Perforated structure

Abstract

Described herein is a perforated structure for use in a simulated respiratory tract, the structure comprising a perforated envelope containing one or more branching channels, each perforation being an open end of a branch. Also described is a pump for moving a volume of gas containing one or more branching channels, such as the perforated structure described herein. Use of the pump in a system for determining the interaction between a test atmosphere and a simulated respiratory tract is also described, along with methods and uses of such a system.

Description

Technical Field

[0001] The present disclosure relates, inter alia, to apparatuses, systems, methods, and uses for simulating experiments on the structure and / or function of the respiratory tract and its respiratory behavior. The present disclosure is useful for examining the interaction between an inspection agent (e.g., an inhalable agent in a test atmosphere) and the respiratory tract, as well as aerosol dynamics and development. In particular, the present disclosure relates to simulating experiments on the human respiratory tract.

Background Art

[0002] The respiratory system runs from the nose and upper airway to the alveolar surface of the lungs where gas exchange takes place. Inhaled aerosol moves from the mouth through the upper airway and ultimately reaches the alveoli. As the aerosol moves deeper into the respiratory tract, more soluble gases are absorbed and certain aerosol particles can deposit deep within the airways and alveoli. One particular aerosol of interest in the context of the present disclosure is smoke, such as cigarette smoke, which can study the effects of smoke on or through a simulated respiratory tract.

[0003] Existing aerosol exposure systems most commonly rely on either a continuous, unidirectional aerosol flow or passive sedimentation. The continuous aerosol flow is generated by positive or negative pressure, and the aerosol is directed vertically into the biological test system or flows out parallel to the plane of the biological test system. The dose delivery efficiency in this exposure mode is mainly a function of the shape of the exposure chamber and the flow rate of the test aerosol. However, the oral cavity, breath-holding times, and the dynamic flow patterns that occur during human respiration (i.e., inhalation and exhalation) cannot be simulated in current aerosol exposure systems. The typical dose delivery in an in vivo situation, particularly the relative delivery of particulate and gaseous aerosol components, is therefore unlikely to be achieved. In addition, the screening action of the conducting airways cannot be simulated by typical means. Thus, in current aerosol exposure systems, aerosols with the same aerosol particle size distribution are used, for example, in bronchial cell cultures and nasal lavage cultures. However, in vivo, larger aerosol particles mainly interact with the aerosol in the upper respiratory tract and are removed from there, while the deeper regions of the respiratory tract are mainly exposed to smaller aerosol particle sizes and gaseous components. In addition, in continuous flow exposure systems, aerosol dilution is achieved by continuously adding dilution air to the aerosol upstream of the exposure chamber. However, when simulating smoking behavior as it occurs in a biological organism, this dilution mode does not address the holding period in the mouth, during which a very high density of aerosol can age for up to several seconds before being drawn into the respiratory tract with a large volume of dilution air, which is not typical of the organism.

[0004] In passive aerosol deposition, the test aerosol is injected into a chamber located at the bottom of the biological test system. Once the aerosol is injected, it can typically sediment onto the test system by gravity. Electrostatic attraction of aerosol particles to the test system may be used to increase the deposition of aerosol particles. Nano-sized aerosol particles may particularly require electrostatic attraction due to their low sedimentation efficiency. Relative delivery by differences in large aerosol particles, small aerosol particles, and gaseous aerosol components is not typical of processes occurring in a living body, and thus exposure to a complex aerosol containing not only gaseous components but also aerosol particles of various sizes and densities cannot be performed in a passive aerosol deposition system.

[0005] There is a continuing need in the art for improved simulation experimental systems for studying the respiratory tract, particularly simulation experimental systems for studying the respiratory tract including a complex airway model. SUMMARY OF THE INVENTION

[0006] Disclosed herein is a structure for simulating the effect of an inhaled aerosol, e.g., on the conducting airway tree from a human or animal. This structure allows increasing the amount of airway branching generations to a biologically more relevant level without simultaneously generating an uncontrollable number of connections. Different versions of the airway model can be used without affecting the overall functionality of the system and without requiring modification to other parts of the simulated respiratory tract system. It is used with a system for simulating the respiratory tract to investigate, for example, how any type of aerosol changes its basic properties (e.g., particle size distribution, particle concentration, or chemical composition, or a combination of two or more thereof) in the airway tree, or how the aerosol deposits on a surface representing the epithelium of the human respiratory tract in the context of aerosol dosimetry, or for any type of analysis or testing of the effect of any type of aerosol on an in vitro biological system (e.g., for use in an in vitro aerosol exposure system).

[0007] In a first aspect, a perforated structure for use in an artificial trachea is provided, the structure comprising a perforated envelope that houses one or more branched channels, each perforation being an open end of a branch.

[0008] In a further aspect, a perforated structure for use in an artificial trachea is provided, the structure comprising a perforated envelope that houses one or more branched channels, each perforation being an open end of one or more of the branched channels.

[0009] It is appropriate for the branches of each channel to be forked.

[0010] It is appropriate for the diameter of each channel that exists after n + 1 consecutive branch points to be less than or equal to the diameter of the channels that exist after n branch points.

[0011] It is appropriate for the total cross-sectional area of all channels that exist after n + 1 consecutive branch points to be greater than or equal to the total cross-sectional area of all channels after n branch points.

[0012] It is appropriate for the structure to be asymmetric or to have radial symmetry.

[0013] It is appropriate for the structure to be modular.

[0014] All or part of the structure is formed from one or more materials selected from the group consisting of non-porous materials including metals and non-porous synthetic materials, porous materials including porous silicon, gel-like materials including thermogels, hydrophobic materials, hydrophilic materials, amphiphilic materials, or combinations of two or more thereof.

[0015] One or more of the branched channels may further include one or more coatings, such as a coating or coatings on part or all of the inner surface of the branched channel to provide water and nutrients to cells grown in all or part of one or more of the branched channels.

[0016] One or more parts or all of the branching channels are suitably coated with a matrix containing cell culture medium, or contain at least one microsensor for monitoring the state within the structure, or a probe for gas sampling or gas characterization.

[0017] One or more of the branching channels are further suitably provided with one or more openings capable of receiving one or more modules containing a matrix containing cell culture medium, and / or at least one microsensor for monitoring the state within the structure, or for gas sampling or gas characterization.

[0018] The cell culture medium suitably includes one or more hydrogels such as gelatin methacryloyl (GelMa).

[0019] In a further aspect, a pump for moving a volume of gas is provided that includes one or more branching channels inside the pump, and the branching channels are connected to ports for receiving and discharging gas.

[0020] In a further aspect, a pump for moving a volume of gas is provided that includes a port and one or more branching channels inside the pump, and the one or more branching channels are connected to ports for receiving and discharging gas.

[0021] In a further aspect, a pump for moving a volume of gas is provided that includes external components and internal components, a port, and one or more branching channels inside the pump, and the one or more branching channels are connected to ports for receiving and discharging gas.

[0022] The branching structure is suitably incorporated within the perforated structure according to the present disclosure.

[0023] One or more of the branching channels are suitably incorporated within the perforated structure according to the present disclosure.

[0024] The bifurcated structure is suitable for reproducing the airway model.

[0025] The pump is suitably configured to comprise a chamber having one or more openings adapted to receive one or more modules for enclosing a volume of gas and for containing at least one microsensor for monitoring the condition in the matrix containing the cell culture medium and / or the chamber, or a probe for gas sampling or gas characterization.

[0026] The pump suitably further comprises a motor for controlling the operation of the pump. The pump is suitably a piston pump.

[0027] The module is suitably located in the base of the chamber.

[0028] The module is suitably screw-type or non-screw-type.

[0029] The module is suitably adapted to enclose a matrix containing the cell culture medium or is configured to store a matrix containing the cell culture medium, or is adapted to monitor the condition in the chamber, or is adapted to monitor the condition in the chamber, or is adapted to sample gas, or is adapted to characterize gas. Alternatively or additionally, the module is adapted to include at least one microsensor or probe, or is configured to store at least one microsensor or probe adapted to monitor the condition in the chamber, or is adapted to monitor the condition in the chamber, or is adapted to sample gas, or is adapted to characterize gas.

[0030] One or more modules are suitably included in a matrix containing cell culture medium. The cell culture medium is suitably included in one or more hydrogels such as gelatin methacryloyl (GelMa). As another method, or additionally, one or more modules include at least one microsensor.

[0031] A matrix containing cell culture medium is suitably provided with or in contact with a cell culture, suitably a two - dimensional or three - dimensional cell culture.

[0032] Modules adapted to include or store cell culture medium and / or at least one microsensor suitably further comprise microfluidic channels and optionally a microfluidic pump connected thereto.

[0033] Modules are suitably adapted to include or include one or more microsensors.

[0034] Modules suitably comprise quartz crystal microbalances.

[0035] The connection structure is suitably joined to a port.

[0036] The connection structure is suitably hollow.

[0037] Pumps suitably further comprise a motor for controlling the operation of the pump.

[0038] The pump pressure is suitably equivalent to atmospheric pressure, or above or below atmospheric pressure.

[0039] The displacement volume of the pump is suitably about 0 - 1000 ml or about 1 - about 100 ml.

[0040] Pumps suitably contain stainless steel.

[0041] The chamber is suitably a cylinder.

[0042] The chamber suitably contains glass.

[0043] The volume of the chamber suitably represents the volume of the lung cavity or a part thereof.

[0044] In a further aspect, a system is provided for determining the interaction between a test atmosphere and a simulated breathing airway. The system includes: (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 capable of opening towards the test atmosphere or the 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 within the chamber, the piston plate including one or more gaps for the intake or inflow of gas into the chamber, one or more or each of the gaps including a valve movable between open and closed positions and capable of regulating the intake or inflow of gas; and (v) a motor for controlling the operation of a first pump, the system further including: (a) a first pump; (b) a second pump as described herein; (c) a connection structure operable to transfer gas from the first pump into the second pump; and (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 thereof, the openings being configured to receive a module including a matrix containing a cell culture medium and / or at least one microsensor, or a matrix containing a cell culture medium and / or for monitoring the state within the chamber, or at least one microsensor adapted to monitor the state within the chamber, or for gas sampling or gas characterization, or for storing a probe adapted to sample or characterize the gas.

[0045] The pump is suitably a piston pump comprising a piston plate and a base.

[0046] The connection structure is suitably hollow.

[0047] The connection structure is preferably branched.

[0048] One end-branch of the connection structure is preferably joined to a second pump, and one or more additional end-branches are preferably joined to separate pumps, each separate pump comprising: (i) a chamber that encloses a volume of gas, the volume of gas being the same volume as a second volume of gas in the second pump; (ii) a port for receiving and discharging gas and for joining to the connection structure; and (iii) a motor for controlling the operation of the pump.

[0049] Each separate pump is preferably the same as the second pump.

[0050] The system is preferably enclosed within a housing, suitably a temperature-controlled housing.

[0051] The temperature of the housing is preferably controlled by a thermostat.

[0052] The temperature within the housing is preferably about 37 °C.

[0053] The different volumes of the first and second pumps preferably represent the internal volumes of different sections of a breathing tube, suitably a human breathing tube.

[0054] The volume of the separate pump preferably represents the internal volume of a different section of a breathing tube, suitably a human breathing tube.

[0055] The displacement volumes of the first and second pumps are preferably at least as large as the maximum achievable volume uptake in the corresponding sections of the breathing tube.

[0056] The displacement volume of the separate pump is preferably at least as large as the maximum achievable volume uptake in the corresponding section of the breathing tube.

[0057] The pump pressure of the motor or pump is suitably equivalent to atmospheric pressure, or above or below atmospheric pressure.

[0058] The displacement volume of the first pump is suitably about 0 to 100 ml or about 1 to 100 ml.

[0059] The displacement volume of the second pump is suitably about 0 to 4000 ml or about 1 to about 4000 ml.

[0060] The pump suitably includes stainless steel.

[0061] The chamber is suitably a cylinder.

[0062] The chamber suitably includes glass.

[0063] The chamber of the first pump suitably has a smaller volume than the chamber of the second pump.

[0064] The volume of the chamber of the first pump suitably represents the volume of the oral cavity and the oropharyngeal cavity.

[0065] The volume of the chamber of the second pump suitably represents the volume of the lung cavity or a part thereof.

[0066] The volume of the connection structure suitably represents the volume of the lungs, suitably the conducting airways of the human lungs.

[0067] The system suitably further comprises a computer controller capable of synchronizing the operation of the system.

[0068] One or more of the first pump, or the second pump, or the connection structure suitably comprises one or more modules including a quartz crystal microbalance.

[0069] The opening in the first pump, or the second pump, or the wall of the connection structure is suitably threaded or non-threaded.

[0070] One or more of the openings suitably contain a module.

[0071] One or more modules are adapted to contain a matrix containing a cell culture medium, or to store a matrix containing a cell culture medium, or to monitor the conditions in the chamber, or to sample a gas, or to characterize a gas. The cell culture medium suitably contains one or more hydrogels such as gelatin methacryloyl (GelMa). Alternatively, or additionally, one or more modules are adapted to contain at least one microsensor, or are configured to store at least one microsensor for monitoring the conditions in the chamber, or are adapted to monitor the conditions in the chamber, or are adapted to sample a gas, or are adapted to characterize a gas.

[0072] The module is suitably located on the base of the piston plate of the first and / or second pump and / or in the wall of the connection structure.

[0073] One or more modules suitably contain a matrix containing a cell culture medium. The cell culture medium suitably contains one or more hydrogels such as gelatin methacryloyl (GelMa). Alternatively, or additionally, one or more modules contain at least one microsensor.

[0074] The matrix containing the cell culture medium suitably comprises or is in contact with a cell culture, suitably a two- or three-dimensional cell culture.

[0075] Modules adapted to contain or store a matrix comprising a cell culture medium and / or at least one microsensor preferably further comprise microfluidic channels and optionally a microfluidic pump connected thereto.

[0076] The module is preferably positioned within a horizontal plane of one or more of the walls of the first pump, or the second pump, or the connection structure.

[0077] The connection structure preferably comprises stainless steel.

[0078] The chamber of the first pump preferably has a volume of about 100 ml.

[0079] The chamber of the second pump preferably has a volume from about 1 liter to about 4 liters.

[0080] Also described is a pump for moving a volume of gas, the pump comprising: (i) a chamber configured to receive one or more apertures for receiving and containing, or storing, a matrix containing a cell culture medium and / or at least one microsensor for monitoring the condition within the chamber, or a probe for gas sampling or gas characterization, and a base; (ii) a first port for receiving and discharging gas when contained within the chamber, the first port comprising a first valve for regulating the flow of gas through the first port, the first valve being movable between open and closed positions, the valve being openable towards a test atmosphere or ambient air in the open position; (iii) a second port for receiving and discharging gas when contained within the chamber, the second port comprising a second valve for regulating the flow of gas through the second port, the valve being movable between open and closed positions; and (iv) a piston plate within the chamber, the piston plate comprising one or more gaps for the intake or inflow of gas into the chamber, one or more or each of the gaps being movable between open and closed positions and including a valve for regulating the intake or inflow of gas.

[0081] The pump is suitably a piston pump.

[0082] The one or more apertures in the chamber are suitably threaded or non-threaded.

[0083] The one or more apertures suitably comprise a module.

[0084] The module is suitably threaded or non-threaded.

[0085] One or more modules are adapted to include a matrix containing cell culture medium and / or at least one microsensor, or to store a matrix containing cell culture medium and / or at least one microsensor, or to be configured to monitor the conditions inside the chamber, or to be adapted to monitor the conditions inside the chamber, or to be adapted to sample a gas, or to be adapted to characterize a gas.

[0086] The module is suitably located on the base of the pump.

[0087] One or more modules are suitably adapted to include a matrix containing cell culture medium. The cell culture medium suitably includes one or more hydrogels such as gelatin methacryloyl (GelMa). Alternatively or additionally, one or more modules include at least one microsensor.

[0088] The matrix containing cell culture medium suitably comprises or is in contact with a cell culture, suitably a two - dimensional or three - dimensional cell culture.

[0089] Modules adapted to include or store a matrix containing cell culture medium and / or at least one microsensor suitably further comprise microfluidic channels and optionally a microfluidic pump connected thereto.

[0090] The module is suitably positioned within a horizontal plane of one or more of the walls of the first pump, or the second pump, or the connection structure.

[0091] The module suitably comprises a quartz crystal microbalance.

[0092] The pump suitably further comprises a motor.

[0093] The pump pressure is suitably equivalent to atmospheric pressure, or above or below atmospheric pressure.

[0094] The displacement volume of the pump is suitably about 0 to 100 ml or about 1 to about 100 ml.

[0095] The pump suitably includes stainless steel.

[0096] The chamber is suitably a cylinder.

[0097] The chamber suitably includes glass.

[0098] The chamber of the pump suitably has a volume of about 100 ml.

[0099] Also described is a piston pump for moving a volume of gas, the piston pump comprising: (i) a chamber configured to include a volume of gas and to include a piston plate having one or more gaps for the intake or inflow of gas into the chamber, wherein one or more or each of the gaps is movable between open and closed positions and includes a valve capable of regulating the intake or inflow of gas; (ii) a first port for receiving gas, the first port having a first valve for regulating the flow of gas through the first port, the first valve being movable between open and closed positions; and (iii) a second port for discharging gas when included within the chamber, the second port having a second valve for regulating the flow of gas through the second port, the valve being movable between open and closed positions. The chamber suitably includes a base and has another opening.

[0100] The opening is suitably threaded or non-threaded.

[0101] The opening suitably includes a module in one or more of the openings.

[0102] The module is suitably either screw-type or non-screw-type.

[0103] The module is suitably adapted to incorporate or hold a matrix containing cell culture medium and / or at least one microsensor for monitoring the conditions in the chamber or a probe for gas sampling or gas characterization.

[0104] One or more modules are suitably adapted to incorporate a matrix containing cell culture medium. The cell culture medium suitably incorporates one or more hydrogels such as gelatin methacryloyl (GelMa). Alternatively or additionally, one or more modules incorporate at least one microsensor.

[0105] The matrix containing cell culture medium suitably comprises a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0106] Modules adapted to incorporate or hold a matrix containing cell culture medium and / or at least one microsensor suitably further comprise microfluidic channels and optionally a microfluidic pump connected thereto.

[0107] The module is suitably provided with a quartz crystal microbalance.

[0108] The connection structure is suitably joined to a second port.

[0109] The connection structure is suitably hollow.

[0110] The pump suitably further comprises a motor.

[0111] The pump pressure of the pump is suitably equivalent to atmospheric pressure or above or below atmospheric pressure.

[0112] The displacement volume of the pump is suitably about 0 to 100 ml or about 1 to about 100 ml.

[0113] The pump suitably includes stainless steel.

[0114] The chamber is suitably a cylinder.

[0115] The chamber suitably includes glass.

[0116] The chamber of the pump suitably has a volume of about 100 ml.

[0117] In a further aspect, a method is provided for performing a simulation experiment of the interaction between a test atmosphere and an artificial respiratory tube, including the use of the pump or system described herein.

[0118] Also disclosed is the use of a pump or system as described herein for performing a simulation experiment of the interaction between a test atmosphere and an artificial respiratory tube.

[0119] Also disclosed is a method for determining the effect of a test atmosphere on a culture of cells or at least one microsensor incorporated in a matrix in an artificial respiratory tube, including the use of the pump or system described herein.

[0120] Also disclosed is the use of a pump or system as described herein for determining the effect of a test atmosphere on a culture of cells or at least one microsensor incorporated in a matrix in an artificial respiratory tube.

[0121] In a further aspect, a method for determining the effect of a test atmosphere on a culture of cells in a matrix or at least one microsensor incorporated in an artificial breathing tube is disclosed, the method comprising: (a) providing a pump or system as described herein, the pump or system comprising a matrix containing a culture of cells or at least one microsensor in one or more of the modules; and (b) comparing the matrix containing the culture of cells or at least one microsensor before and / or after exposure to the test atmosphere, wherein a difference in the matrix containing the culture of cells and / or at least one microsensor before and / or after exposure of the cells or microsensor to the test atmosphere indicates that the test atmosphere has an effect on the culture of cells or at least one microsensor.

[0122] A further aspect relates to a method for simulating an interaction between a test atmosphere and an artificial breathing tube with the system described herein, the method comprising: (a) opening a first valve of a first pump and closing a second valve of the first pump to provide a gas containing the test atmosphere to the first pump via a first port; (b) closing the first valve of the first pump, opening the second valve, and closing a valve on a piston plate of the first pump; (c) operating a second pump to draw the test atmosphere into a connection structure and flowing the chamber of the first pump and the connection structure with ambient air; (d) opening the first valve of the first pump towards the ambient air to form a sealed connection between a first port and a second port of the first pump; and (e) after a certain time, using the second pump to move the test atmosphere through the connection structure and through the first valve of the first pump.

[0123] The present specification further discloses a method for determining the effect of a test atmosphere on a simulated breathing trachea. The method includes: (a) opening a first valve of a first pump and closing a second valve of the first pump to provide a gas containing the test atmosphere to the first pump through a first port; (b) closing the first valve of the first pump, opening the second valve, and closing a valve on the piston plate of the first pump; (c) operating a second pump to draw in the test atmosphere through a connection structure and flowing the chamber of the first pump and the connection structure with ambient air; (d) opening the first valve of the first pump towards the ambient air to form a sealed connection between the first port and the second port of the first pump; (e) after a certain time, using the second pump to move the test atmosphere through the connection structure and through the first valve of the first pump. The test atmosphere is in contact with a matrix containing a cell culture or at least one microsensor located in one or more modules located in the first pump, or the connection structure, or the second pump, or a combination of two or more of them. The method further includes a step of determining the effect of the test atmosphere on the cell culture and / or the microsensor, and the difference in the cell culture and / or the microsensor before and / or after exposure to the test atmosphere indicates that the test atmosphere has an effect on the cell culture and / or the microsensor.

[0124] The module is adapted to monitor the system state and / or for gas sampling and / or for gas characterization, and the method suitably includes obtaining one or more measurements from the module.

[0125] A method for performing a simulation experiment of the interaction between a test atmosphere and a simulated breathing trachea is also disclosed, the method comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) removing the test atmosphere from the first pump into a connection structure that joins the first pump to a second pump; (c) flowing ambient air over the first pump and at least a portion of the connection structure; (d) holding the test atmosphere in the second pump and the connection structure for a defined period of time; (e) using the second pump to move the test atmosphere into the connection structure and the first pump; and (f) performing one or more pump cycles of ambient air in the second pump, wherein the test atmosphere contacts a cell culture located in the first pump, or the connection structure, or the second pump, or a combination of two or more thereof.

[0126] Step (d) suitably includes holding the test atmosphere in the second pump and a portion of the connection structure that still contains the test atmosphere for a defined period of time.

[0127] The pump is suitably a piston pump comprising a piston plate and a base.

[0128] The first pump is suitably as defined in embodiments of the present disclosure.

[0129] The second pump is suitably as defined in embodiments of the present disclosure.

[0130] The connection structure is suitably as defined in embodiments of the present disclosure.

[0131] The method is suitably carried out in a housing, suitably a temperature-controlled housing.

[0132] The temperature of the housing is suitably controlled by a thermostat.

[0133] The temperature in the housing is suitably about 37 °C.

[0134] The different volumes of the first and second pumps are suitably representative of the internal volumes of different compartments of the respiratory tract, suitably of the human respiratory tract.

[0135] The displacement volumes of the first and second pumps are suitably at least as large as the maximum achievable volume uptake in the corresponding compartments of the respiratory tract.

[0136] The pump pressure is suitably equivalent to atmospheric pressure, or above or below atmospheric pressure.

[0137] The displacement volume of the first pump is suitably about 0 - 100 ml or about 1 - about 100 ml.

[0138] The displacement volume of the second pump is suitably about 0 - 4000 ml or about 1 - about 4000 ml.

[0139] The chamber of the first pump suitably has a smaller volume than the chamber of the second pump.

[0140] The volume of the chamber of the first pump is suitably representative of the volume of the oral cavity and the oropharyngeal cavity, suitably of the human oral cavity and oropharyngeal cavity.

[0141] The volume of the chamber of the second pump is suitably representative of the volume of the lung cavity or a part thereof, suitably of the human lung cavity or a part thereof.

[0142] The connection structure is suitably representative of the volume of the conducting airways of the lung, suitably of the human lung.

[0143] A matrix containing a cell culture and / or at least one microsensor is suitably located on the base of the piston plate of the first and / or second pump and / or in the wall of the connection structure.

[0144] The cell culture is suitably a two-dimensional or three-dimensional culture.

[0145] The method further suitably includes monitoring for the state and / or for gas sampling and / or for gas characterization using one or more modules included in one of the first pump, or the second pump, or the connection structure.

[0146] The chamber comprising the matrix containing the cell culture and / or at least one microsensor further suitably comprises a microfluidic channel and optionally a microfluidic pump connected thereto.

[0147] The connection structure suitably includes stainless steel.

[0148] The chamber of the first pump suitably has a volume of about 100 ml.

[0149] The chamber of the second pump suitably has a volume of from about 1 liter to about 4 liters.

[0150] In a further aspect, a method for determining the effect of a test atmosphere on an artificial breathing airway is described, the method comprising: (a) providing the test atmosphere to a chamber of a first pump; (b) removing the test atmosphere from the first pump into a connection structure that joins the first pump to a second pump, the second pump being the pump described herein; (c) flowing ambient air over the first pump and at least a portion of the connection structure; (d) holding the test atmosphere in the second pump and the connection structure for a defined period of time; (e) using the second pump to move the test atmosphere through the connection structure and the first pump; (f) performing one or more pump cycles of ambient air in the second pump, the test atmosphere comprising a matrix comprising a cell culture located in one or more modules located in the first pump, or the connection structure, or the second pump, or a combination of two or more thereof, and / or in contact with at least one microsensor, the method further comprising determining the effect of the test atmosphere on the cell culture and / or the microsensor, wherein a difference in the cell culture and / or the microsensor before and / or after exposure to the test atmosphere indicates that the test atmosphere has an effect on the cell culture and / or the microsensor.

[0151] The module is adapted to monitor the system state and / or for gas sampling and / or for gas characterization, and the method suitably includes obtaining one or more measurements from the module.

[0152] Also described is a connection structure adapted to join at least two pumps for gas transfer therebetween, the connection structure having a hollow channel and / or more threaded or non-threaded openings in the wall thereof.

[0153] The screw-type opening contains a screw-type module in one or more of the openings, and the module is adapted to contain a cell culture medium or is configured to store a cell culture medium, or is adapted to monitor the state inside the chamber, or is adapted to monitor the state inside the chamber, or is adapted to sample a gas, or is adapted to characterize a gas.

[0154] One or more modules suitably include a matrix containing a cell culture medium and / or at least one microsensor. The cell culture medium suitably includes one or more hydrogels such as gelatin methacryloyl (GelMa). Alternatively, or additionally, one or more modules include at least one microsensor.

[0155] The cell culture medium suitably comprises or is in contact with a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0156] The module adapted to contain or store the cell culture medium further suitably comprises microfluidic channels and optionally a microfluidic pump connected thereto.

[0157] The connection structure is suitably hollow.

[0158] The connection structure is suitably branched.

[0159] Each terminal branch of the connection structure is suitably joinable to a separate pump.

[0160] The connection structure suitably represents the volume of the conducting airways of the lung, suitably the human lung.

[0161] The module is suitably positioned in a horizontal plane in the wall of the connection structure.

[0162] The module is suitably adapted to include a matrix containing a cell culture, suitably a two-dimensional or three-dimensional cell culture. Alternatively or additionally, the module is adapted to include at least one microsensor.

[0163] The module is a chamber for containing a matrix containing a cell culture and / or at least one microsensor, and the chamber suitably comprises a microfluidic channel and optionally a microfluidic pump connected thereto.

[0164] The module is suitably adapted for monitoring the state in a connection structure and / or for gas sampling and / or for gas characterization.

[0165] The connection structure suitably includes stainless steel.

[0166] A system comprising the pump described herein is also described.

[0167] The system suitably further comprises a connection structure as described herein.

[0168] The pump is suitably joined by a connection structure.

[0169] An apparatus configured or adapted to perform the method described herein is also disclosed.

[0170] Also disclosed is a method of manufacturing a perforated structure as described herein.

[0171] The perforated structure is suitably manufactured at least in part by three-dimensional printing.

[0172] The perforated structure is suitably formed by modeling.

[0173] The perforated structure is suitably cast from a template.

[0174] The perforated structure is suitably i) substantially mimicking the branching structure of the mammalian bronchial airway, ii) an idealized branching structure, or iii) a combination of i) and ii).

[0175] A further aspect relates to one or more in-silico branching channels within the pump, the branching channels being connected to ports for receiving and discharging gas.

[0176] The branching structure is suitably incorporated within the perforated structure according to the present disclosure.

[0177] The branching structure suitably reproduces an airway model.

Brief Description of the Drawings

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Figure 5

Figure 6

[0179] Some advantages The present disclosure can be used in in vitro simulation tests of the in vivo airway effects on aerosol properties related to fields such as inhalation toxicology and inhalation therapy that enable the evaluation of inhalable consumer products.

[0180] The present disclosure can provide an experimental model for determining in vivo aerosol dosimetry for the respiratory trachea under conditions that are stable, well-characterized, reproducible, and not ethically critical. The perforated structure as the airway model of the present disclosure can increase the amount of airway bifurcation generations to biologically relevant levels without simultaneously generating an uncontrollable number of connections such as the connection between the bronchial model and the second pump.

[0181] Different versions of the airway model can be used without affecting the overall function of the system and without requiring modifications to other parts of the simulated respiratory trachea system. This maximizes the flexibility in investigating how the respiratory trachea affects the aerosol or how the aerosol affects the respiratory trachea. Since the airway has (i) relevant effects on the properties of the aerosol reaching the alveolar cavity and (ii) exhibits significant inter-individual variability (also referred to as diseased state versus healthy state), the flexibility and adaptability in specific parts of the respiratory trachea are of great scientific value.

[0182] When conducting simulation experiments of the process that brings about the physicochemical properties of the test atmosphere in the respiratory trachea, not only the mode of interaction (e.g., dose delivery) between the two, but also clinically relevant dosing information may be obtained according to embodiments of the present disclosure.

[0183] The effects of one or more test atmospheres on one or more compartments of the respiratory trachea can be studied according to embodiments of the present disclosure.

[0184] The effects of one or more test atmospheres on one or more compartments of the respiratory tract can be studied, according to embodiments of the present disclosure, simultaneously or stepwise as needed.

[0185] Modules that can be used in the system provide flexibility in relation to the test system being exposed, experimental endpoints, and exposure parameters that are monitored according to embodiments of the present disclosure.

[0186] The modules can be redesigned or modified according to specific requirements without the need to change the overall structure and function of the system, according to embodiments of the present disclosure.

[0187] In many applications, aerosol generation is driven by the system itself, meaning that, according to embodiments of the present disclosure, an aerosol generator / smoking machine is not required. This can help to simplify the structure of the system.

[0188] In certain embodiments, the system can inherently be modular. This means that various components, such as pumps and connection structures, can be individually and easily replaced, for example, as needed. This can facilitate partial redesign, improvement, or replacement of the system according to specific requirements.

[0189] The mouth, breath-holding times, and dynamic flow patterns that occur during breathing can be simulated in experiments according to embodiments of the present disclosure.

[0190] According to certain embodiments, the present disclosure can address the holding period in the mouth, which enables high-density aerosols to be aged for up to several seconds before being drawn into the respiratory tract with a large amount of dilution air.

DETAILED DESCRIPTION OF THE INVENTION

[0191] In the practice of the present disclosure, in certain embodiments, conventional techniques of engineering, microbiology, cell biology, and biochemistry are used. For biological techniques, 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 (J.E. Celis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E.R. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, I.B. Griffiths, and D.G. Newell, eds., 1993 - 8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994). Procedures for employing commercially available kits and reagents are generally used according to the protocols defined by the manufacturer, unless otherwise described.

[0192] Technical terms and expressions used herein are generally given the meanings commonly applied to them in the relevant art. Any definitions of terms used herein apply to the entire content of this application.

[0193] The term "comprising" does not exclude other elements or steps.

[0194] The indefinite articles "a" or "an" do not exclude a plurality.

[0195] The term "and / or" means, for example, (a) or (b), or (a) and (b).

[0196] As used herein, the terms "comprising" and "comprised of" are synonymous with "including" or "containing", and 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 further elements are present.

[0197] 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 + / −5% or less, more preferably + / −1% or less, even more preferably + / −0.1% or less of and from a particular value, to the extent that the variations are appropriate for carrying out the present disclosure. It should be understood that the value to which the modifier "about" refers is also specifically and preferably disclosed in itself.

[0198] Before discussing the embodiments in more detail, an overview is first provided. The embodiments provide devices and methods that can be used in various applications for studying the respiratory tract. For example, the embodiments find utility when studying the deposition and / or condensation of one or more components present in a test atmosphere on the inner surface of a device, including re-evaporation. The embodiments also provide an evaluation of a test atmosphere that can be examined as it passes through a device to study changes in aerosol concentration, and / or the growth of aerosol particles, and / or the shrinkage of aerosol particles. The effect of a test atmosphere present inside a device / system on a biological test system can be studied in the embodiments of the present disclosure.

[0199] Airway model In one aspect, a perforated structure 1 used in a simulated respiratory tract system 10 is described, the structure comprising a perforated envelope that houses one or more branch channels, each perforation being an open end of a branch. In use, each perforation can be an open end of a branch. As shown in the accompanying figures, each perforation is an empty perforation.

[0200] FIG. 1 shows a simulated airway system 10 including a perforated structure 1 according to an embodiment of the present disclosure.

[0201] The perforated structure 1 can have a substantially solid body and an opening 2 including a socket suitable for providing an airtight and stable connection at a relevant part of the laboratory instrument in which the structure is used. A suitable system is described below and illustrated in FIG. 4, where the structure 1 fits into a port 83 for receiving and discharging gas. In use, the opening 2 is located on the bottom surface of the structure and is centrally fitted onto a base plate 84 by simple insertion using a clamping mechanism or by screw locking. The airtightness of the connection can be established by a seal. The pump 80 can represent the lung volume to a hollow structure representing the most proximal intrathoracic airways, i.e., the trachea and main bronchi (see FIG. 4).

[0202] The body of Structure 1 houses one or more branch channels 4. The outer surface of Structure 1 is perforated, and each perforation 6 is the open end of a branch 4. Inside the perforated structure 1, the channels originating from the proximal opening 2 branch continuously. The angle between the channels 4 originating from a given branch point, their diameter and length, and the number of branch points are thereby designed to result in a branching tree that is structurally similar to an airway tree, such as the human or animal airway tree. The last generation of channels 4, i.e., the distal end openings or perforations 6 of the structures representing the small airways, open towards the surroundings. In the case of the system 10 described herein, it is the internal volume of the second pump 80. Structure 1 thereby connects a model of the proximal airway of the system 10 (e.g., the trachea and main bronchi) to a model of the lung lumen, providing, for example, the branching structure in the human respiratory tract and separating the two regions.

[0203] The branching pattern of the channels 4 inside Structure 1 can follow (i) exactly the branching pattern of a complete animal airway, (ii) an idealized (e.g., with increased symmetry and regularity) version of a complete animal airway, or (iii) a part of either or both of these, i.e., only a part of the human or animal airway is reproduced. Regarding how Structure 1 affects aerosol properties, the idealization requires a computational or experimental equivalence to the human or animal airway being tested.

[0204] The radial symmetry of Structure 1 can be achieved either by idealizing the branching pattern of the airway or by symmetrically arranging multiple replicas of a part of the airway.

[0205] The structure 1 that strictly follows the branching pattern of the human or animal airway may be based on an airway cast or a digital 3D model obtained using tomography or similar techniques. A model with an idealized human or animal branching pattern may be based on relevant literature. For example, the Weibel model is a widely accepted model regarding the structure of the human airway (see Fishman’s Pulmonary Diseases and Disorders (2015) Ed. M.A. Grippi, ISBN: 0071807284), or it may be newly developed by computational or experimental approaches.

[0206] According to the Weibel model, important parameters that can be considered include that the diameter of the channels existing after n + 1 consecutive branch points is less than or equal to the diameter of the channels existing after n branch points, the total cross-sectional area of all channels existing after n + 1 consecutive branch points is greater than or equal to the total cross-sectional area of all channels after n branch points, and the branches are forked.

[0207] The number of branch generations depends on the size of the structure 1, the material from which it is made, and the performance of the techniques available for additive manufacturing. The more generations there are, the closer the simulation experiment of the effects of the airways in vivo approximates.

[0208] The airway structure 1 may consist of a single block of one material that encloses the complete airway tree internally, or it may consist of sub-components 1a, 1b that can be assembled based on experimental requirements (see Figure 2). The connection 7 between the sub-components 1a, 1b needs to be airtight and can be achieved, for example, by a plug-in mechanism or screw thread locking.

[0209] The overall shape and size of the airway module depend on its intended use. In the case of the airway structure used in the system 10 described hereinbelow, rotational symmetry is typically required to uniformly deliver the aerosol to the second pump volume. Rotational symmetry is required, for example, when the distribution of the cell culture in the exposure chamber is rotationally symmetric.

[0210] Radial symmetry can be omitted in certain situations, such as when only one culture is present, or when the distribution of the cultures does not require the equivalence of replicated cell cultures exposed in the same experiment to other symmetries, aerosol deposition as a function of the symmetry of the object (e.g., in the field of aerosol research), or when the complete system is asymmetric.

[0211] For example, in a 3D-replicated human lung, asymmetry is required due to the inherent biological asymmetry of the respiratory tract.

[0212] The spatial dimensions of Structure 1 depend on the simulated respiratory tract system in which Structure 1 is used in combination, because i) the types of airways that the structure is thought to represent can vary greatly in size (e.g., between humans and rodents), and ii) it must fit into the relevant parts of the system.

[0213] In the case of the use in System 10 described herein, the maximum horizontal size (diameter of the module, see Figure 4) is defined by the position of the cell culture modules 113, 213 (described below herein) in the pump 80 (described below herein), because the diameter of Structure 1 cannot be larger than the diameter of the circle in which the cell culture modules 113, 213 are located, since otherwise the modules 113, 213 would be covered by Structure 1. The maximum vertical size of the module (its height, the size in the direction of the axis of radial symmetry, see Figure 4) is typically defined by the residual volume of air present in the second pump 80 between inhalation cycles under a given experimental protocol (i.e., by the lowest position of the piston during the inhalation cycle). For example, in humans, the residual volume is in the range of 1200 mL. The stroke length to achieve an approximate compatible volume of 600 mL in each pump defines the maximum height of Structure 1.

[0214] The airway structure 1 can be designed in silico, taking into account relevant literature on the lung morphology of humans or animals, or the available digital 3D shapes of the respiratory tract of humans or animals, or their idealized shapes. Using the designed 3D shape, the structure 1 can then be generated by 3D printing to form an airway model. The airway structure 1 is typically an artificial or synthetic airway structure, forming an artificial or synthetic airway model. As another method, available airway casts or 3D airway models can be used as physical templates by embedding them in a material suitable for the formation of structure 1. The airway cast can be removed by dissolving it using a suitable solvent, by digesting it using a suitable enzyme, or by heating if the airway cast is made of a material with a lower melting point than the material selected for the airway structure 1. Examples of methods for designing and generating airway models are described in Rinboson et al (The Anatomical Record (2009) 292:1028 - 1044), Zopf et al (New England J. Med. (2013), 365(21); 2043 - 2045), and Zhang et al (Annals of Biomedical Engineering, (2008), 36(12), 2095 - 2110).

[0215] Accordingly, a further aspect relates to a method of manufacturing a perforated structure, the method comprising: (i) embedding one or more branched channels, each including an open end portion, within a material suitable for forming the perforated structure; and (ii) removing the cast of the one or more branched channels from the material. The perforated structure is suitably manufactured, at least in part, by three-dimensional printing. The perforated structure is suitably formed by modeling. The perforated structure is suitably cast from a template. The perforated structure is suitably (i) substantially mimicking the branching structure of the mammalian bronchial airway, (ii) an idealized branching structure, or (iii) a combination of (i) and (ii). The airway cast is suitably removed by dissolving using a suitable solvent, by digesting using a suitable enzyme, or, where the airway is made of a material having a lower melting point than the material selected for the airway structure, by heating.

[0216] The airway structure 1 can be manufactured from any material from which a composite structure can be formed, by additive manufacturing or by one of the above techniques, such as, for example, embedding the airway cast in a metal, ceramic or synthetic material. The exact type of material depends on the intended application.

[0217] For example, when investigating the effect of an airway model on the size distribution and concentration of the non-volatile portion of solid and / or liquid particles in a test aerosol, a non-porous material can be used. Examples include metals (e.g., stainless steel), non-porous synthetic materials (e.g., polyethylene terephthalate (PET), or polyether ether ketone (PEEK) or a combination of two or more of them). As a further example, when investigating the effect of an airway model on the size distribution and concentration of the volatile portion of solid and / or liquid particles and on volatile compounds in a test aerosol, a porous material can be used. Volatile compounds generally do not condense on non-porous surfaces but can enter the pores of a porous material and adhere to it due to the large available surface area. (How activated charcoal works). An example is porous silicon (e.g., polydimethylsiloxane (PDMS)). As a further example, when the effect of an airway model on the size distribution and concentration of the volatile portion of solid and / or liquid particles and on volatile compounds in a test aerosol is investigated, or when cells are cultured on the inner surface of an airway model, a gel-like material can be used. In this case, the gel-like material may be immersed in a cell culture medium or may include the cell culture medium as a substrate in an additive manufacturing process. Examples include thermogels such as poly(vinyl methyl ether) (PVME) or poly(N-vinylcaprolactam) (PNVC) or a combination of two or more of them. As a further example, when enhancing the interaction between a hydrophobic compound in an aerosol and an airway model, a hydrophobic material can be used (e.g., PDMS). As a further example, when enhancing the interaction between a hydrophilic compound in an aerosol and an airway model, a hydrophilic material can be used. Examples include hydrogels such as PVME or PNVC or a combination of them. As a further example, when enhancing the interaction between hydrophobic and hydrophilic compounds in an aerosol and an airway model, an amphiphilic material can be used. Amphiphilicity can be achieved, for example, by coating the surface with a surfactant.Furthermore, the hydrophilicity, hydrophobicity, and absorption characteristics of the airway structure 1 can be adjusted by coating the inner channel surface with a suitable material (e.g., hydrogel, wax, or protein, or a combination of two or more of them). For example, coating with a cell culture fluid containing hydrogel may be used to provide a surface on which cells can be cultured. The possibility of exposing cell cultures to a test aerosol passing through the airway model can be provided by including an exposure chamber 8 within the structure (see FIG. 3). Such an exposure chamber 8 can be formed, for example, by a local enlargement of the channel 4 that is one-dimensional (perpendicular to the channel axis). The enlargement matches the size and shape of one or more (e.g., all) of the available cell culture modules 113, 213 that can be cultured on the hydrogel or within the microfluidic structure that provides a small amount of nutrients. The cell culture and hydrogel may be present at the tip of the "exposure plug" 9, which is inserted into the airway structure 1, thereby transporting the cell culture to the exposure chamber 8, whereby the surface of the cell culture is in the plane of the inner surface of the exposure chamber 8.

[0218] When a probe or sensor is inserted into the airflow channel 4, a similar approach can continue, for example, coating all or a part of the channel 4 within the complete airway structure 1 with a hydrogel that delivers sufficient water and nutrients to the cells grown directly in the channel 4 of the structure 1. This approach typically requires that the airway structure 1 be made of, or include, a highly porous material that can store a large amount of culture fluid, or that the airway structure 1 be formed from a gel-like material that provides nutrients and water.

[0219] Accordingly, in a further aspect, one or more (e.g., a plurality of) branching channels that reproduce an airway model are disclosed, the branching channels including a coating of cell culture fluid on at least a portion or all of the branching channels, providing a surface on which cells can be cultured. The coating may suitably contain cells. In a further aspect, one or more (e.g., a plurality of) branching channels that reproduce an airway model are disclosed, the branching channels including one or more enlarged portions within the channel or including one or more matrices for culturing cells. The matrix may suitably contain cells.

[0220] System In a further aspect, a system 10 for determining the interaction between a test atmosphere and an artificial respiratory tract is described. The system comprises: (i) a chamber configured to contain a first volume of gas that includes the test atmosphere; (ii) a first port adapted to receive and discharge gas and comprising a valve for regulating the flow of gas through the first port, the valve being movable between an open and a closed position, 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 comprising a valve for regulating the flow of gas through the second port, the valve being movable between an open and a closed position; and (iv) a motor for controlling the operation of a first pump. (a) The first pump comprises: (i) a chamber configured to contain a second volume of gas, the first and second volumes of gas being different; (ii) a port adapted to receive and discharge gas; (iii) a piston plate within the chamber, the piston plate comprising one or more gaps for the intake or inflow of gas into the chamber, one or more or each of the gaps being movable between an open and a closed position and including a valve capable of regulating the intake or inflow of gas; and (iv) a motor for controlling the operation of a second pump, the second pump being as described herein. (c) A connection structure operable to transfer gas from the first pump into the second pump; and (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 thereof, the openings being capable of receiving a module for monitoring the state within the chamber containing the cell culture medium and / or matrix and / or for gas sampling or gas characterization and including or holding at least one microsensor.

[0221] The gas can be or can include the test atmosphere.

[0222] FIG. 4 shows a system 10 according to an embodiment 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 certain 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 an individual motor 41, 81 of the pump itself, 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 a respiratory tract, such as the respiratory tract 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 respiratory tract 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 of a human or animal. Another second pump 80 can represent the volume of the pulmonary lumen or a part thereof, such as an individual lung lobe or the lumen of a smaller subunit, which is particularly the pulmonary lumen or a part thereof of a human or animal. The branched hollow connection structure 50 can represent the dimensions of an induction 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 induction airway of a human or animal. The branched hollow connection structure 50 can represent the dimensions of an induction 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 diameter and length, can resemble the tree shape of the induction airway.

[0223] As shown in FIG. 4, the branched hollow structure 50 can be connected to the central openings 43, 83 in the bases 44, 84 of the chambers 42, 82 of each pump 40, 80. In certain 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. A valve 44a can be used directly in the connection between the pump 40 and the connection structure 50 to enable sealing of the pump 40 from all other system components.

[0224] The pump 40 representing the oral cavity can have one or more openings 43 through which the test atmosphere and the dilution air can pass when leaving the pump 40 towards the branched hollow structure 50. The entry and exit points of the test atmosphere 90 are usually located on the piston plate 45 of the pump 40, suitably at its center. This can pass through the hollow piston shaft 46, and there may be a valve 44a, such as a three-way valve, above it. In an embodiment, the valve 44a can be closed or opened towards the test atmosphere source or the ambient air. An arrangement of one or more (e.g., a plurality of) gaps 47 through which ambient air can enter the system is optionally arranged radially and is arranged on the piston plate 45. One or more valves 48 (e.g., a plurality) above one or more or all of the gaps can be used to enable the opening or closing of one or more of these gaps 47. In certain embodiments, each gap 47 is controlled by a valve 48. In certain embodiments, an arrangement of one or more (e.g., a plurality of) gaps through which ambient air can enter the system is optionally arranged radially and can be arranged on the piston plate 84 of the second pump. One or more valves (e.g., a plurality) can be used to enable the opening or closing of one or more of these gaps. In certain embodiments, each gap is controlled by a valve. In certain embodiments, an arrangement of one or more (e.g., a plurality of) gaps through which ambient air can enter the system is 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 the pumps 40, 80. It is advantageous that the branched hollow structure 50 can be disassembled into its main components. This can allow for easy access for placing or removing the test system and / or for cleaning.

[0225] The bases 44, 84 of the pumps 40, 80 can be removed for placing / removing the test system and for cleaning.

[0226] Not only different parts of the connection structure 50, but also in the bases 44, 84 of each of the pumps 40, 80, there can be positioned 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. The openings, holes, or sockets 51 can be placed at 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, suitably on the lower side of the branched hollow structure 50, or in any combination thereof.

[0227] The openings, holes, or sockets 51 can be used to mount devices onto or into various modules 112, 212 that can be used to monitor the operation of the system 10 and / or conduct experiments and / or collect samples and the like. Examples of such modules 112, 212 or devices are shown in FIGS. 5 and 6 and described herein.

[0228] 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.

[0229] FIGS. 5 and 6 show an embodiment of the disclosure where the openings, holes, or sockets 51 are in any form of threaded openings, holes, or sockets 112, 212. One or more of the openings, holes, or sockets 51 can include one or more modules 112, 212. The use of threads facilitates the simple insertion and replacement of the modules 112, 212. The use of threads in the openings, holes, or sockets 51 is optional for constructing the modules, so that the modules can be inserted or pushed into the openings, holes, or sockets 51 to make a sealed engagement. The modules can be of the push-in type. The tightening can be achieved using O-rings and the like.

[0230] The modules 112, 212 to be used can be adapted for various purposes according to the requirements of the system to be configured. For example, the modules 112, 212 can be adapted to contain or store a cell culture medium, or to monitor the conditions in the chambers 42, 82, or to sample a gas or a liquid, or to characterize a gas, and the like. The modules 112, 212 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 of the modules 112, 212 can be configured to contain or store a matrix containing a cell culture medium. According to this embodiment, one or more of the modules 112, 212 can be a container capable of holding a liquid or a solution. The cell culture medium can comprise, or be in contact with, a cell culture, such as a two-dimensional or three-dimensional culture of cells. In certain embodiments, one or more of the modules 113, 213 can hold or position at least one microsensor as an alternative to, or in addition to, the cell culture medium matrix. In certain embodiments, the modules 113, 213 adapted to contain or store a matrix containing a cell culture medium and / or at least one microsensor further comprise microfluidic channels, and optionally, a microfluidic pump connected thereto. The modules 113, 213 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.

[0231] As can be seen in FIGS. 5 and 6, the opening, hole, or socket 51 can be optionally configured to include threaded holes or sockets 112, 212. The threaded holes or sockets 113, 213 can comprise one or more probes 114, 214 of one or more devices 116, 216. Such devices 116, 216 can be used for monitoring internal system states, or for characterizing test atmospheres, or for sampling and the like. The operation of devices 116, 216 can be controlled by computers 117, 217. The threaded holes or sockets 113, 213, and / or modules 112, 212 can be adapted to be used as culture chambers 115, 215 in which a biological test system (e.g., an organotypic cell culture of human respiratory tracheal epithelium as described herein) can be placed for exposure to a test atmosphere. The threaded openings, holes, or sockets 113, 213 can be adapted to include capture agents that can sample the test atmosphere for analysis. Sampling of the cell culture fluid or capture agent during exposure to the test atmosphere can be accomplished by various means, including the use of a microfluidic pump system 211 and / or one or more microsensors. Modules 112, 212 holding quartz crystal microbalances (QCMs, 219) can be used. The modules 112, 212 to which probes 214, chambers 215, or QCMs 219 can be attached can be inserted into any chamber 42, 82 of the system or into the interior of the branched hollow structure 50. The operation of QCM 219 can be controlled by computer 220. System 10 can be fully or partially controlled by computer 13 as needed. System 10 can be partially or fully automated.

[0232] System 10 can comprise one or more (e.g., a plurality of) first pumps. System 10 can comprise one or more (e.g., a plurality of) second pumps. System 10 can comprise one or more (e.g., a plurality of) first pumps and one or more second pumps.

[0233] Pump In a further aspect, a pump for moving a volume of gas, preferably a piston pump, is disclosed that includes one or more branch channels inside the pump, and the branch channels are connected to ports for receiving and discharging gas. The pump may have external and internal components. The one or more branch channels may be inside the pump. The ports may be located on the pump. The pump may include the ports. The ports may be located inside or outside the pump. The branch channels are suitably incorporated within the perforated structures described herein. The branch channels are suitably capable of reproducing an airway model. The pump is configured to include an opening for receiving one or more modules for containing a volume of gas and for containing at least one or more microsensors for monitoring the state in a base, a matrix containing a cell culture medium, and / or a chamber, or for gas sampling or gas characterization, and is suitably provided with a chamber. The pump is suitably further provided with a motor for controlling the operation of the pump. As described above, the branching structure may follow the branching pattern of the human or animal airway and may be based on a digital 3D model obtained using airway casting or tomography or similar techniques. The pump may substantially correspond to a second pump as described below herein.

[0234] A system for determining the interaction between a test atmosphere and a simulated breathing trachea including a pump is also contemplated. The use of a pump in a system for determining the interaction between a test atmosphere and a simulated breathing trachea is also contemplated.

[0235] A method for culturing cells in a pump is also contemplated. The use of a pump for culturing cells is also disclosed.

[0236] First pump In another aspect, a pump for moving a volume of gas, preferably a piston pump, is disclosed, the pump comprising: (i) a chamber configured to enclose a volume of gas and to have one or more openings for receiving a module for containing or storing cell culture fluid, or for monitoring a condition within the chamber, or for gas sampling or gas characterization, and a base; (ii) a first port for receiving and discharging gas when contained within the chamber, the first port having a first valve for regulating the flow of gas through the first port, the first 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 for receiving and discharging gas when contained within the chamber, the second port having a second valve for regulating the flow of gas through the second port, the valve being movable between open and closed positions; and (iv) a piston plate within the chamber, the piston plate having one or more gaps for the intake or inflow of gas into the chamber, one or more or each of the gaps having a valve movable between open and closed positions and capable of regulating the intake or inflow of gas.

[0237] A piston pump for moving a volume of gas is also disclosed, the piston pump comprising: (i) a chamber configured to include a piston plate that includes a volume of gas and one or more gaps for intake or inflow of gas into the chamber, wherein one or more or each of the gaps includes a valve movable between open and closed positions and capable of regulating intake or inflow of gas; (ii) a first port for receiving gas, the first port comprising a first valve for regulating flow of gas through the first port, the first valve being movable between open and closed positions; and (iii) a second port for discharging gas when included within the chamber, the second port comprising a second valve for regulating flow of gas through the second port, the valve being movable between open and closed positions.

[0238] As shown in FIG. 4, system 10 can include a first pump 40 for moving a volume of gas. The first pump 40 is disclosed herein as a separate aspect of the present disclosure and its use is not limited to use in the system 10 described herein.

[0239] The first pump can be a primary pump, so called because of its location in the system as an entry point for the gas. The first pump comprises a chamber 42 (e.g., a cylinder) configured to enclose the volume of the gas, and as shown in FIGS. 5 and 6 and as described herein, a base 44 and one or more openings 43 capable of receiving modules such as threaded or non-threaded modules 113, 213 in holes or sockets 112, 212. The first pump also includes a first port 90 for receiving and discharging the gas when enclosed within the chamber 42, and a first valve 44a, such as a three-way valve, for regulating the flow of gas through the first port 90. The first valve 44a is capable of moving between open and closed positions, and in the open position, the valve 44a can be opened towards the test atmosphere or ambient air. The first pump also includes a second port 43 for receiving and discharging the gas when enclosed within the chamber 42. The second port 43 is suitably configured as an opening. A connection between the first pump 40 and the connection structure 50 enables the pump 40 to be sealed from other system components by a second valve 49 located at the location of the second port 43. The second valve 49 can be used to regulate the flow of gas through the second port 43 and is capable of moving between open and closed positions. As can be seen in FIG. 4, the pump 40 can be a piston pump comprising a piston plate 45. One or more of the openings, holes, or sockets 51 in the chamber 42 can be threaded 112, 212 or non-threaded. One or more of the openings, holes, or sockets 51 in the chamber 42 can comprise modules 113, 213, such as threaded or non-threaded modules 113, 213, as discussed herein. The pump 40 further comprises a motor 41 for controlling the operation of the pump. The pump pressure of the motor 41 can correspond to atmospheric pressure or, if necessary, be above or below atmospheric pressure. In certain embodiments, the pump pressure of the motor 41 can be above or below atmospheric pressure to move the test atmosphere. The displacement volume of the pump 40 can be about 0 to 100 ml or about 1 to about 100 ml. The chamber 42 of the pump 40 can have a volume up to a maximum of about 100 ml.The pump 40 can be manufactured from various materials known in the art, such as stainless steel. The chamber 42 is suitably a cylinder. The chamber 42 is suitably made of glass. The piston plate 45 of the pump 40 has one or more gaps 47 for the intake or inflow of gas. One or more of the gaps 47 can include a valve 48 that is movable between an open and closed position and can regulate the intake or inflow of gas.

[0240] Also, as shown in FIG. 4, the pump 40 can be a piston pump for moving a volume of gas. The piston pump comprises a chamber 42 (e.g., a cylinder such as a glass cylinder) configured to enclose a volume of gas and to enclose a piston plate 45 having one or more gaps 47 for the intake or inflow of gas into the chamber 42. One or more of the gaps 47, or each of the gaps 47, comprises a valve 48 for regulating the intake or inflow of gas through the gap 47.

[0241] This can include a first port 90 for receiving gas and a first valve 44a, such as a three-way valve, for regulating the flow of gas through the first port 90. The first valve 44a is capable of moving between an open and a closed position. This also includes a second port 43 for discharging gas when contained within the chamber 42, and the second port 43 optionally includes a second valve for regulating the flow of gas through the second port 43, and the valve is capable of moving between an open and a closed position. The chamber 42 can include a base 44a and another opening 43. The base can additionally include one or more openings, holes, or sockets 51, which can be threaded or non-threaded, and / or they can include modules 113, 213 as discussed herein. A connection structure 50, such as a hollow connection structure, can be joined to the second port 43. The pump 40 can further include a motor 41 where the pump pressure corresponds to atmospheric pressure, or is above or below atmospheric pressure. The displacement volume of the pump 40 can be about 0 - 100 ml or about 1 - about 100 ml. The chamber 42 of the pump 40 can have a volume of about 100 ml.

[0242] A system with two or more first pumps is contemplated. The use of two or more first pumps in a system for determining the interaction between a test atmosphere and a simulated breathing tube is also contemplated.

[0243] A method for culturing cells in a first pump is also contemplated. The use of a first pump for culturing cells is also disclosed.

[0244] Second pump In another aspect, a pump, preferably a piston pump, is disclosed, the pump comprising: (i) a chamber configured to receive one or more modules to enclose a volume of gas and to receive a base and at least one microsensor for monitoring the state in a matrix containing a cell culture fluid and / or in the chamber, or for gas sampling or gas characterization; and (ii) a port for receiving and discharging gas; and (iii) one or more branching channels located within the port. The branching channels may be incorporated within a perforated structure as described herein.

[0245] As shown in FIG. 4, the simulated breathing tracheal system 10 described hereinafter in this specification may include a pump 80 for moving a volume of gas, the pump comprising a chamber 82, such as a cylinder, configured to enclose a volume of gas, the chamber 82 comprising a base 84 and one or more modules 113, 213 for containing or storing at least one microsensor for monitoring the state in a matrix containing a cell culture fluid and / or in the chamber 82, or for gas sampling or gas characterization and the like. The pump 80 may be a secondary pump.

[0246] The pump 80 is disclosed herein as a separate aspect of the present disclosure and its use is not limited to use in the system 10 described herein.

[0247] The pump 80 further includes a port 83 operable to receive and discharge gas, within which the perforated structure 1 described herein may be fitted.

[0248] As shown in FIG. 4, the pump 80 can be a piston pump including a piston plate 83. There may be no gaps or openings in the piston plate 83. Modules 113, 213, such as screw or non-screw type modules 113, 213 as discussed herein, can be located within the base 84 of the chamber 82. A connection structure 50, such as a hollow connection structure, can be joined to the port 83. The pump 80 further includes a motor 81. The pump pressure of the pump 80 generally corresponds to atmospheric pressure, or is above or below atmospheric pressure. The displacement volume of the pump 80 can be about 0 to about 1000 ml, or about 0 to about 4000 ml, or about 1 to about 1000 ml, or about 1 to about 4000 ml. The volume of the chamber 82 can represent the volume of the lung lumen or a part thereof. In certain embodiments, an array of one or more (e.g., a plurality of) gaps through which ambient air can enter the system can optionally be arranged radially and disposed on the piston plate 84. One or more (e.g., a plurality of) valves can be used to enable opening or closing of one or more of these gaps. In certain embodiments, each gap is controlled by a valve.

[0249] A system comprising two or more pumps 80 as described herein is contemplated. Use of two or more pumps 80 in a system for determining the interaction between a test atmosphere and a simulated breathing airway is also contemplated.

[0250] A method for culturing cells within a pump 80 as described herein is also contemplated. Use of a pump 80 for culturing cells is also disclosed.

[0251] Connection structure Also disclosed is a connection structure operable to transfer or carry gas between two or more pumps. The connection structure can be a tube, or pipe, or conduit, or the like, through which gas can be directed or conveyed. The connection structure can be adapted to connect at least two pumps to transfer or convey gas therebetween. By means of the connection structure, the first port of the first pump can be joined to the port of the second pump described herein at the second port of the first pump. The connection structure can include a hollow channel and one or more openings, such as threaded or non-threaded openings, in the wall of the connection structure. The connection structure can be used in the systems and methods described herein. The system can include a connection structure adapted to join at least two pumps to transfer gas therebetween.

[0252] An embodiment of the connection structure 50 is shown in FIG. 4. The connection structure will generally include a hollow channel. The connection structure will generally be branched. In an embodiment, each end branch of the connection structure can be joined to a separate pump 40, 80 when incorporated into the system. The connection structure 50 can include one or more openings, particularly threaded or non-threaded openings, in the wall of the connection structure. The threaded or non-threaded openings can accommodate modules 112, 212. The modules 112, 212 can be adapted for the inclusion of culture fluid and / or monitoring of the system state and / or gas sampling and / or gas characterization as discussed herein. The connection structure can be branched with two or more branches in certain embodiments. Each end branch of the connection structure can be joined to a separate pump. The connection structure can represent the volume of the conducting airways of the lung. The connection structure 50 can be made of various materials. In certain embodiments, the use of stainless steel is preferred.

[0253] Also contemplated is a method for culturing cells that includes a connection structure. Also disclosed is the use of a connection structure for culturing cells.

[0254] System operation and function The system can be fully or partially controlled by a computer as necessary. This enables synchronization of some or all of the operations of the pumps and valves. This enables synchronization of some or all of the operations of the elements of the system. The computer can be used to set the stroke length of one or more of the pumps and / or the stroke speed of one or more of the pumps. The computer can be used to control the temperature of the system.

[0255] Here, the operation of an embodiment of the system 10 as depicted in FIG. 4 will be described. In the stationary state, the piston of the first pump 40 is in the downstroke position, the position of the second pump 80 is in a position to maintain a defined gas volume in the chamber 82, and the valves 48, 49 are closed. The test atmosphere is absorbed into the pump 40 which can represent the oral cavity. This inflow can be driven by the pump 40 and can enter the chamber 42 via the hollow piston shaft 46. The hollow piston shaft 46 can be directly connected to the test atmosphere source. When the pump 40 completes the upstroke, the valve 44a which can be a three-way valve at the central opening at the upper part of the hollow piston shaft 46 closes, the valve 48 regulates the inflow of ambient air through the gap 47 in the piston plate 45, and the valve 49 at the inlet to the connection structure 50 opens.

[0256] The test atmosphere is removed from the chamber 42 which can represent the oral cavity through the connection structure 50 which can represent the induction airway. This can be driven by the upstroke of the second pump 80 which can represent the lung lumen or a part thereof. Since the total displacement volume of the second pump 80 can be a multiple of the volume of the pump 40, not only at least a part of the connection structure 50 but also the chamber 42 of the pump 40 can be flushed with ambient air, and the ambient air can enter the first pump 40 through the gap 47 in the piston plate 45.

[0257] In the first pump 40, the valve 44a at the upper part of the piston shaft 46 can open towards the surroundings. The valve 48 on the piston plate 45 can close, and the pump 40 performs a downstroke. In the downstroke position, this pump 40 can form a sealed connection between the hollow piston shaft 46 and the opening 43 in the base 44. The sealed connection can be achieved by the gasket 53. The gasket 53 can be located on either the base 44 or the piston plate 45. After a defined "breathing hold time", the second pump 80 can then perform a downstroke, thereby moving the test atmosphere directly to the surroundings through the connection structure 50 and through the piston shaft 46 of the first pump 40. With the pump 40 remaining in the downstroke position and the valve 44a kept open towards the surroundings, before the valve 44a opens again towards the test atmosphere source and the next inhalation cycle of the test atmosphere begins, the second pump 80 can perform one or more (e.g., several) cycles of breathing ambient air.

[0258] In a further aspect, there is provided a method comprising: (a) providing a pump, such as the first pump described herein, having a chamber; (b) removing a gas, such as a test atmosphere, into a connection structure that joins the pump to a further pump, such as the second pump described herein; (c) flowing at least a portion of the pump provided in step (a) and the connection structure with ambient air; (d) holding the gas in the further pump and the connection structure for a defined time; (e) using the further pump to move the gas into the connection structure and the pump provided in step (a); and (f) performing one or more pump cycles of ambient air in the further pump.

[0259] In a further aspect, there is provided a method comprising: (a) providing a gas, such as a test atmosphere, to a chamber of a pump, such as the first pump described herein; (b) removing the gas from the pump of step (a) into a connection structure that joins the pump to a further pump, such as the second pump described herein; (c) flowing at least a portion of the pump and the connection structure of step (a) with ambient air; (d) holding the gas within the further pump and the connection structure for a defined time; (e) using the further pump to move the gas through the connection structure and the pump of step (a); and (f) performing one or more pump cycles of ambient air within the further pump.

[0260] Generation of test atmosphere Test atmospheres, such as aerosols, to be studied using the disclosed system 10 and method can be generated by a variety of means. For many applications, for example, to test tobacco products, or common medical inhalers and the like, the generation of the test atmosphere can be driven by the system itself, i.e., the primary or secondary pump itself generates the negative pressure necessary for the generation and extraction of the test aerosol, which advantageously means that the use of an aerosol generator / smoking machine is not required.

[0261] The test atmosphere can be, for example, an environmental sample of a gas or aerosol for monitoring indoor air quality, occupational exposure, or environmental pollution in close proximity to industrial sites. In certain such cases, the test atmosphere is not generated and is sampled by the action of the system.

[0262] 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 produced 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.

[0263] When the aerosol is smoke, the system 10 can optionally be provided by the system with a smoking machine connectable to a smoking machine or by a pump of the present disclosure, and it is appropriate to hold and light the cigarette. 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 may 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 smoking may be carried out on the smoking machine substantially in accordance with the smoking regimen of the International Organization for Standardization (ISO 2000).

[0264] The use of a control atmosphere, such as an atmosphere that does not include the test atmosphere, is also contemplated. The use of a control atmosphere can serve to determine the effect of the test atmosphere in comparison to the control atmosphere.

[0265] System 10 can be connected to a smoking machine by suitable conduits that provide a flow path for smoke to System 10. The smoke can be transmitted through the conduits 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 flow of smoke. The conduit can include at least one inlet for the introduction or injection of standard reference materials, such as nicotine, into System 10 for calibration purposes. The smoke flow will generally be controlled by the systems or pumps of the present disclosure.

[0266] The smoking machine can be a linear or rotary smoking machine. The smoking machine is preferably operative 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.

[0267] The system 10 and method described herein can be used to perform an analysis of mainstream smoke generated by a smoking article during a smoking test. "Mainstream smoke" refers to the smoke that is drawn through the smoking article and would be inhaled by a consumer during use.

[0268] The test atmosphere may be from an "aerosol generating device", which is a device that interacts with an aerosol forming substrate to generate an aerosol. An example of an aerosol is smoke. The aerosol forming substrate may be part of the aerosol generating article. The aerosol generating device may comprise one or more components suitable for generating an aerosol from the aerosol generating substrate. The aerosol generating device may be an electrically heated aerosol generating device, which is an aerosol generating device comprising a heater operated by electricity to heat the aerosol forming substrate of the aerosol generating article to generate an aerosol. The aerosol generating device may be a gas heated aerosol generating device, a device heated by a carbonaceous heat source, another exothermic chemical reaction, or a heat sink. Other suitable means for generating an aerosol are well known in the art. The aerosol generating device may 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 the user's lungs through the user's mouth.

[0269] Another example of an "aerosol generating device" is an inhaler, which is generally 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 respiratory tract. Inhalers 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 drug 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.

[0270] As used herein, the term "aerosol-forming substrate" relates to a substrate having the ability to release volatile compounds capable of forming an aerosol. Such volatile compounds may be released by heating the aerosol-forming substrate. The aerosol-forming substrate may be adsorbed, coated, impregnated or otherwise loaded onto a carrier or support. Advantageously, the aerosol-forming substrate may 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, for example, an aerosol-generating article or a heated cigarette. The aerosol-generating article is of a size and shape suitable for engaging with an aerosol-generating device to bring the aerosol-forming substrate into contact with a heater.

[0271] The aerosol-forming substrate may contain a medically active compound or a drug such as an antibiotic or an anti-inflammatory agent that can be delivered to a patient via the respiratory tract. A number of medical inhalation devices (inhalers) are known and routinely prescribed for the treatment of various respiratory tract-related and non-respiratory tract-related diseases.

[0272] The aerosol-forming substrate may contain nicotine. The aerosol-forming substrate may contain tobacco. The aerosol-forming substrate may include a tobacco-containing material containing 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 more than 5% on a dry weight basis. Alternatively, the homogenized tobacco material may have an aerosol-forming agent content of 5 to 30% by weight on a dry mass basis. The homogenized tobacco material sheet 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 homogenized tobacco material sheet 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 homogenized tobacco material sheet 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 homogenized tobacco material sheet may include, but is not limited to, other additives including tobacco and non-tobacco fibers, aerosol-forming agents, humectants, plasticizers, flavorants, fillers, aqueous and non-aqueous solvents, and combinations thereof.

[0273] Cell culture Cell cultures for use in the present disclosure include two-dimensional and three-dimensional cell cultures. As described herein, cell cultures will generally be contained or cultured within one or more modules of one or more pumps and / or connection structures. The cell cultures can be exposed to a test atmosphere so as to be able to determine the effect of the test atmosphere on the cell culture. Two or more cell cultures will suitably be at different locations around one or more pumps and / or connection structures and / or the system so that the effect of the test atmosphere on the cell cultures at these different locations can be determined, mimicking the respiratory tract. Two-dimensional cell cultures involve the growth of cells in a flat layer on a plastic surface, which enables the study of some aspects of cell physiology and responses to stimuli such as a test atmosphere, but do not reflect the actual structure and architecture of an organ. In a two-dimensional monolayer, the extracellular matrix, cell-cell interactions, and cell-matrix interactions, which are essential for differentiation, proliferation, and cell function, are lost. Three-dimensional culture systems can form functional tissues with characteristics similar to those observed in vivo. Compared to two-dimensional culture systems, three-dimensional cell culture enables cells to interact with all three directions of their surroundings, resulting in a 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 generate specific tissue-like structures in a way that is more physiologically relevant than conventional two-dimensional cell monolayers and can mimic the function and response of actual tissues. Several three-dimensional tissues mimicking human organs are commercially available. The three-dimensional organ tissue of the lung, which is a particular subject in the context of the present disclosure, can be prepared using primary human cells grown at an air-liquid interface (ALI) where the cells differentiate to form a functional tissue. These three-dimensional tissues possess morphological similarities and metabolic characteristics similar to those of human bronchial tissue. They are composed of basal cells, goblet cells, and ciliated cells arranged in a multi-columnar structure. Similar to the lung, there are actively waving cilia, thereby enabling studies on its function and activity.Compared to human lungs, it has been found that in these 3D ALI cultures, the mRNAs encoding xenobiotic enzymes are at comparable levels. In addition, these tissues can be maintained in vitro for extended periods. This 3D model of lung tissue is an appropriate model for exploring the effects of test atmospheres and the like in accordance with the present disclosure.

[0274] 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. A number of different three-dimensional cell culture methods have been developed, including spheroid culture and organotypic culture.

[0275] Sphroid The term "spheroid" is considered to have the meaning commonly understood in the art as a single cell that divides into either a three-dimensional cell sphere or an aggregation of multiple three-dimensional cells, with or without the use of a matrix or scaffold to support the growth of three-dimensional cells within the spheroid. The three-dimensional spheroid may be an adherent spheroid or a spheroid grown in suspension. Several different systems are available for culturing spheroids for use in the present disclosure, for example, on nano-culture plates, in suspension culture, on gels, on plastics coated with poly-HEMA, by cell encapsulation, or by a hanging drop system, such as spheroids grown as aggregates. Other methods include the use of spinner flasks, rotating systems, concave methods, and liquid overlays. A bioreactor may be adapted for use in three-dimensional spheroid cell culture. In one embodiment, the method used is a hanging drop system, such as the GravityPLUS Hanging Drop System (InSphero). This method involves the use of a GravityTRAP ULA Plate, a non-adherent coated microtiter plate designed for the production of spheroids. The maturation of the spheroid typically occurs within 2 to 5 days from seeding, depending on the cell type and culture conditions. The spheroid is suitably cultured in a volume of 100 μl or more, or 200 μl or more, or 300 μl or more. The spheroid is suitably cultured in a Corning® spheroid microplate.

[0276] 3D cell culture matrices or scaffolds can be used for spheroid culture. These are often porous substrates that can support the growth and differentiation of 3D cells. A variety of materials have been developed, resulting in 3D scaffolds that differ in appearance, porosity, permeability, mechanical properties, and nanoscale surface morphology. Examples of such materials include collagen gels, sponges, or biogels; fibrin; fibronectin; laminin; alginate, hydrogels; cross-linked glycosaminoglycan; polymer-based scaffolds, synthetic scaffolds; peptide scaffolds; and chitosan composite scaffolds.

[0277] 3D spheroids closely resemble in vivo tissues in terms of their intracellular information transmission and extracellular matrix development. These matrices assist in the movement of cells within the spheroid in the same way that cells move in living tissues. Thus, spheroids are greatly improved models for differentiation, survival, cell migration, cell polarization, gene expression, and growth.

[0278] Spheroids can be harvested and studied using a variety of methods well-known in the art, including colorimetric, fluorescence, and luminescence assays measured using a plate reader, or can be easily observed by microscopy. Additional techniques include Western blot, Northern blot, or Southern blot, histological techniques (e.g., immunohistochemistry, in situ hybridization, immunofluorescence), and the like. The use of optical imaging methods - for example, inverted bright-field microscopy, fluorescence microscopy, single-photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging (MRI), and Cherenkov luminescence imaging (CLI) - is also contemplated.

[0279] Uses for the use of three-dimensional spheroids include research on in vitro cell and tissue growth, compound and test atmosphere, toxicity assays, and screening of clinical trials, and the like, in an environment that closely approximates that of in vivo cells and tissues that are known.

[0280] The use of spheroids in three-dimensional cell culture is generally outlined in Expert Opin. Drug Discov.. (2015) 10, 519-540. In vitro, lung spheroid cells can be grown in large numbers, form alveolar-like structures, and acquire a mature lung epithelial phenotype.

[0281] Cell source Lung cells and cell lines for use in the present disclosure can be isolated from tissues or fluids using methods well known in the art. Lung cells and cell lines 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 may be from human or animal subjects, or human or animal cells, or be derived therefrom, and include any of a number of mammalian species, suitably human, but also rats, mice, pigs, rabbits, and non-human primates, and the like. The cells and cell lines can also be obtained from commercial sources. In certain embodiments, the use of human cells is desirable.

[0282] Lung cells, including lung epithelial cells, are the cell type of interest. Epithelial cells of the bronchi and / or airways are particularly useful in the present disclosure. Human bronchial epithelial cells can be obtained by brushing the donor's lung during a bronchoscopy procedure. In one embodiment, the lung cells are normal human bronchial epithelial (NHBE) cells. Lung epithelial cells can be cultured as a monolayer of undifferentiated cells or further differentiated into organotypic lung epithelial-like tissue at the air-liquid interface. Cells can be established at the air-liquid interface using the following methodology. Briefly, epithelial cells are cultured in a flask 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 the apical side and the basal side. 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 way for a further period. 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.

[0283] Lung epithelial cells can be obtained from human or animal subjects with different pathologies, including subjects classified as smokers or non-smokers.

[0284] Microsensor Various microsensors have been developed and described. For example, Routkevitch et al (NSTI-nanotech 2005 ISBN 0-9767954-1-4; Vol.2) provide an overview of a nanostructured gas microsensor platform based on nanostructured alumina ceramics. As another example, Nigam and Shukla (J. Microbiol. Biotechnol. (2015), 25(11) From 1773 to 1781, studies the detection, quantification, and decomposition or conversion of organic and inorganic pollutants in the atmosphere using microbial biosensors. The biosensors include single-celled microorganisms such as bacteria, fungi, and cyanobacteria and enzymes, as outlined in Appl. Biochem. Biotechnol. (2015) 175, 3093 - 3119. Various types of biosensors are available, such as enzyme-based biosensors that can be based on measurements of enzyme inhibition or direct measurements of compounds involved in enzyme reactions. Other examples of biosensors can involve the use of aptamers, molecularly imprinted polymers, biochips or nanotechnology, or combinations thereof.

[0285] Biosensors based on molecularly imprinted polymers can specifically bind to target components of the test aerosol. The target components are present in the test aerosol in known amounts, such as nicotine in tobacco smoke, thereby enabling the calculation of the overall aerosol mass deposition inside the simulated respiratory tract. Alternatively, toxicologically active or physiologically active compounds can be targeted, for example, during environmental monitoring. In particular, when developing toxic aerosol compounds such as polyaromatic hydrocarbons or therapeutic aerosols, pharmacologically active aerosol components, such as corticosteroids like budesonide used in the treatment of asthma, can be targeted.

[0286] Optionally, the microsensor can be arranged, for example, in combination within a perforated structure, or within an artificial trachea, or within a branching channel of a perforated structure. As an example, some or all of the one or more branching channels contain at least one microsensor for monitoring the state within the structure or for gas sampling or gas characterization. As a further example, the one or more branching channels can include one or more openings capable of receiving one or more modules for containing a microsensor for monitoring the state within the structure. As a further example, the microsensor can be arranged at a site where a cell culture is present, such as within one or more exposure plugs, or within one or more probes, or within one or more quartz crystal microbalances, or within one or more holders associated therewith. In any case, of course, the targeted chemical species and the selected microsensor depend on the aerosol being tested.

[0287] Assay The present disclosure can be used for various applications to study the effects of a test atmosphere on an artificial trachea. For example, the present disclosure can be used in in vitro inhalation toxicity studies, investigations of the dynamics of aerosols in the trachea (e.g., deposition and absorption of aerosol particles into a gas cell culture), or investigations of the metabolic activity or transport of a test atmosphere (e.g., aerosol molecules) across the epithelium of the trachea. 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 a medical inhaler on cells of one or more parts of the trachea.

[0288] 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 and / or micro-sensors included in an artificial breathing trachea. The method includes: (a) providing a system as described herein, the system including a cell culture and / or micro-sensor in one or more of the modules; and (b) comparing the cell culture and / or micro-sensor before and / or after exposure to the test atmosphere, wherein a difference in the cell culture and / or micro-sensor before and / or after exposure of the cell to the test atmosphere indicates that the test atmosphere has an effect on the cell culture and / or micro-sensor.

[0289] In an embodiment where the difference in the cell culture and / or micro-sensor is determined after exposure of the cell and / or micro-sensor to the test atmosphere, the cell culture and / or micro-sensor exposed to the test atmosphere can be compared to a cell culture and / or micro-sensor exposed to a control atmosphere, such as a cell culture and / or micro-sensor not exposed to the test atmosphere or an atmosphere that does not include the test atmosphere. According to this embodiment, the difference between the cell culture and / or micro-sensor exposed to the test atmosphere and the cell culture and / or micro-sensor not exposed to the test atmosphere, or the difference between the cell culture and / or micro-sensor exposed to the test atmosphere and the cell culture and / or micro-sensor exposed to a control atmosphere, such as an atmosphere that does not include the test atmosphere, indicates that the test atmosphere has an effect on the cell culture and / or micro-sensor.

[0290] Another aspect relates to a method for conducting a simulation experiment of the interaction between a test atmosphere and a simulated breathing trachea in the system described herein. The method includes: (a) opening a first valve of a first pump and closing a second valve of the first pump to provide a gas containing the test atmosphere to the first pump through a first port; (b) closing the first valve of the first pump, opening the second valve, and closing a valve on the piston plate of the first pump; (c) operating a second pump to draw the test atmosphere into a connection structure and flowing the chamber of the first pump and the connection structure with ambient air; (d) opening the first valve of the first pump towards the ambient air to form a sealed connection between the first port and the second port of the first pump; and (e) after a certain time, using the second pump to move the test atmosphere through the connection structure and through the first valve of the first pump.

[0291] Another aspect relates to a method for determining the effect of a test atmosphere on a simulated breathing trachea in the system described herein. The method includes: (a) opening a first valve of a first pump and closing a second valve of the first pump to provide a gas containing the test atmosphere to the first pump via a first port; (b) closing the first valve of the first pump, opening the second valve, and closing a valve on a piston plate of the first pump; (c) operating a second pump to draw in the test atmosphere through a connection structure and flowing the chamber of the first pump and the connection structure with ambient air; (d) opening the first valve of the first pump towards the ambient air to form a sealed connection between the first port and the second port of the first pump; (e) after a certain time, using the second pump to move the test atmosphere through the connection structure and through the first valve of the first pump. The test atmosphere is in contact with a cell culture and / or a microsensor located in one or more modules located in the first pump, or the connection structure, or the second pump, or a combination of two or more of them. The method further includes the step of determining the effect of the test atmosphere on the cell culture and / or the microsensor, and the difference in the cell culture and / or the microsensor before and / or after exposure to the test atmosphere indicates that the test atmosphere has an effect on the cell culture and / or the microsensor.

[0292] In an embodiment where the difference between the cell culture and / or the microsensor is determined after exposure of the cells to the test atmosphere, the cell culture and / or the microsensor exposed to the test atmosphere can be compared with the cell culture and / or the microsensor exposed to a control atmosphere, such as a cell culture and / or a microsensor not exposed to the test atmosphere or an atmosphere excluding the test atmosphere. According to this embodiment, the difference between the cell culture and / or the microsensor exposed to the test atmosphere and the cell culture and / or the microsensor not exposed to the test atmosphere, or the difference between the cell culture and / or the microsensor exposed to the test atmosphere and the cell culture and / or the microsensor exposed to a control atmosphere, such as an atmosphere excluding the test atmosphere, indicates that the test atmosphere has an effect on the cell culture and / or the microsensor.

[0293] A further aspect relates to a method for performing a simulation experiment of the interaction between a test atmosphere and a simulated trachea. The method includes: (a) providing the test atmosphere to a chamber of a first pump; (b) removing the test atmosphere from the first pump into a connection structure that joins the first pump to a second pump; (c) flowing the first pump and at least a portion of the connection structure with ambient air; (d) holding the test atmosphere in the second pump and the connection structure for a defined time; (e) using the second pump to move the test atmosphere into the connection structure and the first pump; and (f) performing one or more pump cycles of ambient air in the second pump, wherein the test atmosphere contacts a cell culture located in the first pump, or the connection structure, or the second pump, or a combination of two or more of them.

[0294] A further aspect relates to a method of determining the effect of a test atmosphere on an artificial breathing trachea, the method comprising: (a) providing the test atmosphere to a chamber of a first pump; (b) removing the test atmosphere from the first pump into a connection structure that joins the first pump to a second pump; (c) flowing ambient air through the first pump and at least a portion of the connection structure; (d) holding the test atmosphere in the second pump and the connection structure for a defined time; (e) using the second pump to move the test atmosphere through the connection structure and the first pump; (f) performing one or more pump cycles of ambient air in the second pump, wherein the test atmosphere is in contact with a cell culture and / or a microsensor located in one or more modules located in the first pump, or the connection structure, or the second pump, or a combination of two or more thereof, the method further comprising the step of determining the effect of the test atmosphere on the cell culture and / or the microsensor, wherein a difference in the cell culture and / or the microsensor before and / or after exposure to the test atmosphere indicates that the test atmosphere has an effect on the cell culture and / or the microsensor. In embodiments where the difference in the cell culture and / or the microsensor is determined after exposure of the cell and / or the microsensor to the test atmosphere, the cell culture and / or the microsensor exposed to the test atmosphere can be compared to a cell culture and / or a microsensor exposed to a control atmosphere, such as a cell culture and / or a microsensor not exposed to the test atmosphere or an atmosphere that does not contain the test atmosphere. According to this embodiment, the difference between the cell culture and / or the microsensor exposed to the test atmosphere and the cell culture and / or the microsensor not exposed to the test atmosphere, or the difference between the cell culture and / or the microsensor exposed to the test atmosphere and the cell culture and / or the microsensor exposed to a control atmosphere, such as an atmosphere that does not contain the test atmosphere, indicates that the test atmosphere has an effect on the cell culture and / or the microsensor.

[0295] The effect of a 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, permeation barrier function, surfactant production, cytokine response, transporter function, cytochrome P450 expression, albumin secretion, toxicity, and the like.

[0296] Multiple assays may be performed in parallel using different concentrations of the test atmosphere and / or agent to obtain different responses to various concentrations.

[0297] The agent can be any test compound of interest and includes small molecule 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. Test compounds may be screened individually, or as a set of compounds or a combinatorial library. 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 may be made using natural or synthetically produced libraries and compounds modified via conventional chemical, physical, and biochemical means. Known pharmacological agents may be directly or randomly subjected to chemical modifications such as acylation, alkylation, esterification, acidification, etc. to create structural analogs for screening.

[0298] 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 a test atmosphere can be measured. The aerosol dynamics in the respiratory tract (e.g., the deposition and absorption of aerosol particles of a gas into a cell culture) can be measured. As a further example, the metabolic activity and / or transport of molecules across the epithelium of the respiratory tract can be studied.

[0299] Computer Also disclosed is a computer program operable to control a computer so as to perform one or more of the methods described herein when executed by a computer / processor.

[0300] One of ordinary skill in the art will readily recognize that the steps of the various methods described above can be implemented by a programmed computer. In this specification, some embodiments also are directed to a program storage device, such as a machine or computer-readable digital data storage medium, intended to encode a machine-executable program or a computer-executable program of instructions that perform some or all of the steps of the methods described above. The program storage device may be, for example, a digital memory, a magnetic storage medium such as magnetic disks and magnetic tapes, a hard drive, or an optically readable digital data storage medium. Embodiments also are intended to be directed to a programmed computer operable to perform some or all of the steps of the methods described above.

[0301] The functionality of various elements including processors or logic may be provided by not only hardware capable of executing software in conjunction with appropriate software, but also by the use of dedicated hardware. When provided by a processor, the functionality may be provided by a single dedicated processor, by a single shared processor, or by a plurality of individual processors some of which may be shared. Further, the explicit use of the terms "processor", or "controller", or "logic" should not be construed to exclusively refer to hardware capable of executing software, but may implicitly include, without limitation, digital signal processor (DSP) hardware, network processors, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), read only memory (ROM) for storing software, random access memory (RAM), and non-volatile storage devices. Other hardware, both conventional and / or custom, may also be included. Similarly, any switches shown in the figures are conceptual only. Their functionality may be accomplished by the operation of program logic, by dedicated logic, by an interaction of program control and dedicated logic, or even manually, and a particular technique may be selectable by an implementer as more specifically understood from the context.

[0302] Further aspects of the disclosure are described in the following numbered paragraphs.

[0303] 1. A perforated structure for use in an artificial breathing tube, the structure comprising a perforated envelope that houses one or more branch channels, each perforation being an open terminal end of a branch. 2. The perforated structure of paragraph 1, wherein the branches of each channel are forked. 3. The perforated structure of paragraph 1 or paragraph 2, wherein the diameter of each channel that exists after n + 1 consecutive branch points is less than or equal to the diameter of the channel that exists after n branch points. 4. The perforated structure of any one of paragraphs 1 to 3, wherein the total cross-sectional area of all channels that exist after n + 1 consecutive branch points is greater than or equal to the total cross-sectional area of all channels after n branch points. 5. The perforation structure according to any one of paragraphs 1 to 4, wherein the structure is asymmetric. 6. The perforation structure according to any one of paragraphs 1 to 4, wherein the structure has radial symmetry. 7. The perforation structure according to any one of paragraphs 1 to 6, wherein the structure is modular. 8. The perforation structure according to any one of paragraphs 1 to 7, wherein all or part of the structure is formed from one or more materials selected from the group consisting of non-porous materials including metals and non-porous synthetic materials, porous materials including porous silicon, gel-like materials including thermogels, hydrophobic materials, hydrophilic materials, amphiphilic materials, or combinations of two or more thereof. 9. The perforation structure according to any one of paragraphs 1 to 8, wherein one or more of the branched channels further comprise one or more coatings on a part or all of the inner surface of the branched channels. 10. The perforation structure according to paragraph 9, wherein one or more of the coatings provide water and nutrients to cells grown in all or part of one or more of the branched channels. 11. The perforation structure according to any one of paragraphs 1 to 10, wherein a part or all of one or more of the branched channels are coated with a matrix containing a cell culture medium and / or at least one microsensor for monitoring the state within the structure, or a probe for gas sampling or gas characterization. 12. The perforation structure according to any one of paragraphs 1 to 11, wherein one or more of the branched channels further comprise one or more openings capable of receiving one or more modules containing a matrix containing a cell culture medium or a microsensor for monitoring the state within the structure, or a probe for gas sampling or gas characterization. 13. The perforation structure according to paragraph 11 or paragraph 12, wherein the cell culture medium includes one or more hydrogels such as gelatin methacryloyl (GelMa). 14. A pump for moving a volume of gas, comprising one or more branched channels inside the pump, wherein the branched channels are connected to ports for receiving and discharging gas. 15. The pump according to paragraph 14, wherein a branching structure is included within the perforated structure according to any one of paragraphs 1 to 13. 16. The pump according to paragraph 14 or 15, comprising a chamber configured to receive one or more modules that include a gas volume and one or more openings for receiving at least one microsensor for monitoring the state in the base and the matrix containing the cell culture fluid and / or the chamber, or a probe for gas sampling or gas characterization. 17. The pump according to any one of paragraphs 14 to 16, further comprising a motor for controlling the operation of the pump. 18. A system for determining the interaction between a test atmosphere and a simulated respiratory tract, the system comprising: (i) a chamber configured to enclose a first volume of gas containing the test atmosphere; (ii) a first port adapted to receive and discharge gas and having 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 capable of opening towards the test atmosphere or ambient air; (iii) a second port adapted to receive and discharge gas and having 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 within the chamber, the piston plate having one or more gaps for the intake or inflow of gas into the chamber, one or more or each of the gaps having a valve movable between open and closed positions and capable of regulating the intake or inflow of gas; and (v) a motor for controlling the operation of a first pump, (a) the first pump, (b) a second pump as described in any of paragraphs 13 - 16, (c) a connection structure operable to transmit gas from the first pump into the second pump, and (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 thereof, the openings being capable of receiving a module containing a matrix comprising a cell culture fluid and / or at least one microsensor for monitoring the condition within the chamber, or a probe for gas sampling or gas characterization. 19. A method for conducting a simulation experiment of the interaction between a test atmosphere and a simulated respiratory tract, including the use of a pump as described in any of paragraphs 14 - 17 or the system as described in paragraph 18. 20. The use of a pump as described in any of paragraphs 14 - 17 or the system as described in paragraph 18 for conducting a simulation experiment of the interaction between a test atmosphere and a simulated respiratory tract. 21. A method for determining the effect of a test atmosphere on a culture of cells contained within an artificial respiratory tract, including the use of the pump described in any of paragraphs 14 to 17 or the system described in paragraph 18. 22. Use of the pump described in any of paragraphs 14 to 17 or the system described in paragraph 18 for determining the effect of a test atmosphere on a culture of cells contained within an artificial respiratory tract. 23. A method for determining the effect of a test atmosphere on a culture of cells contained within an artificial respiratory tract, comprising: (a) providing a pump as described in any of paragraphs 14 to 17 or a system as described in paragraph 18, the pump or system including a culture of cells and / or at least one microsensor within one or more of the modules; and (b) comparing the culture of cells and / or at least one microsensor before and / or after exposure to the test atmosphere, wherein a difference in the culture of cells and / or at least one microsensor before and / or after exposure of the cells and / or at least one microsensor to the test atmosphere indicates that the test atmosphere has an effect on the culture of cells and / or at least one microsensor. 24. A method for conducting a simulation experiment on the interaction between a test atmosphere and an artificial respiratory tract using the system described in paragraph 18, comprising: (a) opening a first valve of a first pump and closing a second valve of the first pump to provide a gas containing the test atmosphere to the first pump via a first port; (b) closing the first valve of the first pump, opening the second valve, and closing a valve on a piston plate of the first pump; (c) operating a second pump to draw the test atmosphere into a connection structure and flowing the chamber of the first pump and the connection structure with ambient air; (d) opening the first valve of the first pump towards ambient air to form a sealed connection between a first port and a second port of the first pump; and (e) after a certain time, using the second pump to move the test atmosphere through the connection structure and through the first valve of the first pump. 25. A method for determining the effect of a test atmosphere on a simulated breathing trachea in the system described in paragraph 18, the method comprising: (a) opening a first valve of a first pump and closing a second valve of the first pump to provide a gas containing the test atmosphere to the first pump via a first port; (b) closing the first valve of the first pump, opening the second valve, and closing a valve on the piston plate of the first pump; (c) operating a second pump to draw in the test atmosphere through a connection structure and flowing the chamber of the first pump and the connection structure with ambient air; (d) opening the first valve of the first pump towards the ambient air to form a sealed connection between the first port and the second port of the first pump; (e) after a certain time, using the second pump to move the test atmosphere through the connection structure and through the first valve of the first pump, wherein the test atmosphere is in contact with a matrix containing a cell culture and / or at least one microsensor located in one or more modules located in the first pump, or the connection structure, or the second pump, or a combination of two or more of them, the method further comprising the step of determining the effect of the test atmosphere on the cell culture and / or the microsensor, and the difference in the cell culture and / or the microsensor before and / or after exposure to the test atmosphere indicates that the test atmosphere has an effect on the cell culture and / or the microsensor. 26. A method for simulating an interaction between a test atmosphere and a simulated breathing tube, comprising: (a) providing the test atmosphere to a chamber of a first pump; (b) removing the test atmosphere from the first pump into a connection structure that joins the first pump to a second pump, the second pump being the pump described in any one of paragraphs 14 to 17; (c) flowing ambient air through at least a part of the first pump and the connection structure; (d) holding the test atmosphere in the second pump and the connection structure for a defined period of time; (e) using the second pump to move the test atmosphere into the connection structure and the first pump; and (f) performing one or more pump cycles of ambient air in the second pump, wherein the test atmosphere contacts a matrix containing a cell culture and / or at least one microsensor located in the first pump, or the connection structure, or the second pump, or a combination of two or more of them. 27. A method for determining the effect of a test atmosphere on a simulated breathing trachea, comprising: (a) providing the test atmosphere to the chamber of a first pump; (b) removing the test atmosphere from the first pump into a connection structure that joins the first pump to a second pump, wherein the second pump is the pump described in any one of paragraphs 14-17; (c) flowing the first pump and at least a portion of the connection structure with ambient air; (d) holding the test atmosphere in the second pump and the connection structure for a defined period of time; (e) using the second pump to move the test atmosphere through the connection structure and the first pump; (f) performing one or more pump cycles of ambient air in the second pump, wherein the test atmosphere is in contact with a matrix comprising a cell culture and / or at least one microsensor located in one or more modules located in the first pump, or the connection structure, or the second pump, or a combination of two or more of them, and the method further comprises the step of determining the effect of the test atmosphere on the cell culture and / or the microsensor, and the difference in the cell culture before and / or after exposure to the test atmosphere indicates that the test atmosphere has an effect on the cell culture and / or the microsensor. 28. An apparatus configured or adapted to carry out the method according to paragraph 26 or paragraph 27. 29. A method for manufacturing a perforated structure according to any one of paragraphs 1-13, comprising: (i) embedding one or more branched channels, each having an open end portion, in a material suitable for forming a perforated structure; and (ii) removing the cast of the one or more branched channels from the material. 30. The method of paragraph 29, wherein the perforated structure is at least partially manufactured by three-dimensional printing. 31. The method of paragraph 29, wherein the perforated structure is formed by modeling. 32. The method of paragraph 29, wherein the perforated structure is cast from a template. 33. The perforation structure is the method according to any one of paragraphs 29 to 32, which i) substantially mimics the branching structure of the mammalian bronchial airway, ii) is an idealized branching structure, or iii) is a combination of i) and ii).

[0304] An in vitro simulation test of the in vivo airway effect on aerosol properties is disclosed herein for the fields of inhalation toxicology and inhalation therapy and for the evaluation of inhalable consumer products. Further, the present disclosure can function as an experimental model for determining in vivo aerosol dosimetry for the respiratory tract under conditions that are stable, well-characterized, reproducible, and not ethically critical. The airway models described herein function as robust models for studying aerosol mechanics in the respiratory tract or similar complex structures, and thus are identified as valuable for basic research and can therefore contribute to the understanding of the mechanics of the respiratory organs and aerosols in such complex structures.

[0305] All publications cited or described herein provide related information disclosed prior to the filing date of this application. The description herein should not be construed as an admission that the inventors were not entitled to antedate such disclosure. All publications mentioned above 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 present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of 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 perforated structure for use with a simulated breathing tube, said structure comprising a perforated envelope containing one or more perforations and accommodating one or more branched channels, each perforation being an open end portion of said one or more branched channels, one or more of said branched channels being (i) a matrix containing a cell culture fluid on part or all of the inner surface of said branched channel, (ii) a microsensor for monitoring the condition within said perforated structure, (iii) one or more openings capable of receiving one or more modules for a probe for gas sampling or gas characterization, The perforated structure comprising.

2. The perforated structure according to claim 1, wherein the branches of each branched channel are forked.

3. The perforated structure according to claim 1 or claim 2, wherein the diameter of each branched channel existing after n + 1 consecutive branch points is less than or equal to the diameter of the branched channel existing after n branch points.

4. The total cross-sectional area of all branched channels existing after n + 1 consecutive branch points is greater than or equal to the total cross-sectional area of all branched channels after n branch points, preferably, The perforated structure according to any one of claims 1 to 3, wherein the perforated structure is asymmetric or the perforated structure has radial symmetry.

5. The perforated structure is modular, and / or All or part of said perforated structure is formed from one or more materials selected from the group consisting of non-porous materials including metals and non-porous synthetic materials, porous materials including porous silicon, gel-like materials including thermogels, hydrophobic materials, hydrophilic materials, amphiphilic materials, or combinations of two or more thereof. The perforated structure according to any one of claims 1 to 4.

6. The perforated structure according to any one of claims 1 to 5, wherein said cell culture fluid provides water and nutrients to cells grown in all or part of one or more of said branched channels.

7. The perforated structure according to any one of claims 1 to 6, wherein said one or more openings comprise said one or more modules for a probe for gas sampling or gas characterization.

8. A pump for moving a volume of gas, comprising: (i) a port for receiving and discharging gas; and (ii) one or more branched channels of the perforated structure according to any one of claims 1 to 7 inside the pump, wherein the one or more branched channels of the perforated structure are connected to the port.

9. The pump is configured to enclose a volume of gas and comprises a chamber with a base, the chamber further comprising one or more openings capable of receiving one or more modules including: (i) a matrix containing a cell culture medium; (ii) at least one microsensor for monitoring the state inside the chamber; or (iii) a probe for gas sampling or gas characterization. The pump according to claim 8, further comprising a motor for controlling the operation of the pump.

10. A system for determining the interaction between a test atmosphere and a simulated trachea, comprising: (a) A first pump, comprising: (i) a chamber configured to enclose a first volume of gas containing a test atmosphere; (ii) a first port adapted to receive and discharge gas and comprising a valve for regulating the flow of the gas through the first port, the valve being movable between an open position and a closed position, and in the open position the valve being capable of opening towards the test atmosphere or ambient air; (iii) a second port adapted to receive and discharge gas and comprising a valve for regulating the flow of the gas through the second port, the valve being movable between open and closed positions; (iv) a piston plate inside the chamber, the piston plate comprising one or more gaps for the intake or inflow of gas into the chamber, one or more or each of the gaps being movable between open and closed positions and capable of regulating the intake or inflow of the gas, the piston plate including a valve; (v) a motor for controlling the operation of the first pump. (b) A second pump, wherein the second pump is defined in claim 8 or 9; (c) A connection structure operable to transfer the 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 thereof, wherein the opening(s) can receive a module containing a matrix comprising a cell culture medium, a module containing at least one microsensor for monitoring the state in the chamber, or a module containing a probe for gas sampling or gas characterization; a system comprising the one or more openings.

11. A method for conducting a simulation experiment of the interaction between a test atmosphere and a simulated trachea, comprising: (a) providing the test atmosphere to the chamber of a first pump; (b) drawing the test atmosphere from the first pump into a connection structure that joins the first pump to a second pump as claimed in claim 8 or 9; (c) flowing ambient air through at least a part of the first pump and the connection structure; (d) holding the test atmosphere in the second pump and the connection structure for a predetermined time interval; (e) using the second pump to move the test atmosphere to the connection structure and the first pump; (f) performing one or more pump cycles of the ambient air with the second pump, such that the test atmosphere comes into contact with a cell culture disposed in the first pump, the connection structure, the second pump, or a combination of two or more thereof; A method comprising the above steps.

12. A method for determining the effect of a test atmosphere on a cell culture contained within a simulated trachea, comprising: (a) providing the pump as claimed in claim 9 or the system as claimed in claim 10, wherein the pump or system contains a cell culture within one or more of the modules; (b) comparing the cell culture before and / or after exposure of the cells to the test atmosphere, wherein a difference in the cell culture before and / or after exposure of the cells to the test atmosphere indicates that the test atmosphere has an effect on the cell culture; a method comprising the above comparing step.

13. A method for determining the effect of a test atmosphere on a cell culture contained within a simulated trachea, comprising: (a) providing the pump according to claim 9 or the system according to claim 10, wherein the pump or system comprises at least one microsensor in one or more of the modules; (b) comparing the at least one microsensor before and / or after exposure of the at least one microsensor to the test atmosphere, wherein a difference in the at least one microsensor before and / or after exposure of the at least one microsensor to the test atmosphere indicates that the test atmosphere has an effect on the at least one microsensor; A method comprising the above steps.

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