Patent Application
20230383224
N/A.
Organ- and tissue-on-a-chip models can be used to recreate complex biological systems in vitro, making these systems amenable to repeatable scientific analyses. There is a need for such a model of the respiratory system, particularly in light of the global SARS-CoV-2 virus outbreak and the resulting impact on the respiratory pathway.
Accordingly, new systems, methods, and apparatus for simulating an airway are desirable.
Thus, in one embodiment the disclosure provides an apparatus for simulating an airway, including: an air channel having a central portion with an air inlet at a first end and an air outlet at a second end opposite the first end; and a vascular channel adjacent to the central portion of the air channel, the vascular channel being separated from an interior of the central portion of the air channel by a porous membrane, the air channel being configured to conduct air from the air inlet through the central portion such that air moves adjacent to the porous membrane.
In another embodiment the disclosure provides a method for simulating an airway, including: providing an airway simulation apparatus including: an air channel having a central portion with an air inlet at a first end and an air outlet at a second end opposite the first end, and a vascular channel adjacent to the central portion of the air channel, the vascular channel being separated from an interior of the central portion of the air channel by a porous membrane; and conducting, using the air channel, air from the air inlet through the central portion such that air moves adjacent to the porous membrane.
Various objects, features, and advantages of the disclosed subject matter can be more fully appreciated with reference to the following detailed description of the disclosed subject matter when considered in connection with the following drawings, in which like reference numerals identify like elements.
In accordance with some embodiments of the disclosed subject matter, mechanisms (which can include systems, methods, and apparatus) for simulating an airway are provided.
The field of organ-on-chip technology arose from the convergence of microfabrication and tissue engineering. The devices built using this technology can recapitulate key aspects of human physiology in a repeatable, three-dimensional form. Organ-on-chip systems can recreate features such as complex organ functions, tissue-barrier properties, parenchymal tissue function, and multi-organ interactions. The Mechanobiology and Soft Materials Laboratory at the University of Arkansas has extensive experience developing organ-on-chip devices (e.g. for the blood-brain-barrier and heart) that can be used in biomedical research. Embodiments of the present disclosure (referred to herein as the Nasal Airway-Lung-on-Chip, or AirLoC, system) arose from efforts to expand the scope of the laboratory's systems during the global SARS-CoV-2 outbreak. It was understood that the virus has major effects on the respiratory system, yet there were no benchtop micro-physiological systems that replicated both the upper and lower systems concurrently on the same device. This lack of technology has presented a major gap in the biomedical research field, as there are few benchtop technologies that can be used to study the physiological effects of any particulate matter on the combined upper and lower airways.
In various embodiments, the AirLoC device includes a multi-layered cell culture platform, including connected nasal and lung sections. The platform includes an air channel spanning the nasal and lung sections and a pair of vascular channels associated with the nasal section and the lung section which contain cell culture media, which may be static or flowing. Thin micro- or nano-porous membranes (e.g. with pore sizes as small as 3 nm and as large as 10 μm) separate the air and media in both sections. in certain embodiments, the air channel may be attached to a pump to provide controlled air flow at a physiological breathing rate. In various embodiments the air flow may move into and out of the air channel at regular or irregular intervals (e.g. depending on what type of breathing pattern is being simulated) at a rate of 12-16 cycles per minute, similar to normal adult human breathing rates, although slower or faster rates may be used in order to simulate various subjects or health conditions (e.g. faster breathing rates of up to 60 breaths per minute to simulate breathing of small children or infants). Human nasal epithelial cells and human bronchial/tracheal epithelial cells can be seeded on respective porous membranes in the airway side of the device, while human microvascular endothelial cells can be seeded on the reverse side of each membrane. Other cell types found in the airways, such as fibroblasts, can also be cultured within the device. Accordingly, the cellular organization within the AirLoC system can mimic the basic functional unit of the human nasal and lung airways.
Embodiments of the AirLoC device have been designed so that cells cultured on the membranes experience physiological fluid flow both in the airway and in the vascular channels. Accordingly, parameters such as Reynolds (Re=UL/v), Strouhal (St=fL/U), and Womersley (α=(ωL/v)1/2) numbers are used to determine the appropriate channel geometries, air volumes, and flow profiles necessary for the device to simulate human breathing mechanics (where: U—flow velocity; L—channel dimension; v—kinematic viscosity of fluid; f—frequency/periodicity of flow waveform; and ω—angular frequency of flow waveform). In the airway channel, the air flow pump can deliver particulate matter (ranging from nano- to micro-meter diameter scale) and other inhaled species to the system, such as viral particles or corticosteroid aerosol droplets. The distribution pattern of introduced particles is similar to that observed in vivo, thus making the AirLoC device a useful tool in studying the effects of airborne particulate matter on the human respiratory system.
Currently, there are no other existing benchtop microphysiological platforms (i.e. organ-on-chip systems) that mimic the physiology and function of the combined upper and lower respiratory systems. The development of the AirLoC device generates an understanding of combined responses of the upper and lower respiratory airways. Additionally, the AirLoC device can serve as a platform for the discovery of potential new therapeutics for respiratory diseases exacerbated by airborne particulate matter. The data generated by using the device will provide a foundational understanding of the cytotoxicity and physiological effects that result from particulate matter deposition in the airways.
In various embodiments, the AirLoC system may also be employed for other toxicological evaluations. For example, airway effects of ambient air from varying geographic locations could be tested on the platform, as well as airway effects of viral exposure. The use of human cells to engineer the AirLoC also is an advantage over preclinical animal-based approaches. In certain embodiments, the AirLoC can be adapted to custom-build patient-specific devices with various disease phenotypes for the purpose of drug safety and efficacy testing (i.e. to provide personalized medicine).
In various embodiments the AirLoC device is a multilayered cell culture platform including interconnected nasal and lung sections, both of which have airway and vascular channels. An air pump controls air flow through the airway channel, while a peristaltic pump controls media flow through the media channels.
Thus, in one embodiment the disclosure provides an apparatus for simulating a nasal and lung airway which includes an air channel including an extended nasal portion with an air inlet at a first end and a lung portion at a second end opposite the first end. A nasal vascular channel is disposed adjacent to the nasal portion of the air channel, where the nasal vascular channel is separated from an interior of the nasal portion of the air channel by a nasal porous membrane. A lung vascular channel is disposed adjacent to the lung portion of the air channel, where the lung vascular channel is separated from an interior of the lung portion of the air channel by a lung porous membrane. The air channel is configured to conduct air from the air inlet through the nasal portion to the lung portion.
In some embodiments the lung portion of the air channel may include at least one branched passageway for air flow, as shown in
Also as shown in
In some embodiments, each of the nasal vascular channel and the lung vascular channel may include an inlet and an outlet for flow of cell culture media through the respective channel, although as noted above certain “static” embodiments of the channels may not include inlets for media flow or the inlets may be covered or plugged.
In some embodiments, each of the nasal porous membrane and the lung porous membrane may include a nano-porous or micro-porous membrane, which may be a polycarbonate membrane. In various embodiments the membrane may be made of other materials including polymers such as polytetrafluoroethylene (PFTE) or polyester (PET) or a polyester such as polyethylene terephthalate (PET), where the particular choice of material and specifications (e.g. thickness, pore size, flexibility) will depend on the application.
In various embodiments the air channel and/or vascular channels include cells growing on one or more surfaces thereof, including on one or both of the nasal porous membrane or the lung porous membrane. In some embodiments, a first side of the nasal porous membrane facing the air channel may include nasal epithelial cells growing thereon, and a second side of the nasal porous membrane opposite the first side and facing the nasal vascular channel may include microvascular endothelial cells growing thereon. In other embodiments, a first side of the lung porous membrane facing the air channel may include at least one of bronchial or tracheal epithelial cells growing thereon, and a second side of the lung porous membrane opposite the first side and facing the lung vascular channel may include microvascular endothelial cells growing thereon. In some embodiments, the apparatus may include fibroblasts growing on at least one of the nasal porous membrane or the lung porous membrane. Other cell types may also be grown on the device, as discussed further below.
In some embodiments, electrodes may be used to measure a potential within the apparatus, for example across the layers of epithelial cells that form on the membranes. Thus, in one embodiment, a first electrode may be disposed within the air channel and in electrical communication with the nasal porous membrane, and a second electrode may be disposed within the nasal vascular channel and in electrical communication with the nasal porous membrane. The first electrode and the second electrode may be coupled to an instrument such as an electrophysiological amplifier and configured to obtain at least one of a trans-epithelial electrical resistance or a trans-endothelial electrical resistance (TEER) across the nasal porous membrane. One or both of the electrodes may be in electrical communication with the membrane by touching a portion of an electrically conductive liquid (e.g. cell culture media) which has continuity with the membrane.
In various embodiments, the apparatus may include an air pump coupled to the air channel (
In certain embodiments, the apparatus pay include a liquid pump (e.g. a peristaltic pump, see
Nasal Section
The nasal portion of the airway may be a hollow rectangular prism (although other shapes such as a circular or oval cross-section are also possible) made from materials such as polymethyl methacrylate (PMMA) and polystyrene and may have inner dimensions of 8 mm×8 mm×50 mm and the walls of the channel may be 1 mm thick. Centered along the interior face is an opening where the membrane is bonded (see
The inlet and outlet of the nasal airway channel may taper to small openings (e.g. using separate ABS plastic pieces bonded to each end, see
Lung Section
In various embodiments, the end of the nasal airway channel may be tapered to increase the flow rate so that the flow rate of air reaching the bronchial/tracheal airway section is at a physiological rate. In some embodiments, the bronchial airway portion has two levels of branching or bifurcation (although further levels of bifurcation could be incorporated), which mimics the anatomical structure of the trachea and bronchi in the lung. In particular embodiments this branching airway structure is made using soft photolithography techniques to create a master mold, after which polydimethylsiloxane (PDMS) is then poured over the master mold and cured to produce flexible, optically clear channels. A porous PDMS membrane is then fabricated and bonded beneath the bronchial airway channels to form the vascular channel, which may be fabricated using PDMS with the same branching pattern as the airway channel. The airway and vascular channels are bonded together using PDMS, with the porous membrane sandwiched in between. The flexible PDMS allows for the branches to deform at a physiological level and rate as air is pulled into and out of the airway channels. The vascular channels have an inlet and outlet hole where silicone tubing can be inserted and connected to a peristaltic pump to circulate media through the vascular channel. In certain “static” embodiments in which no media is circulated in the lung vascular channel, the inlet and outlet holes may either be plugged up or eliminated altogether.
Previous airway organ- or lab-on-a-chip models have not included the connected nasal and lung portions as disclosed herein. Embodiments of the disclosed design provide an advantage of recapitulating the physiological breathing and subsequent particle distribution for the upper and lower respiratory airways. Furthermore, the AirLoC system uses human cells and thus can reduce the need for animal models/testing while providing relevant data.
Prototypes of the nasal section of AirLoC have been fabricated and tested with cultured cells to validate cell viability, phenotype, and function.
Once the AirLoC system has been fully validated, further studies will be performed to determine the effect of particulate matter inhalation on normal and diseased nasal and lung epithelia, including both healthy cell phenotypes as well as an asthma phenotype. Healthy and diseased AirLoCs will be exposed to various sizes and concentrations of PM-like particles and observed for cytotoxic effects.
Results from these validation studies will contribute to the development of novel in vitro benchtop tools for the study of diseases that affect the upper and lower respiratory systems. Various embodiments of the AirLoC platform can be used to engineer patient-specific nasal airway and lung systems. The platform can also be utilized to study the pathological effects of other airborne pathogens such as viruses on the upper and lower respiratory systems.
Particulate matter (PM) exposure represents a significant risk factor for patients suffering from respiratory illnesses. Unfortunately, effective benchtop “humanized” models that can model PM exposure and their resulting pathological effects on the nasal and lung airways systems do not exist. Lack of these models significantly hinder efforts toward developing therapies for PM exposure-related pathologies.
Accordingly, the disclosed system provides a combined nasal airway and lung-chip platform which mimics the breathing mechanics and air-liquid interface (ALI) of the nasal and lung epithelium. Embodiments of the platform will also include capillary blood flow to mimic transport across the ALI.
PM exposure and drug treatment will be tested on embodiments of the AirLoC platform and the platform will help with understanding disease mechanisms and developing therapeutics for people who suffer from respiratory diseases following PM exposure. In certain embodiments, the AirLoC system can be used to test the effects of particulate matter (smoke, fuel fumes, dust), viral infections (SARS-CoV-2) on nasal and lung function/disease as well as intra-nasal drug delivery testing applications and development of patient-specific nasal/lung airway systems for the purposes of personalized medicine.
Alternate Designs
The following is a description of several alternative embodiments of the AirLoC system:
Different cell types: Several combinations of respiratory cells can be cultured on the device. Instead of, or in addition to, cell types that have been disclosed herein, other examples are as follows:
Change material to model disease: The material stiffness can be altered to model disease states. The lung portion could be made stiffer to simulate fibrotic scar tissue in the lungs or changes in lung mechanical properties with age. The nasal portion could be made flexible to mimic micro-scale expansions/contractions of the nasal tissue during breathing.
Air flow profile/channel dimensions: The dimensions of the device and air flow patterns can be adjusted to model several different aspects of the respiratory passage. Some examples are as follows:
Media channel: The media channel of the device can incorporate flow or be a static reservoir for media. The flow can be connected or separate between both nasal and lung sections. If static, the media reservoir can have caps on openings to seal in media (as shown in diagrams above).
TEER: As disclosed herein, the AirLoC system includes the ability to measure transepithelial electrical resistance (TEER). The electrodes that are needed for making these measurements can be integrated into the device in a number of ways: electrode wires can be fabricated into the device, ports can be included which are compatible with commercially available TEER chopstick electrodes, or a micro-electrode array can be added to the membrane.
Extracellular Matrix: Cells can be seeded in varying ECM environments to produce more two-dimensional (2D) or more three-dimensional (3D) cultures. ECM hydrogels can be added on top of the membrane for a 3D environment.
Disease Phenotypes: Disease phenotypes can be induced on cells by adding different drugs, chemokines, cytokines, etc. These may induce disease such as asthma or rhinitis.
Membrane: The membrane separating the channels of the device does not have to be a specific geometry. It can be altered for a larger/smaller surface area depending on what is desired (i.e. how many cells should be seeded).
Device Coating: Chemical coatings can be applied to the channels within the device to help simulate PM capture or deposition and reduce electrostatic effects.
The following are non-limiting examples of embodiments of the disclosure.
The following are examples of images and other data obtained cells grown on a membrane of an AirLoC system. Among other things, the images and data depict cultured cells that have established an air-liquid interface (ALI) and which have also established a transepithelial electrical resistance (TEER). These Examples demonstrate that the AirLoC system facilitates the culture of cells which functionally and morphologically replicate cells of the human airway and therefore indicate that this system can be used to study the normal and disturbed or diseased state of this organ.
The AirLoC platform embodiment of
Validation by Non-dimensional Analyses and CFD Simulations
The dimensions for the nasal portion of the chip can be optimized based on non-dimensional numbers. From literature, it was found that the three most important numbers used to characterize air flow through the nasal passageways were Reynold's, Womersley, and Strouhal numbers shown in Table 1. The Reynold's number is a function of velocity, chip diameter, and air viscosity and defines the flow as being either laminar or turbulent; the target number was less than 2300 to ensure a laminar flow profile. The Womersley number is a function of chip diameter, breathing frequency, and air viscosity and describes the pulsatile nature of the flow; the target value was between 1.0 and 1.68. The Strouhal number is a function of chip diameter, breathing frequency, and air velocity, and describes the oscillatory or steady nature of the airflow; the target value was less than 1. Since the Reynold's, Womersley, and Strouhal numbers all have common variables, the equations were used to create curves so that the chip dimensions could be selected to satisfy the requirements of the non-dimensional numbers, as well as maintain a physiological level for a resting breathing rate (12-16 cycles/minute) and flow rate (˜200-300 mL/s). All calculations were based on a desired wall shear stress (WSS) of 0.5 dyn/cm2, which has previously been shown to induce mucous secretions at normal physiological levels.
Based on the analysis shown in
More in-depth computational fluid dynamics (CFD) simulations using ANSYS Fluent have been completed. These new simulations incorporate the shortened taper design, and also include Wall Shear Stress (WSS), an important parameter to consider when subjecting airway epithelial cells to airflow. In
WSS at this flow rate was analyzed in the simulation and found to be approximately 0.45 dyne/cm2 at the peak, which is within 10% of the target value (0.5 dyne/cm2).
To ensure that cells can survive on the chip platform and exhibit normal nasal epithelial cell phenotypes, cells were cultured on chips and transwells for 14 days at the ALI and an MTS cell viability assay as well as immunostaining were performed.
Physical Airflow Setup and Preliminary Flow Results
To test chips under airflow conditions, the setup shown in
The mass flow controller can be programmed to control air flow by inputting a sine wave function as previously described. For the preliminary flow experiments, the mass flow rate was set to 13.5 SLPM at a breathing rate of 12 cycles per minute in order to achieve a WSS of 0.5 dyne/cm2 on the cells. Additional validations of flow rate and humidity levels are being conducted.
Preliminary flow experiments at 0.5 dyne/cm2 are beginning to be conducted. Chips that had been in culture for 14 days at the ALI were exposed to oscillatory air flow for 30 minutes, then measured for cell viability and stained for ZO-1 to visualize any changes in the cell monolayer. The initial results, shown in
Thus, while the invention has been described above in connection with particular embodiments and examples, the invention is not necessarily so limited, and that numerous other embodiments, examples, uses, modifications and departures from the embodiments, examples and uses are intended to be encompassed by the claims attached hereto.
The present application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/345,527, as filed May 25, 2022, the contents of which are incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63345527 | May 2022 | US |
