Systems and methods for multichannel blood vessels

Abstract

This invention discloses a system and method for multi-channel vascular and multi-organ vascular-vascular fluid interfaces in in vitro models. The method includes: embedding at least one tissue in a 3D structure; forming a vascular interface for perfusing nutrients into the one or more tissues; modulating the one or more vascular interfaces, together or separately, by altering vascular properties to possess characteristics of a natural biological target tissue or target environment; and lining the fluid interface channels to provide growth fluid in a physiologically generalized manner. The 3D structure includes vascular regeneration elements for vascular regeneration or vascular formation.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 648,209, filed March 26, 2018, the entire contents of which are incorporated herein by reference.

[0002] Statement on Federally Funded Research This invention was completed with government funding granted by the National Institutes of Health (NIH) in the United States (approval number 1UG3TR002198-01). The government holds certain rights to this invention. Background Technology

[0003] Some on-chip organ devices may include microengineered biological cell culture compartments in which tissue and organ-level elements of human physiology can be generalized. These compartments can allow for in vitro physiological modeling of functional biological units of the organ system, such as islet units that mimic insulin secretion in the pancreas or gas-liquid interfaces that mimic oxygen transport in the lungs. Such models can be tested on living human tissues without the need for living human subjects.

[0004] However, some organ-on-a-chip systems cannot create functional and / or realistic multi-organ networks and cannot generalize complex physiological responses and multi-organ interactions at the system level. In 2D models, a monolayer of endothelial cells grown on a Petri dish represents the vascular lumen, but this approach can be limited by the lack of other cells typically involved in vascular behavior, such as pericytes and smooth muscle cells. Some organ-on-a-chip systems have diffusion constraints due to insufficient perfusion efficiency, which limit the size of 3D tissues grown in hydrogels or other scaffolds. Larger tissues may require more efficient perfusion to promote tissue development, survival, regulation, and homeostasis. Some tissues require microenvironmental cues from vascular tissue or vessels to differentiate into physiological states or perform biological functions in vitro.

[0005] Therefore, there is still a need to create multi-organ networks and their interfaces with improved perfusion efficiency for on-chip organ devices used for therapeutic applications and drug screening. Summary of the Invention

[0006] The disclosed subject matter provides systems and methods for generating multichannel vessels or perfusionable vascular fluid interfaces that travel back and forth within or between single or multiple in vitro tissues in or between human or animal living tissues.

[0007] In some embodiments, a method for culturing tissues using microphysiological devices or organ-on-a-chip devices may include embedding at least two tissues within a 3D structure, wherein the 3D structure includes angiogenesis elements. Vascular interfaces for nutrient perfusion are formed within the at least two tissues and are modulated to possess characteristics of a natural biological target tissue or target environment by altering at least one vascular property. Fluid interface channels may be lined and coupled to the 3D structure to provide growth fluids to promote angiogenesis. A corresponding system is similarly disclosed herein.

[0008] In some embodiments, a method for culturing tissue using microphysiological devices or organ-on-a-chip devices may include embedding a tissue type within a 3D structure and one or more vascularization elements. Vascular interfaces for nutrient perfusion are formed within, outside, or both within and outside the tissue and are modulated to possess characteristics of a natural biological target tissue or target environment by altering at least one vascular property. Fluid interface channels may be lined and coupled to the 3D structure to provide growth fluids to promote angiogenesis. A corresponding system is similarly disclosed herein.

[0009] In some embodiments, the method may further include introducing segments, elements, or layers of angiogenic tissue between at least two tissues to create a vascular interface. Angiogenic elements and layers of angiogenic tissue may include hypoxia-inducible factor (HIF), fibroblast growth factor (FGF), and / or vascular endothelial growth factor (VEGF). In some embodiments, the method may further include linking multiple vascular interfaces to form a multichannel vascular culture system.

[0010] In some embodiments, the method may include introducing segments, elements, or layers of angiogenic tissue upstream, downstream, or both upstream and downstream of a tissue to create one or more vascular interfaces, thereby enabling fluid communication with the tissue via vascular structures. Angiogenic elements and layers of angiogenic tissue may include hypoxia-inducible factor (HIF), fibroblast growth factor (FGF), and / or vascular endothelial growth factor (VEGF). In some embodiments, the method may further include linking multiple vascular interfaces to form a multichannel vascular culture system.

[0011] In some implementations, the vascular properties of a blood vessel can be modulated to possess the characteristics of a natural biological target tissue by altering one or more of the following: vascular density, vascular diameter, vascular barrier function, vascular disease condition, or material properties (including stiffness, pore size, or exposed chemical portions or chemical groups) that indirectly achieve one or more of these alterations (e.g., through mechanosensory feedback of vascular cells or vascular tissue). Attached Figure Description

[0012] Other features and advantages of this disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying drawings illustrating illustrative embodiments of the present disclosure, wherein: Figure 1 This is an illustration of an exemplary engineered blood vessel in a 3D fibrin hydrogel within a 3D microphysiological tissue culture apparatus. Scale bar is 200 μm.

[0013] Figure 2 This is an illustration of an exemplary perfusion of 1 μm fluorescent beads (from a point at the bottom) through the hollow lumen of an engineered vascular endothelial cell expressing RFP in a microphysiological device.

[0014] Figure 3 This is an illustration of an exemplary diagram showing the interface between an engineered vascular system and a transplanted human islet tissue in a 3D ECM hydrogel within a microphysiological device.

[0015] Figure 4 This is an illustration of an exemplary engineered 3D vascular interface between the endothelial lining vascular bundle and GFP-expressing islets isolated from a mouse.

[0016] Figure 5 This is an illustration of an exemplary schematic diagram of a bone marrow model, in which human bone marrow tissue can be fluidly introduced via a 3D vascular interface, which in turn connects to a large blood vessel fluid interface formed by coating all sides of the fluid channel with vascular endothelial cells to form a vascular vessel of a defined size.

[0017] Figure 6 This is an illustration of an exemplary perfusion of leukocytes from a central bone marrow tissue compartment through an engineered 3D vascular fluid interface, the perfusion being driven by fluid flow and size-based selectivity determined by the diameter of the lumen of the blood vessel formed therein.

[0018] Figure 7 This is an illustration (center) of an exemplary 3D vascular interface (endothelium) of leukocytes captured in a fluid suspension, showing the inflow and outflow sides.

[0019] Figure 8 The illustration is of an exemplary system with two 3D vascular interfaces, based on the disclosed content.

[0020] Figure 9 This is a diagram illustrating exemplary stages in a method based on the disclosed content.

[0021] Figure 10 This is a diagram illustrating exemplary stages in the method according to the disclosed content. The tissue forms a continuous and perfusionable vascular connection between the flank flow channels.

[0022] Figure 11This is a diagram illustrating an exemplary stage in the method according to the disclosed content. Multiple separate tissues are fluidly connected via various vascular interface materials disposed between the separate tissues.

[0023] Figure 12 This is a diagram illustrating exemplary stages in the method according to the disclosed content. Multiple adjacent tissue types are connected to the microcavities of microfluidic channels via vascular interface materials to simulate a biological interface with the circulatory system.

[0024] Figure 13 This is a diagram illustrating an exemplary stage in the method according to the disclosed content. Multiple separate tissues are internally vascularized through multiple placements of vascular interface material. This process accelerates the formation of a perfusionable multi-tissue network, thereby mimicking the biological connections in blood circulation.

[0025] Figure 14 This is a diagram illustrating an exemplary stage in the method according to the disclosed content. Multiple adjacent tissues are internally vascularized through a vascular interface material to form a perfusionable multi-tissue material.

[0026] Figure 15 This is a diagram illustrating exemplary stages in the method according to the disclosed content. Discrete tissue flows into channels and is then vascularized.

[0027] Figure 16 This is a diagram illustrating an exemplary stage in the method according to the disclosed content. Discrete tissue is suspended in a pregelated vascular material and placed within a cavity. A vascular interface material forms an interface with the encapsulated tissue.

[0028] Figure 17 This is a diagram illustrating exemplary stages in a method according to the disclosed content. Increased vascular interfaces are created by lining microchannels or fluid chambers with a monolayer of cells.

[0029] Figure 18 This is a diagram illustrating exemplary stages in the method according to the disclosed content. Discrete tissue flows into channels and is then vascularized. Tissue capable of forming a monolayer is embedded in the flow channels.

[0030] Throughout the accompanying drawings, unless otherwise stated, the same reference numerals and characters are used to denote similar features, elements, components, or portions of the illustrated embodiments. Furthermore, although this disclosure will now be described in detail with reference to the accompanying drawings, the description is also made in conjunction with illustrative embodiments. Detailed Implementation

[0031] This paper discloses a technique for generating body organs or systems on organ-on-a-chip using multiple microfluidic devices. The disclosed subject matter allows for the use of organ-on-a-chip to perform fully or partially automated organ culture without the need for specialized personnel by modeling feedforward and feedback effects from the connection between one functional unit and another on organ-on-a-chip.

[0032] In some embodiments, the disclosed subject matter provides a microphysiological tissue culture system. This system may include an engineered vascular network. For example, vascular endothelial cells, fibroblasts, pericytes, mesenchymal stem cells, and / or smooth muscle cells can be seeded together in a 3D ECM scaffold or hydrogel and provided with an endothelial cell culture medium containing angiogenic factors, including VEGF, FGF, and endothelial growth hormone. Figure 1 As shown, 3D fibrin hydrogels, collagen hydrogels, or biocompatible hydrogels, or combinations thereof, can be used for vascular engineering. In the presence of these growth factors, cells can form patterned vascular structures with hollow, perfusion-compatible lumens through processes such as angiogenesis, angiogenesis, or a combination of angiogenesis and angiogenesis.

[0033] In some embodiments, the microphysiological tissue culture system may include an endothelial cell liner on the wall of the vascular gel. For example, a monolayer endothelial cell liner can drive vascular regeneration and budding into the hydrogel. A monolayer microphysiological tissue culture system can connect blood vessels formed within a 3D gel matrix to the outer lumen of the gel endothelial liner. In some embodiments, after 2-7 days of growth, the 3D microphysiological tissue culture system may have a perfusionable vascular network comprising hollow endothelial cell-lined lumens surrounded by pericytes or fibroblasts or a combination thereof in the surrounding stroma. Figure 2 and Figure 10 ).

[0034] In some implementations, the microphysiological tissue culture system may have a vascular interface between multiple engineered or natural tissue types. The microphysiological tissue culture system may be a 3D cell culture model that generalizes human-scale or organ-scale tissue dynamics by linking two or more tissue types, including vascular tissue generated in the vascular interface. Data collected via microscopy, effluent sampling, integrated biosensing, or physical / structural measurements can be used to create 3D microphysiological tissue culture systems or 3D cell culture systems. The disclosed culture systems can be used for therapeutic applications, culture of human tissue biopsies (e.g., cancer biopsies, culture of sampled microorganisms, and / or infections), or drug screening.

[0035] In some implementations, 3D microphysiological microfluidic systems can perfuse tissues for development, survival, regulation, and homeostasis by promoting blood flow within the tissue. Blood can carry nutrients, oxygen, hormones, various cell types (including erythrocytes, platelets, leukocytes, and stem cells), as well as metabolic waste and carbon dioxide. For perfusing human tissues, the 3D microphysiological microfluidic system can have a vascular system similar to that of the human body, branching from the large aorta and exiting the heart (e.g., 20–30 mm in diameter) via arteries (e.g., 0.1–10 mm in diameter), arterioles (e.g., 0.01–0.1 mm in diameter), and capillaries (e.g., 0.005–0.01 mm in diameter), where diffusion and transport of blood-derived elements into and out of the surrounding tissues may occur. Blood can circulate back from the capillary network via venules (0.008–0.1 mm in diameter) and veins (0.1–15 mm in diameter). Blood vessels may include an internal endothelium formed by endothelial cells surrounded by nerve-innervated smooth muscle cells, perivascular cells, and fibroblasts in connective tissue.

[0036] In some implementations, 3D microphysiological microfluidic systems can provide mutual signaling, which can occur between biological tissue and the blood vessels perfusing it. Local hypoxia or nutrient deficiency in human tissue may lead to the secretion of signaling molecules, including hypoxia-inducible factor (HIF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). The secretion of signaling molecules can promote angiogenesis, i.e., the formation of blood vessels by precursor cells; and vascular regeneration, i.e., the sprouting of blood vessels from existing endothelial tissue. For example, neovascularization formed by angiogenic endothelial cells may be associated with tissue modeling and induced signaling during embryonic development, resulting in densely vascularized islets of Langerhans from which insulin and other homeostatic hormones are secreted. These islets exist in close contact with porous capillaries and sample the glucose concentration of the blood therein, thereby releasing insulin in response; creating an in vitro model of the islets without including their vascular environment is unforeseen. In addition, endothelial cells and smooth muscle cells can be targets of damage due to drug-induced vascular injury, but the resulting damage can be localized to specific structures, such as branch points in the coronary arteries.

[0037] In some implementations, the 3D microphysiological microfluidic system may include various vascular structures. For example, the vascular structure may include endothelial cells lining blood vessels in the brain, which may form tight junctions with each other to control molecules that allow entry into the brain (i.e., the blood-brain barrier). Alternatively, the 3D microphysiological microfluidic system may include vascular structures whose walls comprise smooth muscle cells, connective tissue, and fine microvessels responsible for alveolar gas exchange.

[0038] In some embodiments, the disclosed microphysiological microfluidic system may include at least one chamber. The shape of the chamber may be varied depending on the purpose. For example, the chamber may be a straight channel. For example, a chamber may completely or partially enclose another chamber. In some embodiments, the chambers may be patterned vertically. The chambers may be placed adjacent to each other. Furthermore, the flow through these patterns may be networked in an adjustable manner. These chambers may be fabricated to create a partitioned model of substances in the bloodstream. For example, conduits may be placed between any two or more ports, entering different regions of tissue to establish direct connections. Fluid effluent flowing out through any of these ports may be collected. For example, one of the tissues may be placed at the center of a 5-channel embodiment, and a flow of candidate drugs may flow from left to right through the tissue, through a first vascular interface on the left and a second vascular interface on the right. Effluent from the vascular ports before and after the tissue may be collected to measure drug metabolism. Figure 13 As shown, a left-handed tissue with a right-handed target organ can be connected to a blood vessel to sample the effluent before, after, but before, and after the target organ; drug metabolism and its effects on secreted products of the target organ can be measured. The disclosed device can be extended to two or more organs with interconnected blood vessels.

[0039] In some embodiments, the disclosed subject matter provides a method for generating 3D vascular interfaces. The method may include connecting at least two solid 3D tissues. The solid tissues may have vascularity or be vascularless. The tissues can be connected to form blood vessels by introducing additional segments, elements, or layers of 3D vascular tissue between them. For example, as... Figure 9 As shown, a vascular interface can be created for tissue placed in a microfluidic device. The adjoining tissue can form a perfusionable vascular structure capable of angiogenesis or regeneration. The adjoining tissue can self-vascularize via angiogenesis and / or regeneration and attach to or connect to existing blood vessels or vasculature in the solid tissue, thereby forming a perfusionable fluid connection interface between opposing original tissues. In some embodiments, the adjoining tissue can vascularize or form new blood vessels in existing tissue via vasculature budding, thereby forming a perfusionable vascular interface fluidly linked to existing solid tissue. Figure 10 In a non-limiting embodiment, the connecting tissue may be connected to certain natural vascular tissue or blood vessels.

[0040] In some implementations, vascularized tissue can be injected before adding 3D tissue modeled from adjacent targets. For example, as... Figure 18As shown, angiogenic tissues can be joined within a scaffold, matrix, or gel (where a vascular interface is formed) to stabilize suitable tissue components subsequently injected or seeded into a tissue culture device. In some embodiments, the joined angiogenic tissues can have structural rigidity to hold injected tissue or a fluid suspension of tissue containing loose or free-flowing elements in place (e.g., after the injection port is plugged). Thus, a scaffold, matrix, or gel with joined angiogenic tissues can prevent loose elements from escaping into the bulk solution by acting as a filter with pore sizes determined by the diameter of the vascular or vessel. In some embodiments, the joined angiogenic tissues can improve the structural rigidity of the gel to hold the injected tissue in place until its own hydrogel, scaffold, or extracellular matrix coagulates, solidifies, and / or crosslinks. For example, injecting adipocytes into a 3D collagen gel can create a “fat-on-sheet” tissue model between two angiogenic hydrogel extracellular matrix (ECM) scaffolds. Adjacent angiogenic tissues can sprout into the fat model in a vascular regeneration manner to create a vascular interface. In a non-limiting embodiment, the angiogenic tissue can further enhance the structural surface of a solidified, coagulated, or cross-linked 3D angiogenic scaffold, matrix, or gel to seed or implant a monolayer of cells, the surface coverage of which is limited to the surface facing the volume enclosed by the angiogenic tissue boundary. For example, a monolayer of endothelial cells can be seeded into a gel chamber to mimic larger blood vessels (such as arteries) to connect with the 3D angiogenic tissue representing a capillary bed. The disclosed system can be used to determine the size of blood vessels at the site of drug-induced vascular injury.

[0041] In some embodiments, the method may include embedding tissue within a scaffold, matrix, or gel containing vascularizing tissue. For example, engineered tissue (e.g., organoids or spheres), natural tissue such as pancreatic β-islets or biopsy cancer, or combinations thereof, may be embedded within the scaffold, matrix, or gel. The tissue embedding system may have vascularizing elements that can form perfusionable vessels that interlock with or connect to existing blood vessels or vasculature within the included tissue. Figure 16 As shown, discrete tissues (e.g., spheres, organoids, biopsy tissue, biopsy tumors, capsules, cell aggregates, or tissue scaffolds) can be suspended in a pre-gelled vascular material and placed in a chamber. The vascular interface material can form an interface with the capsule tissue. In some embodiments, vascularization elements in the scaffold, matrix, or gel form perfusionable vessels that sprout into the contained tissue via vascular regeneration, thereby forming a vascular interface. In a non-limiting embodiment, engineered vessels in the scaffold, matrix, or gel can attach to existing vessels embedded in the tissue, while additional vessels sprout into the tissue via vascular regeneration to enhance its vascularity.

[0042] In some implementations, the scaffold, matrix, or gel of the vascularization tissue interface can spatially confine the adjoining tissue while still allowing fluid inflow for nutrient perfusion. For example, the scaffold, matrix, or gel of the vascularization tissue interface can prevent the merging of multiple discrete tissues, such as two pancreatic islets or two kidney organoids or multiple cancer spheres. The scaffold, matrix, or gel of the vascularization tissue interface can further prevent tissue elements (e.g., spheres or organoids) from dissociating or loosening into multiple parts or from flattening in response to adhesion to a single 2D surface.

[0043] In some embodiments, the method may include modulating the engineered vascular interface to possess properties associated with natural biological target tissues. Modulation can be performed by altering vascular properties, including the density of the engineered vascular network, the diameter of the engineered vessels, the barrier function of the engineered vessels (e.g., tight endothelial junctions, porous junctions, or sinusoidal endothelium), the health of the engineered vessels (e.g., for use in in vitro disease models or vascular injury models), the timing of vascular perfusion (e.g., for rapidly providing fluid inflow to perfuse nutrients into the transplanted tissue), and / or combinations thereof. In some embodiments, for biological research, therapeutic testing, and / or screening purposes, the modified vascular interface may allow adjoining tissues to differentiate into physiological states or to behave in a physiologically relevant manner. Furthermore, the modified vascular interface can be used to determine or influence the fluid distribution coefficients flowing into two or more adjacent tissues. For example, a tissue interface with narrower vessels exhibits increased flow resistance than an interface with larger or denser vessels, thereby increasing the fluid flow into the interface with larger vessels relative to an interface with narrower, restrictive vessels.

[0044] In some implementations, environmental, mechanical, and / or biological conditions can be modulated. For example, environmental conditions may include: the density of angiogenic cells placed within the angiogenic tissue, which can modulate aspects including vascular density; and the ratio of different cells or cell types within the angiogenic tissue, which can modulate aspects including vessel diameter. Biological conditions may include biochemical characteristics consistently or dynamically provided to the cells via a growth medium. For example, biological conditions can be modulated by varying the concentrations of factors including VEGF to accelerate or halt angiogenicity, Ang1 to promote vascular regeneration, corticosteroids to promote tight junction formation, and / or TNF-α to increase vascular permeability. Mechanical conditions may include a fluid environment and a structural environment. For example, the fluid environment may be altered to expose the tissue to continuous fluid perfusion to produce tighter endothelial junctions and an efficient vascular network with fewer redundant branches. The structural environment can be altered to create an angiogenic tissue interface within a rigider ECM scaffold or gel to drive the formation of more rigid blood vessels, or to create an angiogenic tissue interface within a more compliant or softer scaffold or gel to mimic dilated blood vessels, aneurysms, or ruptures.

[0045] In some embodiments, the method may include lining a fluid interface channel coupled to a tissue. The fluid interface channel may include parallel, side-by-side, vertically stacked, or upstream / downstream spatial orientations or configurations. For example, as... Figure 17 and Figure 18 As shown, vascular interfaces with increased surface area can be created by lining microchannels or fluid chambers with a monolayer of cells (e.g., endothelial cells). Microphysiological tissue culture systems with vascular endothelial cells can mimic vascular lumens and create intertissue vascular interfaces. Intertissue vascular interfaces can have the diameter or cross-sectional area of ​​a blood vessel, which can be specifically defined by the geometry of the fluid channel. Intertissue vascular interfaces can have patterned blood vessels. In some embodiments, the vessel wall can be mechanically actuated to simulate vascular contraction or dilation. For example, capsular contraction can be simulated without requiring the blood vessel to be innervated smooth muscle cells.

[0046] In some embodiments, the method may further include linking multiple vascular interfaces to form an "interface-to-interface" structure. In this multi-interface structure, fluid comprising a growth medium can be perfused through a series of vessels with different vascular properties (e.g., diameter, density, connectivity, matrix stiffness) that vary according to the natural physiological characteristics of multiple tissues. For example, a large vessel with a diameter of 500 μm is formed to model an artery, which is then connected to adjacent 3D vascular interfaces of smaller arteries with an average diameter of 100 μm, and subsequently to a network of microvessels with an average diameter of 10-20 μm via a fixed injection. The connected structure can be further connected to target parenchyma or organ tissue or other vascular types included in the model to form a complete circulatory model from large-scale to small-scale vessels. Figure 11 and Figure 12 This illustrates multiple separate and adjacent tissues fluidly connected via various vascular interface materials. In some embodiments, fluid can be perfused to create a series of ordered vascular interfaces connecting to larger or smaller blood vessels within a circulatory system of different scales in an in vitro model. For example, the structure may include a series of 2D or 3D vascular interfaces, one of which can connect to the next interface without substantial tissue. Figure 13 and Figure 14 In the non-limiting embodiment shown, multiple tissues can be vascularized by repeatedly placing vascular interface material, thereby forming a continuously perfusionable multi-tissue material.

[0047] Example 1: Multi-tissue vascular interface (pancreatic islet model) The disclosed invention is used to develop an islet model to create a vascular interface for transplanted islets, which encapsulates the densely vascularized environment of human islets in vivo. The aim of this model is to prolong the viability of transplanted islets in vitro and to allow for studies of islet dynamics, including the assessment of glucose-stimulated insulin secretion, a measure of islet function.

[0048] To achieve these goals, such as Figure 3 As shown, dense and perfusion-enabled vascular interfaces are rapidly engineered from angiogenic tissue, including primary human vascular endothelial cells and fibroblasts. These vessels intersect with the natural microvessels of primary human islets, thereby encompassing the rich vascular system of the human pancreas and the paracrine signaling generated between the appropriate endothelial and endocrine tissues inherent in the islets. This is crucial for in vivo islet function and therefore represents a promising method for maintaining islet function in vitro in our microphysiological 3D tissue culture apparatus.

[0049] Engineered vascular interfaces allow for transient testing of islet function with high temporal resolution; by perfusing the engineered vascular interface, any hormonal secretions from the islet tissue (including insulin) are directly introduced into the perfusion fluid and can be sampled in the device effluent, rather than remaining on the periphery of the islet tissue or diluted in a liquid suspension. Furthermore, as... Figure 4 As shown, fluorescence imaging technology is used to visualize the engineering vascular interface.

[0050] Example II: Multi-tissue vascular interface (bone marrow model) In this example, a mobilization model of leukocytes into the bloodstream is created by spatially patterning a segment or layer of human bone marrow between two vascular interfaces. This mobilization may be a response to inflammatory cytokines released in response to infection in other parts of the body. In this model, the spatial patterning of the two 3D vascular interfaces includes the space between them to allow for subsequent injection of whole human bone marrow suspended in 3D collagen and hyaluronic acid gel or ECM scaffolds, which can be spatially anchored by the vascular interfaces. After injection, angiogenic tissue in the vascular interfaces sprouts into the bone marrow tissue in a vascular regeneration manner to vascularize it and also engages with natural blood vessels in the bone marrow tissue. A second perfusionable vascular interface is created by patterning a fluid inlet channel to form a large vessel model, which is then coated with a monolayer of endothelial cells to form the vascular interface. The vascular interface engages with the previously formed 3D vascular interface to connect with the bone marrow tissue.

[0051] like Figure 5 As shown, the created model utilizes two 3D vascular interfaces to anchor the subsequent bone marrow tissue injection between them. The vascular interfaces are self-vascularized and form microvessels that selectively allow leukocytes to pass through their lumens while preventing larger chunks of bone marrow tissue from shifting due to fluid flow. Furthermore, an externally patterned flow channel is seeded with a monolayer of vascular endothelial cells to create a large-vessel vascular interface that can connect to smaller vessels within the 3D vascular interface. This interface chain with bone marrow tissue allows for modeling of leukocyte extravasation (i.e., migration into the blood vessels) into the bone marrow capillaries, such as in response to exposure to inflammatory cytokines, followed by transport of leukocytes to larger vessels, to model their transport through the circulatory system to inflamed tissue. Figure 6 As shown, leukocyte perfusion from the central bone marrow tissue compartment was detected via an engineered 3D vascular fluid interface. Perfusion was selectively controlled by the lumen diameter of the vessels formed therein. Figure 7 A 3D vascular interface with endothelium is shown on the inflow and outflow sides of leukocytes captured in a fluid suspension. Figure 8An alternative use of the apparatus for creating a bone marrow model is illustrated. In this experiment, two dense 3D vascular interfaces are seeded into patterned channels, between the channels, and on the outer surface seeded with a monolayer of vascular endothelial cells to create an additional layer of vascular interfaces. On the outer channels (upper and lower ends), these monolayer vascular interfaces model large blood vessels (arteries and veins). On the inner channels, the monolayer vascular interfaces sprout to the 3D vascular interfaces in a vascular regeneration manner, allowing for the formation of larger, stronger connections to the vascular lumen within the 3D vascular interfaces. After these connections are established, bone marrow tissue is seeded into the central channel, where vascular regeneration buds from the monolayer can form to create a perfusionable vascular interface. The perfusionable vascular interface extends from the top channel of the model to the bottom.

[0052] It should be understood that the foregoing only illustrates the principles of this disclosure, and those skilled in the art can make various changes without departing from the scope and spirit of this disclosure.

Claims

1. A microphysiological device for culturing tissues, comprising: The first channel includes at least one of hydrogel, cell, or tissue; The second channel includes at least one of a hydrogel, a cell, or a tissue, wherein at least one of the hydrogel, cell, or tissue in the second channel contacts one or more walls of the second channel and at least one of the hydrogel, cell, or tissue in the first channel; as well as A third channel includes at least one of a hydrogel, a cell, or a tissue, wherein at least one of the hydrogel, cell, or tissue in the third channel contacts one or more walls of the third channel and at least one of the hydrogel, cell, or tissue in the first channel.

2. The apparatus of claim 1, wherein at least one of the hydrogel, cell, or tissue in the first channel comprises a hydrogel, the hydrogel comprising at least one of a fibrin hydrogel or a collagen hydrogel.

3. The device of claim 1, wherein at least one of the hydrogel, cell, or tissue in the first channel comprises a cell, said cell comprising at least one of vascular endothelial cells, fibroblasts, pericytes, mesenchymal stem cells, or smooth muscle cells.

4. The device of claim 1, wherein at least one of the hydrogel, cell, or tissue in the first channel comprises tissue, said tissue comprising at least one of organoids, spheroids, bone marrow, pancreatic islets, β-islets, or biopsy carcinoma.

5. The apparatus of claim 1, wherein at least one of the hydrogel, cell, or tissue in the second channel comprises a hydrogel, the hydrogel comprising at least one of a fibrin hydrogel or a collagen hydrogel.

6. The device of claim 1, wherein at least one of the hydrogel, cell, or tissue in the second channel comprises a cell, said cell comprising at least one of vascular endothelial cells, fibroblasts, pericytes, mesenchymal stem cells, or smooth muscle cells.

7. The device of claim 1, wherein at least one of the hydrogel, cell, or tissue in the second channel comprises tissue, said tissue comprising at least one of organoids, spheroids, bone marrow, pancreatic islets, or biopsy carcinoma.

8. The apparatus of claim 1, wherein at least one of the hydrogel, cell, or tissue in the third channel comprises a hydrogel, the hydrogel comprising at least one of a fibrin hydrogel or a collagen hydrogel.

9. The device of claim 1, wherein at least one of the hydrogel, cell, or tissue in the third channel comprises a cell, said cell comprising at least one of vascular endothelial cells, fibroblasts, pericytes, mesenchymal stem cells, or smooth muscle cells.

10. The device of claim 1, wherein at least one of the hydrogel, cell, or tissue in the third channel comprises tissue, said tissue comprising at least one of organoids, spheroids, bone marrow, pancreatic islets, or biopsy carcinoma.

11. The apparatus according to claim 1, wherein The hydrogel, cells, or tissue in the first channel include at least one of the following: hydrogel and cells. The second channel is disposed on a first side of the first channel, and at least one of the hydrogel, cell, or tissue in the second channel includes cells, said cells including endothelial cells. The third channel is disposed on the second side of the first channel, and at least one of the hydrogel, cells, or tissue in the third channel includes cells, including endothelial cells, and The vascular cells in the second channel and the vascular cells in the third channel each form a vascular interface with the hydrogel and the cells in the first channel, allowing culture medium to be perfused through the hydrogel and the cells.

12. The apparatus of claim 11, wherein the cells in the first channel comprise at least one of vascular endothelial cells, fibroblasts, pericytes, mesenchymal stem cells, or smooth muscle cells.

13. The apparatus of claim 11, wherein the hydrogel in the first channel comprises at least one of a fibrin hydrogel or a collagen hydrogel.

14. The apparatus of claim 11, wherein the culture medium comprises hypoxia-inducible factor HIF, fibroblast growth factor FGF and / or vascular endothelial growth factor VEGF.

15. The apparatus of claim 11, wherein the first channel further comprises tissue.

16. The device of claim 15, wherein the tissue comprises at least one of an organoid, a spheroid, bone marrow, pancreatic islets, β-islets, or biopsy carcinoma.

17. The apparatus of claim 11 further comprises a fourth channel disposed on the side of the second channel opposite to the first channel, the fourth channel comprising hydrogel and cells.

18. The apparatus of claim 17 further includes a fifth channel disposed on the side of the fourth channel opposite to the second channel.

19. The apparatus of claim 11 further includes a sixth channel disposed on the side of the third channel opposite to the first channel.

20. The apparatus of claim 19 further includes a seventh channel disposed on the side of the sixth channel opposite to the third channel.

21. The apparatus of claim 1, wherein at least one of the hydrogel, cell, or tissue in the first channel comprises a cell, said cell comprising at least one of vascular endothelial cells, fibroblasts, pericytes, mesenchymal stem cells, or smooth muscle cells.

22. The apparatus of claim 1, wherein at least one of the hydrogel, cell, or tissue in the first channel comprises a hydrogel, the hydrogel comprising at least one of a fibrin hydrogel or a collagen hydrogel.

23. The apparatus of claim 1, wherein the first channel further comprises tissue.

24. The device of claim 23, wherein the tissue comprises at least one of an organoid, a spheroid, bone marrow, pancreatic islets, β-islets, or biopsy carcinoma.

25. The apparatus of claim 1 further includes a fourth channel disposed on the side of the second channel opposite to the first channel.

26. The device of claim 25, wherein the fourth channel comprises hydrogel and cells.

27. The apparatus of claim 25 further includes a fifth channel disposed on the side of the fourth channel opposite to the second channel.

28. The apparatus of claim 1 further includes a sixth channel disposed on the side of the third channel opposite to the first channel.

29. The apparatus of claim 28 further includes a seventh channel disposed on the side of the sixth channel opposite to the third channel.

30. A microphysiological device for culturing tissues, comprising: First passage; The second channel includes at least one of a hydrogel, a cell, or a tissue, wherein at least one of the hydrogel, cell, or tissue in the second channel contacts one or more walls of the second channel and is in fluid communication with the first channel. as well as The third channel is in fluid communication with at least one of the hydrogel, cell, or tissue in the second channel.

31. A method for forming a microphysiological device, comprising: Deposit at least one of hydrogel, cell or tissue in the first channel; At least one of a hydrogel, cell, or tissue is deposited in the second channel, and at least one of the hydrogel, cell, or tissue in the second channel contacts one or more walls of the second channel and at least one of the hydrogel, cell, or tissue in the first channel. as well as At least one of a hydrogel, cell, or tissue is deposited in the third channel, wherein at least one of the hydrogel, cell, or tissue in the third channel contacts one or more walls of the third channel and at least one of the hydrogel, cell, or tissue in the first channel.

32. A microphysiological device, comprising: Tissue pathways, including bone marrow tissue; The first and second vascularization channels are located on opposite sides of the tissue channel; A first flow channel and a second flow channel are fluidly coupled to one of the first vascularization channel and the second vascularization channel, respectively, and each of the first flow channel and the second flow channel includes an endothelial cell; as well as The inlet and outlet ports are configured to establish perfusion from the first flow channel to the second flow channel via the first vascularization channel, the tissue channel, and the second vascularization channel. The first and second vascularization channels include vascularization materials to form a perfusionable, endothelial-lined microvascular network, which is selectively connected to the bone marrow tissue in the tissue channel.

33. A method for forming a microphysiological device, comprising: Deposit the solid tissue into the tissue channels; The vascularization material is deposited into the first and second vascularization channels located on opposite sides of the tissue channel; An endothelial monolayer is established within a first flow channel and a second flow channel, each of the first flow channel and the second flow channel being fluidly coupled to a corresponding fluid of the first vascularization channel and the second vascularization channel. as well as The growth medium is perfused from the first flow channel into the second flow channel through the vascularization material and the solid tissue.

34. A microphysiological device, comprising: The central corridor, including its substantive organization; Each of the first adjacent channel and the second adjacent channel includes vascular generating material that forms a vascular interface; as well as The first fluid flow channel and the second fluid flow channel are configured to transport the growth medium from the first adjacent channel to the second adjacent channel via the central channel.

35. A method for culturing tissues in a microphysiological device, comprising: The substantial tissue is deposited into the central channel; Deposit the vascularization material into the first adjacent channel and the second adjacent channel; The vascularization material is allowed to form a perfusion-compatible, endothelial-lined microvascular network within each of the first adjacent channel and the second adjacent channel; as well as The growth medium is infused from the first adjacent channel into the second adjacent channel through the central channel.

36. A microphysiological device, comprising: A first vascular pathway includes a vascularization material configured to form a perfusionable vascular interface. The first organizational channel and the second organizational channel are located on opposite sides of the central channel, each of the first organizational channel and the second organizational channel includes substantial tissue; The second and third vascular channels are fluidly coupled to a corresponding first and second tissue channels, each of the second and third vascular channels including a vascular generation material for forming a perfusionable vascular interface. as well as A first fluid flow channel and a second fluid flow channel are fluidly coupled to a corresponding second vascular channel and a third vascular channel, and are configured to deliver growth medium from the first fluid flow channel to the second fluid flow channel via the second vascular channel, the first tissue channel, the first vascular channel, the second tissue channel, and the third vascular channel.

37. A method for perfusion-linking multiple tissues in a microphysiological device, comprising: Deposit vascularization material within the first vascular channel to form a perfusionable vascular interface therein; Substantial tissue is deposited in the first and second tissue channels on opposite sides of the central channel; A vascularization material is deposited within a second and a third vascular channel that are fluidly coupled to the first and second tissue channels, respectively, to form a perfusionable vascular interface. as well as Fluid is infused from the first fluid flow channel into the second fluid flow channel via the second vascular channel, the first tissue channel, the first vascular channel, the second tissue channel, and the third vascular channel, wherein the first fluid flow channel and the second fluid flow channel are fluid-coupled to a corresponding fluid of the second vascular channel and the third vascular channel.

38. A microphysiological device, comprising: The first organizational channel includes substantive organizations; The second and third tissue channels are located on opposite sides of the first tissue channel, each of the second and third tissue channels comprising substantial tissue. A first vascular channel and a second vascular channel, fluidly coupled to a corresponding second tissue channel and a third tissue channel, each of the first vascular channel and the second vascular channel including a vascularization material for forming a perfusionable vascular interface; as well as A first fluid flow channel and a second fluid flow channel are fluidly coupled to a corresponding first vascular channel and a second vascular channel, and are configured to deliver growth medium from the first fluid flow channel to the second fluid flow channel via the first vascular channel, the second tissue channel, the first tissue channel, the third tissue channel, and the second vascular channel.

39. A microphysiological device, comprising: The solid tissue is deposited into the first tissue channel; The solid tissue is deposited into the second and third tissue channels on opposite sides of the first tissue channel; Vascular agglomerating material is deposited into a first vascular channel and a second vascular channel that are fluidly coupled to the second tissue channel and the third tissue channel, wherein the vascular agglomerating material in each of the first vascular channel and the second vascular channel is configured to form a perfusionable vascular interface. as well as Fluid is infused from a first fluid flow channel into a second fluid flow channel via the first vascular channel, the second tissue channel, the third tissue channel, and the second vascular channel, wherein the first fluid flow channel and the second fluid flow channel are fluid-coupled to a corresponding fluid of the first vascular channel and the second vascular channel.

40. A microphysiological device, comprising: A tissue receiving chamber having a surface configured to facilitate the attachment of discrete tissue aggregates transported in suspension form. The covered vascularization area is configured to be deposited on top of the attached tissue aggregates; as well as The perfusion path is connected to the covered vascularization region. The covered vascularization region is configured to develop an endothelialized microvascular network to perfuse attached tissue aggregates.

41. A method for vascularizing attached tissue aggregates in a microphysiological device, comprising: A suspension of discrete tissue aggregates is introduced into the tissue receiving chamber, allowing the aggregates to settle and adhere. The vascularization material is placed on the attached aggregate to form a covered vascularization area; A perfusionable endothelialized microvascular network is formed within the covered area; and Perfusion of growth medium provides vascular access for attached aggregates by covering the area.

42. An on-chip tissue device, comprising: A chamber is configured to receive a pregel composition comprising a vascularization material and discrete solid tissue units; as well as The inlet and outlet ports perfuse the culture medium through microvessels formed within the curing composition, wherein the composition is configured to generate a perfusing endothelialized microvascular network upon curing, the microvascular network being connected to each discrete solid tissue unit.

43. A method for embedding and vascularizing discrete tissue in a microphysiological device, comprising: Discrete solid tissue units are suspended in pregelated vascularization materials; The suspension is placed into the chamber, and the vascularization material is solidified; Formation of a perfusionable endothelialized microvascular network within the solidified material; and The growth medium is perfused through the microvascular network to provide vascular access for each tissue unit.

44. A vascular interface device, comprising: The tissue pathway includes at least one of solid tissue and vascular-forming material; The first vascular channel and the second vascular channel include vascular generating material, and the first vascular channel and the second vascular channel are arranged on opposite sides of the tissue channel. as well as A first flow channel and a second flow channel are fluidly coupled to a corresponding first vascular channel and a corresponding second vascular channel, the first flow channel and the second flow channel comprising endothelial cells.

45. A method for creating a large-area vascular interface in a microphysiological device, comprising: Deposit at least one of solid tissue and vascularization material within the tissue channel; A vascularization material is deposited within a first vascular channel and a second vascular channel arranged on opposite sides of the tissue channel. Endothelial cells are deposited within a first flow channel and a second flow channel that are fluidly coupled to the first vascular channel and the second vascular channel, respectively. as well as Fluid is infused from the first flow channel into the second flow channel via the first vascular channel, the tissue channel, and the second vascular channel.

46. ​​An extracorporeal circulation model device, comprising: The capillary bed chambers are configured as a microvascular network developed from vascularization materials and endothelialized. The first flow channel and the second flow channel are located on opposite sides of the capillary bed chamber, each having a surface configured to support a cell monolayer for simulating a large blood vessel interface. A fluid interface located between each flow channel and the capillary bed chamber allows for perfusion fluid exchange while retaining the bulk gel. as well as The controller is configured to drive directional flow from the first flow channel through the capillary bed chamber to the second flow channel to model orderly macrovessel-capillary-macrovessel transport.

47. A method for modeling extracorporeal circulation in a microphysiological device, comprising: Introduce vascularization materials into the capillary bed chambers to form an endothelialized microvascular network; A cell monolayer is formed on the inner surface of the first and second flow channels on opposite sides of the capillary bed chamber to simulate a large blood vessel interface; and The growth medium is directionally perfused from the first flow channel through the capillary bed chamber to the second flow channel to simulate arterial-capillary-venous transport.

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