NL2035770B1

NL2035770B1 - Titerplate

Info

Publication number: NL2035770B1
Application number: NL2035770A
Authority: NL
Inventors: Yannick Michel Nicolas Arnaud; Peter Burton Todd; Johannes Trietsch Sebastiaan; Vulto Paul
Original assignee: Mimetas B V
Priority date: 2023-09-08
Filing date: 2023-09-08
Publication date: 2025-03-14
Also published as: WO2025051969A1

Abstract

M A titerplate configured for unidirectional flow in a plurality of microfluidic networks disposed on the titerplate is described. The at least one microfluidic network comprises a microfluidic layer comprising a first flow channel and a second flow channel; and a reservoir layer disposed above the microfluidic layer and comprising first and second reservoirs, wherein the first reservoir and the second reservoir each have an access port to the first flow channel and an access port to the second flow channel so that the first flow channel and the second flow channel form a flow circuit with the first reservoir and the second reservoir; wherein the access ports in the first reservoir and the second reservoir are spaced apart so that, in use, tilting the titerplate at a first angle induces a volume of fluid in the first reservoirto flow from the first reservoir to the second reservoir primarily via the first flow channel, and adjusting the tilt so that the titerplate is titled at a second angle causes a volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir primarily via the second flow channel.

Description

TITERPLATE

FIELD OF THE INVENTION

[0001] The present invention relates to microfluidic titerplates, more particularly titerplates having a plurality of microfluidic networks through which unidirectional flow is achieved, and methods for causing unidirectional flow in a microfluidic titerplate.

BACKGROUND OF THE INVENTION

[0002] Microfluidic chips are typically perfused to induce fluid flow. In single chip setups this is typically done by means of pumps allowing active flow in a single direction. Microtiter plate microfluidics are typically not compatible with active pump-based perfusion, due to the parallel nature of the setup and associated complex usability and reliability of parallel pump setups. For this reason microtiter plate-based microfluidic chips are typically perfused by passive leveling.

[0003] However, a downside of microtiter plate microfluidics thus far is that passive leveling introduces bi-directional fluid flow, i.e. the reciprocal tilting of the microtiter plate leads to levelling in one direction, followed by reverse levelling into the other direction.

[0004] Literature provides some guidelines as to how unidirectional flow can be achieved through passive leveling:

[0005] US 2020/070165 A1 (165) describes uniflow by passive leveling between reservoirs in a single microfluidic chip device. However, ‘165 does not solve the problem of providing unidirectional flow in a microtiter plate.

[0006] KR 101803325 B1 (325) described a gravity induced unidirectional microfluidic chip, again by passive leveling, with the microfluidic channel in different layer to the reflow channel. However, ‘325 does not solve the problem of providing unidirectional flow in a microtiter plate.

[0007] WO 2023/001831 Ai (831) provides a concept of providing unidirectional flow alongside a semi permeable membrane in a circular motion.

However, ‘831 does not solve the problem of providing unidirectional flow in a microtiter plate.

SUMMARY OF THE INVENTION

[0008] T he present invention solves a crucial and critical issue in microfluidics that allowsunidirectional application of fluid flow in a multitude of microfluidic chips or networks embedded in a microtiter plate format by a combination of tilting and passive levellingbetween reservoirs. The invention relates to a device and method for achieving the same and provides a solution to the problem of lack of scalability, compatibility and automation of unidirectional flow microfluidics.

[0009] According to a first aspect of the invention there is provided a titerplate configured for unidirectional flow in a plurality of microfluidic networks disposed on the titerplate, at least one microfluidic network comprising: a microfluidic layer comprising a first flow channel and a second flow channel; and a reservoir Jayer disposed above the microfluidic layer and comprising first and second reservoirs, wherein the first reservoir and the second reservoir each have an access port to the first flow channel and an access port to the second tlow channel so that the first flow channel and the second flow channel form a flow circuit with the first reservoir and the second reservoir; wherein the access ports in the first reservoir and the second reservoir are spaced apart so that, in use, tilting the titerplate at a first angle induces a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir primarily via the first flow channel, and adjusting the tilt so that the titerplate is titled at a second angle causes a volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir primarily via the second flow channel.

[0010] According to a second aspect of the present invention there is provided a method for causing unidirectional flow in a microfluidic titerplate having a plurality of microfluidic networks, at least one microfluidic network comprising: a microfluidic layer comprising a first flow channel and a second flow channel; and a reservoir layer comprising first and second reservoirs, wherein the first reservoir and the second reservoir each have an access port to the first flow channel and an access port to the second flow channel so that the first flow channel and the second flow channel form a flow circuit with the first reservoir and the second reservoir; wherein the method comprises: tilting the titerplate at a first angle to induce a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir predominantly via the first flow channel; and adjusting the tilt so that the titerplate is titled at a second angle, causing the volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir predominantly via the second flow channel. foou] Other preferred embodiments are defined in the description and dependent claims which follow.

BRIEF DESCRIPTION OF DRAWINGS

[oo12] The present invention will now be described by way of example only, with reference to the Figures.

[0013] Figure 1 shows a microfluidic titerplate according to the present invention. The embodiment depicted includes 64 microfluidic networks on the microfluidic titerplate.

[0014] Figure 2(a) shows a plan view of an exemplary microfluidic network of the microfluidic titerplate of Figure 1.

[0015] Figures 2(b) and 2(c) show in close up (cross-sectional views) the exemplary microfluidic network of Figure 2(a).

[0016] Figure 3(a) and Figure 3(b) show cross-sectional (Figure 3(a)) and plan (Figure 3(b)) views of the microfluidic network of Figure 2(a) at a first tilting angle, and the effect that the tilting has on liquid contained within the microfluidic network.

[0017] Figure 3(c) and Figure 3(d) show cross-sectional (Figure 3(c)) and plan (Figure 3(d)) views of the microfluidic network of Figure 2(a) at a second tilting angle, and the effect that the tilting has on liquid contained within the microfluidic network.

[0018] Figure 4 shows the microfluidic network of Figure 2 with the particular layout of access ports.

[0019] Figure 5 shows an alternative configuration for a microfluidic titerplate according to the present invention. The embodiment depicted includes 64 microfluidic networks. [0020] Figure 6 shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 5.

[0021] Figure 7(a) shows a microfluidic network according to Figure 6 containing a liquid, and the liquid distribution in two end states of a two-state tilting protocol to achieve unidirectional flow.

[0022] Figure 7(b) shows cross-sectional views and a plan view of the microfluidic network of Figure 7(a) during the liquid levelling transition from the first state to the second state.

[0023] Figure 7(c) shows cross-sectional views of the microfluidic network of

Figure 7(a) during the reverse liquid levelling transition from the second state to the first state.

[0024] Figure 7(d) shows a microfluidic network according to Figure 6 and having a liquid therein being subjected to a four-state tilting protocol to achieve unidirectional flow.

[0025] Figure 8 shows an alternative microfluidic titerplate according to the present invention. The embodiment depicted includes 48 microfluidic networks on the microfluidic titerplate.

[0026] Figure 9 shows in close up (plan view) an exemplary microfluidic 5 network of the microfluidic titerplate of Figure 8.

[0027] Figure 10 shows an alternative configuration for a microfluidic titerplate according to the present invention. The embodiment depicted includes 20 microfluidic networks. [0028] Figure 11 shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 10.

[0029] Figure 12 shows an alternative configuration for a microfluidic titerplate according to the present invention. The embodiment depicted includes 32 microfluidic networks. [0030] Figure 13 shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 12.

[0031] Figure 14 shows an alternative configuration for a microfluidic titerplate according to the present invention. The embodiment depicted includes 48 microfluidic networks on the microfluidic titerplate.

[0032] Figure 15 shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 14.

[0033] Figure 16 shows an alternative configuration for a microfluidic titerplate according to the present invention. The embodiment depicted includes 24 microfluidic networks on the microfluidic titerplate.

[0034] Figure 17 shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 17.

[0035] Figure 18 shows an alternative configuration for a microfluidic titerplate according to the present invention. The embodiment depicted includes 32 microfluidic networks on the microfluidic titerplate.

[0036] Figure 19 shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 18.

0

[0037] Figure 20 shows an alternative configuration for a microfluidic titerplate according to the present invention. The embodiment depicted includes 32 microfluidic networks on the microfluidic titerplate.

[0038] Figure 21(a) and Figure 21(b) show a microfluidic network of the configuration of Figure 20, with arrows indicating the direction of fluid flow when the microfluidic network is subjected to a method as described herein.

[0039] Figure 22 shows an alternative configuration for a microfluidic titerplate according to the present invention. The embodiment depicted includes 48 microfluidic networks on the microfluidic titerplate.

[0040] Figure 23 shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 22.

[0041] Figure 24(a) shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 22. The microfluidic network contains a vascular network, and is depicted at a first tilting angle, with arrows indicating the direction of fluid flow.

[0042] Figure 24(b) shows in close up (plan view) the vascular network region of the embodiment of Figure 24(a).

[0043] Figure 24(c) shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 22. The microfluidic network contains a vascular network, and is depicted at a second tilting angle, with arrows indicating the direction of fluid flow.

[0044] Figure 24(d) shows in close up (plan view) the vascular network region of the embodiment of Figure 24(c).

[0045] Figure 25 shows an alternative configuration for a microfluidic titerplate according to the present invention. The embodiment depicted includes 40 microfluidic networks on the microfluidic titerplate.

[0046] Figure 26(a) shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 25.

[0047] Figure 26(b) shows in close up (plan view) an exemplary microfluidic network of the configuration of Figure 25, containing a vascular bed.

[0048] Figure 27(a) shows fluid flow through the microfluidic network of

Figure 26(a) at a first tilting angle, while Figure 27(b) shows fluid flow through the same microfluidic network at a second (inverse) tilting angle.

[0049] Figure 27(c) shows fluid flow through the microfluidic network of

Figure 26(b) at a first tilting angle, while Figure 27(d) shows fluid flow through the same microfluidic network at a second (inverse) tilting angle.

[0050] Figure 28 shows images from experiments on human brain microvascular endothelial cells (HBMEC) after eight days of culture and two days in unidirectional flow using a unidirectional flow titerplate according to the present invention compared with a traditional bidirectional flow experiment.

[0051] Figure 29 and Figures 30(a) and 30(b) show the results of unidirectional flow on fibronectin deposit in human coronary artery endothelial cells (HCAEC) after six days of culture and two days in unidirectional flow using a unidirectional flow titerplate according to the present invention compared with a traditional bidirectional flow experiment.

[0052] Figure 31 and Figures 32(a) and 32(b) show the results of unidirectional flow on fibronectin deposit in human coronary artery endothelial cells (HCAEC) after six days of culture and two days in unidirectional flow using a unidirectional flow titerplate according to the present invention compared with a traditional bidirectional flow experiment.

[0053] Figure 33 shows unidirectional flow of peripheral blood mononuclear cells through a vascular bed using a unidirectional flow titerplate according to the present invention.

[0054] Figure 34(a) shows the cellular morphology of a vascular bed formed in a unidirectional flow titerplate according to the present invention, without unidirectional flow.

[0055] Figure 34(b) shows the cellular morphology of a vascular bed formed in a unidirectional flow titerplate according to the present invention, with unidirectional flow.

[0056] With specific reference to the Figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0057] DEFINITIONS

[0058] Various terms relating to the devices, and methods, of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0059] As used herein, the “a,” “an,” and “the” singular forms also include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0060] As used herein, “substantially”, “about” and “approximately” when referring to a measurable value such as an amount, a temporal duration, and the like, are meant to encompass variations of £20% or £10%, more preferably £5%, even more preferably +1%, and still more preferably £0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0061] As used herein, “comprising” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0062] As used herein, "exemplary" means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein.

[0063] As used herein, the term “microfluidic network” or “microfluidic chip” refers to one or more channels (which may be termed flow channels or microfluidic channels) on or through a layer of material that is covered by a top- substrate or cover, with at least one of the dimensions of length, width or height being in the Jow (for example less than 5 mm or Jess than 2 mm) or sub-millimeter range. It will be understood that the term encompasses channels which are linear channels, as well as channels which are branched, or have bends or corners within their path. A microfluidic network typically comprises at least one access port, for example an inlet for administering a volume of liquid, but may comprise multiple access ports, for example multiple inlets for administering volumes of liquid to different regions of the microfluidic network and one or more outlets located downstream from an inlet. The volume enclosed by a microfluidic network is typically in the microliter or sub-microliter range. A microfluidic networl typically comprises a base, which may be the top surface of an underlying material, at least two side walls, and a ceiling, which may be the lower surface of a top substrate overlying the microfluidic network, with any configuration of inlets, outlets and/or vents as required. The base, side walls and ceiling may each be referred to as an inner surface of the microfluidic network, and collectively may be referred to as the inner surfaces. In some examples, the microfluidic network may have a circular or semi-circular cross-section, which would then be considered to have one or two inner surfaces respectively.

[0064] As used herein, the term “unidirectional flow” refers to flow through a microfluidic network having at least two reservoirs and two underlying flow channels, connected so as to form a flow circuit. One of the flow channels may be referred to or considered as an “active” channel, while the other of the flow channels may be considered as a “reflow” channel. As used herein, the active channel serves as a region into which cells can be introduced from a first reservoir and subsequently cultured, such as for the formation of cellular tubules such as blood vessels, and through which a liquid such as culture media or test solutions can be perfused. The reflow channel then returns the liquid to the first reservoir to be re-perfused through the active channel.

[0065] As used herein, the term “capillary pressure barrier” refers to features of an apparatus that keep a liquid-air meniscus pinned at a certain position by capillary forces. A capillary pressure barrier can be considered to divide a microfluidic network having a volume Vo into two regions or sub-volumes V1 and

V2 into which different fluids can be introduced. Put differently, a capillary pressure barrier generally defines a boundary between first and second regions of the microfluidic network. It will be understood that while the capillary pressure barrier generally defines a boundary between regions of a microfluidic network, a resultant pinned meniscus of a liquid in one region may not be pinned at the exact location of the capillary pressure barrier, and may stretch or bulge beyond the capillary pressure barrier into an adjacent region while still being pinned. For example, a liquid meniscus may be convex in shape, and be pinned by a capillary pressure barrier, with the convex liquid front extending beyond the footprint of the capillary pressure barrier. The liquid meniscus may also be concave, with the solvent front pinned by the capillary pressure barrier and stretching beyond the footprint of the capillary pressure barrier on the surface of the microfluidic network opposite the capillary pressure barrier.

[0066] As used herein, the term “endothelial cells” refers to cells of endothelial origin, or cells that are differentiated into a state in which they express markers identifying the cell as an endothelial cell.

[0067] As used herein, the term “epithelial cells” refers to cells of epithelial origin, or cells that are differentiated into a state in which they express markers identifying the cell as an epithelial cell.

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[0068] As used herein, the term “biological tissue” refers to a collection of identical, similar or different types of functionally interconnected cells that are to be cultured and/or assayed in the methods described herein. The cells may be a cell aggregate, or a particular tissue sample from a patient. For example, the term “biological tissue” encompasses organoids, tissue biopsies, tumor tissue, resected tissue material, spheroids and embryonic bodies.

[0069] As used herein, the term “cell aggregate” refers to a 3D cluster of cells in contrast with surface attached cells that typically grow in monolayers. 3D clusters of cells are typically associated with a more in vivo-like situation. In contrast, surface attached cells may be strongly influenced by the properties of the substrate and may undergo de- differentiation or undergo transition to other cell types.

[0070] As used herein, the term “organoid” refers to a miniature form of a tissue that is generated in vitro and exhibits endogenous three-dimensional organ architecture.

[0071] As used herein, the term “lumened cellular component” refers to a biological tissue (i.e. constituted of cells) having a lumen, for example a microvessel having apical and basal surfaces.

[0072] As used herein, the term “co-culture” refers to two or more different cell types being cultured in a microfluidic network described herein. The different cell types can be cultured in the same region of the network (e.g., first region or second region) and/or in different regions (e.g., one cell type in a first region and another cell type in a second region).

[0073] For example, a network as described herein may have endothelial cells grown as a tubule having an open lumen in a first region or flow channel, organ-specific (parenchymal) cells in a second region or cell culture chamber, separated by a thin layer of gel. In some examples, a network may comprise at least one lumened gel structure lined with endothelium in a flow channel and tissue specific cells in a second region or cell culture chamber.

[0074] TITERPLATES

[0075] A titerplate in accordance with the invention includes a plurality of microfluidic networks disposed on the titerplate. In some examples, the titerplate comprises at least 8 microfluidic networks, more preferably at least 16 microfluidic networks, more preferably at least 20 microfluidic networks, more preferably at least 32 microfluidic networks, more preferably at Jeast 40 microfluidic networks, more preferably at least 64 microfluidic networks. In some examples, the plurality of microfluidic networks disposed on the titerplate are identical microfluidic networks. In some examples, the plurality of microfluidic networks disposed on the titerplate are not identical, that is, at least one microfluidic network may have a different layout to at least one other microfluidic network.

[0076] The titerplate may conform to the ANSI SLAS Microplate

Standards. Specifically, the titerplate may have a footprint in accordance with

ANSI SLAS 1-2004 (R2012); a height dimension in accordance with ANSI SLAS 2- 2004 (R2012); a bottom outside flange dimension in accordance with ANSI SLAS 3-2004 (R2012); well positions in accordance with ANSI SLAS 4-2004 (R2012) and/or a well bottom elevation in accordance with ANSI SLAS 6-2012. That is, in some examples, the titerplate may correspond to a 32 well plate, a 48 well plate, a 96 well plate, or a 384 well plate, for example.

[0077] The microfluidic network of the titerplate generally comprises a reservoir layer overlaving a microfluidic layer, which may be fabricated on a base, and can be fabricated in a variety of manners.

[0078] The base, also referred to herein as the base layer, or bottom substrate, is preferably formed from a substantially rigid material, such as glass or plastic, and serves to provide a supporting surface for the rest of the microfluidic network. A typical method of fabrication of a microfluidic channel is to cast a mouldable material such as polydimethylsiloxane onto a mould, so imprinting the microfluidic channel into the silicon rubber material thereby forming a microfluidic layer. The rubber material with the channel embedded is subsequently placed on a base layer of glass or of the same material to thus create a seal. Alternatively, the channel structure could be etched in a material such as glass or silicon, followed by bonding to a top or bottom substrate (also referred to herein as a cover layer and base layer). Injection moulding or embossing of plastics followed by bonding is another manner to fabricate the microfluidic network. Yet another technique for fabricating the microfluidic layer is by photo lithographically patterning the microfluidic layer in a photopatternable polymer, such as SU-8 or various other dry film or liquid photoresists, followed by a bonding step. When referred to bonding it is meant the closure of a microfluidic channel by a cover or base. Bonding techniques include anodic bonding, covalent bonding, solvent bonding, adhesive bonding, and thermal bonding amongst others.

[0079] In some examples, at least one or each microfluidic network of the plurality of microfluidic networks has a footprint corresponding to a plurality of, for example two or more, well positions of the titerplate. In some examples, the or each microfluidic network has a footprint corresponding to three or more well positions of the titerplate, for example four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more or ten or more well positions of the titerplate. As an example, if a microfluidic network has two reservoirs, each corresponding to three well positions that have been fused together (meaning six well positions in total), then it would be understood that a 384 well microtiter plate would have 64 wells (384 divided by (2x3)). In examples in which the microfluidic network has a footprint corresponding to three or more well positions, the three or more well positions may be in a linear arrangement, or a non-linear arrangement. In one example, the plurality of microfluidic networks are fluidically disconnected from each other; in other words, each microfluidic network operates independently of any other microfluidic network present on the microfluidic titerplate.

[0080] At least one or each microfluidic network of the plurality of microfluidic networks comprises a reservoir layer. In some examples, the reservoir layer comprises a plurality of reservoirs, with at least a first reservoir and/or a second reservoir of a microfluidic network each positioned so as to correspond to the position of at least one well of an ANSI/SLAS microplate. In examples in which a reservoir corresponds to more than one well position, for example two or more, for example three or more well positions of a standard titerplate, it will be understood that the reservoir has the same footprint as those well positions of a standard titerplate, but without the division of that footprint into separate well positions. That is, that reservoir is dimensioned so as to correspond to the position and volume of a plurality of wells of an ANSI/SLAS microplate.

[0081] The reservoir Jayer may contain all reservoirs for each microfluidic network of the plurality of microfluidic networks. At Jeast one microfluidic network comprises first and second reservoirs in the reservoir layer. Each reservoir, for example at least the first and second reservoirs of the at least one microfluidic network comprises walls extending downward to a base, and access ports within each reservoir are provided in the base.

[0082] The access ports are spaced apart so that, in use, tilting the titerplate at a first angle induces a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir primarily via the first flow channel, and adjusting the tilt so that the titerplate is titled at a second angle causing a volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir primarily via the second flow channel.

[0083] In some examples, in at least one of the first and second reservoirs, the access ports are irregularly spaced relative to each other and/or relative to corresponding well positions or central portions of the wells of an ANSI/SLAS microplate. For example, in at least one of the first and second reservoirs, at least one access port, referred to for ease of explanation as a first access port, is spaced in an offset position relative to a position, or a central portion, of a corresponding well of an ANSI/SLAS microplate. The first access port which is spaced in an offset position may be a peripheral or distal access port relative to a centre point of the microfluidic network. The first access port may be spaced from an end wall and/or the intersections of the end wall with the side walls such that it is located toward a central portion of the reservoir relative to a central position of a corresponding well of an ANSI/SLAS microplate. That is to say, the first access port may be located in the reservoir such that upon inclination at a tilt angle the distance from the first access port and an end wall (and the intersection of the end wall with the adjacent side walls) is sufficient to avoid contact between fluid in the first access port and fluid wicked by the end wall. Put differently, the position and dimension of the first access port may be such that at a first tilting angle the liquid connection between the fluid in the first access port and the fluid wicked by the end wall (or its corners) is broken.

[0084] This asymmetric spacing of the first access port relative to the end wall and/or the internal corners that it forms with the side walls provides for an in use disconnect or separation between liquid which is present in the underlying flow channel and the liquid in the reservoir when the titerplate is tilted as described above. In this orientation, liquid in the flow channel underlying the first access port will form a meniscus at the access port (by being pinned by the access port) while, or as, a majority of the liquid in the reservoir flows by gravity away from the first access port to the other (for example second) access port in the reservoir. Unwanted back-flow through the flow channel connected to the first access port can be prevented by way of this disconnect. In some examples, the at least one access port is spaced from the end wall by at least 1 mm, for example, at least 1.25mm, for example at least 1.5 mm, for example at least 2 mm, for example at least 2.5 mm, for example at least 3 mm. In examples in which a reservoir has a footprint corresponding to three or more well positions, the three or more well positions may be in a linear arrangement, or a non-linear arrangement.

[0085] The reservoir layer is disposed above a microfluidic layer of the microfluidic network. The microfluidic layer comprises a first flow channel and a second flow channel, each of which is fluidly connected to the first reservoir and the second reservoir by an access port at each end of each of the first flow channel the second flow channel.

[0086] The microfluidic layer comprises a first flow channel and a second flow channel but is not limited to two flow channels. That is, in some examples, the microfluidic layer of a microfluidic network may comprise more than two flow channels, for example three, four, five or more flow channels. In some examples, the microfluidic layer comprises a first flow channel and a second flow channel which together with a first reservoir and a second reservoir forms a first flow circuit, and further comprises a third flow channel and a fourth flow channel which together with a third reservoir and a fourth reservoir form a second flow circuit. References herein to “the flow channels” may be to “the first flow channel and the second flow channel” but may also be to any number of additional flow channels that may be present in the microfluidic network.

[0087] In some examples, the third flow channel connects at one end to an access port in a third reservoir and at another end to an access port in a fourth reservoir and the fourth flow channel connects at one end to an access port in the third reservoir and at another end to an access port in the fourth reservoir, with the microfluidic network having a plane of reflectional symmetry perpendicular to the plane of the microfluidic network and extending along the lane such that access ports of the first and third flow channels mirror each other and the access ports of the second and fourth flow channels mirror each other.

[0088] Alternatively, the microfluidic network may be configured so that the third flow channel connects at one end to an access port in a third reservoir and at another end to an access port in a fourth reservoir and the fourth flow channel connects at one end to an access port in the third reservoir and at another end to an access port in the fourth reservoir with a centre of symmetry of the microfluidic network being located in the lane such that the access ports of the fourth flow channel are the inversion of the access ports of the first flow channel through the centre of symmetry and the access ports of the third flow channel are the inversion of the access ports of the second flow channel through the centre of symmetry.

[0089] In some examples, each or any flow channel as described herein is independently a linear, i.e. straight or nearly straight, flow channel or a non-linear flow channel. In some examples, the flow channels, for example the first flow channel] and the second flow channel, are configured so that the access ports for each flow channel are each located at the centre of a well position and are aligned.

In other examples, the flow channels, for example the first flow channel and the second flow channel, are configured so that at least one access port is not aligned with an ANSI/SLAS microplate well or the centre position of such a well. In some examples, the first flow channel and the second flow channel are of the same or different lengths, and may contain one or more bends or directional changes along a length thereof to accommodate the offset of an access port. In some examples, one of the first flow channel and second flow channel can be operated as an “active” channel, in which cells are introduced and cultured, while the other of the first flow channel and second flow channel operates as a “reflow” channel through which liquid can return after it has passed through the active channel, as will be described in more detail below in connection with the methods of the present invention.

[0090] In some examples, the first flow channel has a greater length than a length of the second flow channel, with the access ports at each end of the first flow channel being disposed at a periphery of the corresponding reservoir or at a periphery of the microfluidic network, and the access ports at each end of the second flow channel being disposed at an inner or central portion of the corresponding reservoir or at an inner or central portion of the microfluidic network, That is, the first flow channel may connect to access ports that are most distal from one another and the second flow channel may connect to access points that are most proximal to one another.

[0001] In some examples, a microfluidic network may also include a lane configured to receive a gel, in direct fluid connection with at least one of the first flow channel and second flow channel. The term “direct” fluid connection, as used herein, means that at least a part of the lane is contiguous with a part of the flow channel rather than being generally part of the entire flow circuit in series with the tlow channel. As the lane is configured to receive a gel, it may also be referred to as a gel lane, or gel region. The lane may be configured to receive a gel by being fluidly connected to a further reservoir by a further access port. In some examples, the lane is provided with an access port at each end thereof, each access port being located in a different reservoir, those reservoirs being different to the reservoirs of the first and second flow circuits. As a result of the lane being in direct fluid connection with at least one of the first flow channel and second flow channel, through which a perfusion medium can flow, nutrients can be provided to a biological tissue within or on the gel, while endothelial and/or epithelial cells can also or alternatively be introduced into the flow channels for blood vessel formation, or smooth muscle formation, through the gel, for example.

[0092] The lane may be present in the microfluidic layer of the microfluidic network and may be defined in part by one or more capillary pressure barriers within the microfluidic network, each generally defining a boundary between a flow channel and the lane. For example, the Jane may comprise a capillary pressure barrier defining a boundary between the lane and the first flow channel.

[0003] The function and patterning of capillary pressure barriers have been previously described, for example in WO 2014/038943 Al. In the context of the present disclosure, the capillary pressure barrier serves to confine liquid gel precursor to the lane or region into which it is introduced, without the liquid precursor spreading out to at Jeast the first flow channel. After a gel has formed trom the gel precursor, neighbouring flow channels can be perfused with media, for example media containing cells. As will become apparent from the exemplary embodiments described hereinafter, the capillary pressure barrier is not to be understood as a wall (or a cavity which can for example be tilled with a liquid), but instead consists of or comprises a structure which ensures that such a liquid does not spread due to the surface tension. This concept is referred to as meniscus pinning. As such, stable confinement of a liquid to a region (such as the gel lane or gel region) of the microfluidic network can be achieved. In one example, the capillary pressure barrier may be referred to as a confining phaseguide, which is configured to not be overflown during normal use of the cell culture device. The nature of the confinement of a liquid is described herein in connection with the description of the methods of the present invention.

[0004] In one example, the capillary pressure barrier is provided on an internal surface of the microfluidic network and comprises a ridge, groove, or line of material that has an increased water-air contact angle with respect to the internal surface of the microfluidic network. In one example, the capillary pressure barrier comprises or consists of a rim or ridge of material protruding from an internal surface of the microfluidic network; or a groove in an internal surface of the microfluidic network. In order to provide a good barrier, the internal angle formed by a sidewall of the rim or ridge and the top of the rim or ridge is preferably less than 110°, for example about 90°, in some examples less than 90°.

The same counts for the angle between the sidewall of the ridge and the internal surface of the microfluidic network on which the capillary pressure barrier is located. Similar requirements are placed on a capillary pressure barrier formed as a groove.

[0095] An alternative form of capillary pressure barrier is a region of material of different wettability to an internal surface of the microfluidic network, which acts as a spreading stop due to capillary force/surface tension. As a result, the liquid is prevented from flowing beyond the capillary pressure barrier and enables the formation of stably confined volumes in a region of the network. In one example, the internal surfaces of the microfluidic network comprise a hydrophilic material and the capillary pressure barrier is a region of hydrophobic, or less hydrophilic material. In one example, the internal surfaces of the microfluidic network comprise a hydrophobic material and the capillary pressure barrier is a region of hydrophilic, or less hydrophobic material.

[0006] Thus, in some examples, the capillary pressure barrier is selected from arim or ridge, a groove, a hole, or a hydrophobic line of material or combinations thereof. In another embodiment capillary pressure barriers can be created by pillars at selected intervals, the arrangement of which defines a first region or area that is to be occupied by a gel, for example a cell culture chamber. In one example, the pillars extend the full height of the microfluidic network.

[0097] In one example, the capillary pressure barrier is a substantially linear capillary pressure barrier which spans the complete width or length of a flow channel and intersects on each end with sidewalls of the microfluidic network. The capillary pressure barrier may span the intersection of a flow channel with the gel lane and terminate on each end with internal walls of the microfluidic network.

[0098] In another example, the capillary pressure barrier is not linear, but comprises one or more bends or arcuate portions. For instance, the capillary pressure barrier may comprise consecutive angled bends or arcuate portions such that a meandering or even a right-angled shape is created. This way the path of a fluid being guided along the capillary pressure barrier is extended with respect to the path a fluid would follow along a linear capillary pressure barrier. The advantage of having a non-linear capillary pressure barrier is that a lumen created in a fluid aligned along such a capillary pressure barrier can have a non-linear shape, e.g. mimicking the crypt-villi structure of the small intestine, and/or that the length of a non-linear lumen is extended with respect to its linear counterpart.

[0099] The intersection of the capillary pressure barrier with a sidewall or more than one sidewall of the flow channel may have an angle on the downstream side of the capillary pressure barrier with respect to the envisioned filling direction of a first fluid that is larger than 70°, more preferably around 90°, more preferably larger than 90°. This angle is preferably as large as possible in order to provide a good barrier, as described in WO 2014/038943.

[0100] In some examples, the microfluidic network of the apparatus is provided with a second capillary pressure barrier, the form and function of which is substantially as described above. For the avoidance of doubt, references to “a capillary pressure barrier” are to be understood as references to “the first capillary pressure barrier” when a second capillary pressure barrier is present in the microfluidic network. [o101] In some examples, the first capillary pressure barrier defines a boundary between the gel lane and the first flow channel and the second capillary pressure barrier defines a boundary between the gel lane and the second flow channel. The gel lane may be in fluid connection with the first flow channel and the second flow channel and provide a potential flow path from the first flow channel to the second flow channel. A microfluidic network configured in this manner is capable of achieving unidirectional flow through any biological tissue, such as a vascular network extending from the first flow channel through a gel contained within the lane to the second flow channel. Since very few, if any, in vivo environments have bidirectional flow, it is particularly desirable to eliminate this from any in vitro systems. In other examples, the lane may be in direct fluid connection with the first flow channel but not the second flow channel.

[01032] In some examples, the second capillary pressure barrier defines a boundary between a gel lane and a third flow channel. The third flow channel may be fluidly connected to a third reservoir. The third flow channel may be fluidly connected to two other reservoirs of the reservoir layer, for example a third reservoir and a fourth reservoir. The third flow channel may be fluidly connected to a third reservoir and a fourth reservoir, which together with a fourth flow channel form an independent flow circuit. In some examples, a first capillary pressure barrier is provided at a position generally defining a boundary between the gel Jane and a first flow channel, while a second capillary pressure barrier is provided at a position generally defining a boundary between the gel lane and a different flow channel, for example the second flow channel or a third flow channel, so that a liquid gel precursor introduced into the gel lane can align itself along the first capillary pressure barrier and the second capillary pressure barrier, thereby forming a gel structure with surfaces facing the first flow channel and the different flow channel.

[0103] It will be understood that multiple capillary pressure barriers can be used to create gel structures within increasingly complex microfluidic networks to recreate an in vivo environment.

[0104] For example, the microfluidic network may comprise a first gel lane in direct fluid connection with a first flow channel, and a second lane or region configured to receive a gel. The second lane or gel region may be in direct fluid connection with the second flow channel and comprise a capillary pressure barrier defining a boundary between the second gel region and the second flow channel.

Having two separate gel regions, which are both in fluid communication with a single flow circuit enables different cell types, for example two different organoids, to be cultured in a manner that allows substance exchange or mass transport via the unidirectional flow through the single flow circuit. These substances may include metabolites, cytokines, chemokines or migrating cells including stem cells, immune cells or cancer cells.

[0105] In other examples, the microfluidic network may comprise a gel region in direct fluid connection with a first flow channel at a first location, and in direct fluid connection with the first flow channel at a second location downstream from the first location. In particular, the microfluidic network may comprise a gel region comprising a first capillary pressure barrier defining a first boundary with the first flow channel, and a second capillary pressure barrier defining a boundary with the first flow channel at a second location downstream from the first location so that opposite surfaces of a gel within the gel region face the same flow channel. In such a system, the gel region can be filled with extracellular matrix, endothelial cells can be introduced into the first flow channel and caused to form a vascular bed extending through the gel from the first location to the second location.

[01006] METHODS

[0107] The present invention also relates to a method for causing unidirectional flow in a microfluidic titerplate having a plurality of microfluidic networks, at least one microfluidic network comprising: a microfluidic layer comprising a first flow channel and a second flow channel; and a reservoir layer comprising first and second reservoirs, wherein the first reservoir and the second reservoir each have an access port to the first flow channel and an access port to the second flow channel so that the first flow channel and the second flow channel form a flow circuit with the first reservoir and the second reservoir; wherein the method comprises:

tilting the titerplate at a first angle to induce a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir via the first flow channel; and adjusting the tilt so that the titerplate is titled at a second angle, causing the volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir via the second flow channel.

[0108] The titerplate used in the method may be as defined herein.

[0109] As used herein, tilting of the titerplate, i.e. the tilt angle of the titerplate is with reference to the horizontal plane and can be alternatively described as a rotation over or about an axis, for example the x and y axis (Rx and

Ry respectively), both of which are to be considered within the context of the present disclosure as being in the horizontal plane unless stated otherwise. In a typical setup, the v-axis corresponds to or is parallel to a longitudinal axis of the titerplate and the x axis corresponds to or is parallel to a lateral axis of the titerplate.

[0110] At least one of the first and second tilting angles may correspond to a rotation between 1 and 90 degrees about the longitudinal axis of the titerplate relative to the horizontal plane, or a rotation between 1 and 90 degrees about a lateral axis of the titerplate relative to the horizontal plane. In some examples, the second angle is the inverse angle of the first angle, i.e. is the inverse rotation about the same axis. For example, if tilting the titerplate at a first angle comprises rotating the titerplate 45° about the longitudinal axis relative to the horizontal plane, then tilting the titerplate at a second angle comprises rotating about the longitudinal axis -45° from the horizontal plane. As will be described below with reference to the Figures, in this single axis or two-state method, whether or not liquid flows through the first flow channel or the second flow channel can be selected based on a tilting of the titerplate at the first angle (which will induce tlow through the first flow channel) or at the second angle (which will induce flow through the second flow channel), resulting in unidirectional flow through the flow circuit.

[o111] Unidirectional flow can be enhanced by offsetting the position of at least one access port in at least one of the first and second reservoirs, as described above. Spacing the access port from an end wall of the reservoir, and from the corners formed by the end wall and the adjacent side walls, minimizes or completely excludes any backflow through the access port to the underlying flow channel when the titerplate is rotated about an axis of rotation. During the tilting or rotating process, even though the majority of the liquid will flow by gravity toward the opposite end of the reservoir, some liquid will at least initially be wicked by capillary forces toward the end wall corners from which the access port is spaced. At the same time, there will be liquid in the underlying flow channel, with the meniscus of that liquid being pinned by the access port (here, functioning as a capillary pressure barrier). The spacing of the access port as described above ensures that the meniscus of the liquid from the underlying flow channel cannot coalesce with the liquid wicked toward the corner(s), thus ensuring minimal back- flow, if any, through the access port. A 384 well plate grid format is particularly useful, as in comparison to a 96 well plate grid it provides for a larger number of microfluidic networks, while the dimensions of wells are shaped such that this disconnect between corner fluid and access port upon tilting can still be achieved. This is less feasible in a 1536 well grid format, for example.

[0112] In some examples, the degree of rotation corresponding to the first tilt angle may be from 1 to 90°, for example up to 80°, for example up to 70°, for example up to 60°, for example up to 50°, for example up to 40°, for example up to 30°, for example about 20° from the horizontal plane. In some examples, the degree of rotation corresponding to the second tilt angle may be from -1 to -90°, for example up to -80°, for example up to -70°, for example up to -60°, for example up to -50°, for example up to -40°, for example up to -30°, for example about -20° from the horizontal plane.

[013] In some examples the method may further comprise: after tilting the titerplate at the first angle as described above, tilting the titerplate at a third angle by rotating the titerplate between 1 and 90 degrees about a second axis of rotation which is orthogonal to the first axis of rotation but still in the horizontal plane; and/or after tilting the titerplate at the second angle as described above, tilting the titerplate at a fourth angle by rotating the titerplate between 1 and 90 degrees about the second axis of rotation which is orthogonal to the second angle but still in the horizontal plane. For example, the method may comprise: firstly rotating the titerplate between 1 and 90 degrees about a longitudinal axis of rotation relative to the horizontal plane, rotating the titerplate between 1 and 90 degrees about a lateral axis of rotation relative to the horizontal plane; inverting the angle of rotation about the longitudinal axis of rotation and then inverting the angle of rotation about the lateral axis of rotation. This four-step process can then be repeated. Thus, the titerplate is subjected to a two-axis rotational operation, taking in four states or orientations of the titerplate. When combined with the access port spacing as described herein, effective unidirectional flow can be achieved. [o114] Alternatively, instead of tilting the titerplate by sequentially rotating about a longitudinal axis and a lateral axis of the titerplate relative to the horizontal plane, the titerplate may be tilted along a diagonal axis of the plate, that is a simultaneous rotation over or about both x- and y- axes. The diagonal axis may be from one corner of the titerplate to the diagonally opposite corner of the titerplate. Thus, in this scenario, adjusting the tilt from a first tilt angle to the second tilt angle (which is a simultaneous rotation about the x- and y-axes relative to the horizontal plane) comprises inversely rotating the titerplate about the x- and y-axes simultaneously relative to the horizontal plane.

[0115] In some examples, and instead of consecutively or simultaneously rotating the titerplate about these axes of rotation at these angles, the method may comprise continuously varying the tilt of the titerplate in a three-dimensional gyratory motion, or rotating the tilted plate about the x- and y-axes and about the z-axis perpendicular or normal to the horizontal plane or the plane of the microtiter plate. That is, if the y-axis corresponds to or is parallel to a longitudinal axis of the titerplate and the x axis corresponds to or is parallel to a lateral axis of the titerplate, with the x- and y-axes being in the horizontal plane, the z-axis extends above and below the horizontal plane.

[0116] In examples in which at least one microfluidic networl of the titerplate comprises a lane in fluid connection with at least the first flow channel, the method may further comprise introducing a gel, or precursor thereof and optionally containing cells, into the lane, and — in the case of a gel precursor — allowing a gel to form in the Jane. In some examples, the cells incorporated into the gel may be include multiple cell types, such as one or more of: endothelial cells for forming blood vessels, fibroblasts and immune cells. Once formed, cells may be cultured in or on the gel contained in the lane and additional cells may be cultured in the first flow channel. The additional cells may be the same as the cells within the gel (for example endothelial cells), or may be different to the cells within the gel, or may be a mixture of different cells.

[0117] As described above, some titerplates according to the invention include microfluidic networks comprising a first capillary pressure barrier defining a boundary between a gel lane/region and the first flow channel and a second capillary pressure barrier defining a boundary between the gel lane and the second flow channel. The gel lane can be filled with a gel containing a vascular network which provides a flow channel through the gel. The vascular network may optionally be connected to a larger blood vessel that is grown or present in the first and second flow channel. In examples in which the method is performed on such a titerplate, tilting the titerplate at the first angle may induce the volume of fluid in the first reservoir to flow from the first reservoir, at least partially through the vascular networl to the second reservoir. [ons] Similarly, in examples in which the method is performed on a titerplate in which at least one microfluidic network comprises a second gel region in fluid connection with the second flow channel, a gel, or precursor can be introduced into the second gel region and tilting the titerplate at the second angle can induce the volume of fluid in the second reservoir to flow from the second reservoir, through the second gel region to the first reservoir.

[ong] In examples in which the microfluidic network comprises a first capillary pressure barrier defining a boundary between the ge] lane and the first flow channel and a second capillary pressure barrier defining a boundary between the gel lane and a third flow channel, the third flow channel forming a fluid circuit with a fourth flow channel, the third flow channel and fourth flow channel each connecting at one end to an access port in a third reservoir and at the other end to an access port in a fourth reservoir, the first to fourth flow channels may be configured such that tilting the titerplate at the first angle induces fluid flow from the third reservoir through the third flow channel to the fourth reservoir and tilting at the second angle induces fluid flow from the fourth reservoir through the fourth channel to the third reservoir. In other examples the first to fourth flow channels may alternatively be configured such that tilting the titerplate at the first angle induces flow from the fourth reservoir through the third flow channel to the third reservoir and tilting at the second angle induces fluid flow from the third reservoir through the third channel to the fourth reservoir.

[0120] The titerplate of the present invention is intended for use in assays which mimic biological environments, and recreating a unidirectional flow. Thus, the method may also comprise culturing a first cell type in or on a gel in the lane and a second cell type in at least one of the first flow channel and the second flow channel.

[0121] The method may also comprise providing a different shear stress to the first cell type and/or the second cell type by configuring the first flow channel and the second flow channel to provide different fluidic resistances in the first flow channel and the second flow channel as described above.

[0192] The method may also comprise providing a different flow to the first cell type and/or the second cell type by tilting the plate at a first angle inducing flow in the first flow channel and tilting the titerplate at a second angle inducing flow in a second flow channel, whereby the first and second angles are not the inverse angles of each other.

[0123] The method may comprise introducing a gel or gel precursor comprising endothelial cells in the ge] lane and/or one or both of the first flow channel and the second flow channel and allowing the endothelial cells to form a tubule that is perfused predominantly in the same direction and/or seeding endothelial cells in a resultant gel in the Jane and allowing the endothelial cells to form a vascular network or tubule having a lumen within the gel, and which connects the first flow channel and the second flow channel. In some examples, the vascular network or tubule is perfused in predominantly the same direction. In some examples, cells are perfused through the endothelial or epithelial tubule within the first flow channel in a predominantly single direction and are recirculated through the second flow channel. The circulating cells may be immune cells, tumor cells or stem cells.

[0124] In examples of the method in which cells are seeded in the gel lane or region, the method may comprise introducing a support scaffold into the gel lane/region so as to form a scatfold surface facing a flow channel. The support scaffold may be introduced via an access port leading to the gel lane/region.

[0125] In some examples, the support scaffold comprises a membrane. In some examples, the support scaffold comprises a gel and introducing the support scaffold into the gel lane/region comprises introducing a volume of a liquid gel- precursor. In some examples, the liquid precursor is pinned in the gel lane by one or more capillary pressure barriers and allowed to cure or gelate. In some examples, the volume of gel or liquid gel- precursor may be a single droplet or droplet-sized volume of a gel or liquid gel-precursor.

[0126] In one example, a sufficient volume of gel precursor is introduced such that the gel precursor is confined by a capillary pressure barrier at the boundary between the gel lane and a flow channel, for example by two capillary pressure barriers, each defining a boundary between the gel lane and a different flow channel, and completely fills the gel lane. In this manner, the resultant cured gel comprises an exposed surface which faces the flow channel, thus forming an inner surface of the flow channel.

[0127] The gel or liquid gel-precursor includes any hydrogel known in the art suitable for cell culture. Hydrogels used for cell culture can be formed from a vast array of natural and synthetic materials, offering a broad spectrum of mechanical and chemical properties. For a review of the materials and methods used for hydrogel synthesis see Lee and Mooney (Chem Rev 2001;101(7):1869- 1880). Suitable hydrogels promote cell function when formed from natural materials and are permissive to cell function when formed from synthetic materials. Natural gels for cell culture are typically formed of proteins and ECM components such as collagen, fibrin, fibrinogen, hyaluronic acid, or Matrigel, as well as materials derived from other biological sources such as chitosan, alginate or silk fibrils. Since they are derived from natural sources, these gels are inherently biocompatible and bioactive. Permissive synthetic hydrogels can be formed of purely non-natural molecules such as poly(ethylene glycol) (PEG), poly(vinyl alcohol), and poly(2-hydroxy ethyl methacrylate). PEG hydrogels have been shown to maintain the viability of encapsulated cells and allow for 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 facile tuning of mechanical properties, and are simply processed and manufactured.

[0128] The gel or gel precursor can be provided to the titerplate, for example to the gel lane as described above. After the gel or gel precursor is provided, it is caused or allowed to gelate, prior to introduction of a further fluid into a flow channel, for example. Suitable (precursor) gels are well known in the art. By way of example, the gel precursor may be a hydrogel, and is typically an extracellular matrix (ECM) gel. The ECM may for example comprise collagen, fibrinogen, tibronectin, and/or basement membrane extracts such as Matrigel or a synthetic gel.

[0120] The gel or gel precursor may comprise a basement membrane extract, human or animal tissue or cell culture-derived extracellular matrices, animal tissue-

derived extracellular matrices, synthetic extracellular matrices, hydrogels, collagen, soft agar, egg white and commercially available products such as Matrigel.

[0130] Basement membranes, comprising the basal lamina, are thin extracellular matrices which underlie epithelial cells in vivo and are comprised of extracellular matrices, such as proteins and proteoglycans. In one example, the basement membranes are composed of collagen IV, laminin, entactin, heparan sulfide proteoglycans and numerous other minor components (Quaranta et al,

Curr. Opin. Cell Biol. 6, 674-681, 1994). These components alone as well as the intact basement membranes are biologically active and promote cell adhesion, migration and, in many cases growth and differentiation. An example of a gel based on basement membranes is termed Matrigel (US 4829000). This material is very biologically active in vitro as a substratum for epithelial cells.

[0131] Many different suitable gels for use in the method of the invention are commercially available, and include but are not limited to those comprising

Matrigel rgf, BME1, BMEugf, BME2, BME2rgf, BME3 (all Matrigel variants)

Collagen I, Collagen IV, mixtures of Collagen I and IV, or mixtures of Collagen I and IV, and Collagen II and III), puramatrix, hydrogels, Cell-Tak™, Collagen I,

Collagen IV, Matrigel® Matrix, Fibronectin, Gelatin, Laminin, Osteopontin, Poly-

Lysine (PDL, PLL), PDL/LM and PLO/LM, PuraMatrix® or Vitronectin. In one preferred embodiment, the matrix components are obtained as the commercially available Corning® MATRIGEL® Matrix (Corning, NY 14831, USA).

[0132] In a typical setup, endothelial cells are cultured as a tube lining the flow channel. Unidirectional flow is important for maintaining a non-inflamed state and also leads to elongation of the endothelial cells in line with the flow.

Epithelial tubules, such as gut or kidney epithelium may also be grown in the same manner, noting that these are typically exposed to lower shear levels and thus lower tlow rates. This latter is typically achieved by a smaller angle of inclination.

Stromal tissue like fibroblasts, myofibroblasts, muscle cells, neurons, hepatocytes, stellate cells, pericytes may be cultured surrounding the endothelial or epithelial tubule, but may also be cultured in a gel adjacent to the tubule. Cultures may also comprise migrating cells, such as immune cells or tumor cells, that can be mixed with the gel, or added to a perfusion channel.

[0133] In one example, a gel or gel-precursor is preloaded with a cell or cells of interest, i.e. the cells are present in the gel or gel-precursor prior to introduction into the gel lane, and prior to gelation. In another example, the cells are inserted into or onto the partially or fully cured gel after it has been introduced into the gel lane. In another example, a gel or gel-precursor is introduced into the gel lane, and following gelation, a cell mixture, tissue or cell aggregate is placed on top of the gel or introduced into a region of a flow channel adjacent to the gel.

[0134] The cell mixture, tissue or cell aggregate in, on or alongside a cured gel may include epithelial or endothelial cells, stromal cells, muscle cells, one or more other cell types selected from pluripotent cells and central nervous, peripheral nervous, immune, urinary, respiratory, reproductive (male and female), gastrointestinal, endocrine, skin, musculoskeletal, cardiovascular, and mammary cell types.

[0135] In one example, the at least one type of cell or cell aggregate present in or on top of the gel or gel-precursor comprises epithelial cells, which during culture can proliferate and/or differentiate depending on the composition of the culture media, other cell types which may be present, and the extracellular matrix.

Thus, after introduction into the microfluidic network, either using an aqueous medium, preferably a growth medium, or by using the gel (precursor), the epithelial cells are then allowed to proliferate and/or differentiate. Culture of the one or more types of cells or cell aggregates, for example epithelial cells or endothelial cells, is achieved by introduction of media into a flow channel under unidirectional flow and continued under suitable conditions so that the cells are cultured.

[0136] The method may include introducing a medium containing cells into a flow channel via a reservoir.

[0137] Once the medium containing cells has been added to a flow channel, the cells present are allowed to settle onto the surface of a gel lining the flow channel, or the surface of the flow channel itself and, in some cases, form a tubule lining the inner surface of the flow channel including the surface of the gel.

[0138] In some examples, the cells comprise epithelial cells, for example lung epithelial cells, skin epithelial cells, gut epithelial cells, corneal epithelial cells, or mucus producing epithelial cells. In some examples, the cells comprise any epithelial cell found at a fluid- fluid interface in vivo.

[0139] In some examples, a supply of nutrients is provided to the attached cells via the flow channels. In some examples, the supply of nutrients provided to the cells can be transported from a flow channel through a gel present in the gel region to the cells in the gel, or in another flow channel via diffusion or interstitial flow through a gel. The gel can be an extracellular matrix pinned by one or more capillary pressure barriers as described herein. In some examples, the attached cells are allowed to form on a surface in a manner so as to extend around the entire boundary of the flow channel. In another example a gel can be introduced in the flow channel that is subsequently lumenized (see WO 2022/258668), leading to a circular or oval cross-section of a tubular lumen through a gel within the flow channel, with the attached cells being allowed to line the lumen and form a complete tubule within the lumenized gel.

[0140] By controlling the composition(s) introduced in the reservoir(s) the cell culture device of the present invention enables different modes of unidirectional flow-controlled cell culture depending on whether those reservoirs are part of the same flow circuit or different flow circuits. For example, the composition of fluids introduced into reservoirs which are part of different flow circuits can be different. This can be used to create gradients, for instance to attract immune cells, or induce angiogenesis, as well as assymetric exposure, eg apical or basal exposure.

[0141] As will be understood from the present disclosure, titerplates which include biological tissue may include sprouts of endothelial cells which extend from a flow channel through an extracellular matrix gel in the gel region to an opposite flow channel, or vice versa, forming a vascular bed. These sprouts may be microvessels that are a result of angiogenesis. Alternatively cells can be mixed in a gel to self-organize in a microvascular network, referred to as vasculogenesis.

[0142] In general endothelial cells are known as the cells that line the interior surface of the entire circulatory system, from the heart to the smallest lymphatic capillaries. When in contact with blood these cells are called vascular endothelial cells and when in contact with the lymphatic system they are called lymphatic endothelial cells. In a particular embodiment the method includes the step of introducing endothelial cells into a flow channel of the microfluidic network, and causing or allowing said endothelial cells to line the flow channel, i.e. causing or allowing the endothelial cells to form a vessel within the flow channel.

[0143] Introducing endothelial cells into a flow channel under the right conditions, including unidirectional flow can result in the formation of authentic vascular tissue lining the internal surfaces of the flow channel and in some cases the internal surfaces of an extracellular matrix gel. In particular, introducing endothelial cells into a flow channel under unidirectional flow allows the endothelial cells to align themselves with the direction of flow, resulting in an elongated, stretched, shape in the direction of the flow. This elongated shape is believed to be more faithfully mimicking the in vivo environment. These endothelial vessels may be in a quiescent state, meaning that they are not inflamed. Also these endothelial vessels may have a better barrier function, meaning that they are less permeable or penetrable to molecules and substances of a certain size. Permeability is typically measured by trans epithelial electrical resistance (TEER) measurement, which means recording an impedance spectrum and deriving the so-called TEER value of an endothelial tube. An endothelial vessel cultured under unidirectional flow is known to typically have a higher TEER value and thus a better barrier function. This is of particular importance when recapitulating the blood-brain barrier, which is known to have a very good barrier function.

[0144] Low inflammatory state and proper recapitulation of the barrier function in vitro are important prerequisites for studying the process of immune cell activation, adhesion and extravasation. In vivo it is known that at the side of inflammation endothelial cells get activated, resulting amongst others in upregulation of VCAM and ECAM as well as other adhesion molecules. Immune cells may bind to these adhesion molecules subsequently extravasate from the blood vessel into the tissue (diapedesis process). During diapedesis it is thought that the endothelium becomes more permeable. In order to recapitulate this process and enlarge the experimental window in vitro it is important that at baseline the endothelium is non inflamed.

[0145] In addition to improved cell alignment, reduced inflammatory state and increased barrier function, unidirectional flow may also cause better polarization of endothelium and epithelium, meaning that the right transporters and receptors are situated at the right side, e.g. apical side or basal side, of the endothelial or epithelial vessel. An influx transporter of the proximal tubule of the kidney for instance would typically be positioned at the basolateral side of an epithelial tube, while an efflux transporter would in many cases be present at the apical side.

[0146] Culture media, or differentiation media may be added to a reservoir, and establishment of a unidirectional fluid flow through the vascular network may be achieved as described above. For example, tilting the titerplate at the first angle induces the volume of fluid in the first reservoir to flow from the first reservoir, at least partially through the vascular network, to the second reservoir and tilting the titerplate at a second angle induces the volume of fluid in the second reservoir to flow from the second reservoir, at least partially through the vascular network, to the first reservoir, whereby the flow through the vascular network at either tilting angle is in predominantly the same direction. Similarly, compositions of fluids can be controlled as described above. Thus, a vascularised, perfusable network established by the unidirectional flow methods described can be perfused with culture media that allows for provision of nutrients and oxygen to and transport of metabolites from the microvessel within the microfluidic channel of the device and the cells or cell aggregates in or on top of the channels or gel regions.

[0147] As is described in the Examples, the unidirectional flow-enabled titerplates allows performing experiments at significant throughput.

[0148] The present invention also provides an assay plate, comprising a titerplate as described herein with a scaffold or other support structure within the gel region. In some examples, the microfluidic network of the assay plate comprises cells or tissue, present for example in or on the scaffold or support structure, and/or in a flow channel. In some examples, the scaffold or support structure comprises a gel, an extracellular matrix and/or a membrane. In some examples, the cells or tissues at Jeast partly line the flow channel. In some examples, the cells are epithelial cells, for example lung epithelial cells, connective tissue, such as fibroblasts or endothelial cells forming a vascular network or vascular bed.

[0149] As has been described, the titerplates and assay plates of the present disclosure are suited for culturing cells, in particular epithelial tubules, co-culture of different cell types, invasion/migration experiments and vascularization of tissues. Consequentially, the titerplates and assay plates of the present disclosure tind use in efficacy or toxicity assays, in which substances of interest can be perfused through a flow channel and their effects on the different tissues observed.

[0150] The present disclosure also provides kits and articles of manufacture for using the titerplates and assay plates described herein. In some examples, the kit may comprise the titerplate or assay plate described herein and one or more of: a gel, gel-precursor composition or other extra-cellular matrix composition; one or more cells or cell types; growth media; and one or more reagent compositions. The matrix and/or cells may be already prepared within the titerplate, so called “assay ready”, or as separate vials delivered with the plate.

[0151] The kit may further comprise a packaging material, and a label or package insert contained within the packaging material providing instructions for use.

[0152] The kits may further include accessory components such as a second container comprising suitable media for introducing cells, and instructions on using the media.

DETAILED DESCRIPTION OF THE DRAWINGS

[0153] The present invention will now be described by way of example only with reference to the drawings.

[0154] Figure 1 shows a titerplate 100, in plan view. Titerplate 100 has a plurality of microfluidic networks, specifically 64, indicated generally at 101.

Although not shown in Figure 1, titerplate 100 includes a microfluidic layer and a reservoir layer overlaying the microfluidic layer. The microfluidic layer may be formed on or in a base (not shown), as described previously.

[0155] Turning to Figure 2(a), which is a magnified plan view of the microfluidic layer of microfluidic network 101, first flow channel 103, present in the microfluidic layer has a first access port 1024 a second assess port 102c, while second flow channel 104 has a first access port 102b a second assess port 102d. First and second flow channels are non- linear in that each includes a bend toward each end so that access ports 102a to 102d are all aligned with one another along the longitudinal y-axis.

[0156] Figure 2(b) shows a cross-sectional view of microfluidic network 101, along first flow channel 103 (as indicated by the thick dashed line) in which reservoir layer 105 and microfluidic layer 106 can be seen. Reservoir layer 105 includes first reservoir 108, and second reservoir 109, sharing a common wall 107b.

Reservoir 108 is bounded by wall 107a while second reservoir 109 is bounded by wall 107c.

[0157] First reservoir 108 and second reservoir 109 each have an end proximal the centre of microfluidic network 101 and an end distal or furthest from the centre of microfluidic network 101. It will be appreciated that the elongate reservoirs each correspond to multiple well positions of a standard microtiter plate which have been merged together. In this example each reservoir 108 and 109 are a merger of 3 wells of a 384 well microtiter plate.

[0158] Underlying reservoir layer 105 is microfluidic layer 106, with first reservoir 108 having an access port 102a to first flow channel 103, and second reservoir 109 having an access port 102c to first flow channel 103.

[0159] Access port 1024, for first flow channel 103, is located toward the distal end of first reservoir 108 while access port 102c, at the opposite end of first flow channel 103, is located at the proximal end of second reservoir 109. Second flow channel 104 and its access ports 102b and 102d are shown in dashed outline only.

[0160] Figure 2(c) shows an alternative cross-sectional view of microfluidic network 101, along second flow channel 104 (as indicated by the thick dashed line).

In this cross- section, the other half of the flow circuit is shown, with first reservoir 108 having an access port 102b to second flow channel 104, and second reservoir 109 having an access port 102d to second flow channel 104. Access port 102b for second flow channel 104 is located toward the proximal end of first reservoir 108 while access port 102d at the opposite end of second flow channel 104 is located toward the distal end of second reservoir 109.

[0161] In this way, first flow channel 103, second flow channel 104, first reservoir 108 and second reservoir 109, together form a flow circuit configured for unidirectional flow, with first and second flow channels 103, 104 being present in microfluidic layer 106 and first and second reservoirs 108, 109 being present in reservoir layer 105.

[0162] Figures 3(a) and 3(b) illustrate the operation of titerplate 100, specifically within microfluidic network 101, when tilted at first angle by being rotated about the x-axis indicated in Figure 2(a). Figure 3(a) illustrates flow of liquid 110, initially present in first reservoir 108, when microfluidic network 101 is is rotated about the rotation axis x (shown in Figure 2(a)).

[0163] When titerplate 100 is tilted at this first tilt angle as in Figure 3(a), liquid 110 is induced to flow from first reservoir 108 via access port 102b, and flows through second flow channel 104 in the microfluidic layer 106 and enters second reservoir 109 via access port 102d. The angle of tilt (i.e. the angle of rotation about the x-axis), combined with the location of access port 102a at the distal end of first reservoir 108 exposes access port 102a to the atmosphere, with liquid 110 collecting at the right hand end of first reservoir 108 and passing through access port 102b. As can be seen in Figure 3{b) though, not all liquid will collect at the right hand end of first reservoir 108. Indeed, a portion of liquid will be wicked toward the corners of the end wall 1074, leaving an air-lifted region 114 of the base of first reservoir 108 uncovered. As used herein, the terms “air-lift” or “air-lifted” refer to a region on the base of the reservoir around an access port which is exposed to air during a tilting operation, and which provides for the disconnect between the wicked liquid and liquid which remains in first flow channel 103 (and which is pinned by access port 102a). This ensures no coalescence of these two liquids, and no resultant back-flow of liquid. A similar air-lifted region 111b of the base of second reservoir 109 surrounding access port 102c is free of liquid 110 in this state, being again the result of liquid 110 being drawn to the corners of second reservoir 109 and liquid in underlying first flow channel 103 being pinned by access port 102c and prevented from entering second reservoir 109. For completeness, the flow channels included in Figure 3(b) are not shown as containing liquid, so that the liquid distribution in the reservoirs can be more clearly depicted. However, it will be appreciated that in operation the flow channels would contain liquid, as shown in Figure 3(a).

[0164] Conversely, and as shown in Figure 3(c) and Figure 3(d), when titerplate 100 is tilted at a second angle (which could be the inverse angle of the tirst angle) by rotating the titerplate about the x-axis to the inverse angle of the first operation, a volume of fluid 118 now present in second reservoir 109 flows out of second reservoir 109 via access port 102c to first reservoir 108 via access port 102a. Based on these two tilting actions, a unidirectional flow through each microfluidic network of titerplate 100 can be achieved. Figure 3(d) illustrates that during this operation, air-lifted region 11c surrounding access port 102d and ajr-

lifted region 11d surrounding access port 102b are formed for corresponding reasons as set out above in connection with Figure 3(a) and Figure 3(b).

[0165] Figure 4 is the same plan view of microfluidic network 101 as in

Figure 2(a), again showing the positioning of access ports relative to flow channels.

Figure 4 illustrates the offset or asymmetric arrangement of the access ports which can be advantageous in achieving unidirectional flow. The dot-dash crosshairs indicate the well positions of an ANSI/SLAS titerplate of the same footprint.

Specifically, titerplate 100 (of Figure 1) has 16 rows (A to P) and 24 columns (1 to 24), and so would correspond to a standard ANSI SLAS 384-well plate, with microfluidic network 101 indicated in Figure 1 corresponding to positions A13 to

A18. As can be seen in Figure 4 though, access port 102a” and access port 102d” are not located at the central position of the cross-hairs corresponding to the ANSI

SLAS well positions (A13 and A18 in Figure 1), and are displaced or offset by an amount §. This offsetting or spacing away from the respective end wall can ensure that there is no coalescence of liquid wicked toward the end wall with liquid in tirst flow channel 103 pinned by either of access ports 102a’ and 102d’, resulting in effective unidirectional flow. It will be appreciated that the absolute value of the offset § will need to be larger than the wicking fluid in the corner of the microtiter plate and therefore depend on the contact angle of the liquid with both the reservoir material and chip material. However, for a titerplate based on a 384 well footprint, the offset § may be in the region of about 1-2 mm.

[0166] In Figures 3(a) and (b) access ports 102a and 102c are both being airlifted. In the context of the invention airlifting of the access port upstream of the channel (access port 102a in Figures 3(a) and (b)) is of particular importance, while the airlifting of the downstream access port (access port 102c in Figures 3(a) and (b)) is of less importance. It is therefore important for understanding of the invention, that an access port with offset & is implied to be used as upstream reservoir with intention to be airlifted during stopflow. Hitherto this same access port in flow direction will be positioned downstream.In the embodiment of Figures 1to 4, each reservoir corresponds to three wells of a standard 384 well plate that have been fused or connected together. This allows effective airlifting (as shown in

Figure 3(b) and Figure 3(d) and as discussed above) while still allowing for a sufficient quantity of liquid to be present in the flow circuit and to be transferred from one reservoir to another thereby inducing unidirectional flow.

[0167]

[0168] Figure 5 shows a schematic layout for an alternative titerplate 200, with a plurality of microfluidic networks, in this case 64 microfluidic networks, with one microfluidic network being shown at 201.

[0169] Figure 6 is a magnified view of microfluidic network 201, showing an alternative arrangement of reservoirs and flow channels to that of titerplate 100 of

Figure 1. First reservoir 208 and second reservoir 209 are again elongated, having footprints corresponding to multiple (in this case three) well positions of a standard titerplate, but are in this case arranged in parallel, separated by elongate common wall 207b, rather than the end-to-end arrangement of microfluidic network 101. Consequently, first flow channel 203 and second flow channel 204 are also in a different orientation to the arrangement of microfluidic network 101.

Here, first flow channel 202 and second flow channel 203 are much shorter in length, and are on opposite sides of rotation axis x, while traversing a second rotation axis y. Although not indicated with reference to cross-hairs corresponding to ANSI SLAS well positions, it can be seen that access ports 2024 and 202d are again offset, brought about by a bend in flow channels 203 and 204. This arrangement is particularly suited to inducing unidirectional flow with the ability to select which flow channel a liquid will flow through, as will be now be described with reference to Figures 7(a) to 7(c).

[0170] Figure 7(a) shows a coordinate map for the rotation at angle 8 and b about the x and y axes respectively, and the end states of liquid within microfluidic network 201 according to a single axis (2-state) tilting operation, with the tilting operations indicated. Rather than the single axis of rotation corresponding exactly with the x and y axes, the axis of rotation is a diagonal axis as indicated by the transition arrows. Rotating about this axis is equivalent to a simultaneous rotation about the x and y axes between a first state or tilt angle and a second state or tilt angle. The top left quadrant of the coordinate map shows a first state or orientation of the microfluidic network after a transition and complete liquid levelling, corresponding to a “+8, -®” (“plus theta”-“minus phi”) orientation or a first tilt angle. Simultaneous rotation about both x and y axes results in a transition to the second state (bottom right quadrant), corresponding to a “-8, +9” (“plus theta”-“minus phi”) orientation or a second tilt angle.

[0171] In the first state or tilt angle, due to the offset positioning of access port 202a (as can be seen in the bend in the channel), air-lifted region 211a forms.

Due to the orientation, a smaller air-lifted region 211b also forms around access port 202c. Upon rotation about the diagonal axis (or simultaneous rotation about the x and y axes) to state 2 (a second tilt angle), and after liquid levelling, it can be seen that the air-lifted regions around access ports 202a and 202c have disappeared, with new air-lifted regions 211c and 21d forming around access ports 202d and 202b respectively. Again, due to the offset positioning of access port 202b, and the “-8, +97 (“plus theta” “minus phi”) orientation, air-lifted region 202d is more pronounced or larger than air-lifted region 202b. Although Figure 7(a) has been described with reference to the end result of these transitions, after full liquid levelling has occurred, it is also possible to operate transition protocols between these tilt angles before full liquid levelling in the reservoirs has occurred.

[0172] Figure 7(b) shows cross-sectional views and a plan view of the microfluidic network 201 at a tilt angle 8 and @® about the x and y axes respectively after a transition from the first state “+8, -®@” to the second state “-8, +9”, but before full liquid levelling has occurred. As can be seen in the uppermost image, in the second state, access port 202d is exposed to the air, or air-lifted, while liquid flows to the far end of the reservoir and exits through access port 2020. In the orthogonal cross-sectional view, it can be seen that liquid levelling after transition to the second state occurs by liquid passing through access port 202c, along first flow channel] 203 to access port 2024 and into the overlying first reservoir (208, number not included in figure). As the liquid levelling is still ongoing, the air-Jifted regions around access ports 202b and 202d are shown as being of equal size, but it will be understood that once the liquid has completely levelled in this second state, the relative dimensions of air-lifted regions 211c and 211d will be as shown in the bottom right quadrant of Figure 7(a). [o173] Figure 7(c) shows cross-sectional views and a plan view of the microfluidic network 201 at a tilt angle relative to the 8 and ¢ axes after a transition from the second state “-8, +%” to the first state “+8, -®7, i.e, the inverse transition of that of Figure 7(b) but before full liquid levelling has occurred.

As can be seen in the uppermost image, in the first state, access port 202a is exposed to the air, or air-lifted, while liquid flows to the far end of the reservoir and exits through access port 202b. In the orthogonal cross-sectional view, it can be seen that liquid levelling after transition to the first state occurs by liquid passing through access port 202b, along second flow channel 204 to access port 202d and into the overlying second reservoir (209, number not included in figure).

As the liquid levelling is still ongoing, the air-lifted regions around access ports 202a and 202c are shown as being of equal size, but it will be understood that once the liquid has completely levelled in this second state, the relative dimensions of the air-lifted regions will be as shown in the top left quadrant of Figure 7(a).

[0174] It will be understood that continuation of this 2-state operation (single axis-rotation about a diagonal axis), combined with the offset location of access ports 202a and 202d provides for effective unidirectional flow through microfluidic network 201.

[0175] Figure 7(d) illustrates a coordinate map for the same microfluidic network 201, but demonstrating liquid locations, in particular with reference to air- lifted regions during a 2- axis transition protocol, that is, adjusting tilt angles 8 and % relative to the x and y axes in a sequential manner, transitioning from a first state or tilt angle (top left quadrant) to a second state or tilt angle (bottom right quadrant) via a third state or tilt angle (state 1b, top right quadrant), and from the second state or tilt angle (bottom right quadrant) back to the first state or tilt angle (top left quadrant) via a fourth state or tilt angle (state 2b, bottom left quadrant). This four-state operation can be viewed as: a first rotation about the x axis; a second rotation, but about the y axis; a third rotation, which is about the x axis and which is to the inverse degree of rotation to the first rotation; followed by a fourth rotation which is about the y axis and which is to the inverse degree of rotation to the second rotation. As can be seen in Figure 7(d), this sequence of transitions, after complete liquid levelling results in each of access ports 2024, 202b, 202c and 202d being air-lifted, or exposed to the air, with respective exposed regions indicated at 2114, 211d, 211b and 211c. An advantage of the four state rocking sequence as depicted in Figure 7(d) is that backflow can be further eliminated while transitioning from one state to another.

[0176] Figure 8 shows an alternative titerplate 300, with a plurality of microfluidic networks, in this case 48 microfluidic networks, with one microfluidic network being shown at 301. Titerplate 300 includes the row and column markings indicative of a standard 384 well titerplate format, with microfluidic network 301 occupying well positions A17 to A24.

[0177] Figure 9 is a magnified view of microfluidic network 301, showing an alternative arrangement of reservoirs and flow channels to the previously described arrangements. As can be seen in Figure 9, first reservoir 308 is again an elongate reservoir, having a footprint corresponding to three well positions A17 to A19 of titerplate 300, with second reservoir 310 corresponding to well positions A22 to

A24. A similar arrangement of first flow channel 303 and second flow channel 304 to that of Figure 2(a) can be seen, with the exception that microfluidic network 301 includes a gel lane 313 in its microfluidic layer, defined in part by capillary pressure barrier 312 which serves to delineate first flow channel 302 from gel lane 313. A region of gel lane 313 is located at a position corresponding to well positions

A20/A21, with A21 typically being a well that is used for optical inspection and is hitherto either open to the environment, or covered with a transparent material.

Gel lane 313 is served by access port 302e, via well position A20.

[0178] The arrangement of microfluidic network 301 enables unidirectional perfusion of a cell culture in or against an extracellular matrix gel within gel lane

313, as has been described generally above. First flow channel 303 serves as an active channel, being perfused with for example a cell culture medium. In a typical setup endothelial or epithelial cells are grown as a tubule within first flow channel 303 that is hitherto perfused with growth medium in a single direction. Second flow channel 204 serves as a reflow channel, returning liquid media from second reservoir 309 to first reservoir 308. It will be appreciated that a single axis rotation or transition between two states or tilt angles such as depicted in Figures 3(a) to 3(d) results in unidirectional flow through the active channel and reflow channel.

[0179] Figure 10 shows an alternative layout for a titerplate 400, with a plurality of microfluidic networks, in this case 24 microfluidic networks, with one microfluidic network being shown at 401.

[0180] Figure 11 is a magnified view of microfluidic network 401, showing an alternative arrangement of reservoirs and flow channels to the previously described arrangements. Microfluidic network 402 comprises first and second reservoirs 408, 409, which form a first flow circuit with first and second flow channels 403 and 404. In addition, microfluidic network 401 comprises third and fourth reservoirs 414, 415, which form a second flow circuit with third and fourth flow channels 416 and 417. The first flow circuit and the second flow circuit are located either side of the indicated y-axis, along which a gel region 413 can be found. Gel region 413 is in direct fluid contact with first flow channel 402 and with third flow channel 416, but delineated from these flow channels by capillary pressure barriers 4124 and 412b respectively. As with microfluidic network 301, cells or cell aggregates can be introduced into gel region 413 by mixing with the gel or gel precursor introduced via access port 402f or access port 402g. While gel region 413 is shown connected to two access ports 402f and 402g, it will be appreciated that a single access port could be equally useful for some applications.

[0181] The gel or precursor will be pinned on both sides by capillary pressure barriers 412a and 412b, but will be in contact with any liquids flowing through first flow channel 403 and third flow channel 416. Alternatively cells can be introduced in flow channel 403 and or flow channel 416. These cells can form a tubule against the extracellular matrix or migrate into the extracellular matrix. It will be appreciated that — due to the similarities in flow channel layout — unidirectional flow through the two separate flow circuits can be achieved via a single axis rotation between two tilt angles or end states as was described in connection with titerplate 100/microfluidic network 101 and Figures 3(a) to 3(d).

First flow channel 403 serves as an active or perfusion channel to gel region 413 on one side while second flow channel 404 serves as the reflow channel. Similarly, third flow channel 416 serves as an active channel to gel region 412 on the opposite side to first flow channel 403, while fourth flow channel 417 serves as a reflow channel for third flow channel 416.

[0182] In microfluidic network 401, the same flow profile is induced in both first flow channel 403 and third 416 flow channel, on each side of the extracellular matrix in gel region 413 due to the line of symmetry running along gel region 413.

In an alternative design, the access ports of the third flow channel 416 and the fourth flow channel 417are swapped, meaning that the access port positions remain the same, but that flow channel 416 is routed towards access ports 402i and 402k and flow channel 417 is routed towards access ports 402h and 402j. In this manner, flow in third flow channel 416 will be in antiphase to flow in first flow channel 403, meaning that one channel is perfused in a first angle of inclination, whereas the other is perfused at a second, typically inverse angle of inclination. This alternative design is particularly useful in cases in which flow requirements in the first and third flow channel are different. For example, a high flow rate over a shorter period of time can be applied in the first flow channel by inclination at a large first angle, whereas a lower flow rate can be applied to the third flow channel by choosing a smaller angle of inclination, over a longer period of time.

[0183] Figure 12 shows an alternative layout for a titerplate 500, with a plurality of microfluidic networks, in this case 32 microfluidic networks, with one microfluidic network being shown at 501. Titerplate 500 and microfluidic network 501 are similar to titerplate 400 and microfluidic network 401, with differences being described with reference to Figure 13.

[0184] As seen in Figure 13, instead of the unidirectional flow circuit formed from third flow channel 403, fourth flow channel 417 and the overlying reservoirs, microfluidic network 501 does not include a fourth flow channel and instead comprises a third flow channel 514’. Under the same single axis rotation between two tilt angles as discussed above for titerplate 400, third flow channel 514’ functions as a bidirectional flow channel in contact with a gel in the gel lane, and through which a liquid can be perfused.

[0185] Figure 14 shows an alternative footprint for a titerplate 600, with a plurality of microfluidic networks, in this case 48 microfluidic networks, with one microfluidic network being shown at 601.

[0186] Figure 15 is a magnified view of microfluidic network 601, which has a different arrangement with respect to the correspondence with well positions of a standard titerplate. Microfluidic networks 101, 201, 301, 401, and 501 are all configured such that the reservoirs are all linear, that is each reservoir is based on a single row (or column) of well positions. In contrast, microfluidic network 601 comprises two L-shaped reservoirs 608 and 609. First reservoir 608 and second reservoir 609 are still fluidly connected by first flow channel 603 and second flow channel 604 to form a flow circuit. Microfluidic network 601 also includes gel region 613, with capillary pressure barrier 612 defining a boundary between gel region 612 and first flow channel 603. Gel region 613 is served by access port 602e.

As with previous configurations, it can be seen by virtue of the bend in first flow channel 603 that access port 602a is offset relative to an ANSI/SLAS well position and to access port 602c at the opposite end of first flow channel 603. As a result, microfluidic networl 601 is suited to unidirectional flow by two-state tilting (single axis rotation), as has been described above. Due to the non-linear configuration of first reservoir 608 and second reservoir 609, microfluidic network 601 is particularly suited to unidirectional flow by continuously varying the tilt of the titerplate in a three-dimensional gyratory motion about the x-, y- and z-axes relative to the horizontal plane of the x- and y- axes.

[0187] Figure 16 shows an alternative schematic footprint for a titerplate 700, with a plurality of microfluidic networks, in this case 24 microfluidic networks, with one microfluidic network being shown at 701.

[0188] Figure 17 is a magnified view of microfluidic network 701, showing an arrangement of reservoirs and flow channels similar to that shown in Figure 11, but designed to follow the basic concept of Figures 5 to 7. Microfluidic network 701 comprises first and second reservoirs 708, 709, which form a first flow circuit with first and second flow channels 703 and 704. In addition, microfluidic network 701 comprises third and fourth reservoirs 714, 715, which form a second flow circuit with third and fourth flow channels 716 and 717. The first flow circuit and the second flow circuit are located either side of a gel region 713. Gel region 713 is in direct fluid contact with first flow channel 702 and with third flow channel 716, but delineated from these flow channels by capillary pressure barriers 712a and 712b respectively. As with microfluidic network 401, cells or cell aggregates can be introduced into gel region 713, for example within an extracellular matrix gel (or precursor). The gel or precursor will be pinned on both sides by capillary pressure barriers 712a and 712b, but will be in contact with any liquids flowing through first tlow channel 703 and third flow channel 716. Cells can alternatively or in addition be introduced in either or both flow channels 703 or 716 enabling tubule formation and/or migration of cells into the ECM. This configuration of access ports and flow channels facilitates unidirectional flow through both flow circuits via a two- state transition protocol, along a diagonal axis as described in connection with

Figure 7(a), or a transition protocol with more states/tilting angles such as the two- axis rotation/4-state protocol described with reference to Figure 7(d), or even an 8- state/tilting angle methodology, or a three-dimensional gyratory motion as described above.

[0189] Figure 18 shows an alternative schematic configuration for a titerplate 800, with a plurality of microfluidic networks, in this case 32 microfluidic networks, with one microfluidic network being shown at 801.

[0190] Figure 19 is a magnified view of microfluidic network 801 and is a variation of microfluidic networl 501 in Figure 13, following the basic concept as introduced in Figures 5 to 7. In microfluidic network 801, first flow channel 803 and second flow channel 804 form a flow circuit with elongate first reservoir 808 and elongate second reservoir 809. First flow channel is delineated from a gel region 813 by capillary pressure barrier 812a and from a third flow channel 816 by capillary pressure barrier 812b. Under the same single axis rotation between two tilt angles as discussed above for titerplate 500, third flow channel 816 functions as a bidirectional flow channel in contact with a gel in the gel lane 813, and through which a liquid can be perfused, while unidirectional flow through the flow circuit comprising first flow channel 803 and second flow channel 804 is achieved.

However, this configuration of access ports and flow channels is also suited to a transition protocol with more states/tilting angles such as the two-axis rotation/4- state protocol described with reference to Figure 7(d), or even an 8-state/tilting angle methodology, or a three-dimensional gyratory motion as described above.

[0101] Figure 20 shows an alternative titerplate 900, with a plurality of microfluidic networks, in this case 32 microfluidic networks, with one microfluidic network being shown at 901.

[0192] Figure 20(a) and Figure 20(b) are magnified views of microfluidic network 901, which has first and second flow channels 903 and 904, linked by first reservoir 908 and second reservoir 909. First flow channel 903 is in direct fluid contact with a first gel region 9134, while second flow channel 904 is in direct fluid contact with a second gel region 913b. In each case, a capillary pressure barrier (9124, 912b) serves to delineate the gel region from the respective flow channel, so that a liquid medium such as a precursor to an extracellular matrix gel can be introduced into the each gel region and pinned along the capillary pressure barrier.

Once in situ, the gel precursor can be allowed or caused to gel, thus providing a scaffold for cells and tissues grown in or against the gel.

[0193] It will be appreciated that microfluidic network 901 is similar to microfluidic network 201 in Figure 5, with short, flow channels underneath elongate reservoirs to form a single flow circuit, with the exception that the reservoirs of Figures 20(a) and (b) do not share a common wall with each other and are instead separated by four end-to-end chamber or well positions. As already mentioned, having two separate gel regions, which are both in fluid communication with a single flow circuit enables culture of different cell types or tissues in and/or against each gel and flow channel. In this manner the setup of network 901 is different from those in Figures 10 to 19 in that both flow channels for flow and reflow have a cell culture function. In this manner interaction between tissues can be studied through exchange of substance or mass transport via the unidirectional flow through the single flow circuit. Examples thereof include cell circulation, such as eg immune cells, cancer cells, or stem cells from a first tissue to a second tissue, as well as impact of metabolites of a first tissue on a second tissue. Figures 20(a) and 20(b) indicate unidirectional fluid flow through the flow circuit based on the same two-state/tilting angle transition protocol with rotation about a diagonal axis similar to that of Figure 7(a), with air- lifted regions 914 and 9ub resulting from the offset placement of access ports, as has already been described.

[0194] Figure 22 shows an alternative schematic for a titerplate 1000, with a plurality of microfluidic networks, in this case 48 microfluidic networks, with one microfluidic network being shown at 1001.

[0195] Figure 23 is a magnified view of microfluidic network 1001, which has similarities to microfluidic network 701 of Figure 17, in that it includes a gel region or lane 1013 disposed between two flow channels: here, first flow channel 1003 and second flow channel 1004, again with capillary pressure barriers 1012a and 1012b defining a boundary between the gel region 1013 and first flow channel 1003 and second flow channel 1004 respectively. First flow channel 1003 is much longer in length than second flow channel 1004, with the access ports (not numbered) for tirst flow channel 1003 being located at the outer peripheries of first and second reservoirs 1008, 1009 and thus the outer peripheries of microfluidic network 1001,

whereas second flow channel 1004 is shorter in length and has its access ports located inwards of the access ports of first flow channel 1003.

[0196] Microfluidic network 1001 differs from microfluidic network 701 in that first flow channel 1003 and second flow channel 1004 form a single flow circuit with first reservoir 1008 and second reservoir 1009. As has been described herein, cells or cell aggregates can be introduced into gel region 1013, dispersed within an

ECM gel precursor introduced via access port 1002e, or directly via an opening in the overlying well in the reservoir layer. Capillary pressure barriers 1012a and 1012b confine the introduced gel precursor to gel region 1013 without any spread into the neighbouring first and second flow channels 1003, 1004. Subsequent to gelation, cells can be introduced into flow channels 1003 and/or 1004. In a first typical example of use endothelial cells are introduced in both of the gel region and the flow channels. Endothelial cells in the flow channels form a tubular structure lining the channel walls and gel surface, whereas endothelial cells in the gel region form microvessels that typically connect to the tubular vessels in the flow channels. In a second typical example of use the gel does not contain endothelial cells, whereas endothelial tubules are allowed to form in either or both of the flow channels. Subsequent induction of angiogenesis, for example by adding a cocktail of pro-angiogenic factors, results in formation of microvessels through the gel that connect one flow channel to the other flow channel.

[0197] Figures 24(a) to 24(d) illustrate the liquid flow through microfluidic network 1001 (in a two-state transition protocol based on rotation about the x-axis only) after cells have been seeded into the microfluidic network and allowed to form microvessels, such as happens when endothelial cells form a vascular bed in or through a gel within gel region 1013. Figure 24(a) shows that rotation about the x-axis to a first tilt angle results in liquid flow through access port 1002b to access port 1002c at the end of second flow channel 1004 and to access port 1002d through both the microvascular bed and second half of flow channel 1003.

[0198] Figure 24(b) is a close up schematic of vascular bed 1018, showing individual vessels 1019 through which liquid can flow. The dashed circle indicates an option access port through which cells or gel can be added, or samples can be taken.

[0199] Figures 24(c) and 24(d) indicate the liquid flow if the angle of rotation about the x- axis is inverted, showing liquid flow in the opposite direction from second reservoir 1009 to first reservoir 1008, with the flow being via second flow channel 1004 as well as flow through vascular bed 1018 and other half of flow channel 1003. It is noticeable that unidirectional flow through vascular bed 1018 can be achieved based only on rotation about the x-axis. Analogue to what has been explained before, improved function of the design is achieved by the offset nature of access ports 1002a and 1002d with respect to the 384 well grid, as can be seen Figures 24(a) and 24(c). Achieving unidirectional flow through vascular bed 1018 is advantageous as it more authentically recreates an in vivo environment for microvasculature, for example, and leads to improved perfusability, barrier function and non inflamed state of microvessels.

[0200] Figure 25 shows an alternative schematic for a titerplate 1100, with a plurality of microfluidic networks, in this case 40 microfluidic networks, with one microfluidic network being shown at 1101.

[0201] Figure 26(a) is a magnified view of microfluidic network 1101, which has first and second flow channels 1103 and 1104, linked by first reservoir 1108 and second reservoir 1109. First flow channel 1103 is in direct fluid contact with a gel region 113. In this case, a first capillary pressure barrier 1112a serves to delineate the gel region 1113 from a first segment of the first flow channel 103, while a second capillary pressure barrier 112b serves to delineate the gel region 1113 from a second segment of the first flow channel 1103 which is on the opposite side of gel region 1103. A liquid medium such as a precursor to an extracellular matrix gel can be introduced into gel region 1113 via an aperture/access port in a reservoir directly above, and pinned along the capillary pressure barrier. While Figure 26 depicts direct loading via an access port directly above the gel region, an alternative layout is equally possible in which the gel (precursor) is applied via an additional access port, through so-called sideloading (such as access port 1002e in Figure 23). Once in situ, the gel precursor can be allowed or caused to gel, thus providing a matrix to support cells. Endothelial cells introduced to gel region 13, either in suspension in the gel precursor, or via first flow channel 1103 can form a vascular bed 118, as can be seen in Figure 26(b). Figures 27(a) and 27(b) show liquid flow through the flow circuit of microfluidic network 1101 ín the absence of a perfusable vascular bed, but with gel in place, when subjected to a 2-state transition or rocking protocol along a diagonal axis such as described above. Figure 27(a) shows that — due to the presence of the gel — only very limited to no fluid flow is present in the gel region due to its high fluidic resistance, instead flowing the length of first flow channel 1103. When the angle of rotation about the diagonal axis inverted, to the second tilt angle, liquid flows via second flow channel 1104, as would be expected.

[0202] Figures 27(c) and 27(d) depict the situation when microfluidic network 1101 now contains the gel-supported vascular bed 1119 in gel region 1113 and is subjected to tilting at the first angle and then the second angle (about the diagonal axis as before). In this case, at the first tilt angle fluid flow not only continues along first flow channel 1103 as before, but is now also able to flow through vascular bed 118, while second flow channel 1104 continue to serve as a reflow channel. Thus, not only is unidirectional flow achieved through microfluidic network 1103 as a whole, but also through vascular bed 1118.

[0203] It will be understood that variations exist in the context of the invention for the fluidic networks of Figures 8 through 26 in which the gel region can be either a closed gel region which is “side loaded” from an access port in an adjacent reservoir, or an open top gel region with an access port directly above the gel region, or a combination of both.

EXAMPLES

[0204] The foregoing description of embodiments of titerplates in accordance with the invention will be understood as being exemplary, and in no way limiting on the scope of protection. The Examples which follow demonstrate that titerplates configured as described herein not only result in unidirectional flow but also more faithfully recreate an in vivo environment when performing lab- on-chip experiments.

[0205] Example 1

[0206] Blood-brain-barrier model

[0207] For this example a unidirectional titerplate in accordance with the invention (Figure 8) was used to compare the effects of unidirectional flow. To create bidirectional flow the same microfluidic design was used but with a normal 384 well titreplate footprint (i.e. without wells merged) so that the second flow channel acting as a bypass or reflow was not in use during the experiment.

[0208] The gel inlet was filled with 1.2p] of 4mg/ml collagen I neutralized with (Cultrex 3D collagen-I Rat Tail, 5 mg/mL, 3447-020-01, AMSbio) 100 mM HEPES (15630-122, Thermo Fisher, Waltham, MA, USA) and 3.7 mg/mL NaHCO3 (S5761,

Sigma). 1 day prior to cell seeding, the first flow channel (alongside the gel and separated by the capillary pressure barrier) was coated with Matrigel-GFR. The plate was placed in a humidity- controlled environment at 37°C and 5% CO2 for 10 minutes for allow for gelation, subsequently 20 pl HBSS was added in the gel inlet reservoir to maintain hydration overnight, and 50ul was added to the observation window.

[0209] For cell seeding, 10K cells/pl of human brain microvascular endothelial cells (HBMECs) were seeded into the first flow channel from the inlet in the first reservoir and allowed to attach for 2.5 hours with 50ul media (Cell

Biologics H1168 with 10uM MMPi). After cell attachment, 50ul of media was added to the first reservoir and 100pl to the second reservoir. Plates were placed on a 7°, 8 minutes rocker to allow cells to expand and form a full tube, this allowed for cell culture media to passively perfuse through the lumen in a bidirectional fashion.

[0210] After 48 hours, media was changed and 40ul was added to first and second reservoir allowing for an air liquid interface to occur above the outermost hole in the chip once the plate was tilted on a rocker, resulting in unidirectional flow. The plates were placed on an asymmetric rocker at a 1 minute, 25° interval for the first flow channel containing cells and 15 seconds 25° for the second flow channel (the reflow channel). Cells were cultured for an additional 6 days before fixing with 3.7% formaldehyde solution, cells were stained for VE-CAD, DAPI and

Actin.

[0211] It can be seen from Figure 28 that the flow direction has an impact on both the actin cytoskeleton and VE-cadherin tight junction proteins. Tight junctions were less fuzzy when formed in the presence of unidirectional flow and actin filaments and cells align with the direction of flow.

[0212] Example 2

[0213] Vascular inflammation model with HCAECs

[0214] For this example, a unidirectional 2-lane plate was used with a bidirectional control using the same method described in example 1.

[0215] For seeding of the ECM, 1.1 uL of gel composed of 4 mg/mL collagen-I (Cultrex 3D collagen-I Rat Tail, 5 mg/mL, 3447-020-01, AMSbio), 100 mM HEPES (15630-122, Thermo Fisher, Waltham, MA, USA) and 3.7 mg/mL NaHCO3 (S5761,

Sigma) was dispensed in the gel inlet and the titerplate was incubated for 15 min at 37 °C. After gel polymerization, 50 ul of HBSS was added to the gel inlet reservoir and observation window. Plat es were placed in the incubator at 37°C, 5% CO2 overnight.

[0216] Human coronary artery endothelial cells (HCAECs) were harvested using Trypsan + 0.25 EDTA (Lonza, CC-5012) and neutralized with Trypsan

Neutralizing solution (Lonza, 5002). Cells were spun down at 220g for 5 minutes.

Cultures were resuspended to have a density of 10,000 HCAECs/ul. To seed the cells, 50 ul of MV2 medium was added to the first reservoir and 1 ul of cell suspension to the first flow channel of the second reservoir to create a passive pumping mechanism to introduce the cells to the first flow channel. Plates were incubated for 2 hours prior to the addition of 50 ul of MV2 medium to the first reservoir and 100 yl to the second reservoir inlet for the unidirectional conditions.

For the bidirectional conditions, 50 ul of MV2 medium was added to the perfusion inlet, bypass inlet and bypass outlet. Plates were placed at 7° 8 minute perfusion rocker for 3 days.

[0217] Medium was changed and replaced with 40 ul of MV2 medium in the first and second reservoir. Unidirectional conditions were placed on asymmetric rocker with 25° 1- minute interval for first flow channel and 25° 15 second interval for second flow channel. Bidirectional conditions were placed on symmetric 25° 1- minute interval rocker. On day 5 of culture, the plates were fixed in 3.7% formaldehyde (Sigma, 252549) and stored at 4°C.

[0218] After fixation, cultures were stained for immunofluorescent markers.

In short, cells were permeabilized using a Triton X-100 solution for 15 min and blocked using a buffer containing FBS, bovine serum albumin, and Tween-20 for 45 min. Primary antibody was incubated overnight, after which secondary antibody was incubated for 2 hours. The following primary antibodies were used to stain fixed cultures: CD31 1:160 (Dako, M0823) and Fibronectin 1:100 (Sigma,

F3648). The following secondary antibodies were used to stain fixed cultures: Goat anti-rabbit IgG (H+L) Alexa Fluor 555 1:250 (Thermo Fischer Scientific, A21428),

Alexa Fluor 647 1:250 (Biotium, 20040). Nuclei were stained using Hoechst (ThermoFisher, H3570). After staining, the OrganoPlate was transferred to a confocal high content imaging microscope for automated imaging (Micro XLS-C,

Molecular Devices). Images were acquired at 10x magnification at 3 pm increments along the height of the flow channel. Analysis was based on Sum Projection (ICAM expression, Fibronectin, Bodipy) of the full vessel or Max projection (CD31, Actin) images bottom 10 z-slices.

[0219] Immunofluorescent staining quantification was measured by analyzing the signal intensity per number of nuclei in a 10X image of a max projection of 10 slices. Directionality was determined using Max projection of bottom 10 slices of CD31. The Directionality tool in software Fiji was used to quantify alignment. Orientation] Analysis was used to create alignment images of

CDs.

[0220] The direction of flow was seen to impact the amount of fibronectin deposited per cell: fibronectin deposition is an indication of inflammation and was seen to increase for cell grown in bidirectional flow (Figure 30(b)) compared to unidirectional flow as seen in Figure 29 and Figure 30(a). It was also seen that cell alignment was significantly impacted by the direction of flow with cells in unidirectional flow aligning strongly with the flow direction (Figure 31 and 32(a)) with cells grown in bidirectional flow showing a polygonal morphology (Figure 32(b)). Tight junctions appear closer together in unidirectional flow and are seen to be more fuzzy in bidirectional flow.

[0221] Example 3 (H-bridge)

[0222] For this example, a titerplate according to Figures 22/23 was used.

[0223] The gel region was seeded with a mixture of 1 U/ml thrombin, 10 mg/ml Fibrinogen, 20,000 cells/pl of liver mixed non-parachymal cells (NPCs,

Lonza) and 20,000 cells/ul Hepatocytes (iCell). HUVEC (Lonza) were added at a density of 10,000 cells/pl: 2.75 pl was seeded in the first flow channel and 1.75ul in the second flow channel. 100ul of ECGM2 was added to the first and second reservoir and soul to the gel region, the plate was incubated at 5% CO2 at 37°C to allow for cell attachment and then placed on a rocker angled at 7° with an 8- minute interval for 4 days, with media exchanged at day 2.

[0224] At day 4 media in the first and second reservoir was replaced with 4opl of fresh media, and the rocker settings changed to 25° with a 1-minute interval to induce unidirectional flow across the vascular bed. At day 8, 8oul of peripheral blood mononuclear cells (PBMCs, Lonza) were suspended in media at a concentration of 10,000 cells/ul and 4oul was added to the first and second reservoir to perfusion of cells through the vascular network, this was recoded with an inhouse built platform enabling live cell imaging whilst rocking resulting is passive perfusion of cell culture media.

[0225] T-cells were shown to flow in a unidirectional manner through the capillary network:

in Figure 33 pathlines of fluorescently labelled PBMCs through the capillary network can be seen. A stagnant region (A) can be seen in the first half of the first flow channel which results in a pressure drop between the second flow channel and the first flow channel, this results in a unidirectional flow through the capillary network of cells exiting via the second half of the first flow channel (B).

The same action occurs when flow in the second flow channel is reversed but with the stagnant region at location B and flow seen in region A.

[0226] Example 4 (H-Bridge, vascular bed)

[0227] For this example, a titerplate according to Figures 22/23 was used.

[0228] The gel region was seeded with a mixture of 0.1 U/ml thrombin, 5 mg/ml Fibrinogen, 6,500 cells/ul of REP-HUVEC and 1,625 cells/ul NHLF (Lonza).

RFP-HUVEC were added at a density of 10,000 cells/ul with 2.75 pl seeded in the first flow channel and 1.75ul in the second flow channel. 100ul of ECGM2 was added to the first and second reservoir and soul to the gel region, and the plate was then incubated at 5% CO2 at 37°C to allow for cell attachment and subsequently placed on a rocker angled at 7° with an 8-minute interval for 4 days, with media exchanged at day 2.

[0229] At day 4 media in the first and second reservoir was replaced with 4oul of fresh media, and a upcyte hepatocyte donor 653 and RFP-HUVEC were seeded in the gel region as a spheroid at a ratio of 20,000:1,000, with the rocker settings changed to 25° with a 1-minute interval to induce unidirectional flow across the vascular bed. Cells were maintained until day 18 of culture and the perfusion of the system was imaged using 1:1000 FITC Dextran 4kDa to assess the lumen and flow through the vascular network.

[0230] Differences were seen in the morphology of the cells as a result of the unidirectional flow present through the vascular network. Figure 34(a) shows a vascular bed with minimal to no perfusion through the vascular network in a bidirectional flow setup. The network lacks direction and shows lumen which are inconsistent and asteroid shaped. In contrast, when the vascular network is subject to unidirectional flow (Figure 34(b), it forms in the direction of flow with consistent lumen with a close resemblance to that of an in vivo system.

[0231] The foregoing description demonstrates for the first time the successful implementation of unidirectional flow in a microfluidic network on a microtiter plate and is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention. It is not intended that the description set out in detail all modifications and variations which will become apparent upon reading the description. It is intended, however, that all such modifications and variations be included within the scope of the present invention, which is defined in and by the following claims.

Claims

Claims L Titer plate configured for unidirectional flow in a plurality of micro fluid networks disposed on the titer plate, at least one micro fluid network comprising: a micro fluid layer comprising a first flow channel and a second flow channel; and a reservoir layer disposed above the micro fluid network and comprising first and second reservoirs, the first reservoir and the second reservoir each having an access port to the first flow channel and an access port to the second flow channel such that the first flow channel and the second flow channel form a flow circuit with the first reservoir and the second reservoir; wherein the access ports in the first reservoir and the second reservoir are spaced apart from each other such that, in use, tilting the titer plate at a first angle causes a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir predominantly via the first flow channel, and adjusting the tilt so that the titer plate is tilted at a second angle so that a volume of fluid in the second reservoir flows from the second reservoir to the first reservoir predominantly via the second flow channel.

2. Titer plate according to claim 1, wherein the titer plate comprises at least 8 micro fluid networks, more preferably at least 16 micro fluid networks, more preferably at least 20 micro fluid networks, more preferably at least 32 micro fluid networks, more preferably at least 40 micro fluid networks.

3. The titer plate of claim 1 or claim 2, wherein the first reservoir and/or the second reservoir are located to correspond to the position of at least one well of an ANSI/SLAS microplate.

4. The titer plate of claim 3, wherein the first reservoir and/or the second reservoir are each sized to correspond to the position and volume of a plurality of wells of an ANSI/SLAS microplate.

5. The titer plate of any one of claims 1 to 4, wherein the titer plate is based on an ANSI/SLAS 384 well grid layout and at least one of the first reservoir and/or the second reservoir corresponds to three wells of the 384 well grid layout added together.

6. A titer plate according to any preceding claim, wherein each of the first and second reservoirs includes first and second end walls and side walls disposed therebetween, the end walls and side walls extending downwardly to a base in which the access ports are provided.

7. The titer plate of claim 6, wherein, in at least one of the first and second reservoirs, the access ports are non-uniformly spaced from each other and/or from the corresponding source positions of an ANSI/SLAS microplate.

8. The titer plate of claim 6 or claim 7, wherein, in at least one of the first and second reservoirs, at least one access port is spaced in an offset position relative to a center position of a corresponding well of an ANSI/SLAS microplate.

9. The titer plate of claim 6, wherein, in at least one of the first and second reservoirs, at least one access port is spaced from an end wall so as to be disposed toward a central portion of the reservoir relative to a center position of a corresponding well of an ANSI/SLAS microplate.

01

10. A titer plate according to any preceding claim, wherein, in at least one of the first and second reservoirs, the access ports are spaced from an end wall by at least 1 mm, preferably at least 1.25 mm, more preferably at least 1.5 mm.

u. A titer plate according to any one of claims 1 to 10, further comprising a strip configured to receive a gel, the strip being in direct fluid communication with at least one of the first flow channel and the second flow channel.

12. The titer plate of claim u, wherein the strip comprises a capillary pressure barrier defining a boundary between the strip and the first and/or second flow channel.

13. The titer plate of claim 11, wherein the strip is in fluid communication with the first flow channel and the second flow channel and provides a flow path from the first flow channel to the second flow channel.

14. The titer plate of claim 12 or claim 13, wherein the first flow channel has a greater length than the length of the second flow channel, with the access ports at each end of the first flow channel disposed at a periphery of the corresponding reservoir or at a periphery of the microfluidic network and the access ports at each end of the second flow channel disposed at an inner or central portion of the corresponding reservoir or at an inner or central portion of the microfluidic network.

15. The titer plate of any one of claims 12 to 14, wherein the strip comprises a first capillary pressure barrier defining a boundary between the strip and the first flow channel and a second capillary pressure barrier defining a boundary between the strip and the second flow channel.

16. The titer plate of claim 12 further comprising a second strip, the second strip being in direct fluid communication with the second flow channel and comprising a capillary pressure barrier defining a boundary between the second strip and the second flow channel.

The titer plate of any one of claims 1 to 12, wherein the strip comprises a first capillary pressure barrier defining a boundary between the strip and the first flow channel and a second capillary pressure barrier defining a boundary between the strip and a third flow channel, the third flow channel forming a second flow circuit with a fourth flow channel provided in the microfluid layer and at least two further reservoirs of the plurality of reservoirs.

18. The titer plate of claim 17, wherein the third flow channel connects at one end to an access port in a third reservoir and at another end to an access port in a fourth reservoir and the fourth flow channel connects at one end to an access port in the third reservoir and at another end to an access port in the fourth reservoir, wherein the microfluidic network has a plane of reflection symmetry transverse to the plane of the microfluidic network and extending along the strip such that access ports of the first and third flow channels mirror each other and the access ports of the second and fourth flow channels mirror each other.

19. The titer plate of claim 17, wherein the third flow channel connects at one end to an access port in a third reservoir and at another end to an access port in a fourth reservoir and the fourth flow channel connects at one end to an access port in the third reservoir and at another end to an access port in the fourth reservoir, wherein the strip includes a center of symmetry of the microfluidic network such that the access ports of the fourth flow channel are the inversion of the access ports of the first flow channel through the center of symmetry and the access ports of the third flow channel are the inversion of the access ports of the second flow channel through the center of symmetry.

20. A method for inducing unidirectional flow in a microfluidic titer plate having a plurality of microfluidic networks, at least one microfluidic network comprising: a microfluidic layer comprising a first flow channel and a second flow channel; and a reservoir layer comprising first and second reservoirs, the first reservoir and the second reservoir each having an access port to the first flow channel and an access port to the second flow channel such that the first flow channel and the second flow channel form a flow circuit with the first reservoir and the second reservoir; the method comprising: tilting the titer plate at a first angle by rotating about a first axis to cause a volume of fluid in the first reservoir to flow from the first reservoir to the second reservoir via the first flow channel; and adjusting the tilt so that the titer plate is tilted at a second angle by rotating about the first axis, causing the volume of fluid in the second reservoir to flow from the second reservoir to the first reservoir via the second flow channel.

21. The method of claim 20, wherein the second angle is the inverse angle of the first angle.

22. The method of claim 20 or 21, wherein tilting the titer plate at the first angle exposes the top access port of the first reservoir to air to thereby decouple fluid in the top access port of the first reservoir and in the second flow channel of fluid in the first reservoir; and/or wherein tilting the titer plate at the second angle exposes the top access port of the second reservoir to air to thereby decouple fluid in the top access port of the second reservoir and the first flow channel of fluid in the second reservoir.

23. The method of claim 20-22, further comprising: after tilting the titer plate at the first angle by rotating about the first axis, tilting the titer plate at a third angle rotating about a second axis orthogonal to the first axis.

24. The method of claim 23, further comprising: after tilting the titer plate at the second angle by rotating about the first axis, tilting the titer plate at a fourth angle by rotating about the second axis that is orthogonal to the first axis.

25. Method according to any one of claims 20 to 23, comprising continuously varying the tilt of the titer plate in a three-dimensional spinning movement.

26. The method of any one of claims 20 to 23, further comprising rotating the tilted plate about an axis normal to the plane of the titer plate.

27. The method of any one of claims 20 to 26, wherein the at least one microfluidic network comprises a strip in fluid communication with at least the first flow channel, and wherein the method further comprises introducing a gel, or precursor thereof, into the strip.

28. The method of claim 27, further comprising: cultivating a first cell type in the strip or in at least the first flow channel.

29. The method of claim 28, wherein the first cell type comprises endothelial or epithelial cells, the method further comprising allowing said cells to form a tubule, the lumen of which is perfused by fluid flow in substantially one direction.

30. The method of claim 29 comprising flowing cells through the tubule within the first flow channel in a substantially single direction and recirculating the cells through the second flow channel.

31. The method of claim 30, wherein the cells comprise immune cells, tumor cells or stem cells.

32. The method of any one of claims 20 to 31, wherein the microfluidic network comprises a first capillary pressure barrier defining a boundary between the gel strip and the first flow channel and a second capillary pressure barrier defining a boundary between the gel strip and the second flow channel, the method comprising introducing a gel or gel precursor comprising endothelial cells into the gel strip and/or one or both of the first flow channel and the second flow channel and allowing the endothelial cells to form a vascular network within the gel connecting the first flow channel to the second flow channel.

33. The method of claim 32, wherein tilting the titer plate at the first angle causes the volume of fluid in the first reservoir to flow from the first reservoir at least partially through the vascular network to the second network and tilting the titer plate at a second angle causes the volume of fluid in the second reservoir to flow from the second reservoir at least partially through the vascular network to the first reservoir, the flow through the vascular network being in substantially the same direction at each tilt angle.

A method according to claim 32 or claim 33, wherein the microfluidic network comprises a first capillary pressure barrier defining a boundary between the gel strip and the first flow channel and a second capillary pressure barrier defining a boundary between the gel strip and a third flow channel, the third flow channel forming a fluid circuit with a fourth flow channel, the third flow channel and fourth flow channel each connecting at one end to an access port in a third reservoir and at the other end to an access port in a fourth reservoir, and the first through fourth flow channels are configured such that tilting the titer plate at the first angle causes fluid from the third reservoir to flow through the third flow channel to the fourth reservoir and tilting at the second angle causes fluid from the fourth reservoir to flow through the fourth channel to the third reservoir.

A method according to claim 32 or claim 33, wherein the microfluidic network comprises a first capillary pressure barrier defining a boundary between the gel strip and the first flow channel and a second capillary pressure barrier defining a boundary between the gel strip and a third flow channel, the third flow channel forming a fluid circuit with a fourth flow channel, the third flow channel and fourth flow channel each connecting at one end to an access port in a third reservoir and at the other end to an access port in a fourth reservoir, and the first through fourth flow channels being configured such that tilting the titer plate at the first angle causes flow through the fourth reservoir through the third flow channel to the third reservoir and tilting at the second angle causes fluid flow from the third reservoir through the third channel to the fourth reservoir.

36. The method of claim 35, wherein a second cell type is introduced into the third channel.

37. The method of any one of claims 20 to 36, wherein the at least one microfluidic network comprises a second strip in fluid communication with the second flow channel, the method further comprising: introducing a gel, or precursor thereof, into the second strip, the gel or precursor thereof optionally comprising cells.

38. The method of any one of claims 20 to 37, wherein the titer plate is as described in any one of claims 2 to 19.