well equipped with a porous membrane having pores from 2 to 10 pm, said epithelial cells, said alveolar dendritic-like cells, and said macrophage-like cells being present at the apical side of the membrane, and said endothelial cells and said plasmacytoid dendritic-like cells being present at the basal side of the membrane, said endothelial cells and said plasmacytoid dendritic-like cells being immerged in the co-culture medium, said exposing leading to activation of the plasmacytoid dendritic-like cells,B) a step of co-culturing said activated plasmacytoid dendritic-like cells with a T lymphoblast cell line.

[0040] In an exemplary embodiment, said T lymphoblast cell line should present a Helper type 2 (TH2) profile if the product to be tested is a suspected respiratory sensitizer.

[0041] In some embodiments, markers for the respiratory sensitization potential may be measured by FACS (such as for example CD40, CD54, CD86, TSLPr, IL- Ira, OX40L, CCR6, GM-CSF, CXCR4 (so-called CD184), ST2L, CD40, HLA-DR, FceRlalpha, and CCR7) and / or by ELISA (TSLP, IL 33, IL-25, RANTES, MCP-1, MIP-3a, IL-6, IL-7, IL-10 and GM-CSF). Further biological endpoints, such as release of interleukins, genotoxicity, biomarkers of sensitization, proteomics, transcriptomics, metabolic activation may also be measured.

[0042] The three-dimensional in vitro alveolar lung model of the invention presents the main following advantages:- Possibility of exposure at the air-liquid interface (ALI), using gases, liquids or powders as materials to be tested;- Possibility of testing chemicals and particles for both aspects: their inflammatory or sensitizing potential, especially for anhydrides, which are easily hydrolyzed within seconds to their corresponding acids;- Possibility of measuring a multitude of biological endpoints (e.g. release of interleukins, genotoxicity, biomarkers of sensitization, proteomics, transcriptomics, metabolic activation, etc.).

[0043] The foregoing description and accompanying figures illustrate the principles, preferred embodiments and modes of operation of the invention. However, the invention should not be construed as being limited to the particular embodiments discussed above. Additional variations of the embodiments discussed above will be appreciated by those skilled in the art.

[0044] Therefore, the above-described embodiments should be regarded as illustrative rather than restrictive. Accordingly, it should be appreciated that variations to those embodiments can be made by those skilled in the art without departing from the scope of the invention as defined by the following claims.EXAMPLESExample 1

[0045] In this example, the following cell lines are used: A549, EA.hy926, THP-1 and MQ-THP-1. Their respective characteristics are as follows:• A549 is a human bronchial epithelial cell line with ability to produce surfactant, used as the alveolar type II epithelial cell model claimed herein;• EA.hy926 is a somatic cell hybrid cell line with endothelial characteristics, used as the endothelial cell model claimed herein;• THP-1 is a human monocytic leukemic cell line, used as both the alveolar dendritic- like cell model and plasmacytoid dendritic-like cell model claimed herein; and• MQ-THP-1 are THP-1 cells differentiated with PMA (Phorbol-12-myristate-13- acetate) or 1,25-dihydroxy vitamin D3, used as the macrophage-like cell model claimed herein.

[0046] The reagents and plasticware used in establishing the three-dimensional in vitro alveolar lung model are listed as follows:Reagents:• Trypsin solution TrypLE Select (Gibco, Cat. # 12563-011)• Accutase (Sigma, Cat. # A6964)• Phosphate buffered saline without Ca2+and Mg2+(Gibco, Cat. # 14190)• HEPES, IM (Sigma, Cat. # H3537-1L)Plasticware:• cellQART inserts, 5 pm pore size (cellQART made by Sabeu; Translucent: Cat.• 9305002; Clear: Cat. # 9305012)• 6-well suspension culture plate (Greiner bio-one, Cat. # 657185) • 6-well cell culture plate (Greiner bio-one, Cat. # 657160)

[0047] The components and procedures to prepare different media used in establishing the three-dimensional in vitro alveolar lung model are listed as follows:A549 mediumComponent:• DMEM, GlutaMAX™ (Gibco, Cat. # 61965)• FBS Superior, heat inactivated 10% (Merck, Cat. # S0615) Preparation:Remove 50 mL of DMEM (Dulbecco's Modified Eagle's medium) from a new bottle (500 mL) and add 50 mL of FBS (10%). Mix by inversion.EA,hy926 mediumComponent:• DMEM, GlutaMAX™ (Gibco, Cat. # 61965)• HEPES, 25 mM (Sigma, Cat. # H3537-1L)• FBS Superior, heat inactivated, 10% (Merck, Cat. # S0615) Preparation:Remove 62.5 mL of DMEM (Dulbecco's Modified Eagle's medium) from a new bottle (500 mL) and add 50 mL of FBS (10%), then 12.5 mL of HEPES stock solution (I M; sterile, filtered) to get a HEPES buffered medium (25 mM). Mix by inversion.THP-1 and MCb-THP-l mediumComponent:• RPMI 1640 medium, GlutaMAX™ (Gibco, Cat. # 72400-021)• FBS Superior, heat inactivated (Merck, Cat. # S0615)• 0-Mercaptoethanol, 0.05mM (Biorad, Cat. # 161-0710) Preparation:Remove 52.5 mL of RPMI 1640 medium from a new bottle (500 mL) and transfer 10 mL into a Falcon tube. Add 7 uL of commercial solution of 0-mercaptoethanol into 10 mL RPMI to get a working solution of 0-mercaptoethanol in RPMI (10 mM). Add 2.5 mL of 0-mercaptoethanol working solution to get 0.05 mM 0-mercaptoethanol in medium. Add 50 mL of FBS (10%) into the medium. Mix by inversion.Coculture medium 10% FBSComponent:• DMEM, GlutaMAX™ (Gibco, Cat. # 61965)• HEPES, 25 mM (Sigma, Cat. # H3537-1L)• RPMI 1640 medium, GlutaMAX (Gibco, Cat. # 72400-021)• IMDM medium (Gibco, Cat. # 21980-065)• FBS Superior, heat inactivated, 10% (Merck, Cat. # S0615)Preparation:Remove 12.5 mL of DMEM media from a new bottle (500 mL) and add 12.5 mL of HEPES stock solution (I M, sterile, filtered) to get a HEPES buffered medium (25 mM). Mix and remove 125 mL the above medium, and transfer it into a new sterile bottle. Then add 50 mL of IMDM medium and 75 mL of RPMI medium to the above medium. Mix and remove 50 mL medium. Add 50 mL of FBS (10%) into the medium. Mix by inversion.Coculture medium 1% FBSComponent:• DMEM, GlutaMAX™ (Gibco, Cat. # 61965)• HEPES, 25 mM (Sigma, Cat. # H3537-1L)• RPMI 1640 medium, GlutaMAX (Gibco, Cat. # 72400-021)• IMDM medium (Gibco, Cat. # 21980-065)• FBS Superior, heat inactivated, 10% (Merck, Cat. # S0615) Preparation:Remove 12.5 mL of DMEM media from a new bottle (500 mL) and add 12.5 mL of HEPES stock solution (I M, sterile, filtered) to get a HEPES buffered medium (25 mM). Mix and remove 125 mL the above medium, and transfer it into a new sterile bottle. Then add 50 mL of IMDM medium and 75 mL of RPMI medium to the above medium. Mix and remove 5 mL medium. Add 5 mL of FBS (1%) into the medium. Mix by inversion.

[0048] In this example, steps for preparing the three-dimensional in vitro alveolar lung model are chronologically listed as follows:1. Dav -2Differentiating macrophage cells (overnight):THP-1 cells are differentiated with PMA(Phorbol-12-myristate- 13 -acetate) or with 1, 25-dihydroxyvitamin D3 (80 pL PMA + 40 mL medium + 16 million cells per flask T125) overnight and then kept in the flask for 1-5 days, medium being renewed at day 0. Differentiated THP-1 cells are named as MQ-THP-1 cells.2. Dav 0Preparation of the EA.hy926 cells:• Add 0.11 million cells per insert (0.24*105cells / cm2),• The volume needed for the seeding in the insert is here 550 pL,• Prepare first the cell suspension for all inserts (add two extra inserts for pipetting mistakes), mix in order to well homogenate the cell suspension, • Properly put the inserts inside the well of the plate,• Return the plate and gently put the 600 pL of EA.hy926 under the insert, • Then place the plate into incubator.At least 4 hours later:Preparation of the A549 cells:• The volume needed for the seeding in the insert is here 2 mL,• The pellet of A549 cells is re-suspended in the EA.hy926 medium: 0.27 million cells per insert (0.6* 105cells / cm2),• Prepare first the cell suspension of A549 cells for all inserts (into EA.hy926 medium, count two extra inserts for pipetting errors). Mix and homogenate well of the cell suspension.Prepare the plate with inserts for the seeding and homogenate the cell suspension from time to time when seeding cells on inserts, to avoid the cells settling down in the tube:• Return plate and add 1 mL of EA.hy926 medium into the well,• Check the endothelial cellular layer under microscope,• Add the 2 mL of A549 cells in the insert.3. Dav 3Changing the medium of the plate of inserts:• Remove old medium of the insert and add 2 mL of Coculture 10% medium, • Remove old medium of the well and add 2 mL of Coculture 10% medium.4. Dav 4Prepare M<l>-THP-1 cell suspension:• Aspirate cell culture medium from flask,• Rinse the cells by adding 15 mL pre-warmed PBS to each T175 flask, • Aspirate PBS from flasks,• Add 3 mL of pre-warmed Accutase to each flask and distribute equally on the cell culture area of the flask,• Incubate flask for 15 min in incubator (37°C, 5% CO2). Check cell detachment using the microscope. Rap the flask against the palm of the hand to help cells release from the surface of the flask,• Recover the cells with 1% Co-culture medium into centrifuge tube and centrifuge at 180*g for 5 min,• Carefully aspirate supernatant,• Gently resuspend cell pellet in a low volume of 1% Co-Culture medium (1 mL), pipette up and down 5-10 times to ensure uniform single-cell suspension,• Count cells.Prepare THP-1 cell suspension:• Transfer THP-1 cell suspension to a centrifuge tube and centrifuge at 180 g for 5 min,• Carefully aspirate supernatant,• Gently resuspend cell pellet in a low volume of 1% Co-Culture medium (3 mL), pipette up and down 5-10 times to ensure uniform single-cell suspension,• Count cells.Seeding of THP-1 cells (plasmacytoid dendritic-like cells, basolateral compartinent):• Cell number / insert: 0.5 * 106cells / well.• Volume needed for the seeding on the insert is 1 mL.• Prepare first the cell suspension for all wells (in 1% Co-culture medium, plus two extra wells for pipetting errors), mix well to obtain a homogeneous cell suspension.• Gently put 1 mL of THP-1 cell suspension in the well of a new 6-well plate (Cat. # 657185).• Move the inserts from the old plate to the new one.Seeding of THP-1 (alveolar dendritic-like cells) and M(l>-THP-1 (macrophage-like cells) cells (apical compartment):• Seed 1 * 106THP-1 cells and 0.11 * 106MQ-THP-1 cells per insert.• Volume needed for the seeding on the insert is 0.5 mL.• Prepare first the cell suspension for all wells (in 1% Co-culture medium, add two extra wells for pipetting errors), mix well to obtain a homogeneous cell suspension.• Aspirate old medium from the insert.• Gently add 500 pL of cell suspension onto the insert.5. Dav 524h later, aspirate the medium from the apical compartment of the insert to create the air-liquid-interface (ALI) in the insert.6. Dav 6Exposure of the insert to the inhalable product (particles or molecules) to be tested on the three-dimensional model of the invention, prepared under the above protocol, can be performed after 10 to 12 hours (time required for the cells to produce the surfactant) with the in vitro lung model as presented.Example 2

[0049] In this example a comparative three dimensional lung model not forming part of the invention has been prepared according to the method disclosed in Example 1 of international patent application WO2018122219 (Prior art 3D model) in order to compare it with the three dimensional lung model prepared above in example 1 of the present application (B: invention 3D model).

[0050] Briefly and as shown on the schematic drawing of figure 5, in the case of the Prior art 3D model, dendritic cells have been seeded in the basolateral compartment only, while in the case of the Invention 3D model of the present invention, dendritic cells have been seeded both in the basolateral compartment and in the apical compartment.

[0051] The purpose of this example was to demonstrate that the dendritic cells seeded in the basolateral compartment do not migrate towards the apical compartment.

[0052] In both cases and with reference to figure 5 annexed, the dendritic cells used in each model and seeded in these two different compartments were stained with two different cell trackers for a further fluorescence activated cell sorting (FACS) test.• Alveolar dendritic-like cells to be seeded in the apical compartment were stained with CellTracker® Deep Red - 630 / 6600 nm (LIFE TECHNOLOGIES SAS, Supplier Ref: C34565), and• Plasmacytoid dendritic-like cells to be seeded in the basolateral compartment were stained with CellTracker® Violet BMQC - 415 / 516 nm (LIFE TECHNOLOGIES SAS, Supplier Ref.: Cl 0094).

[0053] Dendritic cells were incubated for 30 min at 37°C with a DMSO solution of CellTracker® Deep Red, respectively CellTracker® Violet BMQC before their seeding in the apical, respectively basolateral, compartments as represented on annexed figure 5:• Prior art 3D model: dendritic cells stained with CellTracker® Violet BMQC were seeded in the basolateral compartment only and no dendritic cells were seeded in the apical compartment;• Invention 3D model: dendritic cells stained with CellTracker® Violet BMQC were seeded in the basolateral compartment and dendritic cells stained with CellTracker® Deep Red were seeded in the apical compartment.

[0054] The detailed protocol of the FACS test is given below.1. CellTracker™ Dye Staining of THP-1 Cells1.1 Dye OverviewEx / ExDye Cat. N° Cell Type Stock Solution (nm)Alveolar THP- CellTracker™ Deep Red C34565 630 / 660 1 mM1 (apical)PlasmacytoidCellTracker™ VioletCl 0094 415 / 516 THP-1 10 mMBMQC(basolateral)1.2 Preparation of Stock SolutionsThe stock solutions were prepared by dissolving the lyophilized dye in DMSO as follows:• CellTracker™ Deep Red (C34565): 20 pL of DMSO were added per vial to give a 1 mM stock solution (1000 ).• CellTracker™ Violet BMQC (C 10094) : 0 1 mg was dissolved in 30 pL of DMSO to give a 10 mM stock solution.1.3 Preparation of Working SolutionThe stock solution 1:1000 was diluted in serum-free medium to obtain a final working concentration of 0.5-25 pM, for example by adding 1 pL of 1 mM stock to 999 pL of serum-free medium to obtain a 1 pM working solution.1.4 Cell Staining Protocol• The culture medium was replaced with the working dye solution.• Cells were incubated for 30 minutes at 37 °C in a humidified CO2 incubator.• The dye solution was aspirated and cells were washed gently with warm serum-free medium (xl).• Culture medium was added and cells were seeded into the experimental plate immediately.2, Analysis: FACS2.1 . FACS on Floating Cells (Basolateral Compartment)• THP-1 cells were collected from the basolateral compartment into a 5 mL tube.• The basolateral insert (containing EAhy926 cells) was rinsed with 1 mL PBS and the wash was added into the same tube.• Centrifugation at 300 x g for 5 min; the supernatant was carefully removed and discarded.• The pellet was washed with 1 mL PBS. Centrifugation at 300 x g for 5 min and then the supernatant was removed.• The pellet was resuspended in 300 pL PBS and transferred to a FACS tube.• Viability staining: SYTOX™ Green solution was prepared at 1 : 10 in PBS. 3 pL were added per tube (1 : 1000 final). The tubes were incubated 20 min at 4 °C in the dark.• The data were then acquired on a flow cytometer.2.2. FACS on Tissue / Adherent Cells (Apical Compartment — Inserts)• Inserts were transferred to a new plate.• 1 mL of PBS was added into the apical compartment.• A549 cells were scraped from the apical surface using a cell scraper.• The cell suspension was collected and transferred to a 5 mL tube.• The tube was centrifuge at 300 x g for 5 min and the supernatant was removed.• The pellet was resuspended in 300 pL PBS and the suspension was transferred to a FACS tube.• Viability staining: Prepare SYTOX™ Green was prepared at 1 : 10 in PBS. 3 pL were added per tube (1 : 1000 final). The tubes were incubated 20 min at 4 °C in the dark.• The data were then acquired on a flow cytometer.Results

[0055] The results of the FACS test are reported in figures 6 and 7 annexed. On these figures, QI corresponds to the population of alveolar dendritic cells, Q3 to the population of plasmacytoid dendritic cells and Q4 to the population of unstrained cells, meaning cells that can have been detached by washing the inserts and which are not either alveolar nor plasmacytoid dendritic cells.

[0056] Figure 6 shows the FACS images of the apical compartment (figure 6a) and of the basolateral compartment (figure 6b) of the Prior art 3D model in which dendritic cells stained with CellTracker® Violet BMQC were seeded in the basolateral compartment only. In the apical compartment (fig. 6a) the image shows no violet stained dendritic cells in Q3 while in the basolateral compartment (fig 6b) the image reflects the presence of violet stained dendritic cells in Q3 thus demonstrating that the dendritic cells seeded in the basolateral compartment do not migrate towards the apical compartment.

[0057] Figure 7 shows the FACS images of the apical compartment (figure 7a) and of the basolateral compartment (figure 7b) of the Invention 3D model in which the dendritic cells stained with CellTracker® Violet BMQC were seeded in the basolateral compartment and the dendritic cells stained with CellTracker® Deep Red were seeded in the apical compartment. In the apical compartment (fig. 7a) the image shows only red stained dendritic cells (QI) and no violet stained dendritic cells in Q3 while in the basolateral compartment (fig 7b) the image reflects the presence of both red (QI) and violet (Q3) stained dendritic cells thus demonstrating that a part of the dendritic cells seeded in the apical compartment do migrate towards the basolateral compartment. The basolateral compartment comprises however in majority violet stained dendritic cells.

[0058] As a whole, these experiments that the so-called “migrating” dendritic cells seeded in the basolateral compartment in the Prior art 3D model described in International patentapplication WO2018122219 cannot reach the apical compartment after seeded and in fact do not migrate.

[0059] These results also demonstrate that the Invention 3D model better mimics the in vivo environment and provides greater histological relevance as illustrated in figure 3.

Claims

CLAIMS1. A three-dimensional in vitro alveolar lung model comprising:without mast cells, five cell types as follows:a) alveolar type II epithelial cells able to secrete surfactant,b) endothelial cells which form an inner lining of capillaries providing a permeable barrier,c) alveolar dendritic-like cells,d) plasmacytoid dendritic-like cells linking innate and adaptive immunity, and e) macrophage-like cells able to participate to defense mechanisms by ingesting foreign materials by phagocytosis, wherein:said three-dimensional in vitro alveolar lung model is in the form of a culture well equipped with a porous membrane separating the well into an apical compartment exposed to an air-liquid interface, and a basolateral compartment submerged in a culture medium, said alveolar type II epithelial cells, said alveolar dendritic-like cells, and said macrophage-like cells being present in the apical compartment, and said endothelial cells and said plasmacytoid dendritic-like cells being present at the basolateral compartment and immerged in the culture medium,said porous membrane has pores from 2 to 10 pm, dimensioned to allow migration of the alveolar dendritic-like cells from the apical compartment to the basolateral compartment.

2. The model according to claim 1 wherein said macrophage-like cells are THP-1 cells, differentiated with either one of Phorbol-12-myristate- 13 -acetate (PMA) or with 1,25-dihydroxy vitamin D3.

3. The model according to claim 1, wherein all cells are immortalized human cell lines.

4. The model according to claim 1, wherein said alveolar type II epithelial cells are A549 cells.

5. The model according to claim 1, wherein said endothelial cells are EA.hy926 cells.

6. The model according to claim 1, wherein said alveolar dendritic-like cells and said plasmacytoid dendritic-like cells are non-differentiated THP-1 cells.

7. The model according to claim 1 , wherein the porous membrane has pores from 3 to 8 pm.

8. The model according to claim 1, wherein the porous membrane is part of a membrane insert is insertable into and removable from the culture well.

9. A process for preparing the three-dimensional in vitro alveolar lung model as defined in claim 1, comprising:co-culturing of cells a)-e) in a culture well that is equipped with a membrane insert, having a porous membrane with pores from 2 to 10 pm, in the following step sequence:i) seeding a lower face of the membrane insert with 0.24 / 105endothelial cells / cm2, said lower face corresponding to a basolateral side of the membrane insert,ii) at least four hours later, seeding an upper face of the membrane insert with 0.6 / 105alveolar type II epithelial cells / cm2, said upper face corresponding to an apical side of the membrane insert,iii) about 4 days later, adding a cell suspension containing 0.5 million plasmacytoid dendritic-like cells / mL to the basolateral side of the membrane insert, andiv) adding 1 million alveolar dendritic-like cells mixed with 0.11 million macrophage-like cells on top of the alveolar type II epithelial cells seed, and finally,v) introducing the membrane insert into a co-culture medium of a culture well in order that said endothelial cells and said plasmacytoid dendritic-like cells are present at a basolateral compartment of said culture well and immerged in the co-culture medium; and said epithelial cells, said alveolar dendritic-like cells and said macrophage-like cells are present in an apical compartment of said culture well, at the air-liquid interface.

10. The process according to claim 9, wherein at least one of said macrophage-like cells are THP-1 cells differentiated with PMA (Phorbol-12-myristate- 13 -acetate), said alveolar type II epithelial cells are A549 cells, said endothelial cells are EA.hy926 cells, or said plasmacytoid dendritic-like cells and said alveolar dendritic-like cells are non-differentiated THP-1 cells.

11. An in vitro process for determining and / or predicting the sensitizing effects, and / or the irritation potential or toxicity of an inhalable product on the alveolar barrier of lungs, comprising:A) a step of exposing by aerosolization and nebulizing the inhalable product over the apical compartment of a three-dimensional in vitro alveolar lung model as defined in claim 1, wherein the porous membrane having pores from 2 to 10 pm, and wherein the exposing leads to an activation of the plasmacytoid dendritic-like cells, andB) a step of co-culturing said activated plasmacytoid dendritic-like cells with a T lymphoblast cell line.

12. The in vitro process according to claim 11, wherein T-cells present a Helper type 2 (TH2) profile if the product to be tested is a suspected respiratory sensitizer.

13. The in vitro process according to claim 11, wherein markers for respiratory sensitization potential are measured by FACS and / or by ELISA.

14. The in vitro process according to claim 13, wherein said markers for respiratory sensitization are measured by FACS are at least one of: CD40, CD54, CD86, TSLPr, IL-lra, OX40L, CCR6, GM-CSF, CXCR4, ST2L, CD40, HLA-DR, FceRl alpha, and CCR7.

15. The in vitro process according to claim 13, wherein markers for the respiratory sensitization measured by ELISA are at least one of: TSLP, IL-33, IL-25, RANIES, MCP-1, MIP-3a, IL-6, IL-7, IL-10, and GM-CSF.

16. The in vitro process according to claim 11, wherein further biological endpoints comprising release of interleukins, genotoxicity, biomarkers of sensitization, proteomics, transcriptomics and metabolic activation are measured.

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