Multilayer microfluidic systems and methods

Abstract

Systems and methods are provided to aid in the development of precision medicine approaches using microfluidic cells with releasable aqueous interface membranes separating tissue channels. This approach can include customized medical treatment plans. The systems and methods emulate cellular communication in disease states in a more accurate aqueous environment, providing data on cell-cell interactions that can be used to develop treatments for subjects in need. The systems and methods can also be used to evaluate the effectiveness of a particular treatment, such as, for example, drug therapy, radiation therapy, or a combination thereof. The systems and methods can show how a particular treatment is affected by any of several known factors, including, but not limited to, the subject's sex, the subject's age, genetic factors or other genetic predispositions, and possibly other physiological conditions of the subject, or a combination thereof.

Description

[Technical Field]

[0001] Cross-reference to Fakhrul Ahsan related applications This application claims priority to U.S. Patent Application No. 17 / 865,021, filed July 14, 2022, and claims the benefit of U.S. Provisional Patent Application No. 63 / 226,733, filed July 28, 2021, each of which is incorporated herein by reference in its entirety.

[0002] background FIELD OF THE INVENTION The teachings herein relate to multi-layer microfluidic systems and methods, including improved microfluidic chips that separate tissue types before allowing controlled contact through the release of interfacial membranes formed between pillars, and methods that employ the devices in providing treatment for disease, encompassing personalized medicine approaches. [Background technology]

[0003] 2. Description of Related Art To improve disease treatment, researchers have used various animal studies to develop disease treatments. The problem is that animals often do not effectively mimic human pathophysiology and only partially emulate important human pathological changes. Furthermore, various animal species are used with various disease-causing drugs or environments, which show great variability in disease severity, progression, and response to treatment. The influence of various factors is not fully represented by animal experiments, and the influence of these various factors affects how we treat diseases. Such factors may include, for example, the biological sex of the subject being treated, including the influence of sex hormones, particularly the role of estrogen. A better understanding of how these factors affect treatment will, for example, enable improved development of gender-based therapies.

[0004] Microfluidic devices have been used to construct physiologically relevant representations of tissue- and organ-level function and disease. Prior art microfluidic devices use a single channel for seeding and growing certain types of cells, and these single-channel devices have limited applications. For example, single-channel devices cannot represent interactions between different cell types, such as, but not limited to, layered vascular tissues.

[0005] Developing an effective multilayer system poses the challenge of creating and maintaining the integrity of adjacent cell layers. Multilayer systems must provide realistic interactions between layers, interactions that are controllable. While some prior art microfluidic devices have attempted to use gels to maintain the integrity of cell layers, the interactions between layers are not realistic because gels are not present physiologically and alter the rate of potential interactions; therefore, the amount of control over the interactions is limited by the presence of the gel. Those skilled in the art will understand that the use of gels does not represent a true physiological condition, as mammalian cells do not transport or signal through gels in the body.

[0006] For at least the above reasons, those skilled in the art will appreciate the value of having microfluidic devices that can more realistically represent true physiological conditions in the development of treatments for diseases, especially those involving multiple tissue types that typically interact as layers of tissue. Such microfluidic systems may, for example, allow for the construction of cellular interactions that occur in a selected pathological process. In some cases, the systems provided herein may be constructed so that cells from one channel can easily send cues to cells in an adjacent channel, allowing for a more accurate representation of the pathological condition in treatment development. In some cases, the systems provided herein may allow diseased cells in those channels to communicate with healthy cells in adjacent channels. In some cases, the systems provided herein may allow healthy cells to become diseased and generate data regarding cellular interactions in a particular pathological process in order to develop effective treatments. In some cases, the systems provided herein may indicate the effect of drug treatments on a disease, including relative changes in drug selection, drug dose, drug tolerance, drug half-life, etc. Those skilled in the art will readily appreciate that there are many other variables that can be introduced using the systems and methods provided herein to develop treatments, such as prophylactic treatments to inhibit or prevent the onset of disease, treatments that inhibit pathological processes after onset, and treatments that ameliorate symptoms of disease.

[0007] Thus, for at least the above reasons, those skilled in the art will recognize the value of microfluidic systems and methods that provide more accurate aqueous physiological conditions as: (i) a means of controlling the cellular interactions that occur; (ii) a means of developing treatments based on cellular interactions; (iii) a means of introducing variables of interest in treatment development that affect pathological processes; (iv) a means of testing therapeutics including drugs, radiation, or combinations thereof; and (v) a means of identifying variables that affect treatment in a subject, such as cellular trafficking and signaling mechanisms associated with the treatment of disease. Summary of the Invention

[0008] overview Systems and methods are provided to aid in the development of disease treatments. The systems and methods can encompass precision medicine approaches to any disease, including personalized medicine approaches. The systems and methods involve the use of microfluidic devices that can be used to reconstruct tissue interactions in disease processes. The reconstructions can encompass interactions between multiple cell types, interactions between cell layers within a tissue, or a combination of these cellular interactions. The interactions provide information about relevant cellular communication in disease states for designing treatments, as well as information about the effects of specific types of treatments or combinations of treatments, such as drug therapy, radiation therapy, or a combination thereof. An aqueous interfacial membrane is used to control tissue interactions; the membrane remains intact for the formation of cell layers but releases when desired to allow interactions between cell "layers" in adjacent channels. The interfacial membrane controls release while avoiding the slow transport that occurs when using hydrogels to stabilize tissue layers.

[0009] In some embodiments, a microfluidic system designed to emulate cellular physiology in a subject is provided. The system includes a microfluidic chip and a first channel adjacent to a second channel. In these embodiments, the first channel and the second channel are connected to the chip and include a first port configured to inject a first aqueous cell culture medium into the first channel; a second port configured to inject a second aqueous cell culture medium into the second channel; and a first wall with an opening, the first wall being shared by the first channel and the second channel, and the opening in the first wall is configured to (i) form a temporary / releasable first aqueous interface membrane across the opening in the first wall upon injection of the first aqueous cell solution, and (ii) allow cellular communication to occur between the first channel and the second channel upon release of the first interface membrane from the opening.

[0010] The interface membrane must be released to allow communication between cells in adjacent channels. In some embodiments, injection of the second aqueous cell solution causes the first interface membrane to be released from the opening.

[0011] The walls with the openings must be configured to establish an interface membrane stable enough to maintain the integrity of the cell layer within each channel long enough to create the emulated tissue structure. In some embodiments, the interface membrane can be formed from the injection of any aqueous solution. In some embodiments, the walls are configured to establish a first interface membrane across the opening when a first aqueous cellular solution is injected. In some embodiments, the openings include a hydrophobic material to establish a first interface membrane across the opening in the first wall when the first aqueous cellular solution is injected. In some embodiments, the openings are delineated by opposing wedge-shaped edges that share a common plane to establish a first interface membrane across the opening when the first aqueous cellular solution is injected. In some embodiments, the openings include a shape selected from the group consisting of a circle and an ellipse. In some embodiments, the openings are delineated by a polygonal structure that provides opposing edges on at least two opposing sides of the opening, the opposing edges sharing a common plane to establish a first interface membrane across the opening in the first wall when the first aqueous cellular solution is injected. In some embodiments, the opening in the first wall is a space between adjacent columns along a series of columns; each column in the series has a central axis that is at least substantially perpendicular to the chip, and each pair of adjacent columns provides a pair of opposing edges that share a plane to establish a first interface membrane across the opening in the first wall when the first aqueous cell solution is injected.

[0012] In some embodiments, the system can contain three or more different cell types to emulate more complex tissue structures. In these embodiments, the system further includes a third channel and a third port configured for injecting a third aqueous cell solution into the third channel. The system includes a second wall with an opening, the second wall being shared by the second channel and the third channel, and the opening in the second wall is configured to (i) form a temporary second aqueous interface membrane across the opening in the second wall upon injection of the third aqueous cell solution, and (ii) allow cellular communication to occur between the second channel and the third channel upon release of the interface membrane from the opening.

[0013] The system can be designed with separate injection ports and channels that can be used to supply growth medium to the cells and administer drugs to test the response of the emulated tissue structure to the drugs. Thus, in some embodiments, the system can further include a growth medium channel having a growth medium port. The interface membrane can contain growth medium.

[0014] Similarly, the system can be designed for multiple growth medium ports, each for supplying cell culture medium for the cells and a selection drug to be tested. Thus, in some embodiments, the system can further include a first growth medium channel having a first growth medium port and disposed in communication with the first channel, and a second growth medium channel having a second growth medium port and disposed in communication with the second channel.

[0015] The system is designed to represent the physiological environment of diseased tissue. Thus, in some embodiments, a method for emulating a disease in a subject is provided. The method may include obtaining one of the systems taught herein; injecting a first aqueous cell solution into a first channel to introduce a first diseased cell, thereby forming a temporary first aqueous interface membrane across the opening in the first wall; and injecting a second aqueous cell solution into a second channel to introduce a second diseased cell. In these embodiments, the first interface membrane is released after the injection of the second aqueous solution; and after the release of the first interface membrane, the first cell and the second cell communicate to emulate a disease state. These methods may further include injecting a drug into the system to identify an appropriate drug, identify an appropriate dosage, and / or identify the desired activity of the drug; and administering the drug to a subject in need of treatment.

[0016] In some embodiments, the selected system has at least three channels and at least three different cell types within the emulated tissue structure. Thus, in some embodiments, a method for emulating a disease includes obtaining a system with at least three channels; injecting a first aqueous cell solution into the first channel to introduce a first cell of the disease, thereby forming a temporary first aqueous interface membrane across the opening of the first wall; injecting a third aqueous cell solution into the third channel to introduce a third cell of the disease, thereby forming a temporary second aqueous interface membrane across the opening of the second wall; and injecting a second aqueous cell solution into the second channel to introduce a second cell of the disease. In these embodiments, the first interface membrane is released after the injection of the second aqueous solution; the second interface membrane is released after the injection of the second aqueous solution; after the release of the first interface membrane, the first cell and the second cell communicate to emulate a disease state; and after the release of the second interface membrane, the second cell and the third cell communicate to emulate a disease state. These methods can further include injecting a drug into the system to identify an appropriate drug, identify an appropriate dosage, and / or identify an appropriate activity; and administering the drug to a subject in need of treatment.

[0017] In some embodiments, gel can be added between layers to further stabilize the integrity of those layers, especially between layers of systems where controllable separation of the interface membrane is not necessary or in some embodiments undesirable.This can be useful, for example, in applications where stable layer separation is required.Thus, in some embodiments, a method for emulating a disease in a subject can include obtaining a system with at least three channels; injecting a first aqueous cell solution into the first channel to introduce a first cell type of the disease, thereby forming a temporary first aqueous interface membrane across the opening of the first wall; injecting a third aqueous cell solution into the third channel to introduce a third cell type of the disease, thereby forming a temporary second aqueous interface membrane across the opening of the second wall; and injecting a second aqueous cell solution in the form of an aqueous gel into the second channel to introduce a second cell type of the disease. In these embodiments, the first interfacial membrane is released after injection of the second aqueous solution; the second interfacial membrane is released after injection of the second aqueous solution; the first cell and the second cell communicate after release of the first interfacial membrane to emulate a disease state; and the second cell and a third cell communicate after release of the second interfacial membrane to emulate a disease state. These methods can further include injecting a drug into the system to identify an appropriate drug, identify an appropriate dosage, and / or identify an appropriate activity; and administering the drug to a subject in need of treatment.

[0018] Methods of emulating tissue structures and emulating diseases naturally lead to methods of treating diseased tissue structures. Accordingly, methods of treating emulated diseases with drugs are provided. The methods can include constructing a disease representation as taught herein; selecting drug candidates for treating the disease; injecting / administering a drug into any one of the channels; and observing drug activity. In some embodiments, the drug can be administered in the first channel or the second channel. In some embodiments, the drug can be administered in the first channel, the second channel, or the third channel. These methods can further include injecting a drug into the system to identify an appropriate drug, identify an appropriate dosage, and / or identify an appropriate activity; and administering the drug to a subject in need of treatment.

[0019] The systems and methods provided herein can be used to emulate and treat various types of diseases. In some embodiments, the methods involve treating the emulated vascular disease with a drug. In these embodiments, the methods include obtaining a system as taught herein, the system having at least three channels for introducing three vascular cell types; injecting a first aqueous cell solution into the first channel to introduce a first diseased cell type, forming an adventitial cell layer and forming a temporary first aqueous interface membrane across an opening in the first wall; injecting a third aqueous cell solution into the third channel to introduce a third diseased cell type, forming an endothelial cell layer and forming a temporary second aqueous interface membrane across an opening in the second wall; and injecting a second aqueous cell solution into the second channel to introduce a second diseased cell type, forming a smooth muscle layer. These methods further include selecting a drug and administering the drug to the first growth medium channel or the second growth medium channel. In these embodiments, the first interfacial membrane is released after injection of the second aqueous solution; the second interfacial membrane is released after injection of the second aqueous solution; the first cell and the second cell communicate after release of the first interfacial membrane to emulate a disease state; and the second cell and a third cell communicate after release of the second interfacial membrane to emulate a disease state. These methods can further include injecting a drug into the system to identify an appropriate drug, identify an appropriate dosage, and / or identify an appropriate activity; and administering the drug to a subject in need of treatment. [Brief explanation of the drawings]

[0020] Brief description of the drawings [Figure 1] FIG. 1 shows a diagram of a microfluidic chip according to some embodiments.

[0021] [Figure 2] FIG. 2 shows a vascular tissue structure formed on a chip within a channel according to some embodiments.

[0022] [Figure 3] FIG. 3 is a top view of a channel of a device according to some embodiments, where the channel has "discontinuous walls," which are walls with openings between pillar-shaped structures that make up part of the channel.

[0023] [Figure 4] 4A-4C show the difference in contact angle between the interfacial film and pillars, comparing circular pillars to hexagonal pillars, according to some embodiments.

[0024] [Figure 5] FIG. 5 is a graph showing that there is a preferred inter-pillar distance between hexagonal pillars that avoids leakage of the interfacial film, according to some embodiments.

[0025] [Figure 6] FIG. 6 shows extracellular remodeling in PAH, according to some embodiments.

[0026] [Figure 7] FIG. 7 shows a flowchart of how the systems and methods taught herein can be used to treat a subject in need, according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description of the Invention The teachings herein generally relate to systems and methods for making and using multi-channel microfluidic devices having releasable layers, which in some embodiments include improved microfluidic chips that separate tissue types before allowing controlled contact through the release of an interfacial membrane, and methods for using the devices in providing treatments for disease, such treatments encompassing personalized medicine approaches.

[0028] Those skilled in the art will understand that in some embodiments, the device can be used to develop treatment regimens. The device allows for the seeding and growth of multiple cell types within the system, providing an improved representation of physiological systems. In some embodiments, the system and method represent complex pathological processes for use in treatment development. The term "interface" refers to one side exposed to air and the other side exposed to a fluid, e.g., a cell growth medium, such as an aqueous fluid cell growth medium. The terms "subject" and "patient" can be used interchangeably and refer to animals, such as, but not limited to, non-primates, e.g., cows, pigs, horses, cats, dogs, rabbits, rats, and mice; and mammals, e.g., primates, e.g., monkeys or humans.

[0029] In some embodiments, treatment development can involve identifying a drug with desired activity, dosage, half-life, and / or tolerance, or a combination thereof, in a subject to be treated. The present systems and methods can be used, for example, to identify optimal dosages of treatment drugs, the efficacy of selected treatment drugs alone or in combination, the stability of treatment drugs, patient sensitivity / tolerance to treatment drugs, and treatment formulations. Drug formulations can include, for example, the use of nanoparticles, coatings, targeting moieties, drug delivery complexes, and combinations thereof. Those skilled in the art will understand that the systems and methods provided herein can be used in precision medicine and personalized medicine, leading to the development of improved treatments, such as treatments tailored to a subject's age, sex, race, known allergies, etc.

[0030] Treatment development can involve using the present system and method to represent the intercellular communication that occurs in any system of the body. In some embodiments, the body system can be selected from the circulatory system, cardiovascular system, digestive system, excretory system, endocrine system, exocrine system, integumentary system, immune system, lymphatic system, muscular system, nervous system, renal system, urinary system, reproductive system, respiratory system, skeletal system, or a combination thereof. Each of these physiological systems has its own pathological diseases and problems, many of which are well known to those skilled in the art, and the systems and methods taught herein can be used to identify factors that may affect treatment, such as age, sex, genetic factors, and the presence of other physiological conditions, such as additional diseases or disorders, in the subject, to create a more realistic physiological environment for diseases that may occur in each of these systems. In some embodiments, the present system and method can also be designed to represent the presence of other therapies, including primary therapies, adjuvant therapies, etc., that may affect treatment. For example, a subject may also be receiving chemotherapy while receiving radiation therapy, and the subject may also have additional genetic factors or disease conditions that may affect treatment development.

[0031] Beneficial contributions of the systems and methods taught herein include the ability to construct tissue systems with separate, distinct tissue layers while controlling interactions between the tissue layers. The interfacial membranes taught herein can be used to isolate tissue layers from one another before bringing them into controlled contact. Any organ system can be represented, and treatments for any disease can be pursued, established, and used in subjects, particularly when the disease involves multiple cell layers, such as vascular tissue, immune tissue, cancerous tissue, etc. For example, treatments for diseases involving angiogenesis, inflammatory response, wound healing, drug response, metastasis of cancer cells between organs, etc. can be identified and administered to subjects in need.

[0032] In some embodiments, the system and method may represent vascular disease and be particularly suitable for developing treatments for vascular disease. A treatment method is provided that includes building a system representing vascular disease, identifying a drug for treating the vascular disease using the system, and administering the drug to treat the vascular disease. Identifying the drug may include identifying the amount, half-life, efficacy, and tolerance of the drug in a subject. The vascular system may be composed of smooth cells, adventitial cells, and endothelial cells, each of which may be involved in vascular disease. The method may include selecting a cell growth medium suitable for the growth of smooth cells, adventitial cells, and endothelial cells. Those skilled in the art will understand that a cell growth medium may be selected to support smooth cells, adventitial cells, and endothelial cells.

[0033] In some embodiments, a treatment for atherosclerosis can be developed.In some embodiments, a treatment for any disease involving endothelial dysfunction, such as diabetes or metabolic syndrome, high blood pressure, smoking, and physical inactivity, can be developed.Such treatment can promote healthy endothelium, and not only mediate endothelium-dependent vasodilation, but also actively suppress thrombosis, vascular inflammation, and hypertrophy.Therefore, in some embodiments, the developed treatment can be used to treat thrombosis, vascular inflammation, and hypertrophy.

[0034] In some embodiments, cancer treatments can be developed using the systems and methods taught herein. Such treatments can be vascular disease treatments, such as angiogenesis, or the inhibition of angiogenesis, a mechanism related to cancer and cancer treatment. Angiogenesis inhibitors can be used, for example, to inhibit or stop the growth of blood vessels that lead to the growth of cancerous tissue. Similarly, treatments for wound healing can be developed using drugs that increase angiogenesis to improve blood flow to tissue healing treatments. Treatments for sarcoidosis, sclerosis, and coagulation disorders can all be developed using the systems and methods taught herein.

[0035] In some embodiments, the cancer treatment can be for solid tumors or liquid tumors such as lymphoma and leukemia. In some embodiments, the leukemia treatment is for acute myeloid leukemia (AML). In some embodiments, the systems and methods taught herein can be used to represent any cancer physiological environment, cancer stage, cancer metastasis between tissues, etc., to develop cancer treatments.

[0036] Releasable interface layer The component of the present system that controls the separation and intermixing of tissue layers is referred to herein as a "releasable interface layer" or "interface layer" or "interface film." It may also be referred to as a "temporary" interface layer or interface film. Such terms may be used interchangeably. The interface layer can be fabricated as a component of one or more walls separating independent tissue channels. In some embodiments, the present systems and methods use a single interface layer to separate tissue layers or channels. In some embodiments, the present systems and methods use multiple interface layers to separate tissue layers or channels. In some embodiments, the present systems can have two interface layers, one on each side of the channel separating the tissue layers or channels. The walls separating independent tissue channels are made of pillars, with an interface layer formed between each adjacent pillar, spanning the space between the pillars.

[0037] Cell culture media are used as carriers for each tissue layer. Cell culture media may be referred to as "cell growth media," "cell culture media," or "cell growth culture media." Of course, cell growth media may be nutrient media, culture media, minimal media, selective media, fractionated media, transport media, or enriched media. In some embodiments, liquid media may be referred to as "broth." Thus, in some embodiments, the interface layer may be composed of a growth medium, which may include, for example, an aqueous liquid cell culture growth medium, which in some embodiments may be referred to as an "aqueous liquid cell growth medium." Such media can be used to create a releasable interface layer. An example of a cell growth medium is rat tail type I collagen solution. In some embodiments, any cell growth medium having a collagen concentration ranging from 10% to 35% can be used. In some embodiments, the collagen concentration may be 10%, 15%, 20%, 25%, 30%, 35%, or any amount in 1% increments therein. In some embodiments, the collagen concentration of the cell culture medium is 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, and any amount therein in 0.1% increments.

[0038] However, in some embodiments, the present systems and methods can also include a semi-solid cell growth medium. In some embodiments, the semi-solid cell growth medium can be a hydrogel. Those skilled in the art will appreciate that any suitable hydrogel cell growth medium can be used, such as a collagen-based hydrogel cell growth medium.

[0039] Those skilled in the art can identify appropriate cell growth media suitable for specific tissue applications. In some embodiments, the cell growth medium may be referred to as a "physiological medium," e.g., a chemically defined culture medium intended to provide controlled cell culture conditions to emulate the composition of blood or other physiological environments necessary to represent physiological conditions. In some embodiments, the cell growth medium may be Basal Medium Eagle (BME), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, McCoy's 5A Modified Medium, or cell culture media for specific cell types, such as SmGM™-2 Smooth Muscle Cell Growth Medium-2 BulletKit™, EGM™-2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKit™, and COMPLETE FIBROBLAST MEDIUM / W KIT-500 ML.

[0040] The interfacial layer can be controllably releasable, thereby enabling control in some embodiments. By "controllably releasable," we mean that the interfacial membrane is sufficiently stable to remain in place until an aqueous fluid contacts the "air side" of the interfacial membrane, thereby releasing the membrane containing the cellular tissue within the channel and allowing communication between the channel and its respective cellular tissue. In some embodiments, the interfacial layer can be released upon introducing an aqueous solution into a channel adjacent to the channel in which the membrane is formed, such as an adjacent channel. In contrast, in some embodiments, the integrity of the channels can be extended as needed by introducing a first aqueous cell solution into the first channel and a second aqueous cell solution into the second channel in the form of a gel, in which case the gel maintains the integrity of the first cell layer formed in the first channel and the second cell layer formed in the second channel. However, it should be understood that the gel in this layered combination of cells does not faithfully represent the actual aqueous cellular environment in mammals. It may be desirable to simply stabilize that portion of a representative layered system of cells, while other portions of the representative system include a releasable interfacial membrane. In some embodiments, for example, a first layer can be an aqueous solution, an adjacent second layer can be an aqueous gel, and a third layer can be an aqueous solution. In some embodiments, the layers can have an alternating aqueous cell culture medium-aqueous gel-aqueous cell culture medium structure, for example.

[0041] The interfacial membrane is a technical feature that provides a controllably releasable mechanism that functions as a barrier between cell layers or channels. Of course, in some embodiments, the tissue structure represented by the present system and method can have a layered orientation between the cell layers, and it is desirable to maintain the integrity of the cell layer structure generated at the seeding stage. However, the interfacial membrane between the layers can be later released as needed to allow representative physiological communication between the layers.

[0042] Figure 1 shows a diagram of a microfluidic chip according to some embodiments. Microfluidic chip 100 has five channels 1, 2, 3, 4, and 5. Channels 2, 3, and 4 are for introducing three different types of cells to emulate tissue. Channels 1 and 5 are added to add growth medium to nourish the cells and extend their lifespan; these channels can also function as growth medium reservoirs that can be periodically replaced as needed.

[0043] The systems and methods taught herein can use multiple channels to represent any layered tissue structure and develop treatments for any disease process. Figure 2 illustrates a vascular tissue structure formed on a chip within channels according to some embodiments. The emulated tissue 200 shows channels 1 through 5. Channel 2 contains endothelial cells to emulate an endothelial cell layer, channel 3 contains smooth muscle cells to emulate a smooth muscle cell layer, and channel 4 contains adventitial cells to emulate an adventitial cell layer, the combination of which emulates a vascular structure. As described above, channels 1 and 5 are configured for adding growth medium to nourish the cells and extend their lifespan.

[0044] In some embodiments, channels 1 and 5 can also function as growth medium reservoirs that can be periodically replaced as needed to allow the system and method to operate over extended periods of time. In some embodiments, the period can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or any period or range of time in 1-minute increments therein.

[0045] In some embodiments, channels 1 and 5 can function as ports for administering drugs to tissues in liquid aqueous growth medium. Such a system can, in some embodiments, be used to identify drug type, drug dosage, drug stability, and drug tolerance.

[0046] In some embodiments, channel 1 and / or 5 can be used to apply hydrostatic pressure. Such pressure application can be used, for example, to emulate the effects of blood pressure changes and stress on blood vessels. This can be useful, for example, to develop treatments for subjects with a range of blood pressure. Any suitable pressure mechanism known to those skilled in the art, such as a peristaltic pump, can be used to apply pressure.

[0047] FIG. 3 shows a top view of a channel of a device according to some embodiments. The channel has "discontinuous walls," i.e., walls with openings between pillar-shaped structures that make up part of the channel. Channel 300 includes a wall 305 with openings 310, which are shared with an adjacent channel (not shown). Because the wall is shared with an adjacent channel (not shown), a first cell 314 in a first aqueous cell solution 315 can communicate with a second cell (not shown) in a second aqueous cell solution (not shown), emulating communication between the first and second cells in an emulated tissue structure. However, before communication can occur, the emulated tissue structure must be created, and therefore the integrity of each cell layer must be maintained. Maintaining the integrity of each cell layer during formation of the tissue structure is facilitated by the creation of a temporary aqueous interfacial membrane 325 across opening 310 via aqueous surface tension generated by contact between first aqueous cell solution 315 and columns 320. The process of creating a cell layer may be referred to as "seeding" 330, which can be done manually or automatically via a pump in the system.

[0048] The microfluidic devices taught herein are uniquely designed to provide temporary, free-standing aqueous interfacial membranes between channels. Indeed, the interfacial membranes are a component of the channels and function to initially maintain the integrity of the channels during the formation of the emulated tissue structures. The temporary interfacial membranes allow for the temporary formation of channels capable of accepting a first cell type contained within the interfacial membrane, while another channel, also having the interfacial membrane as a component, accepts a second cell type. Over time, the interfacial membranes are released, allowing cells to communicate between adjacent channels that were previously separated by the interfacial membrane. In some embodiments, these temporary interfacial membranes may be referred to as "sacrificial walls," which initially hold the cell layer in place and then, upon release of the interfacial membrane, allow communication between the cells, eventually releasing when desired to allow cells to communicate, forming a separation representative of the cell arrangement present in the body.

[0049] The pillars can have any desired configuration that influences the interfacial membrane in some embodiments. To form the desired interfacial membrane between the pillars and contain the cell layer formed in the channel, the pillars can be configured with edges that face each other (preferably coplanar). The pillars can have hexagonal bases, as shown in Figure 3, for example. As mentioned above, the hexagonal pillars must have edges oriented between two points, preferably coplanar. The aqueous interfacial membrane forms a temporary wall that maintains the integrity of the cell layer during seeding of the channel, and then the interfacial layer is released, allowing cells to communicate.

[0050] In some embodiments, hydrophilic materials are not used to form the pillars because they cannot form a stable interfacial film with the cell culture medium.

[0051] Those skilled in the art will understand that in some embodiments, the pillars can have a cross section that is circular, elliptical, polygonal, or a combination thereof. In some embodiments, the polygonal shape consists of 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 sides. In some embodiments, the pillars are cylinders. In some embodiments, the pillars are regular cylinders. In some embodiments, the pillars are elliptical cylinders. In some embodiments, the pillars are polygonal prisms, such as the hexagonal prisms of Figure 3.

[0052] The opening 310 is defined by the distance between the pillars, the "bridge distance" of the interfacial membrane of the systems and methods taught herein. The distance between the pillars, sometimes referred to as the "interpillar distance," is a feature of the device design that must be carefully configured in the range of 30 uM to 250 uM. The lower end of the range, 30 uM, is limited by the size of the tissue, while the upper end, 250 uM, is limited by the stability of the interfacial membrane.

[0053] In some embodiments, the inter-pillar distance can be 30 uM to 200 uM, 30 uM to 100 uM, or any range therein in 1 uM increments. In some embodiments, the inter-pillar distance can be about 25 uM, 30 uM, 35 uM, 40 uM, 45 uM, 50 uM, 55 uM, 60 uM, 65 uM, 70 uM, 75 uM, 80 uM, 85 uM, 90 uM, 100 uM, 105 uM, 110 uM, 115 uM, 120 uM, 125 uM, 130 uM, 135 uM, 140 uM, 145 uM, 150 uM, 155 uM, 1 It may be 60uM, 165uM, 170uM, 175uM, 180uM, 185uM, 190uM, 195uM, 200uM, 205uM, 210uM, 200uM, 215uM, 220uM, 225uM, 230uM, 235uM, 240uM, 245uM, 250uM, 255uM, 260uM, or any distance or range of distances therein in 1uM increments.

[0054] The inter-pillar distance is determined by the ability to form an interfacial film between the pillars for cell culture media used in the systems and methods taught herein. The pillar height is also limited by similar surface tension constraints, and the pillar height also forms the bridging distance of the interfacial film, so the pillar height can also be about 25 uM, 30 uM, 35 uM, 40 uM, 45 uM, 50 uM, 55 uM, 60 uM, 65 uM, 70 uM, 75 uM, 80 uM, 85 uM, 90 uM, 100 uM, 105 uM, 110 uM, 115 uM, 120 uM, 125 uM, 130 uM, 135 uM, 140 uM, 150 uM, 160 uM, 170 uM, 180 uM, 190 uM, 200 uM, 210 uM, 220 uM, 230 uM, 240 uM, 250 uM, 260 uM, 270 uM, 280 uM, 290 uM, 300 uM, 310 uM, 320 uM, 330 uM, 340 uM, 350 uM, 360 uM, 370 uM, 380 uM, 390 uM, 400 uM, 410 uM, 420 uM, 430 uM, 440 uM, 450 uM, 460 uM, 470 uM, 48 The distance between pillars can be 145 uM, 150 uM, 155 uM, 160 uM, 165 uM, 170 uM, 175 uM, 180 uM, 185 uM, 190 uM, 195 uM, 200 uM, 205 uM, 210 uM, 200 uM, 215 uM, 220 uM, 225 uM, 230 uM, 235 uM, 240 uM, 245 uM, 250 uM, 255 uM, 260 uM, or any distance or distance range therein in 1 uM increments. Similar to the inter-pillar distance, pillar height is also a feature of device design and must be carefully configured to be in the range of 30 uM to 250 uM. In some embodiments, pillar height can be 30 uM to 200 uM, 30 uM to 100 uM, or any range therein in 1 uM increments.

[0055] In some embodiments, a multichannel microfluidic device can have three channels. In some embodiments, a multichannel microfluidic device can have two, three, four, five, six, seven, eight, nine, ten, or more channels. In some embodiments, a user preselects a device designed with two, three, four, five, six, or more channels. In many embodiments, the multichannel device allows each cell to grow within its own respective one of the multiple channels, each channel communicating with at least one adjacent channel of the multiple channels, and each cell type remains within its own respective channel during the seeding stage but communicates via cell signaling mechanisms, which can include, for example, paracrine signaling, which does not involve cell contact, and junctional signaling, which is ligand-receptor binding that involves cell contact. This arrangement can allow for a wide variety of configurations to be selected and designed for use in emulating cellular communication in pathological processes.

[0056] Thus, in some embodiments, a microfluidic system designed to emulate cellular physiology in a subject is provided. The system can include a microfluidic chip; and a first channel adjacent to a second channel. In these embodiments, the first channel and the second channel are connected to the chip and include a first port configured to inject a first aqueous cellular solution into the first channel; a second port configured to inject a second aqueous cellular solution into the second channel; and a first wall with an opening, the first wall being shared by the first channel and the second channel, and the opening in the first wall is configured to (i) form a temporary first aqueous interface membrane across the opening in the first wall upon injection of the first aqueous cellular solution, and (ii) allow cellular communication to occur between the first channel and the second channel upon release of the first interface membrane from the opening.

[0057] The interface membrane must be released to allow communication between cells in adjacent channels. In some embodiments, injection of the second aqueous cell solution causes the first interface membrane to be released from the opening.

[0058] The walls with the openings must be configured to establish an interface membrane stable enough to maintain the integrity of the cell layer within each channel long enough to create the emulated tissue structure. In some embodiments, the interface membrane can be formed from the injection of any aqueous solution. In some embodiments, the walls are configured to establish a first interface membrane across the opening when a first aqueous cellular solution is injected. In some embodiments, the openings include a hydrophobic material to establish a first interface membrane across the opening in the first wall when the first aqueous cellular solution is injected. In some embodiments, the openings are delineated by opposing wedge-shaped edges that share a common plane to establish a first interface membrane across the opening when the first aqueous cellular solution is injected. In some embodiments, the openings include a shape selected from the group consisting of a circle and an ellipse. In some embodiments, the openings are delineated by a polygonal structure that provides opposing edges on at least two opposing sides of the opening, the opposing edges sharing a common plane to establish a first interface membrane across the opening in the first wall when the first aqueous cellular solution is injected. In some embodiments, the opening in the first wall is a space between adjacent columns along a series of columns; each column in the series has a central axis that is at least substantially perpendicular to the chip, and each pair of adjacent columns provides a pair of opposing edges that share a plane to establish a first interface membrane across the opening in the first wall when the first aqueous cell solution is injected.

[0059] In some embodiments, the system can include three or more different cell types to emulate more complex tissue structures. In these embodiments, the system further includes a third channel and a third port configured for injecting a third aqueous cell solution into the third channel. The system includes a second wall with an opening, the second wall being shared by the second and third channels, and the opening in the second wall configured to (i) form a temporary second aqueous interface membrane across the opening in the second wall upon injection of the third aqueous cell solution, and (ii) allow cellular communication to occur between the second and third channels upon release of the interface membrane from the opening. In some embodiments, the multichannel microfluidic device can be used to study cellular interactions within blood vessels. For example, the device can be used to study interactions between the three major arterial cell types: endothelial cells, smooth muscle cells, and adventitial cells.

[0060] The teachings herein demonstrate that we can emulate pulmonary arterial hypertension (PAH), for example, in the development of PAH treatments. In some embodiments, the systems and methods taught herein can emulate PAH-induced right ventricular hypertrophy for the development of improved treatments. As those skilled in the art will appreciate, these results demonstrate that we can also emulate the cellular communication that leads to pulmonary embolism and the environment of the embolism itself. And, as with any modeling of the human system, we can examine the influence of any of several factors that may affect the effectiveness of a treatment. For example, the systems and methods taught herein focus on the relative influence of whether the subject is male or female, young or adult, suffering from another disease or physiological disorder, suffering from a genetic disorder, or genetically predisposed to a condition. Any factor known to those skilled in the art can be emulated using the systems and methods taught herein.

[0061] Using the provided systems and methods, one skilled in the art can emulate any interaction of various cellular tissues, but vascular tissue was used herein as a model. In some embodiments, cellular interactions occur between vascular tissue layers. In some embodiments, cellular interactions occur between vascular cell types. In some embodiments, cellular interactions occur between vascular tissue layers. In some embodiments, cellular interactions occur between vascular cells and the vascular extracellular matrix.

[0062] Treatment development may include drug selection, amount of drug administered, drug tolerance, drug stability, etc. Thus, the system may be designed with separate injection ports and channels that can be used to supply growth medium to the cells and administer drugs to test the response of the emulated tissue structure to the drug. Also, because cell culture medium may need to be replenished, in some embodiments, the system may further include a growth medium channel with a growth medium port.

[0063] The term "drug" can be used interchangeably with "active agent," "bioactive agent," etc. Bioactive agents include, but are not limited to, small molecules, nucleotides, oligonucleotides, polynucleotides, amino acids, oligopeptides, polypeptides, and proteins. Bioactive agents can include, but are not limited to, antiproliferative agents, antineoplastic agents, antimitotic inhibitors, anti-inflammatory agents, antiplatelet agents, anticoagulants, antifibrins, antithrombins, antibiotics, antiallergic agents, antioxidants, and any prodrugs, codrugs, metabolites, analogs, homologs, congeners, derivatives, salts, and combinations thereof. Of course, one of ordinary skill in the art will understand that in some embodiments of the present invention, some groups, subgroups, and individual bioactive agents may not be used.

[0064] Antiproliferative agents include, for example, actinomycin D, actinomycin IV, actinomycin I1, actinomycin X1, actinomycin C1, and dactinomycin (Cosmegen.RTM., Merck & Co., Inc.). Antineoplastic or antimitotic agents include, for example, paclitaxel (TAXOL, Bristol-Myers Squibb Co.), docetaxel (TAXOTERE, Aventis SA), methotrexate, irinotecan, SN-38, azathioprine, vincristine, vinblastine, fluorouracil, doxorubicin hydrochloride (ADRIAMYCIN, Pfizer Inc.), and mitomycin (MUTAMYCIN, Bristol-Myers Squibb Co.), as well as any prodrugs, codrugs, metabolites, analogs, homologs, congeners, derivatives, salts, and combinations thereof. Antiplatelet agents, anticoagulants, antifibrins and antithrombins include, for example, heparin sodium, low molecular weight heparin, heparins, hirudin, argatroban, forskolin, bapiprost, prostacyclin and prostacyclin analogs, dextran, D-phe-pro-arg-chloromethylketone (synthetic antithrombin), dipyridamole, glycoprotein IIb / IIIa platelet membrane receptor antagonist antibody, recombinant hirudin and thrombin inhibitor (ANGIOMAX, Biogen, Inc.), and any prodrugs, codrugs, metabolites, analogs, homologs, congeners, derivatives, salts and combinations thereof.Cytostatic or antiproliferative agents include, for example, angiopeptin, captopril (CAPOTEN and CAPOZIDE, Bristol-Myers Squibb Co.), cilazapril, or lisinopril (PRINVIL ​​and PRINZIDE, Merck & Co.); calcium channel blockers such as nifedipine; colchicine; fibroblast growth factor (FGF) antagonists, fish oil (omega-3 fatty acids); histamine antagonists; lovastatin (Mevacor, Merck & Co.); monoclonal antibodies, such as, but not limited to, antibodies specific for the platelet-derived growth factor (PDGF) receptor; nitroprusside; phosphodiesterase inhibitors; prostaglandin inhibitors; suramin; serotonin blockers; steroids; thioprotease inhibitors; PDGF antagonists, such as, but not limited to, triazolopyrimidines; and nitric oxide, as well as any prodrugs, codrugs, metabolites, analogs, homologs, congeners, derivatives, salts, and combinations thereof. Anti-allergy agents include, but are not limited to, pemirolast potassium (ALAMAST, Santen, Inc.), and any prodrugs, codrugs, metabolites, analogs, homologs, congeners, derivatives, salts, and combinations thereof.

[0065] It can be understood from the teachings herein that the system and method taught herein can be used to provide treatment for PAH.In some embodiments, the drugs used to treat PAH can include, but are not limited to, epoprostenol, riociguat, bosentan, macitentan, ambrisentan, treprostinil, sildenafil, tadalafil, selexipag, and iloprost, and combinations thereof.In some embodiments, each of these drugs can be administered alone or in combination.In some embodiments, the drugs can be administered as shown in Table 1 below. [Table 1]

[0066] Among the above drugs, we have found that female PAH patients are more responsive to epoprostenol, treprostinil, selexipag, ambrisentan, bosentan and macitentan.Therefore, in some embodiments, a method for treating female PAH patients is provided.The method can include, for example: introducing vascular endothelial cell tissue from a subject having PAH into a first channel of a microfluidic device taught herein, the introducing comprising seeding the vascular endothelial cell tissue in an aqueous cell growth medium to create an emulated vascular endothelial cell layer and injecting the emulated vascular endothelial cell layer into the first channel; introducing vascular adventitial cell tissue from the subject into a third channel of a microfluidic device taught herein, the third channel being separated from the first channel by a second channel, the introducing comprising seeding the vascular adventitial cell tissue in an aqueous cell growth medium to create an emulated vascular adventitial cell layer and injecting the emulated vascular adventitial cell layer into the third channel; · introducing (i) an interface membrane separating the first channel from the second channel, and (ii) an aqueous cell growth medium into the second channel such that a third channel is released from the second channel, wherein the release of the interface membrane allows communication between the emulated vascular endothelial tissue and the emulated vascular adventitia tissue; Identifying the best drug candidate for the subject by administering each of a plurality of drugs selected from the group consisting of epoprostenol, riociguat, bosentan, macitentan, ambrisentan, treprostinil, sildenafil, tadalafil, selexipag, and iloprost, and combinations thereof, and measuring the therapeutic effects, such as activity, efficacy, dose-response, stability, toxicity, and / or combinations thereof, to identify the best drug candidate or formulation for the subject; and · Administering the best drug or formulation to the subject when a therapeutic effect is desired.

[0067] In some embodiments, the methods include using a microfluidic device taught herein having at least five channels, where emulated vascular endothelial tissue is injected into the second channel, emulated vascular adventitial tissue is injected into the fourth channel, and cell growth medium is injected into the first and fifth channels, where the cell growth medium can include one or more drug candidates to identify the best drug candidate or formulation. These methods can include forming a releasable interface membrane between the first and second channels when the emulated endothelial vascular tissue is introduced into the second channel, forming a releasable interface membrane between the fourth and fifth channels when the emulated endothelial vascular tissue is introduced into the fourth channel, and releasing the interface membrane when the cell growth medium is injected into the first and fifth channels, allowing communication between the first and second channels and between the fourth and fifth channels. In some embodiments, the third channel is filled with an emulated vascular smooth muscle tissue layer that may be present in an aqueous gel cell growth medium.

[0068] We have also found that male PAH patients are more responsive to sildenafil and tadalafil. In some embodiments, a method for treating male PAH patients is provided. The method can include, for example: introducing vascular endothelial cell tissue from a subject having PAH into a first channel of a microfluidic device taught herein, the introducing comprising seeding the vascular endothelial cell tissue in an aqueous cell growth medium to create an emulated vascular endothelial cell layer and injecting the emulated vascular endothelial cell layer into the first channel; introducing vascular adventitial cell tissue from the subject into a third channel of a microfluidic device taught herein, the third channel being separated from the first channel by a second channel, the introducing comprising seeding the vascular adventitial cell tissue in an aqueous cell growth medium to create an emulated vascular adventitial cell layer and injecting the emulated vascular adventitial cell layer into the third channel; · introducing (i) an interface membrane separating the first channel from the second channel, and (ii) an aqueous cell growth medium into the second channel such that a third channel is released from the second channel, wherein the release of the interface membrane allows communication between the emulated vascular endothelial tissue and the emulated vascular adventitia tissue; Identifying the best drug candidate for the subject by administering each of a plurality of drugs selected from the group consisting of epoprostenol, riociguat, bosentan, macitentan, ambrisentan, treprostinil, sildenafil, tadalafil, selexipag, and iloprost, and combinations thereof, and measuring the therapeutic effects, such as activity, efficacy, dose-response, stability, toxicity, and / or combinations thereof, to identify the best drug candidate or formulation for the subject; and · Administering the best drug or formulation to the subject when a therapeutic effect is desired.

[0069] In some embodiments, the methods include using a microfluidic device taught herein having at least five channels, where emulated vascular endothelial tissue is injected into the second channel, emulated vascular adventitial tissue is injected into the fourth channel, and cell growth medium is injected into the first and fifth channels, where the cell growth medium can include one or more drug candidates to identify the best drug candidate or formulation. These methods can include forming a releasable interface membrane between the first and second channels when the emulated endothelial vascular tissue is introduced into the second channel, forming a releasable interface membrane between the fourth and fifth channels when the emulated endothelial vascular tissue is introduced into the fourth channel, and releasing the interface membrane when the cell growth medium is injected into the first and fifth channels, allowing communication between the first and second channels and between the fourth and fifth channels. In some embodiments, the third channel is filled with an emulated vascular smooth muscle tissue layer that may be present in an aqueous gel cell growth medium.

[0070] The systems and methods provided herein are fully functional for developing antibody treatments for subjects in need. Antibody therapy provides additional bioactive agents that may be useful when administered in combination with the methods taught herein. For example, AVASTATIN, a human monoclonal antibody against VEGF, has shown beneficial results in colorectal cancer when used in combination with the standard Saltz regimen of irinotecan, 5-fluorouracil, and leucovorin, increasing survival time by more than 30%. Those skilled in the art will appreciate that several monoclonal antibodies are useful. Below are further examples of cancers that can be addressed using the systems and methods taught herein and any of the bioactive agents taught herein, along with several monoclonal antibody therapies that can also be tested alone or in combination with any of the bioactive agents taught herein. [Table 2]

[0071] Generally speaking, cancers that can be addressed using the systems and methods taught herein include any known cancer. In addition to or inclusive of the cancers listed above, the following cancers are of interest: lung cancer, non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, small cell lung cancer, bronchial carcinoma, colon cancer, rectal cancer, colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia, acute myeloid leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, liver cancer, intrahepatic cholangiocarcinoma, ovarian cancer, and esophageal cancer.

[0072] Of course, bioactive agents can be administered alone or in combination with other bioactive agents with the compositions and methods taught herein.For example, chemotherapeutic agents may be most effective when administered in combination as a combination chemotherapy regimen.The rationale for combination chemotherapy is to use drugs that act by different mechanisms of action, thereby reducing the possibility of resistant cancer cells developing.When drugs with different effects are combined, each drug can be used at its optimal dose, and may not be accompanied by intolerable side effects or may be reduced.

[0073] The active agent can be, for example, included in a pharmaceutically acceptable carrier, and the active agent is conjugated to the delivery system in an amount sufficient to exert a therapeutically useful effect without undesirable side effects in the treated patient. The therapeutically effective concentration can be empirically determined by testing the compound in the described in vitro and in vivo systems, and then extrapolated therefrom for human administration.

[0074] In some embodiments, a therapeutically effective dosage should produce a serum concentration of the active ingredient of about 0.1 ng / ml to about 50-100 μg / ml. In other embodiments, pharmaceutical compositions should provide a dosage of about 0.001 mg to about 2000 mg of compound per kilogram of body weight per day. Similarly, pharmaceutical dosage forms are prepared to provide about 0.01 mg, 0.1 mg, or 1 mg to about 500 mg, 1000 mg, or 2000 mg of the active ingredient or combination of essential ingredients per dosage unit, in one embodiment about 10 mg to about 500 mg.

[0075] In some embodiments, a therapeutically or prophylactically effective amount of a composition can be in the concentration range of about 0.001 nM to about 0.10 M; about 0.001 nM to about 0.5 M; about 0.01 nM to about 150 nM; about 0.01 nM to about 500 μM; about 0.01 nM to about 1000 nM, 0.001 μM to about 0.10 M; about 0.001 μM to about 0.5 M; about 0.01 μM to about 150 μM; about 0.01 μM to about 500 μM; about 0.01 μM to about 1000 nM, or any range therein in increments of 0.1 μM. In some embodiments, the composition may be administered in an amount ranging from about 0.001 mg / kg to about 500 mg / kg; from about 0.005 mg / kg to about 400 mg / kg; from about 0.01 mg / kg to about 300 mg / kg; from about 0.01 mg / kg to about 250 mg / kg; from about 0.1 mg / kg to about 200 mg / kg; from about 0.2 mg / kg to about 150 mg / kg; from about 0.4 mg / kg to about 120 mg / kg; from about 0.15 mg / kg to about 100 mg / kg, from about 0.15 mg / kg to about 50 mg / kg, from about 0.5 mg / kg to about 10 mg / kg, or any range of amounts in increments of 0.1 mg / kg therein.

[0076] Typically, the compositions taught herein can be administered by subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Local administration, in some embodiments, can include direct injection of the agent into the tissue to be treated, for example, the area of ​​a solid tumor. In some embodiments, intravenous administration is used, which can be continuous intravenous infusion for a period of several minutes to an hour or more, for example, about 15 minutes. The amount administered can vary widely depending on the type of formulation, the size of the unit dose, the type of excipients, and other factors well known to those skilled in the art. The formulation can contain, for example, about 0.0001% to about 10% (w / w), about 0.01% to about 1%, about 0.1% to about 0.8%, or any range therein, with the remainder comprising excipients or vehicles.

[0077] In some embodiments, this composition can be administered in combination with at least one other therapeutic agent for the disease state to be treated, particularly another agent that can treat cancer, such as a chemotherapeutic agent.The amount of required agent can be further substantially reduced, so that the amount of one or more required agents is reduced to the extent that significant response is observed from the subject.Significant response can include, but is not limited to, the reduction or elimination of nausea, the increase in apparent tolerance, faster response to treatment, more selective response to treatment, or a combination thereof.

[0078] The method can further comprise administering an effective amount of an antiproliferative agent, an effective amount of radiation therapy, surgical therapy, or a combination thereof.The present teachings also relate to a method for treating cancer.In some embodiments, the method comprises administering a drug to a subject in need of cancer treatment, wherein the drug dose is selected to reduce or eliminate immunosuppression that would occur when a substantially higher dose of the drug is administered in the subject; and administering radiation therapy in combination with the drug, wherein the reduction or elimination of immunosuppression enhances the effectiveness of radiation therapy compared to the effectiveness of radiation therapy observed when administered in combination with a substantially higher dose of the drug in the subject.In some embodiments, the drug comprises one or more chemotherapeutic agents in combination with the drug provided herein.In these embodiments, the drug can be selected from the group consisting of dacarbazine, paclitaxel, doxorubicin, or a combination thereof.

[0079] In some embodiments, an effective amount can be, for example, from about 1 mg / day to about 1000 mg / day, from about 10 mg / day to about 500 mg / day, from about 50 mg / day to about 250 mg / day, or any range or amount in 1 mg / day increments therein, for a human of average weight.

[0080] In some embodiments, the average human body weight can be estimated at 60 kg, 65 kg, 70 kg, 75 kg, 80 kg, or any weight in 1 kg increments therein. For treating solid tumors, a similar amount would be therapeutically effective. Those skilled in the art, given their skill and this disclosure, would be able to determine the therapeutically effective amount of the composition of the present invention for a given disease without undue experimentation.

[0081] In some embodiments, G-CSF is administered in combination with the compositions taught herein using any amount, time, and administration method known to be effective by those skilled in the art. The G-CSF can be NEUPOGEN, and can be administered in an amount ranging from about 0.1 μg / kg to about 1 mg / kg, from about 0.5 μg / kg to about 500 μg / kg, from about 1 μg / kg to about 250 μg / kg, from about 1 μg / kg to about 100 μg / kg, from about 1 μg / kg to about 50 μg / kg, or any range or amount therein in increments of 1 μg / kg.

[0082] In some embodiments, radiation therapy can be administered in a single local high dose, for example, in the range of about 20 Gy to about 100 Gy. In some embodiments, radiation therapy can be administered at a total dose ranging from about 20 Gy to about 100 Gy using a modified hypofractionated radiation regimen with administration comprising about two to about five doses over a one-week time frame. In some embodiments, radiation therapy can be administered at a total dose ranging from about 20 Gy to about 100 Gy using a modified hypofractionated radiation regimen with administration comprising two to three doses over a time frame ranging from about two to about three days. Radiation therapy can also be administered at a total dose ranging from about 45 Gy to about 60 Gy using a modified hypofractionated radiation regimen with administration comprising a single dose ranging from about 15 Gy to about 20 Gy every day over a three-day time frame.

[0083] The compositions and treatments taught herein can be administered in combination. For example, the combination can be administered for, for example, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 3 months, 6 months, 1 year, any combination thereof, or any other time period deemed necessary by those skilled in the art. The agents can be administered to a subject simultaneously, sequentially, or cyclically. Cycling therapy involves administering a first agent for a predetermined period of time, and a second agent or treatment for a second predetermined period of time, and repeating this cycle for any desired purpose, such as, for example, to enhance the effectiveness of treatment. The agents can also be administered simultaneously. The term "simultaneously" is not limited to administering the agents at exactly the same time, but rather means that the agents can be administered in an order and at time intervals that allow them to work together to provide additional benefits. Each agent can be administered separately or together in any suitable form, using any suitable means of administering one or more agents.

[0084] Similarly, the system can be designed for multiple growth medium ports, each for supplying cell culture medium for the cells and a selection drug to be tested. Thus, in some embodiments, the system can further include a first growth medium channel having a first growth medium port and disposed in communication with the first channel, and a second growth medium channel having a second growth medium port and disposed in communication with the second channel.

[0085] In developing treatments, the system can be designed to emulate diseased tissues or disease processes. Thus, in some embodiments, a method for emulating a disease in a subject is provided. The method can include obtaining one of the systems taught herein; injecting a first aqueous cell solution into a first channel to introduce a first diseased cell, thereby forming a temporary first aqueous interface membrane across the opening in the first wall; and injecting a second aqueous cell solution into a second channel to introduce a second diseased cell. In these embodiments, the first interface membrane is released after the injection of the second aqueous solution; and after the release of the first interface membrane, the first cell and the second cell communicate to emulate a diseased state.

[0086] In some embodiments, the selected system has at least three channels and at least three different cell types within the emulated tissue structure. Thus, in some embodiments, a method for emulating a disease includes obtaining a microfluidic system as taught herein, wherein the system has at least three channels; injecting a first aqueous cell solution into the first channel to introduce a first cell of the disease, and forming a temporary first aqueous interface film across the opening of the first wall by the injection; injecting a third aqueous cell solution into the third channel to introduce a third cell of the disease, and forming a temporary second aqueous interface film across the opening of the second wall by the injection; and injecting a second aqueous cell solution into the second channel to introduce a second cell of the disease. In these embodiments, the first interfacial membrane is released after injection of the second aqueous solution; the second interfacial membrane is released after injection of the second aqueous solution; the first cell and the second cell communicate after release of the first interfacial membrane to emulate a disease state; and the second cell and the third cell communicate after release of the second interfacial membrane to emulate a disease state.

[0087] In some embodiments, an aqueous gel cell growth medium can be used. The gel can be used in alternating channels to further stabilize the integrity of the at least three channels and slow the rate at which tissues interact. Thus, in some embodiments, a method for emulating a disease in a subject can include obtaining a system with at least three channels; injecting a first aqueous cell solution into the first channel to introduce a first cell of the disease, thereby forming a temporary first aqueous interface film across an opening in the first wall; injecting a third aqueous cell solution into the third channel to introduce a third cell of the disease, thereby forming a temporary second aqueous interface film across an opening in the second wall; and injecting a second aqueous cell solution in the form of an aqueous gel into the second channel to introduce a second cell of the disease. The first interface membrane can be released after injection of the second aqueous solution; the second interface membrane can be released after injection of the second aqueous solution; after release of the first interface membrane, the first cell and the second cell communicate to emulate a disease state; and after release of the second interface membrane, the second cell and a third cell communicate to emulate a disease state.

[0088] Methods of emulating tissue structures and emulating diseases naturally result in methods of treating diseased tissue structures. Accordingly, methods of treating emulated diseases with drugs are provided. In some embodiments, the methods include performing the methods of emulating diseases taught herein; selecting a drug for treating the emulated disease; and administering the drug through any one of the channels. In some embodiments, the drug can be administered through any channel. In some embodiments, the drug can be administered through the first channel or the second channel. In some embodiments, the drug can be administered through the first channel, the second channel, or the third channel. In some embodiments, the drug can be administered through the first channel, the second channel, the third channel, the fourth channel, or the fifth channel. In some embodiments, the drug can be administered through the first channel or the third channel. In some embodiments, the drug can be administered through the first channel or the third channel. In some embodiments, the drug can be administered through the first channel or the fifth channel.

[0089] The systems and methods provided herein can be used to emulate and treat various types of diseases. In some embodiments, the methods involve treating the emulated vascular disease with a drug. In these embodiments, the methods can include obtaining a system as taught herein, the system having at least three channels for introducing three vascular cell types; injecting a first aqueous cell solution into the first channel to introduce a first diseased cell type, forming an adventitial cell layer and forming a temporary first aqueous interface membrane across the opening in the first wall; injecting a third aqueous cell solution into the third channel to introduce a third diseased cell type, forming an endothelial cell layer and forming a temporary second aqueous interface membrane across the opening in the second wall; and injecting a second aqueous cell solution into the second channel to introduce a second diseased cell type, forming a smooth muscle layer. These methods further include selecting a drug and administering the drug to the first growth medium channel or the second growth medium channel. In these embodiments, the first interfacial membrane is released after injection of the second aqueous solution; the second interfacial membrane is released after injection of the second aqueous solution; the first cell and the second cell communicate after release of the first interfacial membrane to emulate a disease state; and the second cell and the third cell communicate after release of the second interfacial membrane to emulate a disease state.

[0090] Example 1. Pillar shape The integrity and stability of the interfacial film include requirements for surface tension and contact angle. Surface tension is dictated by the composition of the pillars and the aqueous material used to form the film, e.g., the cell growth medium.

[0091] Figures 4A-4C show the difference in contact angle between the interfacial film and the pillars, comparing circular pillars with hexagonal pillars, according to some embodiments. The pillars were fabricated by monolithic molding of polydimethylsiloxane (PDMS). A cover glass was attached to the device using ambient plasma treatment (PDC-001-HP series, Harrick, NY) at medium RF power for 2 minutes. The device was then placed in an oven at 80°C for 2 hours to strengthen the bond. The chip was then sterilized by UV treatment (UV Light Box Benchtop Decontamination Chambers, Air Science Inc.) for 45 minutes before proceeding with cell seeding. The pillars were then coated with a poly-D-lysine (PDL) solution (2 mg / ml) to enhance adhesion to the pillars. Chips can also be fabricated using a wide variety of thermoplastic polymers, such as poly(methyl methacrylate) (PMMA), polycarbonate (PC), polystyrene (PS), polyvinyl chloride (PVC), polyimide (PI), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), and cyclic block copolymer (CBC). Chips fabricated from these polymers can be fitted with a coverslip made of the same polymer.

[0092] Cell culture medium was then introduced into the channel, and the contact angle was measured. Figure 4A shows the contact angle between the interfacial film and the hexagonal pillar. Figure 4B shows the contact angle between the interfacial film and the circular pillar. Figure 4C shows an overlay of a hexagonal pillar and a circular pillar to demonstrate the difference in contact angle. Figures 4A-4C show the difference in pressure ΔPw across the channel and the pressure ΔP between the pillars. Pillars with round, hexagonal, and trapezoidal shapes were compared.

[0093] Example 2: Pillar distance Surface tension and contact angle can be optimized to enhance the integrity and stability of the interfacial film, and the distance the film is stretched is also a factor to consider.

[0094] 5 is a graph showing that there is a preferred inter-pillar distance between hexagonal pillars that avoids leakage of the interfacial film, according to some embodiments. It can be seen that for hexagonal pillars, the leakage threshold is above 150 uM, meaning that the pillars can be spaced at least 150 uM apart to maintain a stable interfacial film without leakage.

[0095] Leakage began to occur as the inter-pillar distance approached 200 μM, as indicated by the line across the leakage threshold in Figure 5. All pillars, whether circular, hexagonal, or trapezoidal, were leak-free below an inter-pillar distance of 100 μM.

[0096] Example 3. Fabrication of a 5-channel multichannel microfluidic device A microfluidic chip can be fabricated with five main channels and micropillars for a three-dimensional (3D) environment, for example, as follows:

[0097] To fabricate this device, a network of five channels was created, each 1 mm wide and 3 cm long. AutoCAD was used for this process. The channels were separated by 150 μm tall trapezoidal pillars spaced 200 μm apart along the entire length of the channel.

[0098] We created a photomask from the CAD file and printed it using a high-resolution printer (CAD Art Services Inc., OR). We then placed the mask on a silicon wafer and spin-coated it (Spin Coater WS-650 series, Laurell Technologies Corporation, PA) using SU-8 photoresist polymer (MicroChem Inc., MA) and baked it at 65 °C for 5 min and 95 °C for 25 min. We then exposed the SU-8-coated silicon wafer to UV light (UV-KUB 2, KLOE Inc., France), baked it again at 65 °C for 5 min and 95 °C for 12 min, cooled it to room temperature, and washed it with SU-8 solvent to obtain a silicon master mold. To fabricate devices using the master mold, we poured an aliquot of polydimethylsiloxane (PDMS, Dow Inc., MI) mixed with curing solution (Dow Inc., MI) in a 10:1 ratio into the mold. We then degassed, fired, and cut out individual devices (35 mm diameter, 3-4 mm height), and finally created inlets and outlets using a Biopsy Punch® (Miltex, PA).

[0099] Fabrication of Microfluidic Chips: For laboratory-scale experiments, polydimethylsiloxane (PDMS)-based chips with micropillars can be used. However, using cyclic olefin copolymers (COCs) and injection molding, it is possible to fabricate large numbers of chips for large-scale applications. Pillars were used to create walls with openings. In this embodiment, rounded pillars were not used because it is desirable to have opposing edges to provide surface tension to hold the interfacial film together. This is an example of an embodiment that does not use circular or spherical "pillars."

[0100] Tip operation: Rather than using a manual pipette, a pumping system is provided that can be implemented to automate pumping. See, e.g., https: / / www.sciencedirect.com / science / article / pii / S2468067220300249?via%3Dihub (downloaded July 27, 2021; teaches a peristaltic pump suitable for this application); also see, e.g., https: / / link.springer.com / article / 10.1140 / epje / s10189-020-00002-9 (downloaded July 27, 2021; teaches a constant pressure pump suitable for this application).

[0101] Manual seeding, growing, and evaluation

[0102] The pathological state can be established using manual seeding of cells, growth of manually placed cells using a manual process, establishment and release of an interface layer to allow cell interaction.

[0103] Device Automation

[0104] We can automate the process for cell seeding and growth in a sterile environment. Automated devices are easier to use and can be used at the patient's bedside to, for example, assess treatment response, investigate the effects of gender and age on treatment response, as well as any of a number of other factors known to those skilled in the art, and develop a customized approach to the treatment of a subject.

[0105] Example 4. Consider the application of pulmonary arterial hypertension. Pulmonary arterial hypertension (PAH) is a rare disease in which pulmonary arteries / arterioles become stiff and blocked. The heart must work harder to pump blood through the blocked arteries to the lungs. This causes the heart to enlarge, leading to patient death from right heart failure. The primary clinical symptom of this disease is elevated mean pulmonary artery pressure (mPAP), which results from a series of active structural changes in the pulmonary arteries called arterial remodeling and muscularization. The leading cause of death in patients with pulmonary arterial hypertension (PAH-RVH), for example, is right ventricular hypertrophy (PAH-RVH). The various cellular and biomolecular processes involved in the development of PAH-RVH are poorly understood and can be better treated using the systems and methods taught herein.

[0106] This pathology develops due to abnormal proliferation, migration, and misplaced growth of pulmonary artery cells (PACs), the development of apoptosis-resistant endothelial cells (ECs), increased extracellular matrix (EC) deposition, thickening of the smooth muscle layer, and the acquisition of a smooth muscle cell (SMC)-like phenotype by ECs. Due to unregulated cell growth, the disease becomes severe, and some pulmonary arteries develop glomerular-like luminal obstructions called plexiform lesions. These lesions are described as plexuses of endothelial-lined slit-like channels separated by hyperpigmented, oval-shaped core cells of uncertain phenotype.

[0107] The effect of gender on PAHs

[0108] One puzzling aspect of PAH is that although it affects women more than men, women with PAH tend to survive longer than men. This discrepancy in prevalence versus survival rates among PAH patients likely arises from inherent differences between the two genders and the opposing roles of sex hormones, particularly estrogen, which appear to have both beneficial and detrimental effects on the development and progression of PAH in women. Furthermore, we found that human and animal studies suggest that the sex of the subject plays a role in PAH treatment response, and thus gender-based tailored treatment may potentially benefit PAH patients.

[0109] Investigating ECM remodeling

[0110] As reported in our recent publication, we first characterized the cells for various markers and compared control and PAH cells with adventitial cells (ADCs) or SMCs at 2 × 10 6 At a density of cells / mL, ECs are 5–10 × 10 6 SMCs were seeded into each channel of the chip at a density of 1000 cells / mL. SMCs were seeded in rat tail type I collagen solution (Corning Inc., NY). We supplied the cells with medium through the two outermost channels. In experiments seeding chips with three different cell types, we first injected SMCs mixed with collagen (2 mg / mL) alone or in the inner channel, allowing the collagen to form a gel for 20 minutes. ECs and ADCs were then seeded after a 20-minute gap. The cell-containing chips thus fabricated were placed in a humidified chamber and grown in a CO2 incubator for 5–8 days. The medium was removed and replaced with 150 μL of fresh medium every day.

[0111] On days 1, 3, 5, 7, and 10, we stained ECs, SMCs, and ADCs with markers for CD31, FSP-1, VEGFR2, α-SMA, SM-22α, and CD90, as well as markers for various ECM proteins. To stain the cells, we first washed the chips with basal medium, treated them with 4% paraformaldehyde, blocking buffer, and primary antibodies, and incubated them overnight. The next day, we washed the chips again with wash buffer, injected them with secondary antibodies, incubated them for 1 hour, washed them again, and finally treated them with mounting medium containing DAPI to stain the nuclei. Similarly, we stained the chips to confirm the presence of human type I collagen (ColT1) and type IV collagen (ColT4), laminin, tenascin-C, fibronectin, and elastin. We captured images of the cells and ECM using epifluorescence (DMi8 epifluorescence, Leica, IL) or multiphoton confocal microscopy (Ti-E, Nikon, NY).

[0112] We quantified various ECM proteins using quantitative PCR (qPCR). To do this, we removed and harvested both control and PAH cells 1, 3, 5, 7, and 10 days after seeding. We isolated mRNA using the Quick Prep Micro mRNA Purification Kit (Amersham, Piscataway) and analyzed it for laminin, tenascin-C, ColT1α, ColT4, fibronectin, and elastin. Based on the manufacturer's protocol, we generated first-strand cDNA using the Superscript III First-Strand Synthesis System (Invitrogen) and performed qPCR on an MX4000 using Brilliant SYBR Green QPCR Master Mix with the threshold cycle number determined by MX4000 software v.4.20 (Stratagene, La Jolla, CA). We normalized Ct values ​​to the internal control GAPDH using the formula: ΔCt = Ct (reference gene) - Ct (gene of interest).

[0113] Assessment of endothelial cell dysfunction on a chip

[0114] To investigate whether endothelial cells undergo apoptosis similar to that observed in PAH, we seeded control endothelial cells, smooth muscle cells, and ADCs and grew them for 3 to 5 days under various conditions, including normal serum in normoxia or reduced serum in hypoxia. We also grew N-ECs in the presence or absence of collagen. At the end of the experiment, we stained the cells for the same cellular and ECM markers listed above. To assess apoptosis, we stained cells for the presence of λ-H2AX, PCNA, and cleaved caspase-3, and finally calculated the apoptotic index as the ratio of the number of caspase-3-positive cells to the total number of cells.

[0115] Assessment of Gender-Specific Therapy Response:

[0116] To evaluate the cytological basis of these gender-specific therapeutic effects, we used bosentan, an endothelin receptor antagonist (ERA), to which female PAH patients respond more than male patients. Chips prepared by seeding PAH-ECs / SMCs / ADCs from male and female patients were infused with 50 μM bosentan starting on day 4 of cell seeding, and infusion continued for an additional 3 days. To examine whether the drug had a preventive effect, cell-containing chips were treated with 50 μM bosentan on day 1 of seeding and cells were grown without treatment for an additional 6 days. To examine the effects of growth hormone and anti-PAH drugs on cell function and remodeling, male and female PAH-ECs, QD705-labeled or unlabeled PAH-SMCs, and PAH-ADCs were seeded. Human ColT1 deposition, aromatase (an enzyme that synthesizes estrogen), and CYP1B1 (an estrogen-metabolizing enzyme) expression were evaluated, followed by assessment of arterial remodeling and muscularization.

[0117] To evaluate the effects of drugs on pulmonary artery thickening, we assumed that ECs and SMCs grow in the intimal and medial layers of each channel of the chip, rather than outside their assigned layers. If ECs or SMCs grow outside their original seeded channel, we considered the out-of-specification channel growth to be intimal or medial thickening, respectively. We also considered SMC migration from the medial to the medial layer to represent muscularization, and the number of SMCs growing in the intimal layer represented the degree of muscularization. These assumptions were based on PAH pathology, in which non-muscularized distal arteries / arterioles become muscularized and exhibit SM-like cells derived from pre-existing SMCs. We used phase-contrast microscopy, immunostaining, and quantum dot labeling to delineate distinct cell growth areas and count and identify cells.

[0118] To demarcate the boundaries of different cell growth regions, we first imaged the chip using a phase-contrast microscope and then stained the cells for imaging using a confocal microscope. To calculate the degree of intimal and medial thickening, we calculated the average length of five to seven diametrically heterogeneous points in the same layer. To examine the degree of muscularization, we grew QD-labeled N-SMCs / PAH-SMCs on the chip, stained the chip for FSP-1 and CD31 antibodies, and counted the number of FSP-1+CD31- or QD-labeled SMCs in all three layers of the chip.

[0119] Fabrication of on-chip plexus-like complex cellular lesions

[0120] To reproduce plexiform lesions on chips, we first prepared spheroids or 3D colonies of various cell combinations using the "water droplet method." See, for example, R. Foty. J Vis Exp. 51:2720 (2011). The spheroids were then seeded onto chips. Briefly, we prepared a methylcellulose-cell mixture by dispersing approximately 5 × 10 ECs, EC / SMC (1:1), EC / ADC (1:1), or EC / SMC / ADC (1:1:1) in 25 mL of serum-free medium containing 20% ​​methylcellulose. Next, using a pipette, we dispensed the cell-methylcellulose mixture into 25 μL droplets, and a series of 25 μL droplets were seeded onto nonadherent cell culture dishes. The dish containing the cell droplets was then inverted, so that the droplets remained suspended from the surface of the dish in the air, thus generating hanging cell droplets. We generated single and multiple cell spheroids or 3D colonies by incubating suspended cell droplets in air at 37°C and 5% CO2 for 24 hours. We prepared QD-labeled PAH-EC, PAH-EC / SMC, or PAH-EC / SMC spheroids, as well as control cell spheroids (N-EC / SMC / ADC). The next day, we harvested the spheroids, washed them with medium to remove the methylcellulose, and then dispersed them in 90 μL of collagen as a spheroid matrix. Finally, we seeded 10–15 spheroids into the inner membrane channel of the chip. Three days after seeding the spheroids on the chip, we treated the chip containing the control N-EC / SMC / ADC spheroids with 50 ng / mL VEGF and 1 ng / mL TGF-β for an additional 3 days. We then stained ECs, SMCs, and ADCs with various markers and observed the cells under a confocal microscope (Ti-E, Nikon, NY).

[0121] result

[0122] PAH pathophysiology is reconstructed on this device by growing three pulmonary artery cells (PACs): endothelial cells (ECs), smooth muscle cells (SMCs), and adventitial cells (ADCs). PAH cells can be obtained, for example, from biopsies or using PACs differentiated from induced pluripotent stem cells derived from PAH patients.

[0123] When grown on the chip, PAH PACs migrated from their designated layer and interacted with cells in adjacent layers, causing phenomena similar to the primary pathology of human PAH: muscularization, deposition of extracellular matrix (ECM) proteins, and arterial remodeling. Results included: Flow-induced stress caused morphological changes in control cells grown on chips and induced arterial remodeling. Estrogen and platelet-derived growth factor (PDGF) treatment caused more extensive arterial remodeling in chips prepared with PAC from female patients than in chips with PAC from male patients. Female chips (chips prepared from cells from female patients) were more responsive to the anti-PAH drug bosentan than male chips (chips prepared from cells from male patients). PAH-EC / SMC spheroids, but not control cells, seeded onto chips formed arteries with plexus-like lesions, and PAH-ECs exhibited clonal proliferation.

[0124] 6 illustrates extracellular remodeling in PAH, according to some embodiments. The systems and methods provided herein have produced PAH representations that show ECM remodeling, such as in the pulmonary arteries of clinical patients.

[0125] Because ECM deposition is a major pathological feature of PAH, we used this system and method to demonstrate that PAH cells grown on chips produce ECM proteins, create their own ECM scaffold, and ultimately cause pulmonary artery remodeling. The basement membrane of a normal human pulmonary artery consists of a thin sheet of fibers underlying the endothelial cell layer, formed by specific interactions between ECM components such as laminin, collagen, elastin, fibronectin, tenascin-C, and proteoglycans. We then measured the levels of ECM proteins in chips prepared with three types of control cells and PAH cells. Compared to control chips, chips prepared with PAH cells showed extensive deposition of various ECM proteins, namely, laminin, tenascin-C, and insoluble collagen (ColT1α), but no deposition of ColT4, fibronectin, or elastin. In chips prepared with PAH cells, a thin layer of laminin developed and extended toward both the intimal and luminal compartments; intimal thickening increased with increasing laminin deposition. The intimal layer of PAH chips contained fibrillar collagen fibers, which increased with increasing numbers of PAH-SMCs in the intimal layer. Collagen fiber deposition was most abundant in the intimal layer of PAH chips. Compartment-specific analysis of pulmonary arteries from IPAH patients revealed that ECM remodeling, particularly changes in collagen expression, were more pronounced in the intimal, followed by the medial, and then the adventitial layers.

[0126] We also examined the effect of gender on the development of plexiform lesions by seeding spheroids of various combinations of PAH cells (PAH-EC / SMC, PAH-EC / ADC, and PAH-EC / SMC / ADC) derived from male and female patients. For ease of identification, we used QD705-labeled PAH-ADC and N-ADC. Male and female PAH-EC spheroids generated angiogenic-like sprouting, with luminal arterial channels lined by a monolayer of ECs, but without lesions. Male and female PAH-EC / SMC spheroids developed similarly into lesion-free arteries. In contrast, PAH-EC / ADC spheroids formed disorganized masses with arterial lumens, while a few cuboidal vessels were observed only in the plexiform-mimicking chips in females. In both sexes, PAH-EC / SMC / ADC spheroids produced complex plexiform cellular lesions. Female chips showed more upregulation of α-SMA than male chips, and female PAH-SMCs contained intermediate filaments, suggesting that the SMCs were in a differentiation stage.

[0127] Example 5. Development of disease treatments The systems and devices presented herein are useful for developing disease treatments because they can emulate the salient pathophysiological features of the disease process, as well as cellular interactions and responses to drug administration. In the case of PAH, the cell types are the three major cell types in the pulmonary artery. Interactions include, for example, cell migration from one layer of the pulmonary artery wall to another, endothelial-to-mesenchymal transition, and the formation of plexus-like lesions. In the case of PAH, we demonstrated differences and similarities in the pathogenesis of PAH in male and female patients and used these differences and similarities to develop gender- and age-specific treatments for PAH. This process may help develop personalized therapies for the disease process. See, for example, Taslim A. Al-Hilal et al., Royal Society of Chemistry, Lab Chip 20:3334-3345 (2020), https: / / doi.org / 10.1039 / D0LC00605J (downloaded July 27, 2021), which is incorporated by reference in its entirety.

[0128] We examined the effect of gender on the development of plexiform lesions by seeding spheroids of various combinations of PAH cells (PAH-EC / SMC, PAH-EC / ADC, and PAH-EC / SMC / ADC) derived from male and female patients. For ease of identification, we used QD705-labeled PAH-ADC and N-ADC. Male and female PAH-EC spheroids generated angiogenic-like sprouting, with luminal arterial channels lined by a monolayer of ECs, but without lesions. Male and female PAH-EC / SMC spheroids developed similarly into lesion-free arteries. In contrast, PAH-EC / ADC spheroids formed disorganized masses with arterial lumens, while a few cuboidal vessels were observed only in the plexiform-mimicking chips in females. In both genders, PAH-EC / SMC / ADC spheroids generated complex plexiform cellular lesions. Female chips showed more upregulation of α-SMA than male chips, and female PAH-SMCs contained intermediate filaments, suggesting that the SMCs were in a differentiation stage.

[0129] Gender differences in PAH and gender-preferential responses to PAH treatment are two puzzling features of PAH pathophysiology. PAH affects more women than men, yet men have a worse survival rate than women. Major factors contributing to gender differences in PAH include sex hormones and growth factors. Some reports suggest that differences in PAC cell cycle regulation predispose women to PAH. Furthermore, both human and animal studies suggest that the sex of the subject plays a role in PAH treatment response, and thus, gender-based personalized therapy may potentially benefit PAH patients.

[0130] Example 6. Treatment Development As taught herein, the systems and methods can be used to emulate cellular interactions in any system of the body in the development of disease treatments, hi some embodiments, the systems and methods provided herein can emulate any vascular disease.

[0131] Pulmonary vascular disease encompasses much more than PAH, and includes several pathological conditions that alter the blood vessels that serve as conduits between the heart and lungs. Regarding the range of pulmonary vascular diseases that can be investigated using the systems and methods herein, any disease that has alterations to the pulmonary vasculature, such as stiffening of the pulmonary vascular bed, can lead to various complications that can be addressed in the development of improved treatments.

[0132] 7 shows a flowchart of a method in which a subject in need can be treated using the systems and methods taught herein, according to some embodiments. In many of the systems taught herein, the microfluidic device has at least three channels. Steps 605, 610, and 615 involve introducing a first cell type, cell type 1, into channel 1 in cell culture medium to create an interface layer adjacent to channel 2; introducing a second cell type, cell type 2, into channel 3 in cell culture medium to create an interface layer adjacent to channel 3; and introducing an aqueous medium into channel 2 to controllably release the interface layer adjacent to channel 2, allowing communication between cell type 1 and cell type 3. These are core steps. Steps 620, 625, 630, 635, 640, and 645 provide a system and method for identifying a subject in need of drug therapy (620), obtaining cell type 1 from the subject (625), obtaining cell type 2 from the subject (630), identifying drug candidates for the subject (635), introducing drugs into channels 1, 2, and / or 3 to identify a therapeutic effect (640), and administering the drug to the subject if a therapeutic effect is desired (645).

[0133] In the case of a vascular model, three cell types can be present: smooth cells, adventitial cells, and endothelial cells. In some embodiments of a three-channel device, smooth cells can be placed in the middle channel, channel 2, and adventitial and endothelial cells can be placed in adjacent channels 1 and 3. In some embodiments of a five-channel device, channels 1 and 5 can serve as cell culture medium reservoirs, which can be periodically replaced as needed and can also be used to administer drugs; channels 2 and 4 can be adventitial and endothelial cells; and channel 3 can be smooth muscle cells. Smooth muscle cells can be introduced into an aqueous gel, cell culture medium, or aqueous fluid, cell culture medium. In some embodiments, channel 1 and / or channel 5 can be used to apply hydrostatic pressure to emulate blood pressure changes and simulate stress on the blood vessel, and an endothelial cell layer can be adjacent to one or both of channels 1 and 5. Considering that capillaries only have endothelial cells and major arteries have all three layers, those skilled in the art will understand how the present system and method can be used to represent physiology in treatment development.

[0134] Gender-specific therapy:Our experiments suggested that chips prepared with PACs from female PAH patients were more responsive to estrogen treatment than chips prepared with PACs from male PAH patients. Here, we used chips to examine gender-specific therapy responses by treating them with the endothelin receptor antagonist bosentan. In line with clinical data showing that female PAH patients were more responsive to bosentan therapy than male patients (p = 0.3), chips prepared with PACs from female patients were more responsive to bosentan than their male counterparts. In female chips, the drug reduced medial layer thickening, delayed PAH-SMC migration into the luminal and intimal layers, and reduced CYP1B1 expression in the medial layer. Compartment-specific analysis showed that laminin and ColT1α deposition was lower in bosentan-treated female chips than in male chips. To examine the preventive effect, we treated male and female chips on day 1 after seeding and then allowed the cells to grow for an additional 6 days without treatment. While the degree of intimal and medial thickening was similar in male and female chips, the presence of PAH-SMCs in the endothelial layer was lower in female chips than in male chips. CYP1B1 expression was more reduced in the medial layer of female chips than in male chips. Compared to male chips, bosentan-treated female chips showed lower levels of muscularization, as measured by counting QD705+PAH-SMCs in the intimal layer. Interestingly, early bosentan treatment reduced intimal deposition of laminin and ColT1α in female chips compared to their male counterparts. As can be seen from this example, the systems and methods described herein can be used to develop gender-specific therapies for disease.

[0135] Efficacy of anti-PAH drugs in improving pulmonary arterial hypertension-induced right ventricular hypertrophy (PAH-RVH) : This system can be used to identify interactions between ECs and CMs affected by PAH and evaluate drug efficacy.

[0136] The modified chip device can be used, for example, to grow both cardiomyocytes (CMs) in the study of cardiovascular disease and pulmonary artery endothelial cells (ECs) in the study of pulmonary vascular disease. Pulmonary artery ECs can be harvested, for example, from the pulmonary arteries of healthy donors (N-ECs) and PAH patients (PAH-ECs). We have found that PAH is an example of a disease that could benefit from improved treatments developed with the systems and methods taught herein. For example, through the growth of CMs and either N-ECs or PAH-ECs in their respective compartments or channels, we can assess the effects of PAH-ECs on CMs by measuring various soluble cell markers, assessing CM contractility, evaluating gene expression by CMs, and examining the release of hypertrophic inducers and hypertrophic markers and the effects of anti-PAH PAH drugs on CM contractility. We found, for example, that (i) chips prepared with CM and N-EC showed anisotropic growth of CM and the presence of bundles similar to physiological CM; and (ii) in chips prepared with CM and PAH-EC, CM hypertrophied, lost their contractility, and showed elevated levels of hypertrophy-related markers.

[0137] We developed a drug therapy using this system and method. We used two anti-PAH drugs, sildenafil and bosentan, on chips prepared with CMs and PAH-ECs. The drugs reduced the degree of CM hypertrophy, improved contractility, and reduced both hypertrophy inducers and hypertrophy markers. From our experiments, those skilled in the art will understand that this system and method can identify interactions between PAH-affected ECs and CMs and evaluate the effectiveness of anti-PAH drugs in improving pulmonary arterial hypertension-induced right ventricular hypertrophy (PAH-RVH).

[0138] Electrical stimulation

[0139] Electricity can be applied to cells to simulate an electrophysiological microenvironment. By modifying this device, for example, we can reproduce the following three diseases: (1) venous pulmonary hypertension, (2) pulmonary embolism, and (3) chronic thromboembolic disease. In these embodiments, we can seed organ-specific cells and create microenvironments to emulate the pathological conditions of the diseases. For example, pulmonary embolism and chronic thromboembolic disease are fatal diseases in which blood clots occur in the pulmonary vasculature, providing particularly important directions for the use of the systems and methods provided herein. The systems and methods taught herein can be used to reconstruct the pulmonary vasculature and then examine the interactions between cells and the effects of specific treatments, such as drug therapy, radiation therapy, or a combination thereof, to evaluate how blood coagulation can change the pathology of the pulmonary vascular bed, and how the treatment is affected by the subject's gender, age, some other physiological condition, or a combination thereof.

[0140] Of course, vascular disease encompasses several pathological conditions, each encompassing pathology of arteries, veins, or lymphatic vessels. The systems and methods taught herein can encompass all common vascular diseases. For example, the systems and methods taught herein can be used to emulate, acquire data from, and improve the treatment of various venous diseases, which may include, for example, neurovascular disease, peripheral artery disease, renal artery disease, carotid artery disease, aortic aneurysms, and varicose veins, spider veins, chronic venous insufficiency, deep vein thrombosis, neurovascular disease, and lymphedema.

[0141] In some embodiments, to use the systems and methods taught herein, we harvest human cells from relevant tissues or organs and grow them in the systems taught herein to emulate the cellular interactions of each disease. As described herein, the system is versatile and can serve multiple functions in the development of disease treatments. For example, we have shown that we can examine gender-dependent differences in disease pathology and screen related drugs for their effectiveness in treating the emulated disease. Naturally, the systems and methods taught herein can aid in the development of precision medicine approaches by examining cell-cell interactions and the effects of specific treatments, such as drug therapy, radiation therapy, or a combination thereof, and how that treatment is affected by the subject's gender, the subject's age, some other physiological condition of the subject, or a combination thereof.

Claims

1. 1. A microfluidic system designed to emulate cellular physiology in a subject, comprising: The system includes a microfluidic chip that includes: a first channel adjacent to a second channel to form an emulated tissue structure, wherein the first channel and the second channel are connected to a chip; and a first port configured to inject a first aqueous cellular solution into the first channel to emulate a first layer of tissue, the first aqueous cellular solution comprising a first physiological medium and not a gel; a second port configured to inject a second aqueous cellular solution into the second channel to emulate a second layer of tissue adjacent to the first layer of tissue, the second aqueous cellular solution comprising a second physiological medium and not a gel, wherein the first aqueous cellular solution and the second aqueous cellular solution are the same or different; and a first wall having an opening that is discontinuous and that forms a portion of the first channel and the second channel, the first wall being shared by the first channel and the second channel, and the opening in the first wall being configured to (i) support a releasable first aqueous interface membrane across the opening in the first wall upon injection of the first aqueous cellular solution, and (ii) emulate a physiological environment with physiological communication between the first aqueous cellular solution and the second aqueous cellular solution, thereby emulating cellular communication between the first aqueous cellular solution in the first channel and the second aqueous cellular solution in the second channel through the opening in the first wall upon release of the first interface membrane from the opening; the first channel comprising: where a first interface membrane retaining a first aqueous cell solution in the first channel; the opening in the first wall has a distance along the length of the discontinuous wall of up to 200 μM; Injection of a second aqueous cell solution into the second channel causes the first interface membrane to be released from the opening; and the non-gel first physiological medium and the non-gel second physiological medium emulate a physiological environment to represent physiological conditions; The microfluidic system.

2. The system of claim 1 , wherein the wall is configured to establish a first interface membrane across the opening upon injecting the first aqueous cell solution.

3. 10. The system of claim 1, wherein the opening comprises a hydrophobic material for establishing a first interface membrane across the opening in the first wall upon injecting the first aqueous cell solution.

4. 10. The system of claim 1, wherein the opening is delineated by opposing wedge-shaped edges that share a common plane to establish a first interface membrane across the opening when the first aqueous cell solution is injected.

5. The system of claim 3 , wherein the opening comprises a shape selected from the group consisting of a circle and an oval.

6. 2. The system of claim 1, wherein the opening is defined by a polygonal structure providing opposing edges on at least two opposing sides of the opening, wherein the opposing edges share a common plane to establish a first interface membrane across the opening in the first wall when the first aqueous cell solution is injected.

7. the openings in the first wall are spaces between adjacent columns along the series of columns; each column in the series of columns having a central axis at least substantially perpendicular to the tip; 7. The system of claim 6, wherein each pair of adjacent columns provides a pair of opposing edges that share a common plane to establish a first interface membrane across the opening in the first wall when the first aqueous cell solution is injected.

8. third channel; a third port configured for injecting a third aqueous cell solution into the third channel; and Second wall with opening further comprising the second wall is shared by the second channel and the third channel; and 2. The system of claim 1, wherein the opening in the second wall is configured to (i) form a releasable second aqueous interface membrane across the opening in the second wall upon injection of a third aqueous cell solution, and (ii) emulate a physiological environment, thereby emulating cellular communication between the second channel and the third channel upon release from the interface membrane opening.

9. 10. The system of claim 1 or 8, further comprising a growth medium channel having a growth medium port, wherein the opening in the first wall is a space between adjacent columns along the series of columns, the space ranging from 30 uM to 250 uM.

10. 10. The system of claim 1, further comprising a first growth medium channel having a first growth medium port and disposed in communication with the first channel, and a second growth medium channel having a second growth medium port and disposed in communication with the second channel.

11. 10. The system of claim 1 or 8, further comprising a growth medium channel having a growth medium port.

12. 10. The system of claim 8, further comprising a first growth medium channel having a first growth medium port and disposed in communication with the first channel, and a second growth medium channel having a second growth medium port and disposed in communication with the second channel.

13. 1. A method of emulating a disease in a subject, the method comprising: Obtaining the system of claim 1; injecting a first aqueous cell solution into the first channel to introduce diseased first cells, thereby forming a releasable first aqueous interface film across the opening in the first wall; and injecting a second aqueous cell solution into the second channel to introduce second cells of the disease; wherein the first interfacial membrane is released after injection of the second aqueous solution; and The method wherein the first cell and the second cell communicate after release of the first interface membrane to emulate a disease state.

14. 1. A method of emulating a disease in a subject, the method comprising: Obtaining the system of claim 8; injecting a first aqueous cell solution into the first channel to introduce diseased first cells, the injection forming a releasable first aqueous interface film across the opening in the first wall; injecting a third aqueous cell solution into the third channel to introduce a third cell of the disease, the injection forming a releasable second aqueous interface membrane across the opening in the second wall; and, injecting a second aqueous cell solution into the second channel to introduce second cells of the disease; Including; where: the first interfacial membrane is released after injection of the second aqueous solution; a second interfacial membrane is released after injection of the second aqueous solution; the first cell and the second cell communicate after the release of the first interface membrane to emulate a disease state; The method wherein the second cell communicates with a third cell after release of the second interface membrane to emulate a disease state.

15. 1. A method of emulating a disease in a subject, the method comprising: Obtaining the system of claim 8; injecting a first aqueous cell solution into the first channel to introduce diseased first cells, the injection forming a releasable first aqueous interface film across the opening in the first wall; injecting a third aqueous cell solution into the third channel to introduce a third diseased cell, the injection forming a releasable second aqueous interface membrane across the opening in the second wall; and injecting a second aqueous cell solution in the form of an aqueous gel into the second channel to introduce second cells of the disease; wherein the first interfacial membrane is released after injection of the second aqueous solution; a second interfacial membrane is released after injection of the second aqueous solution; the first cell and the second cell communicate after the release of the first interface membrane to emulate a disease state; The method wherein the second cell communicates with a third cell after release of the second interface membrane to emulate a disease state.

16. 1. A method of treating an emulated disease with a drug, the method comprising: Carrying out the method of claim 13; Selecting a drug to treat the emulated disease; and Administering a drug in the first channel or the second channel The method comprising:

17. 1. A method of treating an emulated disease with a drug, the method comprising: Carrying out the method of claim 14; Selecting a drug to treat the emulated disease; and Administering a drug in the first channel, the second channel, or the third channel The method comprising:

18. 1. A method of treating an emulated disease with a drug, the method comprising: Carrying out the method of claim 15; Selecting a drug to treat the emulated disease; and Administering a drug in the first channel, the second channel, or the third channel The method comprising:

19. 1. A method of treating emulated vascular disease with a drug, the method comprising: Obtaining the system of claim 12; injecting a first aqueous cell solution into the first channel to introduce diseased first cells to form an adventitial cell layer, the injection forming a releasable first aqueous interface membrane across the opening in the first wall; injecting a third aqueous cell solution into the third channel to introduce diseased third cells to form an endothelial cell layer, thereby forming a releasable second aqueous interface membrane across the opening in the second wall; and injecting a second aqueous cell solution into the second channel to introduce second diseased cells to form a smooth muscle layer; Selecting a drug; and administering a drug to the first growth medium channel or the second growth medium channel; the first interfacial membrane is released after injection of the second aqueous solution; a second interfacial membrane is released after injection of the second aqueous solution; the first cell and the second cell communicate after the release of the first interface membrane to emulate a disease state; and The method wherein the second cell communicates with a third cell after release of the second interface membrane to emulate a disease state.

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