Method and apparatus for forming microfluidic gel structures - Patents.com

Abstract

A method for making a lumenalized gel structure is described. The method includes the steps of introducing a first liquid including a gel precursor solution into a microfluidic network, the microfluidic network including a capillary pressure barrier at a location that generally defines a boundary between a first region and a second region of the microfluidic network, allowing the first liquid to enter the first region of the microfluidic network and align itself along the capillary pressure barrier, thereby forming a liquid-air meniscus of the first liquid at the boundary between the first region and the second region of the microfluidic network, forming a lumen through the first liquid by contacting the first liquid with a second liquid, the second liquid having a lower viscosity than that of the first liquid, and allowing or causing gelation of the first liquid to form a gel structure including a lumen therethrough. Also described are devices including a gel with a lumen extending therethrough, and the use of the devices and lumenalized gel structures in assays.

Description

[Technical field]

[0001] The present invention relates to a method and device that allows 3D cell culture, allowing controlled and reliable vascularization and / or perfusion of organoid assays and / or cell cultures.The present invention also relates to the use of the device and method obtained for investigating cell responses to stimulants. [Background technology]

[0002] In a move to emulate ever more physiologically relevant conditions in cell culture, numerous models have been developed to allow, for example, perfusion flow and co-culture in cell-based preclinical models for evaluating drug efficacy and / or ADME safety.

[0003] Microfluidics has become a popular platform technology for such in vitro cell culture models due to the inherent flow of liquids or media during use, together with advances in microengineering techniques that facilitate and enable the creation of complex microfluidic networks. However, there is still great interest in creating models that simulate or replicate the cellular environment of various organs of the human or animal body.

[0004] Organoid culture, or more generally speaking 3D cell culture, can be carried out in various ways. 3D spheroids can be formed in so-called hanging drop plates (see, for example, WO 2010 / 031194) or in low-attachment microtiter plates. Although these spheroids are claimed to have significantly improved predictability over standard cell culture, they are not used for most organoid cultures. The reason is that organoids require extracellular matrix components such as matrigel or collagen, which are usually not present in spheroids in hanging drop plates or low-attachment plates. Parallel efforts have led to the development of 3D cell culture models in which cells are embedded and grown in an extracellular matrix. This approach enhances the expression of differentiated functions and improves tissue organization (Pampaloni et al. (2007). Nat Rev Mol Cell Biol 8:839-84).

[0005] Typical platforms for growing organoids include standard Petri dishes, microtiter plates, and sometimes Transwell® plates made by Corning. In these cases, organoids are grown in extracellular matrix (ECM) or on ECM-coated wells. As already addressed above, these organoids lack the presence of vasculature, and therefore their growth is limited, since beyond a certain size, hypoxia and, at a later stage, necrotic cores may form. It is also hypothesized that the presence of endothelium is important for development into physiologically appropriate tissues, since endothelium excretes important factors for target tissues.

[0006] Microfluidic cell culture has become an increasingly important technology, finding applications in drug screening, tissue culture, toxicity screening, and biological research.

[0007] Numerous microfluidic systems, devices, methods and manufacturing techniques are known, including those disclosed in patent publications such as WO 2008 / 079320, WO 2013 / 151616, WO 2010 / 086179, WO 2012 / 120101, or commercially available, for example, from Mimetas, Leiden, The Netherlands (e.g., OrganoPlate, www.mimetas.com). No specific limitations should be read into any claims presented herein from these applications and publications, but these publications provide useful background material.

[0008] A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip. S. Deosarkar, B. Prabhakarpandian, B. Wang, JB Sheffield, B. Krynska, M. Kiani. In PLOS ONE, 2015, a microfluidic device was developed to generate vasculature that uses sieve-like structures to separate endothelium from astrocytes in an attempt to generate a blood-brain barrier type structure. In WO 2007 / 008609 A2, similar sieve-like structures are used to form cell aggregates to create a tissue morphology that better mimics, for example, liver physiology. Summary of the Invention [Problem to be solved by the invention]

[0009] There remains a need for methods and devices that allow perfusion culture of ECM-supported biological tissues that more closely resemble the in vivo situation. Of particular interest are methods and devices that allow the patterning of multiple cell types in adjacent constructs while minimizing spatial separation and physical barriers between such constructs. The technology must also be compatible with current readout and processing equipment.

[0010] It is an object of the present invention to address some or all of the above needs. [Means for solving the problem]

[0011] According to a first aspect of the present invention, there is provided a method for making a lumenalized gel structure, comprising the steps of: introducing a first liquid comprising a gel precursor solution into a microfluidic network, the microfluidic network comprising a capillary pressure barrier at a location generally defining a boundary between a first region and a second region of the microfluidic network; Allowing a first liquid to enter a first region of the microfluidic network and align itself along a capillary pressure barrier, thereby forming a liquid-air meniscus of the first liquid at the interface between the first and second regions of the microfluidic network; forming a lumen through the first liquid by contacting the first liquid with a second liquid, the second liquid having a lower viscosity than the viscosity of the first liquid; allowing or causing the first liquid to gel to form a gel structure including a lumen therethrough; A method is provided that includes:

[0012] According to a second aspect of the invention there is provided an apparatus comprising a microfluidic network, the microfluidic network comprising: At least two entrances; a capillary pressure barrier positioned to define a boundary between a first region and a second region of the microfluidic network; a gel provided in a first region extending between two of the at least two inlets and held in the first region by a capillary pressure barrier; Including, the gel includes a lumen extending therethrough between two of the at least two inlets, the gel having a first surface facing the lumen and a second surface facing a second region of the microfluidic network, the thickness of the gel between the first surface and the second surface being 200 μm or less; An apparatus is provided.

[0013] According to a third aspect there is provided use of a lumenalised gel structure produced by the method of the first aspect in an assay, such as an assay selected from one or more of a barrier function assay, a transepithelial electrical resistance (TEER) assay, an immune cell adhesion assay, an immune cell migration assay, a transporter assay and a vasodilation or vasoconstriction assay.

[0014] According to a fourth aspect of the invention there is provided use of a device as defined in the second aspect in an assay, such as an assay selected from one or more of a barrier function assay, a transepithelial electrical resistance (TEER) assay, an immune cell adhesion assay, an immune cell migration assay, a transporter assay, and a vasodilation or vasoconstriction assay.

[0015] Further preferred embodiments are defined in the following description and the dependent claims.

[0016] The inventors have unexpectedly found that strategic positioning of a capillary pressure barrier in combination with a viscous fingering technique allows the formation of a lumenized gel structure in a first region of a microfluidic network with an exposed surface facing another region of the microfluidic network. Previously, lumenized gel structures in a microfluidic network have either filled a microfluidic channel and contacted the channel walls on all sides or been supported by a membrane to allow diffusion into and out of the gel. Because the viscous fingering technique relies on one liquid forming a lumen through another liquid, it is unexpected that the viscous fingering lumenization did not destroy the surface tension of the first liquid anchored to the capillary pressure barrier, which could lead to the collapse of the anchored meniscus. While one might expect the need for a supporting wall to prevent the lumenized liquid from flowing out to the side, the present invention shows an unexpected method that allows the formation of a lumen in close proximity to an open space without a constraining wall.

[0017] The methods and devices of the present invention allow the formation of 3D constructs in an extracellular matrix with a thin interstitial space, thus mimicking the in vivo situation in a way that was previously only achievable using membranes. This allows for the controlled co-culture of endothelialized lumens in close proximity to monolayer and / or three-dimensional cultures of tissue-specific cells in a physiologically realistic environment, for example, co-culture of endothelium, pericytes, astrocytes and neurons in a more realistic arrangement and matrix than existing microfluidic blood-brain barrier models.

[0018] definition Various terms relating to the devices, methods, uses and other aspects of the invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains unless otherwise specified. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the invention, the preferred materials and methods are described herein.

[0019] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0020] As used herein, "about" and "approximately": these terms, when referring to measurable values ​​such as amounts, durations, and the like, are meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value, as such variations are appropriate for carrying out the disclosed methods.

[0021] As used herein, "comprising" is intended to be inclusive, open-ended, and not exclusive. Specifically, this term and variations thereof mean that the specified features, steps, or components are included. These terms should not be interpreted to exclude the presence of other features, steps, or components.

[0022] As used herein, "exemplary" means "serving as an example, instance, or illustration," and should not be interpreted as excluding other configurations disclosed herein.

[0023] As used herein, the term "microfluidic network" refers to one or more channels on or through a layer of material covered by a top substrate or cover, with at least one of the dimensions length, width or height being in the low millimeter (e.g., less than 5 mm or less than 2 mm) or sub-millimeter range. The term will be understood to encompass channels that are straight channels and channels that are branched or have bends or corners in their path. A microfluidic network typically includes at least one inlet for dispensing a volume of liquid, but may include multiple inlets for dispensing a volume of liquid to different regions of the microfluidic network. The volume enclosed by a microfluidic network is typically in the microliter or sub-microliter range. A microfluidic network typically includes a base, which may be the top surface of an underlying material, at least two sidewalls, and a ceiling, which may be the bottom surface of a top substrate covering the microfluidic network, with inlets, outlets and / or vents in any arrangement as needed. The base, sidewalls and ceiling may each be referred to as the inner surface of the microfluidic network, or collectively as the inner surfaces. In some instances, the microfluidic network may have a circular or semicircular cross-section, in which case it may be considered to have one or two interior surfaces, respectively.

[0024] As used herein, the term "capillary pressure barrier" refers to a feature of a device that holds a liquid-air meniscus pinned at a particular location by capillary forces. The capillary pressure barrier can be thought of as dividing a microfluidic network having a volume V0 into two regions or sub-volumes V1 and V2 into which different fluids can be introduced. In other words, the capillary pressure barrier generally defines a boundary between a first region and a second region of the microfluidic network. Although the capillary pressure barrier generally defines a boundary between regions of the microfluidic network, it will be understood that the resulting meniscus of liquid pinned in one region may not be pinned at the exact location of the capillary pressure barrier, but may remain pinned and extend or bulge beyond the capillary pressure barrier into an adjacent region. For example, the liquid meniscus may be convex in shape, pinned by the capillary pressure barrier with a convex liquid front extending beyond the footprint of the capillary pressure barrier. The liquid meniscus may also be concave, with the solvent front being pinned by the capillary pressure barrier and extending beyond the footprint of the capillary pressure barrier at the surface of the microfluidic network opposite the capillary pressure barrier.

[0025] As used herein, a "straight" capillary pressure barrier should not be construed as being limited to a straight line. Instead, it should be construed as having two ends, but having a configuration that may include one or more bends or angles. A straight capillary pressure barrier typically intersects with a sidewall of the microfluidic network at each end.

[0026] As used herein, the term "endothelial cell" refers to a cell of endothelial origin or a cell that has differentiated to a state in which it expresses markers that identify the cell as an endothelial cell. As used herein, the term "epithelial cell" refers to a cell of epithelial origin or a cell that has differentiated to a state where it expresses markers that identify the cell as an epithelial cell.

[0027] As used herein, the term "biological tissue" refers to a collection of functionally interconnected cells of the same, similar or different types that are cultured and / or assayed by the methods described herein. The cells may be or take the form of cell aggregates, tubular structures or monolayers. Biological tissues may be composed of multiple subtypes of cells. For example, the term "biological tissue" encompasses cells derived from or including cell lines, organoids, tissue biopsies, tumor tissues, excised tissue materials, and embryonic bodies.

[0028] As used herein, the term "cell aggregate" refers to a 3D cluster of cells, in contrast to surface-attached cells that usually grow in a monolayer. 3D clusters of cells usually relate to a more in vivo-like situation. In contrast, surface-attached cells can be strongly influenced by the properties of the substrate and can undergo dedifferentiation or transition to other cell types.

[0029] As used herein, the term "organoid" refers to miniature forms of tissue generated in vitro that exhibit endogenous three-dimensional organ structure.

[0030] As used herein, the term "co-culture" refers to the culturing of two or more different cell types in the devices described herein. The different cell types can be cultured in the same region of the device (e.g., a first region or a second region) and / or in different regions (e.g., one cell type in the first region and another cell type in the second region).

[0031] For example, the devices described herein may have endothelial cells in a first region grown as tubules with an open lumen and organ-specific (parenchymal) cells in a second region separated by a thin layer of gel in the first region. In some examples, the device may include at least one lumenized gel structure lined with endothelium in the first region and tissue-specific cells in the second region. The tissue-specific cells may be disposed throughout the gel structure in the second region and cover the second region to form tubules that contact the gel structure of the first region (including the lumen lined with endothelium) or cover a lumen extending through the gel structure of the second region.

[0032] As used herein, the term "lumenized gel structure" refers to a biocompatible gel, more preferably a biologically relevant gel, such as an extracellular matrix, having a lumen through the gel, allowing for the formation of, for example, microvessels having an apical and basal surface. It should be understood that "lumenized" and "lumened" can be used interchangeably to have the same meaning.

[0033] As used herein, the term "luminalized cellular component" refers to a living tissue (ie, made up of cells) that has a lumen, such as a microvessel having an apical surface and a basal surface.

[0034] As used herein, the term "implant" or "transplantation" refers to the transfer of tissue, e.g., tissue explants, or cell aggregates, from one location to another, e.g., from a storage container to a cell culture device.

[0035] As used herein, and unless otherwise stated, references to viscosity are to dynamic viscosity, determined as described by Kane et al. (AIP Advances 8, 125332 (2018)). The relationship between lumen formation and viscosity is understood by those skilled in the art as Saffman-Taylor instability, and was described by Saffman and Taylor in 1958 (Proceedings of the Royal Society of London. Series A. Mathematical and Physical Sciences. 245(1242):312-329). The relationship between microchannel dimensions and other relevant properties related to the extracellular matrix is ​​described, for example, in Bischel et al. (Journal of Laboratory Automation 17(2)96-103).

[0036] The invention will now be described, by way of example only, with reference to the drawings in which: [Brief description of the drawings]

[0037] [Figure 1A] 1A-1D show cross-sectional views of a sequence of steps according to the method described herein for forming a lumenalized gel structure. [Figure 1B] 1A-1D show plan views of a sequence of steps according to the methods described herein for forming a lumenalized gel structure. [Figure 1C] 1A-1D show cross-sectional views of a sequence of steps according to the method described herein for forming a lumenalized gel structure. [Diagram 2] 1A-1D show cross-sectional views of a sequence of steps according to the method described herein for forming a lumenalized gel structure. [Diagram 3] 1A-1D show cross-sectional views of a sequence of steps according to the method described herein for forming a lumenalized gel structure. [Figure 4A] 1A-1D show cross-sectional views of a sequence of steps according to the method described herein for forming a lumenalized gel structure. [Figure 4B]1A-1D show cross-sectional views of a sequence of steps according to the method described herein for forming a lumenalized gel structure. [Diagram 5] A cross-sectional view of a device containing two adjacent luminalized cellular components and remodeling of the extracellular matrix is ​​shown. [Figure 6A] 1 shows a cross-sectional view of an exemplary device of the present disclosure including a luminalized cell component. [Figure 6B] FIG. 1 shows a cross-sectional view of an exemplary device of the present disclosure that utilizes a luminalized gel structure to form a luminalized cellular component. [Figure 7] FIG. 1 shows a cross-sectional view of an exemplary device of the present disclosure containing three luminalized cell components. [Figure 8] FIG. 1 shows a cross-sectional view of an exemplary device of the present disclosure containing three luminalized cell components. [Figure 9A] FIG. 8 shows an experimentally obtained confocal microscope cross-sectional view of the device according to the present invention. [Figure 9B] Figure 5 shows an experimentally obtained confocal microscope plan view of the device according to the present invention. [Figure 10] 13 shows an experimentally derived confocal microscope cross-section of a further device according to the present disclosure. [Figure 11A] FIG. 13 shows a cross-sectional view of a further exemplary device of the present disclosure comprising two luminalized cell components. [Figure 11B] FIG. 11B shows an experimentally obtained phase contrast microscope plan view of the device according to FIG. [Figure 12] FIG. 9 shows an experimentally derived phase contrast microscopy plan view image of the device of FIG. 8 containing three adjacent luminalized cellular components. [Figure 13] 13 shows an experimentally derived high resolution image of a plan view of a further device according to the present disclosure. [Figure 14] FIG. 1 shows an experimentally derived phase contrast cross-section of a blood-brain barrier model created in accordance with the present disclosure. [Figure 15] FIG. 1 shows an experimentally derived confocal microscopy cross-section of a blood-brain barrier model created in accordance with the present disclosure. [Figure 16A] FIG. 8 shows a cross-sectional view of an embodiment of the device of FIG. 7 containing one luminalized cell component. [Figure 16B]FIG. 16B shows an experimentally obtained phase contrast microscope plan view of the device according to FIG. 16A. [Figure 17A] FIG. 8 shows a cross-sectional view of an embodiment of the device of FIG. 7 containing two luminalized cell components. [Figure 17B] FIG. 17B shows an experimentally obtained phase contrast microscope plan view of the device according to FIG. 17A. [Figure 18A] 1 shows an experimentally derived confocal microscopy cross-section of a coronary artery model created in accordance with the present disclosure. [Figure 18B] 1 shows an experimentally derived confocal microscopy cross-section of a coronary artery model created in accordance with the present disclosure. [Figure 18C] 1 shows an experimentally derived confocal microscopy cross-section of a coronary artery model created in accordance with the present disclosure. [Figure 19] FIG. 1 shows an experimentally derived phase contrast cross-section of a T cell migration model created in accordance with the present disclosure. [Figure 20] 1 shows experimentally obtained phase contrast microscopy cross sections of a comparative T cell migration model. [Figure 21] Quantitative data obtained from the model shown in Figures 19 and 20 are presented. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Device An apparatus is described. The apparatus may be a microfluidic device, also referred to herein as a microfluidic device. The apparatus includes at least one microfluidic network, e.g., a plurality of microfluidic networks. The microfluidic device is preferably in a multi-array / multi-well format, particularly a high-throughput screening format, to enable its use in in vitro cell-based assays, drug screening assays, toxicity assays, and the like. Such multi-well culture plates are available with 6, 12, 24, 48, 96, 384, and 1536 sample wells arranged in a rectangular matrix, and in the context of the present invention, the multi-array arrangement of the microfluidic networks described herein is present in a microfluidic device. In one example, the microfluidic device conforms to one or more dimensions of a standard ANSI / SLAS microtiter plate format. In an alternative embodiment, the microfluidic device is a multi-array format having the dimensions of a microscope glass slide. In some examples, the microfluidic device is provided with one or more features, including one or more electrodes for performing electrical experiments, transparent materials for enabling optical measurements to be performed, windows or other modifications, and the like.

[0039] Thus, the microfluidic device preferably comprises a plurality of microfluidic networks as described herein. In one example, the multiple microfluidic networks are fluidly disconnected from one another, i.e., each microfluidic network operates independently of any other microfluidic networks present on the microfluidic device. In another example, the microfluidic networks may be connected by one or more connecting channels, as described below.

[0040] Generally, a microfluidic device is one that includes at least a microfluidic network having microfluidic channels. Different configurations of the microfluidic channels or networks are possible within the scope of the invention, but may include, for example, one or more regions for receiving and retaining a gel, such as an extracellular matrix.

[0041] A microfluidic device generally comprises a microfluidic network, which is now described in more detail.

[0042] Microfluidic Networks The microfluidic network of a microfluidic device generally comprises a base, a microfluidic channel or layer, and a cover, also referred to herein as a cover layer, and can be fabricated in a variety of ways.

[0043] The base, also referred to herein as the base layer or lower substrate, is preferably formed from a substantially rigid material such as glass or plastic and serves to provide a support surface for the remainder of the microfluidic network. In one example, the base has dimensions that are the same as or similar to the well area of ​​a standard ANSI / SLAS microtiter plate.

[0044] A microfluidic device or network includes a microfluidic channel or layer disposed on a base. In some examples, the microfluidic network includes different regions or may be divided into different regions, for example, by the presence of a capillary pressure barrier as described herein. In some examples, the microfluidic network may include a first region and a second region. The capillary pressure barrier may generally define a boundary between adjacent regions of the network, for example, between the first region and the second region. In some examples, the microfluidic network includes one or more microfluidic channels, each forming a region of the microfluidic network and fed by its own dedicated inlet and / or outlet.

[0045] The first and second regions can each have a width dimension between about 100 μm and about 10 mm, or between about 200 μm and about 500 μm, or between about 300 μm and about 400 μm.

[0046] A typical method for making a microfluidic network is to cast a moldable material such as polydimethylsiloxane onto a mold to imprint the microfluidic network into a silicone rubber material, thereby forming a microfluidic layer. The rubber material with embedded network or channels is then placed onto a base layer of glass or the same material, thus sealing. Alternatively, channel structures can be etched into a material such as glass or silicon, and then bonded to a top or bottom substrate (also referred to herein as a cover layer and a base layer). Injection molding or embossing of plastics, followed by bonding, are other methods for making a microfluidic channel network. Yet another technique for making a microfluidic channel network is by photolithographically patterning the microfluidic channel network into a photopatternable polymer, such as SU-8 or various other dry film or liquid photoresists, followed by a bonding step. When referring to bonding, it means the closure of the channels by a cover or base. Bonding techniques include, among others, anodic bonding, covalent bonding, solvent bonding, adhesive bonding, and thermal bonding.

[0047] As can be deduced from the various manufacturing methods described above, the microfluidic layer may include a sublayer including a microfluidic channel disposed on the base layer or patterned in either the cover or base layer. In the in-use position, the microfluidic sublayer is disposed on the top surface of the base layer. The microfluidic channel may be formed as a channel through the sublayer of material disposed on the base layer. In one example, the material of the sublayer is a polymer disposed on the base layer, in which the microfluidic channel is patterned. In some examples, the microfluidic layer includes two or more microfluidic networks that may be in fluid communication with each other.

[0048] As required for any particular use of the microfluidic network of the microfluidic device, the microfluidic channel may be provided with one or more fluid inlets and one or more outlets or vents. To allow filling, draining and perfusion of fluids through the microfluidic network, the microfluidic channel is preferably provided with at least two inlets. In one example, each of the at least two inlets is preferably an opening in the cover layer. It will be understood that there is usually no geometrical distinction between inlets and outlets, and in many cases they can be used interchangeably as inlets or outlets. In particular, it will be understood that the inlet opening fluidically connected to the area opposite the inlet opening where the liquid is introduced will then function as an outlet (or vent), allowing the evacuation of air or excess liquid.

[0049] The microfluidic network may include at least two inlets, each of which is configured to allow the introduction of liquid into a first region of the microfluidic network or the removal of liquid from the first region. In some examples, the microfluidic network may include at least three inlets, e.g., at least four inlets, with at least two of the inlets configured to allow the introduction of liquid into a second region of the microfluidic network or the removal of liquid or the evacuation of air from the second region. In some examples, the microfluidic network may further include two additional inlets, each of which is configured to allow the introduction of liquid into a third region of the microfluidic network or the removal of liquid or the evacuation of air from the third region. It will be understood that an inlet configured to allow the introduction of liquid into any given region of the microfluidic network means a fluidic connection to that region, e.g., by a fluidic interface that allows the injection or pipetting of liquid into the region or into a microfluidic channel communicating with the region in question. The location of the capillary pressure barrier may indicate a boundary between two different regions of the microfluidic network. It is to be understood that although the boundary between the two regions is generally aligned in a plane with such capillary pressure barrier, the interface between the two regions may curve in any direction away from the projection of the capillary pressure barrier.

[0050] In some examples, the microfluidic device further includes a top layer disposed on the cover layer described above, the top layer having one or at least one well or reservoir in fluid communication with the remainder of the microfluidic device. In some examples, the top layer has a plurality of such wells, with at least one, e.g., at least two, e.g., at least three, wells in communication with a microfluidic network or channel of the device. For example, the top layer can include a well or reservoir in fluid communication with the microfluidic network through an inlet opening provided in the cover layer of the microfluidic network, thereby forming a SLAS-compliant well plate. In some examples, the top layer with at least one well and the microfluidic layer are integrally formed. For example, a microfluidic channel can be patterned on the underside of an injection molded microtiter plate having at least one well.

[0051] Capillary Pressure Barrier The microfluidic network of the device includes a capillary pressure barrier that generally defines a boundary of the microfluidic network between a first region of the network and a second region of the network.

[0052] The function and patterning of the capillary pressure barrier has already been described, for example, in WO 2014 / 038943 A1. As will become clear from the exemplary embodiments described below, the capillary pressure barrier should not be understood as a wall (or a cavity, for example, that can be filled with a liquid), but instead consists of or includes a structure that ensures that such liquid does not spread due to surface tension. This concept is called meniscus pinning. Thus, a stable confinement of the liquid to a region of the microfluidic network can be achieved. In one example, the capillary pressure barrier, which may also be called a confining phase guide, is configured not to overflow during normal use of the cell culture device. The nature of the confinement of the liquid is described herein in connection with the description of the method of the invention.

[0053] In one example, the capillary pressure barrier is provided on the inner surface of the microfluidic network and comprises a ridge, groove, or line of material having an increased water-air contact angle with respect to the inner surface of the microfluidic network. In one example, the capillary pressure barrier comprises or consists of a rim or ridge of material protruding from the inner surface of the microfluidic network, or a groove in the inner surface of the microfluidic network. To provide a good barrier, the interior angle formed by the sidewall of the ridge or ridge and the top of the ridge or ridge is preferably less than 110°, for example about 90°, in some examples less than 90°. The same is true for the angle between the sidewall of the ridge and the inner surface of the microfluidic network on which the capillary pressure barrier is disposed. Similar requirements are imposed on capillary pressure barriers formed as grooves.

[0054] An alternative form of capillary pressure barrier is a region of material with different wettability relative to the inner surface of the microfluidic network, which serves to prevent spreading due to capillary forces / surface tension. As a result, liquid is prevented from flowing beyond the capillary pressure barrier, allowing the formation of a stable trapped volume within the region of the network. In one example, the inner surface of the microfluidic network comprises a hydrophilic material and the capillary pressure barrier is a region of hydrophobic or relatively less hydrophilic material. In one example, the inner surface of the microfluidic network comprises a hydrophobic material and the capillary pressure barrier is a region of hydrophilic or relatively less hydrophobic material.

[0055] Thus, in some examples, the capillary pressure barrier is selected from edges or ridges of material, grooves, holes, or hydrophobic lines, or combinations thereof. In another embodiment, the capillary pressure barrier can be made of posts at selected intervals, the arrangement of which defines a first region, i.e., the area occupied by the gel. In one example, the posts extend the entire height of the microfluidic network.

[0056] In one example, the capillary pressure barrier is a substantially straight capillary pressure barrier that spans the entire width or length of the microfluidic channel or network and intersects the sidewalls of the microfluidic network at each end.

[0057] In another example, the capillary pressure barrier is not linear, but includes one or more bends or arcs. For example, the capillary pressure barrier may include a series of angled bends or arcs, such that a serpentine or even right-angled shape is created. In this way, the path of the fluid guided along the capillary pressure barrier extends along the path that the fluid could flow along a linear capillary pressure barrier. The advantage of having a non-linear capillary pressure barrier is that the lumen created in the fluid aligned along such a capillary pressure barrier may have a non-linear shape, for example, mimicking the crypt-villus structure of the small intestine, and / or the length of the non-linear lumen is extended compared to its linear counterpart.

[0058] The intersection of the capillary pressure barrier with one or more side walls of the microfluidic channel may have an angle of greater than 70°, more preferably about 90°, more preferably greater than 90° with respect to the expected filling direction of the first fluid downstream of the capillary pressure barrier, preferably as large as possible to provide a good barrier, as described in WO 2014 / 038943.

[0059] In some examples, the capillary pressure barrier is patterned on the inner surface of the microfluidic network to mimic biological structures. Forming the capillary pressure barrier in a shape or configuration to mimic biological structures facilitates the formation of an in vitro system that more closely resembles an in vivo system. For example, the capillary pressure barrier may include a sinusoidal shape to mimic the crypt-villus structure.

[0060] Second Capillary Pressure Barrier In some examples, the microfluidic network of the device is provided with a second capillary pressure barrier, the form and function of which is substantially as described above. For the avoidance of doubt, when a second capillary pressure barrier is present in the microfluidic network, references to a "capillary pressure barrier" should be understood as references to a "first capillary pressure barrier."

[0061] In some examples, the second capillary pressure barrier defines a boundary between the first region and the third region of the microfluidic network, or between the second region and the third region of the microfluidic network. Thus, a microfluidic network having two capillary pressure barriers can include a first region, a second region and a third region, each of which can be supplied by at least one dedicated fluid interface, such as its own dedicated inlet and outlet or vent. In some examples, the second capillary pressure barrier is provided at a location that generally defines a boundary between the first region and the third region of the microfluidic network, such that the liquid can align itself along the first capillary pressure barrier and the second capillary pressure barrier, thereby forming a third surface of the gel structure facing the third region of the microfluidic network. In some further examples, a second capillary pressure barrier is provided at a location generally defining the boundary between the second region and a third region, optionally allowing for patterning and potentially allowing for the formation of a second gel lumen in the third region. It will be appreciated that multiple capillary pressure barriers, regions and gel structures can be combined into increasingly complex microfluidic networks and gel structures.

[0062] gel The gel is provided in a first region of the microfluidic network, extending between the two inlets and constrained to the first region by a capillary pressure barrier. The gel includes a lumen extending therethrough between the two inlets and has a first surface facing the lumen and a second surface facing the second region of the microfluidic network, and the thickness of the gel between the first and second surfaces can be 200 μm or less. In some examples, the lumen is substantially cylindrical, e.g., has a substantially circular cross-section.

[0063] Formation of a lumen through the gel can be carried out according to the methods described herein. The combination of a capillary pressure barrier and lumen formation by the gel provides two surfaces of the gel, thus enabling or allowing the formation of 3D construct tissue with thin interstitial spaces that mimic the in vivo situation in a way that has not been achieved before.

[0064] A gel can be provided to a first region of the microfluidic network by introducing a gel precursor solution to the first region, for example via an inlet feeding the first region, and a lumen is formed through the gel precursor solution according to methods described herein.

[0065] Gels or gel precursors include any hydrogel known in the art to be suitable for cell culture. Hydrogels used for cell culture can be formed from a myriad of natural and synthetic materials, offering a wide range of mechanical and chemical properties. Suitable hydrogels are either facilitative for cell function when formed from natural materials, or permissive for cell function when formed from synthetic materials. Natural gels for cell culture are typically formed from proteins and ECM components such as collagen, fibrin, fibrinogen, fibronectin, hyaluronic acid, laminin, or Matrigel®, as well as materials derived from other biological sources such as chitosan, alginate, or silk fibers. Because they are derived from natural sources, these gels are inherently biocompatible and bioactive. Permissive synthetic hydrogels can be formed from purely non-natural molecules such as polyethylene glycol (PEG), poly(vinyl alcohol), and poly(2-hydroxyethyl methacrylate). PEG hydrogels have been shown to maintain the viability of encapsulated cells and allow ECM deposition as they degrade, demonstrating that synthetic gels can function as 3D cell culture platforms even in the absence of integrin binding ligands. Such inert gels are highly reproducible, allow for easy tuning of mechanical properties, and are simply engineered and manufactured. Gels or gel precursors can include commercially available products such as basement membrane extracts, extracellular matrices derived from human or animal tissues or cell cultures, extracellular matrices derived from animal tissues, synthetic extracellular matrices, hydrogels, collagen, soft agar, egg white, and Matrigel.

[0066] Basement membranes, including the basal layer, are thin extracellular matrices that underlie epithelial cells in vivo and are composed of extracellular matrices such as proteins and proteoglycans. In one example, basement membranes are composed of collagen IV, laminin, entactin, heparan sulfate proteoglycans, and numerous other minor components (Quaranta et al, Curr. Opin. Cell Biol. 6, 674-681, 1994). These components alone, as well as intact basement membranes, are biologically active and promote cell adhesion, migration, and in many cases proliferation and differentiation. Matrigel is an example of a gel based on basement membranes, which is highly biologically active in vitro as a substratum for epithelial cells.

[0067] Many different gels suitable for use in the methods and devices described herein are commercially available, including, but not limited to, Matrigel rgf, BME1, BME Irgf, BME2, BME2rgf, BME3 (all Matrigel variants), collagen I, collagen IV, a mixture of collagen I and IV, or a mixture of collagen I and IV and collagen II and III), puramatrix, hydrogel, Cell-Tak, collagen I, collagen IV, Matrigel® matrix, fibronectin, gelatin, laminin, osteopontin, polylysine (PDL, PLL), PDL / LM and PLO / LM, PuraMatrix®, or vitronectin. In one preferred embodiment, the matrix components are obtained as commercially available Corning® Matrigel® matrix (Corning, NY 14831, USA).

[0068] Due to the meniscus pinning effect of the capillary pressure barrier and the lumenization of the gel (or precursor), the gel in the microfluidic network (e.g., the first region of the network) has two surfaces that may be exposed to air (e.g., by withdrawing the second liquid from the formed lumen) and / or available for the introduction of other liquids to contact the surfaces. For convenience, the inner surface of the gel facing the lumen will generally be described as the first surface, and the outer surface of the gel facing an adjacent region of the microfluidic network (e.g., the second region) will generally be described as the second surface. It will be appreciated that the second surface corresponds to the meniscus of the gel precursor solution pinned by the capillary pressure barrier when the gel precursor solution is introduced into the first region, and thus may extend the length of the capillary pressure barrier. It will be understood that the first surface can extend along the entire length of the microfluidic network, including the portion of the network leading up to and away from the capillary pressure barrier, and thus may be longer than the second surface.

[0069] The gel may have a thickness of 200 μm or less between the first and second surfaces. It will be understood that the thickness of the gel may not be uniform along the entire length between the first and second surfaces, meaning that a thickness of 200 μm or less may be a minimum thickness or a maximum thickness. In some examples, the thickness of the gel between the first and second surfaces may be 150 μm or less, such as 100 μm or less, such as 50 μm or less, such as 40 μm or less, such as 30 μm or less, such as 20 μm or less, such as 10 μm or less, such as about 1 μm. In some examples, the gel may have a thickness approaching zero between the first and second surfaces, such as less than 1 μm, such as less than 500 nm, such as less than 250 nm, such as less than 100 nm, such as less than 50 nm, to replicate the in vivo basal layer. In some examples, the gel has a thickness between the first surface and the second surface that is between 20 nm and 200 μm, such as between 100 nm and 150 μm, such as between 500 nm and 100 μm, such as between 1 μm and 50 μm.

[0070] As described above, the gel may be present in a first region of the microfluidic network and have a surface facing a second region of the microfluidic network with the gel immobilized by a capillary pressure barrier. In some examples, the gel is also immobilized by a second capillary pressure barrier that defines a boundary between the first and third regions of the microfluidic network. In these examples, the gel includes a third surface facing the third region. It will be appreciated that the third surface, like the second surface, may be concave or convex in shape and may extend or protrude beyond the physical location of the capillary pressure barrier while remaining immobilized.

[0071] In other examples, a second capillary pressure barrier is present, which defines a boundary between the second and third regions of the microfluidic network. In these examples, the first region containing the luminalized gel is separated from the third region by the second region.

[0072] In some examples, the microfluidic network includes an opening, which may be a different opening than any inlet opening used for filling or venting liquid, and the gel structure forms a surface facing and / or substantially sealing the opening. The opening may itself act as a capillary pressure barrier, preventing liquid flow from the opening. In some examples, the lumen does not extend to the opening, but instead extends through the gel that is below the opening (when the microfluidic network including the gel is in a plane below the opening during use). Thus, the gel structure may have a surface facing and / or substantially sealing the opening, which may contact the liquid of interest and provide access to the gel at locations between the beginning and end of the lumen along its length.

[0073] In some examples, the second region also contains a gel or gel precursor solution and forms a gel-to-gel contact with the gel in the first region. In some examples, the microfluidic network includes a third region, indicated by a second capillary pressure barrier, the third region also contains a gel or gel precursor solution. In these examples, the third region may be adjacent to the first region or the second region. Thus, the third region may contain a gel that forms a gel-to-gel contact with the gel in the first region, or a gel that forms a gel-to-gel contact with the gel in the second region, or a gel with no gel-to-gel contact between the two in the first and third regions. If present, the gel in the second region and the gel in the third region may be a lumenized gel or gel structure formed using the methods described herein.

[0074] While the present discussion focuses on the presence of one or two capillary pressure barriers followed by the formation of a gel structure in the first, second and third regions of the microfluidic network, it will be understood that the present disclosure is not limited to such an arrangement and may include still further capillary pressure barriers providing still further regions of the microfluidic network. Thus, a microfluidic network may generally be composed of N regions or lanes with N-1 capillary pressure barriers dividing each region or lane. Thus, the described method may be used to form gels in any N lanes where a free-standing meniscus is formed, and N lumens may be created for the N lanes in a microfluidic network.

[0075] Once the gel structure within a microfluidic network has been luminalized by the methods described herein, one or more cells or cell types can be introduced into the microfluidic network, for example, to form gel-supported tubules or blood vessels, as described below in connection with the methods of the present disclosure.

[0076] Methods for making lumenalized gel structures 1. A method for making a lumenalized gel structure, comprising: introducing a first liquid comprising a gel precursor solution into a microfluidic network, the microfluidic network comprising a capillary pressure barrier at a location generally defining a boundary between a first region and a second region of the microfluidic network; allowing a first liquid to enter a first region of the microfluidic network and align itself along a capillary pressure barrier, thereby forming a liquid-air meniscus of the first liquid at a boundary between the first and second regions of the microfluidic network; forming a lumen through the first liquid by contacting the first liquid with a second liquid, the second liquid having a lower viscosity than the viscosity of the first liquid; allowing or causing the first liquid to gel to form a gel structure including a lumen therethrough; Described herein are methods comprising:

[0077] Figures 1A, 1B and 1C show a series of steps according to the method described herein, where Figure 1A shows a side or cross-sectional view of a device containing a microfluidic network, Figure 1B shows the same device in plan view, and Figure 1C shows the same device in a cross-section perpendicular to the view of Figure 1A.

[0078] The device 100 comprises a microfluidic network 102 , in this example provided with two inlets (not numbered) at either end of the network, accessed from above through a cover layer 103 containing wells 105 . In one example, a first liquid comprising a gel precursor solution is introduced into the microfluidic network through an inlet that provides access to the microfluidic network, for example by injection or insertion. More specifically, the microfluidic network and the inlet are configured such that the inlet communicates with a first region of the microfluidic network, i.e., the inlet and the microfluidic network define a flow path from the inlet to the first region. When the first liquid is introduced into the microfluidic network, for example by injection, it can enter the first region, for example through the flow path, and align itself along the capillary pressure barrier. Typically, capillary forces are sufficient to cause the first liquid to flow through the microfluidic network, and no continuous injection pressure or back pressure is required. In some examples, back pressure is applied to allow the first liquid to enter the first region and align itself along the capillary pressure barrier.

[0079] The second image in Figures 1A-1C shows a first liquid 104 comprising a gel precursor solution being introduced into the microfluidic network 102. As seen in Figures 1B and 1C, a capillary pressure barrier 112 is provided in the microfluidic network 102, which generally defines a boundary between a first region 114 and a second region 116 of the microfluidic network. The capillary pressure barrier 112 is provided as a ridge of material that protrudes from the floor of the microfluidic network 102 into the body or channel (shown in dashed lines in Figure 1B). Upon being introduced into the microfluidic network 102, the first liquid 104 fills the first region 114, aligns itself along the capillary pressure barrier 112, and forms a liquid-air meniscus that runs along or parallel to the capillary pressure barrier 112, so that it is generally located at the boundary between the first region 114 and the second region 116. It can be seen in Figure 1C that the meniscus is pinned by the capillary pressure barrier 112, but the portion of the meniscus in contact with the ceiling of the microfluidic network extends partially into the second region 116. However, it will be appreciated that the liquid-air meniscus of the first liquid is still generally disposed at the boundary due to the meniscus pinning effect of the capillary pressure barrier 112.

[0080] Suitable gel and precursor solutions include, but are not limited to, those containing Matrigel, Matrigel gfr, BME1, BME1 gfr, BME2, BME2 gfr, BME3 (all Matrigel variants), collagen I, collagen IV, a mixture of collagen I and IV, or a mixture of collagen I and IV and collagen II and III), puramatrix, hydrogel, Cell-Tak, Matrigel matrix, fibronectin, gelatin, HA, laminin, osteopontin, polylysine (PDL, PLL), PDL / LM and PLO / LM, PuraMatrix, or vitronectin. In one preferred embodiment, the matrix components introduced as the gel precursor solution are obtained as commercially available Corning Matrigel matrix (Corning, NY 14831, USA).

[0081] A lumen is then formed through the first liquid by contacting the first liquid with a second liquid at a first location within the microfluidic network, where the second liquid has a lower viscosity than that of the first liquid. This technique is known as viscous fingering, or Saffman-Taylor instability, and relies on the different relative viscosities of the two liquids for the formation of the lumen. Methods for creating three-dimensional luminal structures in permeable matrices are known in the art (Bischel et al. J Lab Autom. (2012) 17:96-103, and Bischel et al. Biomaterials (2013) 34:1471-1477).

[0082] For lumenization to occur, resulting in the displacement of a first, more viscous liquid by a second, less viscous liquid, a pressure gradient must be created, which can be achieved by a number of methods, including but not limited to Laplace forces due to the surface tension of the droplets (passive pumping), hydrostatic pressure, pneumatic pressure, or mechanical pressure (e.g., via a syringe pump).

[0083] A first liquid, i.e., a gel precursor solution, may have a high enough viscosity to form a defined structure, yet still allow a second liquid, having a lower viscosity, to diffuse through the first liquid, e.g., by passive pumping based on surface tension without the need for external pressure driven flow, and remove a portion of the first liquid, thereby creating a lumen extending through or within the first liquid.

[0084] In some examples, the first liquid has a viscosity of about 5 cP to about 10 6 The first liquid may have a viscosity of about 0.5 cP to about 5 cP, for example about 5 cP to 5000 cP, for example about 30 cP to 1000 cP. The composition and / or viscosity of the second liquid dispersing through the first liquid may vary with the viscosity of the first liquid. In general, the higher the viscosity of the first liquid, the higher the viscosity of the second liquid may need to be in order for the second liquid to extend through the first liquid and create a lumen therethrough. In some examples, the second liquid may have a viscosity of about 0.5 cP to about 5 cP. It is to be recognized that the absolute value of the viscosity of commonly used hydrogels depends on many factors, including dynamic properties such as shear, strain and viscoelastic properties, as a result of the fact that many gel precursors are non-Newtonian fluids. However, within these ranges, the gel precursor will always have a significantly higher viscosity, for example >5 cP, than the fluid used for lumenization, such as water or cell culture medium (e.g., about 1 cP). As long as the fluctuating and dynamic viscosity of the gel precursor can be expected to always be higher than that of the second fluid, the proposed method is applicable even when the exact viscosity of the gel is varying or only approximately defined.

[0085] That is, methods of altering the viscosity of a gel precursor solution to form a gel matrix (ECM) are known in the art and include, for example, adjusting the concentration of the polymer (e.g., collagen) in the solution (higher concentrations result in higher viscosity), inducing partial gelation of the solution (more gelation results in higher viscosity), changing the temperature (lower temperatures slower gelation but increase the viscosity of the ungelled gel precursor), or introducing a viscosity modifier such as polyethylene glycol into the solution.

[0086] The third image of Figures 1A-1C shows a droplet of a second liquid 106 introduced on top of the first liquid 104 filling the microfluidic network 102. For purposes of illustration, the second liquid 106 is introduced through the same inlet as the first liquid 104, which can be considered a first location. To force the second liquid 106 through the first liquid 104 and lumenize the first liquid 104, a third liquid 108 can be applied to a second location spaced apart from the first location, for example, an inlet to the microfluidic network 102 spaced apart from the inlet through which the second liquid 106 was introduced. The third liquid 108, like the second liquid 106, can have a viscosity lower than that of the first liquid 104.

[0087] The third liquid 108 may be introduced when the second liquid 106 is constrained to the inlet by surface tension at the inlet interface. The introduction of the third liquid 108 may help to break the surface tension, thus requiring a lower second liquid 106 pressure. The third liquid 108 may have the same or different composition as the second liquid 106, and may be a liquid selected from, but not limited to, HBSS, cell culture media, PBS, TRIS, water, HEPES, albumin solution, balanced salt solution, second gel or gel precursor solution, or a buffer. The third liquid may be the same as the second liquid. The viscosity of the third liquid may be lower, similar, or higher than the first liquid. In some examples, the second liquid 106 is introduced before the third liquid 108 is introduced, while in other examples, the order of addition is reversed.

[0088] In some examples, contacting the gel precursor solution 104 with the second liquid 106 includes forming a meniscus of the second liquid 106 that is convex in shape and has a first principal radius of curvature, and contacting the gel precursor solution 104 with the third liquid 108 includes forming a meniscus of the third liquid 108 that is concave in shape or convex in shape and has a second principal radius of curvature smaller than the first principal radius of curvature. By providing a third liquid 108 that forms a meniscus that is concave in shape or convex in shape and has a smaller principal radius of curvature than the principal radius of curvature of the second liquid 106 that may form the lumen 110, advantageously the surface tension of the first liquid 104 is lowered or reduced, allowing the second liquid 106 to perforate through the first liquid 104 and forming the lumen 110.

[0089] In this way, the passive pressure-driven flow of the second liquid 106, which has a lower viscosity than that of the first liquid gel precursor 104, can pattern a roughly circular or elliptical lumen 110 in the first liquid 104, as seen in the lower images of Figures 1A-1C. In some examples, the passive pumping can be facilitated by using gravity to apply a pressure differential from one inlet to another, for example, by adjusting the tilt of the microfluidic network. In other examples, no third liquid 108 is used, and an external pressure can be applied to force the second liquid 106 through the first liquid 104.

[0090] The dimensions of the lumen may vary depending on several factors, including, but not limited to, the channel dimensions, the relative viscosities between the first and second liquids, the volumetric flow rate and / or pressure of the second fluid flowing through the first liquid, and combinations thereof. In some embodiments, the cross-sectional area of ​​the lumen may be greater than 50% of the cross-sectional area of ​​the channel. In some embodiments, the cross-sectional area of ​​the lumen may be greater than 90% of the cross-sectional area of ​​the channel, e.g., 98% or 99%. In some examples, the lumen may have a dimension of about 10 μm to about 1000 μm. In some embodiments, the lumen may have a dimension of 20 μm to about 500 μm, e.g., 50 μm to about 250 μm.

[0091] Once the first liquid 104 is luminalized, i.e., a lumen 110 is formed through the first liquid using the second liquid 106, gelation or polymerization of the gel precursor solution 104 results in a gel structure, e.g., an extracellular matrix gel structure, that includes the lumen 110 extending therethrough.

[0092] In some instances, the first liquid 104, which comprises a gel precursor solution, undergoes partial gelation prior to luminalization. In other instances, luminalization occurs prior to gelation of the first liquid 104. The second liquid 106 can be withdrawn from the lumen 110 by applying positive or negative pressure to any openings forming an inlet or outlet, either before or after gelation occurs, thus exposing the inner surface of the gel structure. As seen in the bottom image of FIG. 1C, the gel structure formed from the first liquid 104 has a first surface facing the lumen 110 and a second surface facing the second region 116, both of which can be modified by the addition of one or more cell types, as described below.

[0093] In some examples, the second liquid 106 may itself comprise a gel precursor solution that can be lumenized by a perforating liquid having a viscosity lower than that of the second liquid 106. Thus, the described method may include forming a lumen through the second liquid by contacting the second liquid with a liquid having a viscosity lower than that of the second liquid, and allowing or causing the second liquid to gel within the first gel structure to form a lamellar gel structure including a lumen through the lamellar gel structure. By varying the composition of the gel precursor solution of the second liquid 106 relative to the composition of the gel precursor solution of the first liquid 104, a lamellar and potentially concentric system can be created. In particular, in some examples, one or both of the first liquid 104 and the second liquid 106 may independently comprise one or more cell types, e.g., one or more mesenchymal derived cells selected from stromal cells, myocytes, pericytes, fibroblasts, and myofibroblasts.

[0094] The above method is described with reference to a microfluidic network having one capillary pressure barrier that defines two regions of the microfluidic network.However, it will be understood that this method can be applied to more complex systems that have two or more capillary pressure barriers that result in three or more regions of the microfluidic network, each with one or more inlets, and can be patterned independently with a matrix gel structure that may or may not be luminalized, and / or with one or more cell types that can form organoid bodies or vasculature that cover the surface of the region of interest.

[0095] Thus, in some examples, the method may include introducing an additional liquid, such as a second gel precursor solution, into a second region of the microfluidic network and contacting the second gel precursor solution with the gel structure along the length of the capillary pressure barrier, as shown in Figures 2 and 3, forming a lumen through the second gel precursor solution by contacting the second gel precursor solution with a liquid having a lower viscosity than that of the second gel precursor solution, and allowing or causing the second gel precursor solution to gel and contact the first gel structure to form a second gel structure including a lumen through the second gel structure. 2 and 3 both show a device 200 including a microfluidic network having two capillary pressure barriers, indicated at 212 and 220. Both capillary pressure barrier 212 and capillary pressure barrier 220 are provided as ridges that protrude into the channels from the floor or base of the microfluidic network, although it will be appreciated that other configurations of the capillary pressure barriers are possible as described herein.

[0096] 2, capillary pressure barrier 212 defines a boundary between first region 214 and second region 216. In this example, capillary pressure barrier 220 defines a boundary between second region 216 and third region 218.

[0097] The second image of Figure 2 shows a gel precursor solution of a first liquid 204a being introduced into the first region 214, and the third image shows the formation of a lumen 210 therethrough. After the first liquid 204a has gelled to form a gel structure, a second gel precursor solution 204b can be introduced into the second region 216, which can also be lumenalized as described herein using a viscous fingering method, as seen in the third and fourth images, to provide a second lumen 222. After the second gel precursor solution 204b has gelled to form a gel structure having a lumen, a third gel precursor solution can be introduced into the third region 218, providing a third lumen 224.

[0098] An alternative sequence of steps is shown in Figure 3, which results in the same sequence as in Figure 2. In Figure 3, a first region 214 can be considered to be in the center of the microfluidic network, with a capillary pressure barrier 212 as the boundary between it and a second region 216 on the left, and a capillary pressure barrier 220 as the boundary between the first region 214 and a third region 218.

[0099] As seen in the third image of Figure 3, the methods and arrangements of the present disclosure allow for the formation of a lumenalized gel structure having three surfaces: a first surface facing the lumen 210, a second surface facing the second region 216, and a third surface facing the third region 218. While such an arrangement may be advantageous for certain applications, Figure 3 also shows that it is also possible to introduce gel precursor solutions 204b and 204c into the second region 216 and the third region 218, respectively, to form lumens 222 and 224 through the second region 216 and the third region 218, respectively.

[0100] Although the sequence of steps in Figures 2 and 3 shows all three regions being filled with gel and all three being luminalized, it will be appreciated that there may be advantages to forming a luminalized gel structure only in the first region 214 and the second region 216, leaving the third region 218 available to culture medium or test solution.

[0101] 4A and 4B show an alternative device 300 in which an opening 340 is provided in addition to any dedicated fluid interface inlets and outlets. The opening 340 can act as a capillary pressure barrier, pinning the first liquid 304 to form a meniscus and a surface extending across the opening 340. Similar to the method above, a first liquid gel precursor solution 304 is introduced into the microfluidic network of the device 300, and a second liquid 306 and a third liquid 308 are introduced at either end of the network to break the surface tension and cause the first liquid 304 to be luminalized.

[0102] As seen in Figures 4A and 4B, the lumen 310 extends from the first liquid 306 to the second liquid 308, but does not extend to the opening 340. Thus, it can be seen that a given region of a microfluidic network can have multiple openings, considered inlets or outlets, and the lumenization can be controlled so that the lumen extends only between desired locations (openings) of the microfluidic network. For example, forming a lumen can include minimizing the principal radius of curvature at one of the multiple openings to cause the lumen to extend to that one opening, and only that one opening. This can be accomplished by applying a second liquid to the opening and minimizing the surface tension at the opening.

[0103] In this way, lumenization can occur below the plane of the aperture, resulting in additional surface area of ​​the gel structure. Figure 13 shows a high-resolution image taken from above such a device, where the lumen can be seen penetrating the gel below the aperture through the aperture. Arrows superimposed on the image indicate the path of the gel and lumen.

[0104] The top image of FIG. 5 shows the device in which a gel structure formed from gel precursor 204a is provided in a first region 214 and secured by a capillary pressure barrier. A lumen 222 extends through the gel structure and is lined with tubules of cells 228, which may be endothelial or epithelial cells. In a second region 216 that does not contain a gel structure, second tubules of cells 230 are present, with interstitial spaces between the tubules indicated by "a". Cells 228 and / or cells 230 may be allowed or stimulated to remodel the gel structure by reducing the thickness of the gel structure between the first and second surfaces and / or by secreting one or more ECM components. Cells may be stimulated by growth factors, glucose and oxygen levels to become more or less metabolically active. More proliferative and more motile cells typically degrade ECM more actively. Activation of ECM degrading and remodeling enzymes, such as MMPs or ADAMTS, by activators such as MMP-3, plasmin, kallikrein, tryptase, furin, etc., will further promote ECM degradation and loss of gel structure thickness. Conversely, ECM remodeling can be inhibited using MMP inhibitors, ROCK inhibitors, etc., which may preserve a thicker gel structure.

[0105] As can be seen in the second image of Figure 5, the gel matrix has remodeled (in this case, disassembled) until the distance between the tubules is nearly zero, resulting in (near) direct cell-cell contact between adjacent tubules.

[0106] It will be appreciated that any cells present in the microfluidic network, for example cells introduced via a gel precursor solution, or cells introduced into the lumen of the gel structure (as or via cell tubules), or cells introduced into a region of the microfluidic network opposite the gel structure, may remodel the gel structure.

[0107] FIG. 5 illustrates the potential of the devices and methods described herein to provide an interstitial distance between tubules that can be zero or near zero by remodeling the extracellular matrix gel. In general, one or more cell types can be introduced into the microfluidic network, and remodeling of the gel structure is permitted or stimulated by reducing the thickness of the gel structure. In some examples, the thickness of the gel structure is remodeled between the first and second surfaces of the gel structure (i.e., between the surface facing the lumen and the outer surface of the gel structure). Remodeling can include deposition or degradation of any ECM components. One or more cells can secrete one or more ECM components that result in remodeling.

[0108] The degradation and remodeling of extracellular matrix gel can promote cell migration or migration, for example by orienting matrix components such as collagen fibers. Remodeled ECM resulting in topographical changes such as reduced thickness or fiber orientation is often observed in areas where cancer epithelial cells invade, but remodeled ECM may also affect the behavior of stromal, endothelial or immune cells in the local environment, and therefore there is a need to investigate the behavior of cells in remodeled extracellular matrix or in response to remodeled extracellular matrix. The methods and devices described herein address this need.

[0109] In the bottom image of Figure 5, cells (in this case epithelial cells) are allowed to remodel the ECM by secreting glycoproteins and collagen to form a new basal layer 236 between the tubules. In this way, a more realistic co-culture model can be obtained for investigation, where the remodeled ECM has a thickness approaching zero between the first and second surfaces, e.g. less than 1 μm, e.g. less than 500 nm, e.g. less than 250 nm, e.g. less than 100 nm, e.g. less than 50 nm.

[0110] The present invention is particularly advantageous for non-lumenalized gel structures in a three-lane microfluidic device, which may be described with reference to Figures 6A and 6B. Figure 6A shows a typical model for investigating molecular transport across an interstitial layer between biological structures in a three-lane system. In this model, a gel structure formed from a gel precursor solution is provided in a central region of a microfluidic network bounded by capillary pressure barriers 214 and 220.

[0111] The gel structure may contain or include an extracellular matrix such as collagen or Matrigel® and may include cells (e.g., stromal cells) 234b dispersed throughout the gel. On the right side of the gel structure is a single-layered tubule of cells 230 (e.g., endothelial or epithelial cells) having a lumen 224, and on the left side of the gel structure is a double-layered tubule formed by cells 226 and 232, which may be, for example, a pericyte / endothelium-lined tubule as described above.

[0112] As an example, here a bilayer and stromal cells are introduced. The invention works equally well for two single layer tubes, where no cells are added to the ECM gel.

[0113] In this setup, any material (test compound, nutrient medium) must be introduced into the microfluidic network via double-layered or single-layered tubules and cannot easily reach the adjacent gel structure and / or other tubules since it must pass through the layer of cells forming the tubules, as shown by the blocked arrows in the double-layered tubule formed by cells 226 and 232. In contrast, when the bilayered tubules are formed with a lumenized gel structure as in Figure 6B, one lane of the three-lane system (e.g., the third region 218 in Figure 6B) can be reserved for adding triggers, drugs, staining reagents, markers or reporter molecules, or can be used to sample, for example, excreted metabolites and basal excreted cytokines. Because the gel structure is fixed by the capillary pressure barrier 220 without the need for a membrane, the surface of the gel faces the third region 218 and can be in contact with any solution introduced into the third region 218, thereby facilitating the exchange and transport of materials through the interstitial gel structure to the tubules, resulting in the free exchange of metabolites, nutrients, compounds, drugs, triggers, chemokines, cytokines and oxygen between the fluid in the third region, the gel structure and the basal side of the tubules.

[0114] Of course, one skilled in the art will recognize that cells within the extracellular matrix environment (i.e., the gel structure) are still motile through the gel network. Motility is enabled by cell-matrix interactions through focal adhesions such as integrins, actin remodeling, collagenases, and many other mechanisms that allow cell motility in the extracellular matrix environment. Cells may also migrate in and out of the ECM gel and / or across epithelial or endothelial barriers during the course of the experiment. All of this is enabled by the luminalized components within the gel structure and the free surface of the gel facing the third region 218.

[0115] As can be seen in FIG. 7, it is also possible to introduce one or more cells into the second or third region of the microfluidic network and allow the cells to at least partially cover the second or third surface of the gel structure and the second or third region of the microfluidic network, thereby at least partially forming tubules in the second or third region, for example the one or more cells selected from endothelial or epithelial cells, cells of mesenchymal origin, such as (smooth) muscle cells, pericytes, podocytes, fibroblasts, myofibroblasts.

[0116] In FIG. 7, the first region 214 is shown to be in the middle of the three regions of the microfluidic network and has single layer tubules (e.g., endothelial tubules) of cells 228. The second region 216 is shown to the right of the first region 214 and is shown to not include a gel structure. In this example, the second region 216 of the microfluidic network and the second surface of the gel formed in the first region 214 opposite the second region 216 include a layer of endothelial or epithelial cells that form tubules. That is, cells 230 coat the surface of region 216 and the surface of the gel structure in the first region 214 to form tubules in the second region 216.

[0117] As a result, the tubules themselves form a lumen 224 extending through the second region 216, which may be perfused with a nutrient medium or test solution. In Figure 7, the third region 218 is provided with a gel structure and a cell line. Although these figures show all of the tubules being substantially surrounded by cells present within the gel structure, e.g., cells of mesenchymal origin, it will be appreciated that alternative models are possible in which only the first tubules and / or the second tubules are substantially surrounded by cells present within the gel structure, e.g., cells of mesenchymal origin.

[0118] In general, introducing one or more cells or cell types into a microfluidic network may include perfusing a cell-containing liquid through the microfluidic network from one inlet to another inlet / outlet. Perfusing the liquid may include, for example, applying a pressure differential from the inlet to the outlet and / or adjusting the slope of the microfluidic network. It will be appreciated that the cell-containing liquid may be perfused through any region of the microfluidic network where a fluid interface is provided and through any lumens and / or cell tubules present in that region. In some examples, introducing one or more cells or cell types into a microfluidic network may include introducing or seeding a cell pellet into a region of the microfluidic network. In some examples, a low viscosity liquid such as culture medium may be introduced prior to seeding the region with the cell pellet.

[0119] In some examples, any of the first to third regions may include a gel structure that includes one or more types of immune cells, which may be selected from, but are not limited to, T cells, monocytes, macrophages, neutrophils, eosinophils, mast cells, natural killer cells, dendritic cells, and B cells. The one or more types of immune cells may be present in a gel precursor solution introduced to the region of the microfluidic network, or may be provided to the lumen of the gel structure, e.g., a cell-lined lumen, e.g., an endothelium or lymphatic vessel, and allowed or stimulated to adhere to the first surface, e.g., an endothelium or lymphatic vessel, and allowed to extravasate from the lumen side to the ECM side. Immune cells may also be added to a region of the microfluidic network that does not include a gel structure at all and is configured as a perfusion conduit for nutrient medium and / or test solution. 8 shows an example device that includes two capillary pressure barriers 214 and 220 that indicate conceptual boundaries between a first region of a microfluidic network 214, a second region of a microfluidic network 216, and a third region of a microfluidic network 218. All three regions are filled with liquid, including gel precursor solutions 204a, 204b, and 204c, respectively, and subsequently lumenalized and gelled to form lumens 210, 222, and 224.

[0120] Looking first at the first region 214, it can be seen that the gel resulting from the gel precursor solution 204a includes cells 234a, preferably introduced as part of the precursor solution, but could also be introduced after gelation and stimulated or allowed to migrate across the epithelial or endothelial vessel wall and into the gel structure. The cell type of the cells 234a can be selected based on the need. In some examples, the cells 234a can include one or more cells selected from endothelial or epithelial cells, cells of mesenchymal, endodermal and ectodermal origin, such as (smooth) muscle cells, pericytes, podocytes, fibroblasts, myofibroblasts, astrocytes, or one or more spheroids or organoids. In an example where the device provides a model for investigating the blood-brain barrier, the cells 234a can include human primary astrocytes dispersed through the gel matrix formed from the precursor solution 204a.

[0121] Similarly, the gel structures formed from the precursor solutions 204b and 204c in the second region 216 and the third region 218 may also comprise one or more cells 234a and 234b dispersed throughout the gel structure, the cells being independently selected from endothelial or epithelial cells, cells of mesenchymal origin, such as (smooth) muscle cells, pericytes, podocytes, fibroblasts, myofibroblasts, astrocytes, or one or more spheroids or organoids.

[0122] Thus, the method may comprise introducing cells of mesenchymal origin, e.g. selected from stromal cells, (smooth) muscle cells, pericytes, fibroblasts and myofibroblasts, into the microfluidic network to substantially surround the tubules in the lumen and / or the tubules in the second region. The cells of mesenchymal origin may be present in the gel precursor solution initially to substantially surround the tubules or may be introduced into the microfluidic network via a transport channel and subsequently migrate through the gel structure to substantially surround the tubules.

[0123] In some examples, the gel structure contains epithelial cells that can grow and / or differentiate during culture depending on the composition of the culture medium, other cell types that may be present, and the extracellular matrix. Thus, after being introduced into the microfluidic network, the epithelial cells can then grow and / or differentiate within the gel structure by using an aqueous medium, preferably a growth medium, or by using a gel (precursor). The culture of one or more cell types or cell aggregates, e.g., epithelial cells, is achieved by the introduction of medium into the microfluidic network and continued under appropriate conditions so that the cells are cultured.

[0124] In a second example, the at least one cell type or cell aggregates present in the gel or gel precursor solution include epithelial cells and cells of mesenchymal origin, such as fibroblasts, smooth muscle cells, myofibroblasts, pericytes, astrocytes, oligodendrocytes, etc. Thus, after being introduced into a microfluidic network using the gel (precursor), the epithelial cells and cells of mesenchymal origin can then interact to form tissue that can optionally proliferate and / or differentiate.

[0125] In further examples, the at least one cell type includes any combination of epithelial cells and cells of mesenchymal origin, immune cells (e.g., T cells, macrophages, Kuppfer cells, dendritic cells, neutrophils, eosinophils, NK cells, B cells, granulocytes, mast cells) and / or endothelial cells.

[0126] The described methods also include introducing one or more cells into the lumen of the gel structure, for example, the one or more cells include endothelial cells, epithelial cells, or cells of mesenchymal origin, which can cover the surface of the lumen and form tubules within the lumen. In some examples, the surface of the gel facing the lumen can include a layer of endothelial or epithelial cells that form first tubules.

[0127] As can be seen in FIG. 8 , the lumen 210 of the first region 214 is lined with two different cell types forming a primary tubule 226 of cells and a secondary tubule 232 within the primary tubule 226, while the lumens 222 and 224 are lined with a monolayer of cells 228 and 230.

[0128] For example, cells 228 and / or 230 may be endothelial cells, and endothelial cells 228 and 230 may populate a first surface of the gel structure facing lumens 222 and 224 to generate endothelial tubules with an open lumen. However, cells 228 and 230 may be selected to be epithelial cells, or cells of mesenchymal origin as described elsewhere, depending on the type of model being investigated.

[0129] In one example, the fluid introduced into the lumen may contain endothelial cells. In general, endothelial cells are known as cells that cover the inner surface of the entire circulatory system, from the heart to the smallest lymphatic capillaries. When in contact with blood, these cells are called vascular endothelial cells, and when in contact with the lymphatic system, they are called lymphatic endothelial cells. In a particular embodiment, the method comprises the steps of introducing endothelial cells into the lumen of the microfluidic network and causing or allowing said endothelial cells to cover the surface of the gel facing the lumen, i.e., causing or allowing the endothelial cells to form tubules within the lumen.

[0130] Alternatively, multi-layered tubules are possible by sequentially introducing solutions of cells into the lumen, similar to the lumen 210 of the first region 214. For example, the cells 226 can be pericytes, and the lumen 210 is first populated with pericytes 226 to allow or stimulate the formation of single or multi-layered tubules in the lumen 210. The cells 232 can be endothelial cells, and can be introduced to generate pericyte / endothelial lined tubes with an open lumen, with the endothelium covering the pericytes. Alternatively, multi-layered tubules can be formed by introducing solutions of two different cell types (e.g., endothelial cells and smooth muscle cells) and allowing the different cell types to self-assemble into multi-layered tubules.

[0131] As described above, the method may include introducing one or more immune cells into the microfluidic network. In some examples, the method may include introducing one or more immune cells, such as T cells, monocytes, macrophages, dendritic cells, and / or B cells, into the lumen and / or gel structure such that the one or more immune cells adhere to a first surface of the gel, or to the tubules, if present, or are provided within the gel structure. Once introduced into the microfluidic network, the immune cells may be stimulated or allowed to adhere to the epithelial or endothelial vascular wall of the tubules and, optionally, subsequently migrate across the vascular wall into the gel structure.

[0132] Alternatively, immune cells may adhere to an endothelium formed within the microfluidic network, and immune cells may be stimulated or allowed to migrate through the gel structure to observe their behavior in different regions of the microfluidic network, as described below in connection with assays that may be performed using microfluidic networks resulting from the methods and devices of the present disclosure.

[0133] In any of Figures 5-8, layer 230 may be, by way of example, an epithelial tubule, including, but not limited to, intestinal tubules (including small intestine, colon, ileum, rectum, and duodenum), retinal pigment epithelium, renal epithelium (including proximal tubules, distal tubules, loops of Henle, and podocytes), skin, gastric epithelium, and bile duct.

[0134] The second tubule 228 or 232 may be a blood or lymphatic vessel that is optionally surrounded (226) by pericytes, podocytes, smooth muscle cells, cells of mesenchymal origin. The cells deposited in the interstitial spaces 234a and b may be, by way of example, cells of mesenchymal origin, such as fibroblasts, myofibroblasts, or muscle tissue, as well as resident or infiltrating immune cells.

[0135] In this way, a complete functional unit of a human organ can be reproduced. In the case of the example of the intestinal model, this model contains all the elements associated with the human intestine, including the epithelium, the submucosa, the interstitial cells, the vascular compartment, the lymphatic compartment, and the possibility of mimicking the immune activation process. The lumen of the epithelial tube can further be supplemented with mucus and bacterial flora.

[0136] Such models are important for disease modeling (e.g., inflammatory bowel disease) and for mimicking compound adsorption and transport. It is well known to those skilled in the art that almost every functional unit of any organ contains these same elements and can be modeled accordingly with the help of the right cells.

[0137] Assay The present invention also relates to one or more assays that use the devices described herein, or the lumenalized gel structures produced by the methods described herein.

[0138] The assays may include barrier function assays, transepithelial electrical resistance (TEER) assays, immune cell adhesion assays, immune cell migration assays, transporter assays, and vasodilation or vasoconstriction assays, however, the present disclosure is not limited to the use of the present device or lumenalized gel structures produced by the present methods in these assays, and it will be apparent to one of skill in the art that the present invention enables any number of assays depending on the cell type, cell origin, and test being performed.

[0139] In some examples, any of the aforementioned one or more cells or cell aggregates introduced into the microfluidic network can include cell lines, including immortalized cell lines or organoid lines, primary cells, cells derived from induced pluripotent stem cells, and can be, but are not limited to, clustered cells, printed cells, organoids, tissue biopsies, tumor tissue, excised tissue material, organ explants, or embryonic bodies.

[0140] The one or more cells, or cell aggregates, may comprise one or more cell types obtained from, derived from, or exhibiting a phenotype associated with a particular biological tissue, such as the liver, kidney, brain, breast, lung, skin, pancreas, intestine, retina, or hair. The one or more cells or cell aggregates may comprise healthy or diseased tissue and may be obtained from or derived from a patient. The cells may be of mesodermal, endodermal, or ectodermal origin.

[0141] In one embodiment, the endothelial cells used to vascularize the lumen can be obtained or derived from the patient, hi one embodiment, the endothelial cells obtained or derived from the patient can include blood-grown endothelial cells or endothelial cells derived from pluripotent stem cells.

[0142] By using autologous endothelial cells in combination with biological tissue containing one or more cells or cell aggregates from the same patient, the vascularized system is particularly suited to the field of personalized medicine and for the development of clinical models and assays to determine or predict a patient's likely response to a particular drug.

[0143] For example, the use of tumor tissue obtained from a patient, together with the vascularization of that tumor tissue with endothelial cells derived from the patient as described above, allows a complete analysis of the patient's expected response to chemotherapy. Furthermore, the introduction of one or more types of the patient's own immune cells into such a system allows a determination to be made about the expected immune response to a given drug.

[0144] Barrier function assay Barrier function assays investigate the properties and behavior of epithelial or endothelial cells, i.e., the cells that form epithelial or endothelial tubules, and their permeability to substances. These assays can involve the addition of a dye substance, such as fluorescein, and observing whether the dye substance diffuses through the epithelium or endothelium.

[0145] The present invention allows for a simple barrier function assay. For example, the device can be provided with a microfluidic network as shown in the top image of Figure 8, where cells 228 and 230 can be epithelial or endothelial cells. A diffusible dye can be introduced into lumen 222 and the system monitored for the presence of the diffusible dye in any one or more of the surrounding gel matrix, the lumen formed by the tubules of cells 230, and the free transport channels.

[0146] Transepithelial Electrical Resistance (TEER) Assay TEER assay is a specific example of a barrier function assay, which investigates barrier permeability by monitoring the electrical resistance across an epithelial cell layer. TEER assays performed on cell layers in microfluidic networks are described, for example, in WO 2019 / 166644, the contents of which are incorporated herein by reference. From FIG. 2 of WO 2019 / 166644, it can be seen that a three-lane microfluidic network as described herein can be provided with up to six electrode pairs to measure electrical activity in a TEER barrier function assay.

[0147] Immune cell adhesion assay As already explained, the method of the invention may include the step of introducing immune cells into a microfluidic network. The immune cells may be as described above and may be introduced into the microfluidic network at any position of such network.

[0148] Immune cell adhesion, e.g., T cell adhesion to epithelial or endothelial tissues, is a key step in the inflammatory response and it is therefore desirable to have a system, e.g., a device comprising a microfluidic network, that allows for the investigation of immune cell adhesion to these tissues.

[0149] Thus, the present invention also relates to the use of the devices and microfluidic networks described herein in assays for investigating immune cell adhesion to biological structures.

[0150] Immune cell migration assay As already explained, the method of the invention may include the step of introducing immune cells into a microfluidic network. The immune cells may be as described above and may be introduced into the microfluidic network at any position of such network.

[0151] The present invention allows investigation into the dynamics and regulatory mechanisms of immune cell migration across epithelia and endothelia.

[0152] There is considerable interest in developing reliable immunotherapies for major diseases, therefore immune cell migration assays, especially those examining regulatory T cell migration, can help understand the fundamental functional role of Tregs in autoimmune diseases including multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and cancer (e.g., lung, colorectal, nasopharyngeal, and breast cancer). For example, to prevent allograft rejection, Tregs must migrate to both the graft and lymph nodes. Furthermore, migration and accumulation of functionally suppressive Tregs at tumor sites is associated with cancer progression. Thus, the present invention also relates to the use of the devices and microfluidic networks described herein in assays for investigating immune cell transmigration.

[0153] Transporter assays As previously explained, the present disclosure relates to the formation of single layer biostructures and more complex multi-layer biostructures using the lumen of a lumenized gel structure of a microfluidic network, which can recreate the in vivo environment of, for example, the blood-brain barrier, the intestine, and other organs such as the kidney or liver.

[0154] Since lack of permeability across any given biological membrane or barrier can reduce the efficacy of a drug candidate, understanding how potential therapeutics are transported across such biological structures is of great clinical importance. The formation of biologically relevant tissues in the lumenized gel structure of the described microfluidic network allows for pharmacokinetic investigations of which transport proteins are involved in any drug transport. For example, compounds can be administered to the apical side of tubules, e.g., intestinal tubules, and their concentration can be measured in blood vessels present in the lumenized ECM. Compounds can be quantified by fluorescence imaging, mass spectrometry, ELISA.

[0155] Vasodilatation or vasoconstriction assays. The described devices and microfluidic networks allow the formation of luminalized biological tissue based on epithelial or endothelial cells, or on myocytes, fibroblasts, cardiomyocytes, resulting for example in endothelial blood vessels, whereas epithelial cells can form intestinal-type lumens or renal tubule-type lumens and cardiomyocytes can form atrial or ventricular-type lumens.

[0156] Microfluidic networks with such tissues are therefore particularly well suited to study processes such as vasodilation / vasoconstriction, intestinal peristalsis, renal tubule compression, blood vessel compression, or cardiac muscle cell operation. The (very thin) ECM supporting the tubules can be easily deformed by contracting or expanding tubules or other moving tissues, thereby allowing more sensitive assays of tissue motility than constructs in which cells are directly attached to more rigid surfaces.

[0157] Thus, the present invention also relates to the use of the devices and microfluidic networks described herein in vasodilation or vasoconstriction assays, as well as assays investigating intestinal peristalsis, renal tubule compression, blood vessel compression, and cardiomyocyte operation to investigate the effects of stimulatory or inhibitory agents on biological systems.

[0158] EXAMPLES The present invention will now be described, by way of example only, with reference to the following examples. EXAMPLES

[0159] Seeding of cells in the lumen and on the gel material: EGM-2 (trade name) medium: EBM-2 basal medium (Lonza, Cat. No. CC-3156) EGM-2 SingleQuots Supplement (Lonza, Cat. No. CC-4176) 1% Penicillin / Streptomycin (Sigma, Cat. No. P4333) EMEM: Eagle's Minimum Essential Medium (EMEM) (ATCC, Cat. No. 30-2003) 10% fetal bovine serum (Gibco, Cat. No. 16140-071) 1% non-essential amino acids (LifeTech, Cat. No. 11140050) 1% Penicillin / Streptomycin (Sigma, Cat. No. P4333) HBSS (Hank's Balanced Salt Solution, Sigma Aldrich, Cat. No. H6648) PBS (Phosphate Buffered Saline, Gibco, Cat. No. 700130656)

[0160] In this example, a three-lane OrganoPlate® (MIMETAS) with 400 μm wide lanes containing a microfluidic network of three lanes and two capillary pressure barriers was used.

[0161] The central lane was filled with neutralized bovine type I atelocollagen solution (PureCol® EZ Gel 5 mg / ml from Advanced BioMatrix) at the inlet; the solution was immobilized by a capillary pressure barrier, leaving a free-standing meniscus at the air-liquid interface in the upper and lower lanes.

[0162] 30 μL of HBSS solution was introduced into the inlet of the center lane, and 1 μL of 5% FBS solution in PBS was introduced into the hole of the outlet of the center lane forming a dome shape. These solutions have a lower viscosity than PureCol® EZ Gel. This created pressure from the outlet to the inlet due to the surface tension of the droplet and the resulting Laplace pressure, initiating lumenization. After 1 min, 50 μL of HBSS was added to the outlet and the OrganoPlate was placed in a humidity-controlled environment of 37° C. and 5% CO2 for 1 h.

[0163] Representative measurements of the rheology of ECMs are published by Fleming et al. (https: / / doi.org / 10.1063 / 1.5067382). Viscosities of commonly used cell culture media are published, for example, by Poon (https: / / doi.org / 10.1101 / 2020.08.25.266221).

[0164] For cell seeding, 1 μL of a cell pellet of primary human umbilical vein endothelial cells (HUVEC) containing 10,000 cells / μL was seeded at the outlet of the central lane.

[0165] Cells were allowed to attach for 2-6 hours, after which EGM-2 cell culture medium was added to the center lane so that the inlet and outlet volumes were 50 µL, and the OrganoPlate® was placed on a rocking platform at 7°, with an interval of 8 min, so that cell culture medium could be passively perfused through the lumen.

[0166] Two days later, Caco-2 cells were added to the inlet of the top lane in the same manner, and a Caco-2 cell pellet containing 6,000 cells / μL was seeded into the outlet of the top lane. The cells were allowed to attach for 2-6 hours. After attachment, complete EMEM medium was added to the top lane such that both the inlet and outlet contained 50 μL of complete EMEM medium. The OrganoPlate® was placed on a rocking platform at 7°, with an interval of 8 min, so that cell culture medium could be passively perfused through the lumen.

[0167] Within 1-3 days, both cell types form tubules in the OrganoPlate®, separated by less than 200 μm of ECM. Cell cultures were maintained for 10 days and then fixed in 3.7% formalin solution. Caco-2 cells were stained for epithelial cell adhesion molecule (EPCAM) and HUVEC for VE-cadherin, and the resulting confocal 3D reconstructions can be seen in Figures 9A and 9B. Figure 9A shows a view along the tubules, while Figure 9B shows a view from above. In both figures, the tubules on the left are formed from Caco-2 cells and those on the right are formed from HUVEC cells. The position of the capillary pressure barrier 214 and the resulting geometry that the tubules, especially the HUVEC tubules, adopt can be clearly seen.

[0168] These figures also show that Caco-2 and HUVEC cells are in close contact, facilitating cell-cell communication in a tissue culture model that more closely resembles the in vivo situation, which was measured to be between 15 and 150 μm depending on the length of storage time.

[0169] In another example, Caco-2 cells were seeded in the bottom lane in the same manner as above to form a construct consisting of three tubules (see Figure 10), illustrating the utility of the third lane in supporting the tubules as well.

[0170] Two further examples show how two or even three channels can subsequently be lumenalized. Figure 11A shows a schematic of two Caco-2 tubules in a lumenalized ECM in a two-lane OrganoPlate, and an experimentally obtained top-view phase contrast microscopy image is shown in Figure 11B. Following the procedure shown above, Purecol EZ solution was introduced into the top channel and a lumen was formed using viscous finger patterning. Purecol EZ solution was then introduced into the bottom channel and a lumen was formed. Caco-2 cells were seeded into the lumen of the top channel and HUVEC cells were seeded into the lumen of the bottom channel. In this way, a construct of two lumenalized ECMs, both containing tubules, was formed.

[0171] In a similar manner, FIG. 12 shows three lumenalized ECM constructs, all three containing tubules composed of Caco-2 cells. EXAMPLES

[0172] Luminalized blood-brain barrier model Endothelial medium: Endothelial cell culture medium (Cell biologics, Ca.No.H1168) Endothelial cell medium supplement kit (Cell biologics, Ca. No. H1168) NSC differentiation medium: Our proprietary formulations include: Neurobasal medium (Gibco, Cat.No.21103049)

[0173] In this example, a three-lane OrganoPlate® (MIMETAS) with 400 μm wide lanes containing a microfluidic network of three lanes and two capillary pressure barriers was used.

[0174] The upper lane was filled at the inlet with neutralized bovine type I atelocollagen solution PureCol® EZ Gel 5 mg / ml (Advanced BioMatrix), which was immobilized by a capillary pressure barrier, leaving a free-standing meniscus at the air-liquid interface in the upper and lower lanes.

[0175] 30 μL of HBSS solution was introduced into the inlet of the top lane, and 1 μL of 5% FBS solution in PBS was introduced into the outlet hole of the top lane forming a dome shape, similar to Example 1. This created pressure from the outlet to the inlet due to the surface tension of the droplet and the resulting Laplace pressure. After 1 minute, 50 μL of HBSS was added to the outlet and the OrganoPlate was placed in a humidity controlled environment at 37° C. and 5% CO2 for 1 hour.

[0176] For seeding of astrocytes in gels, 2 μL of an astrocyte suspension in collagen I 4 mg / mL (Cultrex) containing 5,000 cells / μL was seeded at the outlet of the central lane.

[0177] Cells were allowed to attach for 2-6 hours in a humidity-controlled environment at 37°C and 5% CO2.

[0178] Then, 2 μL of neuronal cell pellet containing 15,000 cells / μL was seeded at the outlet of the bottom lane.

[0179] Cells were allowed to attach for 2-6 hours, after which time NSC differentiation medium (Gibco) was added to the bottom lane so that the inlet and outlet volumes were 50 µL, and the OrganoPlate® was placed on a rocking platform at 7° for 8 min intervals so that cell culture medium could be passively perfused through the lumen.

[0180] Within 1–7 days, astrocytes formed a cell network in the gel in the middle lane, and neurons formed a cell network in the bottom lane. After 7 days, human brain microvascular endothelial cells (HBMEC) were seeded in the top lane in the same manner by seeding 1 µL of HBMEC cell pellet containing 10,000 cells / µL into the outlet of the top lane. Cells were allowed to attach for 2–6 h.

[0181] Once the cells had attached, cell culture medium (Cell Biologics) was added to the top lane such that both the inlet and outlet contained 50 μL of cell culture medium (Cell Biologics). The OrganoPlate® was placed on a rocking platform at 7° with an interval of 8 min so that cell culture medium could be passively perfused through the lumen. Replenishment of medium in the top and bottom lanes was performed every 2-3 days.

[0182] Within 1-3 days, HBMEC formed tubules within the OrganoPlate® in direct contact with the astrocytes in the gel. Cell cultures were maintained for 14 days and a barrier integrity assay was performed on HBMEC using sodium fluorescein (dilution 1:100) and 4.4 kDa TRITC (dilution 1:50).

[0183] Figure 14 (astrocytes in collagen I) and Figure 15 (astrocytes in PureCol®) are high resolution images showing the results of these experiments. Both figures show that HBMECs and astrocytes are in close contact, facilitating cell-cell communication in a tissue culture model that more closely resembles the in vivo situation. EXAMPLES

[0184] Tube-in-tube endothelial cell / pericyte co-culture MV2 medium: MV2 (Promocell, Cat. No. C22022) Supplement Mix (Promocell, Cat. No. C39226) 1% Penicillin / Streptomycin (Sigma, Cat. No. P4333) Pericyte medium: Pericyte growth medium (Promocell, Cat. No. C-28041)

[0185] In this example, a three-lane OrganoPlate® (MIMETAS) with 400 μm wide lanes containing a microfluidic network of three lanes and two capillary pressure barriers was used.

[0186] The upper lane was filled at the inlet with neutralized bovine type I atelocollagen solution PureCol® EZ Gel 5 mg / ml (Advanced BioMatrix), which was immobilized by a capillary pressure barrier, leaving a free-standing meniscus at the air-liquid interface in the upper and lower lanes.

[0187] 30 μL of HBSS solution was introduced into the inlet of the top lane, and 1 μL of 5% FBS solution in PBS was introduced into the outlet hole of the top lane forming a dome shape, similar to Example 1. This created pressure from the outlet to the inlet due to the surface tension of the droplet and the resulting Laplace pressure. After 1 minute, 50 μL of HBSS was added to the outlet and the OrganoPlate was placed in a humidity controlled environment at 37° C. and 5% CO2 for 1 hour.

[0188] For cell seeding of pericytes and human umbilical vein endothelial cells (HUVECs), 1 μL of cell pellet containing 15,000 cells / μL HUVECs and 5,000 cells / μL placental pericytes was seeded at the outlet of the top lane. The cell suspension was driven into the microfluidic network by Laplace pressure due to the surface tension of the cell pellet.

[0189] Cells were allowed to attach for 2-6 h, after which a 1:1 mixture of MV2 medium and pericyte growth medium was added to the top lane such that the inlet and outlet volumes were 50 µL, and the OrganoPlate® was placed on a rocking platform at 7°, with an interval of 8 min, so that cell culture medium could be passively perfused through the lumen.

[0190] Within 1-3 days, the cells organized themselves in the co-culture. HUVECs formed tubules within the OrganoPlate®, and placental pericytes migrated into the ECM and surrounded the cell tubules. The middle and bottom channels were filled with medium. Figure 16A shows a schematic cross-section, and Figure 16B shows an experimentally derived confocal microscopy cross-section of a tube-in-tube HUVEC / pericyte co-culture. EXAMPLES

[0191] Triple co-culture with stromal cells in gels MV2 medium: MV2 (Promocell, Cat. No. C22022) Supplement Mix (Promocell, Cat. No. C39226) 1% Penicillin / Streptomycin (Sigma, Cat. No. P4333) EMEM: Eagle's Minimum Essential Medium (EMEM) (ATCC, Cat. No. 30-2003) 10% fetal bovine serum (Gibco, Cat. No. 16140-071) 35 1% Non-essential amino acids (LifeTech, Cat. No. 11140050) 1% Penicillin / Streptomycin (Sigma, Cat. No. P4333)

[0192] In this example, a three-lane OrganoPlate® (MIMETAS) with 400 μm wide lanes containing a microfluidic network of three lanes and two capillary pressure barriers was used.

[0193] The center lane was filled at the inlet with neutralized bovine type I atelocollagen solution PureCol® EZ Gel 5mg / ml (Advanced BioMatrix) containing intestinal fibroblasts at a concentration of 5,000 cells / μL. The solution was immobilized with a capillary pressure barrier, leaving a free-standing meniscus at the air-liquid interface of the upper and lower lanes. 30μL of HBSS solution was introduced at the inlet of the center lane, and 1μL of 5% FBS solution in PBS was introduced at the hole of the outlet of the center lane forming a dome shape as in Example 1. This created a pressure from the outlet to the inlet due to the surface tension of the droplet and the resulting Laplace pressure. After 1 min, 50μL of HBSS was added to the outlet, and the OrganoPlate was placed in a humidity-controlled environment of 37°C and 5% CO2 for 1 h.

[0194] For cell seeding, 1 μL of primary human umbilical vein endothelial cell (HUVEC) cell pellet containing 10,000 cells / μL was seeded at the outlet of the middle lane. The cells were allowed to attach for 2-6 hours. After this, MV2 cell culture medium was added to the middle lane so that the inlet and outlet volumes were 50 μL, and the OrganoPlate® was placed on a rocking platform at 7°, 8 min intervals, so that the cell culture medium could be passively perfused through the lumen. After 2 days, Caco-2 cells were added in the same manner to the inlet of the top lane, and a Caco-2 cell pellet containing 6,000 cells / μL was seeded at the outlet of the top lane. The cells were allowed to attach for 2-6 hours. After attachment, complete EMEM medium was added to the top lane so that both the inlet and outlet contained 50 μL of complete EMEM medium. The OrganoPlate® was placed on a rocking platform at 7° for 8 min intervals so that cell culture medium could be passively perfused through the lumen.

[0195] Within 1-3 days, both cell types formed tubules within the OrganoPlate®, separated by less than 200 μm of ECM. The gel contained fibroblasts surrounding the HUVEC tubules and located between the HUVEC and Caco-2 tubules. Figure 17A shows a schematic cross-section of the triple co-culture.

[0196] Cell cultures were maintained for 7 days and then fixed with 3.7% formalin solution. Caco-2 cells were stained for epithelial cell adhesion molecule (EPCAM), HUVECs for VE-cadherin, and all three cell types, including fibroblasts, were stained for actin. The resulting confocal 3D reconstructions are shown in Figure 17B. EXAMPLES

[0197] Visualization of the contractile lumen MV2 medium: MV2 (Promocell, Cat. No. C22022) Supplement Mix (Promocell, Cat. No. C39226) 1% Penicillin / Streptomycin (Sigma, Cat. No. P4333)

[0198] In this example, a three-lane OrganoPlate® (MIMETAS) with a 400 μm wide lane was used, containing a microfluidic network of three lanes and two capillary pressure barriers. To provide additional humidity to the plate, the network was filled with 50 μL of HBSS at the observation window (the point where the three lanes converge).

[0199] The central lane was filled with neutralized bovine type I atelocollagen solution PureCol® EZ Gel 5 mg / ml (Advanced BioMatrix) at the inlet and the solution was immobilized by a capillary pressure barrier, leaving a free-standing meniscus at the air-liquid interface in the upper and lower lanes.

[0200] 30 μL of HBSS solution was introduced into the inlet of the center lane, and 1 μL of 5% FBS solution in PBS was introduced into the hole of the outlet of the center lane forming a dome shape as in Example 1. This created pressure from the outlet to the inlet due to the surface tension of the droplet and the resulting Laplace pressure. After 1 minute, 50 μL of HBSS was added to the outlet and the OrganoPlate was placed in a humidity controlled environment at 37° C. and 5% CO2 for 1 hour.

[0201] For cell seeding, 1 μL of cell suspension containing 10,000 cells / μL primary human coronary artery endothelial cells (HCAECs) and 2,500 cells / μL primary human coronary artery smooth muscle cells (HCASMCs) was seeded at the outlet of the center lane. The cell suspension was driven into the microfluidic network by Laplace pressure due to the surface tension of the cell pellet.

[0202] Cells were allowed to attach for 2-6 hours. After this time, MV-2 cell culture medium was added to the center lane so that the inlet and outlet volumes were 50 μL, and the OrganoPlate® was placed on a rocking platform at 7°, intervals of 8 min, so that cell culture medium could be passively perfused through the lumen. Within 1-3 days, cells form tubules within the lumen created within the OrganoPlate®.

[0203] Since the ECM in the lumen is less than 200 μm, it is possible to visualize the contraction of the tubules when exposed to a stimulus. For this, cells were first exposed to norepinephrine at concentrations of 1 μM, 10 μM and 100 μM on day 3, and then to sildenafil at concentrations of 5 μM, 50 μM and 500 μM on day 6, with medium only and vehicle controls included. Images were taken at 1 min intervals for 30 min using a phase contrast ImageExpress Nano microscope (Molecular devices).

[0204] Figures 14A-C show the contraction of HCAEC and HCASMC co-cultures after exposure to sildenafil. Figure 18A was taken before exposure, Figure 18B was taken 30 minutes after exposure, and Figure 18C shows a composite image showing the change in lumen diameter upon exposure to the stimulus. This was made possible by the low force required as a result of the thin ECM generated during lumenization. EXAMPLES

[0205] Migration of T cells from epithelial tubules to tumor cells MCDB131 medium: MCDB131(Thermo Fischer,Cat.No.10372019) 10ng / ml hEGF (Sigma, Cat. No. E9644) 1 μg / ml hydrocortisone (Sigma, Cat. No. H0135) 10 mM L-glutamine (Sigma, Cat. No. G7513) 10% fetal bovine serum (ATCC, Cat. No. 30-2020) 1% Penicillin / Streptomycin (Sigma, Cat. No. P4333) AIM-V medium: AIM-V(Thermo Fischer,Cat.No.12055091)

[0206] In this example, a three-lane OrganoPlate® (MIMETAS) with 400 μm wide lanes containing a microfluidic network of three lanes and two capillary pressure barriers was used. The center lane was filled at the inlet with neutralized bovine type I atelocollagen solution PureCol® EZ Gel 5 mg / mL (Advanced BioMatrix), which was immobilized by a capillary pressure barrier, leaving a free-standing meniscus at the air-liquid interface in the upper and lower lanes.

[0207] 30 μL of HBSS solution was introduced into the inlet of the center lane, and 1 μL of 5% FBS in PBS was introduced into the hole of the outlet of the center lane forming a dome shape as in Example 1. This created pressure from the outlet to the inlet due to the surface tension of the droplet and the resulting Laplace pressure. After 1 minute, 50 μL of HBSS was added to the outlet and the OrganoPlate was placed in a humidity controlled environment at 37° C. and 5% CO2 for 1 hour.

[0208] For cell seeding, 1 µL of primary human mammary epithelial cell (HMEC) pellet containing 10,000 cells / µL was seeded into the top outlet. Cells were allowed to attach for 2-6 h. After this, MCDB131 cell culture medium was added to the top lane such that the inlet and outlet volumes were 50 µL, and the OrganoPlate® was placed on a rocking platform at 7°, with an interval of 8 min, so that cell culture medium could be passively perfused through the lumen.

[0209] Five days later, A375 tumor cells were added to the central inlet in the same manner. For this purpose, 2.5 μL of A375 tumor cell pellet containing 10,000 cells / μL was seeded in the central lane outlet. The cells were left for 2-6 hours. Once the cells had attached, AIM-V cell culture medium (Thermo Fischer) was added to the central lane so that both the inlet and outlet contained 50 μL of AIM-V cell culture medium (Thermo Fischer). The OrganoPlate® was placed on a rocking platform at 7°, intervals of 8 min, so that the cell culture medium could be passively perfused through the lumen. Within 1-3 days, the epithelial cells formed tubules within the OrganoPlate® and were separated from the tumor cells by less than 200 μm of ECM.

[0210] Since the ECM separating the two cell types is less than 200 μm, it is possible to perform a T cell migration assay to investigate the number of T cells migrating from the epithelial tubules to the tumor cell compartment. For this, T cells isolated from the buffy coat on day 6 were fluorescently labeled by resuspending the T cell pellet in 2.5 μM CellTracker CMRA (Invitrogen). The labeled T cells were added to the upper inlet, i.e., the epithelial tubule in the upper lane. First, 50 μL of a T cell suspension in AIM-V cell culture medium containing 400,000 cells / mL was added to the upper lane inlet. Then, 50 μL of AIM-V cell culture medium (Thermo Fischer) was added to the upper lane outlet. The cells were left to enter the epithelial tubules.

[0211] Images were taken 24, 48 and 72 hours after T cell seeding using a phase contrast ImageExpress Nano microscope (Molecular devices). Figure 19 shows that the epithelial and tumor cell compartments closely resemble the in vivo situation, with T cells migrating through the ECM into the tumor cell compartment.

[0212] For comparison, see FIG. 20, where the same experiment was performed in a regular 3-lane OrganoPlate®, but the top lane containing epithelial tubules (as in FIG. 19) was separated from the tumor cells (this time present in the bottom lane) by approximately 400 μm of ECM in the middle lane.

[0213] Figure 21 shows a graph of quantification of T cell migration in the systems of Figures 15 and 16, respectively. As can be seen in Figure 21, twice the amount of T cells was observed in the tumor compartment formed using the method of the present invention ("Luminized ECM" in Figure 21) after 24 and 48 hours compared to the experiment in which epithelial and tumor cells were separated by a 400 μm wide ECM ("Normal 3 lane" in Figure 21).

[0214] Thus, the present invention allows the co-culture of multiple primary cell types, such as human brain-derived vascular cells (e.g., endothelial cells, pericytes and astrocytes), with tumor models in a microfluidic network, creating a system in which their normal 3D spatial relationships (e.g., a thin stromal layer of only a few tens of microns, e.g., 50 μm or less) are preserved. All this is achieved without the use of structural membranes, and thus the behavior of these cells can be investigated in vitro, faithfully mimicking the in vivo environment.

[0215] The above examples are intended to teach those skilled in the art how to practice the invention, and are not intended to detail all modifications and variations that may become apparent upon reading this description, but all such modifications and variations are intended to be included within the scope of the invention as defined in the claims.

Claims

1. A method for creating an internal cavity gel structure, comprising: introducing a first liquid containing a gel precursor solution into a microfluidic network, wherein the microfluidic network includes a capillary pressure barrier at a position generally defining a boundary between a first region and a second region of the microfluidic network; allowing the first liquid to enter the first region of the microfluidic network and align itself along the capillary pressure barrier, thereby forming a liquid-air meniscus of the first liquid at the boundary between the first region and the second region of the microfluidic network; forming a lumen passing through the first liquid by contacting the first liquid with a second liquid, wherein the second liquid has a lower viscosity than the viscosity of the first liquid; allowing or causing the first liquid to gel to form the gel structure including a lumen passing through the gel structure; A method comprising the above steps.

2. The gel structure includes a first surface facing the lumen and a second surface facing the second region of the microfluidic network, and the gel structure has a thickness between the first surface and the second surface of 200 μm or less, or less than 100 μm. The method according to claim 1.

3. A second capillary pressure barrier is provided at a position generally defining a boundary between the first region and a third region of the microfluidic network, and the first liquid aligns itself along the second capillary pressure barrier, thereby forming a third surface of the gel structure facing the third region of the microfluidic network. The method according to claim 2.

4. The step of forming the lumen is Contacting the gel precursor solution with the second liquid at a first position within the microfluidic network; Contacting the gel precursor solution with a third liquid at a second position spaced from the first position, the third liquid having a lower viscosity than the viscosity of the first liquid; The method according to claim 1, comprising: **Claim 5** The step of contacting the gel precursor solution with the second liquid forming a meniscus of the second liquid that is convex in shape and has a first principal radius of curvature; The method according to claim 1, comprising: **Claim 6** The step of contacting the gel precursor solution with the third liquid forming a meniscus of the third liquid that is concave in shape or convex in shape and has a second principal radius of curvature smaller than the first principal radius of curvature; The method according to claim 5, comprising: **Claim 7** The gel structure is a first gel structure, introducing a second gel precursor solution into the second region of the microfluidic network and allowing the second gel precursor solution to contact the first gel structure along the length of the capillary pressure barrier; forming a lumen through the second gel precursor solution by contacting the second gel precursor solution with a liquid having a lower viscosity than the viscosity of the second gel precursor solution; allowing or causing the second gel precursor solution to gel to form the second gel structure including a lumen passing through the second gel structure and contacting the first gel structure; The method according to claim 1, further comprising: **Claim 8** The method according to claim 1, wherein the second liquid comprises a gel precursor solution. **Claim 9**: The method according to claim 1, wherein the gel precursor solution comprises one or more mesenchyme-derived cells selected from, for example, stromal cells, muscle cells, pericyte cells, fibroblasts, and myofibroblasts, or one or more immune cells, such as T cells, monocytes, macrophages, dendritic cells, and / or B cells. **Claim 10** The step of introducing one or more cells into the lumen of the gel structure The method according to claim 1, further comprising this step. **Claim 11** The one or more cells include endothelial cells or epithelial cells, The step of enabling the one or more cells to cover the surface of the lumen and form capillaries within the lumen The method according to claim 10, further comprising this step. **Claim 12**: The method according to claim 2, further comprising the step of introducing one or more cells into the second region of the microfluidic network. **Claim 13** The step of enabling the cells to at least partially cover the second surface of the gel structure and the second region of the microfluidic network, wherein, for example, the one or more cells are selected from endothelial cells or epithelial cells, mesenchyme-derived cells, such as (smooth) muscle cells, pericyte cells, podocytes, fibroblasts, myofibroblasts, stellate cells, or one or more spheroids or organoids; the method according to claim 12, further comprising this step. **Claim 14**: The method according to claim 10, wherein the one or more cells include mesenchyme-derived cells selected from, for example, stromal cells, muscle cells, pericyte cells, fibroblasts, tumor cells, and myofibroblasts. **Claim 15** Introducing one or more immune cells, such as T cells, monocytes, macrophages, dendritic cells, and / or B cells, into the lumen such that the one or more immune cells can adhere to the first surface of the gel or, if present, to the capillary when the one or more immune cells are present; optionally stimulating or allowing the one or more immune cells to cross the epithelial or endothelial vascular wall of the capillary and optionally migrate through the gel structure The method according to claim 1, further comprising.

16. The method according to claim 1, wherein the capillary pressure barrier is provided on the inner surface of the microfluidic network and includes ridges, grooves, or lines of a material having different wettabilities with respect to the inner surface of the microfluidic network.

17. An apparatus comprising a microfluidic network, wherein the microfluidic network at least two inlets; a capillary pressure barrier at a position defining a boundary between a first region and a second region of the microfluidic network; a gel provided in the first region extending between two of the at least two inlets and retained in the first region by the capillary pressure barrier; and including The gel includes a lumen extending therethrough between the two of the at least two inlets. The gel has a first surface facing the lumen and a second surface facing the second region of the microfluidic network, and the thickness of the gel between the first surface and the second surface is 200 μm or less. Device.

18. The apparatus according to claim 17, wherein the microfluidic network includes an opening, and the gel forms a surface facing the opening and / or substantially sealing the opening.

19. The apparatus according to claim 17, wherein the surface of the gel facing the lumen includes a layer of endothelial or epithelial cells forming a first capillary. **Claim 20**: The device according to claim 17, wherein the inner cavity contains one or more mesenchymally-derived cells selected from, for example, interstitial cells, muscle cells, pericytes, fibroblasts, tumor cells, and myofibroblasts, or one or more immune cells, such as T cells, monocytes, macrophages, dendritic cells, and / or B cells. **Claim 21** The device according to claim 17, wherein the second region of the microfluidic network and the second surface of the gel facing the second region include a layer of endothelial or epithelial cells that form a second capillary. **Claim 22**: The device according to claim 17, wherein the microfluidic network includes a second capillary pressure barrier that defines a boundary between the first region and the third region of the microfluidic network or a boundary between the second region and the third region of the microfluidic network, and a gel is provided in the third region of the microfluidic network and retained in the third region by the second capillary pressure barrier. **Claim 23** The device according to claim 17, wherein the gel contains one or more cells or one or more types of cells, such as immune cells or mesenchymally-derived cells. **Claim 24** One or more types of immune cells, such as T cells, monocytes, macrophages, dendritic cells, and B cells, are provided in the inner cavity and / or the gel such that the one or more immune cells adhere to the first surface of the gel, or, if present, to the first capillary, or are provided within the gel structure.

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