BR112020000828B1 - SYSTEM FOR DETERMINING THE INTERACTION BETWEEN A TEST ATMOSPHERE AND A SIMULATED RESPIRATORY TRACT, USE OF THE SYSTEM, METHOD FOR DETERMINING THE EFFECT OF A TEST ATMOSPHERE ON A CELL CULTURE, AND METHOD FOR SIMULATING THE INTERACTION BETWEEN A TEST ATMOSPHERE AND A RESPIRATORY TRACT SIMULATED - Google Patents

Abstract

The present invention relates to a system for determining the interaction between a test atmosphere and a simulated respiratory tract, comprising: (a) first pump: (i) chamber for containing a first volume of gas, with a test atmosphere; (ii) first port adapted to receive and emit gas, with a valve to regulate the flow of gas through the first port, where said valve is movable between open and closed positions; and open to the test atmosphere or surrounding air; (iii) second door adapted for gas outlet and reception, with valve, movable between open and closed positions, to regulate the gas flow through the second door; (iv) piston plate in the chamber comprising one or more openings for capturing gas in the chamber wherein one or more, or each of the openings, includes a movable valve, and is capable of regulating gas capture; (b) second pump comprising: (i) chamber for containing second volume of gas, wherein the first and second volumes of gas are different; (ii) port adapted to receive and emit gas; and (iii) motor to control the operation of the first pump; (c) connecting structure operable to transmit the gas from the first to the (...).

Description

FIELD OF INVENTION

[001] The present invention relates to, inter alia, apparatus, systems, methods and uses related to simulating the structure and/or function of a respiratory tract and its respiratory behavior. The present invention is useful for investigating interactions between test agents (e.g., inhalable agents in test atmospheres) and the respiratory tract. In particular, the present invention relates to the simulation of the human respiratory tract.

BASICS OF THE INVENTION

[002] The respiratory system passes 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 reach the alveoli. As the aerosol moves deeper into the respiratory tract, more soluble gases are adsorbed and aerosol particles are deposited deeper into the airways and alveoli. A particular aerosol of interest in the context of the present invention is smoke - such as tobacco smoke - as the impact of smoke on or through a simulated respiratory tract can be studied.

[003] Existing aerosol exposure systems generally rely on a continuous, unidirectional flow of aerosols or passive sedimentation. Continuous aerosol streams are generated by positive or negative pressure and the aerosols are directed perpendicular to the biological test system or transmitted in a direction parallel to the plane of the biological test system. The efficiency of dose delivery in this exposure mode is primarily a function of the geometry of the exposure chamber and the flow velocity of the test aerosol. The mouth, breath-holding time, and dynamic flow patterns that occur during human breathing (i.e., inhalation and exhalation) cannot be simulated by current aerosol exposure systems, however. A dose delivery representative for the in vivo situation, especially the relative delivery of particulate and gaseous aerosol constituents is therefore unlikely to be achieved. Furthermore, the filtering action of the conducting airways cannot be representatively simulated. Thus, with current aerosol exposure systems, the same aerosol particle size distribution is used, for example, with bronchial cell cultures and nasal cultures. In living organisms, however, larger aerosol particles primarily interact with and are removed from the aerosol in the upper respiratory tract, while deeper regions of the respiratory tract are primarily exposed to smaller aerosol particle sizes and gaseous constituents. Furthermore, in continuous flow exposure systems, aerosol dilution is achieved by continuously adding dilution air to the aerosol upstream, close to the exposure chambers. However, if smoking behavior is to be simulated as in a biological organism, this mode of dilution is not representative of the organism, as it does not take into account a period of retention in the mouth during which the highly dense aerosol can age. for up to several seconds followed by being drawn into the respiratory tract along with a large volume of diluting air.

[004] In passive aerosol sedimentation, a test aerosol is injected into a chamber at the bottom of which biological test systems are located. Once the aerosol is injected, the aerosol can settle in the test system, usually by gravitation. The electrostatic attraction of aerosol particles to the test system can be used to enhance aerosol particle deposition. Nanosized aerosol particles, in particular, may require electrostatic attraction due to their low sedimentation efficiency. Exposures to aerosol particles containing complex aerosols of various sizes and densities, as well as gaseous constituents, cannot be conducted in passive aerosol sedimentation systems, as the relative differential administration of large aerosol particles, small aerosol particles, and gaseous constituents aerosol sprays are not representative of the processes occurring in a living organism.

[005] There is a continued need in the technique to improve simulated systems for studying the respiratory tract.

SUMMARY OF THE INVENTION

[006] In a first aspect, a system is provided for determining the interaction between a test atmosphere and a simulated respiratory tract, said system comprising: (a) a first pump comprising: (i) a chamber configured to contain a first volume of gas comprising a test atmosphere; (ii) a first port adapted to receive and emit gas and comprising a valve for regulating the flow of gas through the first port, said valve being movable between open and closed positions, wherein in the open position said valve is opened to a test atmosphere or surrounding air; (iii) a second port adapted for exiting and receiving gas and comprising a valve for regulating the flow of gas through the second port, said valve being movable between open and closed positions; (iv) a piston plate in the chamber, said piston plate comprising one or more openings for capturing or entering gas into the chamber wherein one or more or each of the openings includes a valve that is movable between the open positions and closed and is capable of regulating the capture or entry of gas; and (v) a motor for controlling the operation of the first pump; (b) a second pump comprising: (i) a chamber configured to contain a second volume of gas, wherein the first and second volumes of gas are different; (ii) a port adapted to receive and emit gas; and (iii) a motor to control the operation of the first pump; (c) a connecting structure operable to transmit gas from the first pump to the second pump; and (d) one or more openings in the first pump or the second pump or in the walls of the connecting structure or a combination of two or more thereof, wherein said openings are capable of receiving a module for containing a cell culture medium or configured to store a cell culture medium or to monitor conditions in the chamber or adapted to monitor conditions in the chamber or for gas sampling or adapted to sample the gas or for gas characterization or adapted to characterize the gas.

[007] Suitably, the pumps are piston pumps comprising a piston plate and a base.

[008] Suitably, the connection structure is hollow.

[009] Suitably, the connection structure is branched.

[0010] Suitably, a terminal branch of the connecting structure is joined to the second pump and one or more terminal branches are joined to a separate pump, wherein each separate pump comprises: (i) a chamber for containing a volume of gas, in that the volume of gas is the same volume as the second volume of gas in the second pump; (ii) a port to receive and emit gas and to join the connecting structure; and (iii) a motor to control the operation of the pump.

[0011] Suitably, each separate pump is the same as the second pump.

[0012] Suitably, the system is contained in an enclosure, suitably a temperature-controlled enclosure.

[0013] Suitably, the temperature of the housing is controlled by a thermostat.

[0014] Suitably, the temperature in the casing is about 37 °C.

[0015] Suitably, the different volumes of the first and second pumps represent the internal volume of different compartments of the respiratory tract, appropriately, the human respiratory tract.

[0016] Suitably, the volume of the separate pumps represents the internal volume of different compartments of the respiratory tract, appropriately, the human respiratory tract.

[0017] Suitably, the displacement volume of the first and second pumps is at least as large as the most achievable volume absorption in the corresponding compartment of the respiratory tract.

[0018] Suitably, the displacement volume of the separate pumps is at least as large as the most achievable volume absorption in the corresponding compartment of the respiratory tract.

[0019] Suitably, the pumping pressure of the engine or pump may correspond to atmospheric pressure or above or below atmospheric pressure.

[0020] Suitably, the displacement volume of the first pump is between about 0 and 100 ml or between about 1 and 100 ml.

[0021] Suitably, the displacement volume of the second pump is between about 0 and 4000 ml or between about 1 and 4000 ml.

[0022] Suitably, the pumps comprise stainless steel.

[0023] Suitably, the chamber is a cylinder.

[0024] Suitably, the chamber comprises glass.

[0025] Suitably, the chamber of the first pump has a smaller volume than the chamber of the second pump.

[0026] Suitably, the volume of the chamber of the first pump represents the volume of the oral and oropharyngeal cavity.

[0027] Suitably, the volume of the chamber of the second pump represents the volume of the lung lumen or a portion thereof.

[0028] Suitably, the volume of the connecting structure represents the volume of the conducting airways of the lung, appropriately the human lung.

[0029] Suitably, the system further comprises a computer controller capable of synchronizing the operation of the system.

[0030] Suitably, one or more of the first pump or the second pump or the connecting structure comprises one or more modules containing quartz crystal microbalances.

[0031] Suitably, the openings in the first pump or the second pump or in the walls of the connecting structure are threaded or unthreaded.

[0032] Suitably, one or more of the openings contains a module.

[0033] Suitably, the one or more modules are adapted to contain a cell culture medium or configured to store a cell culture medium or adapted to monitor conditions in the chamber or adapted to sample the gas or adapted to characterize the gas .

[0034] Suitably, the modules are located at the base of the piston plate of the first and/or second pump and/or on the walls of the connecting structure.

[0035] Suitably, the one or more modules contain a cell culture medium.

[0036] Suitably, the cell culture medium comprises or is in contact with a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0037] Suitably, modules adapted to contain or store a cell culture medium further comprise a microfluidic channel and, optionally, a microfluidic pump connected thereto.

[0038] Suitably, the modules are positioned in a horizontal plane on one or more of the first pump or the second pump or the walls of the connecting structure.

[0039] Suitably, the connecting structure comprises stainless steel.

[0040] Suitably, the chamber of the first pump has a volume of about 100 ml.

[0041] Suitably, the chamber of the second pump has a volume of about 1 liter to about 4 liters.

[0042] In a further aspect, a pump is provided for displacing a volume of gas 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 contain or store a cell culture medium or to monitor conditions in the chamber or for gas sampling or for gas characterization; (ii) a first port for receiving and emitting the gas, when contained in the chamber, and comprising a first valve for regulating the flow of gas through the first port, wherein said first valve is movable between the open and closed positions, in that, in the open position, said valve can be opened towards a test atmosphere or surrounding air; and (iii) a second port for emitting and receiving gas when contained in the chamber, and comprising a second valve for regulating the flow of gas through the second port, said valve being movable between open and closed positions; and (iv) a piston plate in the chamber, wherein said piston plate comprises one or more openings for the absorption or inflow of gas into the chamber wherein one or more, or each, of the openings includes a valve that is movable between the open and closed positions and is capable of regulating the absorption or influx of gas.

[0043] Suitably, the pump is a piston pump.

[0044] Suitably, the one or more openings in the chamber are threaded or unthreaded.

[0045] Suitably, the one or more openings comprise a module.

[0046] Suitably, the module is threaded or non-threaded.

[0047] Suitably, the one or more modules are adapted to contain a cell culture medium or configured to store a cell culture medium or adapted to monitor conditions in the chamber or adapted to sample the gas or adapted to characterize the gas .

[0048] Suitably, the modules are located at the base of the pump.

[0049] Suitably, the one or more modules contain a cell culture medium.

[0050] Suitably, the cell culture medium comprises or is in contact with a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0051] Suitably, modules adapted to contain or store a cell culture medium further comprise a microfluidic channel and, optionally, a microfluidic pump connected thereto.

[0052] Suitably, the modules are positioned in a horizontal plane on one or more of the first pump or the second pump or the walls of the connecting structure.

[0053] Suitably, the module comprises quartz crystal microbalances.

[0054] Suitably, the pump additionally comprises a motor.

[0055] Suitably, the pumping pressure corresponds to atmospheric pressure or above or below atmospheric pressure.

[0056] Suitably, the displacement volume of the pump is between about 0 and 100 ml or between about 1 and 100 ml.

[0057] Suitably, the pump comprises stainless steel.

[0058] Suitably, the chamber is a cylinder.

[0059] Suitably, the chamber comprises glass.

[0060] Suitably, the pump chamber has a volume of about 100 ml.

[0061] In a further aspect, there is provided a pump for displacing a volume of gas comprising: (i) a chamber configured to contain a volume of gas and containing a piston plate comprising one or more openings for the absorption or influx of gas in the chamber, wherein one or more, or each, of the openings includes a valve that is movable between the open and closed positions and is capable of regulating the absorption or inflow of gas; (ii) a first port for receiving and comprising a first valve for regulating the flow of gas through the first port, said first valve being movable between open and closed positions; and (iii) a second port for emitting gas when contained in the chamber, and comprising a second valve for regulating the flow of gas through the second port, said valve being movable between open and closed positions. Suitably, the chamber includes a base and comprises a further opening.

[0062] Suitably, the openings are threaded or unthreaded.

[0063] Suitably, the openings comprise a module in one or more of the openings.

[0064] Suitably, the module is threaded or non-threaded.

[0065] Suitably, the module is adapted to contain or store a cell culture medium or monitor conditions in the chamber or for gas sampling or for gas characterization

[0066] Suitably, one or more modules contain a cell culture medium.

[0067] Suitably, the cell culture medium comprises a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0068] Suitably, modules adapted to contain or store a cell culture medium further comprise a microfluidic channel and, optionally, a microfluidic pump connected thereto.

[0069] Suitably, the module comprises quartz crystal microbalances.

[0070] Suitably, a connection structure is attached to the second port.

[0071] Suitably, the connecting structure is hollow.

[0072] Suitably, the pump additionally comprises a motor.

[0073] Suitably, the pumping pressure of the pump corresponds to atmospheric pressure or above or below atmospheric pressure.

[0074] Suitably, the displacement volume of the pump is between about 0 and 100 ml or between about 1 and 100 ml.

[0075] Suitably, the pump comprises stainless steel.

[0076] Suitably, the chamber is a cylinder.

[0077] Suitably, the chamber comprises glass.

[0078] Suitably, the pump chamber has a volume of about 100 ml.

[0079] In a further aspect, a pump is provided for displacing a volume of gas comprising: (i) a chamber configured to contain a volume of gas, said chamber comprising a base and one or more modules for containing or storing a cell culture medium or for monitoring conditions in the chamber or for gas sampling or for gas characterization; and (ii) an operable port for receiving and emitting the gas.

[0080] Suitably, the pump is a piston pump.

[0081] Suitably, the modules are located at the base of the chamber.

[0082] Suitably, the modules are threaded or non-threaded.

[0083] Suitably, the modules are adapted to contain a cell culture medium or configured to store a cell culture medium or adapted to monitor conditions in the chamber or adapted to sample the gas or adapted to characterize the gas.

[0084] Suitably, the one or more modules contain a cell culture medium.

[0085] Suitably, the cell culture medium comprises or is in contact with a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[0086] Suitably, modules adapted to contain or store a cell culture medium further comprise a microfluidic channel and, optionally, a microfluidic pump connected thereto.

[0087] Suitably, the module comprises quartz crystal microbalances.

[0088] Suitably, a connection structure is attached to the door.

[0089] Suitably, the connection structure is hollow.

[0090] Suitably, the pump additionally comprises a motor.

[0091] Suitably, the pumping pressure corresponds to atmospheric pressure or above or below atmospheric pressure.

[0092] Suitably, the displacement volume of the pump is between about 0 and 1000 ml or between about 1 and 100 ml.

[0093] Suitably, the pump comprises stainless steel.

[0094] Suitably, the chamber is a cylinder.

[0095] Suitably, the chamber comprises glass.

[0096] Suitably, the chamber volume represents the volume of the lung lumen or a portion thereof.

[0097] In a further aspect, there is provided a connecting structure adapted to join at least two pumps for transmitting a gas between them, wherein said connecting structure comprises a hollow channel and one or more threaded or unthreaded openings therein. walls of the connecting structure.

[0098] Suitably, the threaded openings contain a threaded module in one or more of the openings, wherein said module is adapted to contain a cell culture medium or configured to store a cell culture medium or adapted to monitor conditions in the chamber or adapted to prove the gas or adapted to characterize the gas.

[0099] Suitably, the one or more modules contain a cell culture medium.

[00100] Suitably, the cell culture medium comprises or is in contact with a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[00101] Suitably, modules adapted to contain or store a cell culture medium further comprise a microfluidic channel and, optionally, a microfluidic pump connected thereto.

[00102] Suitably, the connection structure is hollow.

[00103] Suitably, the connection structure is branched.

[00104] Suitably, each terminal branch of the connecting structure is capable of being joined to a separate pump.

[00105] Suitably, the connecting structure represents the volume of the conducting airways of the lung, appropriately the human lung.

[00106] Suitably, the modules are positioned in a horizontal plane on the walls of the connecting structure.

[00107] Suitably, the modules are adapted to contain a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[00108] Suitably, the module is a chamber for containing a cell culture, said chamber comprising a microfluidic channel and, optionally, a microfluidic pump connected thereto.

[00109] Suitably, the modules are adapted to monitor conditions in the connecting structure and/or for gas sampling and/or for gas characterization.

[00110] Suitably, the connecting structure comprises stainless steel.

[00111] A system comprising the pumps described in this document is also described.

[00112] Suitably, the system further comprises the connection structure as described in this document.

[00113] Suitably, the pumps are joined by the connecting structure.

[00114] In a further aspect, a method for simulating the interaction between a test atmosphere and a simulated respiratory tract is provided that comprises the use of the system described herein.

[00115] Also described is the use of the system, as described in this document, to simulate the interaction between a test atmosphere and a simulated respiratory tract.

[00116] Also described is a method for determining the effect of a test atmosphere on a culture of cells contained in a simulated respiratory tract comprising the use of the system described in this document.

[00117] Also described is the use of the system, as described herein, to determine the effect of a test atmosphere on a culture of cells contained in a simulated respiratory tract.

[00118] In a further aspect, there is described a method for determining the effect of a test atmosphere on a cell culture contained in a simulated respiratory tract comprising: (a) providing the system, as described herein, wherein the system contains a cell culture in one or more of the modules; and (b) comparing the cell culture before and/or after exposure to the test atmosphere, wherein a difference between the cell culture before and/or after exposing the cells to the test atmosphere is indicative that the test atmosphere test affects cell culture.

[00119] A further aspect relates to a method for simulating the interaction between a test atmosphere and a simulated respiratory tract in the system described herein comprising: (a) with the first valve of the first pump open and the second valve of the first closed pump, supplying a gas comprising a test atmosphere to the first pump through the first port; (b) closing the first valve and opening the second valve of the first pump and closing the valves on the piston plate of the first pump; (c) operating the second pump to draw the test atmosphere into the connecting structure and filling the chamber of the first pump and the connecting structure with the surrounding air; (d) opening the first valve of the first pump toward the surrounding 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 displace the test atmosphere through the connecting structure and through the first valve of the first pump.

[00120] There is further described a method for determining the effect of a test atmosphere on a simulated respiratory tract in the system described herein comprising: (a) with the first valve of the first pump open and the second valve of the first pump closed, providing a gas comprising a test atmosphere for the first pump through the first port; (b) closing the first valve and opening the second valve of the first pump and closing the valves on the piston plate of the first pump; (c) operating the second pump to extract the test atmosphere through the connecting structure and flushing the chamber of the first pump and the connecting structure with the surrounding air; (d) opening the first valve of the first pump toward the surrounding 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 move the test atmosphere through the connecting structure and through the first valve of the first pump; wherein the test atmosphere comes into contact with a cell culture located in one or more modules located in the first pump or in the connecting structure or in the second pump or in a combination of two or more thereof and said method comprises the additional step of determining the effect of the test atmosphere on the cell culture, wherein a difference in the cell culture before and/or after exposure to the test atmosphere is indicative that the test atmosphere affects the cell culture.

[00121] Suitably, the modules are adapted for monitoring system conditions and/or for gas sampling and/or for gas characterization and said method comprises taking one or more measurements from the modules.

[00122] Also described is a method for simulating the interaction between a test atmosphere and a simulated breathing interval comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) withdrawing the test atmosphere from the first pump to a connecting structure joining the first pump to a second pump; (c) filling the first pump and at least a portion of the connecting structure with the surrounding air; (d) retain the test atmosphere in the second pump and connecting structure for a defined period of time; (e) moving the test atmosphere to the connecting structure and the first pump using the second pump; and (f) performing one or more cycles of pumping the surrounding air into the second pump; wherein the test atmosphere comes into contact with a cell culture located in the first pump or connecting structure or in the second pump or a combination of two or more.

[00123] Suitably, step (d) comprises maintaining the test atmosphere in the second pump and the portion of the connecting structure still containing the test atmosphere for a defined period of time.

[00124] Suitably, the pumps are piston pumps comprising a piston plate and a base.

[00125] Suitably, the first pump is defined in the embodiments of the present description.

[00126] Suitably, the second pump is defined in the embodiments of the present description.

[00127] Suitably, the connection structure is defined in the embodiments of the present description.

[00128] Suitably, the method is carried out in an enclosure, suitably a temperature-controlled enclosure.

[00129] Suitably, the temperature of the housing is controlled by a thermostat.

[00130] Suitably, the temperature in the casing is about 37 °C.

[00131] Suitably, the different volumes of the first and second pumps represent the internal volume of different compartments of the respiratory tract, appropriately, the human respiratory tract.

[00132] Suitably, the displacement volume of the first and second pumps is at least as large as the most achievable volume absorption possible in the corresponding compartment of the respiratory tract.

[00133] Suitably, the pumping pressure corresponds to atmospheric pressure or above or below atmospheric pressure.

[00134] Suitably, the displacement volume of the first pump is between about 0 and 100 ml or between about 1 and about 100 ml.

[00135] Suitably, the displacement volume of the second pump is between about 0 and 4000 ml or between about 1 and 4000 ml.

[00136] Suitably, the chamber of the first pump has a smaller volume than the chamber of the second pump.

[00137] Suitably, the volume of the chamber of the first pump represents the volume of the oral and oropharyngeal cavity, appropriately the human oral and oropharyngeal cavity.

[00138] Suitably, the volume of the chamber of the second pump represents the volume of the lung lumen or a part thereof, suitably the human lung lumen or a part thereof.

[00139] Suitably, the connecting structure represents the volume of the conducting airways of the lung, appropriately the human lung.

[00140] Suitably, the cell culture is located at the base of the piston plate of the first and/or second pump and/or on the walls of the connecting structure.

[00141] Suitably, cell culture is a two-dimensional or three-dimensional culture.

[00142] Suitably, said method further comprises monitoring conditions and/or for gas sampling and/or for gas characterization and in one or more of the first pump or the second pump or the connection structure using one or more modules contained therein.

[00143] Suitably, the chamber comprising the cell culture additionally comprises a microfluidic channel and, optionally, a microfluidic pump connected thereto.

[00144] Suitably, the connecting structure comprises stainless steel.

[00145] Suitably, the chamber of the first pump has a volume of about 100 ml.

[00146] Suitably, the second pump chamber has a volume of about 1 liter to about 4 liters.

[00147] In a further aspect, a method is described for determining the effect of a test atmosphere on a simulated respiratory tract comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) withdrawing the test atmosphere from the first pump to a connecting structure joining the first pump to a second pump; (c) filling the first pump and at least a portion of the connecting structure with the surrounding air; (d) maintain the test atmosphere in the second pump and the connecting structure for a defined period of time; (e) displacing the test atmosphere through the connecting structure and the first pump using the second pump; and (f) performing one or more cycles of pumping the surrounding air into the second pump; wherein the test atmosphere comes into contact with a cell culture located in one or more modules located in the first pump or in the connecting structure or in the second pump or in a combination of two or more thereof and said method comprises the step further of determining the effect of the test atmosphere on the cell culture, wherein a difference in the cell culture before and/or after exposure to the test atmosphere is indicative that the test atmosphere affects the cell culture.

[00148] Suitably, the modules are adapted for monitoring system conditions and/or for gas sampling and/or for gas characterization and said method comprises taking one or more measurements from the modules.

[00149] An apparatus configured or adapted to perform the method(s) described in this document is also described.

BRIEF DESCRIPTION OF FIGURES

[00150] Figure 1 is an illustration of a system 10 according to an embodiment of the present description. The first pump 40 according to an embodiment of the present disclosure is also described. The second pump 80 in accordance with an embodiment of the present disclosure is also described. The connection structure 50 according to an embodiment of the present description is also described.

[00151] Figures 2 and 3 are illustrations of modules 113, 213 according to the embodiments of the present description.

SOME ADVANTAGES

[00152] How the processes that affect the physicochemical properties of a test atmosphere in the respiratory tract, as well as the mode of interaction between the two (e.g., dose administration), are simulated by the physical and functional properties of the system , clinically relevant dosage information can be obtained in accordance with the embodiments of the present disclosure.

[00153] The effects of one or more test atmospheres in relation to one or more compartments of the respiratory tract can be studied according to the embodiments of the present description.

[00154] The effects of one or more test atmospheres in relation to one or more compartments of the respiratory tract can be studied simultaneously or in stages, as necessary, in accordance with the embodiments of the present description.

[00155] The modules that can be used in the system offer flexibility with respect to the test system exposed, the experimental evaluation criteria and the exposure parameters to be monitored according to the modalities of the present description.

[00156] Modules can be redesigned or changed according to specific requirements, without having to change the general structure and function of the system according to the embodiments of the present description.

[00157] For many applications, aerosol generation is driven by the system itself, which means that aerosol generators/smoking machines are not required according to the embodiments of the present description. This can help simplify the system structure.

[00158] In certain embodiments, the system may be modular in nature. This means that the various components - such as the pumps and the connecting frame, for example, can be exchanged individually and easily as required. Partial redesign, improvement or exchange of system parts according to specific requirements can be facilitated.

[00159] The mouth, air retention time and dynamic flow patterns that occur during breathing can be simulated according to embodiments of the present description.

[00160] The present description may, according to certain embodiments, take into account a retention period in the mouth during which the highly dense aerosol may age for up to several seconds before being drawn into the respiratory tract, together with a large volume of dilution air.

DETAILED DESCRIPTION

[00161] The practice of the present description employs, in certain embodiments, conventional techniques of engineering, microbiology, cell biology and biochemistry. Biological techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. CeIMs, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, IB. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994). Procedures employing commercially available kits and reagents will typically be used in accordance with manufacturer-defined protocols unless otherwise indicated.

[00162] The technical terms and expressions used in this document must, in most cases, be given the meaning that commonly applies to them in the relevant art. All definitions of terms used in this document apply to the entire contents of this application.

[00163] The term "comprising" does not exclude other elements or steps.

[00164] The indefinite article "one" or "an" does not exclude a plurality.

[00165] The term "and/or" means, for example, (a) or (b) or both (a) and (b).

[00166] The terms "comprising", "comprises" and "comprised", as used herein, are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open and do not exclude members , additional elements or method steps not mentioned. The term "consisting of" means that additional components are excluded and it has only the recited elements and nothing else.

[00167] The term "about", as used herein, when referring to a measurable value, such as a parameter, a quantity, a temporal duration, and the like, is intended to encompass variations to and from the specified value, in specific variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, and most preferably +/0.1% or less of and from the specified value, to the extent such variations are appropriate to carry out the description. It should be understood that the value to which the "about" modifier refers is also described, specifically and preferably.

[00168] Before discussing the embodiments in more detail, first an overview will be provided. The embodiments provide apparatus and methods that can be used in a variety of applications to study the respiratory tract. For example, the embodiments find utility in studying the deposition and/or condensation of one or more constituents present in a test atmosphere on internal surfaces of the apparatus, including their re-evaporation. Embodiments also provide for evaluation of test atmospheres that can be investigated during their 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 test atmospheres in relation to biological test systems present within the apparatus/system can be studied in the embodiments of the present description.

System

[00169] In one aspect, described herein is a system 10 for determining the interaction between a test atmosphere and a simulated respiratory tract, wherein said system comprises: (a) a first pump comprising: (i) a chamber configured to contain a first volume of gas comprising a test atmosphere; (ii) a first port adapted to receive and emit gas and comprising a valve for regulating the flow of gas through the first port, wherein said valve is movable between open and closed positions, wherein in the open position said valve is open to a test atmosphere or surrounding air; (iii) a second port adapted for exiting and receiving gas and comprising a valve for regulating the flow of gas through the second port, said valve being movable between open and closed positions; and (iv) a motor to control the operation of the first pump; a second pump comprising: (i) a chamber configured to contain a second volume of gas, wherein the first and second volumes of gas are different; (ii) a port adapted to receive and emit gas; (iii) a piston plate in the chamber, wherein said piston plate comprises one or more openings for the absorption or inflow of gas into the chamber, wherein one or more or each of the openings includes a valve that is movable between the open and closed positions and is capable of regulating the absorption or influx of gas; and (iv) a motor to control the operation of the first pump; (c) a connecting structure operable to transmit gas from the first pump to the second pump; and (d) one or more openings in the first pump or the second pump or in the walls of the connecting structure or a combination of two or more thereof, wherein said openings are capable of receiving a module for containing or storing a culture medium of cells or to monitor conditions in the chamber or for gas sampling or for gas characterization.

[00170] The gas may be a test atmosphere or may include a test atmosphere.

[00171] Figure 1 illustrates a system 10 according to an embodiment of the present description. The system 10 includes at least two pumps 40, 80. The 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 branched hollow structure 50. Each pump 40, 80 may be operated by its own individual motor 41, 81 or two or more pumps may be operated by the same engine 41, 81 as required. The complete system 10 may be located in a climate housing 11 equipped with a thermostat 12 to control the temperature in the housing 11. Pump chambers 40, 80 may be configured to represent the internal volumes of different compartments of a respiratory tract - such as a human or animal respiratory tract. They can be configured to provide a displacement volume at least as large as the most achievable volume absorption in the respective respiratory tract compartment. In particular, a (first) pump 40 may represent the volume of the oral and oropharyngeal cavity - such as a human or animal oral and oropharyngeal cavity. Another second pump 80 may represent the volume of the lung lumen or parts thereof - such as the lumen of individual lung lobes or smaller subunits, especially human or animal lung lumen or parts thereof. The branched hollow connecting structure 50 may represent the dimensions of the conducting airways - such as one or more of the nasopharyngeal cavity, hypopharynx, larynx, trachea, bronchus and bronchial structures to the respiratory bronchioles, especially the human conducting airways or animals. The branched hollow connecting structure 50 can represent the dimensions of the conducting airways - including the nasopharyngeal cavity, hypopharynx, larynx, trachea, bronchus and bronchial structures to the respiratory bronchioles. The dimensions, for example, the diameters and lengths, as well as the branching pattern of the different sub-parts of the connecting structure 50 may resemble the tree of the conducting airways.

[00172] As shown in Figure 1, the branched hollow structure 50 can be connected to a central opening 43, 89 in the base 44, 84 of the chamber 42, 82 of each pump 40, 80. In certain embodiments, multiple openings, holes or sockets 51 may be present at the base 44, 84 of the chamber(s) 42, 82, which may be arranged symmetrically around the central opening 43, 89. Directly at the connection between the pump 40 and the connection structure 50, a valve 44a may be used to allow sealing of the pump 40 from all other parts of the system.

[00173] The pump 40 representing the oral cavity may have one or more openings 43, through which the test and air dilution atmosphere(s) may exit the pump 40 toward the hollow branched structure 50. The entry and exit point of a test atmosphere 90 is normally located on the piston plate 45 of the pump 40, suitably in the center thereof. The same may be carried out via a hollow piston shaft 46, upon which a valve 44a - such as a three-way valve may be present. In embodiments, valve 44a may be closed or opened toward a source of test atmosphere or surrounding air. An array of one or more (e.g., a plurality) openings 47 through which surrounding air can enter the system is disposed in the piston plate 45, optionally in a radial arrangement. One or more valves 48 (e.g., a plurality) in one or more, or all, of the openings may be used to allow opening or closing of one or more of those openings 47. In certain embodiments, each opening 47 is controlled by a valve 48. In certain embodiments, an array of one or more (e.g., a plurality) openings through which surrounding air can enter the system may be disposed in the piston plate 84 of the second pump, optionally in a radial arrangement. One or more valves (e.g., a plurality) may be used to allow opening or closing of one or more such openings. In certain embodiments, each opening is controlled by a valve. In certain embodiments, an array of one or more (e.g., a plurality) openings through which surrounding air can enter the system may be disposed in the piston plate of the first and second pumps, optionally in a radial arrangement. Advantageously, the branched hollow structure 50 can be disconnected from the pumps 40, 80. Advantageously, the branched hollow structure 50 can be disassembled into its primary parts. This can allow easy access for placing or removing test systems and/or for cleaning.

[00174] The bases 44, 84 of the pumps 40, 80 can be removed to place/remove test systems and for cleaning.

[00175] In the base 44, 84 of each pump 40, 80, as well as in the different parts of the connecting structure 50, the openings, holes or sockets 51 - such as threaded or unthreaded openings, threaded or unthreaded holes or threaded sockets or non-threaded 113, 213 - can be located in it. The openings, holes or sockets 51 may be located in various positions - such as in the bases 44, 84 of one or more of the pumps 40, 80, or they may be arranged around the central opening 43, 89 or in the hollow branch structure 50 at various locations of choice, suitably on the underside of the branched hollow structure 50 or any combination thereof.

[00176] The openings, holes or sockets 51 may be used to allow mounting of various modules 112, 212 or devices thereon that may be used to monitor the operation of the system 10 and/or to conduct experiments and/or to collect samples and others. Examples of such modules 112, 212 or devices are shown in Figure 2 and Figure 3 and are described herein.

[00177] Advantageously, the pumps 40, 80 that are used in system 10 are therefore capable of functioning not only to transport test atmospheres, but they can also function as exposure chambers.

[00178] Figures 2 and 3 illustrate an embodiment of the description in which the openings, holes or sockets 51 are in the optional form of threaded openings, holes or sockets 112, 212. The one or more openings, holes or sockets 51 may contain a or more modules 112, 212. The use of the thread facilitates direct insertion and replacement of the module(s) 112, 212. The use of a thread in the openings, holes or sockets 51 is optional, as the modules can be configured so that they can be plugged or pushed into openings, holes or sockets 51 in sealing engagement therewith. Modules can be press fit. Tightness can be achieved through the use of O-rings and the like.

[00179] The modules 112, 212 that are used can be adapted for various purposes depending on the requirements of the system being configured. For example, modules 112, 212 may be adapted to contain or store a cell culture medium or to monitor conditions in chamber 42, 82 or for gas or liquid sampling or for gas characterization and the like. The modules 112, 212 may be located in the base 44, 84 of the first 40 and/or second 80 pump and/or in the walls of the connecting structure 50. In a particular embodiment, the one or more modules 112, 212 may be configured to contain or store a cell culture medium. According to this embodiment, the one or more modules 112, 212 may be receptacles capable of holding a liquid or solution. The cell culture medium may comprise or be in contact with a cell culture - such as a two-dimensional or three-dimensional cell culture. In certain embodiments, the module(s) 113, 213 adapted to contain or store a cell culture medium further comprises a microfluidic channel and, optionally, a microfluidic pump connected thereto. (s). The modules 113, 213 will generally be positioned in a horizontal plane on one or more of the first pump 40 or the second pump 80 or the walls of the connecting structure 50.

[00180] As can be seen in Figures 2 and 3, the openings, holes or sockets 51 can optionally be configured to contain 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 may be used to monitor internal system conditions or for test atmosphere characterization or for sampling and the like. The operation of the device(s) 116, 216 may be controlled by a computer 117, 217. The threaded hole or socket 113, 213 and/or the modules 112, 212 may be adapted for use as a cultivation chamber 115 , 215 in which biological test systems (e.g., cultures of organotypic human respiratory tract epithelium cells, as described herein), can be placed for exposure to the test atmosphere. The threaded openings, holes or sockets 113, 213 can be adapted to contain capture agents in which the test atmosphere can be sampled for analysis. Sampling of cell culture medium or capture agent during exposure to the test atmosphere can be achieved by various means - including through the use of a microfluidic pump system 211. Modules 112, 212 holding quartz crystal microbalances (QCM, 219) can be used. Modules 112, 212 to which probes 214, chambers 215 or QCMs 219 can be mounted can be inserted into any chamber 42, 82 or within the hollow branched structure 50 of the system. The operation of the QCM(s) 219 may be controlled by a computer 220. The system 10 may be controlled completely or partially by a computer 13 as needed. System 10 may be partially or completely automated.

[00181] System 10 may include one or more (e.g., a plurality of) first pumps. System 10 may include one or more (e.g., a plurality of) second pumps. System 10 may include one or more (e.g., a plurality of) first pumps and one or more second pumps.

First bomb

[00182] In one aspect, there is described a pump - suitably a piston pump - for displacing a volume of gas 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 a cell culture medium or for monitoring conditions in the chamber or for gas sampling or for gas characterization; (ii) a first port for receiving and emitting the gas, when contained in the chamber, and comprising a first valve for regulating the flow of gas through the first port, wherein said first valve is movable between the open and closed positions, in that, in the open position, said valve can be opened towards a test atmosphere or surrounding air; (iii) a second port for emitting and receiving gas when contained in the chamber, and comprising a second valve for regulating the flow of gas through the second port, said valve being movable between open and closed positions; and (iv) a piston plate in the chamber, wherein said piston plate comprises one or more openings for the absorption or inflow of gas into the chamber wherein one or more, or each, of the openings includes a valve that is movable between the open and closed positions and is capable of regulating the absorption or influx of gas.

[00183] Also described is a pump for displacing a volume of gas comprising: (i) a chamber configured to contain a volume of gas and containing a piston plate comprising one or more openings for the absorption or inflow of gas into the chamber, wherein one or more, or each, of the openings includes a valve that is movable between the open and closed positions and is capable of regulating the absorption or influx of gas; (ii) a first port for receiving and comprising a first valve for regulating the flow of gas through the first port, said first valve being movable between open and closed positions; and (iii) a second port for emitting gas when contained in the chamber, and comprising a second valve for regulating the flow of gas through the second port, said valve being movable between open and closed positions.

[00184] As shown in Figure 1, system 10 may contain the first pump 40 to displace a volume of gas. The first pump 40 is described herein as a separate aspect of the present disclosure and its use is not limited to use in the system 10 described herein.

[00185] The first pump may be a primary pump, so called because of its location in the system as the entry point for a gas. It comprises a chamber 42 (e.g., a cylinder) configured to contain a volume of gas and comprises a base 44 and one or more openings 43 capable of receiving a module - such as a threaded or non-threaded module 113, 213 in a hole or a socket 112, 212, as shown in Figures 2 and 3 and as described herein. It also contains a first port 90 for receiving and emitting the gas when contained in the chamber 42, and comprises a first valve 44a - such as a three-way valve for regulating the flow of gas through the first port 90, wherein the Said first valve 44a is movable between open and closed positions, wherein in the open position valve 44a can be opened towards a test atmosphere or surrounding air. It also contains a second port 43 for emitting and receiving gas when contained in chamber 42. Suitably, the second port 43 is configured as an opening. In the connection between the first pump 40 and the connecting structure 50, a second valve 49 at the location of the second port 43 allows sealing of the pump 40 from other parts of the system. Second valve 49 may be used to regulate the flow of gas through second port 43, wherein said second valve 49 is movable between open and closed positions. As can be seen in Figure 1, the pump 40 may be a piston pump comprising a piston plate 45. One or more of the openings, holes or sockets 51 in the chamber 42 may be threaded 112, 212 or unthreaded. One or more of the openings, holes or sockets 51 in the chamber 42 may include a module 113, 213 - such as a threaded or non-threaded module 113, 213 as discussed herein. The pump 40 further comprises a motor 41 for controlling the operation of the pump. The pumping pressure of the engine 41 may correspond to atmospheric pressure or it may be above or below atmospheric pressure as required. In certain embodiments, the pumping pressure of the engine 41 may be above or below atmospheric pressure to displace a test atmosphere. The displacement volume of the pump 40 may be between about 0 and 100 ml or between about 1 and 100 ml. The chamber 42 of the pump 40 may have a volume of up to about 100 ml. The pump 40 can be manufactured from various materials that are known in the art - such as stainless steel. Appropriately, chamber 42 is a cylinder. Suitably, the chamber 42 may be made of glass. The piston plate 45 of the pump 40 comprises one or more openings 47 for the absorption or inflow of gas. The one or more of the openings 47 include a valve 48 that is movable between open and closed positions and is capable of regulating the absorption or influx of gas.

[00186] As also shown in Figure 1, pump 40 may be a piston pump for displacing a volume of gas. The piston pump comprises a chamber 42 (e.g., a cylinder - such as a glass cylinder) configured to contain a volume of gas and containing a piston plate 45 comprising one or more openings 47 for the absorption or influx of gas into the chamber 42. One or more of the openings 47 or each of the openings 47 comprises a valve 48 for regulating the absorption or influx of gas through the openings 47.

[00187] It 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 open positions and closed. It also includes a second port 43 for emitting the gas when contained in the chamber 42, wherein the second port 43 further comprises a second valve for regulating the flow of gas through the second port 43, wherein said valve is movable between open and closed positions. The chamber 42 may include a base 44 and a further opening 43. The base may additionally include one or more openings, holes or sockets 51 which may be threaded 119 or non-threaded and/or may include a module 113, 213, as discussed in this document. A connecting structure 50 - such as a hollow connecting structure - may be attached to the second port 43. The pump 40 may additionally include a motor 41 in which the pumping pressure corresponds to atmospheric pressure or above or below atmospheric pressure. The displacement volume of the pump 40 may be between about 0 and 100 ml or between about 1 and 100 ml. The chamber 42 of the pump 40 may have a volume of about 100 ml.

[00188] A system comprising two or more first pumps is contemplated. The use of the first two or more pumps in a system to determine the interaction between a test atmosphere and a simulated respiratory tract is also contemplated.

[00189] A method for culturing a cell in the first pump is also contemplated. The use of the first pump to grow a cell is also described.

Second bomb

[00190] In a further aspect, there is described a pump for displacing a volume of gas comprising: (i) a chamber configured to contain a volume of gas, said chamber comprising a base and one or more modules for containing or storing a cell culture medium or for monitoring conditions in the chamber or for gas sampling or for gas characterization; and (ii) an operable port for receiving and emitting the gas. As shown in Figure 1, the system may contain a second pump 80 for displacing a volume of air comprising a chamber 82 - such as a cylinder - configured to contain a volume of gas, said chamber 82 comprising a base 84 and a or more modules 113, 213 for containing or storing a cell culture medium or for monitoring conditions in chamber 82 or for gas sampling or for gas characterization and the like. The second pump may be a secondary pump.

[00191] The second pump 80 is described herein as a separate aspect of the present description and its use is not limited to use in the system 10 described herein.

[00192] The second pump 80 additionally includes a port 89 operable to receive and emit the gas. As shown in Figure 1, the second pump 80 may be a piston pump comprising a piston plate 83. The piston plate 83 may be free of any openings or holes. Modules 113, 213 - such as threaded or non-threaded modules 113, 213 as discussed herein - may be located in the base 84 of the chamber 82. A connection structure 50 - such as a hollow connection structure - may be attached to the port 89. The second pump 80 further comprises a motor 81. The pumping pressure of the second pump 80 will generally correspond to atmospheric pressure or above or below atmospheric pressure. The displacement volume of the pump may be between about 0 and about 1000 ml or between about 0 and about 4000 ml or between about 1 and about 1000 ml or between about 1 and about 4000 ml. The volume of chamber 82 may represent the volume of the lung lumen or a portion thereof. In certain embodiments, an array of one or more (e.g., a plurality) openings through which surrounding air can enter the system may be disposed in the piston plate 84, optionally in a radial arrangement. One or more valves (e.g., a plurality) may be used to allow opening or closing of one or more such openings. In certain embodiments, each opening is controlled by a valve.

[00193] A system comprising two or more second pumps is contemplated. The use of two or more second pumps in a system to determine the interaction between a test atmosphere and a simulated respiratory tract is also contemplated.

[00194] A method for culturing a cell in the second pump is also contemplated. The use of the second pump to cultivate a cell is also described.

Connection structure

[00195] A connecting structure operable to transmit or transport a gas between two or more pumps is also described. The connecting structure may be a tube or a tube or a pipe or the like through which a gas may be conducted or transported. The connecting structure can be adapted to connect at least two pumps to transmit or transport a gas between them. The connecting structure may attach to a first pump at the second port of the first pump described herein and at the port of the second pump described herein. The connecting structure may include a hollow channel and one or more openings - such as threaded or unthreaded openings - in the walls of the connecting structure. The connection framework can be used in the systems and methods described in this document. The system may include a connecting structure adapted to join at least two pumps to transmit a gas therebetween.

[00196] An embodiment of the connection structure 50 is shown in Figure 1. The connection structure will generally comprise a hollow channel. The same will generally be branched. In embodiments, each terminal branch of the connecting structure may be joined to separate pumps 40, 80 when contained in a system. The connecting structure 50 one or more openings - especially threaded or non-threaded openings - in the walls of the connecting structure. The threaded or non-threaded openings may contain a module 112, 212. The module 112, 212 may be adapted to contain a culture medium and/or monitor system conditions and/or for gas sampling and/or for gas characterization. , as discussed in this document. The connecting structure may, in certain embodiments, be branched with two or more branches. Each terminal branch of the connecting structure can be joined to a separate pump. The connecting structure may represent the volume of the lung's conducting airways. The connecting structure 50 can be made of various materials. In certain embodiments, the use of stainless steel is preferred.

[00197] A method for cultivating a cell comprising the connection structure is also contemplated. The use of the connection structure for culturing a cell is also described.

System operation and function

[00198] The system can be controlled completely or partially by a computer as necessary. This may allow synchronized operation of some or all pumps and valves. This may allow synchronized operation of some or all elements of the system. The computer can be used to set the stroke length of one or more of the pumps and/or the stroke speed of one or more of the pumps. The computer can be used to control the system temperature.

[00199] The operation of an embodiment of the system 10, as described in Figure 1, will now be described. In the rest state, the piston of the first pump 40 is in a downward stroke position, the position of the second pump 80 is in a position to retain a defined volume of gas in the chamber 82, and the valves 48, 49 are closed. A test atmosphere is brought to pump 40, which may represent the oral cavity. This inflow may be driven by pump 40 and may enter chamber 42 via hollow piston shaft 46. Hollow piston shaft 46 may be connected directly to the test atmosphere source. Once the pump 40 has completed the upward stroke, the valve 44a, which may be a three-way valve in the central opening on top of the hollow piston shaft 46 closes, and the valves 48 which regulate the inflow of surrounding air through of the openings 47 in the piston plate 45 and the valve 49 at the inlet to the connecting structure 50 open.

[00200] The test atmosphere is withdrawn from the chamber 42, which may represent the oral cavity through the connecting structure 50, which may represent the conducting airways. This may be driven by the upward stroke of the second pump 80 which may represent the lung lumen or parts thereof. Since the total displacement volume of the second pump 80 may be a multiple of the volume of the pump 40, the chamber 42 of the pump 40 as well as at least parts of the connecting structure 50 may be filled with the surrounding air, which may enter the first pump 40 through openings 47 in piston plate 45.

[00201] In the first pump 40, the valve 44a on top of the piston shaft 46 can open towards the surroundings. The valves 48 in the piston plate 45 can close and the pump 40 makes a downward stroke. In its downstroke position, this pump 40 may form a sealed connection between the hollow piston shaft 46 and the opening 43 in the base 44. The sealed connection may be achieved by means of a gasket 53. The gasket 53 may be located in the base 44 or in the piston plate 45. After a defined "breath hold time", the second pump 80 can then perform a downward stroke, thereby moving the test atmosphere through the connecting frame 50 and through the shaft piston 46 of the first pump 40, directly to the surroundings. With the pump 40 remaining in its downstroke position and the valve 44a remaining open toward the surroundings, the second pump(s) 80 may perform one or more (e.g., several) cycles of breathing surrounding air before valve 44a opens again towards the test atmosphere source and the next test atmosphere inhalation cycle begins.

[00202] In a further aspect, there is provided a method of comprising: (a) providing a pump - such as the first pump described herein - comprising a chamber; (b) withdrawing a gas - such as a test atmosphere - from the pump in a connecting structure that joins the pump to an additional pump - such as the second pump described herein; (c) filling the pump provided in step (a) and at least a portion of the connecting structure with the surrounding air; (d) maintain gas in the additional pump and connecting structure for a defined period of time; (e) displacing the gas in the connecting structure and in the pump provided in step (a) using the additional pump; and (f) performing one or more cycles of pumping the surrounding air into the additional pump.

[00203] In a further aspect, a method is provided comprising: (a) supplying a gas - such as a test atmosphere - to a chamber of a bomb - such as the first bomb described herein; (b) withdrawing gas from the pump described in step (a) into a connecting structure that joins the pump to an additional pump - such as the second pump described in this document; (c) filling the pump of step (a) and at least a portion of the connecting structure with the surrounding air; (d) maintain gas in the additional pump and connecting structure for a defined period of time; (e) displacing the gas through the connecting structure and into the pump of step (a) using the additional pump; and (f) performing one or more cycles of pumping the surrounding air into the additional pump.

Test atmosphere generation

[00204] Test atmospheres - such as aerosols - to be studied using system 10 and described methods can be generated through various means. For many applications, for example for testing tobacco products or common medical inhalers and the like, the test atmosphere generation can be driven by the system itself, i.e. the primary or secondary pumps themselves generate the negative pressure necessary for generation and extraction of the test aerosol, which means that the use of aerosol generators/smoking machines is advantageously not necessary.

[00205] The test atmosphere may be an environmental sample of gases or aerosols, for example, to monitor room air quality, occupational exposures, or environmental pollution in the vicinity of industrial sites. In this particular case, the test atmosphere is not generated, but proved by the action of the system.

[00206] The test atmosphere may be an aerosol - such as smoke or may be derived from smoke. As used herein, the term 'smoking' is used to describe a type of aerosol that is produced by smoking articles, such as cigarettes, or by combustion of an aerosol-forming material. Smoke includes several agents, which can be provided as individual compounds for study if necessary. 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'- nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl, nitrogen monoxide (NO), nitrous oxide (NOx), hydrocyanic acid, ammonia, arsenic, cadmium, chromium, lead, nickel, selenium and mercury.

[00207] When the aerosol is smoke, the system 10 may optionally be connected to a smoking machine. Suitably, the smoking machine holds and lights cigarettes with the pumps provided by the system or pumps of the present description. A set number of puffs per cigarette and a set number of puffs per minute of exposure could be used and the number of cigarettes was varied to adjust for exposure times. Reference cigarettes - such as 3R4F reference cigarettes - can be used as the source of smoke and smoked in the smoking machine in basic compliance with the smoking regime of the International Organization for Standardization (ISO 2000).

[00208] The use of a control atmosphere is also contemplated - such as an atmosphere that does not contain the test atmosphere. Using the control atmosphere can help determine the effect of the test atmosphere compared to the control atmosphere.

[00209] System 10 may be connected to a smoking machine via a suitable conduit, which provides a flow path for smoke to system 10. Smoke may be transferred through the conduit with or without a carrier gas, just like air. When a carrier gas is used, the conduit preferably comprises an inlet for introducing the carrier gas into the conduit for mixing with the smoke stream. The conduit may include at least one inlet for introducing or injecting a reference standard into the system 10) - such as nicotine - for calibration purposes. The smoke flow will generally be controlled by the system or pumps of the present description.

[00210] The smoking machine can be a linear or rotary smoking machine. Suitably, the smoking machine is operated to smoke a plurality of smoking articles simultaneously, so that the cumulative smoke from the plurality of smoking articles can be collected and analyzed. Smoking machines suitable for use in the present disclosure are well known to the person skilled in the art.

[00211] The system 10 and the method described herein can be used to perform an analysis of mainstream smoke generated by a smoking article during the smoke test. "Mainstream smoke" refers to smoke that is drawn through the smoking article and would be inhaled by the consumer during use.

[00212] The test atmosphere may be from an "aerosol generating device", which is a device that interacts with an aerosol-forming substrate to generate an aerosol. An example of an aerosol is smoke. The aerosol-forming substrate may be part of an aerosol-generating article. An aerosol generating device may include one or more components suitable for generating an aerosol from an aerosol generating substrate. An aerosol generating device may be an electrically heated aerosol generating device, which is an aerosol generating device comprising a heater that is operated by electrical power to heat an aerosol forming substrate of an aerosol generating article to generate an aerosol. The aerosol-generating device may be a gas-heated aerosol-generating device, a device heated by a carbonaceous heat source, another exothermic chemical reaction, or a heat sink. Other suitable means for generating an aerosol are well known in the art. An aerosol-generating device may be a device that interacts with an aerosol-forming substrate of an aerosol-generating article to generate an aerosol that is directly inhalable into a user's lungs through the user's mouth.

[00213] Another example of an "aerosol generating device" is an inhalation device (inhaler) which is generally used to administer an aerosol containing an active ingredient - such as a medically active compound. Such inhalation devices are generally used for delivering aerosolized medications to the respiratory tract. They can be used to treat respiratory diseases and other diseases. Such inhalers are well known in the art and are generally of the pressurized metered type, dry powder type or nebulizer type. Generally, the drug is in the form of a pressurized formulation containing fine particles of one or more medicinal compounds suspended in a liquefied propellant, or a solution of one or more compounds dissolved in a propellant/co-solvent system. Such formulations are well known in the art.

[00214] As used herein, the term 'aerosol-forming substrate' refers 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. An aerosol-forming substrate may be adsorbed, coated, impregnated, or otherwise loaded onto a carrier or support. The aerosol-forming substrate may conveniently be part of an aerosol-generating article or a smoking article. In certain applications, the aerosol-forming substrate is contained in an aerosol-generating article, for example, a column-shaped aerosol-generating article, such as a heated aerosol-generating article or heated cigarette. The aerosol-generating article is of a suitable size and shape to engage with the aerosol-generating device so as to bring the aerosol-forming substrate into contact with the heater.

[00215] An aerosol-forming substrate can comprise medically active compounds - such as antibiotic or anti-inflammatory agents that can be administered to a patient via the respiratory tract. Numerous medical inhalation devices (inhalers) are known and routinely prescribed to treat various respiratory and non-respiratory tract-related illnesses.

[00216] An aerosol-forming substrate may comprise nicotine. An aerosol-forming substrate may comprise tobacco. The aerosol-forming substrate may comprise, for example, a tobacco-containing material containing volatile tobacco flavoring compounds, which are released from the aerosol-forming substrate upon heating. In certain embodiments, an aerosol-forming substrate may comprise homogenized tobacco material, e.g., reconstituted tobacco. As used herein, "homogenized tobacco material" refers to the material formed by agglomerating particulate tobacco. The homogenized tobacco may be in the form of a sheet. The homogenized tobacco material may have an aerosol-forming content greater than 5% by dry weight. Alternatively, the homogenized tobacco material may have an aerosol former content of between 5% and 30% by weight based on dry weight. Sheets of homogenized tobacco material may be formed by an agglomeration of tobacco particles obtained by grinding, or otherwise crushing, the blades and stems of the tobacco leaf. Alternatively or additionally, the sheets of homogenized tobacco material may contain one or more of tobacco dust, tobacco residue and other particulate tobacco by-products formed during, for example, the treatment, handling and shipping of tobacco. Sheets of homogenized tobacco material may comprise one or more intrinsic binders, i.e., endogenous tobacco binders, one or more extrinsic binders, i.e., exogenous tobacco binders, or a combination thereof, in order to help agglomerate tobacco particles. tobacco; Alternatively or additionally, sheets of homogenized tobacco material may contain other additives including, but not limited to, fibers and tobacco and non-tobacco, aerosol formers, humectants, plasticizers, flavorings, fillers, aqueous and non-aqueous solvents and combinations thereof.

Cell cultures

[00217] Cell cultures for use in the present description include two-dimensional and three-dimensional cell cultures. As described herein, the cell culture will generally be contained or grown in the one or more modules of one or more pumps and/or connection structure. The cell culture 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 in different positions around the one or more pumps and/or the connecting structure and/or the system, so that the effect of the test atmosphere on the cell cultures in these different locations - that mimic the respiratory tract - can be determined. Two-dimensional cell cultures involve the growth of cells in flat layers on plastic surfaces, which allows the study of various aspects of cellular physiology and responses to stimuli - such as test atmosphere(s), but they do not reflect the true structure and architecture of an organ. In two-dimensional monolayers, the extracellular matrix, cell-cell and cell-matrix interactions, which are essential for cellular differentiation, proliferation and functions, are lost. Three-dimensional culture systems can form functional tissue with features similar to those observed in vivo. Compared to two-dimensional culture systems, three-dimensional cell culture allows cells to interact with their surroundings in all three dimensions and be more physiologically relevant. Such cells can show improvements in viability, proliferation, differentiation, morphology, response to stimuli, drug metabolism, gene expression and protein synthesis and the like. Three-dimensional cell culture can produce specific tissue-like structures and mimic the functions and responses of true tissues in a way that is more physiologically relevant than traditional two-dimensional cell monolayers. Several three-dimensional tissues that mimic human organs are commercially available. Three-dimensional organotypic lung tissues, which are of specific interest in the context of the present description, can be prepared using primary human cells cultured at an air-liquid interface (ALI), where these cells will differentiate and form a functional tissue. . These three-dimensional tissues have close morphological similarity and metabolic characteristics to human bronchial tissues. They are composed of basal, goblet and ciliated cells arranged in a pseudostratified structure. Similar to the lung, actively beating cilia are present, allowing study of their function and activity. Similar levels of xenobiotic mRNA-decoding enzyme were found in these three-dimensional ALI cultures compared to human lungs. Furthermore, these tissues can be maintained in vitro for a long period of time. This three-dimensional model of lung tissue is an appropriate model for exploring the effects of test atmospheres and the like in accordance with the present description.

[00218] The term "three-dimensional cell culture" includes any method that provides for the culture of a cell in three dimensions, with or without the use of a matrix or skeleton. A number of different three-dimensional cell culture methods have been developed, including spheroid cultures and organotypic cultures.

Spheroids

[00219] The term "spheroid" takes on the meaning, as commonly understood in the art, that it is a single cell that divides into a ball of cells in three dimensions, or an aggregation of multiple cells in three dimensions, with or without the use of a matrix or skeleton to support the growth of three-dimensional cells within the spheroid. The three-dimensional spheroid can be an adherent spheroid or a spheroid grown in suspension. Several different systems for growing spheroids for use in the present disclosure are available, including spheroids grown as aggregates, e.g., in nanoculture plates, in suspension culture, in gels, on poly-HEMA coated plastic, through encapsulation of cells or as aggregates through a suspended droplet system. Other methods include the use of centrifuge flasks, spin systems, concave plate methods, and liquid overlay. Bioreactors can also be adapted for use in three-dimensional spheroid cell culture. In one embodiment, the method used is a hanging droplet system - such as the GravityPLUS Hanging Drop System (InSphero). This method involves the use of the GravityTRAP ULA Plate which is a non-adhesive coated microtiter plate designed for the production of spheroids. Spheroid maturation generally occurs within 2 to 5 days after seeding, depending on 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, spheroids are grown in Corning® spheroid microplates.

[00220] Three-dimensional cell culture matrices or skeleton can be used for spheroid culture. These are often porous substrates that can support three-dimensional cell differentiation and growth. A variety of materials have been developed to produce three-dimensional scaffolds with differences in physical appearance, porosity, permeability, mechanical characteristics, and surface morphology at the nanometer scale. Examples of such materials include: collagen gels, sponges or biogels; fibrin; fibronectin; laminin; alginates, hydrogels; cross-linked glycosaminoglycan; polymer-based skeletons, synthetic skeletons; peptide scaffolds; and chitosan composite scaffolds.

[00221] Three-dimensional spheroids more closely resemble in vivo tissue in terms of their cellular communication and development of extracellular matrices. These matrices help cells move within the spheroid similar to the way cells move in living tissue. Spheroids are therefore greatly improved models for differentiation, survival, cell migration, cell polarization, gene expression and growth.

[00222] Spheroids can be collected and studied using several methods well known in the art, including colorimetric assays, fluorescence and luminescence measured with a plate reader or can be easily observed by microscopy. Additional techniques include western, northern or southern blot, histological techniques (e.g., immunohistochemistry, in situ hybridization, immunofluorescence) and the like. The use of optical imaging methods - such as inverse bright field microscopy, fluorescence microscopy, single photon emission computed tomography (SPECT), positron emission topography (PET), magnetic resonance imaging (MRI) and luminescence imaging techniques Cerenkov (CLI) is also included.

[00223] Applications of the use of three-dimensional spheroids include the study of the proliferation of cells and tissues in vitro in an environment that approximates that found in vivo, the screening of compounds and test atmospheres, toxicology assays and clinical trials and the like.

[00224] 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 in large numbers and can form alveoli-like structures and acquire mature lung epithelial phenotypes.

Cellular sources

[00225] Lung cells and cell lines for use in the present description can be isolated from a tissue or fluid using methods that are well known in the art. They can be differentiated from stem cells - such as embryonic stem cells or induced pluripotent stem cells or directly differentiated from somatic cells. Cells and cell lines may be derived from humans or animals or from human or animal cells, including any of several mammalian species, suitably human, but including rat, mouse, pig, rabbit and non-human primates and the like. Cells and cell lines can be obtained from commercial sources. In certain embodiments, the use of human cells is desirable.

[00226] Lung cells - including lung epithelial cells - are a cell type of interest. Bronchial and/or airway epithelial cells are of particular use in the present disclosure. Human bronchial epithelial cells can be collected by brushing donor lungs during a bronchoscopy procedure. In one embodiment, the lung cells are Normal Human Bronchial Epithelial Cells (NHBE). Lung epithelial cells can be cultured as a monolayer of undifferentiated cells or further developed into a tissue similar to organotypic lung epithelium at an air-liquid interface. Cells can be established at an air-liquid interface using the following methodology. Briefly, epithelial cells can be grown in a flask to increase cell numbers. After a period of incubation, the cells are separated from the flask, counted and seeded onto inserts. In these inserts, the cells are incubated with the medium on the apical and basal sides. This phase will ensure that the cells divide and completely cover the insert to form an epithelium. Then the apical medium is removed, the basal medium is retained and replaced with a more complete medium. The cultures are incubated in this way for a further period of time. Meanwhile, the cells will differentiate into 3 cell types: basal cells, goblet cells, and ciliated cells. At the end of maturation, the crops are ready for use. The use of the air-liquid interface for culturing human nasal epithelial cells is described in J Vis Exp.2013; (80): 50646.

[00227] Lung epithelial cells can be obtained from humans or animals with different pathologies, including individuals classified as smokers or non-smokers.

Essay

[00228] The present description can be used for a variety of applications to study the impact of test atmospheres on a simulated respiratory tract. For example, the present disclosure may be used in the study of in vitro inhalation toxicology, investigation of aerosol dynamics in the respiratory tract (e.g., deposition and absorption of aerosol particles from gases in cell cultures), or investigation of metabolic activity or transport of test atmosphere(s) (e.g., aerosol molecules) across the epithelium of the respiratory tract. The present disclosure may be used to test the effect of aerosol(s), smoking or tobacco products or the effect of inhalers - such as medical inhalers. The present disclosure may be used to test the effect of aerosol(s), smoking products or tobacco or the effect of medical inhalers on the cells of one or more parts of the respiratory tract.

[00229] One aspect relates to a method for determining the effect of a test atmosphere on a cell culture - such as one or more cell cultures - contained in a simulated respiratory tract comprising: (a) providing the system, described herein, wherein the system contains a cell culture in one or more of the modules; and (b) comparing the cell culture before and/or after exposure to the test atmosphere, wherein a difference between the cell culture before and/or after exposing the cells to the test atmosphere is indicative that the test atmosphere test affects cell culture.

[00230] In the embodiment where a difference between the cell culture is determined after exposing the cells to the test atmosphere, the culture of cells exposed to the test atmosphere can be compared to a culture of cells that were not exposed to an atmosphere of test or to a culture of cells that have been exposed to a control atmosphere - such as an atmosphere that does not contain the test atmosphere. According to this embodiment, a difference between the culture of cells exposed to the test atmosphere and the culture of cells not exposed to the test atmosphere or a difference between the culture of cells exposed to the test atmosphere and a culture of cells exposed to a Control atmosphere - such as an atmosphere that does not contain the test atmosphere - is indicative that the test atmosphere affects the cell culture.

[00231] Another aspect relates to a method for simulating the interaction between a test atmosphere and a simulated respiratory tract in the system described herein comprising: (a) with the first valve of the first pump open and the second valve of the first pump closed, supplying a gas comprising a test atmosphere to the first pump through the first port; (b) closing the first valve and opening the second valve of the first pump and closing the valves on the piston plate of the first pump; (c) operating the second pump to draw the test atmosphere into the connecting structure and filling the chamber of the first pump and the connecting structure with the surrounding air; (d) opening the first valve of the first pump toward the surrounding 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 displace the test atmosphere through the connecting structure and through the first valve of the first pump.

[00232] Another aspect relates to a method for determining the effect of a test atmosphere on a simulated respiratory tract in the system described herein comprising: (a) with the first valve of the first pump open and the second valve of the first pump closed, supplying a gas comprising a test atmosphere to the first pump through the first port; (b) closing the first valve and opening the second valve of the first pump and closing the valves on the piston plate of the first pump; (c) operating the second pump to extract the test atmosphere through the connecting structure and flushing the first pump chamber and the connecting structure with the surrounding air; (d) opening the first valve of the first pump toward the surrounding 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 displace the test atmosphere through the connecting structure and through the first valve of the first pump; wherein the test atmosphere comes into contact with a cell culture located in one or more modules located in the first pump or in the connecting structure or in the second pump or in a combination of two or more thereof and said method comprises the additional step of determining the effect of the test atmosphere on the cell culture, wherein a difference in the cell culture before and/or after exposure to the test atmosphere is indicative that the test atmosphere affects the cell culture.

[00233] In the embodiment where a difference between the cell culture is determined after exposing the cells to the test atmosphere, the culture of cells exposed to the test atmosphere can be compared to a culture of cells that were not exposed to an atmosphere of test or to a culture of cells that have been exposed to a control atmosphere - such as an atmosphere that does not contain the test atmosphere. According to this embodiment, a difference between the culture of cells exposed to the test atmosphere and the culture of cells not exposed to the test atmosphere or a difference between the culture of cells exposed to the test atmosphere and a culture of cells exposed to a Control atmosphere - such as an atmosphere that does not contain the test atmosphere - is indicative that the test atmosphere affects the cell culture.

[00234] A further aspect relates to a method for simulating the interaction between a test atmosphere and a simulated respiratory tract comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) withdrawing the test atmosphere from the first pump to a connecting structure joining the first pump to a second pump; (c) filling the first pump and at least a portion of the connecting structure with the surrounding air; (d) retain the test atmosphere in the second pump and connecting structure for a defined period of time; (e) moving the test atmosphere to the connecting structure and the first pump using the second pump; and (f) performing one or more cycles of pumping the surrounding air into the second pump; wherein the test atmosphere comes into contact with a cell culture located in the first pump or connecting structure or in the second pump or a combination of two or more.

[00235] Another aspect relates to a method for determining the effect of a test atmosphere on a simulated respiratory tract comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) withdrawing the test atmosphere from the first pump to a connecting structure joining the first pump to a second pump; (c) filling the first pump and at least a portion of the connecting structure with the surrounding air; (d) maintain the test atmosphere in the second pump and the connecting structure for a defined period of time; (e) displacing the test atmosphere through the connecting structure and the first pump using the second pump; and (f) performing one or more cycles of pumping the surrounding air into the second pump; wherein the test atmosphere comes into contact with a cell culture located in one or more modules located in the first pump or in the connecting structure or in the second pump or in a combination of two or more thereof and said method comprises the step further of determining the effect of the test atmosphere on the cell culture, wherein a difference in the cell culture before and/or after exposure to the test atmosphere is indicative that the test atmosphere affects the cell culture. In the embodiment where a difference between the cell culture is determined after exposing the cells to the test atmosphere, the culture of cells exposed to the test atmosphere can be compared to a culture of cells that were not exposed to a test atmosphere or to a culture of cells that have been exposed to a control atmosphere - such as an atmosphere that does not contain the test atmosphere. According to this embodiment, a difference between the culture of cells exposed to the test atmosphere and the culture of cells not exposed to the test atmosphere or a difference between the culture of cells exposed to the test atmosphere and a culture of cells exposed to a Control atmosphere - such as an atmosphere that does not contain the test atmosphere - is indicative that the test atmosphere affects the cell culture.

[00236] The effect of the test atmosphere(s) can be studied in the presence of one or more agents. The agent(s) may include, but are not limited to, a drug, a toxin, a pathogen, a protein, a nucleic acid, an antigen, an antibody, and a chemical compound etc. Examples of the effects that can be measured include oxygen consumption, carbon dioxide production, cell viability, expression of a protein, enzyme activity, penetration, permeability barrier function, surfactant production, cytokine response, transporter function, expression of cytochrome P450, albumin secretion, toxicology and the like.

[00237] A plurality of tests can be performed in parallel with different concentrations of the test atmosphere and/or agent to obtain a differential response to the various concentrations.

[00238] The agent can be any test compound of interest and includes small organic compounds, polypeptides, peptides, higher molecular weight carbohydrates, polynucleotides, fatty acids and lipids, aerosol or one or more components of an aerosol and the like. Test compounds can be screened individually or in sets or combinatorial libraries of the compounds. Test compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be used. Naturally or synthetically produced libraries and compounds that are modified through conventional chemical, physical, and biochemical means can be used to produce combinatorial libraries. Known pharmacological agents can be subjected to targeted or random chemical modifications such as acylation, alkylation, esterification, acidification to produce structural analogues for screening.

[00239] One or more variables that can be measured include cell elements, subcellular material, subcellular components or cellular products. By way of example, the toxicology of the test atmosphere can be measured. Aerosol dynamics in the respiratory tract (e.g. deposition of aerosol particles and absorption of gases in cell cultures) can be measured. As another example, metabolic activity and/or transport of molecules across respiratory tract epithelia can be studied.

Computer

[00240] Also described is a computer program that, when executed by a computer/processor, is operable to control the computer to perform one or more of the methods described herein.

[00241] One skilled in the art will readily recognize that the steps of several of the methods described above can be performed by programmed computers. In this document, some embodiments are also intended to cover program storage devices, e.g., digital data storage media, which are machine- or computer-readable and encode machine-executable or computer-executable instruction programs, wherein said instructions perform some or all of the steps of said methods described above. Program storage devices may be, for example, digital memories, magnetic storage media such as magnetic disks and magnetic tapes, hard drives or optically readable digital data storage media. Embodiments are also intended to cover computers programmed to perform some or all of the steps of the methods described above.

[00242] The functions of the various elements, including the processors or logic, can be provided through the use of dedicated hardware, as well as hardware capable of executing software in association with appropriate software. When provided by a processor, functions may be provided by a single dedicated processor, a single shared processor, or a plurality of individual processors, some of which may be shared. Furthermore, explicit use of the term "processor" or "controller" or "logic" should not be construed to refer exclusively to hardware capable of executing software and may implicitly include, without limitation, digital signal processor (DSP) hardware. , network processor, application-specific integrated circuit (ASIC), field-programmable gate array (FPGA), read-only memory (ROM) for software storage, random access memory (RAM), and non-volatile storage. Other hardware, conventional and/or customized, can also be included. Likewise, any switches shown in the figures are conceptual only. Its function can be accomplished through the operation of program logic, through dedicated logic, through the interaction of program control and dedicated logic, or even manually, the particular technique being selectable by the implementer as more specifically understood from the context.

[00243] Additional aspects of the description, as set forth in the following numbered paragraphs:

[00244] 1. A system for determining the interaction between a test atmosphere and a simulated respiratory tract, wherein said system comprises: (a) a first pump comprising: (i) a chamber configured to contain a first volume of gas comprising a test atmosphere; (ii) a first port adapted to receive and emit gas and comprising a valve for regulating the flow of gas through the first port, said valve being movable between open and closed positions, wherein in the open position said valve is opened to a test atmosphere or surrounding air; (iii) a second port adapted for exiting and receiving gas and comprising a valve for regulating the flow of gas through the second port, said valve being movable between open and closed positions; (iv) a piston plate in the chamber, said piston plate containing a plurality of openings herein, wherein the one or more openings comprise valves adapted to regulate the flow of gas through the openings; and (v) a motor for controlling the operation of the first pump; (b) a second pump comprising: (i) a chamber configured to contain a second volume of gas, wherein the first and second volumes of gas are different; (ii) a port adapted to receive and emit gas; and (iii) a motor to control the operation of the first pump; (c) a connecting structure operable to transmit gas from the first pump to the second pump; and (d) one or more openings in the first pump or the second pump or in the walls of the connecting structure or a combination of two or more thereof, wherein said openings are capable of receiving a module for containing a cell culture medium or configured to store a cell culture medium or to monitor conditions in the chamber or adapted to monitor conditions in the chamber or for gas sampling or adapted to sample the gas or for gas characterization or adapted to characterize the gas.

[00245] 2. The system according to paragraph 1, wherein the pumps are piston pumps comprising a piston plate and a base.

[00246] 3. The system according to paragraph 1 or paragraph 2, in which the connecting structure is hollow.

[00247] 4. The system according to any of the preceding paragraphs, in which the connection structure is branched.

[00248] 5. The system according to paragraph 4, wherein a terminal branch of the connecting structure is joined to the second pump and one or more terminal branches are joined to a separate pump, wherein each separate pump comprises: (i ) a chamber for containing a volume of gas, wherein the volume of gas is the same volume as the second volume of gas in the second pump; (ii) a port to receive and emit gas and to join the connecting structure; and (iii) a motor to control the operation of the pump.

[00249] 6. The system according to paragraph 5, wherein each separate pump is the same as the second pump.

[00250] 7. The system according to some of the preceding paragraphs, wherein the system is contained in an enclosure, suitably a temperature controlled enclosure.

[00251] 8. The system according to paragraph 7, wherein the temperature of the housing is controlled by a thermostat.

[00252] 9. The system according to paragraph 8, wherein the temperature in the housing is about 37 °C.

[00253] 10. The system according to any of the preceding paragraphs, wherein the different volumes of the first and second pumps represent the interior volume of different compartments of the respiratory tract, suitably, the human respiratory tract.

[00254] 11. The system according to any one of paragraphs 6 to 10, wherein the volume of the separate pumps represents the interior volume of different compartments of the respiratory tract, suitably the human respiratory tract.

[00255] 12. The system according to any of the preceding paragraphs, wherein the volume displacement of the first and second pumps is at least as large as the maximum volume uptake achievable in the corresponding compartment of the respiratory tract.

[00256] 13. The system according to any one of paragraphs 5 to 12, wherein the displacement volume of the separate pumps is at least as large as the maximum volume uptake achievable in the corresponding compartment of the respiratory tract.

[00257] 14. The system according to any of the preceding paragraphs, wherein the pumping pressure of the motor or pump may correspond to atmospheric pressure or above or below atmospheric pressure.

[00258] 15. The system according to any of the preceding paragraphs, wherein the displacement volume of the first pump is between about 0 and 100 ml or between about 1 and 100 ml.

[00259] 16. The system according to any of the preceding paragraphs, wherein the displacement volume of the second pump is between about 0 and 4000 ml or between about 1 and about 4000 ml.

[00260] 17. The system according to any of the preceding paragraphs, wherein the pumps comprise or consist of stainless steel.

[00261] 18. The system according to any of the preceding paragraphs, wherein the chamber is a cylinder.

[00262] 19. The system according to any of the preceding paragraphs, wherein the chamber comprises glass.

[00263] 20. The system according to any of the preceding paragraphs, wherein the first pump chamber has a smaller volume than the second pump chamber.

[00264] 21. The system according to any of the previous paragraphs, wherein the volume of the chamber of the first pump represents the volume of the oral and oropharyngeal cavity.

[00265] 22. The system according to any of the preceding paragraphs, wherein the volume of the second pump chamber represents the lung lumen volume or part thereof.

[00266] 23. The system according to any of the preceding paragraphs, wherein the volume of the connecting structure represents the airway conduction volume of the lung, suitably, the human lung.

[00267] 24. The system according to any of the preceding paragraphs, wherein the piston plate of the first pump comprises one or more openings for capturing or inletting gas.

[00268] 25. The system according to any of the preceding paragraphs, wherein the openings of each include a valve that is movable between open and closed positions and is capable of regulating the capture or entry of gas.

[00269] 26. The system according to any of the preceding paragraphs, wherein the system further comprises a computer controller capable of synchronizing the operation of the system.

[00270] 27. The system according to any of the preceding paragraphs, wherein one or more of the first pump or the second pump or the connecting structure comprises one or more modules containing quartz crystal microbalances.

[00271] 28. The system according to any of the preceding paragraphs, wherein the openings in the first pump or the second pump or the walls of the connecting structure are threaded or unthreaded.

[00272] 29. The system according to any of the preceding paragraphs, wherein one or more of the openings contains a module.

[00273] 30. The system according to paragraph 29, wherein the one or more modules are adapted to contain a cell culture medium or configured to store a cell culture medium or adapted to monitor conditions in the chamber or adapted to prove the gas or adapted to characterize the gas.

[00274] 31. The system according to paragraph 29 or 30, wherein the modules are located at the base of the piston plate of the first and/or second pump and/or at the walls of the connecting structure.

[00275] 32. The system according to any one of paragraphs 29 to 31, wherein the one or more modules contain a cell culture medium.

[00276] 33. The system according to any of the preceding paragraphs, wherein the cell culture medium comprises or is in contact with a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[00277] 34. The system according to any one of paragraphs 29 to 32, wherein the modules adapted to contain or store a cell culture medium further comprise a microfluidic channel and optionally a microfluidic pump connected thereto.

[00278] 35. The system according to any one of paragraphs 29 to 34, wherein the modules are positioned in a horizontal plane on one or more of the first pump or the second pump or on the walls of the connecting structure.

[00279] 36. The system according to any of the preceding paragraphs, wherein the connecting structure comprises stainless steel.

[00280] 37. The system according to any of the preceding paragraphs, wherein the first pump chamber has a volume of about 100 ml.

[00281] 38. The system according to any of the preceding paragraphs, wherein the second pump chamber has a volume of about 1 liter to about 4 liters.

[00282] 39. A pump for displacing a volume of gas 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 or storing a medium for cell culture or to monitor conditions in the chamber or for gas sampling or for gas characterization; (ii) a first port for receiving and emitting the gas, when contained in the chamber, and comprising a first valve for regulating the flow of gas through the first port, wherein said first valve is movable between the open and closed positions, in that, in the open position, said valve can be opened towards a test atmosphere or surrounding air; and (iii) a second port for emitting and receiving gas when contained in the chamber, and comprising a second valve for regulating the flow of gas through the second port, said valve being movable between open and closed positions; and (iv) a piston plate containing a plurality of openings therein, wherein one or more of the openings comprise valves adapted to regulate the flow of gas through the openings.

[00283] 40. The pump according to paragraph 39, wherein the pump is a piston pump comprising a piston plate.

[00284] 41. The pump according to paragraph 39 or 40, wherein the one or more openings in the chamber are threaded or unthreaded.

[00285] 42. The pump according to any one of paragraphs 39 to 41, wherein the openings of one or more comprise a module.

[00286] 43. The pump according to paragraph 42, wherein the module is threaded or non-threaded.

[00287] 44. The pump according to paragraphs 42 or 43, wherein the one or more modules are adapted to contain a cell culture medium or configured to store a cell culture medium or adapted to monitor conditions in the chamber or adapted to prove the gas or adapted to characterize the gas.

[00288] 45. The pump according to any one of paragraphs 42 to 44, wherein the modules are located in the base of the pump.

[00289] 46. The pump according to any one of paragraphs 42 to 45, wherein the one or modules contain a cell culture medium.

[00290] 47. The pump according to paragraph 46, wherein the cell culture medium comprises or is in contact with a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[00291] 48. The pump according to any one of paragraphs 44 to 47, wherein the modules adapted to contain or store a cell culture medium further comprise a microfluidic channel and optionally a microfluidic pump connected thereto.

[00292] 49. The pump according to any one of paragraphs 42 to 48, wherein the modules are positioned in a horizontal plane on one or more of the first pump or the second pump or on the walls of the connecting structure.

[00293] 50. The pump according to any one of paragraphs 43 to 49, wherein the module comprises quartz crystal microbalances.

[00294] 51. The pump according to any one of paragraphs 39 to 50, wherein the pump further comprises a motor.

[00295] 52. The pump according to any one of paragraphs 39 to 51, wherein the pumping pressure corresponds to atmospheric pressure or above or below atmospheric pressure.

[00296] 53. The pump according to any one of paragraphs 39 to 52, wherein the displacement volume of the pump is between about 0 and 100 ml or between about 1 and about 100 ml.

[00297] 54. The pump according to any one of paragraphs 39 to 53, wherein the pump is composed of stainless steel.

[00298] 55. The pump according to any one of paragraphs 39 to 54, wherein the chamber is a cylinder.

[00299] 56. The pump according to any one of paragraphs 39 to 55, wherein the chamber comprises glass.

[00300] 57. The pump according to any one of paragraphs 39 to 56, wherein the pump chamber has a volume of about 100 ml.

[00301] 58. The pump according to any one of paragraphs 39 to 57, wherein the pump piston plate comprises one or more openings for capturing or inletting gas.

[00302] 59. The pump according to paragraph 58, wherein the one or more of the openings include a valve that is movable between the open and closed positions and is capable of regulating the capture or entry of gas.

[00303] 60. A piston pump for displacing a volume of gas comprising: (i) a chamber configured to contain a volume of gas and containing a piston plate comprising one or more openings for the absorption or influx of gas into the chamber , wherein one or more, or each, of the openings includes a valve that is movable between the open and closed positions and is capable of regulating the absorption or influx of gas; (ii) a first port for receiving and comprising a first valve for regulating the flow of gas through the first port, said first valve being movable between open and closed positions; and (iii) a second port for emitting gas when contained in the chamber, and comprising a second valve for regulating the flow of gas through the second port, said valve being movable between open and closed positions.

[00304] 61. The pump according to paragraph 60, wherein the chamber includes a base and comprises a further opening.

[00305] 62. The pump according to paragraph 61, wherein the openings are threaded or unthreaded.

[00306] 63. The pump according to paragraph 60 or 61, wherein the openings comprise a module in one or more of the openings.

[00307] 64. The pump according to paragraph 63, wherein the module is threaded or non-threaded.

[00308] 65. The pump according to paragraph 63 or 64, wherein the module is adapted to contain or store a cell culture medium or to monitor conditions in the chamber or for gas sampling or for gas characterization

[00309] 66. The pump according to any one of paragraphs 63 to 65, wherein the one or modules contain a cell culture medium.

[00310] 67. The pump according to paragraph 66, wherein the cell culture medium comprises a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[00311] 68. The pump according to any one of paragraphs 63 to 67, wherein the modules adapted to contain or store a cell culture medium further comprise a microfluidic channel and optionally a microfluidic pump connected thereto.

[00312] 69. The pump according to any one of paragraphs 63 to 67, wherein the module comprises quartz crystal microbalances.

[00313] 70. The pump according to any one of paragraphs 60 to 69, wherein a connecting structure is attached to the second port.

[00314] 71. The pump according to any one of paragraphs 70, wherein the connecting structure is hollow.

[00315] Suitably, the pump additionally comprises a motor.

[00316] 72. The pump according to any one of paragraphs 60 to 71, wherein the pumping pressure of the pump corresponds to atmospheric pressure or above or below atmospheric pressure.

[00317] 73. The pump according to any one of paragraphs 60 to 72, wherein the displacement volume of the pump is between about 0 and 100 ml or between about 1 and about 100 ml.

[00318] 74. The pump according to any one of paragraphs 60 to 73, wherein the pump is composed of stainless steel.

[00319] 75. The pump according to any one of paragraphs 60 to 74, wherein the chamber is a cylinder.

[00320] 76. The pump according to any one of paragraphs 60 to 75, wherein the chamber comprises glass.

[00321] 77. The pump according to any one of paragraphs 60 to 76, wherein the pump chamber has a volume of about 100 ml.

[00322] 78. A pump for displacing a volume of gas comprising: (i) a chamber configured to contain a volume of gas, said chamber comprising a base and one or more modules for containing or storing a culture medium of cells or to monitor conditions in the chamber or for gas sampling or for gas characterization; and (ii) an operable port for receiving and emitting the gas.

[00323] 79. The pump according to paragraph 78, wherein the pump is a piston pump comprising a piston plate.

[00324] 80. The pump according to paragraph 79, wherein the piston plate is free from any openings or openings.

[00325] 81. The pump according to any one of paragraphs 78 to 80, wherein the modules are located at the base of the chamber.

[00326] 82. The pump according to any one of paragraphs 78 to 81, wherein the modules are threaded or non-threaded.

[00327] 83. The pump according to any one of paragraphs 78 to 82, wherein the one or more modules are adapted to contain a cell culture medium or configured to store a cell culture medium or adapted to monitor conditions in the chamber or adapted to prove the gas or adapted to characterize the gas.

[00328] 84. The pump according to any one of paragraphs 78 to 83, wherein the one or modules contain a cell culture medium.

[00329] 85. The pump according to paragraph 84, wherein the cell culture medium comprises or is in contact with a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[00330] 86. The pump according to paragraphs 84 or 85, wherein the modules adapted to contain or store a cell culture medium further comprise a microfluidic channel and optionally a microfluidic pump connected thereto.

[00331] 87. The pump according to any one of paragraphs 78 to 82, wherein the module comprises quartz crystal microbalances.

[00332] 88. The pump according to any one of paragraphs 78 to 87, wherein a connecting structure is attached to the port.

[00333] 89. The pump according to any one of paragraphs 78 to 88, wherein the connecting structure is hollow.

[00334] 90. The pump according to any one of paragraphs 78 to 89, wherein the pump further comprises a motor.

[00335] 91. The pump according to any one of paragraphs 78 to 90, wherein the pumping pressure corresponds to atmospheric pressure or above or below atmospheric pressure.

[00336] 92. The pump according to any one of paragraphs 78 to 91, wherein the displacement volume of the pump is between about 0 and 1000 ml or between about 1 and about 100 ml.

[00337] 93. The pump according to any one of paragraphs 78 to 92, wherein the pump is composed of stainless steel.

[00338] 94. The pump according to any one of paragraphs 78 to 93, wherein the chamber is a cylinder.

[00339] 95. The pump according to any one of paragraphs 78 to 94, wherein the chamber comprises glass.

[00340] 96. The pump according to any one of paragraphs 78 to 95, wherein the volume of the chamber represents the volume of the lung lumen or a portion thereof.

[00341] 97. A connecting structure adapted to join at least two pumps for transmitting a gas between them, wherein said connecting structure comprises a hollow channel and one or more threaded or unthreaded openings in the walls of the connecting structure .

[00342] 98. The connecting structure according to paragraph 97, wherein the threaded openings contain a module threaded into one or more of the openings, said module being adapted to contain a cell culture medium or configured to store a cell culture medium or adapted to monitor conditions in the chamber or adapted to monitor conditions in the chamber or adapted to sample gas or adapted to characterize gas.

[00343] 99. The connecting structure according to paragraph 98, wherein the one or modules contain a cell culture medium.

[00344] 100. The connecting structure according to paragraph 99, wherein the cell culture medium comprises or is in contact with a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[00345] 101. The connecting structure according to any one of paragraphs 98 to 100, wherein the modules adapted to contain or store a cell culture medium further comprises a microfluidic component

[00346] 102. The connection structure according to any one of paragraphs 97 to 102, wherein a channel and optionally a microfluidic pump are connected thereto.

[00347] 103. The connecting structure according to any one of paragraphs 97 to 102, wherein the connecting structure is hollow.

[00348] 104. The connecting structure according to any one of paragraphs 97 to 103, wherein the connecting structure is branched.

[00349] 105. The connecting structure according to paragraph 104, wherein each terminal branch of the connecting structure is capable of being joined to a separate pump.

[00350] 106. The connecting structure according to any one of paragraphs 97 to 105, wherein the connecting structure represents the airway conduction volume of the lung, suitably the human lung.

[00351] 107. The connecting structure according to any one of paragraphs 98 to 106, wherein the modules are positioned in a horizontal plane on the walls of the connecting structure.

[00352] 108. The connecting structure according to any one of paragraphs 98 to 107, wherein the modules are adapted to contain a cell culture, suitably a two-dimensional or three-dimensional cell culture.

[00353] 109. The connecting structure according to any one of paragraphs 98 to 108, wherein the module is a chamber for containing a cell culture, said chamber comprising a microfluidic channel and, optionally, a microfluidic pump connected to it.

[00354] 110. The connecting structure according to any one of paragraphs 98 to 103, wherein the modules are adapted to monitor conditions in the connecting structure and/or for gas sampling and/or for gas characterization.

[00355] 111. The connecting structure according to any one of paragraphs 97 to 110, wherein the connecting structure comprises stainless steel.

[00356] 112. A system comprising the pumps one or more of the pumps as defined in any one of paragraphs 39 to 96.

[00357] 113. The system according to paragraph 112, wherein the system further comprises the connection structure as defined in any of paragraphs 97 to 111.

[00358] 114. The system according to paragraph 112 or paragraph 113, in which the pumps are joined by the connecting structure.

[00359] 115. A method for simulating the interaction between a test atmosphere and a simulated respiratory tract, comprising using the system in accordance with any one of paragraphs 1 to 38.

[00360] 116. Use of the system according to any one of paragraphs 1 to 38 wherein is to simulate the interaction between a test atmosphere and a simulated respiratory tract.

[00361] 117. A method for determining the effect of a test atmosphere on a culture of cells contained in a simulated respiratory tract comprising the use of the system in accordance with any one of paragraphs 1 to 38.

[00362] 118. Use of the system according to any one of paragraphs 1 to 38 to determine the effect of a test atmosphere on a culture of cells contained in a simulated respiratory tract

[00363] 119. A method for determining the effect of a test atmosphere on a cell culture contained in a simulated respiratory tract comprising: (a) providing the system as described herein, wherein the system contains a cell culture in one or more of the modules; and (b) comparing the cell culture before and/or after exposure to the test atmosphere, wherein a difference between the cell culture before and/or after exposing the cells to the test atmosphere is indicative that the test atmosphere test affects cell culture.

[00364] 120. A method for simulating the interaction between a test atmosphere and a simulated respiratory tract in the system described herein comprising: (a) with the first valve of the first pump open and the second valve of the first pump closed, providing a gas comprising a test atmosphere for the first pump through the first port; (b) closing the first valve and opening the second valve of the first pump and closing the valves on the piston plate of the first pump; (c) operating the second pump to draw the test atmosphere into the connecting structure and filling the chamber of the first pump and the connecting structure with the surrounding air; (d) opening the first valve of the first pump toward the surrounding 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 displace the test atmosphere through the connecting structure and through the first valve of the first pump.

[00365] 121. A method for determining the effect of a test atmosphere on a simulated respiratory tract in the system described herein comprising: (a) with the first valve of the first pump open and the second valve of the first pump closed, providing a gas comprising a test atmosphere for the first pump through the first port; (b) closing the first valve and opening the second valve of the first pump and closing the valves on the piston plate of the first pump; (c) operating the second pump to extract the test atmosphere through the connecting structure and flushing the first pump chamber and the connecting structure with the surrounding air; (d) opening the first valve of the first pump toward the surrounding 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 displace the test atmosphere through the connecting structure and through the first valve of the first pump; wherein the test atmosphere comes into contact with a cell culture located in one or more modules located in the first pump or in the connecting structure or in the second pump or in a combination of two or more thereof and said method comprises the additional step of determining the effect of the test atmosphere on the cell culture, wherein a difference in the cell culture before and/or after exposure to the test atmosphere is indicative that the test atmosphere affects the cell culture.

[00366] 122. The method of paragraph 121, wherein the modules are adapted to monitor system conditions and/or for gas sampling and/or for gas characterization and that method comprises taking one or more measurements of the modules .

[00367] 123. A method for simulating the interaction between a test atmosphere and a simulated respiratory tract comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) withdrawing the test atmosphere from the first pump to a connecting structure joining the first pump to a second pump; (c) filling the first pump and at least a portion of the connecting structure with the surrounding air; (d) retain the test atmosphere in the second pump and connecting structure for a defined period of time; (e) moving the test atmosphere to the connecting structure and the first pump using the second pump; and (f) performing one or more cycles of pumping the surrounding air into the second pump; wherein the test atmosphere comes into contact with a cell culture located in the first pump or connecting structure or in the second pump or a combination of two or more.

[00368] 124. The method according to paragraph 123, wherein step (d) comprises retaining the test atmosphere in the second pump and the portion of the connecting structure still containing test atmosphere for a defined period of time.

[00369] 125. The method according to paragraph 123 or 124, wherein the pumps are piston pumps comprising a piston plate and a base.

[00370] 126. The method according to any one of paragraphs 123 to 125, wherein the first pump is as defined in any one of paragraphs 39 to 77.

[00371] 127. The method according to any one of paragraphs 123 to 126, wherein the second pump is as defined in any one of paragraphs 78 to 96.

[00372] 128. The method according to any one of paragraphs 123 to 127, wherein the connecting structure is as defined in any one of paragraphs 97 to 111.

[00373] 129. The method according to any one of paragraphs 123 to 128, wherein the method is carried out in an enclosure, suitably a temperature-controlled enclosure.

[00374] 130. The method according to any one of paragraphs 123 to 129, wherein the temperature of the enclosure is controlled by a thermostat.

[00375] 131. The method according to any one of paragraphs 123 to 130, wherein the temperature in the enclosure is about 37 °C.

[00376] 132. The method according to any one of paragraphs 123 to 131, wherein the different volumes of the first and second pumps represent the interior volume of different compartments of the respiratory tract, suitably the human respiratory tract.

[00377] 133. The method according to any one of paragraphs 123 to 132, wherein the volume displacement of the first and second pumps is at least as large as the maximum volume uptake achievable in the corresponding compartment of the respiratory tract.

[00378] 134. The method according to any one of paragraphs 123 to 133, wherein the pumping pressure corresponds to atmospheric pressure or above or below atmospheric pressure.

[00379] 135. The method according to any one of paragraphs 123 to 134, wherein the displacement volume of the first pump is between about 0 and 100 ml or between about 1 and about 100 ml.

[00380] 136. The method according to any one of paragraphs 123 to 135, wherein the displacement volume of the second pump is between about 0 and 4000 ml or between about 1 and about 4000 ml.

[00381] 137. The method according to any one of paragraphs 123 to 136, wherein the first pump chamber has a smaller volume than the second pump chamber.

[00382] 138. The method according to any one of paragraphs 123 to 137, wherein the volume of the chamber of the first pump represents the volume of the oral and oropharyngeal cavity, suitably the human oral and oropharyngeal cavity.

[00383] 139. The method according to any one of paragraphs 123 to 138, wherein the volume of the chamber of the second pump represents the lung lumen volume or a portion thereof, suitably the human lung lumen or a portion thereof. same.

[00384] 140. The method according to any one of paragraphs 123 to 139, wherein the volume of the connecting structure represents the airway conduction volume of the lung, suitably the human lung.

[00385] 141. The method according to any one of paragraphs 123 to 140, wherein the piston plate comprises one or more openings for capturing or entering gas into the chamber.

[00386] 142. The method according to any one of paragraphs 123 to 141, wherein the one or more openings each include a valve that is movable between open and closed positions and is capable of regulating the capture or entry of gas .

[00387] 143. The method according to any one of paragraphs 123 to 142, wherein the cell culture is located at the base of the piston plate of the first and/or second pump and/or on the walls of the connecting structure.

[00388] 143. The method according to any one of paragraphs 122 to 142, wherein the cell culture is a two-dimensional or three-dimensional culture.

[00389] 144. The method according to any one of paragraphs 122 to 143, wherein the method further comprises monitoring conditions and/or for gas sampling and/or for gas characterization and in one or more of the first pump or the second pump or the connecting structure using one or more modules contained therein.

[00390] 145. The method according to any one of paragraphs 122 to 144, wherein the chamber comprising the cell culture further comprises a microfluidic channel and, optionally, a microfluidic pump connected thereto.

[00391] 145. The connection method according to any one of paragraphs 122 to 145, wherein the connection structure comprises stainless steel.

[00392] 146. The method according to any one of paragraphs 122 to 145, wherein the chamber of the first pump has a volume of about 100 ml.

[00393] 147. The method according to any one of paragraphs 122 to 146, wherein the second pump chamber has a volume of about 1 liter to about 4 liters.

[00394] 148. A method for determining the effect of a test atmosphere on a simulated respiratory tract comprising: (a) providing a test atmosphere to a chamber of a first pump; (b) withdrawing the test atmosphere from the first pump to a connecting structure joining the first pump to a second pump; (c) filling the first pump and at least a portion of the connecting structure with the surrounding air; (d) maintain the test atmosphere in the second pump and the connecting structure for a defined period of time; (e) displacing the test atmosphere through the connecting structure and the first pump using the second pump; and (f) performing one or more cycles of pumping the surrounding air into the second pump; wherein the test atmosphere comes into contact with a cell culture located in one or more modules located in the first pump or in the connecting structure or in the second pump or in a combination of two or more thereof and said method comprises the step further of determining the effect of the test atmosphere on the cell culture, wherein a difference in the cell culture before and/or after exposure to the test atmosphere is indicative that the test atmosphere affects the cell culture.

[00395] 149. The method according to paragraph 148, wherein the modules are adapted to monitor system conditions and/or for gas sampling and/or for gas characterization and said method comprises taking one or more measurements of the modules.

[00396] Any publication cited or described in this document provides relevant information disclosed prior to the filing date of this application. Statements in this document should not be interpreted as an admission that the inventors do not have the right to backdate such descriptions. All publications mentioned in the descriptive report above are incorporated into this document by way of reference. Various modifications and variations of the invention will become apparent to individuals skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention, as claimed, should not be unduly limited to such embodiments. Indeed, various modifications of the described modes of carrying out the invention that are obvious to those skilled in the art in question should fall within the scope of the following claims.

Claims (11)

1. System (10) for determining the interaction between a test atmosphere and a simulated respiratory tract, characterized in that the system (10) comprises: (a) a first pump (40) comprising: (i) a chamber (42) configured to contain a first volume of gas comprising a test atmosphere; (ii) a first port (90) adapted to receive and emit gas and comprising a valve (44a) for regulating the flow of gas through the first port (90), said valve (44a) being movable between open and closed positions , wherein in the open position said valve (44a) is opened to a test atmosphere or surrounding air; (iii) a second port (43) adapted for exiting and receiving gas and comprising a valve (49) for regulating the flow of gas through the second port (43), said valve (49) being movable between positions open and closed; (iv) a piston plate (45) in the chamber (42), said piston plate (45) comprising one or more openings (47) for capturing or entering gas into the chamber (42) wherein one or more, or each of the openings (47) includes a valve (48) that is movable between open and closed positions and is capable of regulating the capture or entry of gas; and (v) a motor (41) for controlling the operation of the first pump (40); (b) a second pump (80) comprising: (i) a chamber (82) configured to contain a second volume of gas, wherein the first and second volumes of gas are different; (ii) a port (89) adapted to receive and emit gas; and (iii) a motor (81) for controlling the operation of the second pump (80); (c) a connecting structure (50) operable to transmit gas from the first pump (40) to the second pump (80); and (d) one or more openings (43, 51) in the first pump (40) or in the second pump (80) or in the walls of the connecting structure (50) or a combination of two or more thereof, wherein said openings (43, 51) capable of receiving a module (112, 113, 212, 213) for containing a cell culture medium or for monitoring conditions in the chamber (42, 82) or for gas sampling or for gas characterization. 2. System according to claim 1, characterized by the fact that the first pump (40) and/or second pump (80) is a piston pump comprising a piston plate (45, 83) and a base (44, 84). 3. System according to claim 1 or 2, characterized by the fact that the system is contained in an enclosure (11), preferably a temperature-controlled enclosure (11, 12). 4. System according to any one of the preceding claims, characterized in that the chamber (42) of the first pump (40) has a smaller volume than the chamber (82) of the second pump (80). 5. System according to any one of the preceding claims, characterized by the fact that the system further comprises a computer controller (13) capable of synchronizing the operation of the system. 6. System according to any one of the preceding claims, characterized by the fact that one or more openings (43, 51) contain a module (112, 113, 212, 213). 7. System, according to claim 6, characterized by the fact that the module (112 113, 212, 213) is located at the base (44, 84) of the piston plate (45, 83) of the first (40) and /or second (80) pump. 8. Use of the system (10), as defined in any one of claims 1 to 7, characterized by the fact that it is to simulate the interaction between a test atmosphere and a simulated respiratory tract. 9. Method for determining the effect of a test atmosphere on a culture of cells contained in a simulated respiratory tract, characterized in that it comprises: (a) providing the system (10), as defined in claim 6 or 7, in that the system (10) contains a cell culture in one or more modules (112, 113, 212, 213); and (b) comparing the cell culture before and/or after exposure to the test atmosphere, wherein a difference between the cell culture before and/or after exposing the cells to the test atmosphere is indicative that the test atmosphere test affects cell culture. 10. Method for simulating the interaction between a test atmosphere and a simulated respiratory tract, characterized by the fact that it comprises: (a) providing a test atmosphere to the chamber (42) of a first pump (40); (b) removing the test atmosphere from the first pump (40) to a connecting structure (50) that joins the first pump (40) to a second pump (80); (c) filling the first pump (40) and at least a portion of the connecting structure (50) with surrounding air; (d) maintaining the test atmosphere in the second pump (80) and the connecting structure (50) for a defined period of time; (e) moving the test atmosphere to the connecting structure (50) and the first pump (40) using the second pump (80); and (f) performing one or more cycles of pumping the surrounding air into the second pump (80); wherein the test atmosphere comes into contact with a cell culture located in the first pump (40) or in the connecting structure (50) or in the second pump (80) or a combination of two or more thereof. 11. Method according to claim 10, characterized in that the test atmosphere comes into contact with a cell culture located in one or more modules located in the first pump (40) or in the connecting structure (50) or in the second pump ( 80) or in a combination of two or more of them; and said method comprising the additional step of determining the effect of the test atmosphere on the cell culture.