Microfluidic cell culture system

Abstract

A microfluidic cell culture device is described. The device includes a microfluidic network including a substrate, a microfluidic channel, and a cover, and at least one perfusion compartment and at least one support compartment within the microfluidic channel. The substrate and cover each include an opening, thereby defining a conduit through the microfluidic channel. The opening is in fluid contact with the at least one support compartment, and through the at least one support compartment, is in fluid contact with the at least one perfusion compartment. Methods for using the device to create a fluid-fluid interface and to investigate cellular responses to stimuli are also described.

Description

Technical Field

[0001] The present invention relates to a microfluidic cell culture device and methods for creating a fluid-fluid interface and investigating cellular responses to stimulants using a microfluidic device.

Background Art

[0002] In the movement to simulate more physiologically appropriate conditions than ever in cell culture, for example, simulating perfusion flow, many models have been developed for co-culturing preclinical cell-based models for assaying drug efficacy and / or ADME safety.

[0003] Microfluidics has become a popular platform technology for such in vitro cell culture models due to the inherent flow of the liquid or medium in use, with the advancement of microengineering techniques that facilitate and enable the fabrication of complex microfluidic networks. However, there remains a great interest in generating models that simulate or reproduce the cellular environment in various organs of the human or animal body.

[0004] There remain challenges in transferring lung tumor characteristics to a high-throughput model. The airway epithelium is responsible for taking up oxygen from the blood and thereby supporting all cells of the human body. These airway epithelial cells are in contact with the outside air at the apical end and are connected basally to the underlying stroma where nutrients are acquired. The lung also forms a physical barrier to the internal environment and is at the forefront of defense against inhaled particles and / or pathogens trapped in the protective mucus layer.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The creation of high-throughput lung models allows for rapid testing of potential therapeutic agents against targets, such as viral or bacterial infections of the lung, while the creation of high-throughput gastrointestinal models is equally important for understanding drug absorption and therapeutic activity. Improved apparatuses and methods for simulating real cellular environments in vitro are needed. [Means for solving the problem]

[0006] In a first embodiment of the present invention, a microfluidic apparatus is provided, which is Microfluidic Networks Including, the microfluidic network is Substrate, microfluidic channels and cover, At least one perfusion compartment inside the microfluidic channel, At least one support compartment inside the microfluidic channel and Includes, The substrate and the cover each include openings, thereby defining conduits through microfluidic channels, and The conduit is in fluid contact with at least one support section and, through at least one support section, is in fluid contact with at least one perfusion section.

[0007] In a second aspect of the present invention, a method is provided for creating a fluid-fluid interface in a microfluidic cell culture apparatus comprising a microfluidic network having a substrate, microfluidic channels and a cover, wherein the microfluidic channels have at least one perfusion compartment and at least one support compartment inside, and the substrate and the cover each include openings that define conduits through the microfluidic channels, and this method is provided. The steps include: introducing a support scaffold to at least one support section in order to form a scaffold surface facing the conduit; The steps include introducing a culture medium containing cells into a xylem through an opening in the cover, while preventing the medium from flowing out of the xylem through an opening in the substrate, A step that allows cells to form layers on the scaffold surface, To expose the cells, the step is to remove the culture medium from the ducts. Includes.

[0008] According to a third aspect of the present invention, an assay plate is provided which includes an apparatus of the first aspect, wherein a support scaffold is provided within a support compartment of a microfluidic channel, and optionally the support scaffold comprises one or more cells or cell aggregates, preferably aligned with its surface facing the conduit.

[0009] A fourth embodiment of the present invention provides a method for investigating the cellular response to a stimulant, the method being A step of creating a fluid-fluid interface in a microfluidic device according to the method of the second embodiment, The step of bringing exposed cells into contact with the fluid flow by advancing the fluid flow through the conduit and Includes.

[0010] A fifth embodiment of the present invention provides a method for investigating the cellular response to a stimulant, the method being The steps include using an assay plate according to the third embodiment, The step of bringing exposed cells into contact with the fluid flow by advancing the fluid flow through the conduit and Includes.

[0011] Other preferred embodiments are provided in the following description and dependent claims.

[0012] Previous research has attempted to create lung models in cell culture environments. However, creating an air-liquid interface without removing seeded cells, and then supplying nutrients to those seeded cells at high throughput, has proven difficult. A device according to any of the above embodiments would unexpectedly enable the creation of an air-liquid interface in lung models, thus opening the way for the development of improved in vitro or ex vivo model systems for assessing drug efficacy or ADME safety.

[0013] definition Various terms relating to the devices, methods, uses, and other embodiments of the present invention are used throughout this specification and the claims. Unless otherwise specified, such terms should be given the common meaning in the art to which the present invention relates. Other particularly defined terms should be interpreted in accordance with the definitions provided herein. Any methods and materials similar to or equivalent to those described herein may be used in practice to test the present invention, but preferred materials and methods are described herein.

[0014] As used herein, the singular forms “a,” “an,” and “it” also include plural references unless the context clearly indicates otherwise. For example, a reference to “a cell” includes combinations of two or more cells, etc.

[0015] As used herein, “substantially,” “about,” and “approximately” mean, when referring to measurable values ​​such as quantity or duration, to include variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from a specified value, and such variations are appropriate for the execution of the disclosed method.

[0016] As used herein, “includes” is to be construed as inclusive, non-exclusive, and non-limiting. Specifically, this term and its variations mean that the specified feature, step, or component is included. These terms should not be construed as excluding the existence of other features, steps, or components.

[0017] As used herein, “exemplary” means “serving as an example, case, or illustration,” and should not be construed as excluding other configurations disclosed herein.

[0018] As used herein, the term "microfluidic channel" refers to a channel that is covered by an upper substrate or cover or passes through a layer of material covered thereby, or a channel that is under or passes through a material disposed on a lower substrate or substrate, where at least one of the length, width or height dimensions is in the submillimeter range. It will be understood that this term encompasses channels that are linear channels and channels that branch, or have bends or corners in their paths. A microfluidic channel typically includes an inlet for administering a volume of liquid. The volume enclosed by the microfluidic channel typically ranges from microliters to submicroliters. A microfluidic channel typically includes a substrate, which can be the upper surface of the underlying material, two sidewalls, and a ceiling, which can be the lower surface of an upper substrate or cover that overlays the microfluidic channel, and optionally includes an inlet, an outlet, and / or a vent of any configuration. The substrate, sidewalls, and ceiling can each be referred to as the inner surface of the microfluidic channel and collectively as the inner surface. In some examples, the microfluidic channel can have a circular or semi-circular cross-section and is thus considered to have one or two inner surfaces, respectively.

[0019] As used herein, the terms "droplet holding structure" and "capillary pressure barrier" are used synonymously and are used in connection with the features of a device that holds a liquid-air or other fluid meniscus in a specific position by capillary forces. A capillary pressure barrier can be considered to divide a microfluidic channel having a volume V0 into two sub-volumes V1 and V2 into which different fluids can be introduced. In short, a capillary pressure barrier at least partially defines one or more sub-volumes of the microfluidic channel by being located at the boundary between the two sub-volumes.

[0020] Specifically with respect to capillary pressure barriers, as used herein, a "closed geometric configuration" is a configuration in which the capillary pressure barrier forms a closed loop, unlike a linear capillary pressure barrier having two ends. For example, when viewed from above, a capillary pressure barrier having a closed geometric configuration can include a circular capillary pressure barrier or a polygonal capillary pressure barrier, such as a triangular capillary pressure barrier, a square capillary pressure barrier, or a pentagonal capillary pressure barrier, etc. In some examples, a closed geometric configuration of a capillary pressure barrier can also refer to two linear capillary pressure barriers that both intersect the same one or more walls of a microfluidic channel, thereby closing or defining a region of the microfluidic channel bounded by the two linear capillary pressure barriers and the walls. As used herein, the term "concentric" refers to any closed geometric configuration and does not include only circular configurations. For example, the term "concentric" can also be understood to refer to two squares, of the same or different dimensions, aligned such that the center of one square is aligned with the center of the other square.

[0021] As used herein, a "linear" capillary pressure barrier should not be interpreted as a straight line, but rather as a line that, in addition to not having a closed geometric configuration, i.e., having two ends, may have one or more bends or corners. A linear capillary pressure barrier typically intersects the sidewall or inner surface of a microfluidic channel at each end.

[0022] As used herein, the term "endothelial cell" refers to a cell of endothelial origin or a cell that has differentiated into a state expressing a marker that identifies the cell as an endothelial cell.

[0023] As used herein, the term "epithelial cell" refers to a cell of epithelial origin or a cell that has differentiated into a state expressing a marker that identifies the cell as an epithelial cell.

[0024] As used herein, the term "droplet" refers to a certain volume of liquid that may or may not exceed the height of a microfluidic channel and does not necessarily represent a round spherical shape. Specifically, when referring to a gel droplet, it refers to a certain volume of gel within the indicated compartment.

[0025] As used herein, the term “biological tissue” refers to an assembly of identical, similar, or different types of functionally interconnected cells cultured and / or assayed in the methods described herein. These cells may be cell aggregates and / or specific tissue samples derived from a patient. For example, the term “biological tissue” includes organoids, tissue biopsies, tumor tissue, excised tissue material, spheroids, and embryoid bodies.

[0026] As used herein, the term “cell aggregate” refers to a 3D cluster of cells, as opposed to surface-adhering cells that typically proliferate in a monolayer. 3D cell clusters are typically associated with more in vivo-like situations. In contrast, surface-adhering cells can be highly influenced by the properties of the substrate and may undergo dedifferentiation or migration to other cell types.

[0027] As used herein, the term “luminal cellular component” refers to a biological tissue having a lumen (i.e., composed of cells), such as microvessels having an apical and basal surface. Here, the present invention will be described with reference to the figures, merely as an example. [Brief explanation of the drawing]

[0028] [Figure 1] A vertical cross-sectional view of a first exemplary configuration of a microfluidic network used in the apparatus described herein is shown. [Figure 2] Figure 1 shows a horizontal top view of the microfluidic network. [Figure 3] Figure 1 shows an enlarged vertical cross-sectional view of the microfluidic network. [Figure 4A]A vertical cross-sectional view of an exemplary configuration of a microfluidic network used in the apparatus described herein is shown. [Figure 4B] A vertical cross-sectional view of an exemplary configuration of a microfluidic network used in the apparatus described herein is shown. [Figure 4C] A vertical cross-sectional view of an exemplary configuration of a microfluidic network used in the apparatus described herein is shown. [Figure 4D] A vertical cross-sectional view of an exemplary configuration of a microfluidic network used in the apparatus described herein is shown. [Figure 5A] This shows a schematic representation of the steps in the method described herein. [Figure 5B] This shows a schematic representation of the steps in the method described herein. [Figure 5C] This shows a schematic representation of the steps in the method described herein. [Figure 5D] This shows a schematic representation of the steps in the method described herein. [Figure 5E] This shows a schematic representation of the steps in the method described herein. [Figure 5F] This shows a schematic representation of the steps in the method described herein. [Figure 5G] This shows a schematic representation of the steps in the method described herein. [Figure 6A] This document outlines the alternative methods described herein. [Figure 6B] This document outlines the alternative methods described herein. [Figure 7] This document outlines the alternative methods described herein. [Figure 8] A plan view of the apparatus described herein, consisting of a multiwell configuration of the microfluidic network described herein, is shown. [Figure 9] This specification shows a vertical cross-sectional view of a device consisting of a multiwell configuration of a microfluidic network. [Figure 10]An example of a microfluidic device with upper and lower enclosures is shown. [Figure 11] An example of a microfluidic device with upper and lower enclosures is shown. [Figure 12A] This shows a microfluidic device with an enclosure that includes a filter. [Figure 12B] This shows a microfluidic device with an enclosure that includes a filter. [Figure 12C] This shows a microfluidic device with an enclosure that includes a filter. [Figure 12D] This shows a microfluidic device with an enclosure that includes a filter. [Figure 13] This shows the airflow through an example of a microfluidic device with an enclosure. [Figure 14] This shows the airflow through an example of a microfluidic device with an enclosure. [Figure 15] An example of a microfluidic device with multiple dedicated fluid inlets is shown. [Figure 16] An example of a microfluidic device with multiple dedicated fluid outlets is shown. [Figure 17] An example of a microfluidic device with multiple dedicated fluid inlets and one common outlet is shown. [Figure 18] An example of a microfluidic device having multiple dedicated fluid inlets and multiple dedicated outlets is shown. [Figure 19A] The image shows a confocal immunostained image of seeded cells obtained using the apparatus described herein. [Figure 19B] The image shows a confocal immunostained image of seeded cells obtained using the apparatus described herein. [Figure 20] This specification shows a confocal microscopy DNA color-coded image of seeded cells obtained using the apparatus described herein. [Modes for carrying out the invention]

[0029] <<Microfluidic Devices>> Microfluidic apparatuses are described. The microfluidic apparatuses are preferably in a multi-array / multi-well format, enabling their use in in vitro cell-based assays, pharmaceutical screening assays, toxicity assays, and particularly in high-throughput screening formats. Such multi-well culture plates are available with 6, 12, 24, 48, 96, 384, and 1536 sample wells arranged in a rectangular matrix, and in relation to the present invention, the microfluidic network of the multi-array configuration described herein resides in the microfluidic apparatus. In one example, the microfluidic apparatus is compatible with one or more dimensions of the standard ANSI / SLAS microtiter plate format. In an alternative embodiment, the microfluidic apparatus is a multi-array format having the dimensions of a microscope glass slide. In some examples, the microfluidic apparatus is provided with one or more functions, including one or more electrodes for performing electrical experiments, transparent materials, windows or other modifications to enable optical measurements.

[0030] Therefore, the microfluidic device preferably has multiple microfluidic networks as described herein. In one example, the multiple microfluidic networks are fluidically isolated from each other, in other words, each microfluidic network operates independently of any other microfluidic networks present on the microfluidic device. In another example, the microfluidic networks may be connected by one or more connection channels, as will be discussed later.

[0031] Generally, a microfluidic device is a microfluidic device that includes at least a microfluidic network having microfluidic channels. Various configurations of microfluidic channels or microfluidic networks are possible within the scope of the present invention, but may include volumes or sub-volumes that are within or in fluid communication with the microfluidic channels in order to receive and confine, for example, a gel, for example, an extracellular matrix.

[0032] Microfluidic devices generally include microfluidic networks, which are described in detail here.

[0033] <Microfluidic Networks> The microfluidic network of a microfluidic apparatus generally includes a substrate, microfluidic channels or microfluidic layers, and a cover, also referred to herein as a cover layer, and can be fabricated in a variety of ways.

[0034] The substrate, also referred herein as the substrate layer or lower substrate, is preferably formed from a substantially rigid material such as glass or plastic and serves to provide a support surface for the rest of the microfluidic network. In one example, the substrate is the same as or similar in dimensions to the well area of ​​a standard ANSI / SLAS microtiter plate. In some examples, the substrate includes an opening to the microfluidic layer or channel of the microfluidic network. In some examples, the substrate includes a connecting channel located inside. The connecting channel connects the microfluidic network to at least one other microfluidic network of the microfluidic apparatus described herein via the opening in the substrate. In some examples, the connecting channel is connected to a conduit of the microfluidic network by a fluid inlet to the opening in the substrate, allowing fluid flow through the conduit.

[0035] A microfluidic device or network includes a microfluidic channel or microfluidic layer disposed on a substrate. In some examples, the microfluidic channel may include or be divided into sub-volumes by the presence of a capillary pressure barrier, for example, as described herein. In some examples, the microfluidic channel may include a first sub-volume, sometimes called a support compartment. In some examples, the support compartment may be defined in part by the presence of a capillary pressure barrier and / or a rim of the opening in the substrate.

[0036] In some examples, the microfluidic channel further includes a second sub-volume containing a flow channel or perfusion compartment. In some examples, the perfusion compartment of the second sub-volume is the flow channel in use.

[0037] A typical method for fabricating microfluidic channels involves pouring a moldable material such as polydimethylsiloxane into a mold and then pressing the microfluidic channels into a silicone rubber material, thereby forming a microfluidic layer. The rubber material with the imprinted channels is then placed on a substrate layer of glass or the same material, thereby creating a seal. Alternatively, the channel structure can be etched into a material such as glass or silicon and then bonded to an upper or lower substrate (also referred to herein as a covering layer and a substrate layer). Plastic injection molding or embossing, followed by bonding, is another way to fabricate a microfluidic channel network. Yet another technique for fabricating a microfluidic channel network involves photolithographically patterning the microfluidic channel network onto a photopatternable polymer such as SU-8 or various other dry films or liquid photoresists, followed by a bonding step. When referring to bonding, it means closing the channels with a covering or substrate. Bonding techniques include, among others, anodic bonding, covalent bonding, solvent bonding, adhesive bonding, and thermal bonding.

[0038] As can be inferred from the various fabrication methods described above, the microfluidic layer may include a sublayer containing microfluidic channels disposed on the substrate layer, or it may be patterned in either the covering or the substrate layer. In the orientation used, the microfluidic sublayer is disposed on the upper surface of the substrate layer. The microfluidic channels may be formed as channels passing through the sublayer of a material disposed on the substrate layer. In one example, the sublayer material is a polymer disposed on the substrate layer, in which the microfluidic channels are patterned. In some examples, the microfluidic layer includes two or more microfluidic channels that can fluidly communicate with each other.

[0039] A microfluidic network includes a cover or covering layer that covers the microfluidic channels. The cover or covering layer can be formed from any suitable material known in the art, for example, a glass layer bonded to a sublayer containing the microfluidic channels. In one example, the covering layer may have pre-formed holes or openings at a defined number of locations. In some examples, the openings in the cover are substantially aligned with openings in the substrate. In some examples, connecting channels are provided in the cover to connect the conduits of at least two of a plurality of microfluidic networks through each opening in the cover. In some examples, the connecting channels are connected to the conduits of the microfluidic networks by fluid inlets to the openings in the cover, allowing fluid flow through the conduits. Openings in the cover and substrate allow fluid communication between the microfluidic channels of the microfluidic layer and other components of the microfluidic apparatus located on or below its surface. Generally, the openings function as interfaces to the outside world or to wells located on top of the cover. In some examples, openings in the cover and substrate are configured to function as capillary pressure barriers. For example, the openings in the cover and substrate may have a rim, which can function as a capillary pressure barrier to hold the liquid in place and prevent it from flowing through the opening.

[0040] In some examples, the cover includes connecting channels disposed internally. The connecting channels connect the microfluidic network to at least one other microfluidic network of the microfluidic apparatus described herein through openings in the cover. Thus, for example, each microfluidic network may individually be supplied with a drug candidate via perfusion channels, but all microfluidic networks may then be supplied with the same fluid flow via connecting channels to individual conduits. In some examples, a connecting channel is provided in the substrate, and a second connecting channel is provided in the cover, providing one or more fluid inlets and / or outlets to openings in the substrate and cover, thereby allowing fluid flow through the conduits of the microfluidic network. In some examples, the connecting channel provides one or more fluid inlets and / or outlets to openings in the substrate and cover, thereby allowing fluid flow through the conduits of multiple microfluidic networks.

[0041] Microfluidic channels may be provided with one or more fluid inlets and one or more outlets or vents as required for any particular application of the microfluidic network in a microfluidic device. To allow fluid filling, discharge, and perfusion through the microfluidic network, microfluidic channels are preferably provided with at least one inlet and at least one outlet or vent. In one example, each of the at least one inlet and at least one outlet or vent is preferably a pre-formed opening in the covering layer. Typically, it will be understood that there is no geometric distinction between the inlets and outlets, and in many cases they can be used interchangeably as inlets or outlets. In some examples, the microfluidic device further includes an upper layer disposed on the covering layer, the upper layer having one or at least one well or reservoir that is in fluid communication with the rest of the microfluidic device. In some examples, the upper layer has a plurality of such wells, and at least one, e.g., at least two, e.g., at least three wells are in communication with the microfluidic network or channels of the device. For example, the upper layer may include a well or reservoir that is in fluid communication with the microfluidic network through an inlet opening provided in the covering layer of the microfluidic network, thereby forming an SLAS-compliant well plate. In some examples, the upper layer having at least one well and a microfluidic layer is integrally formed. For example, a microfluidic channel may be patterned on the underside of an injection-molded microtiter plate having at least one well.

[0042] <Support section> A microfluidic channel includes at least one support compartment. References to support compartments herein will be understood as applicable both to at least one support compartment and to two or more support compartments in general. Support compartments can generally be located near openings in the substrate and covering within the microfluidic channel. In some examples, support compartments are located within the microfluidic channel between a perfusion compartment and a conduit formed by openings in the substrate and covering. In connection with this disclosure, a support compartment should be understood as a region of the microfluidic channel that is open to and fluidly communicates with other components of the microfluidic apparatus, such as the perfusion channel and conduit. Thus, a support compartment may be partially defined by the inner surface of the microfluidic channel (e.g., two side walls, an upper substrate and a lower substrate), but to some extent "open" to other regions of the microfluidic channel and other regions of the apparatus, and its boundary is defined as described below.

[0043] In some examples, the support compartment is defined in at least part by one or more capillary pressure barriers located in the substrate, the cover, and the microfluidic channel. In some examples, the openings in the substrate and cover act as capillary pressure barriers, fixing or confining the fluid within the support compartment so that it does not overflow through any of the openings. In some examples, the support compartment includes a capillary pressure barrier that defines the boundary between the support compartment and the perfusion compartment. In this way, the fluid within the support compartment can be confined internally and not overflow into the perfusion compartment. It will be understood, of course, that the capillary pressure barrier is not a structure that completely seals the microfluidic channel to prevent all fluid flow between the support compartment and the perfusion compartment. Instead, the capillary pressure barrier functions to control the position of the fluid meniscus through meniscus fixation, thereby demarcating different regions of the microfluidic channel.

[0044] In some examples, the support compartment includes an inlet and an outlet, which may be located in close proximity to the support compartment. In other examples, the inlet and outlet are spatially separated from the support compartment and linked by a flow channel.

[0045] In some examples, the support compartment is defined as a region of microfluidic channels demarcated by openings in the substrate and covering, and by capillary pressure barriers within the microfluidic channels. In some examples, the support compartment does not include a membrane. In other examples, the support compartment includes a porous membrane, which may have a gel disposed in the pores of the membrane. In yet another example, the support compartment does not include a membrane and is configured to receive a gel or gel precursor. The gel or gel precursor may be as described herein and may, for example, be a gel containing an extracellular matrix capable of supporting cells.

[0046] In some examples, the support compartment provides or contains a structural support or support scaffold, allowing a layer of cells or cell aggregates to form and create a fluid-fluid interface. In some examples, the support compartment is configured to receive and confine a gel internally, and a layer of cells or cell aggregates is formed on the surface of the gel facing a conduit, and the gel performs the function of a support scaffold. In some examples, the support compartment includes a porous membrane in which a gel is disposed within the pores, and cells or cell aggregates form a layer on the surface of the gel-containing porous membrane using a combination of membrane and gel that performs the function of a support scaffold. Thus, the support compartment having a gel provides a surface on which cells can grow and provides structural and functional support to the cell layer by allowing nutrients to flow to the cells through the gel.

[0047] In some examples, the support compartment provides initial structural support to seeded cells through a gel or other extracellular matrix present in the compartment until the cells have been cultured long enough that support is no longer needed. In these examples, the gel or other extracellular matrix may be sacrificial or degradable material that degrades over time, leaving behind cellular tubules that it supports. Thus, in some examples, the cultured cells themselves provide a structural network within the support compartment that separates perfusion channels and conduits, thereby enhancing the structural integrity or function of the support scaffold. These examples allow for the formation of direct air-to-air or direct air-to-liquid interfaces, as may be required.

[0048] In some examples, a microfluidic channel includes two or more support compartments. For example, a microfluidic channel may include one support compartment on each side of an opening formed in the cover and substrate. Each support compartment may be in fluid communication with its own reservoir for fluid supply, or each support compartment may be in fluid communication with a common reservoir for fluid supply.

[0049] <Perfusion section> A microfluidic channel includes at least one perfusion compartment. References to perfusion compartments herein will be understood as references to at least one perfusion compartment and as generally applicable to two or more perfusion compartments. Perfusion compartments are generally located near support compartments within the microfluidic channel. In some examples, perfusion compartments are located within the microfluidic channel with support compartments positioned between the perfusion compartment and conduits formed by openings in the substrate and covering. In some examples, the perfusion compartments and support compartments are adjacent to each other and lie on the same plane of the microfluidic network.

[0050] In some examples, the perfusion compartment is defined at least partially by one or more capillary pressure barriers located within the microfluidic channel. In some examples, one or more capillary pressure barriers define a boundary that controls (but does not obstruct) the fluid flow between the support compartment and the perfusion compartment. In this way, the fluid meniscus in the support compartment can be fixed to the capillary pressure barrier, thereby confined within the capillary pressure barrier and not overflowing into the perfusion compartment, and vice versa. It will be understood that the fixation of the first fluid in the support compartment and the introduction of the second fluid into the perfusion compartment fuse the two meniscuses, allowing fluid to flow between the support compartment and the perfusion compartment. Thus, one or more capillary pressure barriers are used to control, but not completely obstruct, the fluid flow within the microfluidic channel.

[0051] In some examples, the perfusion compartment includes an inlet and an outlet, which may be located in close proximity to the compartment. In some examples, the inlet and outlet are spatially separated from the perfusion compartment and linked by a flow channel.

[0052] In some examples, the perfusion compartment is configured to provide a flow of nutrients to the support compartment, allowing a layer of cells or cell aggregates to form and grow, thereby creating a fluid-fluid interface.

[0053] In some examples, the perfusion compartment is configured to receive a culture medium containing one or more types of cells or cell aggregates, forming a layer of cells or cell aggregates on the surface of the perfusion compartment. In some examples, the perfusion compartment is configured to receive a culture medium containing one or more types of cells or cell aggregates, forming a layer of cells on the surface of a support compartment adjacent to the perfusion compartment.

[0054] In some examples, one or more types of cells or cell aggregates may be selected from: epithelial or endothelial cells for lining perfusion compartments that potentially form tubes or blood vessels; epithelial or endothelial cells placed within a gel, extracellular matrix, or scaffold that preferably form luminal structures, and more preferably form a vascular bed; stromal cells within or on the surface of a gel, extracellular matrix, or scaffold; muscle cells within or on the surface of a gel, extracellular matrix, or scaffold; or any other type of cell that may be required to create a desired in vitro model system.

[0055] In some examples, a microfluidic channel includes two or more perfusion compartments. For example, a microfluidic channel may include one perfusion compartment on each side of an opening formed in the cover and substrate. Each perfusion compartment may be in fluid communication with its own reservoir for fluid supply, or each perfusion compartment may be in fluid communication with a common reservoir for fluid supply.

[0056] <Opening> As described herein, the substrate layer and the covering layer each include openings. The openings may be openings to the microfluidic layer of the apparatus. In some examples, the openings are substantially aligned with each other. In some examples, the openings are substantially concentric with each other. In some examples, the openings are arranged to define conduits through the microfluidic apparatus.

[0057] In one example, the diameter or area defined by the opening in the cover is greater than the diameter or area defined by the opening in the substrate; in other words, the opening in the cover is circumferential to the opening in the substrate and is larger than the opening in the substrate - or the diameter of the opening in the substrate is smaller than the diameter of the opening in the cover. In another example, the diameter or area defined by the outer circumference of the opening in the substrate is greater than the diameter or area defined by the outer circumference of the opening in the cover; in other words, the opening in the substrate surrounds the opening in the cover and is larger than the opening in the cover. Regardless of shape, one or both of the openings in the substrate and the opening in the cover can define the allowable contact area of ​​the liquid or gel composition introduced into the support compartment.

[0058] In some examples, the opening in the substrate is smaller than the opening in the covering, thereby the defined conduit has a frustoconical cross-section that tapers toward the substrate. In this example, the gel in the support compartment has a surface facing the conduit that slopes downward toward the opening in the substrate, thereby providing a non-vertical surface from which a layer of cells can be formed.

[0059] In some examples, the openings in the substrate and the cover have a diameter or maximum dimension of approximately 2 mm or less, e.g., less than approximately 1.5 mm, e.g., less than approximately 1 mm, e.g., less than approximately 500 μm, e.g., less than approximately 250 μm. In some examples, the openings in the substrate and the cover have a diameter or maximum dimension of approximately 1 mm or 500 μm, respectively. In some examples, the opening in the substrate has a diameter of 1 mm or 500 μm, and the opening in the cover has a diameter or maximum dimension of 1 mm. Preferably, the opening in the substrate is small enough to form a stable capillary pressure barrier, but large enough to prevent it from becoming filled with flow through the gap, e.g., a diameter of approximately 500 μm or less.

[0060] In some examples, one or both of the openings in the substrate and / or the cover may define the contact area of ​​the liquid or gel composition introduced into the support compartment by functioning as a capillary pressure barrier. In some examples, the rim of the opening may be configured to function as a capillary pressure barrier without further modification. In some examples, the rim of the opening may be provided with a raised section in the form of a lip extending into the microfluidic channel, which functions as a capillary pressure barrier. Capillary pressure barriers of other configurations formed on the rim of the opening, such as those described herein, are also contemplated.

[0061] In some cases, openings in the substrate act as capillary pressure barriers, preventing liquid introduced into the ducts from flowing through the openings into the substrate. In this way, a culture medium containing cells can be introduced into the ducts through openings in the covering without the medium (and cells) being lost through openings in the substrate.

[0062] <Conduit> As described herein, the substrate layer and the covering layer each include openings. In some examples, the openings are arranged as described herein to define conduits extending through the microfluidic apparatus. In some examples, the conduits are perpendicular or orthogonal to the surface of the microfluidic layer. For example, in the orientation used, the microfluidic layer may be substantially horizontal, and the conduits may be substantially vertical. It will be understood that the openings may be offset from each other to define conduits at an oblique angle to the surface of the microfluidic layer. In some examples, the conduits have a frustoconical cross-section, for example, tapering toward the openings in the substrate. Conduits having this configuration are advantageous for initial seeding and for imaging the seeded surface during subsequent experiments, due to the tapering of the gel surface.

[0063] In some examples, conduits are configured to receive a flow of fluid, such as air, gas, or liquid, through openings in the cover and / or in the substrate. The fluid flow may be via a well or reservoir positioned above the opening in the cover. The fluid flow may be via one or more inlets or outlets of a device that communicates with the opening. The device is generally configured to connect to a supply of fluid (e.g., a flow of compressed air or gas or dispersed particles). For example, the device may be connected to a removable manifold and configured to provide a flow of fluid to one or more conduits of one or more microfluidic networks. In some examples, the manifold may have one or more branches from a single fluid inlet, connecting a single fluid supply to each conduit of a plurality of microfluidic networks. In some examples, the manifold may have one or more branches connecting a plurality of fluid supplies to conduits of a single microfluidic network. In some examples, the apparatus may be configured to connect to two or more manifolds, each having branched flow paths, as described above, where the flow path of one manifold terminates at an opening in the covering of the microfluidic network, and the flow path of the second manifold terminates at an opening in the substrate of the microfluidic network. The fluid supply includes a positive or negative pressure supply, which produces a flow in one direction or in the opposite direction depending on the nature of the pressure. Thus, fluid flow through the conduit in both directions can be achieved.

[0064] <Capillary pressure barrier> The microfluidic network of a microfluidic device may include a capillary pressure barrier.

[0065] In some examples, the capillary pressure barrier is substantially aligned with the opening in the covering. In some examples, the capillary pressure barrier divides the microfluidic channel into a first sub-volume and a second sub-volume, e.g., a perfusion compartment and a support compartment or a support compartment and a conduit. In some examples, the capillary pressure barrier at least partially defines the sub-volume of the microfluidic channel.

[0066] The function and patterning of capillary pressure barriers have already been described, for example, in International Publication No. 2014 / 038943 A1. As will be apparent from the exemplary embodiments described below, a capillary pressure barrier, also referred to herein as a droplet-holding structure, should not be understood as, for example, a wall or cavity that can be filled with liquid, but rather consists of or includes a structure that ensures that such droplets do not diffuse due to surface tension. This concept is called meniscus fixation. Thus, stable containment of liquid into a region of the microfluidic channel of the apparatus, for example, a support compartment, can be achieved. In one example, the capillary pressure barrier may be called a containment phase guide, which is configured to prevent overflow during normal use of the cell culture apparatus or while the first liquid is being initially filled into the cell culture apparatus. The properties of liquid containment will be described later in conjunction with the description of the method of the present invention. In some examples, openings can function as capillary pressure barriers. For example, an opening in a substrate can function as a capillary pressure barrier to fix liquid, or an opening in a cover can function as a capillary pressure barrier.

[0067] In one example, a capillary pressure barrier includes or consists of a rim or edge of material protruding from the inner surface of a microfluidic channel, or a groove on the inner surface of the microfluidic channel. The sidewalls of the rim or edge may preferably have an angle α with the top of the rim or edge, which is as large as possible. To provide a good barrier, the angle α should be greater than 70°, typically around 90°. The same applies to the angle α between the sidewall of the edge and the inner surface of the microfluidic channel where the capillary pressure barrier is located. Similar requirements are also imposed on capillary pressure barriers formed as grooves.

[0068] An alternative form of capillary pressure barrier is an area of ​​material with a different wettability from the inner surface of the microfluidic channel, acting as a diffusion stop due to capillary forces / surface tension. In one example, the inner surface of the microfluidic channel contains a hydrophilic material, and the capillary pressure barrier is an area of ​​hydrophobic material or a less hydrophilic material.

[0069] In one example, the capillary pressure barrier is selected from rims or edges, grooves, holes, hydrophobic series of materials, or combinations thereof. In another example, the capillary pressure barrier can be created by widening the microfluidic channel or by columns at selected intervals, the arrangement of which can define the support area occupied by the gel. In one example, the columns extend to the entire height of the microfluidic channel.

[0070] As a result of the presence of a capillary pressure barrier, the liquid is prevented from flowing beyond the barrier, allowing the formation of a stably confined volume in one or more of the first, second, or third sub-volumes, which may be called a support compartment, perfusion compartment, or conduit, or may function as such.

[0071] In one example, the capillary pressure barrier is located beneath the covering layer and spaced apart from the opening. In another example, the capillary pressure barrier is provided on the substrate of the microfluidic layer or on the inner surface of a microfluidic channel opposite or facing the opening in the covering. In yet another example, the capillary pressure barrier is located on the substrate layer and spaced apart from the opening. In yet another example, the capillary pressure barrier exists as previously defined and confines fluid droplets to a secondary volume of the microfluidic layer aligned with the opening in the covering.

[0072] In one example, the capillary pressure barrier defines at least a portion of the surface, e.g., the floor, of the first secondary volume of the microfluidic channel, sometimes called the support compartment. The capillary pressure barrier is configured to confine the fluid to the first secondary volume of the microfluidic channel. In one example, the capillary pressure barrier has a closed geometric configuration. In one example, the capillary pressure barrier is concentric with the opening of the covering layer.

[0073] In one example, the capillary pressure barrier is a substantially linear capillary pressure barrier that extends across the entire width of the microfluidic channel and intersects with the side walls of the microfluidic channel at each end.

[0074] <Second capillary pressure barrier> In some examples, the microfluidic network of the device is provided with a second capillary pressure barrier, the form and function of which is substantially as described above. To avoid misunderstanding, references to “capillary pressure barriers” should be understood as references to “first capillary pressure barriers” when a second capillary pressure barrier is present in the device. In some examples, the second capillary pressure barrier is configured and located within the microfluidic network, for example, within a support compartment, to restrict the diffusion of fluid droplets.

[0075] In one example, the second capillary pressure barrier is provided on the inner surface of the microfluidic channel. For example, the second capillary pressure barrier is located on the substrate of the microfluidic layer on the inner surface of the microfluidic channel or on the covering of the microfluidic layer on the inner surface of the microfluidic channel. In one example, the second capillary pressure barrier is located as previously defined to confine fluid droplets in a region of the microfluidic layer aligned with the opening.

[0076] In one example, a second capillary pressure barrier, in combination with the first capillary pressure barrier, defines at least a portion of the surface of the support compartment on the substrate of the microfluidic channel. The second capillary pressure barrier, in combination with the first capillary pressure barrier, is configured to confine the fluid within the support compartment.

[0077] In one example, the second capillary pressure barrier is a substantially linear capillary pressure barrier extending across the entire width of the microfluidic channel and intersecting the side walls of the microfluidic channel at each end. In this example, the first and second capillary pressure barriers, together with the walls where they intersect, can define a region that aligns with and may be concentric with the openings of the covering layer. Alternatively or additionally, the first and second capillary pressure barriers, together with the walls where they intersect, can define a region that aligns with and may be concentric with the openings of the substrate layer. In these examples, the first capillary pressure barrier can be considered to divide the microfluidic network into a first sub-volume containing a support compartment and a second sub-volume containing a first perfusion compartment, and the second capillary pressure barrier divides the microfluidic network into a first sub-volume containing a support compartment and a third sub-volume containing a second perfusion compartment. In other words, one capillary pressure barrier defines, at least partially, the boundary between at least one support section and at least one perfusion section, and / or one capillary pressure barrier defines, at least partially, the boundary between at least one support section and a conduit.

[0078] <Reservoir> In some examples, the microfluidic network includes a reservoir or well that is in fluid communication with the culture medium inlet to the microfluidic channel. The reservoir may be located in a reservoir layer positioned above the covering layer of the microfluidic network. The reservoir may be there to hold a volume of liquid, such as culture medium. In a typical embodiment, the reservoir is capable of holding a volume of fluid larger than that held or that can be held by the microfluidic channel. The reservoir may be a neighbor well to a well aligned with a conduit on a bottomless microtiter plate positioned above the microfluidic layer, and may be in fluid contact with the perfusion compartment. The reservoir may be a neighbor well to a well aligned with a conduit on a bottomless microtiter plate positioned above the microfluidic layer, and may be in fluid contact with the support compartment. In other examples, the reservoir may be a well on the same microtiter plate but spatially separated from the conduit well. It will be understood that the proximity of the reservoir to the conduit well is not critical to the operation of the device, as long as the conduit well and the reservoir are in fluid communication with the microfluidic layer, respectively.

[0079] In some examples, the microfluidic network includes two or more reservoirs, for example, two or three or more, that are in fluid communication with the microfluidic layer through openings in the covering layer, which may be appropriately called inlets or outlets of the microfluidic layer. In embodiments in which at least two reservoirs are present in the microfluidic network, the first reservoir may be used to introduce a fluid, such as a culture medium, into the microfluidic network, while the second reservoir may function as a vent or an overflow compartment for receiving fluid during the execution of the method of the present invention.

[0080] <Enclosure> In some examples, a microfluidic cell culture apparatus includes an enclosure that at least partially surrounds or covers the outside of the substrate, the outside of the cover, or both. In some examples, the enclosure at least partially surrounds or covers a reservoir layer, e.g., a microtiter plate disposed on the cover of a microfluidic network. The enclosure allows for the isolation of the environment within the microfluidic network, and for the exclusion of dust and other small / ultrafine particles, so that the cell culture can be kept sterile. In some examples, the enclosure has the form of a plate with a rim extending from its edge. In some examples, the enclosure is configured to engage with the contact surface of the apparatus to form a seal. In some examples, the rim of the enclosure engages with the contact surface of the apparatus. In some examples, the enclosure is configured to engage with the apparatus in a non-airtight manner. In some examples, the enclosure engages with the apparatus in a non-airtight manner, but is configured to engage with the apparatus to provide a difficult or winding flow path to the enclosure and / or the apparatus.

[0081] In some examples, a microfluidic device includes a first enclosure that at least partially encloses or covers the outside of a substrate, and a second enclosure that at least partially encloses or covers the outside of the cover or a reservoir layer disposed on the cover. In some examples, the enclosure is a single enclosure that completely encloses the outside of the substrate and the outside of the cover or reservoir layer. In some examples, the enclosure is a single enclosure that completely encloses the entire microfluidic device. In some examples, all or part of the enclosure is removable from the device. To avoid misunderstanding, any reference herein to an enclosure, that enclosure or at least one of its enclosures refers to the enclosure that covers the substrate, the enclosure that covers the cover, or both enclosures.

[0082] In some examples, the enclosure may be provided with ports for fluid exchange. In some examples, the enclosure may be provided with tubes for guiding the fluid flow. In some examples, the enclosure may be provided with windows or transparent sections for light access. In some examples, the enclosure may be provided with filters for exchanging fluid without contamination. In some examples, the enclosure may be provided with bends for exchanging gaseous fluids without contamination. When the enclosure engages with a microfluidic apparatus, bends may be provided between the enclosure and the outside of the substrate or cover or reservoir layer. In some examples, bends are provided within the enclosure itself. The bends effectively restrict the flow of bacteria, particulate matter, and other substances that may adversely affect cell culture experiments being performed in the microfluidic cell culture apparatus.

[0083] In some examples, the enclosure includes two or more fluid inlets and two or more fluid outlets. In some examples, the two or more fluid inlets and two or more fluid outlets are connected to conduits of multiple microfluidic networks via connecting channels. In some examples, the number of fluid inlets differs from the number of outlets and / or the number of connected conduits, thereby allowing for the division and / or merging of flow paths. Such configurations allow for the routing of fluid from a single inlet of the device through multiple conduits and / or outlets or from multiple inlets and / or conduits to a single outlet. In some examples, the enclosure is connected to a positive or negative pressure source as described herein.

[0084] <<Method>> <How to create a fluid-fluid interface> In one example, a method is provided for creating a fluid-fluid interface in a microfluidic cell culture apparatus comprising a microfluidic network having a substrate, microfluidic channels, and a cover, wherein the microfluidic channels have at least one perfusion compartment and at least one support compartment inside, and the substrate and cover each include openings that define conduits through the microfluidic channels, and this method The steps include: introducing a support scaffold to at least one support section in order to form a scaffold surface facing the conduit; The steps include introducing a culture medium containing cells into a xylem through an opening in the cover, while preventing the medium from flowing out of the xylem through an opening in the substrate, A step that allows cells to form layers on the scaffold surface, To expose the cells, the step is to remove the culture medium from the ducts. Includes.

[0085] In some examples, the support scaffold comprises a membrane. In some examples, the support scaffold comprises a gel, and the step of introducing the support scaffold into at least one support compartment comprises the step of introducing a liquid gel precursor. In some examples, the liquid precursor is fixed to the support compartment to form a scaffold surface facing the conduit.

[0086] Therefore, in one example, a method is provided for creating a fluid-fluid interface in a microfluidic cell culture apparatus comprising a microfluidic network having a substrate, microfluidic channels and a cover, wherein the microfluidic channels have at least one perfusion compartment and at least one support compartment, and the substrate and cover each include openings that define conduits through the microfluidic channels, and this method is A step of introducing a gel or gel precursor into at least one support compartment, The steps include fixing a gel or gel precursor to at least one support section in order to form a gel surface facing a conduit, The steps include introducing a culture medium containing cells into a xylem through an opening in the cover, while preventing the medium from flowing out of the xylem through an opening in the substrate, A step that allows cells to form a layer on the gel surface, To expose the cells, the step is to remove the culture medium from the ducts. Includes.

[0087] In some cases, the methods described herein are The steps include introducing a gel or liquid gel precursor into at least one support compartment, The steps include: fixing a gel or liquid gel precursor within at least one support compartment and allowing the gel or liquid gel precursor to harden or gel in volume in order to form a hardened gel surface facing a conduit; Includes.

[0088] In some examples, the methods described herein may include the steps of introducing a volume of gel or liquid gel precursor into a support compartment and allowing the volume of gel or liquid gel precursor to be confined by a capillary pressure barrier, and allowing the volume of gel or liquid gel precursor to harden or gel in order to form a hardened gel surface facing the conduit. In some examples, the volume of gel or liquid gel precursor may be a single droplet or a volume of gel or liquid gel precursor the size of a droplet.

[0089] Gels or liquid gel precursors include any hydrogel known in the art that is suitable for cell culture. Hydrogels used in cell culture can be formed from a wide range of natural and synthetic materials and offer a wide range of mechanical and chemical properties. For an overview of materials and methods used in hydrogel synthesis, see Lee and Mooney (Chem Rev 2001;101(7):1869-1880). Suitable hydrogels, when formed from natural materials, promote cell function, and when formed from synthetic materials, tolerate cell function. Natural gels for cell culture are typically formed from proteins and ECM components such as collagen, fibrin, hyaluronic acid, or Matrigel, as well as materials derived from other biosources such as chitosan, alginates, or silk fibers. Because they are derived from natural sources, these gels are inherently biocompatible and bioactive. Acceptable synthetic hydrogels can be formed from pure non-natural molecules, such as poly(ethylene glycol) (PEG), poly(vinyl alcohol), and poly(2-hydroxyethyl methacrylate). PEG hydrogels have been shown to maintain the viability of encapsulated cells and allow ECM deposition as they degrade, demonstrating that synthetic gels can function as 3D cell culture platforms even without integrin-binding ligands. Such inert gels are highly reproducible, allow for easy adjustment of mechanical properties, and are easily processed and manufactured.

[0090] The gel or gel precursor can be provided to the support compartment of a microfluidic cell culture apparatus, such as the apparatus described above. After the gel or gel precursor is provided, gelation is induced or occurs, for example, before further fluid is introduced into the perfusion compartment. Suitable (precursor) gels are well known in the art. For example, the gel precursor may be a hydrogel, typically an extracellular matrix (ECM) gel. The ECM may contain, for example, collagen, fibrinogen, fibronectin and / or basement membrane extracts such as Madrigel or synthetic gels. The gel precursor can be introduced into the support compartment using a pipette, for example, via a well or reservoir that is in fluid communication with the support compartment.

[0091] The gel or gel precursor 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, egg white, and commercially available products such as Madrigel.

[0092] The basement membrane, including the basement plate, is a thin extracellular matrix located beneath epithelial cells in vivo and is composed of extracellular matrix components such as proteins and proteoglycans. For example, the basement membrane is composed of collagen IV, laminin, entactin, heparan sulfate proteoglycans, and numerous other trace components (Quaranta et al., Curr. Opin. Cell Biol. 6, 674-681, 1994). Along with the intact basement membrane, these components are individually biologically active, promoting cell adhesion, migration, and often proliferation and differentiation. An example of a basement membrane-based gel is called Matrigel (U.S. Patent No. 4,829,000). This material is highly biologically active in vitro as a basement layer for epithelial cells.

[0093] Many different gels suitable for use in the method of the present invention are commercially available, and such gels include, but are not limited to, Matrigel rgf, BME1, BME1rgf, BME2, BME2rgf, BME3 (all Matrigel manifolds), collagen I, collagen IV, mixtures of collagen I and IV, mixtures of collagen I and IV, and mixtures of collagen II and III. thing, Examples include PuraMatrix, hydrogel, Cell-Tak®, collagen I, collagen IV, Matrigel® matrix, fibronectin, gelatin, laminin, osteopontin, polylysine (PDL, PLL), PDL / LM and PLO / LM, PuraMatrix®, or vitronectin. In one preferred embodiment, the matrix component is obtained as commercially available Corning® MATRIGEL® matrix (Corning, NY14831, USA).

[0094] The gel or gel precursor is introduced into the apparatus described herein and confined, for example, in a support compartment by a capillary pressure barrier within the microfluidic apparatus, and then gelation is induced or occurs as necessary.

[0095] In one example, a sufficiently large volume of droplets is introduced so that the cured gel is substantially entirely located within the support compartment in the microfluidic layer. In one example, the gelled droplets do not close the opening in the microfluidic covering layer, in which case the unclosed or open area of ​​the opening can be used as a vent. Thus, the vent generally includes an opening or opening in the covering that allows exhaust when loaded into the microfluidic channel through the inlet. In one example, a sufficiently large volume of droplets is introduced so that the droplets are confined at the boundary between the support compartment and the perfusion compartment by a capillary pressure barrier, and / or by the rim of the opening in the covering, and / or by the rim of the opening in the substrate, each of which can also function as a capillary pressure barrier.

[0096] In some examples, the rim of the substrate opening acts as a capillary pressure barrier to prevent the flow of culture medium from the conduit when culture medium is added. In some examples, a removable polymer film may cover the substrate opening by contacting the underside of the substrate layer while culture medium containing cells is introduced into the conduit. It will be understood that when the microfluidic device is in the "in use" orientation, it is most convenient to fill the conduit, meaning that the conduit is most conveniently filled through the covering opening while flow through the substrate opening is blocked. However, it will be immediately apparent to those skilled in the art that it is equally possible to invert the microfluidic device and fill the conduit through the substrate opening while preventing flow through the covering opening.

[0097] The method includes the step of introducing a culture medium containing cells into a xylem vessel through an opening in the cover, while preventing the flow of the medium from the xylem vessel through an opening in the substrate. However, it will become clear that it is equally easy to introduce a culture medium containing cells into a xylem vessel through an opening in the substrate, while preventing the flow of the medium from the xylem vessel through an opening in the cover.

[0098] In some cases, at least a portion of the culture medium is introduced into the ducts separately from the cells, and the cells are added separately to at least a portion of the medium via substrate openings and / or covering openings. Cells can be introduced into the ducts using any liquid handling technique, including non-contact dispensing techniques such as acoustic droplet dispensers. In some cases, especially when introducing a small amount of medium containing cells, the ducts are pre-wetted with a portion of the medium prior to adding the cells.

[0099] When a culture medium containing cells is added to a duct, the present cells can be fixed onto the surface of the gel lining the duct. In some examples, cells can form layers through gravity and / or inclination of a microfluidic apparatus. The degree of inclination may depend on the relative positions of the substrate opening and the covering opening and the resulting gradient of the gel surface between the two. In some examples, the degree of inclination at any given time may range from horizontal to 90°, e.g., 80°, e.g., 70°, e.g., 60°, e.g., 50°, e.g., 40°, e.g., 30°, e.g., 20°.

[0100] In some examples, the cells include epithelial cells, such as lung epithelial cells, skin epithelial cells, gastrointestinal epithelial cells, corneal epithelial cells, or myxogenic epithelial cells. In some examples, the cells include any epithelial cells found in vivo at a fluid-fluid interface.

[0101] Once cells adhere to the gel surface, the remaining culture medium can be carefully removed from the conduits. Removal of the medium can be carried out cautiously, avoiding the removal of any adhered cells. In some cases, the medium is removed from the conduits by one or more of the following: pressure pulses, inertia, changes in the surface tension of the medium fixed to the opening of the substrate, changes in the contact angle between the medium and the solid part of the conduit, suction, contact of the medium with a receiving receptacle from above or below the apparatus, mechanical, acoustic, electrostatic, electromagnetic or other actions, and evaporation. The surface tension of the medium and the contact angle between the medium and the solid part of the conduit can be altered, for example, by adding a surfactant or changing the surface charge of the substrate (electrowetting). Upon removal of the medium, an air-air interface is first created, as the cells are exposed and both the conduits and perfusion compartments are empty. Thus, in some cases, the fluid-fluid interface is an air-air interface. In other cases, the fluid-fluid interface is an air-liquid interface or a liquid-liquid interface, depending on the nature of the experiment and whether the perfusion compartments and / or conduits are filled with liquid or air / gas.

[0102] In some examples, the supply of nutrients is provided to the attached cells via a perfusion compartment. For example, the supply of nutrients to the cells can be transported from at least one perfusion channel through at least one support compartment to the cells via diffusion or pore flow through the permeable interface of the support compartment. The permeable interface may be an extracellular matrix fixed by a permeable membrane which may have one or more capillary pressure barriers (including rims of openings) or pores as described herein. In some examples, the attached cells may form on the surface of the support compartment so as to extend around the entire boundary of the duct. For example, in situations where the duct has a circular cross-section, the attached cells may form a complete tubule surrounding the duct.

[0103] In some examples, one or more types of cells can be introduced into the perfusion compartment to form layers or cell aggregates. In some examples, one or more types of cells or cell aggregates may be selected from epithelial or endothelial cells lining the perfusion compartment, potentially forming tubes or blood vessels; epithelial or endothelial cells placed inside a gel, extracellular matrix, or scaffold, preferably forming tubular structures, and more preferably forming a vascular bed; stromal cells in or on the gel, extracellular matrix, or scaffold; or any other type of cell that may be necessary to create the desired in vitro model system. The use of a capillary pressure barrier allows for the formation of a gel that is stably confined, for example, within a support compartment of a microfluidic network, thereby enabling the addition of a second fluid to the perfusion compartment without displacing the gel or the contents of the perfusion compartment. Thus, the apparatus of the present invention is configured to perform spatially controlled co-culturing with other cells, as described above, and provides a means for controlling the composition of the surrounding culture medium. In the described method, the fluid loaded into the reservoir (also referred to herein as a well) is any of the following: cell culture medium, test solution, buffer, further hydrogel, etc., and may optionally contain cells or cell aggregates.

[0104] By controlling the composition introduced into the reservoir, the cell culture apparatus of the present invention enables various cell culture modes. For example, the composition of the fluid introduced into the reservoir or well can be changed. Such an exchange may be a gradient exchange by introducing a new composition into one of the reservoirs and simultaneously removing fluid from another reservoir in the same microfluidic network until a complete exchange is achieved. Such an exchange may also be discontinuous by aspirating fluid from a reservoir and filling it with the new composition. The fluid volume in the reservoir is much larger than the fluid volume in the microfluidic channels, and leveling between reservoirs occurs almost instantaneously, thereby ensuring that the microfluidic network is flushed with new fluid without the need to empty the microfluidic channel network during the procedure.

[0105] <Methods for investigating cellular responses to stimulants> For example, a method for investigating a cellular response to a stimulant includes, according to the method described herein, the steps of creating a fluid-fluid interface in a microfluidic device and exposing exposed cells to the fluid flow by advancing a fluid flow through a conduit.

[0106] For example, a method for investigating a cellular response to a stimulant includes the steps of using an assay plate described herein and exposing exposed cells to a fluid flow by advancing a fluid flow through a conduit.

[0107] As described above, once a fluid-fluid interface is created, it becomes possible to perform experiments on cultured cells to investigate the cellular response to stimulants. For example, the flow of fluid through conduits containing cellular tubules can be air, smoke, vapor, fog, pathogens, ultrafine particles, analytes, It may contain one or more drug candidates or other compounds of interest. The fluid flow can be a gas flow or a liquid flow. Typically, if a lung model is created and under investigation to reproduce the lung environment, the interface is an air-liquid interface, so the fluid flow is usually a gas flow.

[0108] In some cases, the fluid flow propagated through the conduit does not have an irritant, but instead collects cell samples from the cell layer or induces shear stress on the cell layer. In other words, the fluid flow can also serve the purpose of sample collection and sample investigation. In some cases, the fluid flow propagated through the conduit is simply the culture medium, which provides nutrients directly to the cells rather than transporting them from the perfusion compartment to the cells via the support compartment.

[0109] In some examples, the experiment may involve exposing cells to an irritant in a first fluid flow and then collecting the cells using a second fluid flow different from the first to identify the effect of the irritant on the cells. In some examples, the cellular response to the irritant can be monitored in situ without removing the cells from the duct. In some examples, identifying the cellular response may involve assaying one or more of the cellular phenotype, cellular morphology, and cellular function, depending on the nature of the investigation.

[0110] In some cases, the fluid flow propagated through the conduit can be collected at least partially and recirculated at least partially through the conduit a second or more times. Recirculation can be achieved by the use of a pump.

[0111] In some examples, the method includes providing a supply of nutrients to the cells through at least one perfusion compartment, in conjunction with a method for forming a fluid-fluid interface, as described above. For example, the supply of nutrients may be provided to the cells from at least one perfusion channel through at least one support compartment via diffusion or pore flow through the permeable interface of the support compartment.

[0112] In some examples, the method includes the step of introducing one or more types of cells into at least one perfusion compartment before or after the optionally exposed cells are struck by a fluid flow, allowing the one or more types of cells to form layers or cell aggregates.

[0113] <<Assay Plate>> A further aspect of the present invention provides an assay plate comprising any of the devices described herein.

[0114] In one example, an assay plate is provided comprising a microfluidic apparatus as described herein, having a scaffold or other support structure within a support compartment. In some examples, the microfluidic network of the assay plate includes one or more cells or cell aggregates present, for example, inside or on the scaffold or support structure facing the conduits and / or within the microfluidic channels. In some examples, the scaffold or support structure includes a gel, extracellular matrix and / or membrane. In some examples, the cells or cell aggregates are at least partially aligned with the conduits. In some examples, the cells are epithelial cells, for example, lung epithelial cells. In some examples, the cells or cell aggregates are at least partially aligned with the conduits, thereby forming a fluid-fluidic interface that can be investigated as described herein.

[0115] Assay plates may contain one or more cells or cell aggregates cultured by the methods described herein. The dimensions of the assay plates conform to or are compatible with the standard ANSI / SLAS microtiter plate format. In particular, the footprint or perimeter dimensions of the assay plates may be consistent with the ANSI / SLAS standard for microtiter plates.

[0116] Also described is a cell culture apparatus manufactured by either an assay plate or a method described herein.

[0117] <Kit> This disclosure also provides kits and products for using the microfluidic devices and assay plates described herein. In some examples, a kit may include the device or assay plate described herein, 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.

[0118] The kit may further include packaging materials and labels or packaging inserts contained within the packaging materials that provide instructions for use.

[0119] The kit may further include assembly components such as a second container containing a culture medium suitable for cell transduction, and instructions on how to use the culture medium.

[0120] <<<Detailed explanation of the diagram>>> Here, the present invention will be described simply as an example with reference to the drawings.

[0121] Figures 1 to 3 schematically show a first example of a microfluidic apparatus. The apparatus 100 shown in Figure 1 generally includes a substrate 101, microfluidic channels 102 in a microfluidic layer, and a cover 103 (all shown by solid lines). An upper layer 104 in the form of a multi-well bottomless plate is disposed on top of the cover layer and includes a well or reservoir 105. A culture medium inlet 106 is located in the cover layer of the microfluidic layer. Two capillary pressure barriers 112a and 112b are located on the substrate 101 of the apparatus and are accessible through an opening 109 in the substrate and an opening 110 in the cover layer 103. In this example, one well or reservoir 105 is located above the substrate opening 109 and the cover opening 110. A separate well is provided above the culture medium inlet 106. A conduit 111 is provided through the microfluidic apparatus and is defined by the substrate opening 109 and the cover opening 110.

[0122] As can be seen from Figures 2 and 3, the microfluidic channel 102 is divided into three sub-volumes by capillary pressure barriers 112a and 112b. To the left of the capillary pressure barrier 112a is the first perfusion compartment 107a, to which the flow channel connects to its dedicated culture medium inlet and outlet. To the right of the capillary pressure barrier 112b, the corresponding second perfusion compartment 107b can be found. The areas of the microfluidic channel 102 within the first and second capillary pressure barriers 112a and 112b define a support compartment 108, which in this example encloses the substrate opening 109 and the cover opening 110 and the conduit 111 defined thereby. The support compartment 108 also has its own culture medium inlet connected by a dedicated flow channel. In this example, a dedicated culture medium outlet for the support compartment 108 is not necessary because the conduit 111 functions as a vent when material is introduced into the support compartment 108.

[0123] In Figure 2, the substrate opening 109 and the cover opening 110 are similarly shown as circular openings in subsequent figures. However, it will be understood that the openings can have any shape, and that circular and square are preferred.

[0124] Figures 4A to 4D show further examples of microfluidic cell culture systems with different configurations regarding the arrangement and opening dimensions of the capillary pressure barrier.

[0125] Figure 4A shows an example of a device in which the substrate opening 109 is much narrower than the cover opening 110. As a result, when the support compartment 108 is filled with gel, the gel surface facing the opening 111 is inclined downward toward the substrate opening 109. In other words, the conduit 111 defined by the openings 109 and 110, the gel surface in the support compartment 108, and the reservoir above has a frustoconical cross-section and tapers toward the substrate opening 109. In the device of Figure 4A, the capillary pressure barriers 112a and 112b are placed on the substrate 101 and protrude into the microfluidic channel. Narrowing the width of the substrate opening as shown in this figure has the effect of reducing leakage (possibility) through the substrate opening, i.e., reducing the downward force due to gravity. A wider cover opening has the advantage of improving access to the conduit.

[0126] Figure 4B shows an example of an apparatus in which the substrate opening 109 is much wider than the cover opening 110. As a result, when the support compartment 108 is filled with gel, the gel surface facing the opening 111 spreads outward toward the substrate opening 109. In other words, the conduit 111, defined by openings 109 and 110, the gel surface in the support compartment 108, and the reservoir above, has an hourglass cross-section. In the apparatus of Figure 4B, the capillary pressure barriers 112a and 112b are placed on the cover 103 and protrude into the microfluidic channel. As shown in this figure, a wider substrate opening makes it easier to access from the bottom of the apparatus, for example, when seeding cells through the bottom opening or when extracting one or more cells from the conduit. Furthermore, having a wide substrate opening can improve imaging quality when there is no bottom substrate between the cells and the imaging device.

[0127] Figure 4C shows that the substrate opening 109 covers much more than the opening 110. wide An example apparatus is shown. As a result, when the support compartment 108 is filled with gel, the gel surface facing the opening 111 is inclined downward toward the substrate opening 109. In other words, the conduit 111 defined by the openings 109 and 110, the gel surface in the support compartment 108, and the reservoir above has a frustoconical cross-section and tapers toward the substrate opening 109. In the apparatus of Figure 4C, the capillary pressure barriers 112a and 112b are placed on the substrate 101 and protrude into the microfluidic channel, but are much closer to the substrate opening 109 than in the example of Figure 4A. Having a substrate opening that is much wider than the covering opening, as in the layout of Figure 4C, allows for a cell layer orientation opposite to that of Figure 5. This may be advantageous because when the fluid flow arrives from below through the substrate opening, there may be different fluid flow patterns around the cell layer or the same fluid flow along the cell layer.

[0128] Figures 5A to 5C illustrate the steps of a method for creating a fluid-fluid interface in a microfluidic apparatus as described herein.

[0129] Figure 5A shows the gel or gel precursor 114 filled in the support compartment 108 and fixed in place by the substrate opening 109, the cover opening 110, and the first and second capillary pressure barriers 112a and 112b.

[0130] Figure 5B shows the culture medium 115 introduced into the conduit 111 (unnumbered). The cells 116 suspended in the culture medium 115 are shown beginning to sink towards the bottom of the medium 115. The substrate opening 109 is shown functioning as a capillary pressure barrier, fixing the culture medium 115 in place within the conduit 111 and preventing it from flowing out.

[0131] Figure 5C shows cells 116 forming a layer on the surface of gel 114 facing the conduit 111, and culture medium. 115The substrate is still secured by the substrate opening 109. Figure 5D shows an alternative setup in which a sacrificial, i.e., temporary film 117 is placed over the substrate opening 109 to prevent the culture medium 115 from flowing through the substrate opening.

[0132] Figure 5E shows the culture medium 115 being removed from the conduit 111 by the aspirator 120. Once the culture medium 115 has been thoroughly removed, the meniscus of the culture medium 115 is withdrawn from the substrate opening 109, as shown in Figure 5F, and the cells 116 are exposed to air. Figure 5F shows the second culture medium 118 introduced into perfusion compartments 107a and 107b. a and 118b Also shown. Second medium 118 a and 118b This can be a cell culture medium for supplying nutrients to cells 116 via transport through the gel 114 in the support compartment 108.

[0133] Figure 5G shows medium 118a in perfusion compartment 107a and medium 118b in perfusion compartment 107b. In this example, mediums 118a / b also contain cells 119, which formed tubular structures within each perfusion compartment.

[0134] Figures 6A, 6B, and 7 illustrate a method for investigating cellular responses in a fluid-fluid interface setup similar to the methods in Figures 5A–5G. In Figure 6A, the fluid 121 is in liquid form and is advanced through a conduit to expose cells 116 to the fluid and its contents. In Figure 6B, the fluid 121 is a gaseous flow advanced through a conduit. Whether the fluid 121 is liquid or gaseous, it may contain an irritant to which cells 116 are exposed for the purpose of investigating the cellular response to the irritant. Figure 7 shows the fluid 121 advanced through a conduit, similar to Figure 6B. In Figure 7, perfusion compartments 107a and 107b are aligned with cell layers, e.g., epithelial or endothelial lumens, a second fluid flow through the perfusion compartment, and lumen components. Thus, the apparatus described herein allows two different fluids to flow through separate channels at different velocities and different compositions, as may be required. Therefore, the flow of culture medium through perfusion compartments 107a and 107b allows cells 116 to be cultured for the duration of the investigation of the cellular response to the stimulant, regardless of the stimulant contained in the fluid 121. These figures show the direction of fluid 121 flow as downward, entering the apparatus through the cover opening 108 and exiting through the substrate opening 107, but it will be understood that it is also possible to reverse the direction of flow if necessary and achieve the same results.

[0135] Figure 8 shows a plan view of a microfluidic apparatus (100) including multiple microfluidic networks described herein in a multiwell configuration. As described herein, the apparatus is preferably compatible with or based on a microtiter plate footprint defined by ANSI / SLAS dimensions, as shown in Figure 15. 8 Figure 9 shows a bottom view of such a plate, which includes, for example, 40 separate microfluidic networks, as shown in Figure 1. A conduit 111 is shown at the center of each microfluidic network. Figure 9 shows a cross-section of the multiwell configuration of Figure 8, where the conduit extends through the covering opening and the substrate opening in each microfluidic network.

[0136] Figure 10 shows the microfluidic apparatus 100 described herein, provided with enclosures 124a and 124b. Enclosure 124a is provided on the upper side of the apparatus 100 and rests on top of the reservoir layer (unnumbered) of the apparatus 100. Enclosure 124b is provided on the lower side of the apparatus 100 and sits within two supports at the outer edge of the substrate. In each case, enclosures 124a and 124b do not form a complete hermetically sealed environment with the apparatus 100, but instead provide a bent channel into the apparatus 100 by an extending rim over each enclosure. The bent channel prevents, or at least minimizes, the entry of bacteria-containing particulate matter into the apparatus, as indicated by the arrows.

[0137] Figure 11 shows another example of a microfluidic apparatus with an enclosure. In this example, the lower enclosure 124b is provided with a transparent surface in the form of a window 125 to allow light access to one or more of the individual microfluidic networks. In this example, the use of the window 125 facilitates visual monitoring of experiments being performed in the individual microfluidic networks.

[0138] Figures 12A to 12D show various examples of microfluidic devices having enclosures as described. In Figure 12A, the upper enclosure 124a is provided with a filter 126, while in Figure 12B, the filter 126 is provided in the lower enclosure 124b. In Figure 12C, the microfluidic device is provided with both an upper enclosure 124a and a lower enclosure 124b, both of which are provided with filters 126, while in Figure 12D, a single enclosure 124 is provided, which completely encloses the microfluidic device and is provided with filters 126 on its top and bottom surfaces. The use of filters, which may be in the form of (selectively permeable) membranes, absorption columns such as carbon filters, water locks, geometric trap structures, or valves, allows for the selective passage of fluid into or out of the microfluidic device. For example, the filter 126 may be selected or configured to exclude organisms, viruses, pathogens, particles, water, or ambient moisture from entering the microfluidic device while allowing gas exchange and / or pressure equilibrium.

[0139] Figures 13 and 14 show the fluid flow, such as a gas flow, through the enclosure and microfluidic apparatus. In Figure 13, the filter 126 in the upper enclosure 124a allows only specific fluids to pass through the apparatus, and these fluids then pass through a single conduit of a single microfluidic network and exit through a second filter 126 from the lower enclosure 124b. However, in the apparatus of this example, all microfluidic networks are interconnected, so the fluid flow is more accurately represented as shown in Figure 14 rather than the simplified flow shown in Figure 13. In Figure 14, the fluid entering the microfluidic apparatus through the filter 126 in the upper enclosure 124a is shown to pass through the conduit of each microfluidic network shown, then coupled in the gap between the microfluidic network substrate and the lower enclosure 124b, and exit through the filter 126 in the lower enclosure 124b. Therefore, a fluid flow introduced into a microfluidic device through a single fluid inlet can reach all individual microfluidic networks, enabling high-throughput screening of the effects of a single test compound on multiple cell samples within individual conduits, for example.

[0140] In contrast, the apparatus example in Figure 15 shows each microfluidic network having its own dedicated fluid inlet to the conduit. In particular, the upper enclosure is provided with multiple sealing members 127 that engage with the reservoir layer to separate the conduits of the individual microfluidic networks from one another. Thus, the individual or dedicated fluid inlets 128 provide fluid only to the cell samples in each microfluidic network. In the example, the dedicated inlets 128 are equipped with pumps to control the fluid flow and allow for automation. The fluid flow exiting the conduit through the opening in the substrate is coupled in the gap between the substrate and the lower enclosure 124b and exits the apparatus through a bend as shown. In this example, the lower enclosure 124b is further provided with a window 125 to provide light access to the underside of the microfluidic apparatus. Such an apparatus enables high-throughput screening of multiple test compounds to cell samples, and waste streams are efficiently coupled and removed, for example, as a single fluid outlet for the waste streams.

[0141] Figure 16 shows another configuration of the apparatus for high-throughput screening. In this apparatus, a single fluid inlet 128 controls the fluid flow through a filter into a common space above the apparatus's reservoir layer. Thus, the fluid flow can pass through all the conduits of the microfluidic network, as shown in Figure 14. In contrast to the apparatus in Figure 14, the apparatus in Figure 16 is provided with multiple sealing members 127 in the lower enclosure 124b, and each conduit has its own dedicated fluid outlet 129. Thus, separate cell samples in different microfluidic networks can be exposed to a common test fluid, ensuring that the outgoing fluid flows do not cross-contaminate each other.

[0142] Figure 17 shows an example of a microfluidic apparatus similar to Figure 15, but without the bends provided at the ends of the apparatus, and enclosures 124a and 124b form an hermetically sealed environment with the reservoir layer and substrate of the apparatus. Multiple sealing members 127 allow individual fluid flows to be introduced into individual conduits via dedicated fluid inlets 128, and the fluid flows leaving the individual conduits are combined and exit through a common fluid outlet 129.

[0143] Figures 15-17 show examples of devices having different numbers of fluid inlets and outlets for efficient multiplexing and reverse multiplexing of fluid flows. Figure 18 shows an alternative configuration of the device in which each microfluidic network of the device is completely isolated from other networks, and multiple sealing members 127 are provided to the upper enclosure 124a and the lower enclosure 124b. Each network is provided with a dedicated fluid inlet 128 and a dedicated fluid outlet 129, i.e., a conduit:inlet:outlet ratio of 1:1:1.

[0144] The apparatus shown in the figure is merely an example of how the microfluidic cell culture apparatus of this disclosure may be constructed, and it should be understood that other configurations are also possible. [Examples]

[0145] The microfluidic apparatus shown in Figures 1-3 was constructed using a substrate opening diameter of 0.5 mm and a cover opening diameter of 1 mm, and was used according to the following protocol.

[0146] <Protocol 1> Step 1 2.5 μm via the culture medium inlet. l Collagen 1 (a mixture of collagen 1 (5 mg / ml), hepes, and NaHCO3 in an 8:1:1 ratio, then diluted to 3 mg / ml with HBSS) was sown in the support plots. The gel is filled through the chamber and fixed within the chamber by an opening and a capillary pressure barrier. Incubate the gel for 10 minutes, then add HBSS to the perfusion compartment. Step 2 • Dissociated 5 in the culture medium × 10 6 A suspension of lung organoids at a concentration of / ml was seeded into a conduit in a volume of 0.5 μl, and a layer was formed on the gel surface. The plate was placed at a 90° angle and allowed to sink to one side of the conduit. Microscopic analysis of the above findings confirmed the presence of a meniscus at the substrate opening, indicating fixation, and also confirmed the absence of any signs of leakage into the substrate layer. The meniscus appeared black due to phase contrast caused by the air-liquid interface that refracts light. Cell seeding on the ECM gel was also visible.

[0147] Protocol 2 Step 1 2.5 μm via the culture medium inlet. l Collagen 1 was seeded in the support plot at a concentration of 3 mg / ml. The gel was filled through the chamber and fixed in place by an opening and a capillary pressure barrier. Incubate the gel for 10 minutes, then add HBSS to the perfusion compartment. Step 2 · 2 in the culture medium ×1 0 6 Add 0.3 μg of Caco2 cell suspension at a concentration of / ml. lThe seeds were sown into the xylem vessels at a certain volume, and a layer was formed on the gel surface. We tested two methods. Method 1: The plate was left flat for 30 minutes. It was observed that most cells did not adhere to the ECM, so the plate was then inverted 90° for 1 hour. Method 2: The plate was rotated 90 degrees for 1 hour, and then flipped over for another hour. • To prevent the plate from being broken, use an electronic pipette at the lowest dispensing speed to dispense 25 μL in two steps, totaling 50 μL. l The culture medium was added to the xylem.

[0148] <Culture medium removal - airlift> From the plate in Protocol 2, all culture medium was completely emptied using a suction device (with the suction tip in the corner) without touching the substrate opening. The plate was placed upside down in the flow cabinet. After a few minutes, it was observed that the conduit was airlifted but did not dry. 20μ l The culture medium was added to the inlet and outlet on all sides to fill the perfusion compartment. The plate was returned to the incubator and the culture medium was perfused through the ECM on the locker.

[0149] After 72 hours, all seeded and airlifted microfluidic networks remained intact. Three days after culturing, cells were stained with Actin-Red (Thermofisher Scientific #R37112) to confirm the presence of Caco2 cells in the peritubules around the ducts. Confocal microscopy was used to create images of the formed tubules, representative examples of which are shown in Figures 19A and 19B.

[0150] <Protocol 3> Step 1 - Hydrogel seeding 2.5 μm via the culture medium inlet. l Collagen I was seeded in the support plots at a concentration of 5 mg / ml. The gel was filled through the chamber and fixed in place by an opening and a capillary pressure barrier. Incubate the gel for 20 minutes, then add HBSS to the perfusion compartment. • The plates were stored at room temperature until use. Step 2 - Cell seeding · 20 in the culture medium × 10 6 A suspension of human bronchial epithelial cells (NHBE) at a concentration of / ml was seeded into ducts in a volume of 0.25 μl, and a layer was formed on the gel surface. 50 μl of culture medium was added to all perfusion channels, and the plate was left at a 90° angle for 2 hours to allow sedimentation to occur on one side of the ducts. The plate was rotated 180° and incubated on the other side for 2 hours. 50 μl of culture medium was added to the conduit chamber (at the lowest rate in the electron multichannel).

[0151] <Culture medium removal - airlift> • All culture medium was emptied from the plate using a suction device without touching the substrate opening. The plate was placed upside down in the flow cabinet for approximately 3 minutes to allow the chips to dry. • We checked under a microscope whether or not the airlift mechanism was established. 50μ l The culture medium was added to the inlet and outlet on all sides to fill the perfusion compartment.

[0152] The airlift remained stable over time. After 28 days of culture, the culture was color-coded by DNA. Confocal microscopy was used to create images of the formed tubes, showing the confluence cell layer with the hydrogel as the background. A representative example is shown in Figure 20.

[0153] The above description is intended to teach those skilled in the art how to carry out the present invention and is not intended to detail all modifications and variations that would become apparent from reading the description. However, all such modifications and variations are intended to fall within the scope of the present invention as defined by the following claims.

Claims

1. A microfluidic cell culture apparatus, wherein the microfluidic cell culture apparatus is It includes at least one microfluidic network, the microfluidic network is Substrate, microfluidic channels and cover, At least one perfusion compartment inside the microfluidic channel, The microfluidic channel includes at least one support compartment inside the microfluidic channel, The substrate and the cover each include an opening, thereby defining a conduit through which the microfluidic channel passes, and The conduit is in fluid contact with the at least one support section and, through the at least one support section, is in fluid contact with the at least one perfusion section. The microfluidic cell culture apparatus is configured to allow the flow of fluid through the conduit.

2. The microfluidic cell culture apparatus according to claim 1, further comprising a support scaffold in at least one support section to form a scaffold surface facing the conduit.

3. The microfluidic cell culture apparatus according to claim 2, further comprising cells that form a layer on the surface of the scaffold.

4. It further includes at least one enclosure that at least partially covers the outside of the substrate and / or the outside of the cover, All or part of the aforementioned at least one enclosure is removable, and / or The aforementioned at least one enclosure is Ports for fluid exchange, A tube for guiding the flow of fluids. Window for light access, A filter for exchanging fluids without contamination, and A curved path for exchanging gaseous fluids without contamination. A microfluidic cell culture apparatus according to any one of claims 1 to 3, comprising one or more of the above.

5. A method for creating a fluid-fluid interface in a microfluidic cell culture apparatus comprising a microfluidic network having a substrate, microfluidic channels and a cover, wherein the microfluidic channels have at least one perfusion compartment and at least one support compartment inside, the substrate and the cover each include openings that define conduits through the microfluidic channels, and the method is as follows: The steps include: introducing a support scaffold into at least one support section in order to form a scaffold surface facing the conduit; A step of introducing a culture medium containing cells into the conduit through the opening in the cover, while preventing the flow of the culture medium from the conduit through the opening in the substrate, The step of allowing the cells to form a layer on the surface of the scaffold, A method comprising the step of removing the culture medium from the duct in order to expose the cells.

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