Patent Application
20230357684
The subject matter disclosed herein relates generally to systems and methods for studying inter-organ communication by co-culturing 3D cell cultures, organoids, or tissue slices in recirculating media. More particularly, the subject matter disclosed herein relates to multi-organ-on-a-chip (MOC) systems and methods for operating such system.
Within the human body, organs are in constant communication via blood and lymphatic circulation to help maintain homeostasis and to respond to external cues.2 Inter-organ cross-talk occurs through the release of bioactive molecules, like cytokines, that enable intercellular communication.2 These small signaling molecules get diluted in the blood and lymph fluid, making it hard to discern where they came from and where they are going. This can be especially challenging when studying specific inter-organ communication in vivo. Through the use of microfluidics, multi-organ-on-a-chip (MOC) systems can separate out these complex interactions and study them directly by connecting specialized tissue culture microenvironments via perfusion channels.3 These MOC devices can be used to culture cells,4-6 organoids,7,8 or tissue slices9,10 in settings that mimic physiologically relevant conditions in a quantifiable way.11
Conventionally, the devices used to study inter-organ communication tend to have fabrication processes that are complex and time-consuming, while limiting the architecture of the device. Soft lithography is a traditional microfluidic fabrication method that is widely used because it is inexpensive and easy to use, but these polydimethylsiloxane (PDMS) devices are limited to layered devices and the fabrication process is lengthy and hard to scale up for mass production.9,12 As devices have increased in complexity, as is common with MOC systems, the reproducibility with soft lithography greatly decreases.9,13 Other PDMS devices include additional materials like PMMA or glass, further increasing the complexity, cost, and fabrication time.13, 14 Micro-machining is another fabrication technique that is highly reproducible, but the resulting devices and their respective real-world connections are incredibly complex and quite expensive to produce.3
One major way MOCs mimic in vivo function is through control of fluid flow through the various microscale organs,19 but simple and robust platforms to control flow rate are still needed. Precise fluid control is needed to mimic the very low flow rates observed for in vivo interstitial flow under physiological (0.1-1 μm/s) and pathological (1-10 μm/s) conditions.20 For example, slower physiological fluid flow rates are found in the interstitium (0.1-1 μm/s) and within lymphatic capillaries (1.4-20.4 μm/s), while faster fluid flow rates are found in the blood vessel capillaries (80-180 μm/s), the lymphatic vessels (870 μm/s, with a peak of 2200-9000 μm/s), the veins (15,000-71,000 μm/s), and the aortic artery (1,000,000 μm/s). As fluid moves, it provides nutrient and waste exchange as well as communication between organs, commonly in the form of cell recirculation. Flow also applies shear stress that impacts cellular function and viability and can result in cellular adhesion, activation, and extravasation. Thus, flow control systems for organs-on-chip must generate flow in a range of physiological and pathological flow rates, while ideally enabling transport of blood-borne cells between organs without damage. In addition to controllable flow rates, additional desirable qualities for flow control systems within OOC platforms include multiplexing capabilities, compatibility with cell culture incubators in terms of temperature output, and ability to recirculate media to enable cell circulation and communication between tissues.
Many MOC devices achieve the required low flow rates by using pumps that are large, expensive, complex, and incompatible with incubators.1,5,9,16,21,22 To avoid these major pitfalls of available pumping techniques, in 2008 Kimura et al. integrated a stirrer-based micropump into a PDMS-based microfluidic device.6 A stir bar was mounted in the center of a circular chamber, controlled by an external magnetic field generated by the spinning magnet within a hotplate.6 The basis of this design can be traced back to the centrifugal water pump, first patented in 1990.23 The centrifugal water pump includes a circular chamber with a rotary impeller that has curved vanes to generate a suction force, inducing fluid flow. This pump was initially designed to recirculate and oxygenate water for fish and live bait on a fishing boat, similar to the purpose of media perfusion on a microfluidic device.23 While the central spinning stir bar provided easy and precise flow control, the device required complex fabrication processes, was not amenable to multiplexing, and was not compatible with incubators.
Accordingly, there exists a need for devices for studying inter-organ communication that can be more readily and repeatedly manufactured, while also providing a more flexible architecture that can be adapted for a variety of desired configurations.
In one aspect, the presently disclosed subject matter provides a system to model inter-organ communication. Such a system can include one or more micro-culture well configured to receive a tissue sample therein and an impeller-based pump in fluid communication with the one or more micro-culture well. In this arrangement, the impeller-based pump can be configured to generate fluid flow through the one or more micro-culture well.
In some embodiments, the one or more micro-culture well is configured to perfuse the fluid transversely with respect to the tissue sample.
In some embodiments, the system can further include a mesh base positioned within each of the one or more micro-culture well and configured to structurally support the tissue sample thereon. The mesh base can include one or more vertical posts that extend out of the one or more micro-culture well and are configured to aid in insertion and removal of the mesh base with respect to the one or more micro-culture well. In some embodiments, the system can further include a membrane positioned between the tissue sample and the mesh base.
In some embodiments, the impeller-based pump includes an impeller positioned within a substantially circular chamber and a magnetic element coupled with the impeller. In this configuration, the impeller can be rotatable within the substantially circular chamber upon application of a magnetic field to the magnetic element to generate fluid flow within the system. A magnetic field generator can be spaced apart from the impeller-based pump, the magnetic field generator being configured to apply a rotating magnetic field to the magnetic element.
In some embodiments, each of the one or more micro-culture well is contained within a sample container having a sample container inlet and a sample container outlet and the impeller-based pump includes a pump inlet and a pump outlet. In this arrangement, each sample container and the pump module are arranged to form a single closed fluid circuit in which fluid is flowed into each respective one of the sample container inlet and the pump inlet and out of each respective one of the sample container outlet and the pump outlet. In addition, one or more fluid conduit elements can be arranged to provide fluid connections among and between the impeller-based pump and the one or more sample container.
In another aspect, the presently disclosed subject matter provides a method for modeling inter-organ communication, the method including positioning a tissue sample into each of one or more micro-culture well and pumping fluid through the one or more micro-culture well.
In some embodiments, pumping fluid through the one or more micro-culture well involves perfusing the fluid transversely with respect to the tissue sample.
In some embodiments, positioning a tissue sample into each of one or more micro-culture well involves arranging a plurality of micro-culture wells in sequence within a single closed fluid circuit.
In some embodiments, pumping fluid through the one or more micro-culture well involves positioning an impeller-based pump in fluid communication with the one or more micro-culture well, the impeller-based pump comprising an impeller and a magnetic element coupled with the impeller, and applying a rotating magnetic field to the magnetic element to rotate the impeller and generate fluid flow through the one or more micro-culture well. In some embodiments, applying a rotating magnetic field can involve activating a magnetic field generator that is spaced apart from the impeller-based pump.
In another aspect, the presently disclosed subject matter provides computer-readable storage medium having computer-executable instructions stored thereon which, when executed by one or more processors, cause one or more computers to perform a method for modeling inter-organ communication, the method comprising regulating the operation of a impeller-based pump to pump fluid through a tissue sample positioned in each of one or more micro-culture well.
Accordingly, it is an object of the presently disclosed subject matter to provide a method and system to model inter-organ communication. An object of the presently disclosed subject matter having been stated hereinabove, and which is achieved in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds hereinbelow.
The presently disclosed subject matter will now be described more fully. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein below and in the accompanying Examples. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.
All references listed herein, including but not limited to all patents, patent applications and publications thereof, and scientific journal articles, are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.
While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs.
Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims.
The term “and/or” when used in describing two or more items or conditions, refers to situations where all named items or conditions are present or applicable, or to situations wherein only one (or less than all) of the items or conditions is present or applicable.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” can mean at least a second or more.
The term “comprising,” which is synonymous with “including,” “containing,” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.
As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
With respect to the terms “comprising,” “consisting of,” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.
Unless otherwise indicated, all numbers expressing quantities of size, temperature, time, weight, volume, concentration, capacitance, specific capacity, discharge capacity, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.
As used herein, the term “about,” when referring to a value is meant to encompass variations of in one example ±20% or ±10%, in another example ±5%, in another example ±1%, and in still another example ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods.
Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes, but is not limited to, 1, 1.5, 2, 2.75, 3, 3.90, 10 4, and 5).
To address the issues with conventional MOC systems, in one aspect, the present disclosure provides a reconfigurable 3D-printed microfluidic device having an integrated impeller-based pump. The novel impeller-based pump integrates elements from both the historic centrifugal water pump and a stirrer-based micropump by inserting a magnetic stir bar into a 3D-printed impeller that rotates in a circular chamber within the modular chip.6,23 In some embodiments, the magnetic impeller is externally controlled using magnets mounted on a motor that is operable to produce controllable low speeds (i.e. flow rates) by changing the voltage the motor receives. The integrated impeller pump is inexpensive, easy to multiplex, cuts down on size relative to other conventional pumping methods, and includes few wires with little to no heat given off. In some embodiments, the device is used to gain a better understanding of the immune system by examining lymph node slice culture and how it interacts with other organs in the body. By way of example and not limitation, in some embodiments, the presently disclosed subject matter can provide communication between two or more tissue samples to create a biomimetic model of inter-organ communication.
Referring to the embodiment shown in
In some embodiments, the microfluidic device 100 comprises distinct modular units that can be assembled into any of a variety of configurations that can be easily reconfigured based on the needs of each specific experiment. As shown in
In some embodiments, the modules of the microfluidic device 100 can be produced using 3D printing techniques. For example, stereolithography (SLA) 3D printing is an additive manufacturing fabrication technique that uses photocurable resins to build up a device layer by layer.15 This process can allow for rapid prototype printing (5-60 min) and to design and print intricate architecture within a single 3D structure.12,16-18 When applied to the microfluidic device 100 discussed herein, the use of stereolithography 3D printing can greatly reduce the fabrication time, increase reproducibility, and make the device easy to use and share with collaborators. That being said, while 3D printing is an easy way to reproducibly fabricate microfluidic devices with complex architecture in a short period of time, the liquid photopolymer resins used for stereolithography (SLA) and digital light processing (DLP) 3D printing are often cytotoxic. In addition, the use of additives such as optical absorbers and plasticizers can enhance the print resolution, enabling smaller internal channel sizes and smaller port diameters, such as with the MiiCraft BV007a resin, but these may result in increased toxicity if these molecules leach out of the device. Pre-leaching the printed parts is one way to reduce cytotoxicity, as is coating with impermeable protective materials such as parylene C. Alternatively, as the impeller pump platform is intended to recirculate fluid and cells within MOC systems and other biological model systems, the material used to fabricate the device and impeller can be selected to be cytocompatible for the timescale of the experiment, similar to polydimethylsiloxane (PDMS) that is used in traditional soft lithography microfabrication techniques. Fabrication in well-characterized cell-culture materials such as polystyrene, polypropylene, acrylic, or cyclic olefin copolymer is feasible by methods such as machining, hot embossing, or extrusion, paired if needed with acrylate or silicone adhesives for multilayer parts.
In one exemplary situation, a prototype modular device was designed using Fusion 360 drawing software and 3D printed using a MiiCraft Ultra 50 SLA Printer with BV007a clear resin (MiiCraft) and FormLabs Clear. Although existing modular microfluidic devices16,21,22 can be composed of independent units that have a particular pattern of channels with a specific function, the device is distinguished by its inclusion of a tissue module to accommodate live tissues or models thereof, among other features. For example, the printed modules were designed to have sufficient tolerance at the connections of each piece to fit together tightly. For example, the internal channel dimensions were optimized to be 500×500 μm, the smallest dimensions that could be printed reproducibly within each module with this combination of printer and resin.
Whether using additive manufacturing or some other fabrication technique, the connections between the modules can be designed to be sealed tightly to provide fluid circulation throughout the microfluidic device 100 without any leaks. As shown in
Regarding the particular configuration of the individual component elements, in some embodiments, the sample container 120 can be optimized to house ex vivo tissue slices in a user-friendly way. In particular, for example, referring to the configuration shown in
Referring in particular to
As discussed above, fluid flow is provided to the one or more sample container 120 by the impeller-based pump 110. Referring again to
Referring to
As shown in
In some embodiments, precise flow rate control is valuable in an MOC device to properly mimic both physiological (0.1-1 μm/s) and pathological (1-10 μm/s) environmental conditions.20 To achieve more biomimetic flow velocities, the design of the impeller-based pump 110 and the impeller 112 positioned therein can be configured to control the fluid flow rate within the device. In some embodiments, the impeller 112 can have a “closed impeller” configuration 112a shown in
To fully achieve a fluid velocity that was within the correct physiological range, the pump well dimensions can be configured alongside the impeller design. When designing the dimensions of the pump well, impeller, and entry points of the microchannel, it can be assumed that the hydrodynamic energy produced in the pump well by the rotating impeller decreases further from the impeller, such as due to viscous energy losses. Consistent with this principle, it was observed that if the impeller filled the majority of the cross-sectional area of the well, then the hydrodynamic energy generated fluid velocities that were able to reach as high as 33,000 μm/s, sufficient to model venous or arterial fluid flow. In contrast, where it is desired to model slower capillary and lymphatic vessel flow velocities, a large well diameter (e.g., about 26 mm) can be chosen relative to the width of the impeller piece (e.g., about 11.5 mm). Similarly, the intersection height of the channels approximately two-thirds up the side of the well (e.g., about 8.5 mm from the base of a 12-mm deep well) can be designed to slow the flow rates through the corresponding channel compared to a lower intersection height. In this manner, a 3-dimensional architecture for the device and impeller can be achieved and optimized through rapid 3D printing, enabling the pump to attain biologically-relevant fluid flow regimes.
As shown in
Despite the increasing pool of innovative organ-on-a-chip platforms, many are designed for cell or organoid culture rather than tissue slices and tend to be specifically tailored for each lab's specific application. In contrast, the modular device and impeller-based pump platform proposed herein is not only user-friendly and customizable, but it is completely accessible to other researchers focusing on inter-organ communication, especially between tissue slices and also, optionally, between 3D cultures in hydrogels or other supportive 3D matrices. Incorporation of tissue slices and 3D cultures is made particularly user-friendly by incorporation of the transwell-style inserts that can be added and removed from the device as needed, e.g. for imaging on a microscope or for switching the tissue sample mid-experiment. An aspect of the presently disclosed subject matter provides, among other things, the innovative of on-board impeller pump that provides continuous recirculating flow, without complex pneumatics, dynamic electronic triggering, or undesirable heat output. Having the pump on-board the MOC shortens the pathlength for fluid flow compared to running tubing to-and-from an external pump system, particularly as many external pumps must be located outside of the cell-culture incubator. This MOC platform can be used to study, among other things, physiological and pathological lymph node function with models of the central nervous system (blood-brain barrier and brain), gut, tumor, and more. In the future, this device can be used to focus on inflammation in the central nervous system and how it relates to autoimmune diseases.
An aspect of an embodiment of the present disclosure includes, but not limited thereto, a computer readable medium having computer-executable instructions stored thereon which, when executed by one or more processors, cause one or more computers to perform a method for modeling inter-organ communication. In this regard, in some embodiments, the impeller-based pump 110 can be computer-controlled to efficiently regulate the rotation of the impeller 112 and thus the fluid flow rate through the system.
The following Examples provide illustrative embodiments. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.
In one tested configuration, the device and impeller were designed using Fusion 360 and 3D printed using a CADWorks 3D MiiCraft Ultra 50 Printer with two resins, MiiCraft BV007a and FormLabs Clear. The impeller housed a magnetic stir bar and was rotated using an external platform which consisted of two magnets mounted on a computer fan. The fan rotation was controlled using a potentiometer with a voltmeter for voltage readout. A computational model of the pump was designed using computational fluid dynamics studies via ANSYS software. Fluid velocity was characterized by tracking dye moving through the device using a Dino-Lite Edge 3.0 digital microscope and the images were analyzed using
DinoXcope software. To observe cell circulation, primary murine splenocytes were stained with Calcein AM and images were captured before and after impeller rotation using a Zeiss AxioZoom macroscope. To measure cell viability, cells were cultured on-chip for 1 hr at 37° C., then stained with Calcein AM and 7-AAD for live and dead cells, respectively, and quantified using flow cytometry.
The impeller pump comprised a magnetic impeller in the center of a large well, integrated into in a 3D-printed device with a looped channel for recirculating fluid flow in an arrangement similar to that shown in
A design goal for the impeller pump was minimal heat emission to allow for extended cell culture within an incubator. Stable temperatures (±1° C.) play a role in maintaining viable cell cultures, and we previously found that a peristaltic pump rapidly raised the temperature inside a culture incubator if not countered with cooling packs. To test this feature of the impeller pump, the temperature within a cell culture incubator was monitored over time with six external pump platforms running at a high rotational speed (>10 V) for 9 hrs. During this time period, there was no change in the temperature reported by the cell culture incubator (37.0° C.), which indicated that the multiplexed external pump platform did not emit a noticeable amount of heat and thus are compatible with extended use inside the incubator.
In a further example, a motor-based impeller pump platform was provided. Instead of a computer fan, a small DC motor was used to rotate the magnets, resulting in magnetic impeller rotation. The voltage the motor received was controlled by a potentiometer (POT) with a voltage readout from a voltmeter in an arrangement substantially similar to the configuration shown in
A computational model was used to predict the levels of shear stress within the device during impeller-driven fluid flow. Shear stress is a major consideration for cell recirculation, as high shear stress can damage the cells and diminish viability. Physiological shear stress spans 0.6-12 dyn/cm2 in lymphatic vessels and 0.35-70 dyn/cm2 in normal blood vasculature. A shear stress values of 100 dyne/cm2 is sometimes considered the threshold for pathological shear, which reaches >1500 dyn/cm2 in diseased or stenotic vessels. Using the computational model, fluid shear stress levels during impeller rotation were estimated at various regions within the device. Looking at the impeller surface, 93.2% of the surface was <100 dynes/cm2 (i.e., within the physiological range). The highest shear stress, 400 dynes/cm2, was found along the edges of the impeller, although it is believed that cells suspended in the circulating media would rarely contact the impeller surface or edges due to centrifugal forces and the large volume of the pump well. Within the channels, the surface shear stress approximations were much lower and well within the physiological range: 0.04-0.10 dynes/cm2 in the 0.5 mm channel, and 0.40-1.22 dynes/cm2 in the 1 mm channel, with the highest stress in the corners of the channel. Based off of these results, it is predicted that the impeller rotation would not have a significant impact on the viability of circulating cells.
Cell recirculation is a key feature of inter-organ communication in vivo, and a new pump for organs on chip should be able to drive cell recirculation without impairing viability. The ability of the impeller pump to drive continuous white blood cell recirculation was tested under fluid velocities found within lymphatic vessels and vasculature in vivo. Given the depth and size of the pump well, it was possible that cells would settle to the bottom of the pump well instead of remaining suspended for recirculation through the microfluidic channel, especially at low RPM. To address this concern, primary splenocytes were stained with Calcein AM and deliberately allowed to settle to the base of the pump well of a pre-filled device while the impeller was off. Imaging at this time confirmed that the cells settled along the base of the pump well and that no cells were present within the channels. Once the impeller began to rotate at a low rotational speed (e.g., about 6.20 V, 419 RPM), the cells resting on the base of the pump well were resuspended and began to recirculate through the channels, where they were visible entering the reservoir. Cells moved much faster through the 1-mm channel, as evidenced by the blurring of fluorescently-labeled cells moving through the center of the reservoir, than through the 0.5 mm channel, consistent with the slower flow rate in narrower channels. Thus, the rotation of the impeller pump successfully resuspended cells even from rest and achieved continuous cell recirculation through the device.
The biocompatibility of the system was tested under a range of biomimetic fluid velocities. Cells were continuously recirculated through the microfluidic device (post-treated, FormLabs Clear) for 1 hr, while the whole system was inside a cell culture incubator, to provide ample time for any mechanical damage from the impeller rotation to impact cell viability. As discussed above, a 0.5 mm channel can be used to achieve low velocities similar to those measured within blood capillaries in vivo, and a 1 mm channel can be used to achieve higher velocities similar to those measured within lymphatic vessels in vivo. Compared to the off-chip control and the cells cultured on-chip with no impeller rotation, there were no significant differences in viability for the cells moving 106 μm/s, 388 μm/s, 1420 μm/s, or 2660 μm/s. It is believed that the impeller-driven micropump did not cause mechanical damage to the cells even at the higher RPM or flow velocities. Thus, the microscale impeller pump was a feasible means for cell recirculation over a wide range of biomimetic flow rates, making it suitable for future use in microscale cultures and OOCs.
In summary, while the presently disclosed subject matter has been described with respect to specific embodiments, many modifications, variations, alterations, substitutions, and equivalents will be apparent to those skilled in the art. The presently disclosed subject matter is not to be limited in scope by the specific embodiment described herein. Indeed, various modifications of the presently disclosed subject matter, in addition to those described herein, will be apparent to those of skill in the art from the foregoing description and accompanying drawings. Accordingly, the presently disclosed subject matter is to be considered as limited only by the spirit and scope of the disclosure (and claims) including all modifications and equivalents.
Still other embodiments will become readily apparent to those skilled in this art from reading the above-recited detailed description and drawings of certain exemplary embodiments. It should be understood that numerous variations, modifications, and additional embodiments are possible, and accordingly, all such variations, modifications, and embodiments are to be regarded as being within the spirit and scope of this application. For example, regardless of the content of any portion (e.g., title, field, background, summary, abstract, drawing figure, etc.) of this application, unless clearly specified to the contrary, there is no requirement for the inclusion in any claim herein or of any application claiming priority hereto of any particular described or illustrated activity or element, any particular sequence of such activities, or any particular interrelationship of such elements. Moreover, any activity can be repeated, any activity can be performed by multiple entities, and/or any element can be duplicated. Further, any activity or element can be excluded, the sequence of activities can vary, and/or the interrelationship of elements can vary. Unless clearly specified to the contrary, there is no requirement for any particular described or illustrated activity or element, any particular sequence or such activities, any particular size, speed, material, dimension or frequency, or any particularly interrelationship of such elements. Accordingly, the descriptions and drawings are to be regarded as illustrative in nature, and not as restrictive. Moreover, when any number or range is described herein, unless clearly stated otherwise, that number or range is approximate. When any range is described herein, unless clearly stated otherwise, that range includes all values therein and all sub ranges therein. Any information in any material (e.g., a United States/foreign patent, United States/foreign patent application, book, article, etc.) that has been incorporated by reference herein, is only incorporated by reference to the extent that no conflict exists between such information and the other statements and drawings set forth herein. In the event of such conflict, including a conflict that would render invalid any claim herein or seeking priority hereto, then any such conflicting information in such incorporated by reference material is specifically not incorporated by reference herein.
The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.
The presently disclosed subject matter claims the benefit of U.S. Provisional Patent Application Ser. No. 63/080,320 filed Sep. 18, 2020, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with government support under Grant No. AI131723 awarded by The National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/051064 | 9/20/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63080320 | Sep 2020 | US |
