Multilayer microfluidic system and method

Abstract

The systems and methods provided facilitate the development of a precision medicine approach using microfluidic cells with releasable water interface membranes to separate tissue channels. The method may include personalized medical treatment regimens. These systems and methods can simulate cell communication in disease states in a more accurate aqueous environment and provide data on cell-cell interactions that can be used to develop treatments for subjects in need. These systems and methods can also be used to evaluate the effectiveness of specific therapies, such as drug therapy, radiation therapy, or a combination thereof. These systems and methods can show how a specific therapy is affected by several known factors, including but not limited to the subject's sex, age, genetic factors or other genetic predispositions, and other physiological states of the subject or a combination thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application No. 17 / 865,021, filed on July 14, 2022, which claims the benefit of U.S. Provisional Application No. 63 / 226,733, filed on July 28, 2021. Background Art

[0003] Field of the Invention

[0004] The teachings herein relate to multilayer microfluidic systems and methods, namely, an improved microfluidic chip that separates tissue types prior to controlled contact by releasing interfacial films formed between pillars, and methods of using the device to provide disease treatment, including personalized medicine approaches.

[0005] Description of the Prior Art

[0006] In order to improve the treatment of diseases, researchers use various animal experiments to develop treatments for diseases. The problem is that animals often cannot effectively mimic human pathophysiology and can only partially mimic important human pathological changes. In addition, various animal species are used for various pathogenic factors or environments, which means that there are great differences in the severity, progression and response to therapies of diseases. Animal studies cannot well reflect the influence of various factors, and the influence of these factors can affect the way we treat diseases. For example, these factors may include the biological sex of the treatment subject, including the influence of sex hormones, especially the role of estrogen. A better understanding of how these factors affect treatment methods can help improve the development of sex-based therapies, etc.

[0007] Microfluidic devices have been used to construct physiologically relevant characterizations of tissue and organ-level functions and diseases. Prior art microfluidic devices use a single channel to seed and grow one type of cell, and these single-channel devices are limited in their applications. For example, single-channel devices cannot represent interactions between different types of cells, including but not limited to layered vascular tissues.

[0008] Developing effective multilayer systems involves the challenge of creating and maintaining the integrity of adjacent cell layers. Multilayer systems must provide controlled, realistic interactions between the layers. Some prior art microfluidic devices attempt to use gels to maintain the integrity of the cell layers, but the interactions between the layers are not realistic because gels do not exist physiologically and would alter the rate at which interactions can occur, so the amount of control over the interactions is limited by the presence of the gel. The skilled artisan will appreciate that mammalian cells do not transport or signal in vivo through gels, so the use of gels is not a true physiological condition.

[0009] At least for the above reasons, skilled technicians would like to have a microfluidic device that can more realistically represent real physiological conditions when developing disease treatment methods, especially diseases involving multiple tissue types that usually interact as tissue layers. For example, such a microfluidic system can construct cell interactions that occur during a specific pathological process. In some cases, the system provided herein can be constructed so that cells in one channel can always give prompts to cells in adjacent channels so as to represent more accurate pathological conditions when developing treatment methods. In some cases, the system provided herein can allow diseased cells in its channel to communicate with healthy cells in adjacent channels. In some cases, the system provided herein can make healthy cells sick to generate data on cell interactions in a specific pathological process, thereby developing effective treatment methods. In some cases, the system provided herein can show the effect of drug treatment on the disease, including relative changes in drug selection, drug dosage, drug tolerance, drug half-life, etc. It is easy for technicians to understand that a large number of other variables can be introduced using the systems and methods provided herein to develop treatment methods, including preventive treatment methods that inhibit or prevent the occurrence of diseases, treatment methods that inhibit pathological processes after the occurrence of diseases, and treatment methods that improve disease symptoms.

[0010] Therefore, for at least the reasons stated above, those skilled in the art will appreciate microfluidic systems and methods that can provide more accurate water physiological conditions to (i) control the cellular interactions that occur; (ii) develop therapeutic approaches based on cellular interactions;

[0011] (iii) introducing variables of interest, i.e., variables that influence pathological processes, when developing therapeutic approaches; (iv) testing therapeutic approaches, including drugs, radiation, or combinations thereof; and (v) determining variables that influence therapeutic approaches in subjects, such as cellular transport and signaling mechanisms relevant to disease treatment. SUMMARY OF THE INVENTION

[0013] Systems and methods are provided to aid in the development of treatments for diseases. These systems and methods may include precision medicine approaches for any disease, or may include personalized medicine approaches for individuals. The systems and methods include the use of a microfluidic device that can be used to reconstruct tissue interactions during a disease process. The reconstruction may include interactions between multiple cell types, interactions between cell layers in a tissue, or a combination of these cell interactions. Such interactions may provide information about the relevant cell communications in a disease state to design treatments, and may also provide information about the effects of a particular type of treatment or combination of treatments (e.g., drug therapy, radiation therapy, or a combination thereof). Aqueous interface membranes are used to control tissue interactions, and the interface membranes remain unchanged when cell layers are formed, but are released when needed to allow interaction between cell "layers" in adjacent channels. The interface membranes control release while avoiding the slow transport that occurs when hydrogels are used to stabilize tissue layers.

[0014] In some embodiments, a microfluidic system is provided for simulating cell physiology of a subject. The system may 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 for injecting a first aqueous cell culture medium into the first channel; a second port for injecting a second aqueous cell culture medium into the second channel; a first wall with an opening, the first channel and the second channel share the first wall, and the opening of the first wall is configured to: (i) form a temporary / releasable first water interface film at the opening of the first wall after the first cell aqueous solution is injected;

[0015] (ii) After the first interface membrane is released from the opening, cell communication between the first channel and the second channel is possible.

[0016] The interfacial membrane needs to be released to allow cells in adjacent channels to communicate. In certain embodiments, injection of a second aqueous cell solution causes the first interfacial membrane to be released from the opening.

[0017] The wall with the opening needs to be configured as an interface membrane with sufficient stability to maintain the integrity of the cell layer in the corresponding channel for a long enough time to form a simulated tissue structure. In some embodiments, the interface membrane can be formed by injecting any aqueous solution. In some embodiments, the wall is configured to form a first layer of interface membrane at the opening when the first cell aqueous solution is injected. In some embodiments, the opening includes a hydrophobic material so that a first interface membrane is formed at the opening of the first wall when the first cell aqueous solution is injected. In some embodiments, the opening is outlined by relative wedge edges sharing a plane so that a first interface membrane is formed at the opening when the first cell aqueous solution is injected. In some embodiments, the shape of the opening is selected from a circle and an ellipse. In some embodiments, the opening is outlined by a polygonal structure that provides relative edges on at least two opposite sides of the opening, wherein the relative edges share a plane so that a first interface membrane is established at the opening of the first wall when the first cell aqueous solution is injected. In certain embodiments, the opening of the first wall is a space between adjacent columns along a series of columns; wherein each column in the series of columns has a central axis at least substantially normal to the chip, and each pair of adjacent columns provides a pair of relative edges sharing a plane so that a first interface membrane is established at the opening of the first wall when the first cell aqueous solution is injected.

[0018] In certain embodiments, the system may include three or more different cell types to simulate more complex tissue structures. In these embodiments, the system further includes a third channel; and a third port configured to inject a third cell aqueous solution into the third channel. The system includes a second wall with an opening, the second wall is shared by the second channel and the third channel, and the opening on the second wall is configured to: (i) form a temporary second water interface film at the opening of the second wall after the third cell aqueous solution is injected; (ii) allow cell communication between the second channel and the third channel after the interface film is released from the opening.

[0019] The system can be designed with separate injection ports and channels for providing growth medium to cells and injecting drugs to test the response of the simulated tissue structure to the drug. Therefore, in certain embodiments, the system can further include a growth medium channel having a growth medium port. The interface membrane can include a growth medium.

[0020] Likewise, the system can be designed with multiple growth medium ports, each port being used to supply cell medium to feed cells and select drugs to be tested. Therefore, in some embodiments, the system may further include a first growth medium channel having a first growth medium port and communicating with the first channel, and a second growth medium channel having a second growth medium port and communicating with the second channel.

[0021] These systems are intended to represent the physiological environment of diseased tissue. Thus, in certain embodiments, a method of simulating a disease in a subject is provided. These methods may include obtaining one of the systems taught herein; injecting a first cell aqueous solution into a first channel to introduce a first cell of the disease, injecting a temporary first water interface film formed across the first wall opening; and injecting a second cell aqueous solution into a second channel to introduce a second cell of the disease. In these embodiments, after the second aqueous solution is injected, the first interface film is released; after the first interface film is released, the first cell and the second cell communicate to simulate the disease state. These methods may further include the steps of injecting a drug into the system to determine a suitable drug, determine a suitable dosage, and / or determine the expected activity of the drug; and administering the drug to a subject in need of treatment.

[0022] In some embodiments, the selected system has at least three channels and has at least three different cell types in the simulated tissue structure. Therefore, in some embodiments, the method of simulating a disease includes obtaining a system having at least three channels; injecting a first cell aqueous solution into the first channel to introduce a first cell of the disease, injecting a temporary first water interface film formed across the opening of the first wall; injecting a third cell aqueous solution into the third channel to introduce a third cell of the disease, injecting the temporary second water interface film formed through the opening of the second wall; and, injecting a second cell aqueous solution into the second channel to introduce a second cell of the disease. In these embodiments, the first interface film is released after the second aqueous solution is injected; the second interface film is released after the second aqueous solution is injected; the first cell and the second cell communicate after the first interface film is released to simulate the disease state; the second cell and the third cell communicate after the second interface film is released to simulate the disease state. These methods may further include the steps of injecting drugs into the system to determine the appropriate drug, determine the appropriate dosage and / or determine the appropriate activity; and administering the drug to a subject in need of treatment.

[0023] In certain embodiments, gels may be added between layers to further stabilize the integrity of the layers, particularly between layers of systems where a controlled release interface membrane is not required or in certain embodiments desired. For example, this may be useful in applications where a stable separation between layers is desired. Thus, in certain embodiments, a method of simulating a disease in a subject may include:

[0024] A system with at least three channels is obtained; a first cell aqueous solution is injected into the first channel to introduce a first cell type of the disease, and a temporary first water interface film is formed at the opening of the first wall after injection; a third cell aqueous solution is injected into the third channel to introduce a third cell type of the disease, and a temporary second water interface film is formed at the opening of the second wall during injection; and a second cell aqueous solution is injected into the second channel in the form of a hydrogel to introduce a second cell of the disease. In these embodiments, the first interface film is released after the second aqueous solution is injected; the second interface film is released after the second aqueous solution is injected; the first cell and the second cell communicate after the first interface film is released to simulate the disease state; the second cell and the third cell communicate after the second interface film is released to simulate the disease state. These methods may further include the steps of injecting drugs into the system to determine the appropriate drug, determine the appropriate dose and / or determine the appropriate activity; and administering the drug to a subject in need of treatment.

[0025] Methods of simulating tissue structures and simulating diseases naturally lead to methods of treating diseased tissue structures. Therefore, methods of treating simulated diseases with drugs are provided. These methods may include constructing a disease representation as taught herein; selecting a candidate drug for treating a disease;

[0026] Injection / drug administration in any channel and observation of drug activity. In certain embodiments, drug administration may be in the first channel or the second channel. In certain embodiments, drug administration may be in the first channel, the second channel, or the third channel. These methods may further include the steps of injecting the drug into the system to determine the appropriate drug, determine the appropriate dosage, and / or determine the appropriate activity; and administering the drug to a subject in need of treatment.

[0027] Various types of diseases can be simulated and treated using the systems and methods provided herein. In some embodiments, these methods are used to treat simulated vascular diseases with drugs. In these embodiments, the method can include obtaining a system taught herein, the system having at least three channels for introducing three vascular cell types; injecting a first cell aqueous solution into the first channel to introduce the first cell of the disease to form a wall cell layer, injecting a temporary first water interface membrane at the opening of the first wall; injecting a third cell aqueous solution into the third channel to introduce the third cell of the disease to form an endothelial cell layer, injecting the formed temporary second water interface membrane through the opening of the second wall; and, injecting a second cell aqueous solution into the second channel to introduce the second cell of the disease to form a smooth muscle layer. These methods further include selecting a drug; and applying the drug to the first growth medium channel or the second growth medium channel. In these embodiments, after the second aqueous solution is injected, the first interface membrane is released; after the second aqueous solution is injected, the second interface membrane is released; after the first interface membrane is released, the first cell and the second cell communicate to simulate the disease state; after the second interface membrane is released, the second cell and the third cell communicate to simulate the disease state. These methods may further comprise the steps of: injecting the drug into the system to determine the appropriate drug, determine the appropriate dosage, and / or determine the appropriate activity; and administering the drug to a subject in need of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A diagram of a microfluidic chip according to certain embodiments is shown.

[0029] Figure 2 A vascular tissue structure is shown in a channel formed on a chip according to some embodiments.

[0030] Figure 3 A top view of a device channel is shown, and according to some embodiments, the channel has "non-continuous walls", that is, walls with openings between columnar structures that form part of the channel.

[0031] Figures 4A-4C The difference in contact angle between an interface film and a pillar is illustrated, comparing circular pillars to hexagonal pillars, according to certain embodiments.

[0032] Figure 5 is a graph showing preferred post spacing between hexagonal posts to avoid interfacial film leakage, according to certain embodiments.

[0033] Figure 6 is a map of PAH extracellular remodeling according to certain embodiments.

[0034] Figure 7 A flow chart is shown showing how the systems and methods taught herein may be used to treat a subject in need thereof, according to certain embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0036] The teachings herein are generally directed to systems and methods for creating and using multi-channel microfluidic devices with releasable layers. These devices include improved microfluidic chips that separate tissue types before allowing controlled contact through a release interface membrane, and methods of using these devices to provide disease treatments, which in certain embodiments include personalized medicine approaches.

[0037] The skilled artisan will appreciate that in certain embodiments, the devices can be used to develop treatment plans. The devices can seed and grow multiple cell types in the system to provide improved characterization of physiological systems. In certain embodiments, the systems and methods represent complex pathological processes and can be used to develop treatment plans. The term "interface" refers to exposure to air on one side and to a fluid, such as a cell growth medium, such as an aqueous fluid cell growth medium, on the other side. The terms "subject" and "patient" are used interchangeably and refer to animals, such as mammals, including but not limited to non-primates, such as cows, pigs, horses, cats, dogs, rabbits, rats, and mice;

[0038] and primates, such as monkeys or humans.

[0039] In some embodiments, the development of treatment methods may include identifying a drug with a desired activity, dosage, half-life, and / or tolerability, or a combination thereof, for use in a subject for treatment / These systems and methods can be used, for example, to determine the optimal dosage of a therapeutic drug, the efficacy of a therapeutic drug alone or in combination, the stability of a therapeutic drug, the sensitivity of a patient to a therapeutic drug /

[0040] Tolerability, and determining the formulation of therapeutic drugs. Drug formulations may include the use of nanoparticles, coatings, targeting molecules, drug delivery complexes, and combinations thereof. The skilled person will appreciate that the systems and methods provided herein can be used for precision medicine and personalized medicine to develop better treatments, such as personalized treatments tailored to the subject's age, gender, race, known allergies, and other factors.

[0041] The development of therapeutic approaches can include the use of systems and methods to characterize the communication between cells that occurs in any system in the human body.

[0042] In some embodiments, the human body system can be selected from the circulatory system, cardiovascular system, digestive system, excretory system, endocrine system, exocrine system, skin 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 technicians, and the systems and methods taught herein can be used to create a more realistic physiological environment for diseases that may occur in each of these systems, identifying factors that may affect treatment, such as age, sex, genetic factors, and whether there are other physiological conditions, such as other diseases or disorders present in the subject. In certain embodiments, the systems and methods can also be used to indicate the presence of other therapies, including primary therapies, adjuvant therapies, etc. that may affect treatment. For example, a subject may be receiving chemotherapy while also receiving radiotherapy, and the subject may also have other genetic factors or disease states that can affect the development of treatment.

[0043] An important contribution of the systems and methods taught herein is the ability to construct tissue systems with independent, discrete tissue layers while controlling the interactions between the tissue layers. The interfacial films taught herein can be used to isolate tissue layers from each other before triggering controlled contact of the tissue layers. Any organ system can be represented, and treatment methods for any disease can be pursued, established, and used in subjects, especially when the disease involves multiple cell layers, such as vascular tissue, immune tissue, cancer tissue, etc. Treatment methods for diseases involving angiogenesis, inflammatory response, wound healing, drug response, metastasis of cancer cells between organs, etc. can be determined and administered to subjects in need.

[0044] In some embodiments, systems and methods may be particularly useful for representing vascular disease and developing treatment methods for vascular disease. A treatment method is provided, the method comprising constructing a system for representing vascular disease, using the system to identify a drug for treating vascular disease, and administering the drug to treat vascular disease. Identifying a drug may include identifying the dosage of the drug, the half-life of the drug, the efficacy of the drug, and the subject's tolerance to the drug. The vascular system may be constructed with smooth cells, adjacent cells, and endothelial cells, because each of these cells may be associated with vascular disease. The method may include selecting a cell growth medium suitable for the growth of smooth cells, adjacent cells, and endothelial cells. It will be appreciated by the technician that selecting a cell growth medium may support smooth cells, vascular endothelial cells, and endothelial cells.

[0045] In some embodiments, methods for treating atherosclerosis can be developed. In some embodiments, treatments can be developed for any disease that includes endothelial dysfunction, such as those that may be caused by diabetes or metabolic syndrome, hypertension, smoking, and lack of exercise. Such treatments can promote a healthy endothelium that not only arbitrates endothelium-dependent vasodilation, but also actively inhibits thrombosis, vascular inflammation, and hypertrophy. Therefore, in certain embodiments, the developed treatments can be used to treat thrombosis, vascular inflammation, and hypertrophy.

[0046] In certain embodiments, cancer treatments can be developed using the systems and methods herein. Such treatments can be vascular disease treatments, such as angiogenesis or angiogenesis inhibition, as a mechanism related to cancer and cancer treatment. For example, drugs that inhibit angiogenesis can be used to inhibit or prevent the growth of blood vessels that nourish the growth of cancerous tissue. Similarly, wound healing treatments can also be developed, such as targeted use of drugs that increase angiogenesis to improve blood flow for tissue healing treatments. Treatments for sarcoidosis, sclerosis, and coagulation disorders can all be developed using the systems and methods taught herein.

[0047] In some embodiments, cancer therapy can be used for solid tumors or liquid tumors, such as lymphomas and leukemias. In some embodiments, leukemia therapy is used for acute myeloid leukemia (AML). In some embodiments, the systems and methods taught herein can be used to represent the physiological environment of any cancer, the cancer stage, the metastasis of cancer between tissues, etc., in order to develop cancer treatment methods.

[0048] Releasable interface layer

[0049] The component that can control the separation of tissue layers and the intermixing of tissue layers in the present system is what is referred to herein as a "releasable interface layer" or "interface layer" or "interface membrane". It may also be referred to as a "temporary" interface layer or interface membrane. These terms can be used interchangeably. The interface layer can be a component of one or more walls that separate independent tissue channels. In certain embodiments, the systems and methods use a single interface layer to separate tissue layers or channels. In certain embodiments, the systems and methods use multiple interface layers to separate tissue layers or channels. In certain embodiments, the system can have two interface layers, one on each side of the channel that separates the tissue layers or channels. The walls that separate the independent tissue channels are composed of struts, with an interface layer between each adjacent strut, and a span between the struts.

[0050] The cell medium is used as a carrier for each tissue layer. The cell medium may be referred to as a "cell growth medium", "cell culture medium" or "cell growth medium". It should be understood that the cell growth medium may be a nutrient medium, a culture medium, a minimal culture medium, a selection medium, a differential culture medium, a transport medium or an enrichment medium. In certain embodiments, the liquid culture medium may be referred to as a "broth". Therefore, in certain embodiments, the interface layer may be composed of a growth medium, for example, the growth medium may include an aqueous liquid cell culture growth medium, which may be referred to as an "aqueous liquid cell growth medium" in certain embodiments. Such a culture medium may be used to form a releasable interface layer. An example of a cell growth medium is a rat tail type I collagen solution. In certain embodiments, any cell growth medium having a collagen concentration between 10% and 35% may be used. In certain embodiments, the collagen concentration may be 10%, 15%, 20%, 25%, 30%, 35% or any amount thereof in increments of 1%. In certain embodiments, the collagen concentration of the cell medium is 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or any amount in increments of 0.1%.

[0051] However, in certain embodiments, the systems and methods may also include a semi-solid cell growth medium. In certain embodiments, the semi-solid cell growth medium may be a hydrogel. The skilled person will appreciate that any suitable hydrogel cell growth medium may be used, such as a collagen-based hydrogel cell growth medium.

[0052] A skilled artisan can determine the cell growth medium that is appropriate for a particular tissue application. In certain embodiments, the cell growth medium may be referred to as a "physiological medium," which is a chemically defined medium designed to provide controlled cell culture conditions, e.g., to mimic the composition of blood or other physiological environments to represent physiological conditions. In certain embodiments, the cell growth medium may be basal medium of Eagle (BME), minimal essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, McCoy's 5A modified medium, a cell culture medium for a particular cell type, such as SmGM. TM -2 Smooth Muscle Cell Growth Medium-2 BulletKit TM 、EGM TM -2MV Microvascular Endothelial Cell Growth Medium-2BulletKit TM And COMPLETE FIBROBLAST MEDIUM / WKIT-500ML, etc.

[0053] In some embodiments, the interface layer can provide control because they are controllably releasable. By "controllably releasable," it is meant that the interface membrane is sufficiently stable to remain intact until the aqueous fluid contacts the "air side" of the interface membrane, thereby releasing the membrane containing the cell tissue in the channel, allowing the channel to communicate with the respective cell tissue. In some embodiments, the interface layer can be released when an aqueous solution is introduced into a channel adjacent to the channel forming the film (such as an adjacent channel). On the contrary, in some embodiments, if necessary, the integrity of the channel can be extended by introducing a first cell aqueous solution into the first channel and a second cell aqueous solution in the form of a gel into the second channel, wherein the gel can maintain 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 cell layering combination does not truly represent the actual cell water environment in mammals. It may be necessary to stabilize only this part of the representative cell layering system, while another part of the representative system contains a releasable interface membrane. For example, in some embodiments, the first layer can be an aqueous solution, the adjacent second layer can be a hydrogel, and the third layer can be an aqueous solution. For example, in some embodiments, each layer can have an alternating aqueous cell medium-aqueous gel-aqueous cell medium structure.

[0054] An interfacial film is a technological feature that provides a controllable releasable mechanism as a barrier between cell layers or channels.

[0055] It should be understood that in some embodiments, the tissue structures represented by the systems and methods can have a laminar orientation between the cell layers, and it is desirable to maintain the integrity of the cell layer structure formed during the seeding phase. However, the interface membrane between the cell layers can be released when necessary to achieve representative physiological communication between the cell layers.

[0056] Figure 1 1 shows a diagram of a microfluidic chip according to some embodiments. The microfluidic chip 100 has five channels 1, 2, 3, 4,

[0057] 5. Channels 2, 3, and 4 are used to introduce 3 different cell types to simulate tissue. Channels 1 and 5 are used to add growth media.

[0058] Providing nutrients to the cells to extend their lifespan, the channels also serve as reservoirs for growth media, which are replaced periodically as needed.

[0059] The systems and methods taught herein can use multiple channels to represent any hierarchical tissue structure and develop treatments for any disease process. Figure 2The channel vascular tissue structure formed on the chip according to certain embodiments is shown. The simulated tissue 200 shows channels 1-5. Channel 2 contains endothelial cells to simulate an endothelial cell layer; channel 3 contains smooth muscle cells to simulate a smooth muscle cell layer; channel 4 contains pericytes to simulate a pericyte cell layer. In addition, as mentioned above, channels 1 and 5 can be used to add growth medium to provide nutrients to the cells to extend the life of the cells.

[0060] In some embodiments, channels 1 and 5 can also serve as growth medium reservoirs that are replaced periodically as needed, allowing the systems and methods to operate over longer periods of time. In some embodiments, the time 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 time period or time range in which the increment is 1 minute.

[0061] In some embodiments, channels 1 and 5 can be used as ports to deliver drugs to tissue in liquid aqueous growth media. In some embodiments, such a system can be used to identify drug type, drug dosage, drug stability, and drug tolerance.

[0062] In certain embodiments, channels 1 and / or 5 can be used to apply hydrostatic pressure. For example, such pressure management can be used to simulate changes in blood pressure and the effects of pressure on blood vessels. For example, this helps develop treatments for subjects with different blood pressures. Any suitable pressure mechanism known to the skilled person can be used to increase pressure, such as a peristaltic pump.

[0063] Figure 3 A top view of a device channel is shown, and according to certain embodiments, the channel has a "non-continuous wall", that is, a wall with openings between the columnar structures that form part of the channel. Channel 300 includes a wall 305 with an opening 310, which is shared with an adjacent channel (not shown), so that a first cell 314 in a first cell aqueous solution 315 can communicate with a second cell (not shown) in a second cell aqueous solution (not shown) to simulate the communication between the first cell and the second cell in the simulated tissue structure. However, before communication, a simulated tissue structure needs to be created, so the integrity of each cell layer needs to be maintained. Through the water surface tension generated by the contact between the first cell aqueous solution 315 and the column 320, a temporary water interface film 325 is formed on the opening 310, which helps to maintain the integrity of each cell layer during the formation of the tissue structure. The process of forming a cell layer can be called "seeding" 330, which can be done manually or automatically by a pump in the system.

[0064] The microfluidic devices taught herein are uniquely designed to provide temporary, independent water interface membranes between channels. In fact, the interface membrane is an integral part of the channel and plays an initial role in maintaining the integrity of the channel during the formation of simulated tissue structures. The temporary interface membrane allows the temporary formation of a channel to accept the first cell type contained in the interface membrane, while another channel that also has the interface membrane as a component accepts the second cell type. After a period of time, the interface membrane is released, and cells can communicate between adjacent channels that were once separated by the interface membrane. In certain embodiments, these temporary interface membranes may be referred to as "sacrificial walls", which first fix the cell layer in place and then allow cells to communicate after the interface membrane is released. The separation formed by the sacrificial wall represents the cell arrangement present in the body, and is finally released when needed to allow cells to communicate.

[0065] In certain embodiments, the pillars can have any desired configuration that affects the interface film. In order to form the desired interface film between the pillars and accommodate the cell layer formed in the channel, the pillars can be configured with opposite edges, preferably in the same plane. For example, Figure 3 As shown, the pillars can be hexagonal. As mentioned previously, the edges of the hexagonal pillars should be point-to-point and preferably in the same plane. The aqueous interfacial film forms a temporary wall to maintain the integrity of the cell layer during channel seeding, and then the interfacial layer is released to allow cells to communicate.

[0066] In some cases, hydrophilic materials are not used to form columns because the hydrophilic materials may not form a stable interface film with the cell culture medium.

[0067] One technique is that in some cases, the cross-section of the column can be circular, elliptical, polygonal, or a combination thereof. In some instances, the polygonal shape is a 3-sided, 4-sided, 5-sided, 6-sided, 7-sided, 8-sided, 9-sided, 10-sided, 11-sided, or 12-sided. In some cases, the column is a cylinder. In some cases, the column is an elliptical cylinder. In some instances, the column is a polygonal column, such as Figure 3 The hexagonal columns in .

[0068] The opening 310 is determined by the distance between the pillars, which is the "bridging distance" of the interfacial film of the systems and methods taught herein. The distance between the pillars may also be referred to as the "inter-pillar distance", which is a device design feature that should be carefully configured to be between 30uM and 250uM. The lower limit of 30uM is limited by the size of the cell tissue, while the upper limit of 250uM is limited by the stability of the interfacial film.

[0069] In some embodiments, the distance between the columns can range from 30uM to 200uM, 30uM to 100uM, or any range in which the increment is 1uM. In some embodiments, the distance between the columns can be about 25uM, 30uM, 35uM, 40uM, 45uM, 50uM, 55uM, 60uM, 65uM, 70uM, 75uM, 80uM, 85uM, 90uM, 100uM, 105uM, 110uM, 115uM, 120uM, 125uM, 130uM, 135uM, 140uM, 145uM, 150uM, 15 5uM, 160uM, 155uM, 155uM, 155uM, 155uM, 155uM, 155uM, 155uM, 155uM, 160uM, 165uM, 170uM, 1 75uM, 180uM, 185uM, 190uM, 195uM, 200uM, 205uM, 210uM, 200uM, 215uM, 220uM, 225uM, 230uM,

[0070] 235uM, 240uM, 245uM, 250uM, 255uM, 260uM or any distance or distance range in 1uM increments.

[0071] The distance between the columns depends on the ability of the columns to form an interfacial film with the cell culture medium used in the systems and methods taught herein.

[0072] Since the column height is also limited by similar surface tension, the column height also forms the bridging distance of the interfacial film, so the column height can also be about 25uM, 30uM, 35uM, 40uM, 45uM, 50uM, 55uM, 60uM, 65uM, 70uM, 75uM, 80uM, 85uM, 90uM, 100uM, 105uM, 110uM, 115uM, 120uM, 125uM, 116uM, 117uM, 118uM, 119uM, 120uM, 125uM 140uM, 145uM, 150uM, 155uM, 160uM, 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 in 1uM increments therein. Like the inter-pillar distance, the pill height is also a device design feature and should be carefully configured to be between 30uM and 250uM. In certain embodiments, the column height can be from 30 uM to 200 uM, 30 uM to 100 uM, or any range in 1 uM increments therein.

[0073] In some embodiments, the multichannel microfluidic device can have 3 channels. In some embodiments, the multichannel microfluidic device can have 2, 3, 4, 5, 6, 7, 8, 9, 10 or more channels. In some embodiments, the device preselected by the user is designed to have 2, 3, 4, 5, 6 or more channels. In many embodiments, the multichannel device allows each cell to grow in its own channel in multiple channels, each channel communicates with at least one adjacent channel in multiple channels, and each cell type remains in its own channel during the seeding phase, but communicates through cell signaling mechanisms, for example, paracrine signals (not involving cell contact) and paracrine signals (ligand-receptor binding involving cell contact) can be included. This arrangement can select and design a variety of configurations for simulating cell communication in pathological processes.

[0074] Therefore, in some embodiments, a microfluidic system is provided for simulating the cell physiology of a subject. The system may 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 for injecting a first cell aqueous solution into the first channel; a second port for injecting a second cell aqueous solution into the second channel; a first wall with an opening, the first channel and the second channel share the first wall, and the opening of the first wall is configured to: (i) form a temporary first water interface film at the opening of the first wall after the first cell aqueous solution is injected; (ii) after the first interface film is released from the opening, cell communication occurs between the first channel and the second channel.

[0075] The interfacial membrane needs to be released to allow communication between cells in adjacent channels. In certain embodiments, injection of a second aqueous cell solution causes the first interfacial membrane to be released from the opening.

[0076] The wall with the opening needs to be configured as an interface membrane with sufficient stability to maintain the integrity of the cell layer in each channel for a long enough time to form a simulated tissue structure. In some embodiments, the interface membrane can be formed by injecting any aqueous solution. In some embodiments, the wall is configured to form a first layer of interface membrane at the opening when the first cell aqueous solution is injected. In some embodiments, the opening includes a hydrophobic material so that a first interface membrane is formed at the opening of the first wall when the first cell aqueous solution is injected. In some embodiments, the opening is outlined by relative wedge edges sharing a plane so that a first interface membrane is formed at the opening when the first cell aqueous solution is injected. In some embodiments, the shape of the opening is selected from a circle and an ellipse. In some embodiments, the opening is outlined by a polygonal structure that provides relative edges on at least two opposite sides of the opening, wherein the relative edges share a plane so that a first interface membrane is established at the opening of the first wall when the first cell aqueous solution is injected. In certain embodiments, the opening of the first wall is a space between adjacent columns along a series of columns; wherein each column in the series of columns has a central axis at least substantially normal to the chip, and each pair of adjacent columns provides a pair of relative edges sharing a plane so that a first interface membrane is established at the opening of the first wall when the first cell aqueous solution is injected.

[0077] In certain embodiments, the system can include three or more different cell types to simulate more complex tissue structures. In these embodiments, the system further includes a third channel; and a third port configured to inject a third cell aqueous solution into the third channel. The system includes a second wall with an opening, the second wall is shared by the second channel and the third channel, and the opening on the second wall is configured to: (i) form a temporary second layer of water interface film at the opening of the second wall after the third cell aqueous solution is injected; (ii) after the interface film is released from the opening, cell communication is allowed to occur between the second channel and the third channel. In certain embodiments, the multichannel microfluidic device can be used to study cell interactions in blood vessels. For example, the device can be used to study the interactions between the three main arterial cells (endothelial cells, smooth muscle cells, and vascular adventitial cells).

[0078] The teachings herein show, for example, that we can simulate pulmonary arterial hypertension (PAH) to develop treatments for PAH. In some embodiments, the systems and methods taught herein can simulate PAH-induced right ventricular hypertrophy to develop improved treatments. The skilled person should understand that these results demonstrate that we can also simulate the communication between cells that lead to pulmonary embolism and the environment of the embolism itself. Moreover, as with the modeling of any human system, we can study the effects of any of several factors that may affect the efficacy of the treatment. For example, the systems and methods taught herein can study the relative effects of factors such as whether the subject is male or female, whether he is a teenager or an adult, whether he suffers from other diseases or physiological disorders, whether he suffers from a genetic disease or has a genetic predisposition. Any factor known to the skilled person can be simulated using the systems and methods taught herein.

[0079] Using the provided system and method, the technician can simulate any of a variety of cell tissue interactions, where vascular tissue is used as a model. In certain embodiments, cell interactions occur between vascular tissue layers. In certain embodiments, cell interactions occur between vascular cell types. In certain embodiments, cell interactions occur between vascular tissue layers. In certain embodiments, cell interactions occur between vascular cells and vascular cell extracellular matrix.

[0080] The formulation of the treatment method may include the selection of drugs, the dosage, the tolerance of the drugs, the stability of the drugs, etc. Therefore, the system can be designed with independent injection ports and channels for providing growth medium and drug administration to cells to test the response of simulated tissue structures to drugs. Moreover, since the cell culture medium may need to be supplemented, in some embodiments, the system can further include a growth medium channel with a growth medium port.

[0081] The term "drug" may be used interchangeably with "active agent", "biologically active agent", etc. Biologically active agents include, but are not limited to, small molecules, nucleotides, oligonucleotides, polynucleotides, amino acids, oligopeptides, polypeptides, and proteins. Biologically active agents may include, but are not limited to, antiproliferative agents, antitumor agents, anti-inhibitors, anti-inflammatory agents, antiplatelets, anticoagulants, antifibrinogens, antithrombins, antibiotics, antiallergic agents, antioxidants, and any of their original drugs, co-drugs, metabolites, analogs, homologues, congeners, derivatives, salts, and combinations thereof. Those skilled in the art will recognize that in certain embodiments of the present invention, certain groups, subgroups, and individual biologically active agents may not be used.

[0082] Antiproliferative agents include, for example, actinomycin D, actinomycin IV, actinomycin I1, actinomycin X1, actinomycin C1, and dactinomycin (Cosmegen.RTM., Merck & Co., Inc.). Antineoplastic agents or anti-inhibitors include paclitaxel (TAXOL, Bristol-Myers Squibb), docetaxel (TAXOTERE, Aventis SA), methotrexate, irinotecan, SN-38, azathioprine, vincristine, vinblastine, fluorouracil, doxorubicin hydrochloride (ADRIAMYCIN, Pfizer, Inc.), and mitomycin (MUTAMYCIN, Bristol-Myers Squibb), and any original drug, co-drug, metabolite, analog, homologue, congener, derivative, salt, and combination thereof. Antiplatelet, anticoagulant, antifibrin and antithrombin include heparin sodium, low molecular weight heparin, heparins, hirudin, argatroban, forskolin, valprost, 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 original drug, co-drug, metabolite, analog, homologue, congener, derivative, salt and combination thereof. Cytostatic or antiproliferative agents include, for example, angiostatins, angiotensin converting enzyme inhibitors such as captopril (CAPOTEN and CAPOZIDE, Bristol-Myers Squibb), cilazapril or lisinpril (PRINVIL ​​and PRINZIDE, Merck); calcium channel blockers such as nifedipine; colchicine; fibroblast growth factor (FGF) antagonists, fish oil (omega 3 fatty acids); histamine antagonists; lovastatin (MEVACOR, Merck); monoclonal antibodies, including but not limited to platelet-derived growth factor (PDGF) receptor-specific antibodies; sodium nitroprusside; phosphodiesterase inhibitors; prostaglandin inhibitors; suramin; serotonin blockers; steroids; thioproteinase inhibitors; PDGF antagonists, including but not limited to triazolopyrimidines; nitric oxide, and any parent drug, co-drug, metabolite, analog, homolog, congener, derivative, salt and combination thereof. Antiallergic drugs include, but are not limited to, pemirolast potassium (ALAMAST, Santen Co., Ltd.) and any original drug, homologue, metabolite, analog, homologue, congener, derivative, salt and combination thereof.

[0083] It should be understood from the teachings herein that methods for treating PAH can be provided using the systems and methods taught herein. In some embodiments, drugs used to treat PAH may include, but are not limited to, epoprostenol, riociguat, bosentan, macitentan, ambrisentan, treprostinil, sildenafil, tadalafil, selexipag, and iloprost, and combinations thereof. In certain embodiments, these drugs may be used alone or in combination. In certain embodiments, the drugs may be administered as shown in Table 1:

[0084] Table 1.

[0085]

[0086] Among the above drugs, we found that female PAH patients were more responsive to epoprostenol, treprostinil, selexipag, ambrisentan, bosentan, and macitentan. Therefore, in some embodiments, methods for treating female PAH patients are provided. These methods may include, for example,

[0087] Introducing vascular endothelial cell tissue from a subject with PAH into a first channel of the microfluidic device taught herein, the introducing comprising seeding the vascular endothelial cell tissue into an aqueous cell growth medium to form a simulated vascular endothelial cell layer, and injecting the simulated vascular endothelial cell layer into the first channel;

[0088] Introducing the subject's vascular endothelial cell tissue into the third channel of the microfluidic device of the present invention, the third channel being separated from the first channel by the second channel, the introducing comprising seeding the vascular endothelial cell tissue into a water cell growth medium to form a simulated vascular endothelial cell layer, and injecting the simulated vascular endothelial cell layer into the third channel;

[0089] Introducing an aqueous cell growth medium into the second channel to release (i) an interface membrane separating the first channel from the second channel and (ii) an interface membrane separating the third channel from the second channel, wherein the release of the interface membrane allows communication between the simulated vascular endothelial cell tissue and the simulated vascular endothelial cell tissue;

[0090] determining the best candidate drug by administering to the subject 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, to measure the therapeutic effect, including activity, efficacy, dose response, stability, toxicity and / or combinations thereof, thereby determining the best candidate drug or formulation for the subject; and

[0091] If a therapeutic effect is desired, the optimal drug or formulation is administered to the subject.

[0092] In some embodiments, the method includes using a microfluidic device having at least 5 channels as taught herein, wherein simulated vascular endothelial tissue is injected into a second channel, simulated vascular adventitial tissue is injected into a fourth channel, and cell growth medium is injected into a first channel and a fifth channel, wherein the cell growth medium may include one or more candidate drugs to determine the best candidate drug or formulation. These methods may include forming a releasable interface membrane between the first channel and the second channel when the simulated endothelial vascular tissue is introduced into the second channel; forming a releasable interface membrane between the fourth channel and the fifth channel when the simulated endothelial vascular tissue is introduced into the fourth channel; and releasing the interface membrane when the cell growth medium is injected into the first channel and the fifth channel, so that the first channel and the second channel and the fourth channel and the fifth channel can communicate. In certain embodiments, the third channel is filled with a simulated vascular smooth muscle tissue layer, which may be a hydrogel cell growth medium.

[0093] We also found that male PAH patients responded more strongly to sildenafil and tadalafil. In some embodiments, methods of treating male PAH patients are provided. These methods may include, for example,

[0094] Introducing vascular endothelial cell tissue from a subject with PAH into a first channel of the microfluidic device taught herein, the introducing comprising seeding the vascular endothelial cell tissue into an aqueous cell growth medium to form a simulated vascular endothelial cell layer, and injecting the simulated vascular endothelial cell layer into the first channel;

[0095] Introducing the subject's vascular endothelial cell tissue into the third channel of the microfluidic device of the present invention, the third channel being separated from the first channel by the second channel, the introducing comprising seeding the vascular endothelial cell tissue into a water cell growth medium to form a simulated vascular endothelial cell layer, and injecting the simulated vascular endothelial cell layer into the third channel;

[0096] Introducing an aqueous cell growth medium into the second channel to release (i) an interface membrane separating the first channel from the second channel and (ii) an interface membrane separating the third channel from the second channel, wherein the release of the interface membrane allows communication between the simulated vascular endothelial cell tissue and the simulated vascular endothelial cell tissue;

[0097] determining the best candidate drug by administering to the subject 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, to measure the therapeutic effect, including activity, efficacy, dose response, stability, toxicity and / or combinations thereof, thereby determining the best candidate drug or formulation for the subject; and

[0098] If a therapeutic effect is desired, the optimal drug or formulation is administered to the subject.

[0099] In some embodiments, the method includes using a microfluidic device having at least 5 channels as taught herein, wherein simulated vascular endothelial tissue is injected into a second channel, simulated vascular adventitial tissue is injected into a fourth channel, and cell growth medium is injected into a first channel and a fifth channel, wherein the cell growth medium may include one or more candidate drugs to determine the best candidate drug or formulation. These methods may include forming a releasable interface membrane between the first channel and the second channel when the simulated endothelial vascular tissue is introduced into the second channel; forming a releasable interface membrane between the fourth channel and the fifth channel when the simulated endothelial vascular tissue is introduced into the fourth channel; and releasing the interface membrane when the cell growth medium is injected into the first channel and the fifth channel, so that the first channel and the second channel and the fourth channel and the fifth channel can communicate. In certain embodiments, the third channel is filled with a simulated vascular smooth muscle tissue layer, which may be a hydrogel cell growth medium.

[0100] The systems and methods provided herein can be well suited for developing antibody therapies for subjects in need. Antibody therapies provide additional bioactive agents that may be useful when used in conjunction with the methods taught herein. For example, AVASTATIN is a human monoclonal antibody directed against vascular endothelial growth factor that, when used in combination with the standard Saltz system (irinotecan, 5-fluorouracil, and streptomycin A), has produced beneficial results for colorectal cancer, extending survival by more than 30%. The skilled artisan will appreciate that a variety of monoclonal antibodies are useful, and further examples of cancers that can be treated using the systems and methods taught herein and any bioactive agents taught herein are provided below, as well as some monoclonal antibody therapies that can also be tested alone or in combination with any bioactive agents taught herein:

[0101] Table 2.

[0102]

[0103] * refers to conjugated monoclonal antibodies

[0104] In general, cancers that can be treated using the systems and methods taught herein include any known cancer. In addition to the cancers listed above,

[0105] The following cancers are also of concern: lung cancer, non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, small cell lung cancer, bronchogenic carcinoma, colon cancer, rectal cancer, colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia, acute myeloid leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, liver cancer, intrahepatic bile duct cancer, ovarian cancer, and esophageal cancer.

[0106] It should be understood that biologically active agents can be used alone or in combination with other biologically active agents and using the compositions and methods taught herein. For example, chemotherapy drugs are sometimes most effective when administered in combination, which is a combination chemotherapy regimen. The principle of combination chemotherapy is to use drugs with different mechanisms of action to reduce the possibility of developing drug-resistant cancer cells. When drugs with different actions are used in combination, each drug can be used at an optimal dose, sometimes without or sometimes with reduced intolerable side effects.

[0107] For example, the active agent may be contained in a pharmaceutically acceptable carrier, conjugated to a drug delivery system, and used in an amount sufficient to exert a therapeutic effect without adverse side effects in the patient being treated. Therapeutically effective concentrations may be determined by testing the compound in the in vitro and in vivo systems described, and then empirically extrapolating the dosage for humans.

[0108] In certain embodiments, a therapeutically effective dose should produce a serum active ingredient concentration of about 0.1 ng / ml to about 50-100 μg / ml.

[0109] In another embodiment, the dosage of the pharmaceutical composition should be about 0.001 mg to about 2000 mg of the compound per kg of body weight per day. Similarly, a pharmaceutical dosage unit form is prepared, each dosage unit form provides about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or 2000 mg, and in one embodiment provides about 10 mg to about 500 mg of the active ingredient or essential ingredient combination in a dosage unit form.

[0110] In some embodiments, the concentration range of a therapeutically or prophylactically effective amount of the composition may be from about 0.001 nM to about 0.10 M; from about 0.001 nM to about 0.5 M; from about 0.01 nM to about 150 nM; from about 0.01 nM to about 500 μM; from about 0.01 nM to about 1000 nM, 0.001 μM to about 0.10 M; from about 0.001 μM to about 0.5 M; from about 0.01 μM to about 150 μM; from about 0.01 μM to about 500 μM; from about 0.01 μM to about 1000 nM, or any range in which the increment is 0.1 μM. In certain embodiments, the composition may be administered in an amount of about 0.001 mg / kg to about 500 mg / kg; about 0.005 mg / kg to about 400 mg / kg; about 0.01 mg / kg to about 300 mg / kg; about 0.01 mg / kg to about 250 mg / kg; about 0.1 mg / kg to about 200 mg / kg; about 0.2 mg / kg to about 150 mg / kg; about 0.4 mg / kg to about 120 mg / kg; about 0.15 mg / kg to about 100 mg / kg, about 0.15 mg / kg to about 50 mg / kg, about 0.5 mg / kg to about 10 mg / kg, or any range in increments of 0.1 mg / kg.

[0111] Typically, the compositions taught herein can be administered by subcutaneous injection, intramuscular injection, intraperitoneal injection or intravenous injection. In certain embodiments, local administration may include injecting the agent directly into the tissue area to be treated, such as a solid tumor. In certain embodiments, intravenous administration is adopted, and intravenous injection can be continued for a few minutes to an hour or longer (such as about fifteen minutes). The dosage may vary greatly due to the type of preparation, the size of the unit dose, the type of excipients, and other factors well known to those of ordinary skill in the art. For example, the preparation may include about 0.0001% to about 10% (weight / weight), about 0.01% to about 1%, about 0.1% to about 0.8%, or any range therein, and the remainder includes excipients.

[0112] In certain embodiments, the composition can be administered with at least one other drug for treating a disease, particularly another drug capable of treating cancer, such as a chemotherapeutic drug. The required dosage of the drug can be reduced, even substantially reduced, so that the required dosage of one or more agents is reduced to the extent that a significant response can be observed from the subject. A significant response may include, but is not limited to, a reduction or elimination of nausea, a significant increase in tolerance, a faster response to treatment, a more selective response to treatment, or a combination thereof.

[0113] The method of the present invention may further include administering an effective amount of an antiproliferative agent, an effective amount of radiotherapy, surgical treatment, or a combination thereof. The present invention also relates to a method for treating cancer. In some embodiments, the method includes administering a medicament to a subject in need of cancer treatment, wherein the dose of the medicament is selected to reduce or eliminate immunosuppression, otherwise immunosuppression occurs when a large dose of the medicament is administered to the subject; and administering radiotherapy in combination with the medicament, wherein the reduction or elimination of immunosuppression enhances the efficacy of radiotherapy compared to the efficacy of radiotherapy observed when a large dose of the medicament is administered to the subject. In certain embodiments, the preparation includes one or more chemotherapeutic preparations used in combination with the preparations provided herein. In these embodiments, the medicament may be selected from dacarbazine, paclitaxel, doxorubicin, or a combination thereof.

[0114] In certain embodiments, for a human of average weight, an effective amount can range, 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 increments of 1 mg / day.

[0115] In certain embodiments, the average body weight of a human body can be estimated as 60 kg, 65 kg, 70 kg, 75 kg, 80 kg, or any mass in 1 kg increments. For the treatment of solid tumors, similar amounts will have therapeutic effects. One of ordinary skill in the art, taking into account the technology and the present disclosure, can determine the therapeutically effective amount of the composition of the present invention for a particular disease without undue experimentation.

[0116] In certain embodiments, G-CSF is administered in conjunction with the compositions taught herein, using any effective administration amount, time, and method known to the skilled person. G-CSF can be NEUPOGEN, for example, in an amount 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 in increments of 1 μg / kg.

[0117] In some embodiments, radiation therapy can be administered as a single, localized high dose, for example, from about 20 Gy to about 100 Gy. In some embodiments, the total dose of radiation therapy can be from about 20 Gy to about 100 Gy, using a modified hypofractionated dosing regimen, including about 2 to about 5 administrations over a period of one week. In certain embodiments, the total dose of radiation therapy can be from about 20 Gy to about 100 Gy, using a modified hypofractionated dosing regimen, including 2 to 3 doses injected over a period of about 2 to about 3 days. Radiation therapy can also be performed using a modified hypofractionated dosing regimen, with a total dose ranging from about 45 Gy to about 60 Gy, including single doses of about 15 Gy to about 20 Gy administered daily over a period of 3 days.

[0118] The compositions and therapies described herein can be administered in combination. For example, the compositions can be administered for 30 minutes, 1 hour, 2 hours,

[0119] 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 amount of time deemed necessary by a skilled person.

[0120] These preparations can be administered to the subject simultaneously, continuously or periodically. Cyclic therapy involves administering a first preparation for a predetermined period of time, administering a second preparation or therapy for a second predetermined period of time, and repeating this cycle to achieve any desired purpose, for example, to improve the efficacy of the treatment. The agents can also be administered simultaneously. The term "simultaneously" is not limited to administering the agents at exactly the same time, but means that the agents can be administered in a certain order and time interval so that the agents work together to provide additional benefits. Each preparation can be used alone or together in any appropriate form using any appropriate method of administering the preparations.

[0121] Likewise, the system can be designed with multiple growth medium ports, each port being used to supply cell medium to culture cells and select drugs to be tested. Thus, in certain embodiments, the system can further include a first growth medium channel having a first growth medium port and communicating with the first channel, and a second growth medium channel having a second growth medium port and communicating with the second channel.

[0122] When developing treatment methods, the system can be designed to simulate diseased tissue or disease processes. Therefore, in certain embodiments, a method of simulating a disease in a subject is provided. These methods may include obtaining one of the systems taught herein; injecting a first cell aqueous solution into a first channel to introduce a first cell of the disease, injecting to form a temporary first water interface film across the first wall opening; and injecting a second cell aqueous solution into a second channel to introduce a second cell of the disease. In these embodiments, after the second aqueous solution is injected, the first interface film is released; after the first interface film is released, the first cell and the second cell communicate to simulate the disease state.

[0123] In some embodiments, the selected system has at least three channels, with at least three different cell types in the simulated tissue structure. Therefore, in some embodiments, the method of simulating a disease includes obtaining a microfluidic system taught herein, the system having at least three channels; injecting a first cell aqueous solution into the first channel to introduce a first cell of the disease, injecting a temporary first water interface film across the opening of the first wall; injecting a third cell aqueous solution into the third channel to introduce a third cell of the disease, injecting the formed temporary second water interface film across the opening of the second wall; and, injecting a second cell aqueous solution into the second channel to introduce a second cell of the disease. In these embodiments, the first interface film is released after the second aqueous solution is injected; the second interface film is released after the second aqueous solution is injected; the first cell and the second cell communicate after the first interface film is released to simulate the disease state; the second cell and the third cell communicate after the second interface film is released to simulate the disease state.

[0124] In some embodiments, a hydrogel cell growth medium may be used. The gel may be used in alternating channels to further stabilize the integrity of at least three channels and slow down the rate of tissue interaction. Therefore, in some embodiments, a method of simulating a disease in a subject may include: obtaining a system having at least three channels; injecting a first cell aqueous solution into the first channel to introduce a first cell of the disease, the injection forming a temporary first layer of water interface film across the opening of the first wall; injecting a third cell aqueous solution into the third channel to introduce a third cell of the disease, and forming a temporary second water interface film at the opening of the second wall during injection; and injecting a second cell aqueous solution into the second channel in the form of a hydrogel to introduce a second cell of the disease. The first interface film may be released after the second aqueous solution is injected; the second interface film may be released after the second aqueous solution is injected; the first cell and the second cell communicate after the first interface film is released to simulate the disease state; the second cell and the third cell communicate after the second interface film is released to simulate the disease state.

[0125] Methods of simulating tissue structures and simulating diseases naturally lead to methods of treating diseased tissue structures. Thus, methods of treating simulated diseases with drugs are provided. In some embodiments, these methods may include performing the methods of simulating diseases taught herein;

[0126] Select a drug for treating a simulated disease; and administer the drug in any channel. In some embodiments, the drug may be administered in any channel. In certain embodiments, the drug may be administered in the first channel or the second channel. In certain embodiments, the drug may be administered in the first channel, the second channel, or the third channel. In certain embodiments, the drug may be administered in the first channel, the second channel, the third channel, the fourth channel, or the fifth channel. In certain embodiments, the drug may be administered in the first channel or the third channel. In certain embodiments, the drug may be administered in the first channel or the fifth channel.

[0127] Various types of diseases can be simulated and treated using the systems and methods provided herein. In some embodiments, these methods are used to treat simulated vascular diseases with drugs. In these embodiments, the method can include obtaining a system taught herein, the system having at least three channels for introducing three vascular cell types; injecting a first cell aqueous solution into the first channel to introduce the first cell of the disease to form a wall cell layer, injecting a temporary, first water interface membrane across the first wall opening; injecting a third cell aqueous solution into the third channel to introduce the third cell of the disease to form an endothelial cell layer, injecting the formed temporary second water interface membrane through the opening of the second wall; and, injecting a second cell aqueous solution into the second channel to introduce the second cell of the disease to form a smooth muscle layer. These methods further include selecting a drug; and, applying the drug to the first growth medium channel or the second growth medium channel. In these embodiments, the first interface membrane is released after the second aqueous solution is injected; the second interface membrane is released after the second aqueous solution is injected; the first cell and the second cell communicate after the first interface membrane is released to simulate the disease state; the second cell and the third cell communicate after the second interface membrane is released to simulate the disease state.

[0128] Example 1. Columnar

[0129] The integrity and stability of the interfacial film include surface tension and contact angle. The surface tension is provided by the composition of the pillars and the composition of the aqueous material (eg, cell growth medium) used to form the film.

[0130] Figures 4A-4C The difference in contact angle between circular and hexagonal pillars is illustrated according to certain embodiments. Figures 4A-4C The difference in contact angle between the interfacial film and the pillars is described, comparing circular pillars and hexagonal pillars according to certain embodiments. The pillars are cast from polydimethylsiloxane (PDMS) and treated with ambient plasma at medium RF power for 2 minutes (PDC-001-HP series, Harrickplasma, New York), and a glass cover slip is added to the device. The device is then stored in an oven at 80°C for 2 hours to enhance adhesion. The chip is then treated with UV light (UV light box benchtop cleanroom, Air science Inc.) for 45 minutes to sterilize before the cell seeding step. A poly-D-lysine (PDL) solution (2 mg / mL) is then coated on the pillars to enhance adhesion to the pillars. The chips can also be made from a variety of thermoplastic polymers, such as 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 made with these polymers can be attached to a cover slip made of the same polymer.

[0131] The cell medium was then introduced into the channel, and the contact angle was measured. Figure 4A The contact angle between the interfacial film and the hexagonal pillars is shown. Figure 4B Shows the contact angle between the interfacial film and the circular pillars. Figure 4C An overlay of hexagonal columns and circular columns is shown to illustrate the difference in contact angles. Figures 4A-4C It is shown that there is a difference in the pressure ΔPw across the channel and the pressure ΔP between the pillars. Circular, hexagonal and trapezoidal pillars were compared.

[0132] Example 2. Distance between columns

[0133] Surface tension and contact angle can be optimized to enhance interfacial film integrity and stability, and the distance the film is stretched is also a factor to consider.

[0134] Figure 5 is a graph showing that according to certain embodiments, there is a preferred inter-pillar distance between hexagonal pillars to avoid leakage of the interface film. It can be seen that when using hexagonal pillars, the leakage threshold exceeds 150 μm, which means that the span of the pillars can reach at least 150 μm and maintain a stable interface film without leakage.

[0135] like Figure 5 As shown by the leakage threshold cross line in , leakage begins to occur when the inter-pillar distance approaches 200μm. All pillar shapes, whether round, hexagonal or trapezoidal, do not leak when the inter-pillar distance is less than 100μm.

[0136] Example 3. Creation of a 5-channel multichannel microfluidic device

[0137] For example, a microfluidic chip with five main channels and microcolumns can be prepared, and its three-dimensional (3D) environment is as follows:

[0138] To fabricate the device, we created a network of five channels, each 1 mm wide and 3 cm long. AutoCAD was used for this process. The channels were separated by 150 μm tall trapezoidal pillars that were 200 μm apart throughout the length of the channel.

[0139] Based on the CAD file, we created a photomask, printed the mask with a high-resolution printer (CAD Art Services Inc., OR), and then placed the mask on a silicon wafer, spin-coated (spin coater WS-650 series, Laurel Technology, PA) with SU-8 photoresist polymer (MicroChem Inc., MA), and baked 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, France), baked again at 65 °C for 5 min and 95 °C for 12 min, cooled to room temperature, and cleaned with SU-8 solvent to obtain a silicon master mold. To fabricate devices using the master mold, we mixed equal amounts of polydimethylsiloxane (PDMS, Dow, MI) and curing solution (Dow, MI) in a ratio of 10:1 and poured them on the mold. Individual devices (35 mm in diameter and 3–4 mm in height) were then degassed, baked, and cut out, and finally used (Miltex, Pennsylvania) to make the inlet and outlet.

[0140] Fabrication of Microfluidic Chip: Micro-pillar chips based on polydimethylsiloxane (PDMS) can be used for laboratory scale experiments. However, cyclic olefin copolymer (COC) and injection molding can be used to produce large numbers of chips for large scale use. Pillars are used to create walls with openings. In this example, round features are not used on the pillars because it is desirable to have opposing edges to obtain surface tension to maintain the interface film. This is an example that does not use round or spherical "pillars".

[0141] Hands-on chip: Get a pumping system that allows for automated pumping rather than using a manual pipette. For example, see https: / / www.sciencedirect.com / science / article / pii / S2468067220300249?via% 3Dihub (downloaded on July 27, 2021; teaches a peristaltic pump suitable for this application); see also, e.g. https: / / link.springer.com / article / 10.1140 / epje / s10189-020-00002-9 (Downloaded on July 27, 2021; teaches a constant pressure pump suitable for this application).

[0142] Artificial inoculation, growth and evaluation

[0143] Pathological conditions can be established by artificial seeding of cells, artificial growth processes of artificially placed cells, and establishment and release of interfacial layers to enable cell interactions.

[0144] Equipment Automation

[0145] We can automate the cell seeding and growth process in a sterile environment. This automated equipment will be easier to use and could be used at the patient's bedside, for example, to assess treatment response, investigate the effects of sex and age on treatment response, and any of a host of other factors known to technicians to develop a personalized treatment approach.

[0146] Example 4. Application research on pulmonary hypertension

[0147] Pulmonary arterial hypertension (PAH) is a rare disease in which the pulmonary arteries / arterial vessels become stiff and blocked. The heart has to work harder to pump blood through the blocked arteries to the lungs. As a result, the heart enlarges and the patient can die from right heart failure. The primary clinical manifestation of the disease is an increase in mean pulmonary artery pressure (mPAP), which is caused by a series of active structural changes in the pulmonary arteries (called arterial remodeling and muscularization). For example, the leading cause of death in patients with pulmonary arterial PAH is right ventricular hypertrophy (PAH-RVH). The various cellular and biomolecular processes that occur in PAH-RVH are poorly understood, and the systems and methods described herein may allow for better treatment of PAH-RVH.

[0148] This lesion occurs due to abnormal proliferation, migration, and misplaced growth of pulmonary arterial cells (PACs), development of apoptosis-resistant endothelial cells (ECs), enhanced deposition of extracellular matrix (ECMs), thickening of the smooth muscle layer, and acquisition of a phenotype resembling smooth muscle cells (SMCs) by ECs. As the cell growth becomes chaotic and the condition becomes severe, some pulmonary arteries develop glomerular-like luminal obliterations, termed plexiform lesions. These lesions are described as plexuses of slit-like channels composed of endothelial cells separated by highly pigmented and oval core cells of indeterminate phenotype.

[0149] Effect of gender on PAH

[0150] One of the perplexing aspects of PAH is that more women are affected than men, yet women with PAH tend to live longer than men. This discrepancy in PAH incidence and survival is thought to result from intrinsic differences between the sexes and from the conflicting effects of sex hormones, especially estrogen, which appear to have both beneficial and detrimental effects on the development and progression of PAH in women. In addition, we are aware of human and animal studies that suggest that the sex of the subject plays a role in the response to PAH treatment, and therefore personalized therapies based on sex may benefit PAH patients.

[0151] Studying ECM remodeling

[0152] As described in our recent publication, we first characterized the cells for various markers and cultured the control and PAH groups at 2 × 10 6Clinical cells (ADCs) or SMCs with a density of 5 to 10×10 cells / mL 6 ECs were seeded at a density of 10 cells / mL onto the corresponding channels of the chip. SMCs were seeded in a rat tail collagen type I solution (Corning, New York). We fed the cells with culture medium through the two outermost channels. In experiments seeding the chip with three different types of cells, we first perfused collagen (2 mg / mL) or SMCs mixed with collagen into the inner channels, and after 20 minutes the collagen formed a gel, and then seeded ECs and ADCs at intervals of 20 minutes. The prepared chips containing cells were placed in a humidified chamber and allowed to grow in a CO2 incubator for 5-8 days. The culture medium was removed and replaced with 150 μL of new culture medium every day.

[0153] 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, CD90, and various ECM proteins. To stain cells, we first washed the chip with basal medium and then treated it with 4% paraformaldehyde, blocking buffer, and primary antibodies and incubated overnight. The next day, we washed the chip again with washing buffer, injected secondary antibodies, incubated for one hour, washed again, and finally stained the nuclei with mounting medium containing DAPI. Similarly, we stained the chip for the presence of human collagen types I (ColT1) and IV (ColT4), laminin, tenascin-C, fibronectin, and elastin. We took images of cells and ECMs using epifluorescence microscopy (DMi8epifluorescence, Leica, IL) or multiphoton confocal microscopy (Ti-E, Nikon, NY).

[0154] We used quantitative PCR (qPCR) methods to quantify various ECMs. To this end, we removed and collected control cells and PAH cells 1, 3, 5, 7, and 10 days after seeding. mRNA was isolated using the QuickPrep Micro mRNA Purification Kit (Amersham, Piscataway) and analyzed for laminin, tenascin-C, ColT1α, ColT4, fibronectin, and elastin. According to the manufacturer's protocol, we used the Superscript III First-Strand Synthesis System (Invitrogen) to generate single-stranded cDNA and performed qPCR in the MX4000 using Brilliant SYBR Green QPCR Master Mix at the threshold cycle number generated by the MX4000 software version 4.20 (Stratagene, La Jolla, CA). Using the formula ΔCt = Ct reference gene - Ct gene of interest, we normalized the Ct values ​​to the internal control GAPDH.

[0155] Assessing Endothelial Cell Dysfunction on a Chip

[0156] To investigate whether endothelial cells undergo apoptosis as observed in PAH, we seeded control endothelial cells, smooth muscle cells, and ADCs and cultured the cells for 3 to 5 days under various conditions (e.g., normal serum under normoxic conditions or reduced serum under hypoxic conditions). We also cultured N-ECs in the presence or absence of collagen. At the end of the experiment, we stained the cells with the same cellular and ECM markers as described above. To assess apoptosis, we stained the cells for λ-H2AX, PCNA, and cleaved caspase-3, and finally calculated the apoptotic index based on the ratio of the number of caspase-3-positive cells to the total number of cells.

[0157] Assessment of sex-specific treatment response:

[0158] To assess the cellular basis for these sex-specific treatment effects, we used bosentan, an endothelin receptor antagonist (ERA), to which female PAH patients respond more strongly than male patients. After seeding PAH-ECs / SMCs / ADCs from male and female patients into the prepared chips, we infused 50 μM bosentan starting on day 4 of cell seeding and continuing the infusion for 3 days. To determine if the drug had a preventive effect, we treated the chips containing cells with 50 μM bosentan on day 1 of cell seeding and allowed the cells to grow for an additional 6 days without treatment. To investigate the effects of growth hormone and anti-PAH drugs on cellular function and remodeling, we seeded male and female PAH-ECs, QD705-labeled or unlabeled PAH-SMCs, and PAH-ADCs into the chips. We assessed the deposition of human ColT1, the expression of aromatase (an enzyme that synthesizes estrogen), and CYP1B1 (an enzyme that metabolizes estrogen), and then measured arterial remodeling and muscularization.

[0159] To evaluate the effects of drugs on PA thickening, we assumed that ECs and SMCs grew within their respective channels, i.e., the intimal and endothelial layers of the chip, and not outside of the designated layers. If ECs or SMCs grew outside of their original seeding channels, we considered growth outside of the designated channels as thickening of the intimal or endothelial layers, respectively. We also considered the migration of SMCs from the medial side to the intima as muscularization, and the number of SMCs growing in the intimal layer as the degree of muscularization. These assumptions were based on the pathology of PAH, where non-muscularized distal arteries / arterial vessels become muscularized and show SM-like cells derived from pre-existing SMCs. We used phase contrast microscopy, immunostaining, and quantum dot labeling to draw lines between the growth zones of different cells and count and identify the cells.

[0160] To demarcate the boundaries of different cell growth areas, we first imaged the chip using phase contrast microscopy and then stained the cells using confocal microscopy. To calculate the extent of intimal and medial thickening, we calculated the average length of five to seven points of varying diameters in the same layer. To determine the extent of muscularization, we cultured QD-labeled N-SMC / PAH-SMC on the chip and stained the chip with FSP-1 and CD31 antibodies, then counted the number of FSP-1+CD31 or QD-labeled SMCs in the three layers of the chip.

[0161] Creating plexiform complex cytopathies on a chip

[0162] To reproduce plexiform lesions on a chip, we first prepared spherical or three-dimensional colonies of various cell combinations using the “hanging drop method”. For example, see R. Foty. J Vis Exp. 51:2720 (2011). The spherical cells were then seeded onto the chip. Briefly, ~5×105 ECs, EC / SMC (1:1), EC / ADC (1:1), or EC / SMC / ADC (1:1:1) were dispersed in 25 mL of serum-free medium containing 20% ​​methylcellulose to prepare a cell mixture in methylcellulose. We then aliquoted the cell-methylcellulose mixture into 25 μL droplets using a pipette and seeded a series of 25 μL droplets into non-adherent cell culture dishes. We then inverted the culture dish containing the cell droplets so that the droplets were suspended in the air on the surface of the culture dish, thus forming hanging cell droplets. After culturing the cell droplets suspended in the air at 37°C and 5% CO2 for 24 hours, we obtained spherical or three-dimensional colonies consisting of single cells and multicells. We prepared QD-labeled PAH-ECs, PAH-EC / SMCs, or PAH-EC / SMC / ADCs spheroids, as well as control cell (N-EC / SMC / ADCs) spheroids. The next day, after collecting the spheroid cells, we washed the spheroid cells with culture medium to remove methylcellulose and then dispersed them in 90 μL of collagen as a spheroid matrix. Finally, we seeded 10-15 spheroids on the inner membrane channel of the chip. Three days after seeding the spheroids on the chip, we treated the chip containing control N-EC / SMC / ADC spheroids with 50 ng / mL VEGF and 1 ng / mL TGF-β for 3 days. Then, we stained EC, SMC, and ADC with various markers and observed the cells under a confocal microscope (Ti-E, Nikon, NY).

[0163] result

[0164] PAH pathophysiology is recreated on the device by culturing three types of pulmonary artery cells (PACs), namely endothelial cells (ECs), smooth muscle cells (SMCs) and clinical cells (ADCs). PAH cells can be obtained from biopsies or using PACs differentiated from induced pluripotent stem cells derived from PAH patients.

[0165] When grown on the chip, lesional (PAH) PACs migrated out of their designated layer, interacted with cells in adjacent layers, and produced phenomena similar to the main pathologies of human PAH: muscularization, extracellular matrix (ECM) protein deposition, and arterial remodeling. The results are as follows:

[0166] Flow-induced stress caused morphological changes in control cells grown on the chip and induced arterial remodeling.

[0167] Estrogen and platelet-derived growth factor (PDGF) treatment induced more extensive arterial remodeling in chips prepared with PACs from female patients than in chips prepared with PACs from male patients.

[0168] The female chip (made with cells from female patients) was more sensitive to bosentan, an anti-PAH drug, than the male chip (made with cells from male patients).

[0169] PAH-EC / SMC / ADC spheroids seeded on the chip, but not control cells, evolved into arteries with plexiform vasculopathy, with PAH-ECs displaying clonal proliferation properties.

[0170] Figure 6 Extracellular remodeling in PAH is demonstrated according to certain embodiments. The systems and methods provided herein create PAH characterizations that show ECM remodeling similar to clinical patient pulmonary arteries.

[0171] Since ECM deposition is a major pathological feature of PAH, we used these systems and methods to show that PAH cells grown on a chip produce ECM proteins, create their own ECM scaffold, and thus contribute to pulmonary artery remodeling. The basement membrane of a normal human pulmonary artery 605 consists of a thin layer of fibers beneath the endothelial cell layer, formed by specific interactions between ECM components such as laminin, collagen, elastin, fibronectin, tenascin-C, and proteoglycans. Therefore, we measured the levels of ECM proteins in chips prepared with 3 control cells and PAH cells. Compared with control chips, chips prepared with PAH cells showed extensive deposition of various ECM proteins: laminin, tenascin-C, and insoluble collagen (ColT1α), but not ColT4, fibronectin, or elastin. In chips prepared with PAH cells, a thin layer of laminin was formed and spread toward the intima and lumen; intimal thickening increased with increased laminin deposition. In the intimal layer of the PAH chip, fibrous collagen fibers appeared, and as the number of PAH-SMCs in the intimal layer increased, the number of collagen fibers also increased. The deposition of collagen fibers was the highest in the intimal layer of the PAH chip. Zonal analysis of pulmonary arteries in IPAH patients showed that changes in ECM remodeling, especially changes in collagen expression, were more obvious in the intimal layer, followed by the tunica intima, and then the adventitia.

[0172] We also investigated the effect of sex on the development of plexiform lesions by seeding different combinations of PAH cells (PAH-ECs / SMCs, PAH-ECs / ADCs, and PAH-ECs / SMCs / ADCs) from male and female patients into spheres. For ease of identification, we used QD705-labeled PAH-ADCs and N-ADCs. Male and female PAH-EC spheroids generated angiogenic-like sprouts with lumen arterial channels lined by a monolayer of ECs, but without lesions. Male and female PAH-EC / SMC spheroids similarly developed arteries but without lesions. In contrast, PAH-EC / ADC spheroids formed disorganized clumps with arterial lumens, but a few cube-like vessels were observed only in the female plexiform-mimetic chip. In both sexes, PAH-ECs / SMCs / ADCs spheroids generated plexiform-like complex cytopathies. Female chips showed more α-SMA upregulation than male chips, and female PAH-SMCs contained intermediate filaments, indicating that the SMCs were in a differentiation stage.

[0173] Example 5. Development of disease treatment methods

[0174] The systems and devices presented in this article are useful in developing treatments for diseases because they can simulate the significant pathophysiological features of the disease process, as well as the interactions between cells and the response to drugs. In the case of PAH, the cell types are the three major cell types of the pulmonary artery. These interactions include cell migration from one layer of the pulmonary artery wall to another, endothelial to mesenchymal transformation, and the formation of plexiform lesions. Using PAH as an example, we show the similarities and differences in the pathogenesis of PAH between male and female patients and use these similarities and differences to develop sex- and age-specific treatments for PAH. This process can help develop personalized treatments for the disease process. For example, see Taslim A. Al-Hilal et al. Royal Society of Chemistry, Lab Chip 20:3334-3345 (2020), https: / / doi.org / 10.1039 / D0LC00605J (downloaded on July 27, 2021), the entire text of which is hereby incorporated herein by reference.

[0175] We investigated the effect of sex on the development of plexiform lesions by seeding different combinations of PAH cells (PAH-ECs / SMCs, PAH-ECs / ADCs, and PAH-ECs / SMCs / ADCs) from male and female patients into spheres. For identification, we used QD705-labeled PAH-ADCs and N-ADCs. Male and female PAH-EC spheroids generated angiogenic-like sprouts with lumen arterial channels lined by a monolayer of ECs, but without lesions. Male and female PAH-EC / SMC spheroids similarly developed arteries, but without lesions. In contrast, PAH-EC / ADC spheroids formed disorganized masses with arterial lumens, but a few cube-like vessels were observed only in the female plexiform-mimetic chip. In both sexes, PAH-ECs / SMCs / ADCs spheroids generated plexiform-like complex cytopathies. Female chips showed more α-SMA upregulation than male chips, and female PAH-SMCs contained intermediate filaments, indicating that the SMCs were in a differentiation stage.

[0176] Sex differences in PAH and sex-biased responses to PAH treatments are two puzzling features of PAH pathophysiology. Although more women than men are diagnosed with PAH, the survival rate of men is lower than that of women. Major factors contributing to sex differences in PAH include sex hormones and growth factors. Some reports suggest that differences in PAC cell cycle regulation predispose women to PAH. In addition, both human and animal studies have shown that the sex of the subject plays a role in the treatment response of PAH, so sex-based personalized therapies may benefit PAH patients.

[0177] Example 6. Development of treatment methods

[0178] As described herein, the systems and methods can be used to simulate cellular interactions in any system of the body to develop treatments for diseases. In some embodiments, the systems and methods provided herein can simulate any vascular disease.

[0179] Pulmonary vascular disease extends beyond PAH and includes a wide variety of pathological conditions that alter the blood vessels that serve as the conduit between the heart and the lungs. With respect to the range of pulmonary vascular disease that can be studied using the systems and methods described herein, any disease in which changes are made to the pulmonary vasculature, such as stiffening of the pulmonary vascular bed, can lead to a variety of complications that can be addressed in the development of improved treatments.

[0180] Figure 7A flow chart showing how the systems and methods taught herein can be used to treat a subject in need thereof, according to certain embodiments. In many systems taught herein, the microfluidic device will have at least 3 channels. Steps 605, 610, and 615 introduce a first cell type (cell type 1) into channel 1 in a cell medium to form an interface layer adjacent to channel 2; introduce a second cell type (cell type 2) into channel 3 in a cell medium to form an interface layer adjacent to channel 3; and introduce an aqueous medium into channel 2 to controllably release the interface layer adjacent to channel 2, allowing communication to occur between cell type 1 and cell type 3. These are core steps. Steps 620, 625, 630, 635, 640, and 645 use the systems and methods to identify a subject in need of drug treatment 620, obtain cell type 1 from a subject 625, obtain cell type 2 from a subject 630, identify candidate drugs for a subject 635, introduce drugs into channels 1, 2, and / or 3 640 to determine the therapeutic effect, and administer drugs to a subject 645 if a therapeutic effect is desired.

[0181] For the vascular model, there can be three cell types: smooth cells, adventitial cells, and endothelial cells. In some embodiments, a 3-channel device can place smooth cells in the middle channel 2, and place adjacent cells and endothelial cells in adjacent channels 1 and 3. In some embodiments of the 5-channel device, channels 1 and 5 can serve as cell medium reservoirs that can be replaced regularly as needed and can also be used for drug delivery; channels 2 and 4 can be vascular adventitial cells and endothelial cells; channel 3 can be smooth muscle cells. Smooth muscle cells can be introduced in an aqueous gel, cell medium, or an aqueous fluid, cell medium. In some embodiments, channel 1 and / or channel 5 can be used to apply hydrostatic pressure to simulate changes in blood pressure, thereby simulating stress on the blood vessel, and placing an endothelial cell layer near one or both of channels 1 and 5. Given that capillaries have only endothelial cells, while large arteries have all three layers, the skilled person will understand how to use systems and methods to represent physiological functions when developing treatment methods.

[0182] Gender-specific therapies:Our study showed that chips prepared with PACs from female PAH patients responded more sensitively to estrogen treatment than those prepared with PACs from male PAH patients. Here, we treated the chips with the endothelin receptor antagonist bosentan to investigate sex-specific treatment responses. Clinical data showed that female PAH patients responded more strongly to bosentan treatment than male patients (p = 0.3). In female chips, the drug reduced the thickening of the intimal layer, slowed the migration of PAH-SMCs into the luminal and intimal layers, and reduced the expression of CYP1B1 in the intimal layer. Interval-specific analysis showed that laminin and ColT1α deposition was lower in female slices treated with bosentan than in male slices. To investigate the preventive effect, we treated male and female chips on the first day after seeding and then allowed the cells to grow for another 6 days without treatment. Although the thickening of the intimal and medial layers was similar in male and female slices, the content of PAH-SMCs in the endothelial layer was lower in female slices than in male slices. CYP1B1 expression decreased more in the intimal layer of the female chip than in the male chip. Female slices treated with bosentan showed lower levels of muscularization compared to male slices, as determined by counts of QD705+PAH-SMCs in the intimal layer. Interestingly, early treatment with bosentan reduced deposition of laminin and ColT1α in the intimal layer of the female chip compared to the male chip. As can be seen from this example, the systems and methods described herein can be used to develop sex-specific therapies for disease.

[0183] Efficacy of anti-PAH drugs in improving pulmonary arterial hypertension and right ventricular hypertrophy (PAH-RVH) : This system can be used to identify interactions between PAH-infected ECs and CMs and evaluate drug efficacy.

[0184] For example, the improved chip device can be used to culture both cardiomyocytes (CMs) for cardiovascular disease research and pulmonary artery endothelial cells (ECs) for pulmonary vascular disease research. For example, pulmonary artery ECs can be harvested from the pulmonary arteries of healthy donors (N-ECs) and from the pulmonary arteries of PAH patients (PAH-ECs). We have seen that PAH is a disease that could benefit from improved treatments using the systems and methods taught herein. For example, by growing CMs and N-ECs or PAH-ECs in separate compartments or channels, we can measure the effects of PAH-ECs on CMs by measuring various soluble cell markers, assess the contractility of CMs, assess the expression of CM genes, and study the effects of anti-PAH drugs on the release of hypertrophy inducers and hypertrophy markers and CM contractility. For example, we found that: (i) chips prepared with CMs and N-ECs showed that CMs grew anisotropically and existed in bundles similar to those of physiological CMs; and (ii) chips prepared with CMs and PAH-ECs showed that CMs were hypertrophic, lost their contractile force, and displayed elevated levels of hypertrophy-related markers.

[0185] We used these systems and methods to develop a drug therapy. We used two anti-PAH drugs, sildenafil and bosentan, on a chip made with CMs and PAH-ECs, and the drugs reduced the extent of CM hypertrophy, improved contractility, and reduced hypertrophy inducers and hypertrophy markers. From our study, people will know that the system and method can identify the interaction between PAH-infected ECs and CMs and evaluate the efficacy of anti-PAH drugs in improving pulmonary arterial hypertension right ventricular hypertrophy (PAH-RVH).

[0186] Electrical stimulation

[0187] The cells can be electrified to simulate the electrophysiological microenvironment. For example, by modifying the device, we can reproduce the following three diseases: (1) venous pulmonary hypertension; (2) pulmonary embolism; (3) chronic thromboembolic disease. In these embodiments, we can inoculate organ-specific cells and create a microenvironment to simulate the pathological conditions of the disease. For example, pulmonary embolism and chronic thromboembolic disease provide particularly important approaches for the use of the systems and methods provided herein, because both diseases are fatal diseases in which thrombi occur in the pulmonary vessels. The systems and methods taught herein can be used to regenerate pulmonary vasculature, and then evaluate how blood coagulation changes the pathology of the pulmonary vascular bed by studying the interactions between cells and the effects of specific treatments (e.g., drug therapy, radiation therapy, or a combination thereof), as well as how the treatment is affected by the subject's gender, age, other physiological states of the subject, or a combination thereof.

[0188] It should be understood that vascular disease includes a variety of pathological conditions, each of which includes lesions of arteries, veins, or lymphatic vessels. The systems and methods taught herein can include all common vascular diseases. For example, the systems and methods taught herein can be used to simulate, obtain data, and improve treatment, for example, neurovascular disease, peripheral arterial disease, renal artery disease, carotid artery disease, aortic aneurysm, and various venous diseases, which can include, for example, varicose veins, spider veins, chronic venous insufficiency, deep vein thrombosis, neurovascular disease, and lymphedema.

[0189] In certain embodiments, in order to use the systems and methods taught herein, we will collect human cells from relevant tissues or organs, culture the cells in the systems taught herein, and simulate the cellular interactions of each disease. As described herein, these systems are widely used and can play a variety of functions in the process of developing disease treatment methods. For example, we have demonstrated that we can determine gender differences in disease pathology and screen the efficacy of related drugs for treating simulated diseases. It should be understood that the systems and methods taught herein can assist in the development of precision medicine methods by studying the interactions between cells and the effects of specific treatments (e.g., drug therapy, radiotherapy, or a combination thereof), as well as how the treatment is affected by the subject's gender, the subject's age, other physiological states of the subject, or a combination thereof.

Claims

1. A microfluidic system for simulating the cellular layered physiological functions of a mammalian subject, the system comprising: Microfluidic chips; as well as A first channel adjacent to a second channel; wherein, The first channel includes a first cell aqueous solution as a first tissue layer, wherein the first cell aqueous solution is not a gel-forming solution; and A controllably releasable first water interface film formed by the first cell aqueous solution; The first channel and the second channel are connected to the chip and include a first port, for injecting the first cell aqueous solution into the first channel; a second port for injecting a second cell aqueous solution into the second channel, wherein the second cell aqueous solution is not a gel-forming solution; and A first wall with an opening, the first channel and the second channel share the first wall, and the opening on the first wall is configured such that: (i) after the first cell aqueous solution is injected, the releasable first water interface film is formed at the opening of the first wall; (ii) when the second cell aqueous solution is introduced, after the first water interface film is controllably released from the opening, cell communication occurs between the first channel and the second channel; Wherein, simulation of cell communication between tissue layers is improved compared to the use of hydrogels in the first channel or in the second channel, because the use of hydrogels cannot simulate real physiological conditions, including cell-drug communication occurring in a non-gel aqueous environment in the mammalian subject.

2. The system of claim 1, wherein injection of the second aqueous cell solution results in controllable release of the first water interface film from the opening.

3. The system of claim 1, wherein the wall is configured to form a controllably releasable first water interface film at the opening when the first aqueous cell solution is injected.

4. The system of claim 1, wherein the opening comprises a hydrophobic material for forming the first water interface film controllably releasable at the opening of the first wall when the first cell aqueous solution is injected.

5. The system of claim 1, wherein the opening is delineated by opposing wedge-shaped edges that share a plane so as to form a controllably releasable first water interface film at the opening when the first aqueous cell solution is injected.

6. The system of claim 4, wherein the opening has a shape selected from the group consisting of a circle and an ellipse.

7. The system of claim 1 , wherein the opening is outlined by a polygonal structure providing opposing edges on at least two opposing sides of the opening, wherein the opposing edges share a plane so as to form the first water interface film at the opening of the first wall when the first cell aqueous solution is injected.

8. The system of claim 7, wherein the opening of the first wall is a space between adjacent columns along a series of columns; Each column of the series of columns has a central axis which is at least substantially parallel to a normal to the chip, Each pair of adjacent pillars provides a pair of opposing edges sharing a common plane so as to form a controllably releasable first water interface film at the opening of the first wall when the first cell aqueous solution is injected.

9. The system of claim 1, further comprising: Third channel; as well as a third port, for injecting a third cell aqueous solution into the third channel; A second wall with an opening, the second wall being shared by the second channel and the third channel, the opening on the second wall being configured to: (i) form a controllably releasable second water interface film at the opening of the second wall after the third cell aqueous solution is injected; and (ii) allow cell communication between the second channel and the third channel after the interface film is released from the opening.

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

11. The system of claim 1 further comprising a first growth medium channel having a first growth medium port and communicating with the first channel, and a second growth medium channel having a second growth medium port and communicating with the second channel.

12. The system of claim 9, further comprising a growth medium channel having a growth medium port.

13. The system of claim 9, further comprising a first growth medium channel having a first growth medium port and communicating with the first channel, and a second growth medium channel having a second growth medium port and communicating with the second channel.

14. A method for simulating a disease using a microfluidic system, the method comprising: Obtain the system according to claim 1; Injecting the first cell aqueous solution into the first channel to introduce the first cells of the disease, and forming the controllably releasable first water interface film at the opening of the first wall after the injection; as well as injecting the second cell aqueous solution into the second channel to introduce the second cells of the disease and release the first water interface film; in, After the second cell aqueous solution is injected, the first water interface film is controllably released; as well as After the first water interface film is controllably released, the first cell and the second cell communicate to simulate a disease state.

15. A method for simulating a disease using a microfluidic system, the method comprising: Obtain the system according to claim 9; Injecting the first cell aqueous solution into the first channel to introduce the first cells of the disease, and forming a controllably releasable first water interface film at the opening of the first wall after the injection; Injecting the third cell aqueous solution into the third channel to introduce the third cells of the disease, and forming a controllably releasable second water interface film at the opening of the second wall during the injection; as well as injecting the second cell aqueous solution into the second channel to introduce the second cells of the disease and controllably release the first water interface film and the second water interface film; in, After the second cell aqueous solution is injected, the first water interface film is controllably released; After the second cell aqueous solution is injected, the second water interface film is controllably released; After the first water interface film is controllably released, the first cell and the second cell communicate to simulate a disease state; and After the second water interface film is controllably released, the second cell and the third cell communicate to simulate a disease state.

16. A method for simulating a disease using a microfluidic system, the method comprising: Obtain the system according to claim 9; Injecting the first cell aqueous solution into the first channel to introduce the first cells of the disease, wherein the injection forms a controllably releasable first water interface film at the opening of the first wall; Injecting the third cell aqueous solution into the third channel to introduce the third cells of the disease, wherein the injection forms a controllably releasable second water interface film at the opening of the second wall; as well as injecting the second cell aqueous solution into the second channel in the form of an aqueous gel to introduce the second cells of the disease and controllably release the first water interface film and the second water interface film; in, After the second cell aqueous solution is injected, the first water interface film is controllably released; After the second cell aqueous solution is injected, the second water interface film is controllably released; After the first water interface film is controllably released, the first cell and the second cell communicate to simulate a disease state; and After the second water interface film is controllably released, the second cell and the third cell communicate to simulate a disease state.

17. A method for establishing a model for treating a simulated disease with drugs in a microfluidic system, the method comprising: Execute the method of claim 14; selecting a drug to treat the disease simulated in the microfluidic system; as well as The drug is administered in the first channel or the second channel of the microfluidic system to establish a model for treating a simulated disease.

18. A method for establishing a model for treating a simulated disease with drugs in a microfluidic system, the method comprising: Execute the method of claim 15; selecting a drug to treat the disease simulated in the microfluidic system; as well as The drug is administered in the first channel, the second channel or the third channel of the microfluidic system to establish a model for treating a disease.

19. A method for establishing a model for treating a simulated disease with drugs in a microfluidic system, the method comprising: Execute the method of claim 16; selecting a drug for treating the simulated disease in the microfluidic system; as well as The drug is administered in the first channel, the second channel or the third channel of the microfluidic system to establish a model for treating a simulated disease.

20. A method for establishing a model for treating a simulated vascular disease with drugs in a microfluidic system, the method comprising: Obtain the system of claim 13; Injecting the first cell aqueous solution into the first channel to introduce the first cells of the disease to form a dying cell layer, wherein the injection forms the controllably releasable first water interface film at the opening of the first wall; Injecting the third cell aqueous solution into the third channel to introduce the third cells of the disease to form an endothelial cell layer, wherein the injection forms the controllably releasable second water interface film at the opening of the second wall; as well as injecting the second cell aqueous solution into the second channel to introduce the second cells of the disease, form a smooth muscle layer and controllably release the first water interface film and the second water interface film; Choose a medication; and injecting the drug into the first growth medium channel or the second growth medium channel to establish a model for treating simulated vascular diseases; in, After the second cell aqueous solution is injected, the first water interface film is controllably released; After the second cell aqueous solution is injected, the second water interface film is controllably released; After the first water interface film is controllably released, the first cell and the second cell communicate to simulate a disease state; and After the second water interface film is controllably released, the second cell and the third cell communicate to simulate a disease state.

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