Patent Publication Number: US-2015064689-A1

Title: System and method for analyzing transmissibility of influenza

Description:
BACKGROUND OF THE DISCLOSURE 
     Some of the most dangerous pathogens are capable of being aerosolized and transmitted through the air. Presently, the transmissibility of a pathogen is evaluated experimentally in animal models by placing an infected animal in close proximity to an uninfected animal and monitoring the health of the uninfected animal. Such experiments are costly and difficult to repeat. Furthermore, due to species-specific differences, the animal models may not accurately predict the transmissibility seen in human subjects. 
     SUMMARY OF THE DISCLOSURE 
     The present disclosure provides systems and methods for determining the transmissibility of a pathogen. The system described herein includes a flow device with a plurality of chambers. Cells, such as those derived from the tracheal, bronchial, or alveolar tissues, are cultured within the chambers of the flow device. Cells within a first chamber are infected with a pathogen. The pathogen&#39;s transmissibility is determined by flowing a gas through the flow device such that the gas flows over the infected cells in a manner that could transport the pathogen to a second chamber of the device. The second chamber contains uninfected cells. By quantifying the amount of virus transferred to the cells of the second cell culture chamber after a predetermined amount of time, the transmissibility of the pathogen is determined. 
     According to one aspect of the disclosure, a method of determining the transmissibility of a pathogen includes disposing at least one infected cell in a first cell chamber disposed toward a proximal end of a gas flow channel. The method also includes disposing at least one uninfected cell in a second cell chamber disposed toward a distal end of the gas flow channel. The second cell chamber is separated from the first cell chamber by a barrier, either physical or spatial. The method also includes flowing gas through the gas flow channel such that the gas flows across the at least one infected cell and then flows across the at least one uninfected cell and then determining an infection rate of the uninfected cells. 
     In certain implementations, the gas is flowed through the gas flow channel in a pulsatile manner. In some implementations, the gas is flowed through the gas flow channel in a flow pattern that simulates a human cough. In some implementations, the method also includes incubating the at least one uninfected cell for a predetermined amount of time before determining the infection rate of the previously uninfected cells. The infection rate is determined by measuring the pathogen load of the at least one uninfected cell. 
     In some implementations, the method also includes introducing a fluorescent protein into the at least one uninfected cell. The fluorescent protein fluoresces when the at least one uninfected cell is infected by a pathogen from the at least one infected cells. In certain implementations, determining the infection rate of the at least one uninfected cells includes measuring a fluorescence level. 
     In some implementations, the pathogen is a virus, such as an influenza virus. In yet other implementations, an airway fluid is applied to the barrier space and at least one condition in the gas flow channel is measured. The conditions measured are one of a temperature, a pressure, a flow rate, and a humidity. 
     According to another aspect of the disclosure, a system for determining the transmissibility of a pathogen includes a gas flow channel having a proximal end and a distal end. The gas flow channel further includes a first cell chamber disposed toward the proximal end of the gas flow channel. The first cell chamber is configured to supply nutrients to a basolateral surface of at least one first cell. The gas flow channel also includes a second cell chamber disposed toward the distal end of the gas flow channel. The second cell chamber is also configured to supply nutrients to a basolateral surface of at least one second cell. A barrier space between the first cell chamber and the second cell chamber is also included in the gas flow channel. The barrier space is configured to allow an aerosolized particle to pass from the first cell chamber to the second cell chamber. The system also includes a gas pump configured to flow gas through the gas flow channel at a controlled flow rate and an incubator configured to maintain a controlled atmospheric condition within the gas flow channel. 
     In some implementations, the gas pump is configured to flow gas through the gas flow channel in pattern that simulates a cough. 
     According to yet another aspect of the disclosure, a microfluidic flow device includes a gas flow channel having a proximal end and a distal end. The gas flow channel further includes a first cell chamber disposed toward the proximal end of the gas flow channel. The first cell chamber is configured to supply nutrients to a basolateral surface of at least one first cell. The gas flow channel also includes a second cell chamber disposed toward the distal end of the gas flow channel. The second cell chamber is also configured to supply nutrients to a basolateral surface of at least one second cell. The gas flow channel further includes a barrier space between the first cell chamber and the second cell chamber. The barrier space is configured to allow an aerosolized particle to pass from the first cell chamber to the second cell chamber. 
     In some implementations, the first cell chamber and the second cell chamber include a permeable membrane disposed atop cellular wells. In certain implementations, the barrier space is linear or non-linear. In other implementations, the barrier space is coated with an airway fluid such as a mucous. 
     In yet other implementations, the gas flow channel is configured to induce a predetermined shear force on the at least one first cell and the at least one second cell. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The skilled artisan will understand that the figures, described herein, are for illustration purposes only. It is to be understood that in some instances various aspects of the described implementations may be shown exaggerated or enlarged to facilitate an understanding of the described implementations. In the drawings, like reference characters generally refer to like features, functionally similar and/or structurally similar elements throughout the various drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the teachings. The drawings are not intended to limit the scope of the present teachings in any way. The system and method may be better understood from the following illustrative description with reference to the following drawings in which: 
         FIG. 1  illustrates a system for determining the transmissibility of a pathogen, in accordance with an implementation of the present disclosure; 
         FIG. 2  illustrates a cross sectional view of a flow channel of the system of  FIG. 1 , in accordance with an implementation of the present disclosure; 
         FIGS. 3A and 3B  illustrate top views of the flow channels of  FIG. 1 , in accordance with an implementation of the present disclosure; 
         FIGS. 4A-4D  illustrate gas flow patterns suitable for use in the system shown in  FIG. 1 , in accordance with an implementation of the present disclosure; and 
         FIG. 5  illustrates a method for determining the transmissibility of a pathogen with the system of  FIG. 1 , in accordance with an implementation of the present disclosure. 
     
    
    
     DETAILED DESCRIPTION 
     The various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the described concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes. 
     Pathogens (e.g., viruses and/or bacteria) are often transmitted person to person. The systems and methods described herein enable the assessment of the transmissibility of pathogens, such as the human-adapted influenza virus. The disclosed systems and methods enable the transmissibility of pathogens to be tested under repeatable, laboratory conditions. 
       FIG. 1  illustrates one implementation of a system  100  for analyzing the transmissibility of pathogens. The system  100  includes a flow chamber  102  that is housed within an incubator  104 . The system  100  also includes a first reservoir  106  and a second reservoir  108 . Fluids from the first reservoir  106  and second reservoir  108  are supplied to the flow chamber  102  by pumps  110 . A plurality of sensors  112  monitor various parameters in the flow chamber  102 . The system  100  further includes a gas pump  114 , which drives gas from the plurality of gas reservoirs  116  through the flow chamber  102 . A collection system  118  collects the gas and/or fluid as it exits the flow chamber  102 . The system  100  also includes a microscope  120  for viewing the cell cultures within the flow chamber  102 . The various components of the system  100  are controlled and/or monitored by the control system  122 . 
     As illustrated, system  100  includes a flow chamber  102 . The flow chamber  102  is described in greater detail in relation to  FIGS. 2 and 3 . Briefly, the flow chamber  102  includes at least one flow channel. Each flow channel includes a first and a second cell culturing area. The cell culturing areas are separated by a barrier space. In some implementations, the cells cultured in the first cell culture are infected with a pathogen and the cells cultured in the second cell culture area are uninfected by the pathogen. The transmissibility of the pathogen is tested by flowing a gas through the flow chamber  102 . The transmissibility (including the rate of transmissibility) is determined by quantifying the amount of pathogen present in the cells of the second cell culture after a predetermined amount of time. 
     The flow chamber  102  is housed within an incubator  104 . The incubator  104  maintains an environment within the flow chamber  102  that is conducive for the culturing of the cells. In some implementations, the incubator  104  controls and/or maintains a predetermined temperature, humidity, carbon dioxide level, oxygen level, or any combination thereof. For example, the incubator  104  may be configurable to maintain conditions within the flow chamber  102  that mimic conditions within the human respiratory system, such as a temperature between about 32° C. and about 37° C. with a humidity between about 40% and about 60%. In some implementations, the incubator  104  maintains a 5% carbon dioxide environment within the flow chamber  102 . The incubator  104  also includes a plurality of access ports (not illustrated). The ports allow sensor connections and flow lines to pass from the outside environment to the interior of the incubator  104  without affecting the controlled environment within the incubator  104 . In some implementations, the incubator  104  is coupled inline with the flow chamber  102  such that the incubator  104  conditions the gas entering the flow chamber  102  as described herein. For example, as gas flows from the gas pump  114  to the flow chamber  102 , an inline incubator  104  can heat and oxygenate the gas before it reaches the flow chamber  102 . 
     Also as illustrated, the system  100  includes fluid reservoirs  106  and  108 . In some implementations, the fluid reservoirs  106  and  108  store growth medium and/or test agents that are passed to the cell culturing areas via the pumps  110 . The fluids supplied to each of the cell culturing areas are distinct from one another (i.e., are stored in separate reservoirs) such that a pathogen may not be passed from the first cell culturing area to the second cell culturing area via the fluid in the reservoirs  106  and  108 . In some implementations, the growth medium (or other fluids) stored in the fluid reservoirs  106  and  108  support cell growth, viability, and metabolism. For example, the fluids may include generic growth mediums, Eagle&#39;s minimal essential medium and derivatives, specific growth and/or differentiation medium for mammalian primary airway cells, phosphate buffered saline, horse serum, fetal calf serum, buffers or any combination thereof. In some implementations, the test agents include antibiotics such as penicillin, antifungals, antivirals, antimicrobial compounds, additional cells (e.g., immune cells), amino acids, varied energy sources, vitamins, growth factors, trace elements (e.g., metals), or any combination thereof. 
     The pumps  110  control the flow of fluid from the reservoirs  106  and  108  into and out of the cell culturing areas of the flow chamber  102 . In some implementations, the pumps continuously flow fluid into the first and/or second cell culturing areas. In another example, the pumps  110  flow fluid into the first and/or second cell culturing areas at intermittent intervals, such as once an hour, every day, every two days, once every three days, or once every week. In yet other implementations, the pumps  110  flow fresh or recirculated fluid through the first and/or second cell culturing areas continuously. 
     As described in greater detail in relation to  FIG. 4 , gas is flowed through the flow chamber  102 . The flow of gas through the flow chamber  102  is managed by the gas pump  114 . The gas pump  114  is configurable to flow gas through the flow chamber  102  in a substantially laminar, substantially non-laminar, continuous and/or pulsatile manner. The system  100  includes a plurality of gas reservoirs  116 . The gas reservoirs  116  may store oxygen, carbon dioxide, nitrogen, argon, neon, methane, compressed air, or any combination thereof. In some implementations, the gas pump  114  receives gas from the one or more gas reservoirs  116  and/or ambient room air and mixes the gases before flowing the gas mixture through the flow chamber  102 . In some implementations, the gas is mixed by a gas mixer prior to the gas pump  114  flowing the gas mixture through the flow chamber  102 . In some implementations, the gas mixture that is flowed through the flow chamber  102  is substantially similar to the composition of gases that are exhaled by a human. For example, about 18% O 2 , about 78% N 2 , and about 4% CO 2 . In some implementations, gases or gas mixtures that may affect transmissibility of a pathogen are introduced into the gas flowed through the flow chamber  102 . For example, one or more aerosolized drugs or volatile organic compounds can be added to the gas flowed through the flow chamber  102 . 
     When gas exits the flow chamber  102 , it is collected by the collection system  118 . The collection system  118  collects exhaust gas from the flow chamber  102  and filters the gas. In some implementations, the gas exiting the flow chamber  102  may include pathogens and/or other toxic (or dangerous) materials. The collection system  118  filters and substantially removes the undesirable materials from the exhaust gas before the gas is vented into the environment. In some implementations, the collection system  118  collects and quantifies the amount of pathogens and/or other toxic (or dangerous) materials are within the gas exiting the flow chamber  102 . As described below, in some implementations, the gas pump  114  can drive gas through the flow chamber  102  in a first direction and then drive gas through the flow chamber  102  in the reverse direction. In these implementations, the collection system  118 , houses the gas before it is returned through the flow chamber  102  during the reverse flow period. 
     In some implementations the flow chamber  102  allows for the visual inspection of the cells seeded within the cell culturing area (and other areas) of the flow chamber  102 . For example, the top of the flow chamber  102  may include a glass cover slip through which the interior of the flow chamber  102  is viewable. The system  100  includes a microscope  120  to view the interior of the flow chamber  102 . In some implementations, the microscope  120  is configured to record still or moving images of the cells within the flow chamber  102 . In some implementations, the microscope  120  is an optical light microscope, confocal microscope, fluorescent microscope, or, in general, any type of microscope used in the field of cellular imaging and analysis. In some implementations, the microscope  120  is equipped with cellular analysis software that allows for the detection and/or classification of cells. 
     The system  100  also includes a control system  122 . The control system  122  controls and manages the various above described components of the system  100 . In some implementations, the control system  122  is a general computing device. In some implementations, the control system  122  includes one or more processors and at least one computer readable medium. Processor executable instructions are stored on the computer readable medium. When executed, the instructions cause the control system  122  to perform the control sequences described herein. For example, the control system  122  controls the flow of fluid into and out of the cell culture chambers by controlling the pumps  110 . Similarly, the control system  122  can control the flow of gas through the flow chamber  102  via the gas pump  114 . In some implementations, the control system  122  controls the pumps  110  and the gas pump  114  via an electrical connection. For example, the control system  122  may transmit a transistor—transistor logic (TTL) pulse, a pulse width modulated signal, or similar signal to the pumps  110  and/or gas pump  114  for control. In some implementations, the control system  122  is configured to control the state (On or Off) of the various pumps in the system  100 . In some implementations, the control system  122  is configured to control the rate gases and/or fluid flows through the flow chamber  102 . In some implementations, the control system  122  is configured to control the gas pump  114  such that the gas pump  114  flows gas through the flow chamber  102  at a rate of 30 L/min. In other implementations, the flow rate is between about 0 L/min and 20, between about 20 L/min and about 40 L/min, between about 40 L/min and about 60 L/min, between about 60 L/min and about 80 L/min, or between about 80 L/min and about 100 L/min. In some implementations, the shear stress is about 0.4-1.0 dynes/cm 2 , about 1.0-2.0 dynes/cm 2 , about 2.0-5.0 dynes/cm 2 , about 5.0-10.0 dynes/cm 2 . In models mimicking breathing conditions experienced during exercising or coughing, the shear stress may be between about 1000-1200 dynes/cm 2 , about 1200-1400 dynes/cm 2 , about 1400-1600 dynes/cm 2 , about 1600-1800 dynes/cm 2 , and about 1800-2000 dynes/cm 2 . 
     The system  100  also includes a plurality of sensors  112  that are coupled to the control system  122 . The sensors  112  are used by the control system  122  to monitor the conditions of the system  100 . Example sensors  112  can include temperature sensors, humidity sensors, gas sensors, pH sensors, and sensors to monitor transepithelial electrical resistance. The sensors  112  are used to monitor the internal environment of the incubator  104  and/or flow chamber  102 . In some implementations, the sensors  112  are flow sensors and monitor the flow of gas and liquid into and out of the flow chamber  102 . The control system  122  uses the sensors  112  as feedback sensors for the various flow and control sequences described above. In some implementations, the data (e.g., temperature and flow rates) recorded with the sensors  112  is saved by the control system  112  for later analysis. 
       FIG. 2  illustrates a cross sectional view of one implementation of a flow channel  200  from the flow chamber  102  of the system  100 . In some implementations, the flow chamber  102  is a microfluidic flow device configured to allow gas flow  218  across one or more cell cultures. As illustrated, the flow channel  200  includes a first cell culture chamber  202  positioned toward a proximal end of the flow chamber  200  and a second cell culture chamber  204  positioned toward a distal end of the flow chamber  200 . The first cell culture chamber  202  is separated from the second cell culture chamber  204  by a barrier space  206 . As illustrated, the barrier space  206  is coated with a barrier fluid  208 . In each cell culture chamber, cells  210 ( a ) and  210 ( b ) (or collectively referred to as cells  210 ) sit atop a permeable membrane  212 . The permeable membranes  212  (or similar support structure) allow molecules (e.g., nutrients in the growth medium) to pass from the fluid reservoirs  214  to the cells  210 . The roof  216  of the flow chamber  102  is configured to allow visualization of the cells  210  within the flow chamber  102 . 
     The flow chamber  102  is made from a material suitable for cell culture. In some implementations, the material is non-toxic to cells, durable enough to support the fluid in the fluid reservoirs  214  and the flow of gas through the flow chamber  102 , and/or substantially non-reactive. For example, the flow chamber  102  can include polymeric and/or non-polymeric materials, including polydimethylsiloxane (PDMS), acrylic, polyethylene, polyolefin polymer, polyurethane, polystyrene, Pyrex, glass, polypropylene, Permanox, or any combination thereof. In some implementations, the flow chamber  102  is manufactured using photolithographic techniques, injection molding, direct micromachining, deep RIE etching, hot embossing, or any combinations thereof. 
       FIG. 2  illustrates one flow channel  200  of flow chamber  102 , however, flow chamber  102  is not limited to having only one flow channel  200 . In some implementations, flow chamber  102  includes a plurality of flow channels  200 , each with a first cell culture chamber  202 , second cell culture chamber  204 , and barrier space  206 . For example, the flow chamber  102  may include 1, 2, 4, 8, 12, 16, 20, or 32 flow channels  200 . 
     The first cell culture chamber  202  and second cell culture chamber  204  include a membrane  212 . In some implementations, the cell culturing chambers  202  and  204  include cell culturing areas with a surface area of approximately 0.01-0.02 cm 2 , 0.02-0.05 cm 2 , 0.05-0.1 cm 2 , 0.1-0.2 cm 2 , 0.2-0.5 cm 2 , 0.5-1 cm 2 , 1-2 cm 2 , 2-5 cm 2 , 5-10 cm 2 , or 10-25 cm 2 . In some implementations, the membrane  212  thickness is between 10 μm and 500 μm. In some implementations, the membrane has a thickness between 100 nm and 500 μm. For instance, the membrane may be about 100-200 nm, about 200-500 nm, about 500 nm-1 μm, about 1-2 μm, about 2-5 μm, about 5-10 μm, about 10-20 μm, about 20-50 μm, about 50-100 μm, about 100-200 μm, or about 200-500 μm. The cells  210  and example methods for cell culturing are described below in relation to the method of  FIG. 5  and the Examples. 
     The membrane  212  is compatible with cell metabolism and cell growth. The membrane  212  includes pores smaller than the diameter of the cells  210 , but large enough to allow nutrients to pass from the reservoirs  214  to the cells  210 . In some implementations, the cells  210  substantially adhere to the membrane  212 , and create an air-liquid interface between the gas flowing through the flow channel  200  and the liquid in the fluid reservoir  214 . In some implementations, the membrane  212  includes polycarbonate (PC), polyester (e.g., polyethylene terephthalate (PET)), collagen-coated polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polysulfone, and natural electrospun ECM proteins (such as collagen), polycarbonates, polyesters, polytetrafluoroethylenes, ethylene-vinyl acetates (EVA), and polyvinyl acetates (PVA). In some implementations the membrane  212  is biodegradable and in some implementations the membrane  212  is non-biodegradable. In some implementations, at least a portion of the membrane  212  includes a topographic feature such as a plurality of grooves. 
     The length of the barrier space  206  can vary responsive to the type of pathogen tested. In some implementations, the length of the barrier space  206  is configured such that the gas flow  218  can carry the pathogen (or aerosolized particle) from the first cell culture chamber  202  to the second cell culture chamber  204 . However, the length of the barrier space  206  is long enough that the pathogen cannot infect the cells  210 ( b ) of the second cell culture chamber  204  in the absence of the gas flow  218 . For example, the length of the barrier space  206  can be about 1-5 cm, about 5-10 cm, about 10-25 cm, about 25-50 cm, about 50-75 cm, or about 75-100 cm. In some implementations, the path of the barrier space  206  is tortuous. For example, and as described below in relation to  FIGS. 3A and 3B , in some implementations the flow chamber  102  includes non-linear barrier spaces  206 . 
     The top of the barrier space  206  is coated with a barrier fluid  208 . In some implementations, only a sub-portion or none of the barrier space  206  is coated with the barrier fluid  208 . In other implementations, substantially all of the barrier space  206  is coated with the barrier fluid  208 . The barrier fluid  208  can be (or mimic by having similar characteristics) mucous or other airway fluids. For example, the barrier fluid  208  can include natural mucus (e.g., purified animal mucus) or artificial mucus synthesized from basic components. In other implementations, the barrier fluid  208  is, or simulates, mucus from the gut. In some implementations, the airway fluid  208  is produced by the cells  210 . In some implementations, the barrier space  206  and/or other surfaces of the flow channel  200  are coated with enzymes, such as those used for ELISA assays, which can be used in the detection of a pathogen. 
     In some implementations, the flow channel  200  includes one or more actuators (not illustrated). The one or more actuators are configured to induce movement in the flow channel  200 . For example, the one or more actuators may mimic the movement of the tracheal or alveolar basal membrane during a cough and/or normal respiration. 
       FIGS. 3A and 3B  illustrate top views of the flow channels within the flow chamber  102  of  FIG. 1 . The flow channels  300  and  350  include a first cell culture chamber  202  separated from a second cell culture chamber  204  by a barrier space  302  and  304 , respectively. As illustrated, the barrier space  302  is substantially linear (i.e., substantially straight). In contrast, the barrier space  304  is non-linear. In some implementations, the non-linear path of the barrier space  304  simulates greater distances between the first cell culture chamber  202  and second cell culture chamber  204  when compared to the linear barrier space  302 . One of ordinary skill in the art will understand the flow chambers  300  and  350  are provided for illustrative purposes and are not intended to limit the scope of the disclosure to the illustrated barrier space designs. 
       FIGS. 4A-4D  are plots of illustrative patterns the gas pump  114  can induce in the gas flow  218 . In each gas flow pattern, the gas pump  114  flows gas through the flow chamber  102  from time t(0) until time t(1). A positive value for the gas flow pattern represents gas flowing from the inlet of the flow chamber  102  to the outlet of the flow chamber  102  (i.e., the gas is flowing from the gas pump  114  to the collection system  118 ). A negative value for the gas flow pattern represents gas flowing from the outlet of the flow chamber  102  to the inlet of the flow chamber  102  (i.e., the gas pump  114  is drawing gas back from the collection system  118  towards the gas pump  114 ). 
     In the gas flow pattern  400  of  FIG. 4A , the gas pump  114  flows gas through the flow chamber  102  with a unidirectional, constant flow rate. Example flow rates may include any combination of the flow rates described above in relation to  FIG. 1 . In some implementations, the flow rate is selected to impart a specific shear rate on the cells. In  FIG. 4B , the gas flow pattern  410  simulates a human breathing pattern. As illustrated, the gas flow pattern  410  includes bi-directional flow. During the “inhalation” phase  411 , the gas pump simulates an inhalation by drawing gas from the outlet side of the flow chamber  102  to the inlet side of the flow chamber  102 . During the “exhalation” phase  412  the gas pump  114  then drives the “inhaled” gas back through the flow chamber  102 . The gas flow pattern  410  can include peak flow rates between about 1-15 L/min, about 15-30 L/min, about 30-45 L/min, about 45-60 L/min, about 60-75 L/min, and about 75-90 L/min. 
     The gas flow pattern  420 , illustrated in  FIG. 4C , is a sinusoidal flow pattern. The gas flow pattern  420  may include flow rates similar to those described above in relation to the gas flow pattern  410 . In some implementations, the gas flow pattern  420  represents a simplified breathing pattern. In some implementations, the “inhalation” and “exhalation” phases of the gas flow pattern  420  may have varying or non-equal flow rates. One of ordinary skill in the art will recognize that other waveforms may be used with the flow chamber  102 . For example, the gas pump  114  may flow square wave flow patterns, saw tooth wave flow patterns, non-continuous wave flow patterns, or any combination thereof through the flow chamber  102 . 
     The gas flow pattern  430 , illustrated in  FIG. 4D , simulates a human cough. In gas flow patter  430 , gas is slowly drawn into the flow chamber  102  from the collection system  118  (or outside source). The gas is then quickly flowed back through the flow chamber  102  in a flow pattern that simulates a cough. Peak flow rates during the gas flow pattern  430  can be about 100-200 L/min, about 200-300 L/min, about 300-400 L/min, or about 400-500 L/min. In some implementations, the “cough” flow pattern  430  lasts about 0.02-0.5 seconds, about 0.5-1 seconds, or about 1-3 seconds. In some implementations, the gas flow pattern  430  is combined with one or more of the above described gas flow patterns. 
       FIG. 5  is a flow chart illustrating a method  500  for determining the transmissibility of a pathogen. Briefly, the method  500  begins with the first set of cells being disposed in a first cell culture chamber (step  501 ) and a second set of cells begin disposed in a second cell culture chamber (step  502 ). Next, gas is flowed through the flow chamber (step  503 ). After a predetermined amount of time, the transmissibility of the pathogen is determined (step  504 ). 
     As set forth above, and referring to  FIG. 2 , the first set of cell are disposed in the first cell culture chamber  202  (step  501 ) and the second set of cells are disposed in the second cell culture chamber  204  (step  502 ). As described above, the cells are disposed on the membrane  212  within their respective cell culture chambers. In some implementations, the first set of cells  210 ( a ) are infected with a pathogen. The infection of the first set of cells  210 ( a ) can occur prior to or after the cells  210 ( a ) are placed in the flow chamber  102 . In some implementations, once disposed within the cell culturing chambers, the first and/or second set of cells are cultured within the flow chamber  102  for a predetermined amount of time prior to beginning any experimentation. For example, the cells  210  may be allowed to create a substantially intact epithelial layer on the membrane  212  before experimentation begins. 
     The cells disposed in the cell culture chambers can include human cells or cells from an animal model (e.g., swine or avian animal models). Example cells include tracheal epithelial cells, bronchial epithelial cells, nasal epithelial cells, alveolar cells, tracheal tissue, bronchial tissue, and/or nasal tissue. In other implementations, the cells are commercially-available cells grown for experimentation purposes. In some implementations, it is beneficial to know the transmissibility of a pathogen within a donor patient. Cells may be harvested from the donor patient and disposed in the flow chamber  102 . 
     After the predetermined amount of culturing time has elapsed, gas is flowed through the flow chamber  102  (step  503 ). As described in relation to  FIGS. 4A-4D , there exists a plurality of gas flow patterns that can be used to flow gas through the flow chamber  102 . In some implementations, a barrier fluid  208  is applied to at least the barrier space  206  of the flow chamber  102  prior to the flowing of a gas through the flow chamber  102 . In some implementations, the gas is flowed through the flow chamber  102  for a predetermined amount of time. For example, the gas may be flowed through the flow chamber  102  for about 15 min to about 30 min, about 30 min to about 1 hr, about 1 hr to about 3 hr, about 3 hr to about 12 hr, about 12 hr to 1 day, or about 1 day to about 3 days. In some implementations, the gas is flowed through the flow chamber  102  until a predetermined amount of the cells are infected. For example, the gas may be flowed through the flow chamber  102  until about 10%, 25%, 50%, 75%, or up to substantially 100% of the second set of cells  210 ( b ) are infected. In some implementations, the first and/or second set of cells are cultured in the flow chamber  102  for a second predetermined amount of time prior to the determination of an infection rate of the second set of cells  210 ( b ). 
     Next, the transmissibility of the pathogen is determined (step  504 ). The transmissibility of the pathogen may be quantified by determining the percent of cells  210 ( b ) that are infected after a given amount of time (or similarly the amount of cells  210 ( b ) not infected after a given amount of time). For example, a pathogen that infects 75% of cells  210 ( b ) after 1 day in the flow chamber  102  is more transmissible than a pathogen that infects 10% of cells  210 ( b ) after 1 day in a second flow chamber  102 . In other implementations, the transmissibility of the pathogen is quantified by the length of time it takes to infect a give percentage of the  210 ( b ) cells. For example, using the flow chamber  102 , it may be determined that a first pathogen infects 50% of the cells  210 ( b ) after 1 day of exposure to the cells  210 ( a ) and a second pathogen infects 87% of the cells  210 ( b ) after 1 day of exposure to the cells  210 ( a ). As described above, test agents, such as antibiotics, antivirals, antimicrobial compounds, antibodies, other cells, and/or mucus-modifying agents may be added to the nutrients provided to the cells  210 ( a ) and/or the cells  210 ( b ). In such an implementation, the above experiments are repeated, but with the test agent provided to the cells  210 ( b ). Comparing the infection rate seen in the first and second experiments, the effectiveness of the antibiotic or other agent in preventing infection may be assessed. 
     Referring back to the step  504 , in some implementations, the infection rate of the second set of cells  210 ( b ) is determined when the cells are still housed in the flow chamber  102 . For example, the cells of the second cell culture chamber  204  may express a fluorescence protein when exposed to the pathogen. The fluorescence can then be detected with the microscope  120 . In other implementations, the second cell culture chamber  204  and/or the cells  210 ( b ) are removed from the flow chamber  102  prior to a viral load being detected in the cells  210 ( b ). The presence of the virus in the cells  210 ( b ) can be detected with real-time PCR (qPCR), Northern blots, Southern blots, ELISA assays or similar detection methods. 
     EXAMPLES 
     The Influenza strain is one example pathogen that may be used with the systems and methods described herein. Influenza strains are able to effectively replicate in the ciliated epithelium of the conducting airways of humans. Human-adapted strains bind with highest affinity to glycans of a specific topology that are terminated by sialic acid (SA) that is α2-6 linked to the penultimate galactose (or Gal) sugar (known as α2-6 sialylated glycans). α2-6 sialylated glycans are mostly present in the human upper respiratory epithelia. Deep lung epithelia of the human alveolae predominantly express α2-3 sialylated glycans. Since transformed cell lines express a multitude of surface glycan receptors, including those with α2-3 linked SA and α2-6 linked SA, they are generally capable of supporting replication of a wide variety of influenza strains. While useful for replicating influenza in vitro and studying certain aspects of the infection process, transformed cell lines do not truly represent the viral-host interactions that occur in the human respiratory tract. 
     Primary human tracheal or bronchial epithelial cells grown on a membrane support with an air-liquid interface (ALI) differentiate into a pseudostratified epithelium with morphological and physiological features representative of the human conducting airway in vivo. For example, the cells differentiate into a heterogenous population of ciliated and non-ciliated cells (e.g., secretory, goblet/mucous-producing, and ciliated basal), which create a more representative model to study respiratory virus infection. In some implementations with human tracheobronchial epithelial cells grown with an ALI, ciliated cells are preferentially infected by avian-adapted influenza strains while human-adapted strains prefer to target non-ciliated cells. 
     To test the transmissibility of influenza strains (e.g., influenza strains PR/8, Brisbane/10 and Brisbane/59), primary normal human bronchial epithelial (NHBE) cells are seeded onto the membrane  212  in the first cell culture chamber  202  for at least 21 days in preparation for viral infection studies. In some implementations, a 21-day culture achieves sufficient differentiation; though longer times of differentiation, up to 35 days, may be used. Immunohistochemistry (IHC) of markers of NHBE differentiation are used to identify goblet/mucosa cells (Muc5AC), ciliated cells (Foxj1) and basal cells (CK5). 
     In general, the cell culture protocol includes expanding frozen cell stock in a T75 flasks using Lonza growth medium. The cell culture chamber is coated with collagen, and the cells are seeded into the cell culture chambers (50K per cell culture chamber). The cells are allowed to adhere and acclimate to the cell culture chamber in growth medium for three days at 37° C. and 5% CO 2 . After three days the growth medium is removed from the apical and basal side of the membrane. The apical side corresponds to the portion of the cell culture chamber above the membrane  212  and the basal side corresponds to the fluid reservoirs  214  below the membrane  212 . A differentiation medium is the added to the basal side (fluid reservoir  214 ) of the cell culture chambers. The cells are then allowed to differentiate for about 21 to about 35 days. The pump  110  exchanges the medium in the fluid reservoir  214  every two days. On average, the cells start producing mucus around day 13. During the differentiation period, the apical surface is washed with sterile PBS once per week. 
     The process of infecting the cells in the first cell culture chamber  202  begins once the cells are suitably differentiated (e.g., a differentiated epithelial monolayer has formed across the membrane  212 ). First, the apical surface is washed three times with PBS. An influenza strain is mixed with 100 μl of PBS and added to the apical surface of the first cell culture chamber. An example MOI for the virus is between about 0.001 to 0.1. The cells are then incubated at 37° C. for 1 hour. The fluid is then aspirated from the apical surface and the cells are washed with 100 μl PBS. The cells were returned to the incubator for about 24 hr to 72 hr. 
     After the cells of the first cell culture chamber are infected, the flow chamber is placed in the system described herein. Gas is flowed through the flow chamber  102  as described above in relation to  FIGS. 4A-4D . After a predetermined flow period, the cells are removed from the flow chamber, and in some implementations allowed to further incubate for 24 hr to 72 hr. The viral load is quantified by plaque assay on the MDCK cells or by qPCR. 
     The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The forgoing implementations are therefore to be considered in all respects illustrative, rather than limiting of the invention.