Distal airway and alveolar model

Abstract

A respiratory simulator including a rigid porous foam is disclosed, the rigid porous foam including (i) macropores, and (ii) an interconnected open cell network of micropores and / or nanopores.

Description

Technical Field

[0001] The present invention discloses a distal airway and alveolar model that can be used to simulate the alveolar ducts and alveolar spaces of the terminal bronchioles, transitional bronchioles, and the alveolar spaces of the airway. In particular, the present invention can be used to simulate the interaction between inhalable agents and that region of the airway.

Background Art

[0002] Inhalable agents such as smoke, fumes, aerosolized dust, gases, pollen, bacteria, or aerosols generated by medical inhalers can have neutral, harmful, or beneficial effects on the airway. The nature and amplitude of the observable effects depend on the delivery dose of the inhalant, which is the amount of the test agent or its individual components that deposit on or diffuse into the epithelium of the airway. The delivered dose is affected by the characteristics of the inhaled test agent and the characteristics of the airway. The relevant characteristics of the test agent include its concentration, its composition, and the respective physical and chemical characteristics of its components, such as particle size, particle density, vapor pressure, diffusivity, or water solubility. The relevant characteristics of the airway include its geometric shape and surface characteristics, which vary gradually from the nose and upper airway to the respiratory zone of the distal lung. Therefore, the dose delivered from a given inhalant to a given region of the airway varies along the airway. Furthermore, the fraction of the inhalant deposited in the upper airway is no longer available for deposition in the distal airway, so the composition of the inhalant changes. Thus, different regions of the airway are exposed to different test agents, which further increases the non-uniformity of the delivery dose across the airway.

[0003] The ability to simulate these deposition patterns in vitro has great value in the field of in vitro inhalation science. This enables the performance of physiologically relevant in vitro exposures of cell cultures of airway epithelia, such as human airway epithelium. Realistic exposure conditions and physiologically relevant dose delivery will increase the translationality of the biological responses to such exposures in vitro and in vivo compared to currently applied methodologies. During the development of inhalable drugs, for example, this reduces the need for expensive, time-consuming, and ethically debated animal studies and, at the same time, reduces the risk of failure of drug candidates during clinical trials.

[0004] Currently, there is no technology that can reliably simulate the interaction between inhalants and the entire airway. Ideal or realistic models of the upper airway up to the first few generations of bronchial branching have been described and constructed and used for the study of particle deposition during breathing. However, these models are generally not designed to hold cell cultures for in vitro exposure, do not capture the dynamics of the distal airways or alveoli, and do not create a physiologically relevant state from the perspectives of breathing dynamics, surface properties of the airway wall and harvest rate, and humidification of inhalants.

[0005] Since the diameters of the distal airways and alveoli are in the sub-millimeter range, the diffusion of gases and small particles present in inhaled air to the epithelium is rapid and efficient. Furthermore, the total cross-sectional area of the airway increases towards the more distal regions and ultimately reaches approximately 1 m 2is reached. This design reduces the flow velocity of the inhaled air towards the more distal regions of the airway from 1 - 10 m / s in the upper airway to less than 1 cm / s close to the breathing zone. The supply of gas (and particles) to the alveoli themselves is driven entirely by diffusion, and convective mass transport does not reach into the alveoli. This has a great effect on the mass transport from the inhaled air to the respiratory epithelium and the bloodstream. Specifically, the flow velocity in the breathing zone is optimized to meet the physiological requirement of balancing the convective transport of inhaled air into and out of the alveolar space with the diffusive transport of oxygen from the alveolar lumen across the alveolar wall. The distal airways and alveoli are difficult to model due to their structural complexity.

[0006] Devices for performing in vitro exposure of cell cultures representing the distal airways and alveoli are described in Tenenbaum-Katan et al. (2018) Biomicrofluidics 12(4), Stucki et al. (2018) Scientific Reports 8, 1 - 3 and Muller et al. (2001) Insciences J.1, 30 - 64, but they generally cannot reproduce the physical conditions and / or deposition patterns. This is because, on the one hand, the main mechanisms governing the geometric shape of the devices and / or the deposition of the test material within these devices are not correctly aligned with the anatomical structure. On the other hand, these devices cannot be connected to a model of the upper airway and thus cannot shape the physicochemical properties of the test agent in a way that is relevant to the distal airways, i.e., by delivering a test agent that is not related to the distal airways. Furthermore, the distal airways and the breathing zone of the airway have a large total surface area (e.g., 60 - 140 m 2It is characterized by (the range of). The respiratory epithelium separates the volume of inhaled air from the blood circulation supplied from the right ventricle through the pulmonary artery. The thickness of this blood-gas barrier in the respiratory zone is below the micrometer range. For example, a complete cardiac output of blood in the range of 4 to 8 liters per minute reaches the respiratory epithelium via the pulmonary artery. For any compound present in the inhaled air and capable of diffusing across the respiratory epithelium, the rapidly exchanged blood acts as a sink that keeps the concentration gradient between the gas and the epithelium at a relatively constant high level. In summary, a large surface area, short diffusion distance, optimized flow rate, and relatively high and somewhat concentration gradient result in a very efficient gas exchange between the inhaled air and the blood. This applies to the two gases intended to be exchanged by the lungs, namely oxygen and carbon dioxide, but clearly also to any other gas present in the inhaled air. The same applies to low vapor pressure particles and compounds, which do not rely on their continuous removal from the lung tissue by the bloodstream delivered at a certain rate. When immersed in the inner layer of the epithelial liquid layer, they enter the gas phase again to a very limited extent. That is, they do not reach equilibrium conditions at all, or reach equilibrium only after a very high mass has been transferred to the epithelium.

[0007] WO2019016094 describes an apparatus that mimics the macroscopic anatomical features of the simulated airway and can simulate human breathing. WO2020 / 148238 describes a perforated structure that can be inserted into / within the simulated airway and further refines the anatomical simulation of the airway by adding models of the lobes as well as the segmental bronchi and bronchi. Both WO2019016094 and WO2020 / 148238 may be designed to hold cell cultures, sensors, or microfluidic or mesofluidic devices, and may also be used to conduct in vitro exposures of a biological test system to test aerosols or gases and determine the deposition of the test aerosol or gas in vitro under physiologically relevant conditions.

[0008] In the art, there is a need for a system that can simulate the mass transport of aerosols and gases in the distal airways and alveoli of the respiratory zone in vitro. The present invention endeavors to address this need and other needs. SUMMARY OF THE INVENTION

[0009] The present disclosure provides a model of the distal airways and alveoli. The model can be used to simulate the terminal, transitional, and respiratory bronchioles, alveolar ducts, and alveolar spaces of the airways. Using the distal airway and alveolar model, the interaction between inhalable agents and this region of the airways can be simulated. To achieve this, the distal airway and alveolar model utilizes a rigid porous foam. The rigid porous foams used in the present disclosure are commercially available and known for use in applications including separation technology and filtration (e.g., desorption, diesel particulate filtration, filtration of liquids), chemical and thermal process engineering (e.g., catalyst supports, porous burners), medical technology (e.g., bone replacement materials), and as carriers for catalysts. It is understood that porous foams, particularly those including an interconnected open-cell network of (i) macropores and (ii) micropores and / or nanopores, have not been described for use in models of the distal airways and alveoli of the airways. One reason for this is that their use for this purpose runs counter to the intuition of those skilled in the art. This is because a flexible structure is understood to be more closely similar to the airways. However, the inventors have found that rigid porous foams are preferred over such flexible structures, such as those made from silicone, rubber, gelatin, or similar materials, because they do not have the stability required for bimodal or hierarchical pore sizes, which can lead to the collapse of the foam. Furthermore, the reproducible handling and cleaning of flexible structures is difficult to achieve in the laboratory, and strategies for attaching cell cultures within such structures have not been identified to date.

[0010] The claimed rigid porous foam is an open-cell hierarchical network of (i) macropores and (ii) micropores and / or nanopores that represent models of the distal airways and alveoli in the respiratory tract. One size of pores (macropores that can mimic the size of alveoli) can allow the gas or aerosol supplied to the model to pass through, while the other pore size (micropores and / or nanopores that can mimic the size of distal airways) can allow an aqueous solution to pass through, hold this aqueous solution within the network of micropores and / or nanopores, and provide a liquid film on the surface of the macropores. By providing an aqueous or liquid film on the surface of the macropores, humidification of the gas generated within the alveoli becomes possible, capturing the characteristics of the evaporative mass flow into the gas phase. The liquid film also has the ability to absorb the gas, which means that the gas can dissolve within the liquid lining. Advantageously, this improved model is configured to match as closely as possible the anatomical and physiological characteristics of the respiratory tract, including a large and wet surface, the average distance between any position within the test atmosphere and the system wall in the sub-millimeter range, an appropriate flow rate, and an optimal active transport of deposited water-soluble materials away from the deposition site. This simulates the conditions within the distal region of the respiratory tract where the diameters of the airways as well as the alveolar sacs are within the sub-millimeter range and the surface is covered by a continuous aqueous or liquid layer. In one aspect, a respiratory simulator is disclosed that includes a rigid porous foam. The rigid porous foam includes (i) macropores and (ii) an interconnected open-cell network of micropores and / or nanopores.

[0011] Preferably, the rigid porous foam is (i) a ceramic or metallic rigid porous foam, or (ii) a rigid porous foam of ceramic and metal, and preferably the rigid porous foam is made of alumina or silicon carbide, or oxygen-bonded silicon carbide or sintered silicon carbide, or a combination of two or more of them.

[0012] Preferably, (i) the macropores contain gas or aerosol, or (ii) the micropores and / or nanopores contain an aqueous solution or liquid, or (iii) the macropores contain gas or aerosol and the micropores and / or nanopores contain an aqueous solution or liquid.

[0013] Preferably, the micropores and / or nanopores containing an aqueous solution or liquid form a water layer or liquid layer covering a part or all of the surface of the macropores.

[0014] Preferably, the rigid porous foam contains one or more cavities, or sockets, or depressions, or a combination of two or more thereof, preferably one or more cavities, or sockets, or depressions, or a combination of two or more thereof contains one or more sensing devices, or probes, or sampling devices, or cell cultures.

[0015] Preferably, at least two rigid porous foams each having a different pore size distribution, or a different porosity, or a different material are used in a respiratory simulator.

[0016] Preferably, the respiratory simulator includes a pump and the rigid porous foam is incorporated in or attached to the pump, preferably the pump is a piston pump.

[0017] In another aspect, the use of a rigid porous foam for simulating the distal airways and alveoli of a respiratory simulator is disclosed, the rigid porous foam including (i) macropores, and (ii) an interconnected open cell network of micropores and / or nanopores.

[0018] Preferably, the rigid porous foam is (i) a ceramic or metal rigid porous foam, or (ii) a ceramic and metal rigid porous foam, and preferably, the rigid porous foam is made of alumina or silicon carbide, or oxygen-bonded silicon carbide or sintered silicon carbide, or a combination of two or more thereof.

[0019] In another aspect, there is disclosed a method for determining the effect of a test atmosphere on the simulated distal airways and alveoli of the airway, comprising (i) providing a breathing simulator according to the present disclosure, (ii) contacting the breathing simulator with the test atmosphere, and (iii) determining the effect of the test atmosphere on the simulated distal airways and alveoli of the airway.

[0020] In another aspect, there is disclosed a rigid porous foam for use in a model of the distal airways and alveoli of the airway, comprising (i) macropores containing a gas or aerosol, and (ii) interconnected open cell networks of micropores and / or nanopores, wherein the micropores and / or nanopores contain an aqueous solution or liquid, and the micropores and / or nanopores containing the aqueous solution or liquid form a water layer or liquid layer covering a part or all of the surface of the macropores.

[0021] Preferably, the rigid porous foam is (i) a ceramic or metal rigid porous foam, or (ii) a ceramic and metal rigid porous foam, and preferably, the rigid porous foam is made of alumina or silicon carbide, or oxygen-bonded silicon carbide or sintered silicon carbide, or a combination of two or more thereof.

[0022] Preferably, (i) the macropores contain a gas or aerosol, or (ii) the micropores and / or nanopores contain an aqueous solution or liquid, or (iii) the macropores contain a gas or aerosol and the micropores and / or nanopores contain an aqueous solution or liquid.

[0023] Preferably, the rigid porous foam includes one or more cavities, or sockets, or depressions, or combinations of two or more thereof, and preferably, one or more cavities, or sockets, or depressions, or combinations of two or more thereof include one or more modules for containing or storing cell culture fluid and / or at least one microsensor for monitoring the state within the chamber and / or for gas sampling or gas characterization.

[0024] Preferably, the rigid porous foam is included in or attached inside a pump, and preferably, the pump is a piston pump.

[0025] Some advantages Advantageously, the present invention provides an improved model of the distal airways and alveoli, as discussed herein.

[0026] Advantageously, models of the distal airways and alveoli can improve the accuracy, reliability, and in vitro-in vivo translatability of in vitro methods applied in the context of inhaled toxicity, inhaled dose measurement, and the development and preclinical testing of agents for inhaled therapy.

[0027] Advantageously, models of the distal airways and alveoli enable determination of how the deposition of inhalable test agents and the interaction with the distal airways and alveoli models affect the physiochemical properties of inhalable test agents, and evaluation of how an in vitro biological test system responds to exposure to inhalable test agents within such models. To achieve this, the distal airways and alveoli models can be used in a system configured to allow introduction of one or more modules, such as sensing devices, probes, cell cultures, and / or sampling devices, including all necessary electrical, optical, and microfluidic and / or mesofluidic connections, for example.

[0028] Advantageously, the distal airway and alveolar model can be configured to match the anatomical and physiological characteristics of the airway as closely as possible, with a large, highly moist surface, an average distance between any position in the test atmosphere and the system wall in the submillimeter range, an appropriate flow rate, and optimal active transport of deposited water-soluble material away from the deposition site. This reproduces the conditions in the distal region of the airway, where the diameter of the airway and the alveolar sacs is in the submillimeter range and the surface is covered by a continuous aqueous or liquid layer.

[0029] Advantageously, the distal airway and alveolar model is not limited to simulating the human airway, but can be extended to any type of airway of interest through appropriate parameterization of the design.

[0030] Advantageously, the distal airway and alveolar model is not limited to being applied in combination with those described in WO2019 / 016094 and WO2020 / 148238. Those skilled in the art can design embodiments of the present disclosure that are compatible with any other device used to simulate the interaction between inhalable agents and the airway.

[0031] Embodiments of the present disclosure are described in more detail with reference to the accompanying drawings.

Brief Description of the Drawings

[0032]

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[0033] In the practice of the present disclosure, in certain embodiments, conventional techniques of engineering, microbiology, cell biology, and biochemistry are used. Biological techniques are well described in the following references. Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE. Celis, ed., 1998) Academic Press; Animal Cell Culture (RI. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP. Mather and PE. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, IB. Griffiths and DG. Newell, eds., 1993 - 8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994). Procedures employing commercially available kits and reagents are generally used according to the protocols determined by the manufacturer, unless otherwise described.

[0034] Technical terms and expressions used herein are generally given the meanings commonly applied to them in the relevant art. The definitions of the terms used herein apply to the entire content of this application.

[0035] The term "comprising" does not exclude other elements or steps.

[0036] The indefinite articles "a" or "an" do not exclude a plurality.

[0037] The term "and / or" means, for example, (a) or (b), or (a) and (b).

[0038] As used herein, the terms "comprising" and "comprised of" are synonymous with "including" or "containing", are inclusive or non-limiting, and do not exclude additional members, elements, or method steps not recited. The term "consisting of" means that additional components are excluded and that only the recited elements are present and no more.

[0039] When referring to measurable values such as parameters, amounts, lengths of time, and the like, the term "about" as used herein includes variations of and variations from a particular value, specifically, + / −10% or less, preferably + / −1 to 5% or less, more preferably + / −1% or less, still more preferably + / −0.1% or less of a particular value and from a particular value, to the extent that such variations are appropriate for carrying out the present disclosure. It should be understood that the values referred to by the modifier "about" are also specifically and preferably disclosed in themselves.

[0040] The present disclosure can be used in various applications for airway research. For example, in embodiments, it has been found useful for studying the deposition and / or condensation of one or more components present in a test atmosphere on the inner surface of the device. Also, according to embodiments, it provides an evaluation of a test atmosphere that can be examined while passing through the device to study changes in aerosol concentration, and / or growth of aerosol particles, and / or shrinkage of aerosol particles. The effect of a test atmosphere present inside the device on a biological test system can be studied in embodiments of the present disclosure.

[0041] The models of the distal airways and alveoli described herein include one or more commercially available rigid porous foams. For example, the foam can be purchased from the Fraunhofer Institute for Ceramic Technologies and Systems IKTS, Winterbergstr 28 01277 Dresden, Germany, and Ultramet, 12173 Montague City, Pacoima, CA 91331, USA. The rigid porous foam can be made of a ceramic foam or a metal foam. These can be manufactured with different pore sizes, different pore size distributions, and different amounts of open and closed porosity. Ceramic foams can be manufactured, for example, by replication methods (Boettge et al. (2013) Journal of Materials Research 28, 2220 - 2233), direct foaming of ceramic slurries (Zheng et al. (2021) Journal of Asia Ceramic Societies 9, 24 - 29), 3D printing (Minas et al. (2016) Advanced Materials 28, 9993 - 9999), or by dealloying (Song et al. (2018) Corrosion Science 134, 78 - 98), by several methods well described in the art. In one such method known as the replication method, components made of open cell polyurethane foam are impregnated with a ceramic slurry. Excess material is extruded so that only the surface of the polymer struts is covered with the ceramic material. The wet ceramic film is dried, the foam part is peeled off, thereby burning out the polymer foam. In the final step, the ceramic is sintered according to specific sintering conditions of the material. As a result, the ceramic foam component has a cellular structure and geometric shape that are nearly similar compared to the starting polymer foam part.

[0042] Materials used in the production of ceramic foams include boron, aluminum, silicon, titanium, and zirconium, carbides and nitrides of cordierite. In certain embodiments, the use of ceramic foams is preferred.

[0043] Methods for manufacturing metal foams are described below. Tatt et al., ASM Sc.J. (2021), 16, and Mooraj et al., (2020) Scripta Materialia 177, 146 - 150. Materials used in the production of metal foams include gold, titanium, or iron.

[0044] Thus, the rigid porous foam can be a ceramic or metal rigid porous foam, or a combination of ceramic and metal rigid porous foams. In one embodiment, the rigid porous foam is made of alumina or silicon carbide or oxygen - bonded silicon carbide or sintered silicon carbide, or a combination of two or more thereof, preferably an alumina ceramic foam.

[0045] Properties of the foam such as pore size distribution, homogeneity, mechanical stability, surface properties, porosity, flow resistance, or capillary activity are well - described in the art and can be fine - tuned through the selection of materials, manufacturing methods, surface treatments, and coatings (e.g., Hammel et al. (2014) Ceramics International 40, 15351 - 15370, and Costacurta et al. (2007) J.Am.Ceram.Soc. 90, 2172 - 2177).

[0046] In one embodiment of the present invention, the rigid porous foam is not made only of carbon.

[0047] The rigid porous foams according to the present disclosure have hierarchical pore sizes in that they include (i) macropores, and (ii) micropores and / or nanopores. The foam has a rigid structure to provide the stability required for the bimodal or hierarchical pore size of the foam. The rigidity must be sufficient to prevent the collapse of the macropores during shrinkage of the foam. The collapse is likely to result in the exudation of the macropores because the difference between the capillary activity of the macropores and the micropores and / or nanopores temporarily disappears. Also, the gas permeability can be reduced. The amount of rigidity can be measured using the Young's modulus. As will be understood by those skilled in the art, the Young's modulus is a mechanical property that measures the tensile rigidity of a solid material. Various methods are available for measuring the Young's modulus of a material's elasticity. For example, the Young's modulus of elasticity can be estimated by taking one or more atomic force microscopy (AFM) images of the material and applying the Derjaguin-Muller-Toporov (DMT) mechanical contact model to the pull-out curve. Alternatively, the Young's modulus can be calculated by measuring the slope of the load-displacement curve of the load-displacement plot obtained by mechanical compression of the material. The minimum value of the Young's modulus of the rigid porous foam (i.e., the value at which significant blockage of the macropores is expected) is about 20 kPa for rigid porous foams of metals and / or ceramics.

[0048] The rigid porous foam is (i) a macroporous, and (ii) an open cell network of micropores and / or nanopores. The macropores can allow gases or aerosols (e.g., inhalable test agents) to pass through to the distal airways and alveolar models. The micropores and / or nanopores can be filled with an aqueous solution or liquid that can be retained and passed through, such as water. The aqueous solution or liquid is retained within the network of micropores and / or nanopores to provide an aqueous or liquid film on the surface of the macropores. The micropores and / or nanopores are present within the ceramic or metal struts of the rigid porous foam that form the macropores. A schematic diagram of the pore structure of the rigid porous foam is shown in FIG. 1. The micropores and / or nanopores can be filled with an aqueous solution or liquid, and the macropores can be filled with a gas or aerosol. The ceramic or metal walls of the macropores of the rigid porous foam can be covered with open micropores and / or open nanopores that open towards the macropores, so that the aqueous solution or liquid present within the micropores and / or nanopores forms an aqueous or liquid layer that covers all or part of the surface of the macropores. Thus, both the macropores and the micropores and / or nanopores form a continuous interconnected network of pores within the foam. In this interconnected network, pairs of macropores are connected by a continuous channel formed by the macropores, pairs of micropores and / or nanopores are connected by a continuous channel formed by the micropores and / or nanopores, and the micropores and / or nanopores are connected to the macropores by a continuous channel formed by the micropores and / or nanopores and the macropores.

[0049] The size of the macro pores can mimic the size of the alveoli, and thus the radius of the macro pores can correspond to the radius of the alveoli. The radius of the macro pores can be about 0.1 to about 0.4 mm, or about 0.1 to about 0.3 mm, or about 0.2 to about 0.4 mm, or about 0.2 to about 0.3 mm, or about 0.25 mm. The radius of the macro pores can be about 100 μm to about 2000 μm or about 200 μm to about 2000 μm.

[0050] The radius of the micro pores and / or nano pores is typically about 0.1 μm to about 50 μm, or about 0.1 μm to about 40 μm, or about 0.1 μm to about 30 μm, or about 0.1 μm to about 20 μm, or about 0.1 μm to about 10 μm, or about 0.25 μm to about 25 μm.

[0051] The pore density in the rigid porous foam can be at least about 30 pores per inch, at least about 45 pores per inch, at least about 60 pores per inch, or at least about 80 pores per inch. In certain embodiments, the pore density in the rigid porous foam is 40 - 50 pores per inch. In certain embodiments, the pore density in the rigid porous foam is about 45 pores per inch.

[0052] The total surface area of the rigid porous foam is at least 70 m 2 , preferably 100 m 2 or more.

[0053] Without wishing to be bound by theory, it is possible to fill the micro pores and / or nano pores with an aqueous solution or liquid, but due to the capillary action of the foam as explained by Equation 1, it is not possible for the macro pores.

Number

[0054] The pressure difference ΔP across the surface of the aqueous liquid present in the capillary vessel with respect to the air on this surface is the surface tension γ (Joule m 2) is a function of the contact angle θ with the material of the capillary container. r is the radius of the capillary container (in meters). ΔP means the negative pressure in the aqueous solution or liquid with respect to the ambient air (atmospheric pressure in the applications described herein). The contact angles of solid materials with water vary widely and should be less than 90° if capillary rise is observed. Glass, for example, has a contact angle of zero, and for many ceramic materials, similar values can be assumed. From Equation 1, in the hierarchical form, when the average diameter of the micropores and / or nanopores is one order of magnitude (10 times) smaller than the average diameter of the macropores, it can be estimated that the negative pressure in the aqueous solution or liquid under the surface of the micropores and / or nanopores is 10 times that of the negative pressure under the aqueous solution or liquid surface across the macropores (the pressure in the ambient air and the contact angle are the same for both pore types) (ignoring the effects of surface roughness and irregularities in the foam). Therefore, when the aqueous solution or liquid supplied to the rigid porous foam is maintained under a negative pressure that is greater in absolute value than the negative pressure resulting from the capillary action of the macropores but smaller than the negative pressure resulting from the capillary action of the micropores and / or nanopores, the micropores and / or nanopores of the rigid porous foam are filled with the aqueous solution or liquid, but the macropores are not filled with the aqueous solution or liquid. In one embodiment, the average diameter of the micropores and / or nanopores is about 10 times smaller than the average diameter of the macropores. In another embodiment, the aqueous solution or liquid supplied to the rigid porous foam is maintained under a negative pressure that is greater in absolute value than the negative pressure resulting from the capillary action of the macropores but smaller than the negative pressure resulting from the capillary action of the micropores and / or nanopores.

[0055] A further aspect of the present disclosure relates to a method of filling the micropores and / or nanopores, but not the macropores, of the rigid porous foams described herein with an aqueous solution or liquid, the rigid porous foam comprising (i) macropores and (ii) an interconnected open cell network of micropores and / or nanopores, the method comprising supplying an aqueous solution or liquid to the rigid porous foam under a negative pressure that, in absolute value, is greater than the negative pressure resulting from the capillary action of the macropores but less than the negative pressure resulting from the capillary action of the micropores and / or nanopores, and (ii) filling the micropores and / or nanopores, but not the macropores, of the rigid porous foam with the aqueous solution or liquid.

[0056] With a sufficiently large difference between the average size of the micropores and / or nanopores and the average size of the macropores, a drain can be connected to the system containing the foam (e.g., a pump). When the aqueous solution or liquid present in the drain is set to a negative pressure that is greater than the negative pressure applied to the supply of the aqueous solution or liquid but less than the negative pressure induced by the capillary action of the micropores and / or nanopores, a flux of the aqueous solution or liquid from the supply to the drain can be induced while keeping the micropores and / or nanopores filled with the aqueous solution or liquid. Such a drain may act as a simulated blood flow and can somewhat stabilize the concentration gradient between the gas or aerosol and the aqueous solution or liquid lining present on the surface of the rigid porous foam.

[0057] The capillary action of metal or ceramic rigid porous foam types with 30, 45, and 60 pores per inch is sufficient to maintain a water column height greater than 20 cm (ΔP greater than 2000 Pascal) without exuding the macropores or increasing the resistance of the rigid porous foam to the airflow passing through the microporous network.

[0058] Optionally, the distal airway and alveolar model can be a combination of one or more rigid porous foams. In certain embodiments, one or more rigid porous foams can be combined with one or more different foams or structures that include a plurality of bifurcating channels. One such structure, described in WO2020 / 148238, relates to a perforated structure consisting of a perforated skin that houses one or more bifurcating channels, where each perforation is an open end of one or more bifurcating channels. The foam structure that provides the plurality of bifurcating channels preferably includes micropores and / or nanopores (not macropores) and is made of a ceramic or metal material that can maintain and / or implement the aqueous solutions or liquids described herein. The bifurcating channels within the structure preferably reproduce the anatomical structure of the lung in terms of channel length, bifurcation pattern, bifurcation angle, and channel diameter. Such structures can be generated by the same methodology as the hierarchical foams described herein. Due to the low complexity of the 3D geometric shape of such structures (e.g., lack of hierarchical structure), 3D printing of ceramic precursors that continue to sinter (see Minas et al., supra) or 3D printing of metal materials (see Mooraj et al., supra) is possible.

[0059] The optimal pore size distribution for a particular application can be determined using computational and empirical approaches. The rigid porous foam may have a high (macro) porosity and provide a resistance to the flow of gas or aerosol passing through it that is the same as or lower than any of Sections 1 - 4 of FIG. 1 described in WO2019016094A1. In particular, the rigid porous foam can provide a resistance to the flow of gas or aerosol passing through it that is the same as or lower than any part of the conductive or extra-thoracic airways of the airway.

[0060] The rigid porous foam may be composed of a plurality of individual units, and the individual units provide means for establishing a leak-free connection between them. In embodiments that include two or more individual units of rigid porous foam, such as the embodiment of FIG. 4, each individual structure may include the same or different structures, such as the same or different pore size distributions. For example, it may be desirable to adjust the pore size distribution based on specific experimental needs (or simulation of a deep lung disease state). In the embodiment of FIG. 4, three individual rigid porous foams 401a, 401b, and 401c are shown. The presence of two or more individual rigid porous foams allows for the introduction of more locations into which one or more modules, such as one or more sensing devices, sampling devices, or cell cultures, can be inserted. The presence of two or more individual rigid porous foams can enable fine-tuning of the properties of an overall model of the distal airways and alveoli, for example, by incorporating rigid porous foams of different pore sizes, porosities, or materials.

[0061] The rigid porous foam and / or individual units of the rigid porous foam may be connected to a source or sources of aqueous solution or liquid present within the micropores and / or nanopores.

[0062] The rigid porous foam and / or individual units of the rigid porous foam can be connected to a drain or drains that can discard the aqueous solution or liquid supplied from the source or sources.

[0063] The rigid porous foam can also be used to enclose or accommodate one or more modules. When multiple modules are used, they can be of the same or different types. Such modules can be used to monitor operations and / or conduct experiments and / or collect samples and / or accommodate cell cultures and the like. For example, a module can enclose or store a cell culture medium containing an optional cell culture, or can enclose or store for monitoring the state during an experiment or for gas or liquid sampling or for gas characterization, etc. A module can be a receptacle capable of holding an aqueous solution or liquid such as a cell culture medium. A module can be a device such as a probe. Such devices can be used for monitoring the internal system state or for characterizing the test atmosphere or for sampling and the like. The operation of the module can be controlled by a computer. A module can be a culture chamber in which a biological test system, such as a cell culture, or airway epithelial cells and / or alveolar epithelial cells can be placed for exposure to a test atmosphere. A module equipped with a quartz crystal microbalance can be used. The module can be placed within the rigid porous foam or in the vicinity of the surface of the rigid porous foam as required. One or more cavities, sockets, or depressions can be included within the rigid porous foam in which one or more of the modules can be placed and held or stored.

[0064] The rigid porous foam can include one or more bifurcated channels. The bifurcated channels can bifurcate. The bifurcated channels can be present within a structure made of a rigid ceramic or metal material containing micropores and / or nanopores. Embodiments including bifurcated channels can be seen in FIG. 3. In this embodiment, the distal airway and alveolar model includes a cylindrical rigid porous foam 301 and a proximal cylindrical or disc-shaped structure 302 that provides the bifurcated channels. There is also an additional more proximal airway model 307, such as that described in WO2020 / 148238, which is connected to the disc-shaped structure 302. The opening of the more proximal airway model 307 bifurcates so as to end in a defined number of smaller distal openings.

[0065] The rigid porous foam may be mounted within the breathing simulator so as to leave sufficient space at the surface of the rigid porous foam in a suitable location, thereby allowing one or more of the various modules to be arranged and held.

[0066] If the models of the distal airways and alveoli include a plurality of rigid porous foams, or a combination of a rigid porous foam and one or more structures providing a plurality of bifurcated channels, they can be connected in a way that leaves sufficient space for arranging and holding one or more of the various modules between the individual parts.

[0067] For ease of use, the rigid porous foam can be formed into any desired shape. This can facilitate the intended use of the rigid porous foam, such as in a breathing simulator. Non-limiting examples of suitable shapes are cylindrical or disc-shaped or cubic. The rigid porous foam shape can contain the foam that is surrounded and shaped by the radial walls. The radial walls are typically composed of a material different from the foam. In one embodiment, the different material does not contain pores. In another embodiment, the different material is a synthetic material such as an epoxy resin or metal or a combination thereof.

[0068] An element capable of contacting a rigid porous foam includes a cross-linked structure capable of contacting the rigid porous foam. This cross-linked structure can be used for any intended purpose, such as facilitating the contact of an aqueous solution or a liquid circuit with the foam. The circuit can be configured to supply an aqueous solution or a liquid or to discharge an aqueous solution or a liquid from the rigid porous foam. The contact can be achieved by adhering or welding the cross-linked structure to the foam or can be part of the 3D printing design of the foam. One can refer to FIG. 5 for an embodiment where there is a connection during the supply of an aqueous solution or a liquid to the rigid porous foam or between the drains of the aqueous solution or the liquid present in the micropores and / or nanopores of the rigid porous foam. The cross-linked structure 503 provides a connection, for example, to a microfluidic or mesofluidic tube 504 that is derived from an aqueous solution or a liquid or another storage part of an aqueous solution or a liquid located outside the pump. The connection to the microfluidic system and / or the mesofluidic system establishes a constant supply of an aqueous solution or a liquid that is driven by the capillary action of the micropores and / or nanopores of the rigid porous foam, the evaporation of water inside the rigid porous foam, and / or the negative pressure applied to the drains.

[0069] An example of the cross-linked structure is a microporous and / or nanoporous cross-link between an aqueous or liquid circuit and the foam. The microporous and / or nanoporous cross-link may be composed of various materials such as fibrous materials like cellulose acetate fibers or spongy materials, and it is preferably flexible and easily deformable so as to conform its shape to the shape of the rigid porous foam at the connection site, thereby maximizing the contact area with the foam. Such an embodiment can be seen in FIG. 6. The cross-linked structure 602 (which can be adhered or welded to the rigid porous foam 601 or can be part of the 3D print design of the foam) enables the connection of the circuit (supply or discharge of an aqueous solution or a liquid) by means of the microfluidic and / or mesofluidic tube 603. The microporous and / or nanoporous cross-link 604 is inserted into the cross-linked structure 602 to establish contact with the rigid porous foam 601.

[0070] An example of a rigid porous foam that can be used in accordance with the present disclosure has a pore density of 45 pores per inch, an accessible pore volume of 19347 mm 3 , a separated pore volume of 5 mm 3 and a total pore volume of 19352 mm 3 is an alumina ceramic rigid porous foam. The accessible porosity is 85.65%, the separated porosity is 0.024%, and the total porosity is 85.67%. The average pore diameter is 0.82 ± 0.15 mm. The total volume of the rigid porous foam is 31487 mm 3 . The rigid porous foam is very homogeneous and the separated porosity is very small. The flow resistance of the rigid porous foam is measured before and after foam wetting. The ΔP across the foam is found to be in the range of 2 - 10 Pa and at physiologically relevant flow rates, which is within the range of the pressure difference that accumulates between the conductive airways and the alveolar space of the human lung during normal breathing. The exudation of macropores is limited. High-efficiency water transfer to the gas phase (10 L / min) with rH from less than 3% to more than 75% is achieved in about 0.6 seconds, indicating excellent air humidification and excellent capillary activity in the foam. The capillary ΔP is greater than 2.5 kPa, indicating efficient water supply for humidification. Particle retention in the rigid porous foam is found to meet the requirements regarding particle size and number.

[0071] A further aspect of the present disclosure relates to a rigid porous foam for use in a model of the distal airways and alveoli of the respiratory tract, comprising (i) macropores containing a gas or aerosol, and (ii) interconnected open cell networks of micropores and / or nanopores, wherein the micropores and / or nanopores contain an aqueous solution or liquid, and the micropores and / or nanopores containing the aqueous solution or liquid form a water layer or liquid layer covering a part or all of the surface of the macropores. The rigid porous foam generally has the characteristics of the aforementioned rigid porous foam. As will be understood, this rigid porous foam can be integrated or incorporated into a respiratory simulator, such as a pump of a respiratory simulator, as described above. The pump can be a piston pump. Also disclosed is the use of this rigid porous foam for simulating the distal airways and alveoli in a respiratory simulator, as described above.

[0072] Models of the distal airways and alveoli that include a rigid porous foam can be conveniently incorporated into a respiratory simulator, such as a component of a respiratory simulator that can be a pump or the like. Respiratory simulators are described in the art, such as WO2019016094, WO2020 / 148238, WO2022040247, WO2016118935. Exemplary embodiments in which the distal airway and alveoli model is incorporated inside the pump of the respiratory simulator are shown in FIGS. 3, 4, 5, and 6. For example, if the respiratory simulator includes a pump, the distal airway and alveoli model can be incorporated inside the pump, which can be a piston pump. Advantageously, this can be achieved without affecting the function of the respiratory simulator or its components. A leak-free connection can be conveniently established between the distal airway and alveoli model and the respiratory simulator. In one embodiment, one or more of the rigid porous foams described herein can be coupled to a lung model, such as those described in WO2019 / 016094A1. According to this embodiment, the rigid porous foam can be arranged in the pump shown as section 5 of FIG. 2, which couples the lung model to the trachea model (section 3 of FIG. 2) to form a single continuous structure and connects to the bronchial tree model and the more proximal airway model (section 4 of FIG. 2). The model can provide means for fixing it within the lung pump (section 5 of FIG. 2) and for establishing a leak-free connection to the bronchial tree model (section 3 of FIG. 2) and between its individual submodels. The distal airway and alveoli model can be connected to the supply of an aqueous solution or liquid and / or the drain of an aqueous solution or liquid.

[0073] Here, an embodiment of the present disclosure will be described where a rigid porous foam is installed inside a piston pump 300. The piston pump 300 can form part of a respiratory simulator. This embodiment is shown in the cross-sectional view of FIG. 3. The distal airway and alveolar model includes a cylindrical rigid porous foam 301 and a cylindrical or disc-shaped structure 302 that provides a bifurcated channel. The distal airway and alveolar model is inserted into the piston pump 300 and mounted within the piston pump 300. Examples of respiratory simulators including such pumps are described in WO201901609. The piston pump 300 has a cylinder wall 303, a base plate 304, and a piston 305. The base plate 304 provides a centrally located opening 306 through which an airway model 307, such as that described in WO2020 / 148238A1, which can be used to simulate the effect of the conductive airway tree, is connected. The airway model 307 provides a proximal opening 308 that can be connected to one or more additional proximal airways, for example, a model of a bronchus, as required. The opening of the more proximal airway model 307 bifurcates such that it ends at a defined number of smaller distal openings at or near the inner surface of the piston pump base plate 304. Optionally, the base plate 304 of the piston pump provides a further opening 309 through which one or more sensors, probes, electrical connections, or microfluidic or mesofluidic connections can be inserted. A plurality of such openings or a single opening may be present. The opening provides a structure that hermetically seals the internal volume of the pump from the surroundings. Such structures are well described in the art and, in the simplest case, may include a perforated silicon plug that seals the perforation in the pump base plate 304 through which the connection passes. Optionally, the base plate 304 of the piston pump may provide yet a further opening 310 through which the rigid porous foam 301 and / or the structure 302 can be connected to a supply of an aqueous solution or liquid, such as water, and / or a drain of the aqueous solution or liquid, and / or electronics required for, for example, heating and / or temperature monitoring.The rigid porous foam 301 is located centrally within the piston pump and is attached, for example, by means of screws 311 that project through the wall 312 of the rigid porous foam 301, the disc-shaped structure 302, and the base plate 304. The screws may, for example, capture a nut 313, whereby the rigid porous foam 301 and the disc-shaped structure 302 are pushed towards the base plate 304. In the illustrated embodiment, the radial wall 312 of the rigid porous foam 301 is preferably of a different texture and / or a different material than the core of the rigid porous foam. For example, the radial wall 312 can be made of the same ceramic or metallic material as the core of the rigid porous foam 301, but provides no macropores and only micropores and / or nanopores, thus providing no passage for aerosol or gas and providing a high capillary action. Alternatively, the radial wall 312 of the rigid porous foam 301 can be made of a synthetic material such as epoxy resin or metal. The radial wall 312 illustrated in FIG. 3 provides mechanical stability for fixing the rigid porous foam by means of the screws 311, but alternative embodiments may omit the radial wall 312 in order to increase the total cross-sectional area of the flow channels through the foam. The contact between the rigid porous foam 301 and the disc-shaped structure 302, and between the disc-shaped structure 302 and the pump base plate 304, can be sealed, for example, by means of a gasket or O-ring 314. In this configuration, the suction stroke of the piston 304 forces gas and aerosol through the more proximal airway model 307, and the distal airway and alveolar models include the rigid porous foam 301 and the distal airway model 302 within the pump cylinder, and the compression stroke of the piston forces the gas and aerosol present within the pump through the rigid porous foam 301, the distal airway model 302, and the more proximal airway model 307. A gasket or O-ring 314 introduces a gap 315 between the pump base plate 304 or the more proximal airway model 306 and the more distal airway model 302, and between the more distal airway model 302 and the rigid porous foam 301.The gap between the more proximal airway model 306 and the more distal airway model 302 functions to connect the channels present in the two models, although they are not necessarily located at the same radial and tangential positions within the two models. The gap between the more distal airway model 302 and the rigid porous foam 301 functions to avoid the entry of the test agent into the rigid porous foam 301 such that the foam is uniformly connected to the channels present in the airway model, i.e., is locally limited to the location of the channels present in the more distal airway model 302. Further, the pump base plate 304 or the gap 315 between the more proximal airway model 306 and the more distal airway model 302, as well as the gap between the more distal airway model 302 and the rigid porous foam 301, provide a location where one or more modules, such as a cell culture, sampling device, probe, or sensing device of appropriate dimensions and shape, can be placed (not shown).

[0074] An alternative embodiment of the displacement pump 400 including three separate rigid porous foams is shown in FIG. 4. The pump body, the more proximal airway model, the radial walls of the rigid porous foam, the gasket or O-ring, and the elements of the gaps between the individual elements are of the same function and structure as those described for the piston pump embodiment 300 shown in FIG. 3. The presence of the three separate rigid porous foams 401a, 401b and 401c allows the insertion of one or more modules such as one or more sensing devices, sampling devices, or cell cultures, and at the same time, for example, by incorporating foams of different pore sizes, porosities, or materials, provides for the introduction of more positions that enable the fine-tuning of the characteristics of the overall model of the distal airway and alveoli. The piston pump 400 has a cylinder wall 403, a base plate 404, and a piston 405. The base plate 404 provides a centrally located opening 406 through which an airway model 407, such as that described in WO2020 / 148238, is connected. The airway model 407 provides a proximal opening 408 that can be connected to one or more additional proximal airways, for example, a model of a bronchus, as required. The opening of the more proximal airway model 407 bifurcates at or near the inner surface of the piston pump base plate 404 to end in a defined number of smaller distal openings. Optionally, the base plate 404 of the piston pump provides a further opening 409 through which one or more sensors, probes, electrical connections, or microfluidic or mesofluidic connections can be inserted. A plurality of such openings or a single opening may be present. The openings provide a structure that seals the internal volume of the pump airtight from the surroundings. Such structures are well described in the art and, in the simplest case, may include a perforated silicon plug through which the connection passes and seals the perforation in the pump base plate 404.Optionally, the base plate 404 of the piston pump may provide additional openings 410 through which the foams 401a, 401b, and 401c and / or the structure 402 can be connected to a supply of aqueous solution or liquid, such as water, and / or a drain of aqueous solution or liquid, and / or electronics necessary for, for example, heating and / or temperature monitoring. The rigid porous foams 401a, 401b, and 401c are located centrally within the piston pump and are attached, for example, by screws 411 that protrude through the walls 412 of the foams 401a, 401b, and 401c, the disk-shaped structure 402, and the base plate 404. The screws may, for example, capture nuts 413 by the foams 401a, 401b, and 401c, and the disk-shaped structure 402 is pushed towards the base plate 404. In the illustrated embodiment, the radial walls 412 of the foams 401a, 401b, and 401c are preferably of a different texture and / or a different material than the core of the rigid porous foam. For example, the radial walls 412 can be made of the same ceramic or metallic material as the core of the foams 401a, 401b, and 401c, but provide no macropores and only micropores and / or nanopores, thus not allowing aerosol or gas to pass through and providing high capillary action. Alternatively, the radial walls 412 of the foams 401a, 401b, and 401c can be made of a synthetic material such as epoxy resin or metal. The radial walls 412 illustrated in FIG. 4 provide mechanical stability for fixing the rigid porous foam by the screws 411, but alternative embodiments may omit the radial walls 412 to increase the total cross-sectional area of the flow path through the foam. The contact between the rigid porous foams 401a, 401b, and 401c and the disk-shaped structure 402, and between the disk-shaped structure 402 and the pump base plate 404, can be sealed, for example, by gaskets or O-rings 414.In this configuration, the suction stroke of the piston 404 forces gas and aerosol into the pump cylinder through the more proximal airway model 407 and the distal airway and alveolar model consisting of foams 401a, 401b, and 401c and the distal airway model 402, and the compression stroke of the piston forces the gas and aerosol present in the pump through the foams 401a, 401b, and 401c, the distal airway model 402, and the more proximal airway model 407. A gasket or O-ring 414 introduces a gap 415 between the pump base plate 404 or the more proximal airway model 406 and the more distal airway model 402, and between the more distal airway model 402 and the rigid porous foams 401a, 401b, and 401c. The gap between the more proximal airway model 406 and the more distal airway model 402 functions to connect the channels present in the two models, although they are not necessarily located at the same radial and tangential positions within the two models. The gap between the more distal airway model 402 and the rigid porous foams 401a, 401b, and 401c functions to uniformly connect the rigid porous foam to the channels present in the airway model, i.e., to be locally restricted to the location of the channels present in the more distal airway model 402, to avoid the test agent from entering the foams 401a, 401b, and 401c. Further, the gap 415 between the pump base plate 404 or the more proximal airway model 406 and the more distal airway model 402, and the gap between the more distal airway model 402 and the rigid porous foams 401a, 401b, and 401c provide a location where one or more modules such as one or more cell cultures, sampling devices, probes, or sensing devices of appropriate dimensions and shapes can be placed (not shown).

[0075] Embodiments of connections during the supply of an aqueous solution or liquid to a rigid porous foam, or of drains of an aqueous solution or liquid present within the micropores and / or nanopores of a rigid porous foam, are shown in FIG. 5. The figure shows an enlarged section of a cross-sectional view of a lung model 500 containing a distal airway and alveolar model. In the illustrated embodiment, the radial wall 501 of the rigid porous foam 502 is made of a porous material (e.g., the same material as the rigid porous foam itself), but contains no macropores and only micropores and / or nanopores. The micropores and / or nanopores form a continuous porous connection between the rigid porous foam and the pump volume located between the pump cylinder wall and the distal airway and alveolar model. The bridging structure 503 provides a connection to a microfluidic or mesofluidic tube 504, for example, originating from a reservoir of an aqueous solution or liquid located outside the pump. The bridging structure can be adhered or welded, for example, to the surface of the radial wall 501 of the rigid porous foam. Alternatively, it can be made part of the 3D printing design of the rigid porous foam. The bridging structure 503 preferably provides an inner cavity 505 that increases the contact area between the fluid present within the bridging structure 503 and the radial wall 501 of the rigid porous foam. The connection to the microfluidic or mesofluidic system establishes a constant supply of an aqueous solution or liquid, driven by capillary action of the micropores and / or nanopores of the foam, evaporation of water within the rigid porous foam, and / or negative pressure applied to the drain. By setting the aqueous solution or liquid within the microfluidic or mesofluidic tube to an appropriate negative pressure with respect to the ambient air, capillary action of the micropores and / or nanopores present in the radial wall 501 of the rigid porous foam 502 and the struts of the rigid porous foam prevents or reduces air from entering the micropores and / or nanopores, and establishes an aqueous or liquid continuum that reaches from the reservoir through the microfluidic or mesofluidic tube of the supply section, the micropores and / or nanopores of the distal airway and alveolar model, and the microfluidic or mesofluidic tube of the drain to the drain reservoir.

[0076] Another possible embodiment of the connection during the supply of an aqueous solution or liquid to a rigid porous foam, or the connection between the drains of an aqueous solution or liquid present in the micro / nano pores of a rigid porous foam, is shown in FIG. 6. This figure shows an enlarged section 600 of a cross-sectional view of a lung model, including an embodiment of a distal airway and alveolar model, where the hierarchical rigid porous foam 601 is not surrounded by a radial wall but is allowed to the gas or aerosol on its entire surface. Such embodiments cannot be connected to the source and / or drain of an aqueous solution or liquid in the manner shown in FIG. 5, because the direct connection of an aqueous solution or liquid to the macropores of a rigid porous foam eliminates the closed contact between the capillary activity of the micro and / or nano pores, resulting in the intake of air into the aqueous solution or liquid circuit. To overcome this limitation, a microporous and / or nanoporous bridge between the aqueous or liquid circuit and the rigid porous foam is required. This can be established, for example, by providing a bridging structure 602 that can be adhered or welded to, for example, the rigid porous foam 601 or can be part of the 3D printing design of the foam. The bridging structure 602 allows the connection of an aqueous or liquid circuit (supply or discharge of an aqueous solution or liquid) by means of microfluidic or mesofluidic tubes 603. The microporous and / or nanoporous bridge 604 is inserted into the bridging structure 602 to establish contact with the rigid porous foam 601. The microporous and / or nanoporous bridge may be composed of, for example, a fibrous or spongy material (e.g., cellulose acetate), and it is preferably flexible and easily deformable so as to conform its shape to the shape of the rigid porous foam at the connection site, thereby maximizing the contact area to the microporous and / or nanoporous network in the foam. A bridge made of a high-density bundle of cellulose acetate fibers about 2 cm in length was successfully tested in combination with an alumina ceramic hierarchical rigid porous foam having a macropore density of 45 pores / inch, an average macropore size of 820 μm, and a micropore and / or nanopore size of less than 30 μm.The bridge can maintain a closed aqueous or liquid circuit at a negative pressure exceeding 2000 Pascals, enabling continuous evaporation-driven water supply to the alumina foam.

[0077] In a further aspect, a system for determining the interaction between a test atmosphere and a simulated airway is described, the system comprising: (i) a chamber configured to enclose a first volume of gas comprising the test atmosphere; (ii) a first port adapted to receive and discharge gas and including a valve for regulating the flow of gas through the first port, the valve being movable between open and closed positions and, in the open position, being openable towards the test atmosphere or ambient air; (iii) a second port adapted to receive and discharge gas and including a valve for regulating the flow of gas through the second port, the valve being movable between open and closed positions; (iv) a motor for controlling the operation of a first pump, the first pump comprising: (a) a chamber configured to enclose a second volume of gas, the first and second volumes of gas being different; (b) a port adapted to receive and discharge gas; (c) a piston plate within the chamber, the piston plate including one or more apertures for the intake or inflow of gas into the chamber, one or more or each of the apertures being movable between open and closed positions and including a valve capable of regulating the intake or inflow of gas; (d) an airway and alveolar model of the present disclosure stored within the second pump; (e) a motor for controlling the operation of the second pump, the second pump comprising: (c) a connection structure operable to transfer gas from the first pump into the second pump; (d) one or more apertures in the first pump or the second pump, or the wall of the connection structure, or a combination of two or more thereof, the apertures being capable of receiving a module for monitoring the state within the chamber containing the cell culture matrix and / or for gas sampling or gas characterization and including or storing at least one microsensor.

[0078] The gas can be the test atmosphere or can include the test atmosphere. FIG. 7 shows the system 10 according to this aspect of the present disclosure. The system 10 includes at least two pumps 40, 80. Two or more pumps 40, 80 are connected to each other. In some embodiments, two or more pumps 40, 80 are connected to each other by a branched hollow structure 50. Each pump 40, 80 may be operated by its own individual motor 41, 81, or two or more pumps may be operated by the same motor 41, 81 as needed. The complete system 10 can be located within a climate-affected housing 11 equipped with a thermostat 12 so as to control the temperature within the housing 11. The chambers of the pumps 40, 80 can be configured to represent the internal volumes of different sections of the airway, such as the airway of a human or animal. The chambers can be configured to provide a capture volume that can be maximally achieved in each section of the airway and a displacement volume that is at least as large. In particular, one (first) pump 40 can represent the volume of the oral cavity and the oropharyngeal cavity, such as that of a human or animal. Another second pump 80 can represent the volume of the lung lumen or a part thereof, such as an individual lung lobe or the lumen of a smaller subunit, which is particularly the lung lumen or a part thereof of a human or animal. The airway and alveolar model of the present disclosure can be housed within the second pump 80. The branched hollow connection structure 50 can represent the dimensions of the conductive airway, such as one or more of the nasopharyngeal cavity, hypopharynx, larynx, trachea, bronchi, and bronchiole structures up to the respiratory bronchioles, particularly the dimensions of the conductive airway of a human or animal. The branched hollow connection structure 50 can represent the dimensions of the conductive airway including the nasopharyngeal cavity, hypopharynx, larynx, trachea, bronchi, and bronchiole structures up to the respiratory bronchioles. Not only the branching pattern of the different sub-components of the connection structure 50, but also the dimensions such as, for example, diameter and length, can resemble the tree of the conductive airway. The branched hollow structure 50 can be connected to the central openings 43, 83 at the bottoms 44, 84 of the chambers 42, 82 of each pump 40, 80. In some embodiments, a plurality of openings, holes, or sockets 51 can be present on the bases 44, 84 of the chambers 42, 82 and can be arranged symmetrically around the central openings 43, 83.By directly using valve 44a in the connection between pump 40 and connection structure 50, it is possible to seal pump 40 from all other system components. Pump 40, which represents the oral cavity, can have one or more openings 43 through which the test atmosphere and dilution air can pass when leaving pump 40 towards the branched hollow structure 50. The entry and exit points of the test atmosphere 90 are typically located on the piston plate 45 of pump 40, preferably at its center. This can pass through the hollow piston shaft 46, and a valve 44a such as a three-way valve may exist above it. In an embodiment, valve 44a can be closed or opened towards the test atmosphere source or ambient air. The arrangement of one or more (e.g., multiple) openings 47 through which ambient air can enter the system can be optionally arranged radially and is arranged on the piston plate 45. One or more (e.g., multiple) valves 48 can be used to enable opening or closing of one or more of these openings 47. In certain embodiments, each opening 47 is controlled by a valve 48. In certain embodiments, the arrangement of one or more (e.g., multiple) openings through which ambient air can enter the system can be optionally arranged radially and can be arranged on the piston plate 84 of the second pump. One or more (e.g., multiple) valves can be used to enable opening or closing of one or more of these openings. In certain embodiments, each opening is controlled by a valve. In certain embodiments, the arrangement of one or more (e.g., multiple) openings through which ambient air can enter the system can be optionally arranged radially and can be arranged on the piston plates of the first and second pumps. It is advantageous that the branched hollow structure 50 can be separated from pumps 40 and 80. It is advantageous that the branched hollow structure 50 can be disassembled into its main components. This can enable easy access for placing or removing the test system and / or for cleaning. The bases 44 and 84 of pumps 40 and 80 can be removed for placing / removing the test system and for cleaning.Not only different parts of the connection structure 50, but also within the bases 44, 84 of each of the pumps 40, 80, openings, holes, or sockets 51 such as threaded or non-threaded openings, threaded or non-threaded holes, or threaded or non-threaded sockets 113, 213 can be located. The openings, holes, or sockets 51 can be placed in various positions, such as on one or more of the bases 44, 84 of the pumps 40, 80, or around the central openings 43, 83, or at various optimal locations within the branched hollow structure 50, preferably on the underside of the branched hollow structure 50, or in any combination thereof. The openings, holes, or sockets 51 can be used to monitor the operation of the system 10 and / or conduct experiments and / or collect samples and the like, and can be used to enable the attachment of various modules 112, 212, or devices onto or into them. Examples of such modules are described herein. Thus, it is also advantageous that the pumps 40, 80 used in the system 10 can not only function to transport the test atmosphere but also function as an exposure chamber.

[0079] The modules to be used can be adapted for various purposes according to the requirements of the system to be configured. For example, the modules can be adapted to contain or store cell culture media, or to monitor the conditions in chambers 42, 82, or to sample gases or liquids, or to characterize gases, and the like. The modules can be located on the bases 44, 84 of the first pump 40 and / or the second pump 80, and / or within the walls of the connection structure 50. In certain embodiments, one or more modules can be configured to contain or store a matrix containing cell culture media. According to this embodiment, one or more modules can be containers capable of holding a liquid or solution. The cell culture media can contain or be in contact with a cell culture, such as a two-dimensional or three-dimensional culture of cells. In some embodiments, one or more modules can hold or position at least one microsensor, as an alternative to or in addition to the cell culture media matrix. In some embodiments, a module adapted to contain or store a matrix containing cell culture media and / or at least one microsensor further includes microfluidic channels, and optionally, a microfluidic pump connected thereto. The modules will generally be positioned within one or more horizontal planes of the walls of the first pump 40, or the second pump 80, or the connection structure 50.

[0080] Test atmospheres such as gases and aerosols can be studied and can be generated via various means. For example, in many applications such as testing tobacco products and general medical inhalers, test atmosphere generation can be driven by a pump present in a breathing simulator to generate the negative pressure required for test atmosphere generation and extraction. That is, the use of an aerosol generator / smoking machine is advantageously not necessary. The test atmosphere can be an aerosol such as smoke or can be derived from smoke. As used herein, the term "smoke" is used to describe the type of aerosol generated by a smoking article such as a cigarette or by burning an aerosol-forming material. Smoke contains various agents which can be provided as individual compounds for research if needed. Examples of such agents include nicotine-free dry particulate matter, carbon monoxide, formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, crotonaldehyde, methyl-ethyl ketone, butyraldehyde, benzo[a]pyrene, phenol, m-cresol, o-cresol, p-cresol, catechol, resorcinol, hydroquinone, 1,3-butadiene, isoprene, acrylonitrile, benzene, toluene, pyridine, quinoline, styrene, N'-nitrosornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl, nitric oxide (NO), nitrogen oxides (NOx), hydrocyanic acid, ammonia, arsenic, cadmium, chromium, lead, nickel, selenium and mercury.

[0081] When the aerosol is smoke, a smoking machine can be used to generate the aerosol. The smoking machine may be attached to a breathing simulator. In this way, the smoking machine holds and ignites the cigarette, and the aerosol is provided directly to the breathing simulator. The defined number of puffs per cigarette and the defined number of puffs per minute of exposure may be used, and the number of cigarettes can be changed to adjust the exposure time. A reference cigarette, for example, 3R4F of the reference cigarette, may be used as the source of smoke, and it may be smoked on the smoking machine substantially in accordance with the smoking regimen of the International Organization for Standardization (ISO 2000).

[0082] The use of a control atmosphere, such as an atmosphere that does not include the test atmosphere, is also intended. The use of a control atmosphere can help to determine the effect of the test atmosphere in comparison with the control atmosphere.

[0083] The breathing simulator can be connected to the smoking machine by a suitable conduit that provides a flow path for the smoke to the breathing simulator. The smoke can be transmitted through the conduit with a carrier gas such as air or without a carrier gas. When using a carrier gas, the conduit preferably includes an inlet for introducing the carrier gas into the conduit so as to mix with the smoke flow. This conduit may include at least one injection port for introducing or injecting a standard reference substance such as nicotine into the breathing simulator for calibration purposes. The smoke flow is generally controlled by the breathing simulator.

[0084] The smoking machine can be a linear or rotary smoking machine. Preferably, the smoking machine operates to simultaneously draw on a plurality of smoking articles so as to collect and analyze the cumulative smoke from the plurality of smoking articles. Suitable smoking machines for use in the present disclosure are well known to those skilled in the art.

[0085] The present disclosure can be used to analyze mainstream smoke generated from a smoking article during a smoking test. "Mainstream smoke" refers to the smoke that is drawn through a smoking article and would be inhaled by a consumer during use.

[0086] The test atmosphere can be from an "aerosol generating device", which is a device that interacts with an aerosol-forming substrate to generate an aerosol so as to generate an aerosol. An example of an aerosol is smoke. The aerosol-forming substrate can be part of an aerosol-generating article. The aerosol generating device can include one or more components suitable for generating an aerosol from an aerosol-generating substrate. The aerosol generating device can be an electrically heated aerosol generating device that includes a heater operated by electricity to heat the aerosol-forming substrate of the aerosol-generating article to generate an aerosol. The aerosol generating device can be a gas-heated aerosol generating device, a device heated by a carbonaceous heat source, other exothermic chemical reactions, or a heat sink. Other suitable means for generating an aerosol are well known in the art. The aerosol generating device can be a device that interacts with the aerosol-forming substrate of the aerosol-generating article to generate an aerosol that can be directly inhaled into a user's lungs through the user's mouth.

[0087] Another example of an "aerosol generating device" is an inhaler (inhalation device) that is commonly used to deliver an aerosol containing an active ingredient such as a medically active compound. Such inhalers are generally used for the delivery of aerosolized drugs to the airways.

[0088] They can be used for the treatment of respiratory and other diseases. Such inhalers are well known in the art and are generally of the pressurized metered type, dry powder type, or nebulizer type. Generally, the medicament is in the form of a pressurized formulation containing fine particles of one or more pharmaceutical compounds suspended in a liquefiable propellant, or a solution of one or more compounds dissolved in a propellant / cosolvent system. Such formulations are well known in the art.

[0089] As used herein, the term "aerosol-forming substrate" relates to a substrate capable of releasing a volatile compound capable of forming an aerosol. Such volatile compounds can be released by heating the aerosol-forming substrate. The aerosol-forming substrate can be adsorbed, coated, impregnated, or otherwise loaded onto a carrier or support. Advantageously, the aerosol-forming substrate can be part of an aerosol-generating article or a smoking article. In certain applications, the aerosol-forming substrate is included within an aerosol-generating article, such as a rod-shaped aerosol-generating article, such as an aerosol-generating article or a heated cigarette. The aerosol-generating article is of a size and shape suitable for engaging an aerosol-generating device to bring the aerosol-forming substrate into contact with a heater.

[0090] The aerosol-forming substrate may contain a medically active compound or an agent such as an antibiotic or an anti-inflammatory agent that can be delivered to a patient via the respiratory tract. A number of medical inhalers are known and routinely prescribed for the treatment of various respiratory and non-respiratory diseases.

[0091] The aerosol-forming substrate may contain nicotine. The aerosol-forming substrate may contain tobacco. The aerosol-forming substrate may include a tobacco-containing material that contains volatile tobacco flavor compounds released from the aerosol-forming substrate upon heating. In certain embodiments, the aerosol-forming substrate may include a homogenized tobacco material, such as cast leaf tobacco. As used herein, "homogenized tobacco material" refers to a material formed by aggregating particulate tobacco. The homogenized tobacco may be in the form of a sheet. The homogenized tobacco material may have an aerosol-forming agent content of greater than 5% on a dry weight basis. Alternatively, the homogenized tobacco material may have an aerosol-forming agent content of from 5% to 30% by weight on a dry weight basis. The sheet of homogenized tobacco material may be formed by aggregating particulate tobacco obtained by grinding or otherwise subdividing one or both of tobacco leaf lamina and tobacco leaf stems. Alternatively or additionally, the sheet of homogenized tobacco material may include one or more of, for example, tobacco dust, tobacco fines, and other particulate tobacco by-products formed during the processing, handling, and transportation of tobacco. The sheet of homogenized tobacco material may include one or more native binders (i.e., tobacco endogenous binders), one or more foreign binders (i.e., tobacco exogenous binders), or combinations thereof to assist in the aggregation of particulate tobacco. Alternatively or additionally, the sheet of homogenized tobacco material may include other additives including, but not limited to, tobacco and non-tobacco fibers, aerosol-forming agents, humectants, plasticizers, flavorants, fillers, aqueous and non-aqueous solvents, and combinations thereof.

[0092] Cell cultures for use in the present disclosure include two-dimensional and three-dimensional cell cultures. As described herein, the cell cultures can be contained in one or more of the foams. The cell cultures can be exposed to a test atmosphere so as to determine the effect of the test atmosphere on the cell cultures. Since two or more cell cultures can be placed at different positions within the rigid porous foam, the effect of the test atmosphere on the cell cultures at these different positions mimicking the airway can be determined. A two-dimensional cell culture is one in which cells are cultured in a flat layer on a plastic surface, and various aspects of cell physiology and responses to stimuli such as a test atmosphere can be studied, but these do not reflect the actual structure and tissue of an organ. In a two-dimensional monolayer, the extracellular matrix, cell-cell interactions, and cell-matrix interactions that are essential for differentiation, proliferation, and cell function are lost. A three-dimensional culture system can form functional tissues with properties similar to those observed in vivo. Compared to a two-dimensional culture system, three-dimensional cell culture allows cells to interact with all three surrounding directions, resulting in higher physiological relevance. Such cells may show improvements in viability, proliferation, differentiation, morphology, stimulus response, drug metabolism, gene expression, and protein synthesis, and the like. Three-dimensional cell culture can create specific tissue-like structures and mimic the functions and responses of actual tissues in a way that is more physiologically relevant than conventional two-dimensional cell monolayers. Several three-dimensional tissues mimicking human organs are commercially available. For example, three-dimensional organotypic lung tissues can be prepared using primary human cells grown at an air-liquid interface (ALI), where these cells differentiate and form functional tissues. These three-dimensional tissues possess morphological similarities and metabolic properties similar to human bronchial tissue. They are composed of basal cells, goblet cells, and ciliated cells arranged in a multi-row structure. Similar to the lung, there are actively beating cilia, which enables studies on their function and activity. It has been found that the mRNA encoding xenobiotic enzymes is at comparable levels in these three-dimensional ALI cultures compared to human lungs. Furthermore, these tissues can be maintained in vitro for extended periods.

[0093] This distal airway and alveolar model is a model suitable for investigating effects such as test atmospheres in accordance with the present disclosure. The term "three-dimensional cell culture" includes any method that provides for the culturing of cells in three dimensions, with or without the use of a matrix or scaffold. Many different three-dimensional cell culture methods have been developed, including spheroid cultures and organotypic cultures.

[0094] Cells for use in the present disclosure can be isolated from tissues or liquids using methods known in the art. They can be differentiated from stem cells such as embryonic stem cells or induced pluripotent stem cells, or directly from somatic cells. The cells and cell lines can be of human or animal origin, or derived therefrom, and can be from any of a number of mammalian species, preferably human, but also including rats, mice, pigs, rabbits, and non-human primates. The cells and cell lines can also be obtained from commercial sources. In certain embodiments, the use of human cells is desirable. Lung cells, including lung epithelial cells, are the cell type of interest. In the present disclosure, bronchial and / or airway epithelial cells can be used. Human bronchial epithelial cells can be obtained by brushing the donor lung during a bronchoscopy procedure. In one embodiment, the lung cells are normal human bronchial epithelial (NHBE) cells. The lung epithelial cells can be cultured as a monolayer of undifferentiated cells or further differentiated into an organotypic lung epithelial-like tissue at the air-liquid interface. The cells can be established at the air-liquid interface using the following methodology. Briefly, the epithelial cells can be cultured in flasks to increase the cell number. After the incubation period, the cells are detached from the flask, counted, and seeded onto inserts. On these inserts, the cells are incubated with medium on both the apical and basal sides. This step ensures that the cells divide and completely cover the insert to form an epithelium. Thereafter, the medium on the apical side is removed and the medium on the basal side is retained and replaced with a more complete medium. The culture is incubated in this manner for a further period of time. During this time, the cells differentiate into three cell types: basal cells, goblet cells, and ciliated cells. Once maturation is complete, the culture can be used. The use of the air-liquid interface for culturing human nasal epithelial cells is described in J Vis Exp. (2013), 80, 50646. The lung epithelial cells can be obtained from human or animal subjects with different pathologies, including subjects classified as smokers or non-smokers.

[0095] Airway and alveolar cell cultures are reviewed in the European respiratory Journal (2019) 54:1900742. Adult tracheal, bronchial, and small airway epithelial cells can be isolated from donor lungs obtained from transplantation programs, surgically resected tissue, or bronchial brushes obtained during bronchoscopy. Nasal epithelial cells can be obtained by nasal brushing. Airway epithelial cells are commercially available as frozen vials or cultures from companies such as Lonza and Epithelix. Epithelial cells can be dissociated by protease treatment (to separate the cells from each other, from the extracellular matrix, and from unwanted cells). Selective media can be used to inhibit the growth of other cell types such as fibroblasts. Most procedures utilize cells immersed in media and culture tissue culture plastic coated with an extracellular matrix such as collagen. Culturing epithelial cells on Transwells on microporous membranes at the air-liquid interface promotes the differentiation of airway basal epithelial cells into mucociliary epithelial cultures similar to the in situ airway epithelium, while at the same time the associated exposure protocols allow for the study of airborne substances. Alveolar epithelial cells can be collected from adult lung tissue from tissue of normal appearance after surgery. Isolation of AEC2 typically involves additional steps such as differential adhesion and magnetic bead sorting to separate these AEC2 from other cells such as macrophages and fibroblasts. Primary alveolar epithelial cells are also available from commercial suppliers. Culturing lung epithelial cells in Transwells allows for culture at the air-liquid interface. Epithelial cells can also grow on a layer of collagen in which fibroblasts are embedded. Various methods can be used to characterize epithelial cell cultures based on the structure, morphology, and expression of specific cell markers, including electron and confocal microscopy, immunostaining, and gene expression analysis by RT-PCR. Intrinsic functional properties include ciliary beat frequency for airway cells and surfactant synthesis for AEC2.Alveolar epithelial cells are derived from induced pluripotent stem cells (iPSCs) and can form 3D structures under organotypic culture (see, for example, Elife, 4 (2015), p.e05098; Stem Cell Rep., 3 (2014), pp. 394 - 403; Cell Stem Cell, 21 (2017), pp. 472 - 488.e10, and Nat.Methods, 14 (2017), pp. 1097 - 1106). iScience (2022), 25, 2, 10378 describes an alveolar epithelial - derived distal lung cell line.

[0096] Three - dimensional lung epithelial cells are obtained from airway basal cells (see Proc Natl Acad Sci USA (2009) 106, 12771 - 5), and from alveolar cells (see J Clin Invest (2013) 123, 3025 - 3036), and from iPSC - derived airway or alveolar cells (see Development (2017), 144, 986 - 997 and Curr Pathobiol Rep (2017) 5, 223 - 231).

[0097] The present disclosure can be used for various applications to study the effects of a test atmosphere on the terminal, transitional and respiratory bronchioles, and / or alveolar ducts and / or alveolar spaces of the airway, and optionally, other parts of the airway if required. For example, the present disclosure can be used in the study of in vitro inhalation toxicity in one or more of the bronchioles and / or alveolar ducts and / or alveolar spaces of the airway. For example, the present disclosure can be used to investigate the dynamics of aerosols, such as particle deposition of aerosols and absorption of gases into cell culture, or to investigate the metabolic activity or transport of a test atmosphere (such as aerosol molecules) passing through the bronchioles and / or alveolar ducts and / or alveolar spaces of the airway. The present disclosure can be used to test the effects of aerosols, smoke, or tobacco products, or the effects of inhalers such as medical inhalers. The present disclosure can be used to test the effects of aerosols, smoke, or tobacco products, or the effects of medical inhalers on cells in one or more parts of the airway.

[0098] One aspect relates to a method for determining the effect of a test atmosphere on a cell culture, such as one or more cultures of cells, contained in an artificial airway, the method comprising: (a) providing a respiratory simulator as described herein, the system being provided to include a cell culture; and (b) comparing the cell cultures before and / or after exposure to the test atmosphere, the comparison being made such that a difference in the cell cultures before and / or after exposure of the cells to the test atmosphere indicates an effect of the test atmosphere on the cell culture.

[0099] In an embodiment where the difference in the cell cultures is determined after exposure of the cells to the test atmosphere, the cell culture exposed to the test atmosphere can be compared to a cell culture not exposed to the test atmosphere or to a cell culture exposed to a control atmosphere, such as an atmosphere not containing the test atmosphere. According to this embodiment, a difference between the cell culture exposed to the test atmosphere and the cell culture not exposed to the test atmosphere, or a difference between the cell culture exposed to the test atmosphere and the cell culture exposed to a control atmosphere, such as an atmosphere not containing the test atmosphere, indicates an effect of the test atmosphere on the cell culture.

[0100] The effect of the test atmosphere can be studied in the presence of one or more agents. Agents can include, but are not limited to, drugs, toxins, pathogens, proteins, nucleic acids, antigens, antibodies, and chemical compounds. Examples of effects that can be measured include oxygen consumption, carbon dioxide production, cell viability, protein expression, enzyme activity, permeability, barrier function, surfactant production, cytokine response, transporter function, cytochrome P450 expression, albumin secretion, toxicity, and the like.

[0101] Multiple assays can be performed in parallel using different concentrations of the test atmosphere and / or agent to obtain different responses to different concentrations.

[0102] The agent can be any test compound of interest, including small organic compounds, polypeptides, peptides, high molecular weight carbohydrates, polynucleotides, fatty acids and lipids, aerosols or one or more components of an aerosol, and the like. The test compounds can be screened individually, or as a set or combinatorial library of compounds. The test compounds can be obtained from a wide variety of sources, including libraries of synthetic compounds or natural compounds. Natural compound libraries in the form of extracts of bacteria, fungi, plants, and animals can be used. Combinatorial libraries can be created using natural or synthetically made libraries, and compounds modified via conventional chemical, physical, and biochemical means. Known pharmacological agents can be directly or randomly subjected to chemical modifications such as acylation, alkylation, esterification, acidification, etc., to create structural analogs for screening.

[0103] One or more variables that can be measured include elements of cells, intracellular substances, intracellular components, or cell products. As an example, the toxicity of the test atmosphere can be measured. The dynamics of the aerosol (e.g., deposition of aerosol particles on cell cultures and absorption of gases) can be measured. Further examples include studying metabolic activity and / or molecular transport.

[0104] Further aspects of the present disclosure are described in the following numbered paragraphs. 1. A respiratory simulator comprising a rigid porous foam, wherein the rigid porous foam comprises (i) macropores and (ii) an interconnected open cell network of micropores and / or nanopores. 2. The respiratory simulator according to paragraph 1, wherein the rigid porous foam is (i) a rigid porous foam of ceramic or metal, or (ii) a rigid porous foam of ceramic and metal. 3. The respiratory simulator according to paragraph 2, wherein the rigid porous foam is made of alumina, silicon carbide, oxygen-bonded silicon carbide, sintered silicon carbide, or a combination of two or more thereof. 4. The respiratory simulator according to any one of paragraphs 1 to 3, wherein the rigid porous foam includes walls and struts, the struts of the rigid porous foam include micropores and / or nanopores, and the walls of the macropores of the rigid porous foam are covered with micropores and / or nanopores that open towards the macropores. 5. The respiratory simulator according to any one of paragraphs 1 to 4, wherein the macropores can allow a gas or aerosol supplied to the rigid porous foam to pass through. 6. The respiratory simulator according to any one of paragraphs 1 to 5, wherein the micropores and / or nanopores can (i) allow an aqueous solution or liquid supplied to the rigid porous foam to pass through, and (ii) hold the aqueous solution or liquid within the micropores and / or nanopores. 7. The respiratory simulator according to any one of paragraphs 1 to 6, wherein (i) the macropores contain a gas or aerosol, or (ii) the micropores and / or nanopores contain an aqueous solution or liquid, or (iii) the macropores contain a gas or aerosol and the micropores and / or nanopores contain an aqueous solution or liquid. 8. The respiratory simulator according to paragraph 7, wherein the aqueous solution or liquid present within the micropores and / or nanopores forms a water layer or liquid layer that covers a part or all of the surface of the macropores. 9. The respiratory simulator according to any one of paragraphs 1 to 8, wherein the rigid porous foam contains a first connector that can establish a leak-free connection with the respiratory simulator. 10. The respiratory simulator according to any one of paragraphs 1 to 9, wherein the rigid porous foam contains a second connector that can connect the rigid porous foam to a source of aqueous solution or liquid. 11. The breathing simulator according to any one of paragraphs 1 to 10, wherein the rigid porous foam contains a third connector that can connect the rigid porous foam to a drain. 12. The breathing simulator according to any one of paragraphs 1 to 11, which includes one or more cavities, or sockets, or depressions, or a combination of two or more thereof in the rigid porous foam, and the one or more cavities, or sockets, or depressions, or a combination of two or more thereof can receive one or more sensing devices, or probes, or sampling devices, or cell cultures. 13. The breathing simulator according to paragraph 12, wherein the one or more cavities, or sockets, or depressions, or a combination of two or more thereof contain one or more sensing device probes, or sampling devices, or cell cultures. 14. The breathing simulator according to any one of paragraphs 1 to 13, wherein the rigid porous foam includes one or more bifurcated channels, preferably the bifurcated channels bifurcate, and more preferably the rigid porous foam forming the bifurcated channels contains micropores and / or nanopores. 15. The interconnected network of macropores and micropores and / or nanopores is (i) a pair of macropores connected by a continuous channel formed by the macropores, (ii) a pair of micropores and / or nanopores connected by a continuous channel formed by the micropores and / or nanopores, (iii) micropores and / or nanopores connected to any macropore by a continuous channel formed by the micropores and / or nanopores and macropores, The breathing simulator according to any one of paragraphs 1 to 14. 16. The radius of the macro pores is about 0.1 to about 0.4 mm in diameter, or about 0.1 to about 0.3 mm in diameter, or about 0.2 to about 0.4 mm, or about 0.2 to about 0.3 mm, or about 200 μm to about 2000 μm in diameter, or about 820 μm ± 150 μm in diameter, the breathing simulator according to any one of paragraphs 1 to 15. 17. The radius of the micro pores and / or nano pores is about 0.01 μm to about 50 μm in diameter, preferably less than about 30 μm, or about 0.25 μm to about 25 μm, the breathing simulator according to any one of paragraphs 1 to 16. 18. The total surface area of the rigid porous foam is at least about 70 square meters, preferably at least about 100 square meters, the breathing simulator according to any one of paragraphs 1 to 17. 19. The breathing simulator according to any one of paragraphs 1 to 18, comprising at least two rigid porous foams each having a different pore size distribution, or each having a different porosity, or each made of a different material. 20. The aqueous solution or liquid below the surface reaching the micro pores and / or nano pores has a negative pressure with respect to the environment that is greater in absolute value than the negative pressure with respect to the environment below the surface of the aqueous solution or liquid reaching the macro pores, the breathing simulator according to any one of paragraphs 7 to 19. 21. The rigid porous foam has a cylindrical shape, or a disc shape, or a cube shape, the breathing simulator according to any one of paragraphs 1 to 20. 22. The breathing simulator according to paragraph 21, wherein the cylindrical or disc-shaped or cube-shaped rigid porous foam includes a radial wall made of a material different from the rigid porous foam. 23. The different material does not include macro pores, or the different material is a synthetic material, preferably an epoxy resin or a metal, or a combination thereof, the breathing simulator according to paragraph 22. 24. The cross-linked structure is in contact with the rigid porous foam, preferably the cross-link is joined to the cross-linked structure, preferably the cross-link is flexible, the breathing simulator according to any one of paragraphs 1 to 23. 25. The breathing simulator according to any one of paragraphs 1 to 24, wherein the breathing simulator includes a pump and a rigid porous foam is accommodated in the pump. 26. The breathing simulator according to any one of paragraphs 1 to 25, wherein the pump is a piston pump. 27. Use of a rigid porous foam for simulating distal airways and alveoli in a breathing simulator, wherein the rigid porous foam includes (i) macropores, and (ii) an interconnected open cell network of micropores and / or nanopores. 28. The use according to paragraph 27, wherein the rigid porous foam is (i) a ceramic or metal rigid porous foam, or (ii) a ceramic and metal rigid porous foam. 29. The use according to paragraph 28, wherein the rigid porous foam is made of alumina or silicon carbide or oxygen-bonded silicon carbide or sintered silicon carbide, or a combination of two or more thereof. 30. The use according to any one of paragraphs 27 to 29, wherein the rigid porous foam includes walls and struts, the struts of the rigid porous foam include micropores and / or nanopores, and the walls of the macropores of the rigid porous foam are covered with micropores and / or nanopores that open towards the macropores. 31. The use according to any one of paragraphs 27 to 30, wherein the macropores can pass a gas or aerosol supplied to the rigid porous foam. 32. The use according to any one of paragraphs 27 to 31, wherein the micropores and / or nanopores can (i) pass an aqueous solution or liquid supplied to the rigid porous foam, and (ii) hold an aqueous solution or liquid in the micropores and / or nanopores. 33. (i) The macropores contain a gas or aerosol, or (ii) the micropores and / or nanopores contain an aqueous solution or liquid, or (iii) the macropores contain a gas or aerosol and the micropores and / or nanopores contain an aqueous solution or liquid. The use according to any one of paragraphs 27 to 32. 34. Use according to paragraph 33, wherein the micropores and / or nanopores containing an aqueous solution or liquid form an aqueous or liquid layer covering a part or all of the surface of the macropores. 35. Use according to any one of paragraphs 27 to 34, wherein the rigid porous foam contains one or more cavities, or sockets, or depressions, or a combination of two or more thereof in the rigid porous foam, and one or more cavities, or sockets, or depressions, or a combination of two or more thereof can receive one or more sensing devices, or probes, or sampling devices, or cell cultures. 36. Use according to paragraph 35, wherein one or more cavities, or sockets, or depressions, or a combination of two or more thereof contain one or more sensing device probes, or sampling devices, or cell cultures. 37. Use according to any one of paragraphs 27 to 36, wherein the rigid porous foam contains one or more bifurcated channels, preferably the bifurcated channels are bifurcated, and more preferably the bifurcated channels are located within the micropores and / or nanopores. 38. The interconnected network of macropores and micropores and / or nanopores is (i) a pair of macropores connected by a continuous channel formed by the macropores, (ii) a pair of micropores and / or nanopores connected by a continuous channel formed by the micropores and / or nanopores, (iii) micropores and / or nanopores connected to any macropore by a continuous channel formed by the micropores and / or nanopores and macropores, and includes the use according to any one of paragraphs 27 to 37. 39. Use according to any one of paragraphs 27 to 38, wherein the radius of the macropores is from about 0.1 to about 0.4 mm in diameter, or from about 0.1 to about 0.3 mm, or from about 0.2 to about 0.4 mm, or from about 0.2 to about 0.3 mm, or from about 200 μm to about 2000 μm. 40. Use according to any one of paragraphs 27 to 39, wherein the radius of the micropores and / or nanopores is from about 0.01 μm to about 50 μm in diameter, or from about 0.25 μm to about 25 μm. 41. Use according to any one of paragraphs 27 to 40, wherein the total surface area of the rigid porous foam is at least about 70 square meters, preferably at least about 100 square meters. 42. Use according to any one of paragraphs 27 to 41, comprising at least two rigid porous foams having different pore size distributions, or different porosities, or different materials. 43. Use according to any one of paragraphs 33 to 42, having a negative pressure with respect to the environment, wherein the aqueous solution or liquid under the surface over the micropores and / or nanopores is greater in absolute value than the negative pressure of the environment under the surface of the aqueous solution or liquid over the macropores. 44. Use according to any one of paragraphs 27 to 43, wherein the rigid porous foam has a cylindrical shape, or a disc shape, or a cube shape. 45. Use according to paragraph 44, wherein the rigid porous foam having a cylindrical shape, or a disc shape, or a cube shape comprises a radially oriented wall made of a material different from the rigid porous foam. 46. Use according to paragraph 45, wherein the different material does not contain macropores, or the different material is a synthetic material, preferably an epoxy resin or a metal, or a combination thereof. 47. Use according to any one of paragraphs 27 to 46, wherein the crosslinked structure is in contact with the rigid porous foam, preferably the crosslinking is bonded to the crosslinked structure, and preferably the crosslinking is flexible. 48. Use according to any one of paragraphs 27 to 47, wherein the breathing simulator includes a pump and the rigid porous foam is housed within the pump. 49. Use according to paragraph 48, wherein the pump is a piston pump. 50. A method for determining the effect of a test atmosphere on the simulated distal airways and alveoli of the mammalian airway, comprising: (i) providing a breathing simulator according to any one of paragraphs 1 to 26; (ii) contacting the breathing simulator with the test atmosphere; (iii) determining the effect of the test atmosphere on the simulated distal airways and alveoli of the mammalian airway of the breathing simulator, a method comprising. 51. Use of a breathing simulator according to any one of paragraphs 1 to 26 for determining the effect of a test atmosphere on the simulated distal airways and alveoli of the mammalian airway. 52. A method of filling the micropores and / or nanopores of a rigid porous foam with an aqueous solution or liquid, but not the macropores, wherein the rigid porous foam comprises (i) macropores and (ii) an open cell network in which the micropores and / or nanopores are interconnected, the method comprising (i) supplying an aqueous solution or liquid to the rigid porous foam under a negative pressure that is greater in absolute value than the negative pressure resulting from the capillary action of the macropores but less than the negative pressure resulting from the capillary action of the micropores and / or nanopores; (ii) filling the micropores and / or nanopores of the rigid porous foam with an aqueous solution or liquid, but not the macropores. 53. (i) Macropores containing gas or aerosol, and (ii) micropores and / or nanopores containing an aqueous solution or liquid, wherein the micropores and / or nanopores containing the aqueous solution or liquid form a water layer or liquid layer covering part or all of the surface of the macropores, a rigid porous foam for use in a model of the distal airways and alveoli of the airway, comprising an open cell network in which the micropores and / or nanopores are interconnected. 54. The rigid porous foam according to paragraph 53, wherein the rigid porous foam is (i) a ceramic or metal rigid porous foam, or (ii) a ceramic and metal rigid porous foam. 55. The rigid porous foam according to paragraph 53, wherein the rigid porous foam is made of alumina or silicon carbide or oxygen-bonded silicon carbide or sintered silicon carbide, or a combination of two or more thereof. 56. The rigid porous foam includes walls and struts, the struts of the rigid porous foam include micropores and / or nanopores, and the walls of the macropores of the rigid porous foam are covered with micropores and / or nanopores that open towards the macropores, the rigid porous foam according to any one of paragraphs 53 to 55. 57. The rigid porous foam includes one or more cavities, or sockets, or depressions, or combinations of two or more thereof, and one or more cavities, or sockets, or depressions, or combinations of two or more thereof can receive one or more sensory devices, or probes, or sampling devices, or cell cultures, the rigid porous foam according to any one of paragraphs 53 to 56. 58. The rigid porous foam according to paragraph 57, wherein one or more cavities, or sockets, or depressions, or combinations of two or more thereof contain one or more sensory device probes, or sampling devices, or cell cultures. 59. The rigid porous foam includes one or more bifurcated channels, preferably the bifurcated channels bifurcate, and more preferably the bifurcated channels are present within micropores and / or nanopores, the rigid porous foam according to any one of paragraphs 53 to 58. 60. The interconnected network of macropores and micropores and / or nanopores is (i) a pair of macropores connected by a continuous channel formed by the macropores, (ii) a pair of micropores and / or nanopores connected by a continuous channel formed by the micropores and / or nanopores, (iii) micropores and / or nanopores connected to any macropore by a continuous channel formed by the micropores and / or nanopores and macropores, the rigid porous foam according to any one of paragraphs 53 to 59. 61. The radius of the macro pores is about 0.1 to about 0.4 mm in diameter, or about 0.1 to about 0.3 mm, or about 0.2 to about 0.4 mm, or about 0.2 to about 0.3 mm, or about 200 μm to about 2000 μm, preferably about 820 μm ± 150 μm in diameter, the rigid porous foam according to any one of paragraphs 53 to 60. 62. The radius of the micro pores and / or nano pores is about 0.01 μm to about 50 μm in diameter, preferably less than about 30 μm in diameter or about 0.25 μm to about 25 μm, the rigid porous foam according to any one of paragraphs 53 to 61. 63. The total surface area of the rigid porous foam is at least about 70 square meters, preferably at least about 100 square meters, the rigid porous foam according to any one of paragraphs 53 to 62. 64. The rigid porous foam according to any one of paragraphs 53 to 63, comprising at least two rigid porous foams with different pore size distributions, or different porosities, or different materials. 65. The rigid porous foam according to any one of paragraphs 53 to 64, having a negative pressure with respect to the environment, such that the aqueous solution or liquid under the surface reaching the micro pores and / or nano pores has a greater absolute value than the negative pressure with respect to the environment under the surface of the aqueous solution or liquid reaching the macro pores. 66. The rigid porous foam according to any one of paragraphs 53 to 65, having a cylindrical shape, or a disc shape, or a cube shape. 67. The rigid porous foam according to paragraph 66, wherein the cylindrical, disc-shaped or cube-shaped rigid porous foam comprises a radially extending wall made of a material different from the rigid porous foam. 68. The rigid porous foam according to paragraph 67, wherein the different material does not contain macro pores, or the different material is a synthetic material, preferably an epoxy resin or a metal, or a combination thereof. 69. The rigid porous foam according to any one of paragraphs 53 to 68, wherein a crosslinked structure is in contact with the rigid porous foam, preferably the crosslinking is joined to the crosslinked structure, and preferably the crosslinking is flexible. 70. The rigid porous foam according to any one of paragraphs 53 to 69, wherein the rigid porous foam is incorporated or attached inside a pump, preferably a piston pump. 71. A pump for moving the volume of gas containing or accommodated in the rigid porous foam in the pump, wherein the rigid porous foam comprises (i) macropores and (ii) an interconnected open cell network of micropores and / or nanopores as described herein, and optionally, the rigid porous foam is connected to a port for receiving and discharging gas. 72. A system for determining the interaction between a test atmosphere and a simulated airway, the system comprising (a) A first pump, (i) A chamber configured to contain a first volume of gas including the test atmosphere, (ii) A first port adapted to receive and discharge gas and including a valve for regulating the flow of gas through the first port, the valve being movable between open and closed positions, and in the open position, the valve being openable towards the test atmosphere or ambient air, (iii) A second port adapted to receive and discharge gas and including a valve for regulating the flow of gas through the second port, the valve being movable between open and closed positions, (iv) A piston plate in the chamber, the piston plate including one or more openings for taking in or flowing in gas into the chamber, and one or more or each of the openings being movable between open and closed positions and capable of regulating the intake or inflow of gas, (v) A motor for controlling the operation of the first pump, (b) A second pump as defined in paragraph 71, (c) A connection structure operable to transmit gas from the first pump into the second pump, (d) One or more openings in the first pump or the second pump, or the wall of the connection structure, or a combination of two or more of them, wherein the openings can receive a module for containing a matrix containing a cell culture medium and / or for monitoring the state in the chamber or for gas sampling or gas characterization, the one or more openings, a system comprising. 73. A method of simulating the interaction between a test atmosphere and a simulated airway, including the use of the breathing simulator according to any one of paragraphs 1 to 26, the rigid porous foam according to any one of paragraphs 53 to 70, the pump according to paragraph 71, or the system according to paragraph 72. 74. The use of the breathing simulator according to any one of paragraphs 1 to 26, or the rigid porous foam according to any one of paragraphs 53 to 70, or the pump according to paragraph 71, or the system according to paragraph 72, for simulating the interaction between a test atmosphere and a simulated airway or for determining the effect of a test atmosphere on a cell culture contained in a rigid porous foam or a pump or a system. 75. A method of determining the effect of a test atmosphere on a cell culture contained in a simulated airway, including the use of the breathing simulator according to any one of paragraphs 1 to 26, or the rigid porous foam according to any one of paragraphs 53 to 70, or the pump according to paragraph 71, or the system according to paragraph 72. 76. A method for determining the effect of a test atmosphere on a cell culture contained within a simulated airway, (a) Providing the breathing simulator according to any one of paragraphs 1 to 26, or the rigid porous foam according to any one of paragraphs 53 to 70, or the pump according to paragraph 71, or the system according to paragraph 72, wherein the rigid porous foam or the pump or the system is provided to contain a cell culture and / or at least one sensor within one or more modules; (b) Comparing a cell culture and / or at least one sensor before and / or after exposure to a test atmosphere, wherein the difference in the cell culture and / or at least one sensor before and / or after exposure of the cell and / or at least one sensor to the test atmosphere is compared such that it indicates that the test atmosphere has an effect on the cell culture and / or at least one sensor, a method comprising this. An apparatus configured or adapted to carry out the method according to any one of paragraphs 50, 73, 75 or 76.

[0105] All publications cited or described herein provide relevant information disclosed prior to the filing date of this application. The description herein shall not be construed as an admission that the inventors were not entitled to antedate such disclosure. All publications mentioned in the above specification are hereby incorporated by reference into this specification. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should of course not be unduly limited to such specific embodiments. Indeed, various changes in the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

1. A respiratory simulator comprising a rigid porous foam, wherein the rigid porous foam comprises (i) macropores and (ii) an open cell network of interconnected micropores and / or nanopores.

2. The rigid porous foam is (i) a rigid porous foam made of ceramic or metal, or (ii) a rigid porous foam made of ceramic and metal, preferably, The breathing simulator according to claim 1, wherein the rigid porous foam is made of alumina, or silicon carbide, or oxygen-bonded silicon carbide, or sintered silicon carbide, or a combination of two or more thereof.

3. (i) the macropores contain a gas or aerosol, or (ii) the micropores and / or nanopores contain an aqueous solution or liquid, or (iii) the macropores contain a gas or aerosol and the micropores and / or nanopores contain an aqueous solution or liquid, according to claim 1.

4. The breathing simulator according to claim 3, wherein the micropores and / or nanopores containing the aqueous solution or liquid form an aqueous or liquid layer covering part or all of the surface of the macropores.

5. The respiratory simulator according to claim 1, wherein the rigid porous foam comprises one or more cavities, sockets, or depressions, or two or more combinations thereof, and preferably, the one or more cavities, sockets, or depressions, or two or more combinations thereof, contain one or more sensory devices, probes, sampling devices, or cell cultures.

6. The breathing simulator according to claim 1, comprising at least two rigid porous foams, each with a different pore size distribution, a different porosity, or made of a different material.

7. The breathing simulator according to claim 1, wherein the breathing simulator includes a pump, the rigid porous foam is contained within or attached to the pump, and preferably the pump is a piston pump.

8. Use of a rigid porous foam for simulating the distal airways and alveoli of a respiratory simulator, wherein the rigid porous foam includes (i) macropores and (ii) an interconnected open cell network of micropores and / or nanopores.

9. The rigid porous foam is (i) a rigid porous foam made of ceramic or metal, or (ii) a rigid porous foam made of ceramic and metal, preferably, The use according to claim 8, wherein the rigid porous foam is made of alumina, or silicon carbide, or oxygen-bonded silicon carbide, or sintered silicon carbide, or a combination of two or more thereof.

10. A method for determining the effect of a test atmosphere on simulated distal airways and alveoli of the airway, (i) To provide the respiratory simulator described in claim 1, (ii) Bringing the respiratory simulator into contact with the test atmosphere, (iii) A method comprising determining the effect of the test atmosphere on the simulated distal airway and alveoli of the airway.

11. A rigid porous foam for use in a model of the distal airway and alveoli of an airway, comprising an interconnected open cell network of (i) macropores containing gas or aerosol, and (ii) micropores and / or nanopores containing aqueous solution or liquid, wherein the micropores and / or nanopores containing aqueous solution or liquid form an aqueous or liquid layer covering part or all of the surface of the macropores.

12. The rigid porous foam is (i) a rigid porous foam made of ceramic or metal, or (ii) a rigid porous foam made of ceramic and metal, preferably, The rigid porous foam according to claim 11, wherein the rigid porous foam is made of alumina, or silicon carbide, or oxygen-bonded silicon carbide, or sintered silicon carbide, or a combination of two or more thereof.

13. (i) the macropores contain a gas or aerosol, or (ii) the micropores and / or nanopores contain an aqueous solution or liquid, or (iii) the macropores contain a gas or aerosol and the micropores and / or nanopores contain an aqueous solution or liquid, according to claim 11.

14. The rigid porous foam according to claim 11, wherein the rigid porous foam comprises one or more cavities, sockets, or depressions, or two or more combinations thereof, preferably the one or more cavities, sockets, or depressions, or two or more combinations thereof, comprising one or more modules for containing or storing cell culture media and / or at least one microsensor for monitoring conditions within a chamber, or for gas sampling or gas characterization.

15. The rigid porous foam according to claim 11, wherein the rigid porous foam is enclosed inside or installed inside a pump, and preferably the pump is a piston pump.

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