Perforating structure

Abstract

Described herein is a perforated structure for a simulated respiratory tract, the structure comprising a perforated envelope housing one or more branch channels, where each perforation is an open end of a branch. Also described is a pump for discharging a volume of gas, the pump comprising one or more branch channels, such as the perforated structures described herein. The use of the pump in a system for determining an interaction between a test atmosphere and a simulated respiratory tract, as well as a method and use of such a system, is also described.

Description

[0001] This divisional application is based on the Chinese patent application with application number 202080007268.1, application date January 3, 2020, and invention name “Perforated Structure”. This Chinese patent application is the Chinese national phase of the international application with international application number PCT / EP2020 / 050725. Technical Field

[0002] The present disclosure particularly relates to devices, systems, methods and uses related to the structure and / or function of simulated respiratory tract and its respiratory behavior. The present disclosure can be used to study the interaction between test agents (e.g., test inhalable agents in the atmosphere) and respiratory tract and aerosol dynamics and evolution. Specifically, the present disclosure relates to simulated human respiratory tract. Background Art

[0003] The respiratory system travels from the nose and upper airways to the alveolar surface of the lungs, where gas exchange occurs. Inhaled aerosols move from the mouth through the upper airways and eventually to the alveoli. As the aerosol moves deeper into the respiratory tract, more soluble gases are absorbed, and some aerosol particles can be deposited deep within the airways and alveoli. A particular aerosol of interest in the context of the present disclosure is smoke, such as tobacco smoke, because the effects of smoke on a simulated respiratory tract can be studied or the effects of smoke passing through a simulated respiratory tract can be studied.

[0004] Existing aerosol exposure systems most often rely on continuous unidirectional aerosol flow or passive sedimentation. The continuous aerosol flow is generated by positive or negative pressure, and the aerosol is directed vertically toward the biological test system or streamed in a direction parallel to the plane of the biological test system. In this exposure mode, the dose delivery efficiency is mainly a function of the geometry of the exposure chamber and the flow rate of the test aerosol. However, the current aerosol exposure system cannot simulate the dynamic flow patterns (i.e., inhalation and exhalation) that occur during oral cavity, breath holding time and human breathing. Therefore, it is unlikely to achieve dose delivery representative of the in vivo situation, especially the relative delivery of particles and gas aerosol components. In addition, the filtering action of the conducting airway cannot be simulated in a representative manner. Therefore, in the current aerosol exposure system, aerosols with the same aerosol particle size distribution are used, for example, with bronchial cell cultures and nasal cultures. However, in the organism, larger aerosol particles mainly interact with and are removed from the aerosol in the upper respiratory tract, while deeper areas of the respiratory tract are mainly exposed to smaller aerosol particle sizes and gas components. Furthermore, in continuous flow exposure systems, aerosol dilution is achieved by continuously adding dilution air to the aerosol upstream near the exposure chamber. However, if one is to simulate smoking behavior in, for example, an organism, this dilution pattern is not representative of the organism, as it does not take into account the oral retention period, during which the high-density aerosol is allowed to age for up to several seconds before being inhaled into the respiratory tract along with a large amount of dilution gas.

[0005] In passive aerosol sedimentation, a test aerosol is injected into a chamber at the base of the biological test system. Once the aerosol is injected, the aerosol is usually settled on the test system by gravity. Electrostatic attraction of aerosol particles to the test system can be used to improve aerosol particle deposition. In particular, nano-sized aerosol particles may require electrostatic attraction because they have low sedimentation efficiency. Exposure to complex aerosols containing aerosol particles of various sizes and densities as well as gas components cannot be performed in a passive aerosol sedimentation system because the differential relative delivery of large aerosol particles, small aerosol particles, and gas aerosol components does not represent the processes that occur in an organism.

[0006] There is a continuing need in the art for improved simulation systems for studying the respiratory tract, particularly simulation systems for studying the respiratory tract that include complex airway models. Summary of the invention

[0007] Disclosed herein is a structure for simulating the effects of an inhaled aerosol from, for example, a conducting airway tree of a human or animal. It allows the number of airway branching generations to be increased to a more physiologically relevant level without simultaneously creating an unmanageable number of connections. Different versions of the airway model can be used without affecting the overall functionality of the system and without requiring any modifications to other parts of the simulated respiratory system. It can be used with a system simulating the respiratory tract to allow analysis or testing of, for example, the effects of any kind of aerosol on an in vitro biological system (e.g., application in an in vitro aerosol exposure system); investigating how the fundamental properties of any kind of aerosol (e.g., particle size distribution, particle concentration, or chemical composition, or a combination of two or more thereof) are altered in the airway tree; or studying how aerosols are deposited on surfaces representing the human respiratory epithelium in the context of aerosol dosimetry.

[0008] In a first aspect, a perforated structure for a simulated respiratory tract is provided, the structure comprising a perforated envelope containing one or more branching channels, wherein each perforation is an open end of a branch.

[0009] In another aspect, a perforated structure for a simulated respiratory tract is provided, the structure comprising a perforated envelope containing one or more branch channels, wherein each perforation is an open end of the one or more branch channels.

[0010] Suitably, the branches of each channel are bipartite.

[0011] Suitably, the diameter of each channel present after n+1 consecutive branching points is less than or equal to said diameter of said channel present after n branching points.

[0012] Suitably, the total cross-sectional area of ​​all channels present after n+1 consecutive branching points is greater than or equal to the total cross-sectional area of ​​all channels after n branching points.

[0013] Suitably, the structure is asymmetric or has radial symmetry.

[0014] Suitably, the structure is modular.

[0015] Suitably, all or part of the structure is formed from one or more materials selected from the following: non-porous materials, including metals and non-porous synthetic materials; porous materials, including porous silicones; colloidal materials, including thermogels; hydrophobic materials; hydrophilic materials; amphiphilic materials or a combination of two or more thereof.

[0016] Suitably, one or more of the branch channels further comprises one or more coatings on part or all of the inner surface of the branch channels, for example a coating that provides water and nutrients to cells growing in all or part of one or more of the branch channels.

[0017] Suitably, part or all of one or more of the branch channels is coated with a matrix comprising a cell culture medium or comprises at least one microsensor for monitoring conditions in the structure or a probe for gas sampling or for gas characterisation.

[0018] Suitably, one or more of the branch channels further comprise one or more openings capable of receiving one or more modules for containing a matrix comprising a cell culture medium and / or at least one microsensor for monitoring conditions in the structure or for gas sampling or for gas characterisation.

[0019] Suitably, the cell culture medium comprises one or more hydrogels - for example gelatin methacryloyl (GelMa).

[0020] In another aspect, a pump for discharging a volume of gas is provided, the pump comprising one or more branch channels inside the pump, wherein the branch channels are connected to a port for receiving and outputting the gas.

[0021] In another aspect, a pump for discharging a volume of gas is provided, the pump comprising a port and one or more branch channels inside the pump, wherein the one or more branch channels are connected to the port for receiving and outputting the gas.

[0022] In another aspect, a pump for discharging a volume of gas is provided, the pump comprising an outer portion and an inner portion, a port and one or more branch channels inside the pump, wherein the one or more branch channels are connected to the port for receiving and outputting the gas.

[0023] Suitably, the branching structure is comprised in the perforated structure according to the present disclosure.

[0024] Suitably, the one or more branch channels are comprised in the perforated structure according to the present disclosure.

[0025] Suitably, the branching structure replicates an airway model.

[0026] Suitably, the pump comprises a chamber configured for containing said volume of gas and comprising a base and one or more openings capable of receiving one or more modules for containing a matrix comprising a cell culture medium and / or at least one microsensor for monitoring conditions in the chamber or a probe for gas sampling or for gas characterisation.

[0027] Suitably, the pump further comprises a motor for controlling operation of the pump.Suitably, the pump is a piston pump.

[0028] Suitably, the module is located in a base of the chamber.

[0029] Suitably, the module is threaded or non-threaded.

[0030] Suitably, the module is adapted to contain or is configured to store a matrix comprising a cell culture medium or is adapted to monitor conditions in the chamber or is adapted to monitor conditions in the chamber or is adapted to sample gases or is adapted to characterize gases. Alternatively or additionally, the module is adapted to contain or is configured to store at least one microsensor or probe, the at least one microsensor or probe being adapted to monitor conditions in the chamber or is adapted to monitor conditions in the chamber or is adapted to sample gases or is adapted to characterize gases.

[0031] Suitably, the one or more modules comprise a matrix comprising a cell culture medium. Suitably, the cell culture medium comprises one or more hydrogels, such as methacrylated gelatin (GelMa). Alternatively or additionally, the one or more modules comprise at least one microsensor.

[0032] Suitably, the matrix comprising a cell culture medium comprises or contacts a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0033] Suitably, said module adapted to contain or store cell culture medium and / or at least one microsensor further comprises a microfluidic channel and optionally a microfluidic pump connected thereto.

[0034] Suitably, the module comprises or is adapted to comprise one or more microsensors.

[0035] Suitably, the module comprises a quartz crystal microbalance.

[0036] Suitably, a connection structure is connected to said port.

[0037] Suitably, the connection structure is hollow.

[0038] Suitably, the pump further comprises a motor for controlling operation of the pump.

[0039] Suitably, the pumping pressure corresponds to atmospheric pressure or to pressure above or below atmospheric pressure.

[0040] Suitably, the pump has a displacement of between about 0 and 1000 ml, or between about 1 and about 100 ml.

[0041] Suitably, the pump comprises stainless steel.

[0042] Suitably, the chamber is a cylinder.

[0043] Suitably, the chamber comprises glass.

[0044] Suitably, the volume of the chamber represents the volume of a lung cavity or a part thereof.

[0045] In another aspect, a system for determining the interaction between a test atmosphere and a simulated respiratory tract is provided, the system comprising: (a) a first pump comprising: (i) a chamber configured to contain a first volume of gas, the first volume of gas comprising the test atmosphere; (ii) a first port adapted to receive and output gas and comprising a valve for regulating gas flow through the first port, the valve being movable between an open position and a closed position, wherein in the open position the valve is open to the test atmosphere or ambient air; (iii) a second port adapted to output and receive gas and comprising a valve for regulating gas flow through the second port, the valve being movable between an open position and a closed position; and (iv) a piston plate in the chamber, the piston plate comprising one or more orifices for ingesting or flowing gas into the chamber, wherein the orifices One or more or each of the ports comprises a valve movable between an open position and a closed position and capable of regulating the intake or inflow of gas; and (v) a motor for controlling the operation of the first pump; (b) a second pump as described herein; (c) a connecting structure operable to transfer the gas from the first pump to the second pump; and (d) one or more openings located in a wall of the first pump or the second pump or the connecting structure or a combination of two or more thereof, the openings being capable of receiving a module for containing a matrix comprising a cell culture medium and / or at least one microsensor, or configured to store a matrix comprising a cell culture medium and / or at least one microsensor for monitoring conditions in the chamber, or a probe suitable for detecting conditions in the chamber or for gas sampling, or suitable for sampling gas or for gas characterization or suitable for characterizing gas.

[0046] Suitably, the pump is a piston pump comprising a piston plate and a base.

[0047] Suitably, the connection structure is hollow.

[0048] Suitably, the connection structure is branched.

[0049] Suitably, one terminal branch of the connection structure is connected to the second pump and one or more other terminal branches are connected to an independent pump, each independent pump comprising: (i) a chamber containing a certain volume of gas, wherein the volume of gas is the same as the volume of the second volume of gas in the second pump; (ii) a port for receiving and outputting gas and for connecting to the connection structure; and (iii) a motor for controlling the operation of the pump.

[0050] Suitably, each independent pump is identical to said second pump.

[0051] Suitably, the system is contained in an enclosure, suitably a temperature controlled enclosure.

[0052] Suitably, the temperature of the housing is controlled by a thermostat.

[0053] Suitably, the temperature in the housing is approximately 37°C.

[0054] Suitably, the different volumes of the first and second pumps represent the internal volumes of different compartments of the airway, suitably the human airway.

[0055] Suitably, the certain volumes of the independent pumps are representative of the internal volumes of different compartments of the airway, suitably the human airway.

[0056] Suitably, the displacement of the first and second pumps is at least as large as the maximum achievable volume uptake in the respective compartments of the respiratory tract.

[0057] Suitably, the displacement of the independent pumps is at least as great as the maximum achievable volume uptake in the respective compartment of the respiratory tract.

[0058] Suitably, the pumping pressure of the motor or pump may correspond to atmospheric pressure or be above or below atmospheric pressure.

[0059] Suitably, the first pump has a displacement of between about 0 and 100 ml, or between about 1 and 100 ml.

[0060] Suitably, the second pump has a displacement of between about 0 and 4000 ml, or between about 1 and about 4000 ml.

[0061] Suitably, the pump comprises stainless steel.

[0062] Suitably, the chamber is a cylinder.

[0063] Suitably, the chamber comprises glass.

[0064] Suitably, the volume of the chamber of the first pump is smaller than the volume of the chamber of the second pump.

[0065] Suitably, the volume of the chamber of the first pump is representative of the volume of the oral and oropharyngeal cavities.

[0066] Suitably, the volume of the chamber of the second pump is representative of the volume of a lung cavity or a part thereof.

[0067] Suitably, the volume of the connected structure represents the volume of the conducting airways of a lung, suitably a human lung.

[0068] Suitably, the system further comprises a computer controller capable of synchronising the operation of the system.

[0069] Suitably, one or more of the first pump or the second pump or the connection arrangement comprises one or more modules comprising a quartz crystal microbalance.

[0070] Suitably, the opening in the wall of the first pump or the second pump or the connecting structure is threaded or unthreaded.

[0071] Suitably, one or more of said openings contains a module.

[0072] Suitably, the one or more modules are adapted to contain or be configured to store a matrix comprising a cell culture medium or are adapted to monitor conditions in the chamber or are adapted to monitor conditions in the chamber or are adapted to sample gases or are adapted to characterize gases. Suitably, the cell culture medium comprises one or more hydrogels - for example gelatin methacrylate (GelMa). Alternatively or in addition, the one or more modules are adapted to contain or be configured to store at least one microsensor for monitoring conditions in the chamber or are adapted to monitor conditions in the chamber or are adapted to sample gases or are adapted to characterize gases.

[0073] Suitably, the module is located on the base of a piston plate of the first and / or second pump and / or in a wall of the connecting structure.

[0074] Suitably, the one or more modules comprise a matrix comprising a cell culture medium. Suitably, the cell culture medium comprises one or more hydrogels - for example gelatin methacrylate (GelMa). Alternatively or in addition, one or more modules comprise at least one microsensor.

[0075] Suitably, the matrix comprising a cell culture medium comprises or contacts a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0076] Suitably, said module adapted to contain or store a matrix comprising a cell culture medium and / or at least one microsensor further comprises a microfluidic channel and optionally a microfluidic pump connected thereto.

[0077] Suitably, the module is located in a horizontal plane in one or more of the first pump or the second pump or a wall of the connection structure.

[0078] Suitably, the connection structure comprises stainless steel.

[0079] Suitably, the chamber of the first pump has a volume of approximately 100 ml.

[0080] Suitably, the chamber of the second pump has a volume of about 1 litre to about 4 litres.

[0081] A pump for displacing a volume of gas is also described, the pump comprising: (i) a chamber configured to contain a volume of gas and comprising a base and one or more openings, the one or more openings being capable of receiving one or more modules for containing or storing a matrix including a cell culture medium and / or at least one microsensor for monitoring conditions in the chamber or a probe for gas sampling or for gas characterization; (ii) a first port for receiving and outputting the gas when contained in the chamber and comprising a first valve for regulating gas flow through the first port, the first valve being switchable between an open position and a closed position. (iii) a second port for outputting and receiving the gas when contained in the chamber and comprising a second valve for regulating the flow of gas through the second port, the valve being movable between an open position and a closed position; and (iv) a piston plate in the chamber, the piston plate comprising one or more orifices for taking in or flowing gas into the chamber, wherein one or more or each of the orifices comprises a valve, the valve being movable between an open position and a closed position and being capable of regulating the intake or inflow of gas.

[0082] Suitably, the pump is a piston pump.

[0083] Suitably, the one or more openings in the chamber are threaded or unthreaded.

[0084] Suitably, the one or more openings comprise a module.

[0085] Suitably, the module is threaded or non-threaded.

[0086] Suitably, the one or more modules are adapted to contain a matrix comprising a cell culture medium and / or at least one microsensor, or are configured to store a matrix comprising a cell culture medium and / or at least one microsensor, or monitor conditions in the chamber, or are adapted to monitor conditions in the chamber, or are adapted to sample gases or are adapted to characterize gases.

[0087] Suitably, the module is located on a base of the pump.

[0088] Suitably, the one or modules comprises a matrix comprising a cell culture medium. Suitably, the cell culture medium comprises one or more hydrogels - for example gelatin methacrylate (GelMa). Alternatively or in addition, one or more modules comprises at least one microsensor.

[0089] Suitably, the matrix comprising a cell culture medium comprises or contacts a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0090] Suitably, said module adapted to contain or store a matrix comprising a cell culture medium and / or at least one microsensor further comprises a microfluidic channel and optionally a microfluidic pump connected thereto.

[0091] Suitably, the module is located in a horizontal plane in one or more of the first pump or the second pump or a wall of the connection structure.

[0092] Suitably, the module comprises a quartz crystal microbalance.

[0093] Suitably, the pump further comprises a motor.

[0094] Suitably, the pumping pressure corresponds to atmospheric pressure or to pressure above or below atmospheric pressure.

[0095] Suitably, the pump has a displacement of between about 0 and 100 ml, or between about 1 and about 100 ml.

[0096] Suitably, the pump comprises stainless steel.

[0097] Suitably, the chamber is a cylinder.

[0098] Suitably, the chamber comprises glass.

[0099] Suitably, the chamber of the pump has a volume of approximately 100 ml.

[0100] A piston pump for displacing a volume of gas is also described, the piston pump comprising: (i) a chamber configured to contain a volume of gas and comprising a piston plate comprising one or more orifices for ingesting or flowing gas into the chamber, wherein one or more or each of the orifices comprises a valve movable between an open position and a closed position and capable of regulating the ingestion or ingestion of gas; (ii) a first port for receiving gas and comprising a first valve for regulating gas flow through the first port, the first valve being movable between an open position and a closed position, and (iii) a second port for discharging gas when contained in the chamber and comprising a second valve for regulating gas flow through the second port, the valve being movable between an open position and a closed position. Suitably, the chamber comprises a base and comprises one or more openings.

[0101] Suitably, the opening is threaded or unthreaded.

[0102] Suitably, said openings comprise a module in one or more of said openings.

[0103] Suitably, the module is threaded or non-threaded.

[0104] Suitably, the module is adapted to contain or store a matrix comprising a cell culture medium and / or at least one microsensor for monitoring conditions in the chamber or a probe for gas sampling or for gas characterisation.

[0105] Suitably, one or more modules comprise a matrix comprising a cell culture medium. Suitably, the cell culture medium comprises one or more hydrogels - for example methacrylated gelatin (GelMa). Alternatively or in addition, one or more modules comprise at least one microsensor.

[0106] Suitably, the matrix comprising a cell culture medium comprises a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0107] Suitably, said module adapted to contain or store a matrix comprising a cell culture medium and / or at least one microsensor further comprises a microfluidic channel and optionally a microfluidic pump connected thereto.

[0108] Suitably, the module comprises a quartz crystal microbalance.

[0109] Suitably, a connection structure is connected to said second port.

[0110] Suitably, the connection structure is hollow.

[0111] Suitably, the pump further comprises a motor.

[0112] Suitably, the pumping pressure of the pump corresponds to atmospheric pressure or is above or below atmospheric pressure.

[0113] Suitably, the pump has a displacement of between about 0 and 100 ml, or between about 1 and about 100 ml.

[0114] Suitably, the pump comprises stainless steel.

[0115] Suitably, the chamber is a cylinder.

[0116] Suitably, the chamber comprises glass.

[0117] Suitably, the chamber of the pump has a volume of approximately 100 ml.

[0118] In another aspect, a method for simulating the interaction between a test atmosphere and a simulated respiratory tract is provided, comprising using the system described herein.

[0119] Also disclosed is the use of a pump or system as described herein for simulating the interaction between a test atmosphere and a simulated respiratory tract.

[0120] Also disclosed is a method for determining the effect of a test atmosphere on a cell culture contained in a matrix or at least one microsensor in a simulated respiratory tract comprising using the pump or system described herein.

[0121] Also disclosed is the use of a pump or system as described herein for determining the effect of a test atmosphere on a cell culture contained in a matrix or at least one microsensor in a simulated respiratory tract.

[0122] In another aspect, a method for determining the effect of a test atmosphere on a cell culture in a matrix or at least one microsensor contained in a simulated respiratory tract is disclosed, the method comprising: (a) providing a pump and system as described herein, wherein the pump and system comprise a matrix comprising a cell culture or at least one microsensor in one or more of the modules; and (b) comparing the matrix comprising a cell culture or at least one microsensor before and / or after exposure to the test atmosphere, wherein a difference between the matrix comprising a cell culture and / or at least one microsensor before and / or after exposure of the cells to the test atmosphere indicates that the test atmosphere affects the cell culture or at least one microsensor.

[0123] On the other hand, a method for simulating the interaction between a test atmosphere and a simulated respiratory tract in the system described herein is provided, the method comprising: (a) providing a gas including a test atmosphere to the first pump through the first port when the first valve of the first pump is open and the second valve of the first pump is closed; (b) closing the first valve of the first pump and opening the second valve of the first pump, and closing the valve on the piston plate of the first pump; (c) operating the second pump to draw the test atmosphere into the connecting structure and fill the chamber of the first pump and the connecting structure with ambient air; (d) opening the first valve of the first pump to the ambient air and forming a sealed connection between the first port and the second port of the first pump; and (e) after a period of time, using the second pump to discharge the test atmosphere through the connecting structure and through the first valve of the first pump.

[0124] Also disclosed is a method for determining the effect of a test atmosphere on a simulated respiratory tract in the system described herein, the method comprising: (a) supplying a gas comprising a test atmosphere to the first pump via the first port with the first valve of the first pump open and the second valve of the first pump closed; (b) closing the first valve of the first pump and opening the second valve of the first pump, and closing the valve on the piston plate of the first pump; (c) operating the second pump to draw in the test atmosphere through the connecting structure and flush the chamber of the first pump and the connecting structure with ambient air; (d) opening the first valve of the first pump to the ambient air and and (e) after a period of time, exhausting the test atmosphere through the connecting structure and through the first valve of the first pump using the second pump; wherein the test atmosphere contacts a matrix including a cell culture or at least one microsensor located in one or more modules, the one or more modules being located in the first pump or the connecting structure or the second pump or a combination of two or more thereof, and the method includes another step of determining an effect of the test atmosphere on the cell culture and / or the microsensor, wherein differences in the cell culture and / or the sensor before and / or after exposure to the test atmosphere indicate that the test atmosphere affects the cell culture and / or the microsensor.

[0125] Suitably, the module is adapted for monitoring system conditions and / or for gas sampling and / or for gas characterisation, and the method comprises acquiring one or more measurements from the module.

[0126] Also disclosed is a method for simulating the interaction between a test atmosphere and a simulated respiratory tract, comprising: (a) providing the test atmosphere to a chamber of a first pump; (b) pumping the test atmosphere from the first pump back into a connecting structure, the connecting structure connecting the first pump to a second pump; (c) filling at least a portion of the first pump and the connecting structure with ambient air; (d) maintaining the test atmosphere in the second pump and the connecting structure for a defined time period; (e) exhausting the test atmosphere into the connecting structure and the first pump using the second pump; and (f) performing one or more pumping cycles of the ambient air in the second pump; wherein the test atmosphere contacts a cell culture located in the first pump or the connecting structure or the second pump or a combination of two or more thereof.

[0127] Suitably, step (d) comprises maintaining the test atmosphere in the second pump and the portion of the connection structure still containing the test atmosphere for a defined period of time.

[0128] Suitably, the pump is a piston pump comprising a piston plate and a base.

[0129] Suitably, the first pump is as defined in the embodiments of the present disclosure.

[0130] Suitably, the second pump is as defined in an embodiment of the present disclosure.

[0131] Suitably, the connection structure is as defined in the embodiments of the present disclosure.

[0132] Suitably, the method is carried out in an enclosure, suitably a temperature controlled enclosure.

[0133] Suitably, the temperature of the housing is controlled by a thermostat.

[0134] Suitably, the temperature in the housing is approximately 37°C.

[0135] Suitably, the different volumes of the first and second pumps represent the internal volumes of different compartments of the airway, suitably the human airway.

[0136] Suitably, the displacement of the first and second pumps is at least as large as the maximum achievable volume uptake in the respective compartments of the respiratory tract.

[0137] Suitably, the pumping pressure corresponds to atmospheric pressure or to pressure above or below atmospheric pressure.

[0138] Suitably, the first pump has a displacement of between about 0 and 100 ml, or between about 1 and about 100 ml.

[0139] Suitably, the second pump has a displacement of between about 0 and 4000 ml, or between about 1 and about 4000 ml.

[0140] Suitably, the volume of the chamber of the first pump is smaller than the volume of the chamber of the second pump.

[0141] Suitably, the volume of the chamber of the first pump is representative of the volume of the oral and oropharyngeal cavities, suitably of a human.

[0142] Suitably, the volume of the chamber of the second pump represents the volume of a lung cavity or part thereof (suitably a human lung cavity or part thereof).

[0143] Suitably, the connected structure represents the volume of a conducting airway of a lung, suitably a human lung.

[0144] Suitably, the matrix comprising the cell culture and / or the at least one microsensor is located on the base of the piston plate of the first pump and / or the second pump and / or in the wall of the connecting structure.

[0145] Suitably, the cell culture is a two-dimensional or three-dimensional culture.

[0146] Suitably, the method further comprises using one or more modules comprised therein for monitoring conditions and / or for gas sampling and / or for gas characterisation in one or more of the first pump or the second pump or the connection arrangement.

[0147] Suitably, the chamber comprising the matrix containing the cell culture and / or the at least one microsensor further comprises a microfluidic channel and optionally a microfluidic pump connected thereto.

[0148] Suitably, the connection structure comprises stainless steel.

[0149] Suitably, the chamber of the first pump has a volume of approximately 100 ml.

[0150] Suitably, the chamber of the second pump has a volume of about 1 litre to about 4 litres.

[0151] In another aspect, a method for determining the effect of a test atmosphere on a simulated respiratory tract is described, the method comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) pumping the test atmosphere from the first pump back into a connecting structure, the connecting structure connecting the first pump to a second pump; (c) filling at least a portion of the first pump and the connecting structure with ambient air; (d) maintaining the test atmosphere in the second pump and the connecting structure for a defined period of time; (e) exhausting the test atmosphere through the connecting structure and the first pump using the second pump; and (f) performing one or more pumping cycles of the ambient air in the second pump; wherein the test atmosphere contacts a matrix including a cell culture and / or at least one microsensor located in one or more modules, the one or more modules being located in the first pump or the connecting structure or the second pump or a combination of two or more thereof, and the method comprises another step of determining the effect of the test atmosphere on the cell culture and / or microsensor, wherein differences in the cell culture and / or microsensor before and / or after exposure to the test atmosphere indicate that the test atmosphere affects the cell culture and / or microsensor.

[0152] Suitably, the module is adapted for monitoring system conditions and / or for gas sampling and / or for gas characterisation, and the method comprises acquiring one or more measurements from the module.

[0153] A connection structure suitable for connecting at least two pumps for transferring gas therebetween comprises a hollow passage and one or more threaded or non-threaded openings in a wall of the connection structure.

[0154] Suitably, the threaded opening comprises a threaded module in one or more of the openings, the module being suitable for containing cell culture medium or being configured to store cell culture medium or being suitable for monitoring conditions in the chamber, or being suitable for monitoring conditions in the chamber, or being suitable for sampling gases or being suitable for characterising gases.

[0155] Suitably, the one or more modules comprise a matrix comprising a cell culture medium and / or one or more microsensors. Suitably, the cell culture medium comprises one or more hydrogels - for example gelatin methacrylate (GelMa). Alternatively or in addition, one or more modules comprise at least one microsensor.

[0156] Suitably, the cell culture medium comprises or contacts a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0157] Suitably, said module adapted to contain or store cell culture medium further comprises a microfluidic channel and optionally a microfluidic pump connected thereto.

[0158] Suitably, the connection structure is hollow.

[0159] Suitably, the connection structure is branched.

[0160] Suitably, each terminating branch of the connection structure is connectable to a separate pump.

[0161] Suitably, the connected structure represents the volume of a conducting airway of a lung, suitably a human lung.

[0162] Suitably, the modules are located in a horizontal plane in a wall of the connecting structure.

[0163] Suitably, the module is adapted to contain a matrix comprising a cell culture, suitably a two-dimensional or three-dimensional cell culture.Alternatively or additionally, the module is adapted to contain at least one microsensor.

[0164] Suitably, the module is for containing a matrix comprising a cell culture and / or at least one microsensor, the chamber comprising a microfluidic channel and optionally a microfluidic pump connected thereto.

[0165] Suitably, the module is adapted for monitoring conditions in the connection structure and / or for gas sampling and / or for gas characterisation.

[0166] Suitably, the connection structure comprises stainless steel.

[0167] Also described are systems comprising the pumps described herein.

[0168] Suitably, the system further comprises a connection structure as described herein.

[0169] Suitably, said pump is connected via said connection structure.

[0170] Also disclosed is a device configured or adapted to perform the method described herein.

[0171] Also disclosed is a method of making a perforated structure as described herein.

[0172] Suitably, the perforated structure is at least partly manufactured by 3-dimensional printing.

[0173] Suitably, the perforated structure is formed by moulding.

[0174] Suitably, the perforated structure is cast from a formwork.

[0175] Suitably, the perforated structure i) substantially mimics the branching structure of a mammalian bronchial airway, ii) is an idealised branching structure, or iii) is a combination of i) and ii).

[0176] Another aspect relates to a plurality of silicon branch channels within the pump, wherein the branch channels are connected to ports for receiving and outputting the gas.

[0177] Suitably, the branching structure is comprised in the perforated structure according to the present disclosure.

[0178] Suitably, the branching structure replicates an airway model. BRIEF DESCRIPTION OF THE DRAWINGS

[0179] Figure 1 is a diagram of a perforated structure 1 for simulating a respiratory tract according to an embodiment of the present disclosure, wherein Figure 1 A is a schematic representation of the base of the chamber in the simulated respiratory tract, and Figure 1 B is a schematic representation of a cross section along section XX.

[0180] Figure 2 yes Figure 1 Schematic representation of a substrate of a chamber in a simulated respiratory tract, showing the modular construction of the perforated structure 1 according to an embodiment of the present disclosure.

[0181] Figure 3A is a schematic representation of a perforated structure 1 for simulating a respiratory tract according to an embodiment of the present disclosure, wherein Figure 3B is an enlarged illustration of a location widening of an exposed chamber into which a module 113, 213 according to an embodiment of the present disclosure may be inserted.

[0182] Figure 4 is a diagram of a system 10 according to an embodiment of the present disclosure. A first pump 40 according to an embodiment of the present disclosure is also disclosed. A second pump 80 according to an embodiment of the present disclosure is also disclosed. A connection structure 50 according to an embodiment of the present disclosure is also disclosed.

[0183] Figure 5 and Figure 6 is a diagrammatic representation of modules 113, 213 according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0184] Some advantages

[0185] The present disclosure can be used to simulate in vitro the effects of the in vivo airway on aerosol properties relevant to fields such as inhalation toxicology and inhalation therapy, thereby enabling the evaluation of inhalable consumer products.

[0186] The present disclosure can provide an experimental model for determining in vivo aerosol dosimetry of the respiratory tract under stable, well-characterized, reproducible and ethical non-critical conditions. As an airway model, the perforated structure of the present invention can allow the airway branching to increase to physiologically relevant levels without simultaneously creating an unmanageable number of connections, such as between the bronchial model and a second pump.

[0187] Different versions of the airway model can be used without affecting the overall functionality of the system and without requiring any modifications to other parts of the simulated respiratory system. This maximizes the flexibility of studying how the airway affects aerosols or how aerosols affect the airway. Because the airway i) has a relevant influence on the properties of aerosols reaching the alveolar space and ii) shows considerable inter-individual variability (also referring to diseased vs. healthy states), the flexibility and adaptability of this specific part of the airway is of great scientific interest.

[0188] Since the processes that affect the physicochemical properties of the test atmosphere in the respiratory tract and the way the two interact (eg, dose delivery) are modeled by the physical and functional properties of the system, clinically relevant dosing information can be obtained according to embodiments of the present disclosure.

[0189] According to embodiments of the present disclosure, the effects of one or more test atmospheres on one or more compartments of the respiratory tract may be studied.

[0190] According to embodiments of the present disclosure, the effects of one or more test atmospheres on one or more compartments of the respiratory tract may be studied simultaneously or in stages as desired.

[0191] According to embodiments of the present disclosure, modules that may be used in the system provide flexibility with respect to the test system exposed, the experimental endpoints, and the exposure parameters to be monitored.

[0192] The modules may be redesigned or changed according to specific requirements without changing the overall structure and function of the system according to the embodiments of the present disclosure.

[0193] For many applications, aerosol generation is driven by the system itself, which means that an aerosol generator / smoking machine is not required according to embodiments of the present disclosure. This can help to simplify the structure of the system.

[0194] In certain embodiments, the system may be modular in nature. This means that, for example, various components, such as pumps and connections, may be individually and easily exchanged as needed. Partial redesign, modification, or replacement of parts of the system according to specific requirements may be facilitated.

[0195] According to embodiments of the present disclosure, the oral cavity, breath hold times, and dynamic flow patterns occurring during breathing may be simulated.

[0196] According to certain embodiments, the present disclosure may contemplate an oral retention period during which a high density aerosol may be allowed to age for up to several seconds before being inhaled into the respiratory tract along with a large volume of dilution air. Specific embodiments

[0198] In certain embodiments, the practice of the present disclosure employs conventional techniques of engineering, microbiology, cell biology, and biochemistry. Biological techniques are fully explained in, for example, the following literature: Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE CeIMs, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, IB. Griffiths and DG Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., ed., 1987); PCR: The Polymerase Chain Reaction: A Review of Previous Studies (2001). Reaction, (Mullis et al., eds., 1994). Unless otherwise indicated, procedures employing commercially available kits and reagents will generally be used according to the manufacturer's established protocols.

[0199] Technical terms and expressions used herein are generally given the meanings commonly applied to them in the relevant art. All term definitions used herein apply to the entire content of this application.

[0200] The term "comprising" does not exclude other elements or steps.

[0201] The indefinite article "a" or "an" does not exclude a plurality.

[0202] For example, the term "and / or" means (a) or (b) or both (a) and (b).

[0203] As used herein, the term "comprising" is synonymous with "including" or "containing", and is inclusive or open-ended and does not exclude other unrecited members, elements, or method steps. The term "consisting of" means excluding other components and having only the recited elements and no other elements.

[0204] As used herein, when referring to measurable values ​​such as parameters, amounts, time durations, etc., the term "about" is meant to cover the specified value and variations therefrom, especially the specified value and variations therefrom of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less, as long as these variations are suitable for implementation in the present disclosure. It should be understood that the value to which the modifier "about" refers is itself also explicitly and preferably disclosed.

[0205] Before discussing the embodiments in more detail, an overview will first be provided. The embodiments provide apparatus and methods for studying the respiratory tract that can be used in a variety of applications. For example, the embodiments have utility in studying the deposition and / or condensation of one or more components present in a test atmosphere on the interior surfaces of the apparatus, including re-evaporation thereof. The embodiments also provide for evaluation of the test atmosphere, which can be investigated during its passage through the apparatus in order to study changes in aerosol concentration and / or aerosol particle growth and / or aerosol particle shrinkage. The effects of the test atmosphere on biological test systems present within the apparatus / system can be studied in embodiments of the present disclosure.

[0206] Airway Model

[0207] In one aspect, a perforated structure 1 for a simulated respiratory system 10 is described, the structure comprising a perforated envelope accommodating one or more branch channels, wherein each perforation is an open end of a branch. In use, each perforation can be an open end of a branch. As shown in the accompanying drawings, each perforation is an empty perforation.

[0208] Figure 1 A simulated respiratory system 10 including a perforated structure 1 is shown according to an embodiment of the present disclosure.

[0209] The perforated structure 1 may have a substantially solid body and an opening 2, said body and opening comprising fittings suitable for providing an airtight and stable connection at a relevant part of the laboratory equipment in which the structure is used. A suitable system is described below and shown in Figure 4 , wherein the structure 1 fits into the port 83 for receiving and outputting gas. In use, the opening 2 is located on the lower surface of the structure and is centrally fitted on the base plate 84 by simple blocking, using a clamping mechanism or by screw locking. Tightness of connection can be established by a seal. The pump 80 can represent the lung volume of the hollow structure representing the most proximal intrathoracic airways, namely the trachea and main bronchi (see Figure 4 ).

[0210] The body of the structure 1 accommodates one or more branch channels 4. The outer surface of the structure 1 is perforated, and each perforation 6 is an open end of a branch 4. Inside the perforated structure 1, the channels originating from the proximal opening 2 branch continuously. The angles between the channels 4 originating from a given branch point, their diameters and lengths, and the number of branch points are thus designed to be structurally similar to a branching tree of an airway tree, such as a human or animal airway tree. The last generation of channels 4, i.e., the distal openings or perforations 6 of the structure representing the small airways, are open to the surroundings. In the case of the system 10 described herein, the internal volume of the second pump 80. The structure 1 thereby connects a model of the proximal airways (e.g., trachea and main bronchi) in the system 10 with a model of the lung cavity, and provides a branching structure that separates two regions, for example, in the human respiratory tract.

[0211] The branching pattern of channels 4 within structure 1 can strictly follow (i) the branching pattern of an intact animal airway; (ii) an idealized version of an intact animal airway (i.e., with increased symmetry and regularity); or (iii) either or a portion of both, i.e., reproducing only a portion of a human or animal airway, the idealization requiring computational or experimental testing of equivalence to a human or animal airway with respect to how structure 1 affects aerosol properties.

[0212] The radial symmetry of structure 1 can be achieved by idealizing the branching pattern of the airway or by symmetrically arranging multiple copies of a portion of the airway.

[0213] A structure 1 that strictly adheres to the branching pattern of the human or animal airway can be based on an airway cast or on a digital 3D model obtained using tomography or similar techniques. A model with an idealized human or animal branching pattern can be based on relevant literature, for example, Weibel's model is a widely accepted model of human airway structure (see Fishman's Pulmonary Diseases and Disorders (2015) ed. MA Grippi, ISBN: 0071807284)), or can be newly developed by computational or experimental methods.

[0214] According to Weibel's model, the key parameters that can be considered include: the diameter of the 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; 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; and the branch points are bipartite.

[0215] The number of branching generations will depend on the size of the structure 1, the material from which it is made, and the capabilities of existing technologies for additive manufacturing. The greater the number of generations, the closer the simulation will be to the airway effects in a living organism.

[0216] The airway structure 1 can consist of a single piece of material containing the complete airway tree, or of subsections 1a, 1b that can be assembled according to experimental requirements (see Figure 2 ). The connection 7 between the sub-parts 1a, 1b needs to be airtight and can be achieved, for example, by an insertion mechanism or by thread locking.

[0217] The overall shape and size of the airway module depends on its intended application. In the case of an airway structure to be used in the system 10 described below, radial symmetry is generally required to uniformly deliver the aerosol to the second pump volume. For example, if the distribution of the cell culture in the exposure chamber is radially symmetric, radial symmetry is required.

[0218] Radial symmetry can be omitted in certain cases, for example if only one culture is present or if the culture distribution follows another symmetry; equivalence of replicate cell cultures exposed in the same experiment is not required; aerosol deposition that depends on symmetry is of concern (e.g., in the field of aerosol research); or the overall system is asymmetric.

[0219] For example, in a 3D rendition of a human lung, asymmetry would be desired due to the inherent biological asymmetry of the respiratory tract.

[0220] The spatial dimensions of Structure 1 depend on i) the type of airway that the structure is supposed to represent, as these airways can vary greatly in size (e.g., humans versus rodents), and ii) the simulated respiratory system with which Structure 1 is to be used, as the structure must fit into the relevant part of the system.

[0221] In the case of the application of the system 10 described herein, the maximum size in the horizontal direction (diameter of the module, see Figure 4 ) can be defined by the position of the cell culture module 113, 213 (described below) in the pump 80 (described below), since the diameter of the structure 1 cannot be larger than the diameter of the circle on which the cell culture module 113, 213 is located, since otherwise the module 113, 213 would be covered by the structure 1. The maximum size of the module in the vertical direction (its height, size in the direction of the radial symmetry axis, see Figure 4 ) is generally defined by the minimum 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 that achieves a corresponding volume of approximately 600 mL in each pump defines the maximum height of the structure 1.

[0222] The airway structure 1 can be designed on a computer, taking into account the relevant literature on the available digital 3D geometry or its ideal geometry of human or animal lung morphology or human or animal respiratory organs. Using the designed 3D geometry, the structure 1 can then be generated by 3-D printing to form an airway model. The airway structure 1 is typically an artificial or synthetic airway structure to form an artificial or synthetic airway model. Alternatively, an available airway cast or 3D airway model can be used as a physical template by embedding them in a material suitable for forming the structure 1. In the case where the airway cast is composed of a material with a melting point lower than the material selected for the airway structure 1, the airway cast can be removed by using enough solvent to dissolve the airway cast, using enough enzyme digestion or by heating. Examples of methods for designing and generating airway models are described by Robinson 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).

[0223] Therefore, another aspect relates to a method of manufacturing a perforated structure, the method comprising: (i) embedding one or more branch channels in a material suitable for forming the perforated structure, each branch channel comprising an open end; and (ii) removing a casting of the one or more branch channels from the material. Suitably, the perforated structure is at least partially manufactured by 3D printing. Suitably, the perforated structure is formed by molding. Suitably, the perforated structure is cast from a template. Suitably, the perforated structure i) substantially mimics the branching structure of a mammalian bronchial airway, ii) is an idealized branching structure, or iii) is a combination of i) and ii). Suitably, in the case where the airway cast is composed of a material having a melting point lower than the material selected for the airway structure, the airway cast is removed by using sufficient solvent to dissolve the airway cast, using sufficient enzyme digestion or by heating.

[0224] The airway structure 1 may be manufactured from any material capable of forming a complex structure by one of the above techniques - for example in metals, ceramics or composite materials, such as additive manufacturing or embedding of airway castings. The exact type of material depends on the intended application.

[0225] For example, if the effect of the airway model on the size distribution and concentration of the non-volatile fraction of solid particles and / or liquid particles in the test aerosol is studied, a non-porous material can be used. Examples include metals (e.g., stainless steel), non-porous synthetic materials (e.g., polyethylene terephthalate (PET) or polyetheretherketone (PEEK) or a combination of two or more thereof). As a further example, if the effect of the airway model on the size distribution and concentration of the volatile fraction of solid particles and / or liquid particles and volatile compounds in the test aerosol is studied, a porous material can be used. Volatile compounds generally do not condense on non-porous surfaces, but may enter the pores of porous materials and adhere there due to the large available surface (the mode of action of activated carbon). An example is a porous silicone (e.g., polydimethylsiloxane (PDMS)). As a further example, if the effect of the airway model on the size distribution and concentration of the volatile fraction of solid particles and / or liquid particles and volatile compounds in the test aerosol is studied, or if cells are cultured on the inner surface of the airway model, a colloidal material can be used. In this case, the colloidal material can be soaked with cell culture medium, or the cell culture medium can be included as a substrate in the additive manufacturing process. Examples include thermogels, such as poly (vinyl methyl ether) (PVME) or poly (N-vinyl caprolactam) (PNVC) or a combination of two or more thereof. As another example, if the interaction between the hydrophobic compound in the aerosol and the airway model is to be enhanced (e.g., PDMS), a hydrophobic material can be used. As another example, if the interaction between the hydrophilic compound in the aerosol and the airway model is to be enhanced, a hydrophilic material can be used. Examples include hydrogels, such as PVME or PNVC or a combination thereof. As another example, if the interaction between the hydrophobic compound and the hydrophobic compound in the aerosol and the airway model is to be enhanced, an amphiphilic material can be used. Amphiphilicity can be achieved, for example, by coating the surface with a surfactant. In addition, the hydrophilicity, hydrophobicity, and absorption properties of the airway structure 1 can be adjusted by coating the inner channel surface with a suitable material (e.g., a hydrogel, wax, or protein, or a combination of two or more thereof). For example, a coating with a cell culture medium comprising a hydrogel can be used to provide a surface on which cells can be cultured. The possibility of exposing the cell culture to a test aerosol passing through the airway model can be provided by including an exposure chamber 8 inside the structure (see FIG. 3 ). Such an exposure chamber 8 can be formed, for example, by a local widening of the channel 4 in one dimension (perpendicular to the channel axis). The widening matches the size and shape of one or more (e.g. all) of the available cell culture modules 113, 213, which can be cultured on a small volume of a nutrient-providing hydrogel or in a microfluidic structure.The cell culture and hydrogel may be present at the end of an “exposure plug” 9 which may be inserted into the airway structure 1 , thereby introducing the cell culture into the exposure chamber 8 , whereby the surface of the cell culture is in the plane of the inner surface of the exposure chamber 8 .

[0226] A similar approach can be used if the probe or sensor is inserted into the airway channel 4, for example, by coating all or part of the channels 4 throughout the airway structure 1 with a hydrogel that delivers sufficient water and nutrients to cells grown directly in the channels 4 of the structure 1. This approach typically requires that the airway structure 1 be composed of or include a highly porous material capable of storing large amounts of culture medium, or by forming the airway structure 1 from a gelatinous material that provides nutrients and water.

[0227] Thus, in another aspect, one or more (e.g., multiple) branch channels replicating an airway model are disclosed, the airway model comprising a cell culture medium coating on at least part or all of the branch channels to provide a surface on which cells can be cultured. Suitably, the coating comprises cells. In another aspect, one or more (e.g., multiple) branch channels replicating an airway model are disclosed, the airway model comprising one or more widenings in the channel comprising one or more matrices for culturing cells. Suitably, the matrices comprise cells.

[0228] system

[0229] On the other hand, a system 10 for determining the interaction between a test atmosphere and a simulated respiratory tract is described, the system comprising: (a) a first pump, the first pump comprising: (i) a chamber, the chamber being configured to contain a first volume of gas including the test atmosphere; (ii) a first port, the first port being suitable for receiving and outputting gas and comprising a valve for regulating gas flow through the first port, the valve being movable between an open position and a closed position, wherein in the open position, the valve is open to the test atmosphere or ambient air; (iii) a second port, the second port being suitable for outputting and receiving gas and comprising a valve for regulating gas flow through the second port, the valve being movable between an open position and a closed position; and (iv) a motor for controlling operation of the first pump; and (b) a second pump as described herein, the second pump comprising: (i) a chamber, the chamber being configured to contain a second volume of gas, wherein the first port a volume of gas that is different from a second volume of gas; (ii) a port adapted to receive and output gas; (iii) a piston plate in the chamber, the piston plate comprising one or more orifices for ingesting or flowing gas into the chamber, wherein one or more or each of the orifices comprises a valve movable between an open position and a closed position and capable of regulating the ingestion or inflow of gas; and (iv) a motor for controlling operation of the second pump; (c) a connecting structure operable to transfer gas from the first pump to the second pump; and (d) one or more openings located in a wall of the first pump or the second pump or the connecting structure or a combination of two or more thereof, the opening being capable of receiving a module for containing or storing a matrix comprising a cell culture medium and / or for monitoring conditions in the chamber or for gas sampling or for gas characterization.

[0230] The gas may be, or may include, the test atmosphere.

[0231] Figure 4A system 10 according to an embodiment of the present disclosure is shown. The system 10 includes at least two pumps 40, 80. Two or more pumps 40, 80 are connected to each other. In certain embodiments, the two or more pumps 40, 80 are connected to each other by a branching hollow structure 50. Each pump 40, 80 can be operated by its own separate motor 41, 81, or two or more pumps can be operated by the same motor 41, 81 as needed. The entire system 10 can be located in a climate housing 11 equipped with a thermostat 12 to control the temperature in the housing 11. The chambers of the pumps 40, 80 can be configured to represent the internal volumes of different compartments of the respiratory tract (e.g., the respiratory tract of a human or animal). They can be configured to provide a displacement at least as large as the maximum achievable volume intake in the corresponding compartment of the respiratory tract. Specifically, one (first) pump 40 can represent the volume of the oral cavity and oropharyngeal cavity (e.g., the oral cavity and oropharyngeal cavity of a human or animal). Another second pump 80 can represent the volume of a lung cavity or a portion thereof (such as the lumen of an individual lung lobe or a smaller subunit), in particular a lung cavity or a portion thereof of a human or animal. The branched hollow connection structure 50 can represent the size of a conducting airway, in particular a conducting airway of a human or animal, such as one or more of the nasopharyngeal cavity, hypopharynx, larynx, trachea, bronchi, and bronchiolar structures that reach the respiratory bronchioles. The branched hollow connection structure 50 can represent the size of a conducting airway, including the nasopharyngeal cavity, hypopharynx, larynx, trachea, bronchi, and bronchiolar structures that reach the respiratory bronchioles. The sizes (e.g., diameters and lengths) of the different sub-portions of the connection structure 50 and the branching pattern can resemble a tree of conducting airways.

[0232] like Figure 4 As shown, the branching hollow structure 50 may be connected to a central opening 43, 83 in the base 44, 84 of the chamber 42, 82 of each pump 40, 80. In certain embodiments, a plurality of openings, holes or sockets 51 may be present on the base 44, 84 of the chamber 42, 82, which may be arranged symmetrically around the central opening 43, 83. A valve 44a directly at the connection between the pump 40 and the connecting structure 50 may be used to allow the pump 40 to be sealed from all other system parts.

[0233] The pump 40 representing the oral cavity may have one or more openings 43 through which the test atmosphere and dilution air can leave the pump 40 toward the branch 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, appropriately located at its center. It can run through the hollow piston axis 46, and a valve 44a, such as a three-way valve, can be present on the top of the hollow piston axis. In an embodiment, the valve 44a can be closed or opened toward the test atmosphere source or ambient air. An array of one or more (e.g., multiple) orifices 47 through which ambient air can enter the system is optionally arranged on the piston plate 45 in a radial arrangement. One or more valves 48 (e.g., multiple) on one or more or all of the orifices can be used to allow one or more of these orifices 47 to be opened or closed. In certain embodiments, each orifice 47 is controlled by a valve 48. In certain embodiments, an array of one or more (e.g., multiple) orifices through which ambient air can enter the system can optionally be arranged in a radial arrangement on the piston plate 84 of the second pump. One or more valves (e.g., multiple) can be used to allow one or more of these orifices to be opened or closed. In certain embodiments, each orifice is controlled by a valve. In certain embodiments, an array of one or more orifices (e.g., multiple) through which ambient air can enter the system can be optionally arranged in a radial arrangement on the piston plates of the first pump and the second pump. Advantageously, the branch hollow structure 50 can be disconnected from the pump 40, 80. Advantageously, the branch hollow structure 50 can be decomposed into its main parts. This can allow easy access to replace or remove the test system and / or for cleaning.

[0234] The base 44, 84 of the pump 40, 80 may be removed for replacement / removal of the test system and for cleaning.

[0235] In the base 44, 84 of each pump 40, 80, and in the various parts of the connection structure 50, 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, may be located therein. The openings, holes or sockets 51 may be located at various locations, such as on the base 44, 84 of one or more of the pumps 40, 80, or may be arranged around the central opening 43, 83 or at various selected locations in the branching hollow structure 50, appropriately on the underside of the branching hollow structure 50, or any combination thereof.

[0236] The opening, hole or socket 51 can be used to allow various modules 112, 212 or devices to be installed therein or thereon, which can be used to monitor the operation of the system 10 and / or conduct experiments and / or collect samples, etc. Examples of such modules 112, 212 or devices are shown in Figure 5 and Figure 6 and described in this article.

[0237] Advantageously, therefore, the pumps 40, 80 used in the system 10 can be used not only to transport the test atmosphere, but can also serve as an exposure chamber.

[0238] Figure 5 and Figure 6 An embodiment of the present disclosure is shown in which the opening, hole or socket 51 is an optional form of a threaded opening, hole or socket 112, 212. One or more openings, holes or sockets 51 may contain one or more modules 112, 212. The use of threads facilitates direct insertion and replacement of modules 112, 212. The use of threads in the opening, hole or socket 51 is optional because the modules can be configured so that they can be inserted or pushed into the opening, hole or socket 51 to seal with it. These modules can be push-fit. Fastening can be achieved by using O-rings, etc.

[0239] The modules 112, 212 used can be adapted to various purposes according to the requirements of the configured system. For example, the modules 112, 212 can be suitable for accommodating or storing cell culture media, or for monitoring conditions in the chambers 42, 82, or for gas or liquid sampling or for gas characterization, etc. 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 in the wall of the connecting structure 50. In a specific embodiment, one or more modules 112, 212 can be configured to contain or store a matrix including a cell culture medium. According to this embodiment, one or more modules 112, 212 can be a container capable of holding a liquid or solution. The cell culture medium can include or can contact a cell culture, such as a two-dimensional or three-dimensional cell culture. In certain embodiments, one or more modules 113, 213 can hold or position at least one microsensor as a substitute or supplement for the cell culture medium matrix. In certain embodiments, the modules 113, 213 suitable for containing or storing a matrix including a cell culture medium and / or at least one microsensor also include a microfluidic channel and a microfluidic pump optionally connected thereto. The modules 113 , 213 will generally be located in a level plane with one or more of the first pump 40 or the second pump 80 or a wall of the connecting structure 50 .

[0240] like Figure 5 and Figure 6As can be seen in the figure, the opening, hole or socket 51 can be optionally configured to include a threaded hole or socket 112, 212. The threaded hole or socket 113, 213 can include one or more probes 114, 214 of one or more devices 116, 216. Such devices 116, 216 can be used to monitor internal system conditions or for testing atmospheric characterization or for sampling, etc. The operation of the device 116, 216 can be controlled by a computer 117, 217. The threaded hole or socket 113, 213 and / or the module 112, 212 can be suitable for use as a culture chamber 115, 215, in which a biological test system (e.g., an organ cell culture of human respiratory epithelium as described herein) can be placed for testing atmospheric exposure. The threaded opening, hole or socket 113, 213 can be suitable for accommodating a capture agent, in which the test atmosphere can be sampled for analysis. Sampling of the cell culture medium or the trapping agent during the test atmosphere exposure can be achieved by various methods, including by using a microfluidic pump system 211 and / or one or more microsensors. A quartz crystal microbalance (QCM, 219) can be held using a module 112, 212. The module 112, 212 on which the probe 214, chamber 215 or QCM 219 can be mounted can be inserted into any chamber 42, 82 or interior of the branched hollow structure 50 of the system. The operation of the QCM 219 can be controlled by a computer 220. The system 10 can be fully or partially controlled by a computer 13 as desired. The system 10 can be partially or fully automated.

[0241] The system 10 may include one or more (eg, a plurality of) first pumps. The system 10 may include one or more (eg, a plurality of) second pumps. The system 10 may include one or more (eg, a plurality of) first pumps and one or more second pumps.

[0242] Pump

[0243] In another aspect, a pump (suitably a piston pump) for discharging a volume of gas is disclosed, the pump comprising one or more branch channels inside the pump, wherein the branch channels are connected to a port for receiving and outputting the gas. The pump may have an external portion and an internal portion. One or more branch channels may be inside the pump. The port may be located on the pump. The pump may include a port. The port may be located inside or outside the pump. Suitably, the branch channel is contained in a perforated structure as described herein. Suitably, the branch structure replicates an airway model. Suitably, the pump comprises a chamber configured to contain a volume of gas and comprising a base and one or more openings, the one or more openings being capable of receiving one or more modules for containing a matrix including a cell culture medium and / or for monitoring conditions in the chamber or for gas sampling or at least one microsensor for gas characterization. Suitably, the pump also comprises a motor for controlling the operation of the pump. As described above, the branch structure complies with the branching pattern of the airway of a human or animal and may be based on an airway cast or on a digital 3D model obtained using tomography or similar techniques. The pump may substantially correspond to the second pump described below.

[0244] Also contemplated is a system for determining an interaction between a test atmosphere and a simulated airway that includes a pump. Also contemplated is the use of a pump in a system for determining an interaction between a test atmosphere and a simulated airway.

[0245] A method of culturing cells in a pump is also contemplated. Also disclosed is the use of a pump for culturing cells.

[0246] First Pump

[0247] In another aspect, a pump for displacing a volume of gas is disclosed, suitably a piston pump, the pump comprising: (i) a chamber configured to contain a volume of gas and comprising a base and one or more openings capable of receiving a module for containing or storing cell culture medium or for monitoring conditions in the chamber or for gas sampling or for gas characterization; (ii) a first port, when contained in the chamber, for receiving and outputting gas and comprising a first valve for regulating gas flow through the first port, the first valve being movable between an open position and a closed position, wherein in the open position the valve is open to a test atmosphere or ambient air; (iii) a second port, when contained in the chamber, for outputting and receiving gas and comprising a second valve for regulating gas flow through the second port, the valve being movable between an open position and a closed position, and (iv) a piston plate in the chamber, the piston plate comprising one or more orifices for ingesting or flowing gas into the chamber, wherein one or more or each of the orifices comprises a valve movable between an open position and a closed position and capable of regulating the ingestion or inflow of gas.

[0248] Also disclosed is a piston pump for discharging a certain volume of gas, the piston pump comprising: (i) a chamber, the chamber being configured to contain a certain volume of gas and comprising a piston plate, the piston plate comprising one or more orifices for taking in or flowing gas into the chamber, wherein one or more or each of the orifices comprises a valve, the valve being movable between an open position and a closed position and being capable of regulating the intake or inflow of gas; (ii) a first port, the first port being used to receive gas and comprising a first valve for regulating gas flow through the first port, the first valve being movable between an open position and a closed position, and (iii) a second port, the second port being used to output gas when contained in the chamber and comprising a second valve for regulating gas flow through the second port, the valve being movable between an open position and a closed position.

[0249] like Figure 4 As shown, the system 10 can include a first pump 40 for displacing 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.

[0250] The first pump may be a primary pump, so called because its location in the system is the entry point for the gas. The first pump includes a chamber 42 (e.g., a cylinder) configured to contain a volume of gas and includes a base 44 and a housing capable of receiving a module (e.g., Figure 5 and Figure 6, 213) in a threaded or non-threaded module 113, 213 as shown in the hole or socket 112, 212 and as described herein. The first pump also includes a first port 90 for receiving and outputting gas when contained in the chamber 42, and includes a first valve 44a, such as a three-way valve, for regulating the flow of gas through the first port 90, and the first valve 44a is movable between an open position and a closed position, wherein in the open position, the valve 44a can be opened to the test atmosphere or ambient air. The first pump also includes a second port 43 for outputting and receiving gas when contained in the chamber 42. Suitably, the second port 43 is configured as an opening. At the connection between the first pump 40 and the connecting structure 50, a second valve 49 at the position of the second port 43 allows the pump 40 to be sealed from other system parts. The second valve 49 can be used to regulate the flow of gas through the second port 43, and the second valve 49 is movable between an open position and a closed position. As Figure 4 As can be seen in the figure, the pump 40 can be a piston pump including 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 include modules 113, 213, for example, threaded or non-threaded modules 113, 213 as discussed herein. The pump 40 also includes a motor 41 to control the operation of the pump. The pumping pressure of the motor 41 can correspond to atmospheric pressure, or can be higher or lower than atmospheric pressure as required. In certain embodiments, the pumping pressure of the motor 41 can be higher or lower than atmospheric pressure to exhaust the test atmosphere. The displacement of the pump 40 can be between about 0 and 100 ml, or between about 1 and about 100 ml. The chamber 42 of the pump 40 can have a volume of up to about 100 ml. The pump 40 can be made of various materials known in the art, such as stainless steel. Suitably, the chamber 42 is a cylinder. Suitably, the chamber 42 can be made of glass. The piston plate 45 of the pump 40 comprises one or more orifices 47 for the intake or inflow of gas. One or more of the orifices 47 may comprise a valve 48 movable between an open position and a closed position and capable of regulating the intake or inflow of gas.

[0251] Also like Figure 4 As shown in , pump 40 can be a piston pump for displacing a volume of gas. The piston pump includes a chamber 42 (e.g., a cylinder, such as a glass cylinder) configured to contain a volume of gas and includes a piston plate 45, which includes one or more orifices 47 for ingesting or flowing gas into chamber 42. One or more of orifices 47 or each of orifices 47 includes a valve 48 for regulating the ingestion or inflow of gas through orifice 47.

[0252] The orifice may 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 movable between an open position and a closed position. It also includes a second port 43 for outputting gas when contained in the chamber 42, and the second port 43 optionally includes a second valve for regulating the flow of gas through the second port 43, which valve is movable between an open position and a closed position. The chamber 42 may include a base 44a and a plurality of openings 43. The base may also include one or more openings, holes or sockets 51, which may be threaded 119 or non-threaded, and / or they may include modules 113, 213, as discussed herein. A connecting structure 50 (such as a hollow connecting structure) may be connected to the second port 43. The pump 40 may also include a motor 41, wherein the pumping pressure corresponds to atmospheric pressure or is higher or lower than atmospheric pressure. The displacement of the pump 40 may be between about 0 and 100 ml, or between about 1 and about 100 ml. The chamber 42 of the pump 40 may have a volume of approximately 100 ml.

[0253] Systems comprising two or more first pumps are contemplated. It is also contemplated to use two or more first pumps in a system to determine the interaction between a test atmosphere and a simulated respiratory tract.

[0254] A method of culturing cells in the first pump is also contemplated. Also disclosed is the use of the first pump for culturing cells.

[0255] Second pump

[0256] In another aspect, a pump is disclosed, suitably a piston pump, comprising: (i) a chamber configured to contain a volume of gas and comprising a base and one or more openings capable of receiving one or more modules for containing a matrix comprising a cell culture medium or for monitoring conditions in the chamber or for gas sampling or at least one microsensor for gas characterization; and (ii) a port for receiving and outputting gas; and (iii) one or more branch channels located in the port. The branch channels may be included in the perforated structure described herein.

[0257] like Figure 4 As shown, the simulated respiratory system 10 described below can include a second pump 80 for displacing a volume of gas, the second pump comprising a chamber 82 (such as a cylinder) configured to contain a volume of gas, the chamber 82 comprising a base 84 and one or more modules 113, 213 for containing or storing a matrix including a cell culture medium and / or for monitoring conditions in the chamber 82 or for gas sampling or for gas characterization, etc. The pump 80 can be a secondary pump.

[0258] 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.

[0259] The pump 80 also includes a port 83 operable to receive and output gas into which a perforated structure 1 as described herein may be installed.

[0260] like Figure 4 As shown, pump 80 can be a piston pump including a piston plate 83. Piston plate 83 can be without any orifices or openings. Modules 113, 213 as discussed herein, such as threaded or non-threaded modules 113, 213, can be located in the base 84 of chamber 82. A connection structure 50 (such as a hollow connection structure) can be connected to port 83. Pump 80 also includes a motor 81. The pumping pressure of pump 80 will generally correspond to atmospheric pressure or above or below atmospheric pressure. The displacement of pump 80 can be between about 0 to about 1000 ml, or between about 0 to about 4000 ml, or between about 1 to about 1000 ml or between about 1 to about 4000 ml. The volume of chamber 82 can represent the volume of a lung cavity or a portion thereof. In certain embodiments, an array of one or more (e.g., multiple) orifices through which ambient air enters the system can be optionally arranged on piston plate 84 in a radial arrangement. One or more valves (e.g., multiple) can be used to allow one or more of these orifices to be opened or closed. In certain embodiments, each orifice is controlled by a valve.

[0261] Systems comprising two or more pumps 80 as described herein are contemplated. It is also contemplated that two or more pumps 80 are used in a system to determine the interaction between a test atmosphere and a simulated respiratory tract.

[0262] Also contemplated are methods of culturing cells in a pump 80 as described herein. Also disclosed is the use of a pump 80 for culturing cells.

[0263] Connection structure

[0264] Also disclosed is a connection structure operable to transmit or carry gas between two or more pumps. The connection structure may be a tube or a pipe or a conduit, etc., through which gas can be conducted or transmitted. The connection structure may be suitable for connecting at least two pumps for transmitting or carrying gas therebetween. The connection structure may connect the first pump at the second port of the first pump described herein and the port of the second pump described herein. The connection structure may include a hollow passage and one or more openings in the wall of the connection structure, such as threaded or non-threaded openings. The connection structure can be used in the systems and methods described herein. The system may include a connection structure suitable for connecting at least two pumps for transmitting gas therebetween.

[0265] An embodiment of the connecting structure 50 is shown in Figure 4 In. The connecting structure will typically include a hollow channel. It is typically branched. In an embodiment, each terminating branch of the connecting structure can be connected to an independent pump 40, 80 when it is included in the system. The connecting structure 50 may include one or more openings in the wall of the connecting structure - especially threaded or unthreaded openings. The threaded or unthreaded openings may include modules 112, 212. As discussed herein, modules 112, 212 may be suitable for accommodating culture media and / or monitoring system conditions and / or for gas sampling and / or for gas characterization. In certain embodiments, the connecting structure may branch out into two or more branches. Each terminating branch of the connecting structure may be connected to an independent pump. The connecting structure may represent the volume of the conducting airways of the lungs. The connecting structure 50 may be made of various materials. In certain embodiments, stainless steel is preferably used.

[0266] Also contemplated are methods for culturing cells comprising the connection structure. Also disclosed is the use of the connection structure for culturing cells.

[0267] System Operation and Functionality

[0268] The system can be controlled in whole or in part by a computer as desired. This can allow for the synchronous operation of some or all of the pumps and valves. This can allow for the synchronous operation of some or all of the system elements. The computer can be used to set the stroke length of one or more pumps and / or the stroke speed of one or more pumps. The computer can be used to control the temperature of the system.

[0269] We will now describe Figure 4 Operation of the embodiment of the depicted system 10. In a stationary state, the piston of the first pump 40 is in a downstroke position, the position of the second pump 80 is in a position to maintain a defined volume of gas in the chamber 82, and the valves 48, 49 are closed. Test atmosphere is drawn 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 axis 46. The hollow piston axis 46 can be directly connected to a source of test atmosphere. Once the pump 40 has completed the upstroke, the valve 44a (which can be a three-way valve) is closed at the central opening on the top of the hollow piston axis 46, and the valve 48 regulates the inflow of ambient air through the orifice 47 in the piston plate 45, and the valve 49 is opened at the entrance of the connecting structure 50.

[0270] The test atmosphere may be drawn from the chamber 42, which may represent the oral cavity, through the connecting structure 50, which may represent the conducting airway. This may be driven by the upstroke of the second pump 80, which may represent the lung cavity or a portion thereof. Since the total displacement of the second pump 80 may be a multiple of the volume of the pump 40, the chamber 42 of the pump 40 and at least part of the connecting structure 50 may be filled with ambient air, which may enter the first pump 40 through the orifice 47 in the piston plate 45.

[0271] At the first pump 40, the valve 44a on the top of the piston axis 46 can be opened to the surrounding environment. The valve 48 on the piston plate 45 can be closed and the pump 40 performs a downstroke. In its downstroke position, the pump 40 can form a sealed connection between the hollow piston axis 46 and the opening 43 in the base 44. The sealed connection can be achieved by a gasket 53. The gasket 53 can be located on the base 44 or the piston plate 45. After a defined "breathing hold time", the second pump 80 can then perform a downstroke, thereby directly exhausting the test atmosphere to the surrounding environment through the connecting structure 50 and through the piston axis 46 of the first pump 40. While the pump 40 remains in its downstroke position and the valve 44a remains open to the surrounding environment, the second pump 80 can perform one or more (e.g., several) cycles of breathing ambient air before the valve 44a is opened again to the test atmosphere source and the next inhalation cycle of the test atmosphere begins.

[0272] In another aspect, a method is provided, comprising: (a) providing a pump comprising a chamber, such as a first pump described herein; (b) drawing a gas, such as a test atmosphere, from the pump into a connecting structure connecting the pump to another pump (such as a second pump described herein); (c) filling at least a portion of the pump and the connecting structure provided in step (a) with ambient air; (d) maintaining the gas in the other pump and the connecting structure for a defined period of time; (e) exhausting the gas into the connecting structure and the pump provided in step (a) using the other pump; and (f) performing one or more pumping cycles of the ambient air in the other pump.

[0273] In another aspect, a method is provided that includes: (a) providing a gas, such as a test atmosphere, to a chamber of a pump (such as a first pump described herein); (b) drawing the gas from the pump described in step (a) into a connecting structure that connects the pump to another pump (such as a second pump described herein); (c) filling at least a portion of the pump and the connecting structure of step (a) with ambient air; (d) maintaining the gas in the other pump and the connecting structure for a defined time period; (e) exhausting the gas through the connecting structure and the pump of step (a) using the other pump; and (f) performing one or more pumping cycles of the ambient air in the other pump.

[0274] Test environment generation

[0275] The test atmosphere, such as an aerosol, studied using the disclosed system 10 and method can be generated by various methods. For many applications, such as for testing tobacco products or commonly used medical inhalers, etc., the test atmosphere generation can be driven by the system itself, that is, the primary pump or the secondary pump itself generates the negative pressure required to generate and extract the test aerosol, which means that advantageously, the use of an aerosol generator / smoking machine is not required.

[0276] The test atmosphere can be an ambient sample of gases or aerosols, for example for monitoring indoor air quality, occupational exposure in close proximity to industrial sites, or environmental pollution. In this specific case, the test atmosphere is not generated, but is sampled by the action of the system.

[0277] The test atmosphere may be an aerosol, such as smoke, or may be derived from smoke. As used herein, the term "smoke" is used to describe a type of aerosol produced by smoking articles such as cigarettes or by burning aerosol-forming materials. Smoke includes a variety of agents, which can be provided as individual compounds for study if desired. 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′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanatabine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl, nitric oxide (NO), nitrous oxide (NOx), hydrocyanic acid, ammonia, arsenic, cadmium, chromium, lead, nickel, selenium, and mercury.

[0278] When the aerosol is smoke, the system 10 can be optionally connected to a smoking machine. Suitably, the smoking machine uses a pump provided by the system or a pump of the present disclosure to hold and light a cigarette. A defined number of puffs per cigarette and a defined number of puffs exposed per minute can be used, and the number of cigarettes can be varied to accommodate the exposure time. A reference cigarette, such as reference cigarette 3R4F, can be used as a source of smoke and smoked on a smoking machine that substantially complies with the International Organization for Standardized Smoking Schemes (ISO 2000).

[0279] The use of a control atmosphere, such as an atmosphere that does not contain the test atmosphere, is also contemplated. The use of a control atmosphere can help determine the impact of the test atmosphere compared to the control atmosphere.

[0280] The system 10 can be connected to a smoking machine by a suitable conduit that provides a flow path for the smoke to the system 10. The smoke can be delivered by the conduit with or without a carrier gas (e.g., air). In the case of using a carrier gas, the conduit preferably includes an inlet for introducing the carrier gas into the conduit to mix with the smoke flow. The conduit may include at least one inlet for introducing or injecting a standard reference (e.g., nicotine) into the system 10 for calibration purposes. The smoke flow will typically be controlled by the system or pump of the present disclosure.

[0281] The smoking machine may be a linear or rotary smoking machine. Suitably, the smoking machine is operated to smoke a plurality of smoking articles simultaneously so that cumulative smoke from the plurality of smoking articles can be collected and analysed. Suitable smoking machines for use with the present disclosure are well known to the skilled person.

[0282] The system 10 and methods described herein may be used to analyze mainstream smoke generated by a smoking article during a smoking test."Mainstream smoke" refers to the smoke drawn through a smoking article and inhaled by a consumer during use.

[0283] The test atmosphere may come 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 an aerosol-generating article. The aerosol generating device may include one or more components suitable for generating an aerosol from an aerosol-generating substrate. The aerosol generating device may be an electrically heated aerosol generating device, which is an aerosol generating device comprising a heater that is operated by electricity to heat the aerosol-forming substrate of an aerosol-generating article to generate an aerosol. The aerosol generating device may be a gas-heated aerosol generating device, i.e., a device heated by a carbon-containing heat source, other exothermic chemical reactions, or a heat sink. Other suitable methods of generating aerosols are known in the art. The aerosol generating device may be a device that interacts with the aerosol-forming substrate of an aerosol-generating article to generate an aerosol that can be directly inhaled into the lungs of a user through the user's mouth.

[0284] Another example of an "aerosol generating device" is an inhalation device (inhaler), which is typically used to deliver an aerosol containing an active ingredient (e.g., a medically active compound). Such inhalation devices are typically used to deliver aerosolized medicaments to the respiratory tract. They can be used to treat respiratory and other diseases. Such inhalers are well known in the art and are typically of the pressurized metered type, dry powder type, or nebulizer type. Typically, the medicament is in the form of a pressurized formulation containing fine particles of one or more drug compounds suspended in a liquefied propellant, or a solution of one or more compounds dissolved in a propellant / cosolvent system. Such formulations are well known in the art.

[0285] As used herein, the term 'aerosol-forming substrate' relates to a substrate capable of releasing volatile compounds that can form an aerosol. Such volatile compounds can be released by heating the aerosol-forming substrate. The aerosol-forming substrate can be adsorbed, coated, impregnated or otherwise loaded onto a carrier or support. The aerosol-forming substrate can suitably be part of an aerosol-generating article or a smoking article. In certain applications, the aerosol-forming substrate is included in an aerosol-generating article, such as a rod-shaped aerosol-generating article, such as a heated aerosol-generating article or a heated cigarette. The aerosol-generating article has a suitable size and shape for engagement with an aerosol-generating device so that the aerosol-forming substrate is in contact with the heater.

[0286] The aerosol-forming substrate may include a medically active compound or agent, such as an antibiotic or anti-inflammatory agent that can be delivered to a patient through the respiratory tract.Numerous medical inhalation devices (inhalers) are known and are routinely prescribed for the treatment of a variety of respiratory and non-respiratory related diseases.

[0287] Aerosol-forming substrate may include nicotine. Aerosol-forming substrate may include tobacco. Aerosol-forming substrate may include, for example, tobacco-containing materials containing volatile tobacco flavor compounds, which are released from aerosol-forming substrate when heated. In certain embodiments, aerosol-forming substrate may include homogenized tobacco material, for example, fallen leaf tobacco. As used herein, "homogenized tobacco material" refers to the material formed by agglomerating particle tobacco. Homogenized tobacco may be in the form of flakes. Homogenized tobacco material may have an aerosol-forming agent greater than 5% by dry weight content. Alternatively, homogenized tobacco material may have an aerosol-forming agent between 5% by weight and 30% by weight by dry weight content. The sheet of homogenized tobacco material may be formed by agglomerating particle tobacco, which is obtained by grinding or otherwise pulverizing one or both of tobacco leaf blades and tobacco stems. Alternatively or in addition, the sheet of homogenized tobacco material may include one or more of tobacco dust, broken smoke and other particle tobacco byproducts formed during the processing, operation and transportation of (for example) tobacco. The homogenized tobacco material sheet may contain one or more inherent binders as tobacco endogenous binders, one or more external binders as tobacco exogenous binders, or combinations thereof to help the particulate tobacco agglomerate; alternatively or additionally, the homogenized tobacco material sheet may include other additives, including but not limited to tobacco and non-tobacco fibers, aerosol formers, humectants, plasticizers, flavorings, fillers, aqueous solvents and non-aqueous solvents, and combinations thereof.

[0288] Cell culture

[0289] Cell cultures for the present disclosure include two-dimensional cell cultures and three-dimensional cell cultures. As described herein, in general, cell cultures are contained or cultured in one or more modules of one or more pumps and / or connection structures. Cell cultures can be exposed to a test atmosphere so that the effect of the test atmosphere on the cell culture can be determined. Suitably, two or more cell cultures will be located at different locations around one or more pumps and / or connection structures and / or systems so that the effect of the test atmosphere on the cell cultures at these different locations can be determined-it simulates the respiratory tract. Two-dimensional cell cultures involve growing cells in a flat layer on a plastic surface, which allows several aspects of cell physiology to be studied and responses to stimuli such as test atmospheres, but they cannot reflect the true structure and architecture of organs. In a two-dimensional monolayer, the extracellular matrix, intercellular and cell-matrix interactions are lost, which are essential for differentiation, proliferation and cell function. Three-dimensional culture systems can form functional tissues with features similar to those observed in vivo. Compared with two-dimensional culture systems, three-dimensional cell cultures allow cells to interact with their surroundings in all three dimensions and are more physiologically relevant. Such cells can show improvements in aspects such as viability, proliferation, differentiation, morphology, response to stimulation, drug metabolism, gene expression and protein synthesis, etc. Three-dimensional cell cultures can produce specific tissue-like structures in a more physiologically relevant way than traditional two-dimensional cell monolayers, and simulate the functions and reactions of real tissues. Several three-dimensional tissues simulating human organs are commercially available. Primary human cells grown at the air-liquid interface (ALI) can be used to prepare lung three-dimensional organotypic tissues of particular interest in the context of the present disclosure, where these cells will differentiate and form functional tissues. These three-dimensional tissues have close morphological similarities and metabolic characteristics with human bronchial tissues. They are composed of basal cells, goblet cells and ciliated cells arranged in a pseudostratified structure. Similar to the lung, there are actively swinging cilia, allowing the study of their function and activity. Compared with human lungs, similar levels of mRNA encoding xenobiotic enzymes have been found in these three-dimensional ALI cultures. In addition, these tissues can be maintained for a long time in vitro. This three-dimensional model of lung tissue is an appropriate model for exploring the effects of test atmospheres, etc. according to the present disclosure.

[0290] The term "three-dimensional cell culture" includes any method that provides cell culture 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.

[0291] spheroids

[0292] The term "spheroid" is used as generally understood in the art, which is a single cell that divides into a cell ball in three dimensions, or an aggregate of multiple cells in three dimensions, with or without the use of a matrix or scaffold for support in the three-dimensional cell growth within the spheroid. The three-dimensional spheroid can be an adherent spheroid or a spheroid grown in suspension. Several different systems for culturing spheroids for the present disclosure can be used, including spheroids grown in aggregate form, such as on nanoculture plates, in suspension cultures, on gels, on plastics coated with poly-HEMA via cell encapsulation, or via a hanging drop system. Other methods include the use of rotating bottles, rotating systems, concave plate methods, and liquid overlays. Bioreactors can also be suitable for use in three-dimensional spheroid cell cultures. In one embodiment, the method used is a hanging drop system-e.g., a GravityPLUS hanging drop system (InSphero). This method involves the use of a GravityTRAP ULA plate, which is a non-adhesive, coated microtiter plate designed for the production of spheroids. Spheroid maturation typically occurs within 2 to 5 days of seeding, depending on the cell type and culture conditions. Suitably, the spheroids are cultured in a volume of 100 μl or more, or 200 μl or more, or 300 μl or more. Suitably, the spheroids are cultured in Corning® Spheroid Microplates.

[0293] Three-dimensional cell culture matrices or scaffolds can be used for spheroid culture. These are often porous substrates that can support three-dimensional cell growth and differentiation. A variety of materials have been developed to produce three-dimensional scaffolds that vary in physical appearance, porosity, permeability, mechanical characteristics, and nanoscale surface morphology. Examples of such materials include: collagen gels, sponges, or biogels; fibrin; fibronectin; laminin; alginates, hydrogels; cross-linked mucopolysaccharides; polymer-based scaffolds, synthetic scaffolds; peptide scaffolds; and chitosan composite scaffolds.

[0294] Three-dimensional spheroids more closely resemble in vivo tissues in terms of cell communication and the development of extracellular matrices. These matrices help cells move within the spheroid, similar to the way cells move in living tissues. Therefore, spheroids are a greatly improved model for differentiation, survival, cell migration, cell polarization, gene expression, and growth.

[0295] Spheroids can be harvested and studied using a variety of methods well known in the art, including colorimetric, fluorescent, and luminescent assays measured with a plate reader, or they can be easily observed by microscopy. Other techniques include Western, Northern, or Southern blotting, histological techniques (e.g., immunohistochemistry, in situ hybridization, immunofluorescence), etc. It is also contemplated to use optical imaging methods such as inverted bright field microscopy, fluorescence microscopy, single photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging (MRI), and Cerenkov luminescence imaging (CLI) techniques.

[0296] Applications using 3D spheroids include studying the proliferation of cells and tissues in vitro in an environment that more closely approximates that found in vivo, screening compounds and testing atmospheres, toxicology assays and clinical trials, among others.

[0297] The use of spheroids in three-dimensional cell culture is generally reviewed in Expert Opin. Drug Discov. (2015) 10, 519–540. In vitro, lung spheroid cells can be expanded to large numbers and can form alveolar-like structures and acquire a mature lung epithelial phenotype.

[0298] Cell source

[0299] Lung cells and cell lines used in the present disclosure can be isolated from tissues or fluids using methods well known in the art. Lung cells and cell lines used in the present disclosure can be differentiated from stem cells (e.g., embryonic stem cells or induced pluripotent stem cells), or directly differentiated from somatic cells. Cells and cell lines can be or can be derived from human or animal subjects or from human or animal cells, including any of a variety of mammalian species, suitably humans, but including rats, mice, pigs, rabbits, and non-human primates, etc. Cells and cell lines can be obtained from commercial sources. In certain embodiments, it is desirable to use human cells.

[0300] Lung cells including lung epithelial cells are a cell type of interest. Bronchial and / or airway epithelial cells are particularly useful in the present disclosure. Human bronchial epithelial cells can be collected by brushing donor lungs during bronchoscopy procedures. In one embodiment, the lung cells are normal human bronchial epithelial (NHBE) cells. Lung epithelial cells can be cultured into a monolayer of undifferentiated cells, or further developed into organotypic lung epithelial cell-like tissues at the air-liquid interface. Cells can be established at the air-liquid interface using the following method. In short, epithelial cells can be cultured in flasks to increase cell number. After the incubation period, cells are detached from the flasks, counted and inoculated onto inserts. On these inserts, cells are cultivated together with culture medium on the top and base sides. This stage ensures that the cells will divide and completely cover the inserts, thereby forming epithelial cells. Next, the top culture medium is removed, the base culture medium is retained, and replaced with a more complete culture medium. The culture is cultivated for a period of time like this. At the same time, the cells will differentiate into three cell types: basal cells, goblet cells and ciliated cells. At the end of maturation, the culture is ready for use. Culturing human nasal epithelial cells using an air-liquid interface is described in J Vis Exp. 2013; (80): 50646.

[0301] Lung epithelial cells can be obtained from human or animal subjects having various pathologies, including subjects classified as smokers or non-smokers.

[0302] Micro Sensor

[0303] Various microsensors have been developed and described. For example, Routkevitch et al. (NSTI-nanotech 2005ISBN 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), 1773–1781) explore the detection, quantification and degradation or conversion of atmospheric organic and inorganic pollutants using microbial biosensors. Biosensors include unicellular microorganisms, such as bacteria, fungi and blue-green algae and enzymes, and are reviewed in Appl. Biochem. Biotechnol. (2015) 175, 3093-3119. Various biosensors are available, such as enzyme-based biosensors, which can be based on the measurement of enzyme inhibition or the direct measurement of compounds involved in the enzyme reaction. Other examples of biosensors may involve the use of aptamers, molecularly imprinted polymers, biochips or nanotechnology or a combination thereof.

[0304] Biosensors based on molecular imprinting polymers can specifically bind to target components of the test aerosol. The target component can be a marker compound present in a known amount in the test aerosol, such as nicotine in tobacco smoke, and thereby allow the calculation of the overall aerosol mass deposition in the simulated respiratory tract. Alternatively, for example, in environmental monitoring processes, compounds with toxicological activity or biological activity can be targeted. In particular, toxic aerosol compounds - such as polyaromatic hydrocarbons, or when developing therapeutic aerosols, pharmacologically active aerosol components can be targeted, such as corticosteroids - such as budesonide for asthma treatment.

[0305] As required, the microsensor can be placed in, for example, a perforated structure or a simulated respiratory tract or combination, such as in a branch channel of a perforated structure. For example, part or all of one or more of the branch channels include at least one microsensor for monitoring conditions in the structure or for gas sampling or for gas characterization. By another example, one or more branch channels may include one or more openings, which can receive one or more modules, and the modules are used to contain microsensors for monitoring conditions in the structure. By another example, the microsensor can be placed at a site where a cell culture is present, such as in or on one or more of the exposure plugs, or in or on one or more of the probes, or in or on one or more of the quartz crystal microbalances, or in or on one or more holders associated therewith. In any case, it should be understood that the targeted chemical substance and the selected microsensor will depend on the aerosol tested.

[0306] analyze

[0307] The present disclosure can be used for various applications of studying the effects of test atmospheres on simulated respiratory tracts. For example, the present disclosure can be used to study in vitro inhalation toxicology, investigate aerosol dynamics in the respiratory tract (e.g., aerosol particle deposition and gas absorption into cell culture) or investigate metabolic activity or the transmission of test atmospheres (e.g., aerosol molecules) in epithelial cells of the respiratory tract. The present disclosure can be used to test the effects of aerosols, smoke, or tobacco products, or the effects of inhalers (e.g., medical inhalers). The present disclosure can be used to test the effects of aerosols, smoke, or tobacco products, or the effects of medical inhalers on one or more parts of the respiratory tract.

[0308] One aspect relates to a method for determining the effect of a test atmosphere on a cell culture (e.g., one or more cell cultures) and / or one or more microsensors contained in a simulated respiratory tract, the method comprising: (a) providing a system as described herein, wherein the system comprises cell cultures and / or microsensors in one or more of the modules; and (b) comparing the cell cultures and / or the microsensors before and / or after exposure to the test atmosphere, wherein differences between the cell cultures and / or the microsensors before and / or after exposure of the cells to the test atmosphere indicate that the test atmosphere affects the cell cultures and / or the microsensors.

[0309] In embodiments where differences between cell cultures and / or microsensors are determined after the cells and / or microsensors are exposed to a test atmosphere, the cell cultures and / or microsensors exposed to the test atmosphere can be compared to cell cultures and / or microsensors that have not been exposed to the test atmosphere or to cell cultures and / or microsensors exposed to a control atmosphere (such as an atmosphere that does not contain the test atmosphere). According to this embodiment, differences between cell cultures and / or microsensors exposed to the test atmosphere and cell cultures and / or microsensors that are not exposed to the test atmosphere, or differences between cell cultures and / or microsensors exposed to the test atmosphere and cell cultures and / or microsensors exposed to a control atmosphere (such as an atmosphere that does not contain the test atmosphere) indicate that the test atmosphere affects the cell cultures and / or microsensors.

[0310] On the other hand, a method for simulating the interaction between a test atmosphere and a simulated respiratory tract in the system described herein is provided, the method comprising: (a) providing a gas including a test atmosphere to the first pump through the first port when the first valve of the first pump is open and the second valve of the first pump is closed; (b) closing the first valve of the first pump and opening the second valve of the first pump, and closing the valve on the piston plate of the first pump; (c) operating the second pump to draw the test atmosphere into the connecting structure and fill the chamber of the first pump and the connecting structure with ambient air; (d) opening the first valve of the first pump to the ambient air and forming a sealed connection between the first port and the second port of the first pump; and (e) after a period of time, using the second pump to discharge the test atmosphere through the connecting structure and through the first valve of the first pump.

[0311] Another aspect relates to a method for determining the effect of a test atmosphere on a simulated respiratory tract in a system described herein, the method comprising: (a) providing a gas comprising a test atmosphere to the first pump via the first port with the first valve of the first pump open and the second valve of the first pump closed; (b) closing the first valve of the first pump and opening the second valve of the first pump, and closing the valve on the piston plate of the first pump; (c) operating the second pump to draw the test atmosphere through the connecting structure and flush the chamber of the first pump and the connecting structure with ambient air; (d) opening the first valve of the first pump to the ambient air and in the first forming a sealed connection between the first port and the second port of the pump; and (e) after a period of time, exhausting the test atmosphere through the connecting structure and through the first valve of the first pump using the second pump; wherein the test atmosphere contacts the cell culture and / or microsensor located in one or more modules, the one or more modules being located in the first pump or the connecting structure or the second pump or a combination of two or more thereof, and the method includes another step of determining the effect of the test atmosphere on the cell culture and / or microsensor, wherein differences in the cell culture and / or sensor before and / or after exposure to the test atmosphere indicate that the test atmosphere affects the cell culture and / or microsensor.

[0312] In embodiments where differences between cell cultures and / or microsensors are determined after the cells are exposed to a test atmosphere, the cell cultures and / or microsensors exposed to the test atmosphere can be compared to cell cultures and / or microsensors that have not been exposed to the test atmosphere or to cell cultures and / or microsensors that have been exposed to a control atmosphere (such as an atmosphere that does not contain the test atmosphere). According to this embodiment, differences between cell cultures and / or microsensors exposed to the test atmosphere and cell cultures and / or microsensors that are not exposed to the test atmosphere, or differences between cell cultures and / or microsensors exposed to the test atmosphere and cell cultures and / or microsensors exposed to a control atmosphere (such as an atmosphere that does not contain the test atmosphere) indicate that the test atmosphere affects the cell cultures and / or microsensors.

[0313] Another aspect relates to a method for simulating the interaction between a test atmosphere and a simulated respiratory tract, the method comprising: (a) providing the test atmosphere to a chamber of a first pump; (b) pumping the test atmosphere from the first pump back into a connecting structure, the connecting structure connecting the first pump to a second pump; (c) filling at least a portion of the first pump and the connecting structure with ambient air; (d) maintaining the test atmosphere in the second pump and the connecting structure for a defined time period; (e) exhausting the test atmosphere into the connecting structure and the first pump using the second pump; and (f) performing one or more pumping cycles of the ambient air in the second pump; wherein the test atmosphere contacts a cell culture located in the first pump or the connecting structure or the second pump or a combination of two or more thereof.

[0314] Another aspect relates to a method for determining the effect of a test atmosphere on a simulated respiratory tract, the method comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) pumping the test atmosphere from the first pump back into a connecting structure, the connecting structure connecting the first pump to a second pump; (c) filling at least a portion of the first pump and the connecting structure with ambient air; (d) maintaining the test atmosphere in the second pump and the connecting structure for a defined time period; (e) exhausting the test atmosphere through the connecting structure and the first pump using the second pump; and (f) performing one or more pumping cycles of the ambient air in the second pump; wherein the test atmosphere contacts a cell culture and / or microsensor located in one or more modules, the one or more modules being located in the first pump or the connecting structure or the second pump or a combination of two or more thereof, and the method comprises another step of determining the effect of the test atmosphere on the cell culture and / or microsensor, wherein differences in the cell culture and / or microsensor before and / or after exposure to the test atmosphere indicate that the test atmosphere affects the cell culture and / or microsensor. In embodiments where differences between cell cultures and / or microsensors are determined after the cells and / or microsensors are exposed to a test atmosphere, the cell cultures and / or microsensors exposed to the test atmosphere can be compared to cell cultures and / or microsensors that have not been exposed to the test atmosphere or to cell cultures and / or microsensors exposed to a control atmosphere (such as an atmosphere that does not contain the test atmosphere). According to this embodiment, differences between cell cultures and / or microsensors exposed to the test atmosphere and cell cultures and / or microsensors that are not exposed to the test atmosphere, or differences between cell cultures and / or microsensors exposed to the test atmosphere and cell cultures and / or microsensors exposed to a control atmosphere (such as an atmosphere that does not contain the test atmosphere) indicate that the test atmosphere affects the cell cultures and / or microsensors.

[0315] The effects of the test atmosphere can be studied in the presence of one or more agents. Agents can include, but are not limited to, drugs, toxins, pathogens, proteins, nucleic acids, antigens, antibodies, and compounds, etc. Examples of effects that can be measured include oxygen consumption, carbon dioxide production, cell viability, protein expression, enzyme activity, permeation, permeability barrier function, surfactant production, response to cytokines, transporter function, cytochrome P450 expression, albumin secretion, toxicity, etc.

[0316] Multiple assays may be run in parallel with varying concentrations of test atmospheres and / or reagents to obtain differential responses to a variety of concentrations.

[0317] The reagent can be any test compound of interest and includes small organic compounds, polypeptides, peptides, higher molecular weight carbohydrates, polynucleotides, fatty acids and lipids, aerosols or one or more components of aerosols, etc. The test compound can be screened individually, or with a collection of compounds or combinatorial libraries. Test compounds can be obtained from various sources including libraries of synthetic or natural compounds. Libraries of natural compounds in the form of bacteria, fungi, plants and animal extracts can be used. Libraries produced naturally or synthetically and compounds modified by conventional chemical, physical and biochemical means can be used to produce combinatorial libraries. Known agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, acidification to produce structural analogs for screening.

[0318] The one or more variables that can be measured include elements of cells, subcellular materials, subcellular components, or cell products. For example, the toxicology of the test atmosphere can be measured. Aerosol dynamics in the respiratory tract (e.g., aerosol particle deposition and gas absorption into cell cultures) can be measured. By way of further example, metabolic activity and / or the transport of molecules in epithelial cells of the respiratory tract can be studied.

[0319] computer

[0320] Also disclosed is a computer program which, when executed by a computer / processor, is operable to control the computer to perform one or more of the methods described herein.

[0321] Those skilled in the art will readily recognize that the steps of the various methods described above can be performed by a programmed computer. In this article, some embodiments are also intended to cover program storage devices (e.g., digital data storage media) that are machine or computer readable and encode a machine executable program or computer executable program of instructions, wherein the instructions perform some or all of the steps of the above methods. For example, the program storage device can be a digital memory, a magnetic storage medium (e.g., a disk and tape), a hard drive, or an optically readable digital data storage medium. The embodiments are also intended to cover computers that are programmed to perform some or all of the steps of the above methods.

[0322] The functions of various elements including processors or logic can be provided by using dedicated hardware and hardware capable of executing software in association with appropriate software. When the function is provided by a processor, the function can be provided by a single dedicated processor, by a single shared processor, or by multiple individual processors (some of which can be shared). In addition, the explicit use of the term "processor" or "controller" or "logic" should not be interpreted as specifically referring to hardware capable of executing software, and may implicitly include but is not limited to digital signal processor (DSP) hardware, network processor, application specific integrated circuit (ASIC), field programmable gate array (FPGA), read-only memory (ROM), random access memory (RAM) and non-volatile storage device for storing software. Other conventional and / or customized hardware may also be included. Similarly, any switch shown in the figure is conceptual only. Its function can be performed by the operation of program logic, by dedicated logic, by the interaction of program control and dedicated logic, or even manually, and the specific technology can be selected by the implementer as more specifically understood from the context.

[0323] Further aspects of the disclosure are set forth in the following numbered paragraphs:

[0324] 1. A perforated structure for a simulated respiratory tract, the structure comprising a perforated envelope accommodating one or more branch channels, wherein each perforation is an open end of a branch.

[0325] 2. A perforated structure according to paragraph 1, wherein the branches of each channel are bifurcated.

[0326] 3. A perforated structure according to paragraph 1 or paragraph 2, wherein the diameter of each channel existing after n+1 consecutive branching points is less than or equal to the diameter of the channel existing after n branching points.

[0327] 4. A perforated structure according to any one of paragraphs 1 to 3, wherein the total cross-sectional area of ​​all channels present after n+1 consecutive branch points is greater than or equal to the total cross-sectional area of ​​all channels after n branch points.

[0328] 5. A perforated structure according to any one of paragraphs 1 to 4, wherein the structure is symmetrical.

[0329] 6. A perforated structure according to any one of paragraphs 1 to 4, wherein the structure has radial symmetry.

[0330] 7. A perforated structure according to any one of paragraphs 1 to 6, wherein the structure is modular.

[0331] 8. A perforated 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 following: non-porous materials, including metals and non-porous synthetic materials; porous materials, including porous silicones; colloidal materials, including thermogels; hydrophobic materials; hydrophilic materials; amphiphilic materials or a combination of two or more thereof.

[0332] 9. A perforated structure according to any one of paragraphs 1 to 8, wherein one or more of the branch channels further comprises one or more coatings located on part or all of the inner surface of the branch channels.

[0333] 10. The perforated structure of paragraph 9, wherein the one or more of the coatings provide water and nutrients to cells growing in all or part of one or more of the branch channels.

[0334] 11. A perforated structure according to any one of paragraphs 1 to 10, wherein part or all of one or more of the branch channels is coated with a matrix comprising a cell culture medium and / or at least one microsensor for monitoring conditions in the structure or a probe for gas sampling or for gas characterization.

[0335] 12. A perforated structure according to any one of paragraphs 1 to 11, wherein one or more of the branch channels further comprises one or more openings capable of receiving one or more modules for containing a matrix comprising a cell culture medium or a microsensor for monitoring conditions in the structure or a probe for gas sampling or for gas characterization.

[0336] 13. A perforated structure according to paragraph 11 or paragraph 12, wherein the cell culture medium comprises one or more hydrogels - such as methacrylated gelatin (GelMa).

[0337] 14. A pump for discharging a volume of gas, the pump comprising one or more branch channels inside the pump, wherein the branch channels are connected to ports for receiving and outputting the gas 15. A pump according to paragraph 14, wherein the branch channels are included in a perforated structure according to any one of paragraphs 1 to 13.

[0338] 16. A perforated structure according to paragraph 14 or paragraph 15, comprising a chamber configured to contain a volume of gas and comprising a base and one or more openings capable of receiving one or more modules for containing a matrix including a cell culture medium and / or at least one microsensor for monitoring conditions in the chamber or a probe for gas sampling or for gas characterization.

[0339] 17. A pump according to any of paragraphs 14 to 16, wherein the pump further comprises a motor for controlling operation of the pump.

[0340] 18. A system for determining the interaction between a test atmosphere and a simulated respiratory tract, the system comprising: (a) a first pump, the first pump comprising: (i) a chamber, the chamber being configured to contain a first volume of gas, the first volume of gas comprising the test atmosphere; (ii) a first port, the first port being adapted to receive and output gas and comprising a valve for regulating gas flow through the first port, the valve being movable between an open position and a closed position, wherein in the open position the valve is open to the test atmosphere or ambient air; (iii) a second port, the second port being adapted to output and receive gas and comprising a valve for regulating gas flow through the second port, the valve being movable between an open position and a closed position; and (iv) a piston plate in the chamber, the piston plate comprising a valve for ingesting or flowing gas into the chamber. one or more orifices in the chamber, wherein one or more or each of the orifices includes a valve that is movable between an open position and a closed position and is capable of regulating the intake or inflow of gas; and (v) a motor that is used to control the operation of the first pump; (b) a second pump according to any of paragraphs 13 to 16; (c) a connecting structure that is operable to transfer the gas from the first pump to the second pump; and (d) one or more openings located in the wall of the first pump or the second pump or the connecting structure or a combination of two or more thereof, the opening being capable of receiving a module that is used to contain a matrix including a cell culture medium and / or at least one microsensor for monitoring conditions in the chamber or a probe for gas sampling or for gas characterization.

[0341] 19. A method for simulating the interaction between a test atmosphere and a simulated respiratory tract, the method comprising using a pump according to any one of paragraphs 14 to 17 or a system according to paragraph 18.

[0342] 20. Use of a pump according to any one of paragraphs 14 to 17 or a system according to paragraph 18 for simulating said interaction between a test atmosphere and a simulated respiratory tract.

[0343] 21. A method for determining the effect of a test atmosphere on a cell culture contained in a simulated respiratory tract comprising using a pump according to paragraphs 14 to 17 or a system according to paragraph 18.

[0344] 22. Use of a pump according to any one of paragraphs 14 to 17 or a system according to paragraph 18 for determining the effect of a test atmosphere on a cell culture contained in a simulated respiratory tract.

[0345] 23. A method for determining the effect of a test atmosphere on a cell culture contained in a simulated respiratory tract, the method comprising: (a) providing a pump according to any of paragraphs 14 to 17 or a system according to paragraph 18, wherein the pump or system contains a cell culture and / or at least one microsensor in one or more of the modules; and (b) comparing the cell culture and / or the at least one microsensor before and / or after exposure to the test atmosphere, wherein differences between the cell culture and / or the at least one microsensor before and / or after exposure of the cells and / or the at least one microsensor to the test atmosphere indicate that the test atmosphere affects the cell culture and / or the at least one microsensor.

[0346] 24. A method for simulating the interaction between a test atmosphere and a simulated respiratory tract in a system according to paragraph 18, the method comprising: (a) providing a gas including a test atmosphere to the first pump through the first port when the first valve of the first pump is open and the second valve of the first pump is closed; (b) closing the first valve of the first pump and opening the second valve of the first pump, and closing the valve on the piston plate of the first pump; (c) operating the second pump to draw the test atmosphere into the connecting structure and fill the chamber of the first pump and the connecting structure with ambient air; (d) opening the first valve of the first pump to the ambient air and forming a sealed connection between the first port and the second port of the first pump; and (e) after a period of time, using the second pump to discharge the test atmosphere through the connecting structure and through the first valve of the first pump.

[0347] 25. A method for determining the effect of a test atmosphere on a simulated respiratory tract in a system according to paragraph 18, the method comprising: (a) providing a gas comprising a test atmosphere to the first pump via the first port with the first valve of the first pump open and the second valve of the first pump closed; (b) closing the first valve of the first pump and opening the second valve of the first pump, and closing the valve on the piston plate of the first pump; (c) operating the second pump to draw the test atmosphere through the connecting structure and flush the chamber of the first pump and the connecting structure with ambient air; (d) opening the first valve of the first pump to the ambient air and in the first valve of the first pump and (e) after a period of time, exhausting the test atmosphere through the connecting structure and through the first valve of the first pump using the second pump; wherein the test atmosphere contacts a matrix including a cell culture or at least one microsensor located in one or more modules, the one or more modules being located in the first pump or the connecting structure or the second pump or a combination of two or more thereof, and the method includes another step of determining an effect of the test atmosphere on the cell culture and / or the microsensor, wherein differences in the cell culture and / or the sensor before and / or after exposure to the test atmosphere indicate that the test atmosphere affects the cell culture and / or the microsensor.

[0348] 26. A method for simulating the interaction between a test atmosphere and a simulated respiratory tract, the method comprising: (a) providing the test atmosphere to a chamber of a first pump; (b) pumping the test atmosphere from the first pump back into a connecting structure, the connecting structure connecting the first pump to a second pump, wherein the second pump is a pump according to any one of paragraphs 14 to 17; (c) filling at least a portion of the first pump and the connecting structure with ambient air; (d) maintaining the test atmosphere in the second pump and the connecting structure for a defined time period; (e) exhausting the test atmosphere into the connecting structure and the first pump using the second pump; and (f) performing one or more pumping cycles of the ambient air in the second pump; wherein the test atmosphere contacts a matrix including a cell culture and / or at least one microsensor located in the first pump or the connecting structure or the second pump or a combination of two or more thereof.

[0349] 27. A method for determining the effect of a test atmosphere on a simulated respiratory tract, the method comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) pumping the test atmosphere from the first pump back into a connection structure, the connection structure connecting the first pump to a second pump, wherein the second pump is a pump according to any of paragraphs 14 to 17; (c) filling the first pump and at least a portion of the connection structure with ambient air; (d) maintaining the test atmosphere in the second pump and the connection structure for a defined period of time; (e) exhausting the test atmosphere through the connection structure and the first pump using the second pump; and (f) performing one or more pumping cycles of the ambient air in the second pump; wherein the test atmosphere contacts a substrate including 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 thereof, and the method comprises a further step of determining the effect of the test atmosphere on the cell culture and / or microsensor, wherein differences in the cell culture before and / or after exposure to the test atmosphere indicate that the test atmosphere affects the cell culture and / or microsensor.

[0350] 28. A device configured or adapted to perform the method of paragraph 26 or paragraph 27.

[0351] 29. A method of making a perforated structure according to any one of paragraphs 1 to 13, the method comprising: (i) embedding one or more branch channels, each branch channel comprising an open end, in a material suitable for forming the perforated structure; and (ii) removing a casting of the one or more branch channels from the material.

[0352] 30. A method according to paragraph 29, wherein the perforated structure is at least partially manufactured by 3D printing.

[0353] 31. A method according to paragraph 29, wherein the perforated structure is formed by molding.

[0354] 32. A method according to paragraph 29, wherein the perforated structure is cast from a template.

[0355] 33. The method of any of paragraphs 29 to 32, wherein the perforated structure i) substantially mimics the branching structure of a mammalian bronchial airway, ii) is an idealized branching structure, or iii) is a combination of i) and ii).

[0356] This article discloses an in vitro simulation of the impact of the airway in vivo on the aerosol properties related to the field of inhalation toxicology, inhalation therapy and inhalable consumer product evaluation. In addition, it can also be used as an experimental model for determining the in vivo aerosol dosimetry of the respiratory tract under stable, fully characterized, reproducible and ethical non-critical conditions. The value of basic research can be identified because the airway model as described herein can serve as a robust model for studying the aerosol dynamics in the respiratory tract or in structures of similar complexity, and therefore can contribute to understanding the dynamics of the aerosol in respiratory organs and such complex structures.

[0357] Any publication cited or described in this article provides relevant information disclosed before the filing date of this application. Statements herein should not be interpreted as admitting that the inventor loses the qualification prior to such disclosure. All publications mentioned in the above specification are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the present invention as claimed should not be unduly limited to such specific embodiments. In fact, multiple variations of the described modes for carrying out the present invention are apparent to those skilled in the relevant art and are intended to be within the scope of the following claims.

Claims

1. A perforated structure for a simulated respiratory tract, the structure comprising a perforated envelope accommodating one or more branch channels, wherein each perforation is an open end of the one or more branch channels.

2. The perforated structure of claim 1, wherein the branches of each channel are bifurcated.

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

4. A perforated structure according to any one of the preceding claims, wherein the total cross-sectional area of ​​all channels present after n+1 consecutive branching points is greater than or equal to the total cross-sectional area of ​​all channels after n branching points; preferably, wherein the structure is asymmetric or wherein the structure has radial symmetry.

5. A perforated structure according to any one of the preceding claims, wherein the structure is modular; and / or wherein all or part of the structure is formed of one or more materials selected from the group consisting of: non-porous materials, including metals and non-porous synthetic materials; porous materials, including porous silicones; gel-like materials, including thermogels; hydrophobic materials; hydrophilic materials; amphiphilic materials or a combination of two or more thereof; and / or wherein one or more of the branch channels further comprises one or more coatings located on a portion or all of an inner surface of the branch channel; and / or wherein one or more of said coatings provide water and nutrients to cells growing in all or a portion of one or more of said branch channels; and / or wherein part or all of one or more of the branch channels is coated with a matrix comprising a cell culture medium and / or at least one microsensor for monitoring conditions in the structure or for gas sampling or for gas characterization; and / or One or more of the branch channels further comprise one or more openings capable of receiving one or more modules for containing a matrix comprising a cell culture medium or a microsensor for monitoring conditions in the structure or a probe for gas sampling or for gas characterization.

6. A pump for discharging a volume of gas, the pump comprising a port and one or more branch channels inside the pump, wherein the one or more branch channels are connected to the port for receiving and outputting the gas.

7. A pump according to claim 6, wherein the one or more branch channels are contained in a perforated structure according to any one of claims 1 to 5; and / or wherein the pump comprises a chamber configured for containing the volume of gas and comprising a base and one or more openings capable of receiving one or more modules for containing a matrix comprising a cell culture medium and / or at least one microsensor for monitoring conditions in the chamber or for gas sampling or for gas characterization; and / or The pump further comprises a motor for controlling the operation of the pump.

8. A system for determining the interaction between a test atmosphere and a simulated respiratory tract, the system comprising: (a) a first pump, the first pump comprising: (i) a chamber configured to contain a first volume of gas, the first volume of gas comprising a test atmosphere; (ii) a first port adapted to receive and output a gas and comprising a valve for regulating gas flow through the first port, the valve being movable between an open position and a closed position, wherein in the open position the valve is open to a test atmosphere or ambient air; (iii) a second port adapted to output and receive gas and comprising a valve for regulating gas flow through the second port, the valve being movable between an open position and a closed position; (iv) a piston plate in the chamber, the piston plate comprising one or more apertures for the intake or flow of gas into the chamber, wherein one or more or each of the apertures comprises a valve movable between an open position and a closed position and capable of regulating the intake or flow of gas; and (v) a motor for controlling the operation of the first pump; (b) a second pump, wherein the second pump is as defined in claim 6 or 7; (c) a connection structure operable to transfer the gas from the first pump to the second pump; and (d) one or more openings located in the first pump or the second pump or in a wall of the connecting structure or in a combination of two or more thereof, the openings being capable of receiving a module for containing a matrix including a cell culture medium and / or at least one microsensor for monitoring conditions in the chamber or for gas sampling or for gas characterization.

9. A method for simulating the interaction between a test atmosphere and a simulated respiratory tract, the method comprising using a pump according to claim 6 or 7 or a system according to claim 8.

10. Use of a pump according to claim 6 or 7 or a system according to claim 8 for simulating the interaction between a test atmosphere and a simulated respiratory tract.

11. A method for determining the effect of a test atmosphere on a cell culture contained in a simulated respiratory tract, the method comprising using a pump according to claim 6 or 7 or a system according to claim 8.

12. Use of a pump according to claim 6 or 7 or a system according to claim 8 for determining the effect of a test atmosphere on a cell culture contained in a simulated respiratory tract.

13. A method for determining the effect of a test atmosphere on a cell culture contained in a simulated respiratory tract, the method comprising: (a) providing a pump according to claim 6 or 7 or a system according to claim 8, wherein the pump or system comprises a cell culture and / or at least one microsensor in one or more of the modules; and (b) comparing the cell culture and / or the at least one microsensor before and / or after exposure to the test atmosphere, wherein differences between the cell culture and / or the at least one microsensor before and / or after exposure of the cells and / or the at least one microsensor to the test atmosphere indicate that the test atmosphere affects the cell culture and / or the at least one microsensor.

14. An apparatus configured or adapted to carry out the method according to claim 11 or claim 13.

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