Patent Application
20230070705
The present disclosure relates to tissue engineering and organ-on-a-chip devices.
The COVID-19 pandemic has raised immense public awareness of respiratory health around the world. Yet, even before COVID-19 emerged in early 2020, chronic lung diseases (CLDs) such as chronic obstructive pulmonary disease (COPD), bronchitis, and asthma were already becoming a major global health challenge responsible for millions of deaths globally each year.[1] Every time we breathe, our lungs are exposed to airborne pathogens (including viruses), environmental toxicants, and indoor and outdoor pollutants that either cause respiratory diseases or exacerbate underlying illnesses.[2-4] New drugs for CLDs are urgently needed, but the development of new therapeutics for CLDs is slow and costly because of high failure rates.[5] The successful development and manufacturing of COVID-19 vaccines have been the exception in the drug development space, owing to the massive collective efforts of the international research community and the urgent need to limit the dangerous spread and lethal impact of COVID-19. Aside from this example, however, high attrition rates during the drug development process are the norm. A major problem in drug development that contributes to high failure rates is that existing experimental models for testing drug delivery and pharmacological activity do not properly represent how real human tissues interact with drugs. In respiratory research specifically, experimental models must overcome two main challenges: (i) accurately modelling the complex microenvironment of lung tissues, and (ii) accurately modelling the delivery and exposure of airborne substances to the tissue.
The respiratory epithelium serves as the first line of defense against environmental agents that enter the respiratory tract. The epithelium lines the inner wall of lung airways and the inner surface of alveoli. To understand the interactions between environmental agents and the epithelial surface, the mechanisms of lung disease, and the effects of drugs on respiratory health, it is necessary to examine how epithelial cells behave, function, and adapt to different insults or stimuli, ideally within a tissue microenvironment that mimics native airways.
Airborne substances enter the lung via inhalation and are carried by airflow into the various branches of the respiratory tree. Airways in different generations of the respiratory tree experience different airflow rates, which impose different shear stresses on the epithelium leading to mechanobiological stimulation of the epithelium. Particulate matter and other airborne substances carried by airflow then deposit on the epithelium via impaction, gravitational sedimentation, and Brownian diffusion depending on the locations of the airway and the characteristics of the particulates.[6] Most of the deposited particles are removed by mucociliary clearance, a transport mechanism involving the coordinated movement of beating cilia on the apical surface of the epithelium and the mucus layer resting above the cilia. However, particle deposition and retention within the airway are much more complex phenomena that involve other factors besides particle size and number. Other factors that have contributing roles include the lung airway surface, dynamics of particles caused by chemical composition or aggregation, and the shape and surface chemistry of the particles.[7] Dysfunction of mucociliary clearance is commonly associated with various CLDs,[8] with various factors including ciliary beating frequency, mucus secretion rates, and mucin composition contributing to the regulation of mucociliary clearance. [9,10] Since goblet cells and ciliated cells within the epithelium are responsible for producing mucin and generating movement of cilia, respectively, the ability to recapitulate accurate proportions of different cell populations within the epithelium is critical to creating and maintaining effective in vitro models of airway epithelium. Importantly, the quality of airflow on the epithelium is vital to the airway microenvironment as it governs both mucociliary transport as well as epithelial barrier function.[11,12] Yet, despite the importance of airflow on the biological relevance of epithelial tissue models, many researchers neglect to apply physiological airflow on epithelium and do not examine the potential impact of airflow absence on the physiological relevance of their in vitro models.
One of the most popular and common in vitro formats for airway epithelial cells is culturing on Transwell membrane inserts and creating an air-liquid interface (ALl) above the epithelial cells to induce apicobasal polarization.[13] Successful ALI culture results in expression of tight junctions, motile cilia, and viscous mucus, which are all indicative of the morphology of airway epithelium.[14][15] However, mucociliary differentiation by ALI has been shown to require approximately three weeks of culture and maintenance; such long-term culture increases the potential for contamination, dehydration, and lower cell viability.[16] In addition, Transwell membrane inserts are 2D polymeric substrates that are not easily amenable to airflow and are also biologically inert, lacking the proper 3D extracellular matrix (ECM) components that comprise the lamina propria of airway tissues. Elad et al. developed a parallel plate flow chamber system that enables airflow exposure over epithelium cultured in a Transwell insert,[17,18] but such large parallel plate flow systems are not scalable and are therefore not suitable for drug screening applications. Moreover, the issue of bioinertness of the membrane still remains even though airflow can be achieved.
The recent emergence of microfluidic “organ-on-a-chip” (OOC) systems have enabled the recapitulation of critical spatiotemporal features of complex tissue microenvironments in vitro. [19-21] Recapitulating lung environment on a microfluidic device was first introduced by Huh et al., who showed a successful fabrication of PDMS-based membrane device to mimic the blood-air barrier of an alveolus.[22] Its main feature is the cyclic stretching of its PDMS membrane to mimic the breathing motion of the lung and stretching of the alveolus; however, the elastomeric membrane does lack the physicochemical properties and bioactivity of the matrix found in the native basement membrane. Recently, Zamprogno et al. developed a “second-generation” lung-on-a-chip using a biodegradable collagen and elastin membrane on a hexagonal golden mesh structure.[23] Although the biocompatibility of this mesh-supported membrane overcomes some constraints of PDMS-based membranes, downstream analyses in this system remain limited to on-chip assessments.
The present inventors previously developed an airway-on-a-chip with a matrix-based hydrogel that could accommodate the coculture of airway epithelial and bronchial smooth muscle cells while also mimicking the lamina propria layer between the two cell types in bronchioles,[24] but this previous design was only tested under static conditions, and did not include any design elements to anchor the hydrogel and prevent gel detachment or leakage during sustained airflow. Note that while a number of lung-on-a-chip devices focus on cyclic stretching as the dominant mechanical stimulus,[24] not many focus on the effect of airflow-induced shear stress. Thus, there is a critical need to develop an in vitro platform for airway studies that has the potential for increased throughput, allows mechano-stimulation via airflow, enables cell-matrix interactions, and is amenable to both on-chip and off-chip downstream analyses for more advanced readouts.
Fluidic devices are provided comprising, and/or configured to form and support, extractable in-situ-formed hydrogels or hydrogel membranes that reside in a hydrogel chamber formed above, and in direct fluid communication with, an underlying fluidic channel, in the absence of an intervening membrane. In some example embodiments, the integrated fluidic device may include a geometrical hydrogel retention structure that provides a restoring force to the hydrogel when fluidic pressure is applied to the hydrogel from the underlying fluidic channel, or a geometrical meniscus-pinning feature that resists flow of a hydrogel precursor solution out of the hydrogel chamber, facilitating the formation of a hydrogel membrane extending over the integrated fluidic channel. The hydrogel or hydrogel membrane may be seeded with cells by delivering a cell-containing liquid to the fluidic channel, optionally while contacting the hydrogel with media provided in a media reservoir residing above the hydrogel layer.
Accordingly, in a first aspect, there is provided a fluidic device comprising:
In some example implementations of the device, the hydrogel chamber and the aperture are configured such that when a hydrogel precursor solution is dispensed such that the hydrogel precursor solution contacts the base surface, the hydrogel precursor solution extends across the aperture without flowing into the fluidic channel, thereby facilitating in-situ formation of the hydrogel within the hydrogel chamber.
In some example implementations of the device, the geometrical hydrogel retention structure comprises a hydrogel retention lip extending from the side wall at a location remote from the base surface, such that when the hydrogel is formed within the hydrogel chamber with an upper surface of the hydrogel contacting a lower surface of the hydrogel retention lip, the hydrogel retention lip provides, at least in part, the restoring force to the hydrogel when fluidic pressure is applied to the lower surface of the hydrogel from the fluidic channel.
In some example implementations of the device, the geometrical hydrogel retention structure comprises one or more protrusions extending from the base surface, such that when the hydrogel is formed within the hydrogel chamber with the hydrogel at least partially surrounding the protrusions, the protrusions provide, at least in part, the restoring force to the hydrogel when fluidic pressure is applied to the lower surface of the hydrogel from the fluidic channel.
In some example implementations of the device, at least one protrusion is a micropost.
In some example implementations of the device, at least a portion of the base surface extends across the fluidic channel, thereby forming a lower lip feature configured to resist flow of a hydrogel precursor solution into the fluidic channel when the hydrogel precursor solution is dispensed into the hydrogel chamber.
In some example implementations, the device includes the hydrogel within the hydrogel chamber.
In some example implementations of the device, the fluidic channel, the hydrogel chamber and the media reservoir define a first fluidic network, the multilayer fluidic structure comprising at least one additional fluidic network.
In another aspect, there is provided a method of forming a hydrogel in-situ within a fluidic device, the method comprising:
In some example implementations, the method further includes providing a cell-containing liquid to the fluidic channel and incubating the fluidic device to facilitate adhesion of cells of the cell-containing liquid to the lower surface of the hydrogel exposed by the aperture.
In some example implementations, the method includes providing liquid media to the media reservoir and incubating the fluidic device.
In some example implementations, the method further includes removing the cell-containing liquid from the fluidic channel; and delivering a fluid to the fluidic channel to expose the cells formed on the lower surface of the hydrogel to the fluid.
In some example implementations of the method, the fluid comprises a gas, and wherein the hydrogel is secured by the geometrical hydrogel retention structure such that a seal is maintained between the hydrogel and the fluidic channel during delivery of the gas.
In some example implementations of the method, the cells are airway epithelial cells, wherein the fluid comprises air, and wherein the air is delivered at a flow rate mimicking a physiological flow rate.
In some example implementations of the method, the fluid comprises particulate matter.
In some example implementations, the method further includes extracting the hydrogel from the hydrogel chamber, thereby obtaining an extracted hydrogel; and performing one or more analytical procedures to characterize the cells of the extracted hydrogel.
In another aspect, there is provided a fluidic system comprising:
In some example implementations, the fluidic system further includes a mixer in fluid communication with a particulate matter source and a fluidic path extending from the fluid delivery apparatus to the fluidic device, the mixer being configured for injection of particulate matter into the fluid delivered to the fluidic device.
In another aspect, there is provided a fluidic device comprising:
In some example implementations of the fluidic device, the geometrical meniscus-pinning feature comprises a ridge.
In some example implementations of the fluidic device, the aperture is a first aperture and the base surface is a first base surface, and wherein the media reservoir is defined in part by a second base surface having a second aperture defined therein, such that the media reservoir is in fluid communication with the hydrogel chamber through the second aperture, and wherein the geometrical meniscus-pinning feature resides on a portion of the second base surface that lies adjacent to the second aperture.
In some example implementations, the fluidic device further includes the hydrogel membrane extending over the aperture.
In some example implementations of the fluidic device, the fluidic channel, the hydrogel chamber and the media reservoir define a first fluidic network, the multilayer fluidic structure comprising at least one additional fluidic network.
In another aspect, there is provided a method of forming a hydrogel membrane in-situ within a fluidic device, the method comprising:
In some example implementations, the method further includes providing a cell-containing liquid to the fluidic channel and incubating the fluidic device to facilitate adhesion of cells of the cell-containing liquid to the lower surface of the hydrogel membrane exposed by the aperture.
In some example implementations, the method further includes providing liquid media to the media reservoir and incubating the fluidic device.
In some example implementations, the method further includes removing the cell-containing liquid from the fluidic channel; and delivering a fluid to the fluidic channel to expose the cells formed on the lower surface of the hydrogel membrane to the fluid.
In some example implementations of the method, the fluid comprises a gas, and wherein the hydrogel membrane is secured by to the base surface of the hydrogel chamber such that a seal is maintained between the hydrogel membrane and the fluidic channel during delivery of the gas.
In some example implementations of the method, the cells are airway epithelial cells, wherein the fluid comprises air, and wherein the air is delivered at a flow rate mimicking a physiological flow rate.
In some example implementations of the method, the fluid comprises particulate matter.
In some example implementations, the method further includes extracting the hydrogel membrane from the hydrogel chamber, thereby obtaining an extracted hydrogel membrane; and performing one or more analytical procedures to characterize the cells of the extracted hydrogel.
In another aspect, there is provided a fluidic system comprising:
In some example implementations, the fluidic system further includes a mixer in fluid communication with a particulate matter source and a fluidic path extending from the fluid delivery apparatus to the fluidic device, the mixer being configured for injection of particulate matter into the fluid delivered to the fluidic device.
A further understanding of the functional and advantageous aspects of the disclosure can be realized by reference to the following detailed description and drawings.
Embodiments will now be described, by way of example only, with reference to the drawings, in which:
Various embodiments and aspects of the disclosure will be described with reference to details discussed below. The following description and drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. Numerous specific details are described to provide a thorough understanding of various embodiments of the present disclosure. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present disclosure.
As used herein, the terms “comprises” and “comprising” are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms “comprises” and “comprising” and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.
As used herein, the term “exemplary” means “serving as an example, instance, or illustration,” and should not be construed as preferred or advantageous over other configurations disclosed herein.
As used herein, the terms “about” and “approximately” are meant to cover variations that may exist in the upper and lower limits of the ranges of values, such as variations in properties, parameters, and dimensions. Unless otherwise specified, the terms “about” and “approximately” mean plus or minus 25 percent or less.
It is to be understood that unless otherwise specified, any specified range or group is as a shorthand way of referring to each and every member of a range or group individually, as well as each and every possible sub-range or sub-group encompassed therein and similarly with respect to any sub-ranges or sub-groups therein. Unless otherwise specified, the present disclosure relates to and explicitly incorporates each and every specific member and combination of sub-ranges or sub-groups.
As used herein, the term “on the order of”, when used in conjunction with a quantity or parameter, refers to a range spanning approximately one tenth to ten times the stated quantity or parameter.
According to various example embodiments of the present disclosure, fluidic devices are provided comprising, and/or configured to form and support, extractable in-situ-formed hydrogels or hydrogel membranes that reside in a hydrogel chamber formed above, and in direct fluid communication with, an underlying fluidic channel, in the absence of an intervening membrane. Such a device is henceforth referred to as an “integrated hydrogel-supporting fluidic device”.
The hydrogel chamber 120, which resides above the fluidic channel 110, is bounded by a side wall 122 and a base surface 124. The base surface 124 includes an aperture 130 that brings the hydrogel chamber 120 in direct fluid communication with the fluidic channel 110. A media reservoir 140 is located above the hydrogel chamber 120. As will be described in further detail below, the base surface 124 of the hydrogel chamber and the aperture 130 are provided with dimensions such that when a hydrogel precursor solution is dispensed into the hydrogel chamber 120 and contacts the base surface 124, the hydrogel precursor solution extends across the aperture 130 without flowing into the underlying fluidic channel 110 due to surface tension forces, thereby facilitating in-situ formation of the hydrogel in the hydrogel chamber 120.
In this embodiment illustrated in
As can be seen in
While
Unlike previously known fluidic devices that interface a hydrogel structure with a fluidic channel within a multilayer fluidic device, each of the example embodiments illustrated in
A first example of a geometrical hydrogel retention structure illustrated in
In the example configuration shown in
In the example embodiments illustrated in
The example multilayer devices shown in
Moreover, other example device configurations may employ alternative combinations of the features shown in
Furthermore, although the preceding examples illustrate devices with a single fluidic network that includes the fluidic channel, hydrogel chamber and media reservoir, it will be understood that one or more of such fluidic networks may be integrated into a single multilayer integrated hydrogel-supporting fluidic device. For example, a plurality of such fluidic networks may be provided in a single device in an arrayed configuration, thereby enabling the in-situ formation and testing of multiple hydrogel constructs per device.
The side walls 122 of the hydrogel chamber 120 may be configured such that the hydrogel precursor solution flows by assisted capillary action. The present inventors found that during the flow of the hydrogel precursor solution into the hydrogel chamber, external forces such as gravity were negligible. However, it was found that an excess of pipetting pressure could exceed the interfacial tension between the side wall 122 and the hydrogel precursor solution, which could lead to the hydrogel precursor passing through the aperture into the underlying fluidic channel.
When all the hydrogel chamber 120 is filled with the hydrogel precursor solution, the device may be incubated to cause hardening of the hydrogel. For example, the device may be placed in a pre-warmed humid chamber and then placed in the CO2 incubator at 37° C. for 1 hour. After the hydrogel is polymerized, the device maybe be removed from the incubator and a cell culture solution may be dispensed into the media reservoir 140. The fluidic channel 110 may also be with the cell culture solution to rehydrate the hydrogel, and optionally to determine whether or not any leakage from the top channel to the bottom channel is present.
In some example applications, the suspended hydrogel may be employed to act as a biological scaffold to mimic the mechanical properties of the extracellular matrix (ECM). Type I collagen solution is comprised of collagen fibres. When conditions are near physiological (i.e., pH of ∼7.4 and temperature -36.5-37° C.) the fibres crosslink and form a gel-like structure. This process is similar to that of Matrigel, which consists mostly of type IV collagen and laminin. In some example implementations, the hydrogel precursor liquid may include, for example, a mixture of type I collagen and Matrigel solutions, which may be prepared in a chilled (< 4° C.) state to prevent rapid polymerization. Example final concentrations of the mixture include 6 mg/ml of Matrigel and 3 mg/ml of collagen. This example and non-limiting ratio of mixture components was tested to promote the adhesion and proliferation of Calu-3 cells in the examples described below.
A cell suspension may then be delivered to the fluidic channel 110, and the device may be subsequently incubated to facilitate cell sedimentation and attachment (optionally with the device inverted to facilitate sedimentation onto the hydrogel surface). Cell culture media may then be delivered to the fluidic chamber 110 to remove unattached cells. Cell culture media may be replenished to the media reservoir 140 and/or the fluidic channel 110 one or more times.
Having formed a cell layer along the bottom surface of the hydrogel, such as the epithelium 180, the cells may be exposed to a fluid delivered to the fluidic channel 110. It will be understood that the fluid may be a liquid or gas. In many of the non-limiting examples described below, the fluid may be air that is humidified and with which particulate matter 182 has been mixed, as illustrated in
After exposing the cell layer of the hydrogel to a fluid, the hydrogel may be extracted from the integrated hydrogel-supporting fluidic device. In example implementations in which a top layer of the device encloses the hydrogel chamber, the device may be disassembled to provide access to the hydrogel. For example, a tool such as a razor blade may be employed to cleave bonded area, or the device may be configured to include two removably attachable layers, such as layers adhered via an adhesive that enables detachment. When the hydrogel chamber 200 is accessible, a tool such as a scalpel may be employed to detach the hydrogel from the hydrogel chamber. The present inventors have found that extraction of the hydrogel can be facilitated by first detaching the hydrogel from the side wall, and subsequently employing tweezers to extract the hydrogel (gel construct). If the hydrogel contains embedded cells, a stream of liquid may be pipetted under the hydrogel one or more times to slip the gel out of the hydrogel chamber. When handling the gel construct, the present inventors found that it was beneficial to minimize use of sharp tweezers to prevent any potential damage. A scooping tool may instead be employed to scoop the hydrogel from the hydrogel chamber when transferring the hydrogel to another location.
The ability to extract the hydrogel from the integrated hydrogel-supporting fluidic device expands the number of analytical methods that may be employed to characterize the hydrogel, especially given the ability of the integrated hydrogel-supporting fluidic system to be exposed to airflow and particulate matter insult. The present inventors found that the extracted hydrogels were sufficiently robust during sample handling and manipulation to permit common procedures such as immunocytochemical staining, histology sectioning, and H&E staining, as well as even more rigorous procedures such as critical point dehydration, which is necessary during sample preparation prior to SEM imaging. Based on experience handling the hydrogel samples, the extracted hydrogels were found to resemble excised tissue in tactility, and thus it is conceivable to perform other biological assays and procedures off-chip that are normally performed on ex vivo tissues, including cell lysis, cell isolation, single-cell RNA sequencing (after cell isolation), flow cytometry, matrix stiffness measurements (e.g., by atomic force microscopy), and many others. These techniques are challenging for existing lung-on-a-chip platforms with embedded polymeric membranes or meshes.
Referring now to
In some example implementations, chemical treatment of the surface of the hydrogel chamber may be performed to facilitate or improve adhesion of the hydrogel membrane to the supporting device. For example, the hydrogel chamber 120 may be sequentially treated with sodium hydroxide and polyethylenimine (PEI) to aminate the plastic surface. The aminated surface may then be treated with glutaraldehyde, which acts as a double-headed linker molecule which covalently bonds with both the PEI and proteins in the subsequently seeded hydrogel membrane.
As shown in
As shown in
The present inventors have found that in order for the hydrogel membrane thickness to be reproducible and uniform for the example hydrogel precursor solutions used in the examples provided below, the height to width aspect ratio of the hydrogel precursor liquid should be sufficiently below one, which can be achieved by employing a hydrogel chamber 120 with a height-to-width aspect ratio of less than one. The present inventors have found that small aspect ratio ensures that the hydrogel matrix preferentially collapses downwards in a pseudo one-dimensional manner when it dehydrates, as the effect of wall adhesion is sufficiently far away from the suspended portion of the hydrogel. A larger aspect ratio results in the matrix being pulled both downwards and towards the wall as it dehydrates, and this results in inconsistent dehydration behaviour. The specific numerical aspect ratio that below which reliable membrane formation occurs is case-dependent and depends on the absolute dimensions of the membrane well and the portion of the well bottom that is open (i.e. the suspended portion of the hydrogel membrane). The skilled artisan can perform experiments to determine a suitable height-to-width aspect ratio for a given choice of hydrogel precursor liquid. In some example implementations, the aspect ratio can be less than 1, less than 0.75, or less than 0.5.
The hydrogel or hydrogel membrane that is formed in-situ within the integrated hydrogel-supporting fluidic device can be employed as a scaffold to mimic biologically active tissues or organs. For example, many of the examples provided within the present disclosure demonstrate applications in which an in-situ fabricated hydrogel is employed to simulate the ECM of the airway tissue, and a set of microanchors and/or lip structures are employed to maintain structural integrity of the hydrogel in the presence of airflow-induced pressure.
In some example embodiments, an integrated hydrogel-supporting fluidic device may be integrated with a fluid delivery system (e.g. airflow system) that permits controlled injection of fluid, optionally with particulate matter, for studies such as air pollution studies.
Referring now to
In some of the example embodiments described below, an airflow system is interfaced with the integrated hydrogel-supporting fluidic device, enabling flow rate control within the channel while delivering humidified air to maintain conditions that are favorable to airway epithelial function.[25] The example airflow systems described below also incorporates particulate delivery via physiological flow on the airway epithelium. The integration of the integrated hydrogel-supporting fluidic device with the airflow system allows accurate representation of the airway microenvironment and shows potential for many applications in respiratory research including air pollution and respiratory infection studies.
As shown in the figure, the example control and processing circuitry 400 may include a processor 410, a memory 415, a system bus 405, one or more input/output devices 420, and a plurality of optional additional devices such as communications interface 425, external storage 430, and a data acquisition interface 435. In one example implementation, a display (not shown) may be employed to provide a user interface for facilitating input to control the operation of the system 400. The display may be directly integrated into a control and processing device (for example, as an embedded display), or may be provided as an external device (for example, an external monitor).
The control and processing system 400 may include or be connectable to a console 480 that provides an interface for facilitating an operator to control one or more of the fluidic control devices, such as the pump/mass flow controller 510, and/or to monitor one or more sensor readings. The console may include, for example, one or more input devices, such, but not limited to, a keypad, mouse, joystick, touchscreen, and may optionally include a display device.
The methods described herein, such as methods for the controlled exposure of an in-situ-formed hydrogel to a test fluid, or other example methods described herein, can be implemented via processor 410 and/or memory 415. As shown in
The methods described herein can be partially implemented via hardware logic in processor 410 and partially using the instructions stored in memory 415. Some embodiments may be implemented using processor 410 without additional instructions stored in memory 415. Some embodiments are implemented using the instructions stored in memory 415 for execution by one or more microprocessors. Thus, the disclosure is not limited to a specific configuration of hardware and/or software.
It is to be understood that the example system shown in the figure is not intended to be limited to the components that may be employed in a given implementation. For example, the system may include one or more additional processors. Furthermore, one or more components of control and processing circuitry 400 may be provided as an external component that is interfaced to a processing device. Furthermore, although the bus 405 is depicted as a single connection between all of the components, it will be appreciated that the bus 405 may represent one or more circuits, devices or communication channels which link two or more of the components. For example, the bus 405 may include a motherboard. The control and processing circuitry 400 may include many more or less components than those shown.
Some aspects of the present disclosure can be embodied, at least in part, in software, which, when executed on a computing system, transforms an otherwise generic computing system into a specialty-purpose computing system that is capable of performing the methods disclosed herein, or variations thereof. That is, the techniques can be carried out in a computer system or other data processing system in response to its processor, such as a microprocessor, executing sequences of instructions contained in a memory, such as ROM, volatile RAM, non-volatile memory, cache, magnetic and optical disks, or a remote storage device. Further, the instructions can be downloaded into a computing device over a data network in a form of compiled and linked version. Alternatively, the logic to perform the processes as discussed above could be implemented in additional computer and/or machine-readable media, such as discrete hardware components as large-scale integrated circuits (LSI’s), application-specific integrated circuits (ASIC’s), or firmware such as electrically erasable programmable read-only memory (EEPROM’s) and field-programmable gate arrays (FPGAs).
A computer readable storage medium can be used to store software and data which when executed by a data processing system causes the system to perform various methods. The executable software and data may be stored in various places including for example ROM, volatile RAM, nonvolatile memory and/or cache. Portions of this software and/or data may be stored in any one of these storage devices. As used herein, the phrases “computer readable material” and “computer readable storage medium” refers to all computer-readable media, except for a transitory propagating signal per se.
The multilayer integrated hydrogel-supporting fluidic devices of the present disclosure may be fabricated based on a wide variety of material platforms and methods. For example, device layers may be formed from materials such as, but not limited to, elastomers (e.g., PDMS), other thermoplastics (e.g., polystyrene, cyclo-olefin polymers (COPs) and cycle-olefin co-polymers (COCs), polytetrafluoroethylene (PTFE, or Teflon(™)), polycarbonate, acrylic or polymethylmethacrylate (PMMA)), and thermoplastic elastomers (“TPEs”). Surface treatment may be performed, depending on the material choice, in order to achieve a desired level of surface tension for supporting the hydrogel precursor solution over the aperture.
In example embodiments that employ anchoring protrusions, the contact angle between the hydrogel precursor solution and anchoring protrusion material should be considerably less than 90 degrees. The present inventors have found that surface treatment is unnecessary when PMMA is used as the device material, as PMMA is already mildly hydrophilic, and the protein content of typical hydrogel precursor solutions serves to further decrease the contact angle of the system. If similar geometry is to be fabricated from PDMS, surface treatment or oxygen plasma treatment may be almost employed to achieve reliable seeding of the hydrogel precursor solution.
While the present examples describe specific hydrogel precursor materials, it will be understood that a wide variety of hydrogel material systems may be employed to form an in-situ hydrogel. Non-limiting examples include biologically sourced gels that are commercially available such as Matrigel®, Cultrex® and Geltrex(™) derived from Engelbreth-Holm-Swarm (EHS) tumors, biologically sourced gels such as gelatin or Collagen I, either on its own or supplemented with other ECM components such as EHS tumor extract, purified laminin, other collagen types (IV, VII, etc.), or elastin, modified biomaterials to add features such as UV crosslinking such as gelatin methacryloyl (GeIMA), and synthetic or otherwise non-reactive hydrogels (polyethylene glycol (PEG), chitosan, alginate).
In some example implementations, one or more biomaterials may be incorporated into the gel composition to enhance the structural integrity of the gel, which may be beneficial, for example, to maintain gel integrity during extraction and handling of thinner gel constructs.
The present examples demonstrate the use of integrated hydrogel-supporting fluidic devices for airway tissue modelling, demonstrating arrayable and scalable devices that are amenable to withstand physiologic airflow. The examples show that device can be combined with a custom airflow system that permits controlled injection of particulate matter for air pollution studies. Results show that airflow is critical to efficiently achieving physiologic mimicry of airway epithelium composition, tight junction expression, mucus production, and cilia formation on epithelial cells. The examples below allow demonstrate how standard on-chip analysis while also permitting complete sample extraction and off-chip analysis via immunocytochemistry, microscopy, and histological sectioning and staining, thereby expanding the number and types of biological assays that can be employed.
Indeed, as demonstrated below, airflow on airway epithelium in the integrated hydrogel-supporting fluidic device was found to produce improved physiologic mimicry in airway epithelium composition, tight junction expression, mucus production, and cilia formation compared to submerged and static ALI cultures. Furthermore, the present examples show that integrated hydrogel-supporting fluidic device can be analyzed both on-chip to study particulate matter deposition as well as off-chip, after gel extraction, to enable immunocytochemistry, fluorescence and scanning electron microscopy, and histological sectioning and staining. The example integrated hydrogel-supporting fluidic devices and its extractability offers significant potential to study lung cell biology in new ways that can advance an understanding of particle-cell-matrix interactions and the effects of air pollution on lung disease.
In some example applications, airway epithelial cells may be exposed to different airflow rates, for longer airflow exposure times, or with different airflow directions (for mimicking breathing patterns) to examine how epithelial cell morphology and cell compositions are affected by various flow parameters. Second, airway smooth muscle cells may be embedded into the floating gel, or cultured on the top side of the gel (similar to previous work by the present inventors[27]), to shed light on epithelial-smooth muscle interactions that may be involved in the regulation of airway thickening and remodeling commonly associated with the onset of various CLDs such as asthma.[41-43] Third, particulate matter deposition onto epithelium and its effects on matrix remodeling and cell morphology may be studied by investigating deposition efficiency based on airflow rates and exposure times, and by analyzing the extracted gel using the various biological assays mentioned above. Such studies may have implications on the impact of air pollution on chronic lung disease development, and aid in the development of therapeutics to manage CLDs under adverse environmental conditions.
In some of the examples described below, an integrated hydrogel-supporting fluidic system was tested with only the Calu-3 cell line and a thick (~300 µm) floating gel, and further advances may include the use of stem-cell-derived or primary lung cells and the integration of thinner floating gels to better mimic the physiological cell and tissue microenvironments of native airways. Calu-3 cells were a convenient option to aid in the development of a cell culture protocol for the new integrated hydrogel-supporting fluidic system, while the thicker gel constructs helped to ensure gel integrity during sample handling.
In terms of mechanotransduction on airway epithelium, airway epithelial cells are constantly exposed to luminal shear stress caused by respiration. Shear stress, as well as other mechanical stimuli such as stretching and compression, is known to affect extracellular adenosine triphosphate (ATP) release, thereby regulating mucus secretion on the airway cilia.[41] In addition, studies have proposed various mechanisms of cilia response to mechano-stimulation, including via curvature-gated channels, strain-sensing molecules, stretch-sensitive channels connected to nearby microvilli, membrane tension-sensing molecules, shear stress-sensing membrane polymers, or internal shear-sensing molecules.[28] Similar phenomenon has been described previously in human umbilical vein endothelial cells (HUVECs), with surface-expressed glycocalyx providing mechanosensitivity to shear flow.[45] However, there remain open questions regarding the role of shear stress in airway epithelial cell differentiation and epithelial damage and repair mechanisms, thus providing an opportunity to apply the present example integrated hydrogel-supporting fluidic devices to explore these and other mechanistic questions.
In addition to the example applications described in the examples below, the present example integrated hydrogel-supporting fluidic devices and associated systems may be employed for a wide variety of applications and studies, including, but not limited to, lung airway modelling with airflow over epithelium and other lung cell types in coculture (e.g., endothelial cells, bronchial smooth muscle cells, mast cells), virus infection studies (e.g., COVID-19) for fundamental understanding of viral-particle-lung tissue interactions, cancer metastasis studies, including cell extravasation and intravasation through endothelial layers and underlying matrix (i.e., cell migration and cell invasion), cell invasion through basement membrane layer (cancer and immune cells), fundamental studies of molecular transport through ECM and through thin basement membranes, pre-clinical drug testing for diseases such as asthma and cancer (for anti-invasion therapy), construction of multilayered thin tissues structures (skin, intestinal wall, etc.) as model tissues for basic research complementing animal and human tissue models (i.e., all types of “organ-on-a-chip” systems), and usage of micro posts made of elastomeric materials such as PDMS to measure the contractile force from smooth muscle cells embedded in matrix.
The following examples are presented to enable those skilled in the art to understand and to practice embodiments of the present disclosure. They should not be considered as a limitation on the scope of the disclosure, but merely as being illustrative and representative thereof.
The integrated hydrogel-supporting fluidic device described in the present non-limiting examples consists of four layers of poly(methylmethacrylate) (PMMA) plastic sheets that are first milled to create desired micro-geometric features and then bonded together by a liquid solvent bonding technique to create reliable bonds that remain leak-free throughout experimentation (
In the present example device, the top layer contains the media reservoir located directly above the suspended gel, and also consists of the inlet and outlet access ports for loading hydrogel precursors, delivering cell culture media, and applying airflow to the microchannels (
The second layer, which was machined to a thickness of 300 µm using a facing operation, provides a protruding “lip” feature positioned above the upper gel surface. This lip feature is beneficial for preventing air leakage, a common occurrence with designs that do not include the lip whenever airflow is applied. Detachment of the gel is further prevented by providing additional surface area (beyond the side wall surfaces) for gel adhesion.
The third layer, which is faced to a thickness of 800 µm, contains the suspended hydrogel itself as well as anchoring microposts or “microanchors” that hold the hydrogel in position and withstand air pressure from the bottom channel while airflow is applied (
In the present experiments, the gel remained intact for > 48 h with flow rates of 0.6 cm3 s-1, which based on the current design allowed the present inventors to essentially apply the full range of flow conditions found in the human lung inside the integrated hydrogel-supporting fluidic device.
Reinforcement of the hydrogel by microanchors is also advantageous because it secures the gel during culture and airflow but still allows the extraction of the gel for off-chip assessment after airflow exposure (
After gel extraction, the cell-coated gel construct can maintain its structural and mechanical integrity, allowing us to perform various sample manipulations including immunocytochemical staining, scanning electron microscopy (SEM), and histological sectioning to characterize the gel sample. Because of its location within the device, the suspended gel serves as an intermediary biological membrane that separates the upper media reservoir from the airway epithelial monolayer. The bottom side of the gel was chosen to be the culture site for the epithelium (
A schematic illustration and photographs of an example implementation of an extractable floating liquid-gel-based organ-on-a-chip system is provided in
To provide airflow-mediated mechanotransduction on the airway epithelium in integrated hydrogel-supporting fluidic devices, an airflow system was custom-built with flow rate control, humidity control, inline particulate matter delivery, and humidity and temperature monitoring (
The example integrated hydrogel-supporting fluidic microdevice was fabricated from poly(methylmethacrylate) sheets (PMMA or acrylic, McMaster-Carr) that were micromachined and then solvent-bonded to create sealed devices. The toolpaths for the milling process were created using computer-aided design software Autodesk Fusion 360 (Autodesk Inc, CA, USA). The design file that contained the G-code for each layer was imported and used to direct the micromilling on an automated 3-axis computer numerical control (CNC) milling machine (P/N: PCNC770 Tormach, Waunakee, Wl, USA) that used different carbide endmills for different microchannels in the device.[46,47] The endmills used for fabrication of the example integrated hydrogel-supporting fluidic device were purchased from Caliber Industrial Supply (Mississauga, ON, Canada) and included the following: diameter 0.381 mm (P/N: 11101500, TuffCut- M.A. Ford), 3.175 mm (P/N: 211-214, MasterCut), 0.7938 mm (P/N: 209-202-1, MasterCut), 1.9844 mm (P/N: 30109, SOWA), and 1.5875 mm (P/N: 211-206, MasterCut). In total, the integrated hydrogel-supporting fluidic device was comprised of four layers of micromilled PMMA with various thicknesses (i.e., 1.5 mm, 0.3 mm, 0.8 mm, and 1.5 mm in order from top to bottom layer). Face operations were used to create the thin layers from original 1.5-mm stock sheets. The four PMMA layers were then bonded with a liquid solvent bonding technique.[48] 99% ethanol was carefully pipetted between the aligned PMMA layers, which were then placed between the platens of a heated hydraulic press (Carver Inc., Wabash, IN, USA). A compressive force of 1000 lbf over 4.9 cm2 (i.e., the surface area of the device; equal to 200 psi pressure) at a temperature of 70° C. was applied for one minute.
Compressed medical-grade air (P/N: 100034, Messer) was used as the air source. The source air flowed through a plastic in-line filter (P/N: 4795K42, McMaster-Carr) that removed airborne particles > 0.01 µm. The tubing was appropriately reduced in diameter to be fitted into the ports of the integrated hydrogel-supporting fluidic device (⅛” OD). The tubes used for the airflow system were purchased from McMaster-Carr: diameter ¼" ID / ⅜" OD, polyurethane rubber tubing (P/N: 5545K14), ⅛" ID / ¼" OD, PVC plastic tubing (P/N: 55485K72), ⅟16" ID / ⅛" OD, PVC plastic tubing (P/N: 5233K51).
After the air was filtered, the tube was connected to a mass flow controller (MFC) (Sierra 100 Smart-Trak, Sierra) to control the flow rate delivered to the device. The tube was locally heated to 70° C. to help increase the airstream temperature from room temperature to 37° C. A custom bubbler humidifier was placed inside the 37° C. incubator to produce saturated air with relative humidity of ~95%. Inside the bubbler humidifier, a porous stone was connected to the tube to produce microscale air bubbles. The tube that exited the humidifier was connected to a calibrated hygrometer (P/N: R6001, REED Instruments, Newmarket, ON, Canada) for real-time monitoring of the temperature and relative humidity. The sensor of the hygrometer was inserted into a custom-designed 3D-printed in-line adapter that was exposed to the oncoming airflow via a T-shaped junction. The tube that extended out of the hygrometer was connected to a 3D-printed manifold that divided the airflow into four smaller tubes that interfaced with the integrated hydrogel-supporting fluidic device.
For particulate matter delivery, a 3-way valve was set up in-line with the airflow system to connect the syringe containing carbon black (Vulcan XC-72R, FuelCell Store) in powder form. The carbon black was applied as a bolus injection into the air stream and then ultimately into the microfluidic channel. To confirm carbon black deposition, the cell-laden gel was extracted from the device and was fixed for scanning electron microscopy (SEM).
To select a representative flow rate, a 75-kg male exchanging 500 ml of tidal volume of air per breath was selected as a model. Dimensions for each of the airways were obtained from Filipovic et al and Weibel et al.[32] To mimic the shear stress on the epithelium in the device, wall shear stresses were calculated for all generations of the native airways, and then these shear stress values were used to back-calculate the average velocity required for the epithelium in the integrated hydrogel-supporting fluidic device to generate the same wall shear stress. Once integrated hydrogel-supporting fluidic device obtained a confluent epithelial monolayer, the airflow system was connected to the device and airflow of 0.0083 cm3 s-1 was applied for either 24 h or 48 h.
A computational fluid dynamics (CFD) simulation was generated in COMSOL to check that the wall shear stress on the epithelium was uniform across the width of the gel. A simple conversion was used to calculate the flow velocity in the integrated hydrogel-supporting fluidic device bottom channels with given volumetric flow Vavg = Q/hw, where Vavg is average velocity in each channel, Q is the volumetric flow rate (m3 s-1) given by the mass flow controller, h is the height of the channel (m), and w is the width of the channel (m).
Calu-3 cells (ATCC® HTB-55TM) were cultured in MEM with Earle’s Salts (P/N: 320-026-CL, Wisent Bioproducts, Quebec, Canada) supplemented with 10% of fetal bovine serum (FBS, P/N: 26140079, Thermo Fisher Scientific, Waltham, MA, USA) and 1% of 10,000 U mL-1 penicillin-streptomycin (P/N: 15140163, Thermo Fisher Scientific). Cell culture media was replenished every 48 hours and cells were subcultured when they reached ~70-80% confluent. The Calu-3 cells used in this study were subcultured up to ~15 passages.
The microchannels within the integrated hydrogel-supporting fluidic device were sterilized with 70% ethanol followed by washes of DPBS (-/-) (P/N: 14190144) and DPBS (+/+) (P/N: 14040133). After channels were dried, the gel pockets were coated with human plasma fibronectin (P/N: F0895-2 MG, Sigma Aldrich, St. Louis, MO, USA) with a concentration of 100 µg ml-1 and incubated for 30 min at 37° C.
The floating hydrogel was prepared with a mixture of 6 mg ml-1 Matrigel (phenol red-free, P/N: 356237, Corning), and 3 mg ml-1 Type I collagen (rat-tail, P/N: CADB354249, VWR International, Radnor, PA, USA) at pH 7.4. The ratio of Matrigel to collagen was previously determined based on optimal cell viability.[27] Immediately after removing the fibronectin solution from the gel pocket, prepolymerized hydrogel solution (7 µl) was carefully pipetted into the gel pocket. The devices were incubated at 37° C. for 1 hour to polymerize the gel. After incubation, all the device channels were filled with cell culture media to rehydrate the gel for at least 1 day.
Calu-3 cells were trypsinized from the tissue culture flask and resuspended at 5 million cells ml-1. 85 µl of cell suspension was pipetted into the bottom microchannel of the device and the device was then immediately flipped upside-down and placed on top of support blocks inside the incubator. After two hours of cell sedimentation and attachment, the cell culture media was replenished to remove unattached cells. Cell culture media was replenished every 24 hours until the end of the experiments.
After initial cell seeding, the suspended gel was closely monitored for cell confluency. Once the cells reached confluency on the gel, each device was tested under three different culture conditions: (i) submerged culture (epithelial cells submerged in liquid media) for 96 hours, (ii) static air-liquid interface (ALI) culture for 96 hours, and (iii) 48 hours of ALI culture followed by either 24 hours or 48 hours of airflow culture. ALI culture was achieved by removing the cell culture media from the bottom channel while maintaining media in the top reservoir channel.
At the end of the culture experiment, the device was disassembled by cleaving each layer with a razor blade. The hydrogels were carefully extracted from the devices and were fixed in 4% paraformaldehyde (PFA) and 1% glutaraldehyde (GA) in 0.1 M phosphate buffer (pH 7.2) for at least 1 hour. The cells were then post-fixed with 1% Osmium Tetroxide and dehydrated using 50%, 60%, 70%, 90%, and 100% ethanol, consecutively. The samples were dried using critical point drying (CPD) machine (P/N: Autosamdri-810, Tousimis, Maryland, USA) and were coated with gold using Gold Sputter Coater (P/N: SC7640, Quorum Technologies, England). Images were taken at the Centre for Nanostructure Imaging (University of Toronto) using a scanning electron microscope (Quanta FEG 250 ESEM, FEI, Oregon) with various magnifications to observe the morphology of the epithelial surface. For the carbon black deposited SEM sample, energy dispersive spectroscopy (EDS) was performed using an EDAX silicon drift detector that scanned the region for carbon black particles.
Cells were fixed with 4% PFA for 20 minutes at room temperature. Cells were permeabilized with 0.1% (v/v) Triton X for 3 minutes followed by blocking solution of 1% bovine albumin serum (BSA) for 30 minutes. Primary antibodies were diluted in the blocking solution and were applied to the cells overnight. Primary antibodies used included: MUC5AC monoclonal antibody (1:100, P/N: MA5-12178, Thermo Fisher Scientific), and ZO-1 polyclonal antibody (1:100, P/N: 40-2200, Thermo Fisher Scientific). The primary antibody was washed with PBS (+/+) 3 times in intervals of 10 minutes. Secondary antibodies (goat anti-rabbit IgG (Alexa Fluor 568, P/N: A11011, Thermo Fisher Scientific), goat antimouse IgG (Alexa Fluor 488, P/N: A11001, Thermo Fisher Scientific)) were applied for 30 minutes along with the Hoechst nuclear dye (1:1000, P/N: 33342, Thermo Fisher Scientific). Images were taken with Olympus® IX-83 inverted microscope with ORCA® Flash 4.0 V2 camera. Images from each fluorescence channel were processed and merged using ImageJ software.
Extracted gels were fixed in 4% PFA for 20 minutes and were submerged in 1X PBS. Samples were embedded in HistoGel™ and placed in 70% ethanol overnight. The samples were then processed in the tissue processor (Histo-Tek VP1, Sakura Finetek, USA). The samples were bisected and embedded using tissue embedder (Tissue-Tek®TECTM 6, Sakura Finetek, USA), which produced a paraffin block. Sections were cut with a thickness of 4 µm using a rotary microtome (P/N: HM 325, Epredia, USA) and were mounted on a glass slide. The sections on the slides were dried at 60∘C for 2 hours. Finally, sections were stained for hemotoxylin and eosin using standard protocols.
For goblet cell differentiation, MUC5AC-expressing cells and total cells were manually counted, and the number of MUC5AC-expressing cells per 100 total cells was plotted. Goblet cell counts were obtained from three independent experiments (n = 3). One-way ANOVA with post hoc Tukey test was used to determine the statistical significance between the three culture conditions: (i) submerged, (ii) static ALI, and (iii) airflow.
Deposition of particulate matter in lung airways depends on the size, density, and chemical composition of the particles. For instance, fine particulate matter with lower density tend to deposit deeper inside of the lungs compared with fine particulate matter with high density. Thus, versatility in the airflow system is necessary to model various situations involving particulate matter delivery, exposure, and deposition. Deng et al. modelled particle deposition and showed that particulate matter of diameter ~3 µm mostly deposited near the 20th airway generation (G20) of the respiratory tree.[28] Given that particulate matter of ~ 2.5 µm or smaller (referred to as PM2.5) can penetrate deep into the respiratory system and can exacerbate respiratory diseases such as asthma and lower respiratory inflammations,[29-31] the present inventors focused on airflow settings that matched the physiological flow conditions in lower respiratory airways (G19 to G22) (
A wall shear stress magnitude of 0.026 dyn cm-1 was targeted on the epithelium, which based on a previous study by Weibel et al. mimics the shear stress in airway generation G20 of a 75-kg human with an estimated 1.0-L tidal volume.[32,33] To achieve 0.026 dyn cm-2 in the integrated hydrogel-supporting fluidic device, the present inventors set the MFC to produce a volumetric airflow rate of 0.0083 cm3 s-1 (or 0.5 cm3 min-1). At this flow rate, the airflow Reynolds number was estimated to be Re ~ 0.2, resulting in laminar flow of air in the microchannel and a predictable parabolic velocity profile. With the 3D construction of the device (
After assembling the airflow system and selecting the desired flow rate for mimicking shear in lower respiratory airways, the integrated hydrogel-supporting fluidic device was connected to the airflow system and tested the impact of airflow on morphology and function of the airway epithelium cultured on the floating gel. It was hypothesized that shear-induced mechanical stimulation caused by airflow over the airway epithelial cells (AECs) would offer a more physiologically relevant physical environment that would lead to improved epithelial monolayer formation, increased tight junction expression (and thus improved barrier function), and more representative airway epithelial cell composition, based on goblet cell population density. Three different culture conditions were compared on the gel-attached AECs: (i) submerged in cell culture media for 96 h (“submerged”); (ii) static air-liquid interface culture without airflow for 96 h (static ALI); and (iii) static ALI culture for 48 h followed immediately by airflow exposure with 0.026 dyn cm-2 for an additional 48 h (ALI + airflow) (
AECs in submerged culture for 96 h showed diffuse cytoplasmic expression of ZO-1 with no localization on epithelial cell borders and did not display any acetylated α-tubulin expression (
To characterize the cilia featured on the surface of AECs in the integrated hydrogel-supporting fluidic device, scanning electron microscopy (SEM) was performed of the cell-coated gel samples for all three culture conditions (
Viability of AECs cultured under all three conditions on the gel of the integrated hydrogel-supporting fluidic device remained high throughout the experiments. However, it was observed that the relative humidity of the air played a crucial role in both AEC viability and cilia expression. If relative humidity of the air dropped to 80%, the gel experienced surface dehydration leading to a significant reduction in cell viability to only ~20% after only 6-h of low-humidity airflow (
To demonstrate the utility of gel extraction for off-chip downstream analyses, the floating gel was extracted from the integrated hydrogel-supporting fluidic device for histology sectioning and hematoxylin and eosin (H&E) staining, with the goal of confirming epithelial morphology and examining the underlying matrix tissue structure under different experimental conditions (
To demonstrate the potential of the platform to facilitate air pollution studies, particulate matter deposition was tested by delivering a dose of carbon black powder into the airstream for transport and deposition onto the airway epithelium in the integrated hydrogel-supporting fluidic device (
The airflow system described in the previous examples provides a unidirectional constant flow for a certain time. It can manipulate its flow rate but will always be flowing in one direction. However, to accurate mimic the breathing inside of the lung airways, it is essential to provide the bidirectional airflow on to the airway epithelial cells. The present example describes an adaptation of the airflow system to provide bidirectional functionality. The system includes the syringe pump, mass flow controller, humidity chamber and a gas cylinder, as shown in
The specific embodiments described above have been shown by way of example, and it should be understood that these embodiments may be susceptible to various modifications and alternative forms. It should be further understood that the claims are not intended to be limited to the particular forms disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure.
WHO, Chron. Respir. Dis. 2007, 1.
S. Fereol, R. Fodil, G. Pelle, B. Louis, D. Isabey, Respir. Physiol. Neurobiol. 2008, 163, 3.
O. D. Chuquimia, D. H. Petursdottir, N. Periolo, C. Ferńndez, Infect. Immun. 2013, 81, 381.
M. E. Kreft, U. D. Jerman, E. Lasič, N. Hevir-Kene, T. L. Rižner, L. Peternel, K. Kristan, Eur. J. Pharm. Sci. 2015, 69, 1.
J. A. Dimasi, L. Feldman, A. Seckler, A. Wilson, Clin. Pharmacol. Ther. 2010, 87, 272.
T. G. O’Riordan, G. C. Smaldone, in Murray Nadel’s Textb. Respir. Med., 2015.
W. H. Chen, K. H. Lee, J. K. Mutuku, C. J. Hwang, Aerosol Air Qual. Res. 2018, 18, 866.
M. A. Mall, J. Aerosol Med. Pulm. Drug Deliv. 2008, 21, 13.
W. Stannard, C. O’Callaghan, J. Aerosol Med. Depos. Clear. Eff. Lung 2006, 19, 110.
M. B. Antunes, N. A. Cohen, Curr. Opin. Allergy Clin. Immunol. 2007, 7, 5.
V. K. Sidhaye, K. S. Schweitzer, M. J. Caterina, L. Shimoda, L. S. King, Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 3345.
A. Pieterse, S. D. Hanekom, Multidiscip. Respir. Med. 2018, 13, 1.
A. A. Pezzulo, T. D. Starner, T. E. Scheetz, G. L. Traver, A. E. Tilley, B. G. Harvey, R. G. Crystal, P. B. McCray, J. Zabner, Am. J. Physiol. - Lung Cell. Mol. Physiol. 2011, 300, L25.
A. G. Buckley, K. Looi, T. losifidis, K. M. Ling, E. N. Sutanto, K. M. Martinovich, E. Kicic-Starcevich, L. W. Garratt, N. C. Shaw, F. J. Lannigan, A. N. Larcombe, G. Zosky, D. A. Knight, P. J. Rigby, A. Kicic, S. M. Stick, Biol. Proced. Online 2018, 20, 1.
D. Jiang, N. Schaefer, H. W. Chu, in Methods Mol. Biol., Humana Press Inc., 2018, 1809, 91-109.
G. Abraham, C. Zizzadoro, J. Kacza, C. Ellenberger, V. Abs, J. Franke, H. A. Schoon, J. Seeger, Y. Tesfaigzi, F. R. Ungemach, BMC Vet. Res. 2011, 7, 26.
N. E. T. Davidovich, Y. Kloog, M. Wolf, D. Elad, Biophys. J. 2011, 100, 2855.
D. Elad, U. Zaretsky, S. Avraham, R. Gotlieb, M. Wolf, I. Katra, S. Sarig, E. Zaady, Biomech. Model. Mechanobiol. 2018, 17, 891.
S. N. Bhatia, D. E. Ingber, Nat. Biotechnol. 2014, 32, 760.
E. W. Esch, A. Bahinski, D. Huh, Nat. Rev. Drug Discov. 2015, 14, 248.
S. Ahadian, R. Civitarese, D. Bannerman, M. H. Mohammadi, R. Lu, E. Wang, L. Davenport-Huyer, B. Lai, B. Zhang, Y. Zhao, S. Mandla, A. Korolj, M. Radisic, Adv. Healthc. Mater. 2018, 7, 2.
D. Huh, B. D. Matthews, A. Mammoto, M. Montoya-Zavala, H. Y. Hsin, D. E. Ingber, Science (80-.). 2010, 328, 1662.
P. Zamprogno, S. Wüthrich, S. Achenbach, G. Thoma, J. D. Stucki, N. Hobi, N. Schneider-Daum, C. M. Lehr, H. Huwer, T. Geiser, R. A. Schmid, O. T. Guenat, Commun. Biol. 2021, 4, 1, 12
A. O. Stucki, J. D. Stucki, S. R. R. Hall, M. Felder, Y. Mermoud, R. A. Schmid, T. Geiser, O. T. Guenat, Lab Chip 2015, 15, 1302.
E. Kilgour, N. Rankin, S. Ryan, R. Pack, Intensive Care Med. 2004, 30, 1491.
A. M. D. Wan, A. Sadri, E. W. K. Young, Lab Chip 2015, 15, 3785.
M. Humayun, C. W. Chow, E. W. K. Young, Lab Chip 2018, 18, 1298.
Q. Deng, L. Deng, Y. Miao, X. Guo, Y. Li, Environ. Res. 2019, 169, 237.
R. Habre, E. Moshier, W. Castro, A. Nath, A. Grunin, A. Rohr, J. Godbold, N. Schachter, M. Kattan, B. Coull, P. Koutrakis, J. Expo. Sci. Environ. Epidemiol. 2014, 24, 380.
A. Zanobetti, F. Dominici, Y. Wang, J. D. Schwartz, Environ. Heal. A Glob. Access Sci. Source 2014, 13, 1.
I. Kloog, B. Ridgway, P. Koutrakis, B. A. Coull, J. D. Schwartz, Epidemiology 2013, 24, 555.
E. R. Weibel, Am. J. Physiol. - Lung Cell. Mol. Physiol. 2013, 305, 6.
E. R. Weibel, B. Sapoval, M. Filoche, in Respir. Physiol. Neurobiol., Respir Physiol Neurobiol, 2005, 148, 1-2 SPEC. ISS, 3-21.
R. R. Mercer, M. L. Russell, V. L. Roggli, J. D. Crapo, Am. J. Respir. Cell Mol. Biol. 1994, 10, 613.
A. Wanner, M. Salathe, T. G. O’Riordan, Am. J. Respir. Crit. Care Med. 1996, 154, 1868.
K. H. Benam, R. Villenave, C. Lucchesi, A. Varone, C. Hubeau, H. H. Lee, S. E. Alves, M. Salmon, T. C. Ferrante, J. C. Weaver, A. Bahinski, G. A. Hamilton, D. E. Ingber, Nat. Methods 2016, 13, 2, 151-157.
J. E. C. Walker, R. E. Wells, Am. J. Med. 1961, 30, 259.
S. M. Jazaeri Farsani, M. Deijs, R. Dijkman, R. Molenkamp, R. E. Jeeninga, M. leven, H. Goossens, L. van der Hoek, Influenza Other Respi. Viruses 2015, 9, 51.
A. Dvorak, A. E. Tilley, R. Shaykhiev, R. Wang, R. G. Crystal, Am. J. Respir. Cell Mol. Biol. 2011, 44, 465.
R. G. Crystal, S. H. Randell, J. F. Engelhardt, J. Voynow, M. E. Sunday, in Proc. Am. Thorac. Soc., American Thoracic Society, 2008, 5, 7, 772-777.
B. Button, R. C. Boucher, Respir. Physiol. Neurobiol. 2008, 163, 189.
K. Yamamoto, T. Sokabe, N. Ohura, H. Nakatsuka, A. Kamiya, J. Ando, Am. J. Physiol. - Hear. Circ. Physiol. 2003, 285, 2 54-2.
D. C. Genetos, D. J. Geist, D. Liu, H. J. Donahue, R. L. Duncan, J. Bone Miner. Res. 2004, 20, 41.
M. J. Mondrinos, Y. S. Yi, N. K. Wu, X. Ding, D. Huh, Lab Chip 2017, 17, 3146.
Y. Zeng, X. F. Zhang, B. M. Fu, J. M. Tarbell, in Adv. Exp. Med. Biol., Springer New York LLC, 2018, 1097, 1-27.
D. Konstantinou, A. Shirazi, A. Sadri, E. W. K. Young, Sensors Actuators, B Chem. 2016, 234, 209.
D. J. Guckenberger, T. E. de Groot, A. M. D. Wan, D. J. Beebe, E. W. K. Young, Lab Chip 2015, 15, 2364.
A. M. D. Wan, T. A. Moore, E. W. K. Young, J. Vis. Exp. 2017, 2017, 55175.
This application claims priority to U.S. Provisional Pat. Application No. 63/241,884, titled “EXTRACTABLE FLOATING LIQUID GEL-BASED ORGAN-ON-A-CHIP” and filed on Sep. 8, 2021, the entire contents of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63241884 | Sep 2021 | US |
