Devices for assessing mechanical strain induced in or by cells

Abstract

A microfluidic device comprising a microfluidic network is described herein. The device comprises a substrate, a microfluidic channel, and a cover, wherein the substrate comprises a membrane forming at least a portion of an inner surface of the microfluidic channel. The device can be used in methods for evaluating mechanical strain induced in cells or cellular strain induced by cells, which methods are also described herein.

Description

Technical Field

[0001] The present invention relates to microfluidic devices, and more particularly to methods of inducing or assessing mechanical strain in cells using microfluidic devices. Background Art

[0002] In an attempt to mimic more physiologically relevant conditions in cell culture, a variety of models have been developed, for example, simulating perfusion flow, co-culture, and mechanical strain in preclinical cell-based models for evaluating drug efficacy, ADME safety.

[0003] Microfluidics has become a common platform technology for such in vitro cell culture models due to the inherent flow of liquid or culture medium during use, and microengineering technology has advantages in manufacturing complex microfluidic networks. However, the art still wants to develop models that simulate or reproduce the mechanical strains imposed on cells in, for example, the lungs or intestines, because of the shear stress induced by the air / liquid flow generated by respiratory motion and peristalsis.

[0004] Currently, there is an Emulate chip lung in the art, in which two microfluidic channels are separated by a porous membrane, human alveolar epithelial cells are cultured on one side of the membrane, and human pulmonary microvascular endothelial cells are cultured on the other side of the membrane (Science (2010) 328, p 1662-1668). The Emulate chip lung is also described in the patent application WO2010 / 009307 of Children's Medical Center Corporation. However, direct cell-cell contact and possible juxtacrine signaling are hindered by the presence of the membrane and the size and distribution of the pores.

[0005] Alveolix (http: / / www.alveolix.com / technology / ) has different types of lungs on a chip. Part of the device is described in Bern's patent application WO2015 / 032889. In this device, epithelial cells are cultured on a membrane that is open from the top. On the bottom side of the membrane, the membrane is in contact with a microfluidic channel that has a diaphragm that applies a stretching force to the first membrane after being stimulated.

[0006] To date, there is no way in the art to culture different cell types in a device structure that simultaneously allows for juxtacrine interactions between, for example, the microvascular network and the epithelial cell layer while still being in a position to apply stretching forces to the epithelial cell layer. This requires a completely different approach that does not involve artificial membranes.

[0007] Therefore, there is a need in the art for improved devices for simulating mechanical strain in cells. Summary of the invention

[0008] In a first aspect of the present invention, there is provided a microfluidic device comprising:

[0009] A microfluidic network, the microfluidic network comprising:

[0010] substrate, microfluidic channels, and lid;

[0011] Wherein, the substrate includes a non-porous membrane forming at least a portion of an inner surface of a microfluidic channel, and the microfluidic channel includes a subspace at least partially defined by the membrane and a capillary pressure barrier in the microfluidic channel.

[0012] In a second aspect of the present invention, there is provided a method for assessing mechanical strain induced by a cell, the method comprising:

[0013] introducing one or more types of cells or cell aggregates into the microfluidic network of the microfluidic device of the first aspect;

[0014] Optionally, culturing the one or more types of cells or cell aggregates; and

[0015] Deformation of the diaphragm is monitored using one or more electrodes, sensors, probes, reference markers for monitoring diaphragm movement, ferromagnetic particles, or antibodies disposed on or operably connected to the diaphragm.

[0016] In a third aspect of the invention, provided herein is a method of subjecting one or more types of cells or cell aggregates to mechanical strain (i.e., inducing mechanical strain in one or more types of cells or cell aggregates), the method comprising:

[0017] introducing one or more types of cells or cell aggregates into the microfluidic network of the microfluidic device of the first aspect;

[0018] Optionally, culturing the one or more types of cells or cell aggregates; and

[0019] One or more types of cells or cell aggregates are subjected to mechanical strain by applying positive or negative pressure to the membrane.

[0020] According to a fourth aspect of the present invention, there is provided an analytical plate comprising the device of the first aspect with a gel confined in a subspace of a microfluidic channel by a capillary pressure barrier, optionally wherein the gel comprises one or more cells or cell aggregates.

[0021] According to a fifth aspect of the present invention, there is provided a kit comprising:

[0022] The analysis plate according to the third aspect of the present invention; and

[0023] One or more pro-angiogenic compounds for inducing angiogenesis.

[0024] Previous studies have shown stimulation of epithelial cells and stimulation of both epithelial cells and endothelial cells. However, mechanical stimulation of vascularized tissue in the form of multiple microvessels interacting with epithelial cells in a near-secretory manner (i.e., direct cell-cell contact) has been considered impossible or unproven so far. In order to achieve this purpose, it is necessary to have an initial model without some forms of artificial barriers such as filter membranes, that is, epithelial cells need to be seeded in a position different from endothelial cells, while also allowing those cells to interact directly. The device according to any of the above-described aspects can unexpectedly mechanically stimulate vascularized tissue, therefore, promoting the development of improved in vitro or ex vivo model systems for evaluating drug efficacy or ADME safety.

[0025] Interpretation

[0026] Various different terms related to equipment, methods, uses and other aspects of the present invention are used throughout the specification and claims. Unless otherwise indicated, these terms have the common meanings of the technical field to which the present invention belongs. Other specifically defined terms are interpreted as being consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used to implement the detection of the present invention, preferred materials and methods are described herein.

[0027] As used herein, the singular articles ("a", "an" and "the") include plural referents as well, unless expressly stated otherwise. Thus, for example, the singular term "a cell" includes a combination of two or more cells, etc.

[0028] As used herein, the terms "about" and "approximately" when referring to measurements such as amounts, time ranges, and the like, are intended to include variations of plus or minus 20%, or plus or minus 10%, more preferably plus or minus 5%, even more preferably plus or minus 1%, and still more preferably plus or minus 0.1% of the particular value, such that these variations are suitable for performing the methods disclosed herein.

[0029] The term "comprising" as used herein is interpreted as inclusive and open-ended, not exclusive. Specifically, the term and its variations refer to specific features, steps or components being included. These terms should not be interpreted as excluding the presence of other features, steps or components.

[0030] The term “exemplary” as used herein means serving as an example, instance, or illustration and should not be construed to exclude other configurations disclosed herein.

[0031] The term "microfluidic channel" as used herein refers to a channel located on a material layer covered by an upper substrate or a cover or a channel passing through the material layer, or refers to a channel located under or passing through a material disposed on a bottom plate or a substrate, and at least one of the length, width or height of the channel is in the submillimeter range. It should be understood that the term includes linear channels and branched channels or channels with bends or turns in their passages. Microfluidic channels typically include an inlet for applying a certain volume of liquid. The volume enclosed by the microfluidic channel is typically in the microliter or sub-microliter range. The microfluidic channel typically includes a substrate, which may be the top surface of the lower material; two side walls and a top, which may be the lower surface of the top substrate on the microfluidic channel, and the microfluidic channel may be configured with an inlet, an outlet and / or a vent as required. The substrate, the side walls and the top may all be referred to as the inner surface of the microfluidic channel, and may be collectively referred to as the inner surface. In some embodiments, the microfluidic channel may have a circular or semicircular cross-section, and the microfluidic channel may be considered to have one or two inner surfaces, respectively.

[0032] As used herein, the term "diaphragm" refers to an elastic and / or porous membrane that is elastically biased so that it is deformable under conditions of applied pressure but returns to a resting state once the applied pressure ceases. The terms "stimulation," "displacement," "deflection," or "distortion" used with respect to a diaphragm should be understood to be equivalent to "deformation" of the diaphragm.

[0033] As used herein, the terms "droplet retention structures" and "capillary pressure barriers" are used interchangeably and refer to the property of a device to maintain a liquid-gas or fluid-fluid meniscus in a certain position by capillary forces. The capillary pressure barrier can be considered to be used to separate a microfluidic channel having a volume V0 into two subvolumes V1 and V2 into which different liquids can be introduced. In other words, the capillary pressure barrier at least partially defines a subspace of the microfluidic channel by a boundary located between the two subspaces.

[0034] The term "substantially aligned" as used herein with particular reference to the capillary pressure barrier (e.g., substantially aligned with the aperture) may be understood to mean that the position of the capillary pressure barrier relative to a point on the aperture periphery is not significantly deviated or displaced when viewing the microfluidic device from above.

[0035] The term "closed geometry" used specifically in relation to a capillary pressure barrier refers to a capillary pressure barrier that is different from a linear capillary pressure barrier having two ends and forming a closed loop. For example, when viewed from above, a capillary pressure barrier having a closed geometry may include a circular capillary pressure barrier or a polygonal capillary pressure barrier, such as a triangular capillary pressure barrier, or a square capillary pressure barrier, or a pentagonal capillary pressure barrier, and the like. In some embodiments, the closed geometry of the capillary pressure barrier may also refer to two linear capillary pressure barriers arranged as follows: the two capillary pressure barriers intersect at the same wall or at the wall of the microfluidic channel and thereby close or define a microfluidic channel region with the two linear capillary pressure barriers and their walls as the periphery.

[0036] The term "coaxial" as used herein may be understood to refer to any closed geometric structure having a central and non-circular capillary pressure barrier or any other shape or structure corresponding to the shape or structure of another capillary barrier or pore that is coaxial with the capillary pressure barrier, i.e., co-centered. For example, the term "coaxial" is also understood to refer to two linear capillary pressure barriers arranged as follows: the two capillary pressure barriers intersect at the same wall or at the wall of the microfluidic channel and thereby enclose or define a microfluidic channel region having a center with the two linear capillary pressure barriers and their walls as the periphery.

[0037] The term "linear" capillary pressure barrier as used herein should not be construed as being straight, but rather as having shapes other than closed geometries, i.e., having two ends like a line segment, but which may include one or more bends or corners. The linear capillary pressure barrier typically intersects the sidewalls of the microfluidic channel at each of its ends.

[0038] As used herein, the terms "strain chamber" and "cell culture chamber" refer to a subspace of a microfluidic network that is at least partially defined by a membrane surface. The subspace may also be at least partially defined by a capillary pressure barrier located in the microfluidic network.

[0039] As used herein, the term "endothelial cell" refers to a cell of an endothelial organ or a cell differentiated to a state in which the cell expresses markers that identify the cell as an endothelial cell.

[0040] As used herein, the term "epithelial cell" refers to a cell of an epithelial organ or a cell differentiated to a state in which the cell expresses markers that identify the cell as an epithelial cell.

[0041] As used herein, the term "droplet" refers to a volume of liquid that may or may not exceed the height of a microfluidic channel and that is not necessarily spherical. Specifically, a gel droplet refers to a volume of gel in a strain chamber.

[0042] The term "biological tissue" as used herein refers to a collection of functionally related cells of the same type, similar type, or different types that are cultured and / or analyzed in the methods described herein. The cells can be cell aggregates, and / or specific tissue samples from patients. For example, the term "biological tissue" includes organoids, tissue biopsies, tumor tissue, excised tissue material, spheres, and embryoid bodies.

[0043] The term "cell aggregate" as used herein refers to a 3D cell cluster that is different from surface-attached cells that are usually grown in a monolayer. 3D cell clusters are generally associated with a situation that is more similar to that in vivo. In contrast, surface-attached cells can be significantly affected by the properties of the substrate and may undergo dedifferentiation or undergo transformation into other cell types.

[0044] As used herein, the term "lumened cellular component" refers to a biological tissue having a lumen (ie, a biological tissue composed of cells), for example, a microvessel having an apical and a basal surface.

[0045] As used herein, the term "non-porous" in relation to a membrane refers to a membrane that is substantially impermeable or completely impermeable to liquids, in particular, liquids containing nutrients or waste products from cell culture experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will now be described by way of example only with reference to the accompanying drawings, in which:

[0047] Figures 1 to 3 A vertical cross-sectional view showing a first possible structure of a microfluidic network for use in the device described herein ( Figure 1 ), horizontal top view ( Figure 2 ) and enlarged vertical cross-section ( Figure 3 ).

[0048] Figures 4 to 6 A vertical cross-sectional view showing a second possible structure of a microfluidic network for use in the device described herein ( Figure 4 ), horizontal top view ( Figure 5 ) and enlarged vertical cross-section ( Figure 6 ).

[0049] 7A to 7C An enlarged vertical cross-sectional view of a microfluidic network used in the device described herein is shown, specifically showing a diaphragm ( Fig. 7A ), the diaphragm in a deformed state under negative stimulation ( Figure 7B ) and the diaphragm in the deformed state under positive stimulation ( Figure 7C ).

[0050] FIG. 8A to FIG. 8F Exemplary representative steps in the methods described herein are shown.

[0051] 9A to 9F Exemplary representative steps in alternative methods described herein are shown.

[0052] FIG. 10A to FIG. 10C An enlarged vertical cross-sectional view of an alternative structure of a microfluidic network used in the devices described herein is shown.

[0053] Fig.11A and Fig. 11B Shown are gels or extracellular matrices immobilized by capillary pressure barriers used in the devices described herein and gels or extracellular matrices immobilized by the edges of wells of microfluidic networks of different structures used in the devices described herein.

[0054] Fig.12 and Fig.13 The use of alternative structures of the microfluidic networks used in the devices described herein is shown.

[0055] Fig.14A and Fig. 14B Shown are alternative ways of securing the membrane to the microfluidic networks or devices described herein.

[0056] Fig.15 A plan view of a device according to the invention and consisting of the porous structure of the microfluidic network described herein is shown.

[0057] Fig.16 and Fig.17 A vertical cross-sectional view of a device described herein and comprised of a porous structure of a microfluidic network is shown. DETAILED DESCRIPTION

[0058] Microfluidic devices

[0059] The microfluidic device is described below. The microfluidic device is preferably in a multi-array format / multi-porous format so that it can be applied to in vitro cell analysis, drug screening analysis, toxicity analysis, etc., specifically, the microfluidic device is a high-throughput screening format. These multi-porous culture plates can provide 6 sample wells, 12 sample wells, 24 sample wells, 48 ​​sample wells, 96 sample wells, 384 sample wells and 1536 sample wells arranged in a rectangular matrix, wherein, in this article, there is a multi-array structure of the microfluidic network described herein in the microfluidic device. In one embodiment, the microfluidic device is compatible with one or more dimensions of a standard ANSI / SLAS microtiter plate. In an optional embodiment, the microfluidic device is a multi-array format with the size of a microscope slide.

[0060] Therefore, the microfluidic device preferably has a plurality of microfluidic networks as described herein.In one embodiment, the plurality of microfluidic networks are non-fluidically connected to each other; in other words, each microfluidic network operates independently of any other microfluidic network present in the microfluidic device.

[0061] In one embodiment, a microfluidic device comprises:

[0062] A microfluidic network, the microfluidic network comprising:

[0063] substrate, microfluidic channels, and lid;

[0064] wherein the substrate comprises a membrane forming at least a portion of an inner surface of the microfluidic channel, and wherein the microfluidic channel comprises a subspace at least partially defined by the membrane and a capillary pressure barrier in the microfluidic channel.

[0065] In one embodiment, a microfluidic device comprises:

[0066] A microfluidic network, the microfluidic network comprising:

[0067] a microfluidic channel including a cell culture chamber;

[0068] a cover positioned over the microfluidic channel; and

[0069] a substrate on which the microfluidic channel is disposed,

[0070] The substrate includes a hole leading to the cell culture chamber and a membrane extending through the hole and thereby forming at least a portion of a floor of the cell culture chamber.

[0071] In one embodiment, a microfluidic device comprises:

[0072] A microfluidic network, the microfluidic network comprising:

[0073] a microfluidic channel including a strain chamber;

[0074] a cover positioned over the microfluidic channel; and

[0075] a substrate having a microfluidic channel disposed thereon,

[0076] The substrate includes a hole leading to a cell culture chamber and a membrane extending through the hole and thereby forming at least a portion of the floor of the strain chamber.

[0077] In one embodiment, a microfluidic device comprises:

[0078] A microfluidic network, the microfluidic network comprising:

[0079] Microfluidic channels;

[0080] a cover positioned over the microfluidic channel, the cover comprising a hole leading to the microfluidic channel; and

[0081] a substrate on which the microfluidic channel is disposed;

[0082] The substrate includes a membrane forming at least a portion of a floor of the microfluidic channel, wherein the membrane is substantially aligned with the aperture.

[0083] In one embodiment, a microfluidic device comprises:

[0084] A microfluidic network, the microfluidic network comprising:

[0085] Microfluidic channels;

[0086] a cover positioned over the microfluidic channel; and

[0087] A substrate on which the microfluidic channel is provided, the substrate including a region having a thinner cross section than a peripheral portion of the substrate.

[0088] In one embodiment, a microfluidic device comprises:

[0089] A microfluidic network, the microfluidic network comprising:

[0090] a substrate having a microfluidic channel on an inner surface, and a cover including a hole leading to the microfluidic channel;

[0091] The microfluidic channel includes first and second capillary pressure barriers, which are respectively arranged on the same inner surface and are substantially aligned with and coaxial with the hole on the cover.

[0092] In general, a microfluidic device is a microfluidic device that includes at least a microfluidic network having microfluidic channels. Different structures of microfluidic channels or networks are within the scope of the present invention, but may also include, for example, spaces or subspaces within or in fluid communication with microfluidic channels that are used to receive or confine a gel, for example, an extracellular matrix.

[0093] Microfluidic devices generally include a microfluidic network, which will be described in detail below.

[0094] Microfluidic Networks

[0095] A microfluidic network of a microfluidic device generally comprises a substrate, microfluidic channels or a microfluidic layer, and a lid (also referred to herein as a cover layer) and can be manufactured in a variety of ways.

[0096] The substrate (also referred to herein as a substrate layer or base plate) is preferably formed of a substantially rigid material (e.g., glass or plastic) and is used to provide a support surface for the remainder of the microfluidic network. In one embodiment, the size of the substrate is the same or similar to the size of the well area of ​​a standard ANSI / SLAS microtiter plate. In some embodiments, the substrate includes a hole leading to a microfluidic layer or microfluidic channel, and the diaphragm described herein extends through the hole. In some embodiments, the substrate is formed of a bulk material having a rigidity sufficient to support the remainder of the microfluidic device, but the bulk material acts as an elastomer in the form of a sheet. In these embodiments, the substrate may include a cross-sectional area thinner than the peripheral portion, for example, the remainder of the thinner cross-sectional area of ​​the substrate having sufficient elasticity can play the role of the diaphragm described herein.

[0097] In one embodiment, the base layer may include a membrane sandwiched and laminated between two sheets of etched, laser drilled, or ground glass.

[0098] In the process of using the microfluidic device, the substrate interacts in a manner that stimulates the diaphragm. For example, the substrate can be configured to operably connect the diaphragm to one or more of a positive pressure source or a negative (air) pressure source (i.e., a pump), a physical actuator, an electromagnetic actuator, and a foamable foam. These methods of stimulating the diaphragm are known in the art and need not be discussed further.

[0099] The microfluidic device or microfluidic network includes a microfluidic channel or a microfluidic layer disposed on a substrate. In some embodiments, the microfluidic channel may include or may be divided into a plurality of subspaces, for example, separated by the presence of a capillary pressure barrier as described herein. In some embodiments, the microfluidic channel may include a first subspace, which may be referred to as a strain chamber or a cell culture chamber. In some embodiments, the strain chamber or the cell culture chamber may be defined in part by a capillary pressure barrier and / or a diaphragm present in the microfluidic channel. In some embodiments, the diaphragm may form at least a portion of a surface or floor of the first subspace.

[0100] In certain embodiments, the microfluidic channel also includes a second subspace, which includes a fluid channel, and a third subspace separated from the second subspace by the first subspace. In certain embodiments, the fluid channel of the second subspace is the fluid channel in use. The third subspace can be the second fluid channel in use near the first subspace, or conceptually, the third subspace can be at least partially located above the first subspace, and once the first subspace has been filled with, for example, gel or extracellular matrix compositions, the third subspace can be left to be filled or occupied. In certain embodiments, the diaphragm forms at least a portion of the surface of the third subspace. The third subspace can be limited by another capillary pressure barrier.

[0101] A typical method for preparing microfluidic channels is to cast a moldable material such as polydimethylsiloxane on a mold, thereby printing the microfluidic channels in a silicone rubber material, thereby forming a microfluid layer. The rubber material with the embedded channel is then placed on a glass substrate layer or on the same rubber material substrate layer, thereby sealing. Alternatively, the channel structure can be etched in a material such as glass or silicon, which is then bonded to a top substrate or a bottom substrate (also referred to as a cover layer and a substrate layer). Plastic injection molding or relief printing performed after bonding is another method for manufacturing a microfluidic channel network. Another technique for manufacturing a microfluidic channel network is to form a microfluidic channel network pattern by photolithography in a polymer (e.g., SU-8 or various other dry films or liquid photoresists) that can be exposed to imaging, followed by a bonding step. When bonding, bonding refers to a closed channel by a lid or substrate. Bonding techniques include anodic bonding, covalent bonding, solvent bonding, adhesive bonding, and thermal bonding, etc.

[0102] It can be inferred from the various different manufacturing methods described above that the microfluidic layer may include a sublayer comprising a microfluidic channel disposed on a substrate layer or a pattern of the microfluidic layer is formed on a cover layer or substrate layer. In the use direction, the microfluidic sublayer is disposed on the top surface of the substrate layer. The microfluidic channel may be formed as a channel passing through a material sublayer disposed on the substrate layer. In one embodiment, the material sublayer is a polymer disposed on the substrate layer and a microfluidic channel pattern is formed in the material sublayer. In some embodiments, the microfluidic layer includes two or more microfluidic channels, which can be fluidically connected to each other.

[0103] The microfluidic network includes a cover or cover layer covering the microfluidic channels. The cover or cover layer can be formed of any suitable material known in the art, for example, a glass layer bonded to a sublayer including the microfluidic channels. In one embodiment, the cover layer is provided with preformed holes at defined points. The holes (also referred to herein as inlet holes) allow the microfluidic channels of the microfluidic layer to be in fluid communication with other elements of the microfluidic device disposed thereon. In general, the inlet holes serve as an interface with the external space or a hole disposed on top of the hole.

[0104] According to the requirements of any specific use of the microfluidic network of the microfluidic device, the microfluidic channel may be provided with one or more additional fluid inlets, and one or more outlets or vents. In order to fill liquid, drain liquid and perfuse liquid through the microfluidic network, the microfluidic channel is preferably provided with at least one inlet and at least one outlet or vent. In one embodiment, each of at least one inlet and at least one outlet or vent is preferably a preformed hole in the cover layer. It should be understood that there is no geometrical difference between the inlet and the outlet and that the inlet and outlet can be used interchangeably in many cases.

[0105] In some embodiments, the microfluidic device further comprises a top layer disposed on the above-mentioned cover layer, the top layer having one or at least one hole in fluid communication with other parts of the microfluidic device. In some embodiments, the top layer has a plurality of such holes, and at least one (e.g., at least two, e.g., at least three) hole is in communication with the microfluidic network or channel of the device. For example, the top layer may include a hole in fluid communication with the microfluidic network through an inlet hole disposed in the cover layer of the microfluidic network, thereby forming a SLAS compatible orifice plate. The hole and the inlet hole may be substantially aligned with the diaphragm of the microfluidic device described herein. In some embodiments, the top layer having at least one hole and the microfluidic layer are integrally formed. For example, a microfluidic channel may be formed on the bottom surface of an injection molded microtiter plate having at least one hole.

[0106] Diaphragm

[0107] In some embodiments, the microfluidic devices disclosed herein generally include a diaphragm that is in the form of an elastic membrane and / or a non-porous membrane. These properties of the diaphragm disclosed herein distinguish it from various types of membranes commonly used for cell culture in microfluidic devices, which are used as a permeable support for the cells being cultured while isolating the cells from perfusion channels that provide nutrients and / or remove waste, or isolating the cells from other co-cultured cells.

[0108] The function of the diaphragm of the device described herein is to simulate muscle stimulation in the body, for example, to deform in a repetitive manner to simulate breathing, peristalsis or heartbeat, or to deform in a non-repetitive manner to simulate the expansion or constriction of blood vessels, or to simulate the contraction / relaxation of muscles such as the iris.

[0109] In some embodiments, the diaphragm at least partially forms the inner surface of the microfluidic channel, for example, the floor. In some embodiments, the diaphragm at least partially forms the inner surface of the microfluidic channel, for example, the floor, a subspace (e.g., the first subspace and / or the second subspace and / or the third subspace) of the microfluidic channel. In some embodiments, the diaphragm at least partially forms the inner surface of the cell culture chamber, for example, the floor. In some embodiments, the diaphragm at least partially forms the inner surface of the strain chamber, for example, the floor. In some embodiments, the diaphragm is substantially aligned with the inlet hole provided in the cover of the microfluidic device.

[0110] In some embodiments, the substrate comprises two sublayers with an elastomeric sheet sandwiched between the two sublayers. In these embodiments, the two sublayers of the substrate have co-aligned holes, wherein the elastomer extends completely through the holes to form a diaphragm. In other embodiments, the elastomeric sheet forming the diaphragm is similar in size to the hole and is adhered to the upper surface of the substrate, the lower surface of the substrate, or the inner sidewall of the hole using standard bonding techniques such as adhesives, clamps, surface stretching, covalent bonding, anchoring, molding, or other manufacturing techniques.

[0111] The membrane may be a biocompatible membrane, which means that the membrane is formed of an elastomer that is biocompatible and suitable for cell culture purposes. Those skilled in the art know what requirements a material meets to be considered biocompatible and suitable for cell culture, but examples of biocompatibility may include good cytokinesis, low gas permeability, low cytotoxicity, chemical inertness, low filtration, and low autofluorescence.

[0112] The diaphragm may include an elastomer selected from the following materials: polyisoprene, polybutadiene, chloroprene, butyl rubber, styrene-butadiene, nitrile, ethylene propylene, ethylene propylene diene, epichlorohydrin, polyacrylic rubber, silicone, polydimethylsiloxane, fluorosilicone rubber, fluoroelastomer, perfluoroelastomer, polyether block amide, chlorosulfonated polyethylene, ethylene-vinyl acetate, polyurethane, polysulfide, polyvinylidene fluoride (PVDF), ultra low density polyethylene (ULDPE), vinyl alcohol (EVOH). Examples of commercially available elastomers include: Dai-ElTM, and Those skilled in the art will appreciate that reversibly deformable elastic materials may be used as diaphragms.

[0113] In some embodiments, the membrane is transparent or optically clear, and preferably has a thickness of less than 1 mm, more preferably less than 250 μm, and more preferably less than 100 μm.

[0114] In some embodiments, the membrane is a functionalized membrane comprising one or more electrodes, sensors, probes, reference markers for monitoring membrane movement, ferromagnetic particles, or adhesion molecules or antibodies for promoting cell adhesion to the membrane surface.

[0115] Functionalizing the membrane in this manner allows experimental monitoring of the mechanical strain exerted by cells placed on the membrane, and control of external stimulation of the membrane by monitoring the extent of deformation relative to the applied stimulation force.

[0116] The shape of the membrane and / or the hole in the substrate through which the membrane extends is not limited to any particular shape, but may, for example, correspond to a circle, an oval, a rectangle, a rounded rectangle, a dog bone or a star.

[0117] The size of the diaphragm is generally 1 mm to 2 mm for 384-well plate arrangements. However, a larger diaphragm may be advantageous for some applications, especially for example in conjunction with a 98-well microtiter plate. In the latter case, a diaphragm of 2 mm to 4 mm or more may be advantageous.

[0118] Capillary pressure barrier

[0119] The microfluidic network of a microfluidic device may include a capillary pressure barrier.

[0120] In some embodiments, the capillary pressure barrier is substantially aligned with the hole in the cover. In some embodiments, the capillary pressure barrier separates the microfluidic channel into a first subspace and a second subspace. In some embodiments, the capillary pressure barrier is combined with the diaphragm to at least partially define the subspaces of the microfluidic channel.

[0121] The function and form of the capillary pressure barrier have been previously described, for example, in WO2014 / 038943A1. It will be clear from the exemplary embodiments described below that the capillary pressure barrier (also referred to herein as a droplet retention structure) should not be understood as a wall or chamber that can be filled with droplets containing one or more cells or cell aggregates, for example, but should be understood as consisting of or including a structure that ensures that such droplets do not spread due to surface tension. This concept is called meniscus fixation. In this way, it is possible to stably confine droplets containing one or more cells or cell aggregates to a subspace of a microfluidic channel of a device. In one embodiment, the capillary pressure barrier may be referred to as a confinement phase, which is configured so that overflow does not occur during normal use of the cell culture device or during initial filling of the cell culture device with a first liquid. The confinement characteristics of the droplets will be described below when describing the method of the present invention.

[0122] In one embodiment, the capillary pressure barrier comprises or consists of an edge of material or a ridge of material protruding from the inner surface of the microfluidic channel or a groove in the inner surface of the microfluidic channel. The side wall of the edge or ridge is at an angle α with the top of the edge or ridge, which angle α is preferably as large as possible. In order to provide a good barrier, the angle α should be greater than 70°, typically about 90°. The same is true for the angle α between the side wall of the ridge and the inner surface of the microfluidic channel on which the capillary pressure barrier is disposed. Similar requirements apply to capillary pressure barriers formed as grooves.

[0123] An optional form of the capillary pressure barrier is a material area with different wettability to the inner surface of the microfluidic channel, which area acts to stop diffusion due to capillary forces / surface tension. In one embodiment, the inner surface of the microfluidic channel includes a hydrophilic material and the capillary pressure barrier is a hydrophobic area or an area of ​​less hydrophilic material. In one embodiment, the inner surface of the microfluidic channel includes a hydrophobic material and the capillary pressure barrier is a hydrophilic area or an area of ​​less hydrophobic material.

[0124] Thus, in a particular embodiment of the invention, the capillary pressure barrier is selected from: an edge or ridge, a groove, a hole or a hydrophobic lining of a material or a combination thereof. In other embodiments, the capillary pressure barrier may be formed by a widening of the microfluidic channel or pillars at selected intervals, the arrangement of the capillary pressure barrier defining a first subspace or region to be occupied by the gel. In one embodiment, the pillars extend the entire height of the microfluidic channel.

[0125] Due to the presence of the capillary pressure barrier, the liquid will not flow beyond the capillary pressure barrier and can form a stable confined volume in the microfluidic channel, for example, one or more of the first, second or third subspaces, any of which can be referred to as or serve as a strain chamber or a cell culture chamber.

[0126] The capillary pressure barrier may be substantially aligned with the aperture in the cover layer, thereby limiting the spreading of fluid droplets in the microfluidic network. In one embodiment, the capillary pressure barrier is located on the bottom surface of the cover layer, substantially adjacent to the aperture. In one embodiment, the capillary pressure barrier is at least partially formed by the aperture itself.

[0127] In one embodiment, the capillary pressure barrier is disposed on the inner surface of the microfluidic channel, facing the hole in the cover. In a more specific embodiment, the capillary pressure barrier is disposed on the substrate of the microfluidic layer or on the inner surface of the microfluidic channel, substantially opposite to or facing the hole in the cover. In one embodiment, the capillary pressure barrier is present as previously described, thereby confining the droplet to a subspace of the microfluidic layer aligned with the hole in the cover.

[0128] In one embodiment, the capillary pressure barrier at least partially defines a surface (e.g., a floor) of a first subspace of the microfluidic channel, which may also be referred to as a cell culture chamber or a strain chamber. The capillary pressure barrier is configured to confine the liquid to the first subspace of the microfluidic channel. In one embodiment, the capillary pressure barrier comprises a closed geometric structure. In one embodiment, the capillary pressure barrier is coaxial with the pores of the cover layer.

[0129] In one embodiment, the diameter or area defined by the periphery of the capillary pressure barrier is greater than the diameter or area defined by the periphery of the hole in the cover, in other words, the perimeter of the capillary pressure barrier is greater than the perimeter of the hole. In another embodiment, the diameter or area defined by the periphery of the hole is greater than the diameter or area defined by the periphery of the capillary pressure barrier, in other words, the perimeter of the hole is greater than the perimeter of the capillary pressure barrier. Regardless of the shape, in a preferred embodiment, the capillary pressure barrier outlines the contact area of ​​the droplet or gel composition including one or more cells or cell aggregates introduced into the microfluidic channel with the substrate of the microfluidic channel (i.e., the periphery of the contact area of ​​the droplet including one or more cells or cell aggregates with the substrate of the microfluidic channel).

[0130] In one embodiment, the capillary pressure barrier is a substantially linear capillary pressure barrier that spans the entire width of the microfluidic channel and each end of which intersects a sidewall of the microfluidic channel.

[0131] As part of the microfluidic network, a capillary pressure barrier separates the network into at least two subspaces.

[0132] Second capillary pressure barrier

[0133] In some embodiments, the microfluidic network of the device is provided with a second capillary pressure barrier, which is substantially as described above in form and function. To avoid confusion, when there is a second capillary pressure barrier in the device, it is understood to be the "first capillary pressure barrier" when referring to the "capillary pressure barrier".

[0134] In some embodiments, the second capillary pressure barrier is substantially aligned with the hole in the cover layer, thereby limiting the spreading of droplets in the microfluidic network. In one embodiment, the second capillary pressure barrier is located on the bottom surface of the cover layer, substantially adjacent to the hole. In one embodiment, the second capillary pressure barrier is at least partially formed by the hole itself.

[0135] In one embodiment, the second capillary pressure barrier is disposed on the inner surface of the microfluidic channel, facing the hole in the cover. In a more specific embodiment, the second capillary pressure barrier is located on the substrate of the microfluidic layer or on the inner surface of the microfluidic channel, substantially opposite to or facing the hole. In one embodiment, the second capillary pressure barrier is present relative to the hole as defined above, thereby confining the droplet to a region of the microfluidic layer aligned with the hole.

[0136] In one embodiment, the second capillary pressure barrier is combined with the first capillary pressure barrier to at least partially define the surface of the strain chamber or cell culture chamber on the substrate of the microfluidic layer, on the substrate of the microfluidic channel and / or on the diaphragm. The second capillary pressure barrier is configured to confine the liquid to a first subspace including the strain chamber and / or the cell culture chamber in combination with the first capillary pressure barrier. In one embodiment, the second capillary pressure barrier comprises a closed geometric structure. In one embodiment, the second capillary pressure barrier is coaxial with the hole of the cover layer and / or the first capillary pressure barrier. In one embodiment, the diameter or area defined by the periphery of the second capillary pressure barrier is greater than the diameter or area defined by the periphery of the hole and / or the periphery of the first capillary pressure barrier, in other words, the perimeter of the second capillary pressure barrier is greater than the first capillary pressure barrier and / or the hole. In one embodiment, the second capillary pressure barrier is coaxial with the first capillary pressure barrier and is located within the periphery of the first capillary pressure barrier. In another embodiment, the diameter or area defined by the periphery of the hole is greater than the diameter or area defined by the periphery of the second capillary pressure barrier, in other words, the periphery of the hole is greater than the periphery of the second capillary pressure barrier. Regardless of the shape, in a preferred embodiment, the second capillary pressure barrier describes the contact area of ​​the droplet or gel composition including one or more cells or cell aggregates introduced into the strain chamber and the substrate of the strain chamber, that is, the periphery of the contact area of ​​the droplet including one or more cells or cell aggregates and the substrate of the strain chamber.

[0137] In one embodiment, the second capillary pressure barrier is a substantially linear capillary pressure barrier that spans the entire width of the microfluidic channel and each end of which intersects the side wall of the microfluidic channel. In this embodiment, the first and second capillary pressure barriers in combination with the walls intersecting therewith can define an area aligned with the holes of the cover layer and the area can also be coaxial with the holes of the cover layer. In this embodiment, the first capillary pressure barrier can be considered to separate the microfluidic network into a first subspace including a strain chamber or a cell culture chamber and a second subspace including the microfluidic channel, wherein the second capillary pressure barrier separates the microfluidic network into a first subspace including the cell culture chamber or the strain chamber and a third subspace including the second microfluidic channel.

[0138] As part of the microfluidic network, the second capillary pressure barrier separates the network into at least two subspaces, the first of which is a first subspace, which refers to the subspace including the strain chamber or the cell culture chamber as described above, and a third subspace. In one embodiment, the third subspace includes a portion of the microfluidic channel separated from the first subspace (i.e., not included in the first subspace). In one embodiment, the third subspace is completely included in the first subspace, i.e., the first and second capillary pressure barriers are both closed geometric structures and the second capillary pressure barrier is completely surrounded by the first capillary pressure barrier.

[0139] In some embodiments, the first and second capillary pressure barriers are both disposed on a substrate or bottom plate of the microfluidic channel, or on an upper surface or top surface of the microfluidic channel. In some embodiments, the first capillary pressure barrier defines a first subspace of the microfluidic channel aligned with the hole. In some embodiments, the first and second capillary pressure barriers define a second subspace of the microfluidic channel, which is coaxial with the first subspace and the hole and closes the first subspace. In some embodiments, the first and second capillary pressure barriers are closed geometric structures (e.g., circular) and the second capillary pressure barrier surrounds the first capillary pressure barrier. In some embodiments, the first capillary pressure barrier includes a first pair of linear capillary pressure barriers, which are disposed on opposite sides of the hole and extend to opposite inner surfaces to define the first subspace, and the second capillary pressure barrier includes a second pair of linear capillary pressure barriers, which are disposed on opposite sides of the hole, extend to opposite inner surfaces and are spaced apart from the first capillary pressure barrier and are located outside the first capillary pressure barrier, thereby defining the second subspace and the third subspace. In these embodiments, external tissue samples (e.g., tissue sections) or organoids can be placed in chambers created within and by a fixing gel or ECM because the plane of the sample is coplanar with the vascularized bed and, therefore, more easily vascularized (once the gel has been vascularized). This configuration also allows for better positioning of the tissue for visualization of the entire system because all elements are in the same focal plane.

[0140] Reservoir

[0141] In some embodiments, the microfluidic network includes a reservoir or well connected to the culture medium fluid entering the microfluidic channel. The reservoir can store a certain volume of liquid, such as culture medium. In a typical embodiment, the reservoir is capable of storing a fluid volume larger than the fluid volume stored by the microfluidic channel or the fluid volume that can be stored by the microfluidic channel. The reservoir can be a well adjacent to the following hole, which is aligned with the entrance hole of the cell culture chamber on the bottomless microtiter plate arranged on the top of the microfluidic layer. In other embodiments, the reservoir can be a well located on the same microtiter plate, but spatially away from the hole of the strain chamber. It should be understood that the proximity of the reservoir to the hole of the strain chamber is not critical to the operation of the device, as long as the two are connected through the microfluidic layer fluid.

[0142] In some embodiments, the microfluidic network includes more than one, such as two or more, reservoirs in fluid communication with the microfluidic layer and in fluid communication with the cell culture chamber or strain chamber and any other reservoirs present in the microfluidic network. Each reservoir may be in fluid communication with the microfluidic layer through a hole in the cover layer of the microfluidic layer, which may be referred to as an inlet or outlet, as appropriate. In embodiments where there are at least two reservoirs in the microfluidic network, a first reservoir may be used to direct liquid, such as culture medium, into the microfluidic network, while a second reservoir may act as a vent or an overflow chamber for receiving liquid during the process of performing the methods of the present invention.

[0143] In some embodiments, the microfluidic network of the device further comprises a biological material or a biomimetic material, which includes one or more of the following components:

[0144] a. a gel, an extracellular matrix or a scaffold disposed in, for example, the first subspace;

[0145] b. epithelial cells or endothelial cells lining microfluidic channels and / or gels, such as those forming conduits or blood vessels;

[0146] c. epithelial cells or endothelial cells located inside, on or against a gel, extracellular matrix or scaffold, preferably forming an endoluminal structure, more preferably forming a vascular bed;

[0147] d. stromal cells located within, on, or against a gel, extracellular matrix, or scaffold;

[0148] e. muscle cells located within, on, or against a gel, extracellular matrix, or scaffold;

[0149] f. One or more types of cells selected from pluripotent cells, central nervous cells, peripheral nervous cells, immune cells, urinary cells, respiratory cells, germ cells (male and female), gastrointestinal cells, endocrine cells, skin cells, musculoskeletal cells, cardiovascular cells and mammary cells.

[0150] Because of the presence of cells, these devices can also be considered as analytical plates, for example in the form of a vascular network and an optional biological tissue disposed on the top surface of the extracellular matrix, and are therefore easy to use in the analysis or methods described herein. As can be understood by the content disclosed herein, the products of these devices can be identified using any of the methods described below. In one embodiment, endothelial cell buds extend into the extracellular matrix gel to form a vascular bed. Optionally, these cell buds are microvessels generated by angiogenesis or vasculogenesis.

[0151] Any one or more forms of biological tissue in the above-mentioned different cell types may include or may be derived from healthy tissue or diseased tissue, and may be obtained or derived from a patient. Endothelial cells forming a vascular network may be obtained or derived from a patient, such as the same patient from whom the biological tissue was obtained or from which the biological tissue was derived. In one embodiment, the endothelial cells include endothelial cells that naturally develop from blood vessels (e.g., as described in Nature Protocols 7, 1709–1715 (2012)) or endothelial cells derived from stem cells, including but not limited to induced pluripotent stem cells.

[0152] method

[0153] In one embodiment, provided herein is a method for assessing mechanical strain induced by a cell, comprising:

[0154] introducing one or more types of cells or cell aggregates into a microfluidic network of a microfluidic device described herein;

[0155] Optionally, culturing one or more types of cells or cell aggregates; and

[0156] The deformation of the diaphragm is monitored using one or more electrodes, sensors, probes, reference markers for monitoring the movement of the diaphragm, ferromagnetic particles or antibodies disposed on or operably connected to the diaphragm.

[0157] In one embodiment, provided herein is a method of subjecting one or more types of cells or cell aggregates to mechanical strain, that is, a method of inducing mechanical strain in one or more types of cells or cell aggregates, comprising:

[0158] introducing one or more types of cells or cell aggregates into the microfluidic network of the microfluidic device of the first aspect;

[0159] Optionally, culturing one or more types of cells or cell aggregates; and

[0160] The one or more types of cells or cell aggregates are subjected to mechanical strain by applying positive or negative pressure to the membrane.

[0161] In some embodiments, the methods described herein include:

[0162] introducing a volume of gel or gel precursor into the microfluidic network;

[0163] causing the volume of gel or gel precursor to solidify or gel to form a solidified gel;

[0164] loading the microfluidic network with fluid; and

[0165] One or more types of cells or cell aggregates are cultured.

[0166] In some embodiments, the methods described herein may include:

[0167] introducing a volume of gel or gel precursor into the first subspace and confining the volume of gel or gel precursor by a capillary pressure barrier;

[0168] causing the volume of gel or gel precursor to solidify or gel to form a solidified gel;

[0169] loading the microfluidic network with fluid, and

[0170] One or more types of cells or cell aggregates are cultured.

[0171] In some embodiments, the volume of gel or gel precursor may be a single droplet or droplet-sized gel or gel precursor.

[0172] In some embodiments, after applying pressure to the diaphragm, a cellular response to mechanical strain is monitored. In some embodiments, the cellular response may come from a monolayer of cells formed on the upper surface of the gel droplet or from a vascular bed formed inside the gel. In some embodiments, the cellular response may come from a lumen cell component contained within a microfluidic channel of the microfluidic device. In some embodiments, the cellular response may come from a lumen cell component formed on the surface of the diaphragm.

[0173] The cellular response can be monitored in any manner known in the art. Methods may include monitoring one or more changes in pH, monitoring changes in secreted factors (e.g., metabolites, growth factors, cytokines), sampling cells and / or tissues and monitoring up-regulation or down-regulation of specific proteins, or monitoring levels of reactive oxygen species. Alternatively, or in addition, the cellular or tissue response can be monitored visually (using a microscope), for example, based on immunohistochemical staining or other hybridization formats.

[0174] In some embodiments, one or more types of cells or cell aggregates can be selected from: epithelial cells or endothelial cells used to line microfluidic channels and possibly form conduits or blood vessels, epithelial cells or endothelial cells located inside a gel, extracellular matrix or scaffold (which preferably forms an endoluminal structure, more preferably a vascular bed), stromal cells located within or on a gel, extracellular matrix or scaffold, muscle cells located within or on a gel, extracellular matrix or scaffold, cell types selected from one or more of pluripotent cells, central nervous cells, peripheral nervous cells, immune cells, urinary cells, respiratory cells, reproductive cells (male and female), gastrointestinal cells, endocrine cells, skin cells, musculoskeletal cells, cardiovascular cells and breast cell types.

[0175] Gels or gel precursors include any gel known in the art that is suitable for cell culture. Gels for cell culture can be formed from a large number of natural and synthetic materials that provide a wide spectrum of mechanical and chemical properties. For a review of materials and methods for gel synthesis, see Lee and Mooney (Chem Rev 2001; 101 (7): 1869–1880). When the gel is formed from natural materials, suitable gels promote cell function, and when the gel is formed from synthetic materials, suitable gels allow cell function to be initiated. Natural gels for cell culture are typically formed from proteins and ECM components (e.g., collagen, fibrin, hyaluronic acid or matrigel) and materials derived from other biological sources (e.g., chitosan, alginate or fibrils). Because they are derived from natural sources, these gels themselves are inherently biocompatible and bioactive. Permitted synthetic gels can be formed from pure non-natural molecules (e.g., polyethylene glycol (PEG), polyvinyl alcohol and poly (2-hydroxyethyl methacrylate)). PEG gels have been shown to maintain the viability of encapsulated cells and allow ECM deposition as they degrade, suggesting that synthetic gels can serve as 3D cell culture platforms, even in the absence of integrin-binding ligands. These inert gels are highly reproducible, their mechanical properties can be easily tuned, and they are easy to process and manufacture.

[0176] Gel precursor can be provided to microfluidic cell culture equipment, for example the strain chamber of equipment described above.After gel is provided, it is gelled, and then another fluid is introduced.Suitable gel (gel precursor) is well known in the art.For example, gel precursor can be a hydrogel, and it is usually an extracellular matrix (ECM) gel.ECM for example can include collagen, fibrinogen, fibronectin and / or basement membrane extract (for example, matrigel or synthetic gel).For example, gel precursor can be introduced into the strain chamber with a pipette.

[0177] Gels or gel precursors may include basement membrane extracts, extracellular matrix derived from human or animal tissue or cell culture, extracellular matrix derived from animal tissue, synthetic extracellular matrix, hydrogels, collagen, soft agar, proteins and commercially available products such as Matrigel.

[0178] The basement membrane comprising a thin layer of basal layer is a thin extracellular matrix, which is the basis of epithelial cells in vivo and is such a protein and proteoglycan consisting of extracellular matrix. In one embodiment, basement membrane is made of type IV collagen, laminin, nidogen, heparan sulfate proteoglycan and multiple other trace components (Quaranta et al, Curr.Opin.Cell Biol.6,674-681,1994). These compositions and whole basement membrane are biologically active, and they promote cell adhesion, migration, and promote cell growth and differentiation in many cases. The example based on the gel of basement membrane is called matrigel (Matrigel, US 4829000). This material has very high biological activity in vitro, as the bottom layer of epithelial cells.

[0179] Many different suitable gels for use in the methods of the invention are commercially available, including, but not limited to, those comprising Matrigel rgf, BME1, BME1 rgf, BME2, BME2 rgf, BME3 (all Matrigel variants), collagen type I, collagen type IV, a mixture of collagen type I and collagen type IV, or a mixture of collagen type I, collagen type IV with collagen types II and III, puramatrix, hydrogel, Cell-Tak TM , collagen type I, collagen type IV, Matrix, fibronectin, gelatin, laminin, osteopontin, polylysine (PDL, PLL), PDL / LM and PLO / LM, In a preferred embodiment, the matrix component is commercially available Matrix (Corning, NY 14831, USA).

[0180] A gel or gel precursor is introduced into a device described herein and confined by a hair cell pressure barrier in a microfluidic device, for example, to a first subspace comprising a strain chamber having a device diaphragm as its substrate, and the gel or gel precursor is subsequently gelled or allowed to gel.

[0181] In one embodiment, a sufficient volume of droplets is introduced such that substantially the entirety of the solidified gel is located in a portion of the strain chamber within the microfluidic layer. In one embodiment, the volume of the gelled droplets is a volume that does not completely block the pores in the microfluidic cover layer, wherein the unblocked or open areas of the pores serve as vents. Thus, the vents typically include openings or holes in the cover layer that allow air to escape when the microfluidic channel is loaded through the inlet. In one embodiment, a sufficient volume of droplets is introduced such that the droplets are confined by the capillary pressure barrier and the majority of the liquid volume is contained in a portion of the strain chamber located outside the microfluidic layer, for example, wherein the majority of the droplet volume is contained within the pores of the top layer.

[0182] In one embodiment, the gel or gel precursor is preloaded with target cells, that is, the cells are present in droplets of the gel or gel precursor, which are then introduced into a microfluidic cell culture device and subsequently gelled. In another embodiment, after droplets of the gel or gel precursor are introduced into a microfluidic cell culture device (e.g., a strain chamber of a device as described herein), the cells are inserted into partially or completely solidified droplets. Therefore, an optional method includes inoculating the target cells in solidified droplets of a cell culture gel. In another embodiment, the gel or gel precursor is introduced into a microfluidic cell culture device and subsequently gelled, and a cell mixture, tissue, or cell aggregate is placed on top of the gel or in an area near the gel of a microfluidic channel.

[0183] The cell mixture, tissue or cell aggregate within, on or next to the solidified gel may include epithelial cells or endothelial cells, stromal cells, muscle cells, one or more other cell types selected from multipotent cells and central nervous cells, peripheral nervous cells, immune cells, urinary cells, respiratory cells, germ cells (male and female), gastrointestinal cells, endocrine cells, skin cells, musculoskeletal cells, cardiovascular cells and breast cell types.

[0184] In one embodiment, at least one type of cell or cell aggregate present in or on top of the droplet of gel or gel precursor includes epithelial cells, other cell types and extracellular matrix that may be present, and the epithelial cells can proliferate and / or differentiate based on the composition of the culture medium during the culture process. Therefore, after epithelial cells are introduced into the microfluidic network using an aqueous culture medium (preferably a growth medium) or using a gel (gel precursor), the epithelial cells will proliferate and / or differentiate. The cultivation of one or more types of cells or cell aggregates (e.g., epithelial cells) is carried out by introducing the culture medium into the microfluidic channel and cultivating under suitable conditions, so that the cells can be cultured. In order to avoid confusion, the use of the term "droplet" should be considered to refer to the volume of the gel introduced into the cell culture device described herein and then confined in the cell culture device.

[0185] In one embodiment, after the gel precursor in the first subspace (e.g., strain chamber) of the microfluidic network is gelled, one or more cells or cell aggregates are introduced into the second subspace of the microfluidic network, e.g., the region adjacent to the gel of the microfluidic channel and the capillary pressure barrier. One or more cells or cell aggregates can be epithelial cells or endothelial cells. In general, it is known that endothelial cells are cells lining the inner surface of the entire circulatory system from the heart to the smallest lymphatic capillaries. When in contact with blood, these cells are referred to as vascular endothelial cells, and when in contact with the lymphatic system, these cells are referred to as lymphatic endothelial cells. In a specific embodiment, the culture method includes introducing endothelial cells into the microfluidic channel of the microfluidic network and making the endothelial cells lined in the microfluidic channel (i.e., making endothelial cells form blood vessels in the microfluidic channel). Cells or cell aggregates can be introduced into the microfluidic network using any suitable culture medium.

[0186] Introducing endothelial cells into the microfluidic channel under appropriate conditions (e.g., conditions suitable for promoting angiogenesis) can not only form vascular tissue lining the inner surface of the channel, but also, in some cases, the inner surface of the extracellular matrix gel that becomes permeable can grow new microvessels. Suitable conditions for promoting angiogenesis include adding pro-angiogenic compounds, such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), phorbol tetradecanoyl-13-acetate (PMA), sphingosine-1-phosphate (S1P), IGFBP-2, hepatocyte growth factor (HGF), prolyl hydrogenase inhibitor (PHi), monocyte chemoattractant protein-1 (MCP-1), basic fibroblast growth factor (bFGF) and ephrin, etc.

[0187] When one or more pro-angiogenic compounds are applied in a gradient, the one or more pro-angiogenic compounds can be considered to act as chemical attractants that promote directional angiogenesis toward and within the confined gel droplets. In this way, endothelial cells are stimulated to form a vascular tissue layer within the microfluidic layer and the gel, which then undergoes permeabilization and leads to the growth of new microvessels. One or more pro-angiogenic compounds can be added to the gel droplets or gel precursor droplets, which are then introduced into the microfluidic network, or one or more pro-angiogenic compounds can be added after the gel is formed, for example, to the top surface of the gel. In another embodiment, one or more pro-angiogenic compounds can be added to the microfluidic network through another inlet of the microfluidic channel, for example, an inlet downstream of the inlet for introducing the culture medium and / or an inlet downstream of the strain chamber.

[0188] In some embodiments, after forming vascular tissue in the microfluidic layer and the gel, the method of the present invention may further include introducing one or more types of cells (preferably including at least one type of epithelial cells) into the third subspace of the microfluidic network through the inlet hole, and forming one or more types of cells into a monolayer or cell aggregates. For example, one or more types of cells can form a monolayer of cells on top of the gel confined in the first subspace.

[0189] In one embodiment, one or more cells or cell aggregates completely cover the top surface of the at least partially solidified gel, thereby forming a tissue barrier layer on the top surface of the at least partially solidified gel. The barrier layer may include a monolayer of cells or multiple layers of cells or cell aggregates. In one embodiment, a monolayer of cells or multiple layers of cells may be cultured so that the cells proliferate and / or differentiate, and then at least one microvessel undergoes angiogenesis into the at least partially solidified gel, or after at least one microvessel undergoes angiogenesis into the at least partially solidified gel, a monolayer of cells or multiple layers of cells may be cultured so that the cells proliferate and / or differentiate. Examples of flat laminar tissues include skin tissue (including, for example, keratinocytes, adipose tissue, and fibroblasts), intestinal epithelial cells, and other epithelial tissues such as lungs and retina.

[0190] Culture medium or differentiation medium can be added to the microfluidic channel described above, and the fluid flow through the vascular network can also be achieved as described above, thereby allowing cell proliferation and / or differentiation. Similarly, the composition of the fluid can also be controlled as described above. Therefore, the vascularized perfusable network established by the method described herein allows metabolites, nutrients and oxygen to be freely exchanged between the fluid in the microvessels in the microfluidic channel of the device and the cells or cell aggregates on the top of the solidified gel.

[0191] As already explained previously, the use of a capillary pressure barrier enables the formation of a gel of a stable confined volume, for example, in a subspace of a microfluidic network, so that a second fluid can be added without having to replace the gel or its contents. Thus, the apparatus of the present invention is configured for spatially controlled co-culture with other cells as described above and provides a means of controlling the composition of the surrounding culture medium. Thus, in the method of the present invention, the fluid loaded into the reservoir (also referred to as a well) is any cell culture medium, detection solution, buffer, other hydrogel, etc., and may optionally include cells or cell aggregates.

[0192] By controlling the components introduced into the reservoir, the cell culture device of the present invention can implement different modes of cell culture. For example, the composition of the fluid introduced into the reservoir or well can be changed. This exchange can be a gradient exchange by introducing a new component in one of the reservoirs and removing the fluid from another reservoir in the same microfluidic network at the same time, until the exchange is completely performed. This exchange can be discontinuous, by extracting fluid from the reservoir and filling the reservoir with a new composition. The fluid volume in the reservoir is much larger than the fluid volume in the microfluidic channel, and the leveling between the reservoirs occurs almost instantly, thereby ensuring that the new fluid flushes the microfluidic network without emptying the microfluidic channel network during operation.

[0193] In one embodiment, the presence of a second capillary cell pressure barrier in the device even allows the formation of a lamellar gel composition. In this embodiment, a first capillary pressure barrier (e.g., a circular capillary pressure barrier) fixes a liquid composition comprising a first gel or a gel precursor as a static droplet on a base layer (e.g., on a diaphragm) of a microfluidic network. After the first liquid composition is fixed, a second gel or gel precursor optionally containing cells is loaded. The second composition will be retained by a second capillary pressure barrier, which is, for example, a circular capillary pressure barrier having a larger diameter than the first capillary pressure barrier and which is coaxial with and surrounds the first capillary pressure barrier. With this configuration, the second capillary pressure barrier prevents the second composition from flowing into the microfluidic channel and encapsulating the first gel. The presence of two capillary pressure barriers accordingly separates the microfluidic network into separate spaces and gives the user the possibility of spatial configuration of the microfluidic network.

[0194] The discussion of the methods so far has described how to incorporate cells or cell aggregates into the microfluidic devices described herein. Once the device has been loaded with cells or cell aggregates and any necessary culturing of the cells has been performed, the methods may include one or more steps of subjecting the cells to mechanical strain and / or detecting mechanical strain emanating from the cells.

[0195] The step of subjecting the cell to mechanical strain may include applying positive or negative pressure to the diaphragm, and in one embodiment, applying alternating positive and negative pressures to the diaphragm. Applying pressure will induce deformation of the diaphragm into the microfluidic channel (when positive pressure is applied from the bottom of the diaphragm) or induce deformation of the diaphragm away from the microfluidic channel into the basal layer (when negative pressure is applied from the bottom of the diaphragm). In these methods, the surface of the diaphragm facing the microfluidic channel may have one or more cells or cell aggregates directly disposed thereon. In addition or alternatively, the one or more cells or cell aggregates may be disposed on the surface of the microfluidic channel near the diaphragm, for example, lining the surface of the microfluidic channel. The one or more cells or cell aggregates may also be disposed in or on a gel limited to the diaphragm surface by one or more capillary pressure barriers in the microfluidic channel. The one or more cells or cell aggregates are typically disposed in a position within the microfluidic network, where the diaphragm can still be displaced. It can be known that the more cells or cell aggregates there are from the diaphragm, the greater the displacement required, thereby exerting a role on the cell. Thus, in one embodiment, one or more cells or cell aggregates are present in or on a gel disposed on a surface of a membrane facing a microfluidic channel.

[0196] In some embodiments of the methods described herein, mechanical strain is varied over time by moving the diaphragm in a single cyclical or repetitive pattern. That is, the diaphragm may be displaced multiple times in a specific rhythm or sequence. For example, the diaphragm may be displaced in a rhythmic pattern similar to breathing, thereby recreating mechanical strain in lung tissue. In another embodiment, the diaphragm may be displaced in a pattern that recreates the peristaltic pattern of intestinal tissue.

[0197] Ways to displace or deform a membrane in a microfluidic device are known in the art and have been discussed above in connection with the microfluidic device. In some embodiments, the device may include a plurality of membranes in contact with the microfluidic channel. The plurality of membranes may be configured to stimulate one or more of the plurality of membranes multiple times in a predetermined pattern to move net fluid through the microfluidic network over multiple stimulation cycles.

[0198] In some embodiments, the extent of membrane displacement is such that mechanical strain can be applied to the monolayer of cells on the upper surface of the gel present on the upper surface of the membrane. As described above, mechanical strain can be applied to the monolayer of cells by applying positive or negative air pressure, or by applying a force from a mechanical actuator.

[0199] In some embodiments of the methods described herein, the diaphragm displacement is not stimulated by an external force, but rather the diaphragm displacement is induced or caused by one or more cells or cell aggregates present in the microfluidic layer, for example, one or more cells or cell aggregates present on the diaphragm, such as one or more cells or cell aggregates in or on the gel on the diaphragm or one or more cells or cell aggregates in or on the ECM on the diaphragm. One or more cells or cell aggregates can also be directly disposed on the diaphragm, optionally, by coating the diaphragm with cell adhesion molecules and one or more cells or cell aggregates are directly disposed on the diaphragm. In these embodiments, the diaphragm is advantageously functionalized by one or more electrodes, sensors, or reference markers for detecting diaphragm movement.

[0200] In one embodiment, the marker is printed in the same material of the diaphragm, i.e., by etching, grinding or by including the marker in a mold that forms the membrane. In another embodiment, the marker, sensor or transducer may be added to the diaphragm material, such as by adding it to the material during the manufacturing process. For example, magnetic beads may be mixed with the polymer from which the diaphragm is made and then used for stimulation or detection. Alternatively, the coil may be embedded in the polymer. In yet another embodiment, the material is applied to the diaphragm by surface deposition of the material (e.g., sputtering, plasma deposition, screen printing or other forms of printing or deposition). These processes can be used to print markers from inks, metals or other materials that can be used to monitor displacement.

[0201] Analysis Plate

[0202] A further aspect of the invention provides an assay plate comprising any of the devices described herein.When referring to a cell culture device comprising a vascular network and optionally also biological components such as a monolayer of cells, an assay plate is also referred to.

[0203] In one embodiment, provided herein is an analytical plate comprising a microfluidic device as described herein and a gel confined to a first subspace of a microfluidic channel by a capillary pressure barrier, wherein the microfluidic network comprises one or more cells or cell aggregates present, for example, within or on the gel and / or within the microfluidic channel.

[0204] The assay plate may include one or more cells or cell aggregates cultured by the methods described herein. In one embodiment, at least a portion of the microfluidic channels of the device of the assay plate includes a layer of vascular tissue including endothelial cells extending into the gel.

[0205] The dimensions of the assay plate may be consistent with or compatible with standard ANSI / SLAS microtiter plates. Specifically, the footprint dimensions or perimeter of the assay plate may be consistent with standard ANSI / SLAS microtiter plates.

[0206] Also described herein are assay plates or cell culture devices produced by any of the methods described herein.

[0207] Reagent test kit

[0208] The present disclosure also provides kits and articles of manufacture using the microfluidic devices and assay plates described herein. In one embodiment, the kit includes the device or assay plate described herein, and one or more pro-angiogenic compounds for inducing angiogenesis. In some embodiments, the kit may include the device or assay plate described herein and one or more of the following: a gel, a gel precursor composition or other extracellular matrix composition, one or more cells or cell types, a growth medium, and one or more reagent compositions including one or more pro-angiogenic compounds.

[0209] The assay plate of the cell culture device or kit preferably comprises a vascular bed, in other words an extracellular matrix gel arranged to receive at least one cell to be vascularized on the top surface of the extracellular matrix gel, and a vascular network of endothelial cells lining the inner surface of the microfluidic channel.

[0210] The kit may also include packaging material and a label or package insert contained within the packaging material providing instructions for inducing angiogenesis using one or more pro-angiogenic compounds in a cell culture apparatus or assay plate.

[0211] The one or more pro-angiogenic compounds may include one or more of fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang2), phorbol tetradecanoyl-13-acetate (PMA), sphingosine-1-phosphate (S1P), IGFBP-2, hepatocyte growth factor (HGF), prolyl hydrogenase inhibitor (PHi), monocyte chemoattractant protein-1 (MCP-1), basic fibroblast growth factor (bFGF) and ephrin.

[0212] The kit may also include accessory components, for example, a second container comprising a suitable culture medium for introducing one or more pro-angiogenic compounds and instructions for using the culture medium.

[0213] The present invention will now be described by way of example with reference to the accompanying drawings.

[0214] A first embodiment of a microfluidic device is Figures 1 to 3 As shown in FIG. Figure 1 As shown, the device 100 generally includes a substrate 101, a microfluidic channel 102 in a microfluidic layer, and a cover 103 (all shown in solid lines). A culture medium inlet 104 is present in the cover layer of the microfluidic layer. A capillary pressure barrier 105 is present on the substrate 101 of the device and is accessible through a hole 107 in the cover 103. In this specific embodiment, a hole is also provided on the substrate 101, through which a diaphragm 106 extends. A top layer 108 in the form of a porous bottomless flat plate is provided on top of the cover layer and includes a hole 109 located above each of the inlet hole 107 and the culture medium inlet 104.

[0215] As shown in the top view ( Figure 2 ), the circular capillary pressure barrier divides the microfluidic network into two subspaces. In this embodiment, one subspace of the central space of the capillary pressure barrier includes a strain chamber or a cell culture chamber, and the second subspace defined by the microfluidic channel 102 leads to the first subspace and surrounds the first subspace. The microfluidic channel 102 is Figure 2 In FIG. 1 , it is shown exemplarily as a solid circle surrounding the capillary pressure barrier, wherein the hole 107 is shown by a dotted line.

[0216] The formation of two subspaces in direct contact with each other without any intervening structures such as walls or membranes is one of the key features of the device and the analytical plate. Furthermore, the pores and microfluidic channels even enable the culture medium surrounding the gel to be controlled and regulated, for example, before or during strain experiments. Figure 3 An enlarged view of a vertical cross section of a portion of a microfluidic network is provided, showing a capillary pressure barrier 105 and a membrane 106 on a substrate. For example, the presence of the capillary pressure barrier 105 prevents the gel or gel precursor from filling the microfluidic channel when loaded, in other words, the gel or gel precursor is fixed on the capillary pressure barrier 105 during use. Figure 3 Also shown is one possible structure for adhering the membrane to the substrate layer, ie, fixing the membrane to the lower surface of the substrate layer.

[0217] Figure 2 The hole 107 in the subsequent figures is also shown as a circular hole. However, it should be understood that the hole can have any shape, preferably a circle and a square.

[0218] Additional branches of the microfluidic channel 102 may also be present in order to control the composition of the culture medium surrounding the gel composition bounded by the capillary pressure barrier. Figures 4 to 6In this embodiment, the central strain chamber is connected to four medium inlets 104 in a cross structure (see Figure 5 ), wherein there are two linear capillary pressure barriers 105, each of which partially defines a first subspace including a strain chamber.

[0219] 7A to 7C The various states that the diaphragm can exist in before or during the strain test are shown. In general, the diaphragm exists in a relatively tense state, such as Fig. 7A As shown, the diaphragm is in a relatively stretched state even at rest. Figure 7B The diaphragm is shown in tension, deflecting away from the microfluidic channel 102 after negative pressure is applied from below the diaphragm (eg, such negative pressure may be applied using a vacuum pump). However, it should be understood that the same effect may be achieved by applying positive pressure from above the diaphragm. Figure 7C The diaphragm is shown in an optional tensioned state, which can be deflected into the microfluidic channel 102 after positive pressure is applied from below the diaphragm, for example, using a pump, a mechanical actuator such as a pin or expandable foam to apply positive pressure. However, it should be understood that the same effect can be achieved by applying negative pressure from above the diaphragm.

[0220] The different steps in the method of using the device described herein are FIG. 8A to FIG. 8F In the first step, a first drop of a gel or gel precursor 110 is introduced, fixed to the capillary pressure barrier and solidified (cured, gelled). Again and as described above, the first liquid composition typically comprises a gel or gel precursor, for example, a hydrogel (or its precursor) for cell culture, and includes any hydrogel known in the art and suitable for the purposes of the present invention. The gel may optionally include suspended cells.

[0221] Once the gel droplet solidifies, the microfluidic channel is loaded with a second liquid, thereby introducing endothelial cells 111 into the microfluidic channel ( Figure 8B ). These may be introduced as a component of the cell culture medium or growth medium, or may be introduced sequentially. After culturing the device thus seeded, the endothelial cells 114 may be vascularized or line the inner surface of the microfluidic channel, i.e., the wall, base and top, and may also be on the ECM gel surface, depending on the composition of the second liquid (the liquid loaded in the microfluidic channel).

[0222] In a further step, adding angiogenic agents to the top of the gel can allow or induce angiogenesis in the blood vessels formed in the microfluidic channels ( Figure 8C), wherein the invasion of the gel droplets and / or the capillaries formed therein forms a vascular bed. Culture conditions that allow angiogenesis to occur are known to those skilled in the art and include, for example, oxygen deprivation, mechanical stimulation, and chemical stimulation using pro-angiogenic agents such as the pro-angiogenic proteins described above.

[0223] A typical spouting mixture includes 37.5 ng / ml to 150 ng / ml of VEGF, MCP-1, HGF, bFGF, PMA and 250 nM to 1000 nM of S1 P. An alternative spouting mixture includes 500 nM of S1 P, 50 ng / ml of VEGF, 20 ng / ml of FGF and 20 ng / ml of PMA.

[0224] In this way, blood vessels connecting the inlet and outlet of the microfluidic channel are formed, lining the surface of the channel and extending into the gel.

[0225] The preferred result of the method is a gel comprising a vascular bed of microvessels connected to larger blood vessels via one or more microfluidic channels through which a flow of growth medium, serum or other substances can be applied. In this way, using the device of the present invention it is possible to co-culture a first type of cells within a first defined subspace of a microfluidic network comprising a gel and an endothelial cell culture within a second subspace comprising microfluidic channels, thereby achieving a vascularized model of cell aggregates present inside or on top of a gel droplet, which is connected to a reservoir via endothelial blood vessels formed inside the microfluidic channels.

[0226] In order to achieve a vascular bed or vascularized tissue, it is not necessary to use a mixture of angiogenic compounds. In an alternative method of creating a vascular bed, the tissue is placed on top of the gel. The tissue itself secretes factors that induce angiogenesis, which leads to the sprouting of major vessels and the formation of a vascular bed or even the formation of vascularized tissue.

[0227] Fig.8D Cells 113 are shown added to the top of gel 110, whereupon the cells form a monolayer. The cells can be of any type, but they are typically epithelial or endothelial cells, depending on the strain experiment being performed. Finally, Fig. 8E and Figure 8F The bidirectional deformation of the membrane 106 during the strain experiment is shown, which results in the subsequent application of strain to the monolayer of cells 114 on top of the gel 110 including the microvessels.

[0228] 9A to 9F Shows FIG. 8A to FIG. 8FAn alternative method to the method shown. In this alternative method, the first two steps and the last three steps are the same as the method of FIG. 8 , with the only difference being that (i) cells 113 are added to the surface of the gel 110 and (ii) the gel 110 is vascularized by endothelial cells 111 .

[0229] FIG. 10A to FIG. 10C An enlarged vertical cross-sectional view of an alternative structure of a microfluidic network is shown. Specifically, Fig. 10A A hole in substrate 101 is shown with diaphragm 106 extending therethrough, wherein capillary pressure barrier 105 is located outside the hole. In this particular embodiment, diaphragm 106 is aligned with inlet hole 107, but the exposed or usable surface of diaphragm 106 is narrower than the cross-section of inlet hole 107, while capillary pressure barrier 105 is away from diaphragm 106 and located outside of inlet hole 107. In contrast, in Fig. 10B In the configuration, the size of the hole extending through the diaphragm 106 is substantially the same as the size of the inlet hole 107, so that the size of the exposed or usable surface of the diaphragm is substantially the same as the size of the cross-sectional area of ​​the inlet hole 107, but wherein the two capillary pressure barriers 105a, b are located below the cover 103. Finally, in Fig. 10C In the embodiment of the present invention, the hole extending through the septum 106 is larger than the inlet hole 107, so that the exposed or usable surface area of ​​the septum is greater than the cross-sectional area of ​​the inlet hole 107, wherein the capillary pressure barrier 105 is adjacent to the septum 106. In general, the larger the septum and / or the larger the hole, the more epithelial cells can be exposed to mechanical strain. In contrast, a smaller septum will not be displaced as much as a larger septum (with the same applied force), and therefore will produce less displacement and / or less damage to the strained tissue, especially to the area around the inlet edge. It should be understood that the overall requirement is to have the septum and the inlet hole substantially aligned to facilitate the introduction of materials into the device.

[0230] Fig.11A and Fig. 11B The capillary pressure barriers 105a, b and Fig. 10B and Fig. 10AThe microfluidic network structures shown have gel or extracellular matrix 108 fixed to the edges of the holes 107. In these structures, the gel is not fixed by any capillary pressure barrier so that the gel is set on the membrane to any meaningful extent. Instead, the gel is mainly fixed in the microfluidic channels. In these structures, external tissue samples (such as tissue slices) or organoids can be placed in the chambers created in the fixed gel or ECM and placed in the chambers created by the fixed gel or ECM, and the external tissue samples (such as tissue slices) or organoids are more easily vascularized when they are located in the same plane as the vascular bed (once the gel is vascularized). This result also enables better positioning of tissues for imaging of the entire system because all components are in the same focal plane.

[0231] Fig.12 and Fig.13 The use of an alternative configuration of a microfluidic network for use in the device is shown, specifically a configuration in which there are no holes in the lid 103 that are aligned with the diaphragm. Fig.12 Shown is a setup for measuring mechanical strain produced by cells 114 or cell-induced movement (e.g., contraction of muscle cells, fibroblasts, cardiomyocytes) or measuring pressure induced on brain cells, bone cells, or compression of other biological tissues. Fig.13 An alternative use of the structure is shown, wherein cells 111 can form a structure with chambers on a membrane, for example, around a gel held in place by a capillary pressure barrier 105. The cells can include endothelial cells forming blood vessels, epithelial cells forming intestinal or tubular lumens, or cardiomyocytes forming atrial or ventricular lumens. Thus, this type of device can monitor or induce mechanical strain resulting from vasodilation / vasoconstriction, intestinal peristalsis, tubular compression, vascular compression, and cardiomyocyte activation or simulate mechanical strain resulting from vasodilation / vasoconstriction, intestinal peristalsis, tubular compression, vascular compression, and cardiomyocyte activation, or this type of device can actually induce these activities.

[0232] Fig.14A and Fig. 14B Shown are alternative ways of securing the membrane to a substrate of a microfluidic network or device described herein. Fig.14A The diaphragm 106 is shown sandwiched between two sublayers 101a, 101b of the substrate layer, and Fig. 14B A membrane 106 is shown secured to the upper surface of the substrate layer 101 .

[0233] Fig.15 1 shows a plan view of a porous device 115 according to the present invention, which is composed of the porous structure of the microfluidic network described herein. Fig.15As shown, the device is preferably compatible with or based on the dimensions of a microtiter plate defined by ANSI / SLAS dimensions, Fig.15 A microfluidic network consisting of 128 individual microfluidic networks (e.g. Figure 1 A bottom view of such a plate with microfluidic networks shown in the figure. A membrane 106 is shown in the center of each microfluidic network. Fig.16 Shows Fig.15 A cross-section of a porous structure, wherein in each microfluidic network, each membrane extends across a pore. Fig.17 Shows Fig.16 An alternative configuration of a porous structure is provided in which a single elastomeric sheet extends across the entire width of the device 115, thereby extending across each of the pores of an independent microfluidic network.

[0234] Example

[0235] A method of constructing a substrate layer for use in a device will be described below.

[0236] In general, the substrate layer includes two layers of ground glass sheets and a flexible membrane (e.g., a polyurethane membrane), wherein each layer of ground glass sheets has a plurality of holes with a diameter of 2 mm. The membrane is located between the two layers of glass sheets and the glass sheets are aligned so that the holes are aligned. The three layers are then placed under high temperature and pressure conditions of 4 bar and 95° C. The final product is a substrate layer for a microfluidic device, which is composed of two sheets of ground glass and a laminated polyurethane membrane that can provide stimulation to the microfluidic channels of the microfluidic network.

[0237] The substrate layer can be connected to a manifold comprising a 10 mm thick polycarbonate sheet and a silicone rubber gasket connected to a pressurized air supply. A pressure of 1 bar is applied. Observation under a microscope and / or photography is performed to confirm that all diaphragms to which pressure is applied are displaced.

[0238] The above description is to teach those skilled in the art how to implement the present invention, and is not intended to detail all improvements and changes that are obvious after reading this specification. However, all improvements and changes are included in the scope of the present invention defined by the appended claims.

Claims

1. A microfluidic device comprising: A microfluidic network, the microfluidic network comprising: substrate, microfluidic channels, and lid; wherein the substrate comprises a non-porous membrane forming at least a portion of an inner surface of the microfluidic channel, and the microfluidic channel comprises a subspace at least partially defined by the membrane and a capillary pressure barrier in the microfluidic channel, wherein the membrane comprises an elastomer.

2. The microfluidic device of claim 1, wherein: The cover includes an inlet hole leading to the microfluidic channel, and the inlet hole is substantially aligned with the diaphragm.

3. The microfluidic device according to claim 1 or 2, wherein: The substrate includes a hole leading to the microfluidic channel, and the membrane extends across the hole.

4. The microfluidic device of claim 1, wherein: The diaphragm includes a region having a thinner cross-section than a peripheral portion of the base.

5. The microfluidic device of claim 1, wherein: The membrane is transparent or optically clear.

6. The microfluidic device of claim 5, wherein: The thickness of the diaphragm is less than 1 mm.

7. The microfluidic device of claim 1, wherein: The membrane is a functionalized membrane comprising one or more electrodes, sensors, probes, reference markers for monitoring membrane movement, ferromagnetic particles, adhesion molecules or antibodies.

8. The microfluidic device of claim 2, wherein: The subspace is a first subspace, and the microfluidic channel further comprises: a second subspace including a fluid channel, and A third subspace, wherein the first subspace separates the third subspace from the second subspace.

9. The microfluidic device of claim 8, wherein: The apparatus further comprises a top layer having a well, and the first subspace and / or the third subspace extends into the well through the inlet aperture.

10. The microfluidic device of claim 2, wherein: The capillary pressure barrier is substantially aligned with the inlet aperture.

11. The microfluidic device of claim 8, wherein: The membrane forms at least part of the surface of the first subspace.

12. The microfluidic device of claim 8, wherein: The membrane forms at least part of the surface of the third subspace, which is optionally delimited by a further capillary pressure barrier.

13. The microfluidic device of claim 1, wherein: The substrate is configured to operably connect the diaphragm to one or more of the following: Positive or negative sources of stress; Physical drives; electromagnetic actuator; and Expandable foam.

14. The microfluidic device of claim 1, wherein: The capillary pressure barrier comprises: ridges of material protruding from the interior surface of a microfluidic channel; a widened portion of a microfluidic channel; a groove in an inner surface of a microfluidic channel; regions of material having different wettability to the interior surface of the microfluidic channel; A plurality of columns are arranged at regular intervals.

15. The microfluidic device of claim 1, wherein: The microfluidic network comprises a biological material or a biomimetic material, which comprises one or more of the following materials: a. a gel, extracellular matrix or scaffold disposed in the first subspace; b. epithelial cells or endothelial cells lining the microfluidic channels; c. epithelial cells or endothelial cells located inside a gel, extracellular matrix or scaffold; d. Stromal cells in or on a gel, extracellular matrix or scaffold; e. Muscle cells in or on a gel, extracellular matrix or scaffold; f. One or more other cell types selected from pluripotent cells and central nervous cells, peripheral nervous cells, lymphoreticular cells, immune cells, urinary cells, respiratory cells, germ cells, gastrointestinal cells, endocrine cells, skin cells, musculoskeletal cells, cardiovascular cells and mammary cell types.

16. A method for evaluating mechanical strain induced by a cell, comprising: introducing one or more types of cells or cell aggregates into a microfluidic network of a microfluidic device according to any one of claims 1 to 15; Optionally, culturing the one or more types of cells or cell aggregates; and Deformation of the diaphragm is monitored using one or more electrodes, sensors, probes, or reference markers disposed on or operably connected to the diaphragm for monitoring movement of the diaphragm.

17. A method of subjecting one or more types of cells or cell aggregates to mechanical strain, comprising: introducing one or more types of cells or cell aggregates into a microfluidic network of a microfluidic device according to any one of claims 1 to 15; Optionally, culturing the one or more types of cells or cell aggregates; and One or more types of cells or cell aggregates are subjected to mechanical strain by applying positive or negative pressure to the membrane.

18. The method of claim 17, comprising applying alternating positive and negative pressures.

19. The method according to claim 17 or 18, wherein: The mechanical strain varies over time in a single cycle or in a repetitive manner.

20. The method of claim 17, wherein: The device includes a plurality of membranes in contact with the microfluidic channel, and the plurality of membranes are configured to stimulate one or more of the plurality of membranes a plurality of times in a predetermined pattern to produce a net fluid movement through the microfluidic network during a plurality of stimulation cycles.

21. The method of claim 16 or 17, comprising: introducing a volume of gel or gel precursor into the microfluidic network; causing the volume of gel or gel precursor to solidify or gel to form a solidified gel; loading a liquid into the microfluidic network; as well as One or more types of cells or cell aggregates are cultured.

22. The method of claim 17, wherein: The method comprises: A volume of gel or gel precursor is introduced into the first subspace and is confined by the capillary pressure barrier.

23. The method of claim 16 or 17, further comprising: One or more types of cells are introduced into the microfluidic channel.

24. The method of claim 21, further comprising: One or more types of cells are introduced into the third subspace, and the one or more types of cells are allowed to form a monolayer of cells or cell aggregates.

25. The method of claim 24, wherein: The third subspace is fluidly connected to the inlet aperture and is optionally at least partially defined by the gel surface.

26. The method of claim 16 or 17, comprising culturing any one or a combination of the following cells: a. epithelial cells or endothelial cells lining the microfluidic channels capable of forming conduits or blood vessels; b. epithelial cells or endothelial cells located within, on, or against a gel, extracellular matrix, or scaffold; c. stromal cells located within, on, or against a gel, extracellular matrix, or scaffold; d. muscle cells located within, on, or against a gel, extracellular matrix, or scaffold; e. One or more other cell types selected from pluripotent cells and central nervous cells, peripheral nervous cells, lymphoreticular cells, immune cells, urinary cells, respiratory cells, germ cells, gastrointestinal cells, endocrine cells, skin cells, musculoskeletal cells, cardiovascular cells and mammary cell types.

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