Microphysiological system

Abstract

A microphysiological device system comprises a base portion and a body portion, wherein one of the base portion and the body portion comprises a cell culture chamber and the other of the base portion and the body portion comprises a mould. A deformable membrane separates the cell culture chamber from the mould. An actuator is configured to cause the deformable membrane to conform to the shape of the mould. The system may be used as an organ-on-chip platform. Also provided is a device, a kit for assembling a microphysiological system, and a method of cell and / or tissue culture.

Description

[0001] Microphysiological system

[0002] Field of the invention

[0003] The invention relates to a microphysiological device and system which may be used as an organ-on-chip platform. The system is configured to cause a deformable membrane to conform to the shape of a solid mould. Also provided is a kit for assembling a microphysiological system, and a method of cell and / or tissue culture.

[0004] Background to the invention

[0005] Drug testing is a long, complex, and expensive process. Current in vitro models are unable to accurately test and screen drug safety and efficacy, whereas animal models are often poor representations of the human condition.

[0006] In recent years, there has been a surge of interest in microphysiological systems (MPSs) such as organ-on-a-chip (OoC) technologies. The chips utilize microfluidics and organ cell cultures to create advanced in vitro test systems. OoC platforms have the potential to offer huge value at various stages of drug development. The key aim of OoC platforms is to provide a more accurate representation of human (patho)physiology than current animal or in vitro models, which will lead to new insights into basic disease mechanisms, help identify pharmacological targets, enable high-throughput screening of compound pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion (ADME) and toxicity of chemical substances), offer significant opportunities for precision and personalized medicine, and could expedite regulatory review.

[0007] OoC platforms have enabled researchers, both academic and industrial, to grow tissues and organ-mimics in either 2D or 3D systems. In 2D systems, cells are grown as a monolayer on an appropriate biocompatible substrate material or membrane. 3D systems involve either seeding / encapsulating cells onto / into a scaffolding biomaterial or generating tissues in a scaffold-free system resulting in the formation of cell aggregates, spheroids, microtissues, or organoids. However, in both approaches, the shape of the resulting cellular environment is fixed, i.e. the cells within 2D environments cannot become 3D within the chip, or cells within a 3D system cannot undergo further complex geometric changes.

[0008] Actively actuated OoCs incorporate active mechanical stimulation to the cell cultures within an OoC. These systems aim to stimulate the cells and cause a desired physiological response, and thus can allow an even better approximation of in vivo conditions. However, active actuation alone does not account for complex geometric changes in the cellular environment. As such, there is an imminent demand for the development of new advanced / complex in vitro model systems that better mimic human tissues in healthy and diseased states as test platforms for therapeutic development.

[0009] Summary of the invention

[0010] According to a first aspect of the invention, there is provided a microphysiological system comprising: a base portion and; a body portion, wherein at least one of the base portion and the body portion comprises a cell culture chamber and at least the other of the base portion and the body portion comprises a mould; a deformable membrane separating the cell culture chamber from the mould; and an actuator configured to cause the deformable membrane to conform to the shape of the mould.

[0011] In some embodiments, the base portion comprises a cell culture chamber and the body portion comprises a mould.

[0012] In some embodiments, the base portion comprises a mould and the body portion comprises a cell culture chamber.

[0013] It will be appreciated that a cell culture chamber and / or a mould may be provided on both sides of the deformable membrane. Thus, in some embodiments, both the base portion and the body portion comprises a cell culture chamber. In some embodiments, both the base portion and the body portion comprises a mould. In some further embodiments, both the base portion and the body portion comprises a cell culture chamber and a mould.

[0014] According to a second aspect of the invention, there is provided a device for a microphysiological system, the device comprising: a body portion for locating on a base portion, the body portion comprising a cell culture chamber or a mould; a deformable membrane; and an actuator configured to cause the deformable membrane to conform to the shape of the mould.

[0015] In some embodiments, the body portion comprises a mould. In some embodiments, the body portion comprises a cell culture chamber.

[0016] In some embodiments, the body portion comprises a cell culture chamber and a mould e.g. a mould may be located in the cell culture chamber.

[0017] According to a third aspect of the invention, there is provided a kit for assembling a microphysiological system, the kit comprising: a body portion or a base portion comprising a mould; and an actuator configured to cause a deformable membrane to conform to the shape of the mould.

[0018] According to a fourth aspect of the invention, there is provided a method of cell and / or tissue culture, the method comprising: culturing cells or tissue on a deformable membrane; and conforming the membrane, and the cells or tissue thereon, to the shape of a mould.

[0019] According to a fifth aspect of the invention, there is provided the use of the system according to the first aspect of the invention, the device according to the second aspect of the invention, the kit according to the third aspect of the invention and / or the method according to the fourth aspect of the invention to model a biological process or a disease, or to generate a cell aggregate, a biological tissue, a spheroid, an organoid or an organ.

[0020] Brief description of figures

[0021] Embodiments of the invention will now be described by way of example with reference to the figures in which:

[0022] Figure 1 shows an exploded view schematic representation of a system according to the invention;

[0023] Figure 2 shows a face-on exploded view schematic representation of the system of Figure 1 ; Figure 3 shows a side-on exploded view schematic representation of the system of Figures 1 and 2;

[0024] Figure 4 shows schematic representations of a system according to the invention, shown in exploded (4A), face-on (4B), and side-on views (4C);

[0025] Figure 5 shows schematic representations of a device according to the invention, shown in assembled (5A), bottom up (5B), and exploded views (5C);

[0026] Figure 6 shows schematic representations of a device according to the invention, shown in assembled (6A), bottom up (6B), and exploded views (6B);

[0027] Figure 7 shows schematic representations of a multi-well configuration in which multiple devices according to the invention are used in series with each other, shown in side views (7A, 7B, 7D) and top view (7C). The devices can be connected so that cell growth medium can be supplied to specific devices e.g. in series;

[0028] Figure 8 shows schematic representations of simple “in-plane” membrane deformations achievable in existing OoC platforms (Figures 8A) and complex “out-of-plane” deformations achievable in embodiments of the invention (Figure 8B). The upper images show top views of the membrane, while the lower images show side views; and

[0029] Figure 9 shows confocal images of a membrane in a microphysiological system according to an embodiment of the invention, in static (2D) (Figures 9A, 9D), low actuation (2.5D) (Figures 9B, 9E) and full actuation (3D configurations) (Figures 9C, 9F) (scale bar = 200 pm). Figures 9A, 9B and 9C show a front view. Figures 9D, 9E and 9F show an isometric view;

[0030] Figure 10 shows an alternative configuration of a system according to the invention, shown in the unactuated (Figure 10A) and actuated (Figure 10B) configurations;

[0031] Figure 11 shows an alternative configuration of a system according to the invention, shown in the unactuated (Figure 11 A) and actuated (Figure 11 B) configurations;

[0032] Figure 12 shows an alternative configuration of a system according to the invention, wherein there are moulds on either side of the membrane, shown in the unactuated (Figure 12A) and actuated (Figure 12B) configurations;

[0033] Figure 13 shows a further configuration of the system of Figure 12 in the actuated configurations;

[0034] Figure 14 shows the system of Figure 13 in use as a lung MPS, depicting exhalation (Figure 14A); inhalation (Figure 14B) and an enlarged view of the membrane (Figure 14C).

[0035] Figure 15 shows schematic representations of a slide format configuration of a system according to the invention. Figure 15A shows an exploded view, Figure 15B shows a side-on exploded view, Figure 15C shows an assembled side-on view, and Figure 15D shows a top- down view of the slide format configuration. Figures 15E-H show the same views as 15A-15D but include a lid on the cell culture chamber;

[0036] Figure 16 shows a microtitre (well) plate format configuration of a system according to the invention (exploded (Figure 16A), side-on exploded (Figure 16B), and assembled side, top down, and front views (Figure 16C)); and

[0037] Figure 17 shows schematic representations of further embodiments of systems according to the invention. Depicted are an actuated system comprising an open cell culture (Figure 17A), and non-actuated systems comprising open cell cultures with various combinations of air and liquid media (Figure 17B, 17C, 17D, 17E). Figures 17F, 17G 17H and 171 show a further embodiment that includes a lid. Figures 17J and 17K show a further embodiment which includes a mould with additional texture. Figure 17L shows a system according to the invention in use. Figure 17M shows a system of the invention having regular (17Mi), textured (17Mii), and porous (17Miii and 17Miv) membranes. In a first aspect, the invention provides a microphysiological system comprising: a base portion and a body portion, wherein one of the base portion and the body portion comprises a cell culture chamber and the other of the base portion and the body portion comprises a mould; a deformable membrane separating the cell culture chamber from the mould; and an actuator configured to cause the deformable membrane to conform to the shape of the mould.

[0038] In use, the base portion may form a bottom or lower component of the system. The body portion may be located on or supported by the base portion.

[0039] In some embodiments, the base portion comprises at least one cell culture chamber. In other embodiments, the body portion comprises at least one cell culture chamber.

[0040] It may be that both the base portion and the body portion comprises at least one cell culture chamber.

[0041] It will be appreciated that a cell culture chamber is a space which is suitable for growing cells or tissue. In use, the cell culture chamber may be filled with a suitable fluid for growing cells, e.g. a liquid such as culture media. In some cases, it may be that in use the cell culture chamber is filled with a gas. The gas composition, humidity, pressure and / or temperature may be selected to simulate certain conditions or environments. For example, the gas may be selected to simulate atmospheric or ambient conditions. In another example, the gas may comprise an additional component e.g. an aerosol, a particulate or a pollutant. In some embodiments, the gas is air, for example to replicate lung tissue which is exposed to air.

[0042] In some embodiments, the base portion and / or the body portion comprises a single cell culture chamber. In other embodiments, the base portion and / or the body portion comprises at least two cell culture chambers, or a plurality of cell culture chambers. The base portion and / or the body portion may comprise at least 2, 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 800 or at least 1000 cell culture chambers. For example, the base portion and / or the body portion may comprise 4, 6, 12, 24, 48, 96, 384, or 1536 cell culture chambers.

[0043] The base portion and / or the body portion may further comprise one or more channels (e.g. microfluidic channels) for delivering fluids into and / or out of the or each cell culture chamber, or for delivering fluids to the membrane. The one or more channels may further enable sampling and / or the delivery of reagents. In some embodiments, the base portion comprises an inlet channel and an outlet channel.

[0044] As is known in the art, a “microfluidic” channel is a tiny channel typically having a diameter of from 1 to 1000 micrometers, or from 10 to 100 micrometers. Microfluidic channels are capable of handling nanolitres to microlitre volumes of liquid.

[0045] In some embodiments, one or more channels (e.g. microfluidic channels) are provided in both the body portion and the base portion. This advantageously provides two independent fluidic networks, one which is in fluid communication with one side of the membrane, while the other is in fluid communication with the other side of the membrane. Thus, one fluidic network can be used to supply materials (e.g. cells, tissue, or soluble factors such as nutrients, drugs or bioactives) above the membrane, while the other can be used to supply materials below the membrane.

[0046] The base portion and / or the body portion may further comprise one or more sensors, such as biosensors. The sensor(s) may enable the detection of molecular and / or cellular changes. In response to a signal from the cell or tissue (e.g. a chemical signal, a movement, a protein), the sensor(s) may generate signals which can then be interpreted by software operated by a computer. The computer may relate the signals to a therapeutic effect. The computer may output information based on the signal(s) detected to a user.

[0047] The base portion and the body portion may be formed from any suitable material. In some embodiments, the base portion and the body portion are formed from a material independently selected from a polymer (e.g. PDMS, polystyrene, polypropylene, polyethylene, or polycarbonate), glass, metal (e.g. stainless steel, titanium, cobalt-chromium alloy, aluminium, platinum, palladium or gold), and ceramic (e.g. alumina, aluminosilicate, hydroxyapatite).

[0048] The base portion and / or the body portion may be at least partially transparent. In some embodiments, the base portion and / or the body portion is wholly transparent. In some embodiments, the base portion and / or the body portion comprises a window through which the cells in the cell culture chamber(s) may be observed, when the system is in use. Conveniently, these features allow for visual evaluation of the ongoing culture, e.g. using microscopy.

[0049] In some embodiments the base portion and / or the body portion comprises one or more cavities or wells therein, each of which defines a cell culture chamber. In other embodiments the base portion and / or the body portion comprises one or more holes therethrough, each of which partially defines a cell culture chamber. The microphysiological system may further comprise one or more covers which, when attached to the base portion of the body portion, form a wall of the cell culture chamber. The cover(s) may be attached by any convenient means, such as adhesive.

[0050] Conveniently, the base portion and / or the body portion may have dimensions which correspond to those of a standard multi-well plate or microscopy slide. This allows compatibility of the system with existing laboratory instrumentation and analytical techniques. However, it will be appreciated that the size and shape of the base portion is not restricted to this particular configuration.

[0051] For example, in some embodiments, the base portion and / or the body portion may have a length and a width which correspond to the length and width of a standard microscopy slide (e.g. approximately 76 mm long and approximately 25 mm wide), thereby enabling the system to be compatible with a microscope.

[0052] In some embodiments, the base portion and / or the body portion may have a length and a width which correspond to the length and width of a standard multi-well plate (e.g. 127.76 mm long and 85.48 mm wide ± 0.5 mm). In some embodiments, the base portion is sized in accordance with one or more of the following ANSI / SLAS microplate standards: ANSI SLAS 1-2004 (R2012): Footprint Dimensions; ANSI SLAS 2-2004 (R2012): Height Dimensions; ANSI SLAS 3-2004 (R2012): Bottom Outside Flange Dimensions; ANSI SLAS 4-2004 (R2012): Well Positions; ANSI SLAS 6-2012: Well Bottom Elevation.

[0053] In some embodiments, the base portion and / or the body portion comprises a plurality of wells, each of which defines a cell culture chamber. It may be that the base portion and / or the body portion comprises at least 2, 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 500, 800 or at least 1000 wells. For example, the base portion and / or the body portion may comprise a plurality of wells in a lay-out similar to that of a standard multi-well plate, e.g. 6, 12, 24, 48 or 96 wells.

[0054] In some embodiments, for each well (i.e. each cell culture chamber) provided in one of the base portion and the body portion, a corresponding mould may be provided in the other of the base portion and the body portion, and a corresponding membrane may be positioned between each well and its corresponding mould. For example, in embodiments wherein the base portion comprises n wells, the body portion may comprise n moulds and n membranes. It may be that a single actuator is configured to cause each membrane to simultaneously conform to the shape of its corresponding mould. A multi-well configuration advantageously increases the experimental output of the cell culturing system.

[0055] Thus, in some embodiments the number of membranes in the system is the same as the number of moulds and the number of cell culture chambers.

[0056] In other embodiments, the number of membranes is different to the number of moulds and / or the number of cell culture chambers.

[0057] In some embodiments, the number of cell culture chambers is greater than the number of membranes and / or moulds. For example, it may be that one or more cell culture chambers (e.g. wells) are occupied by biological material such as cells or tissue (e.g. a patient biopsy) that are not cultured on a membrane, but are required in the microphysiological model. In this context, the system may comprise an arrangement in which one or more pharmacologically relevant cells / tissues / organs are cultured on a membrane and connected to one or more cell culture chambers (without a corresponding membrane) containing the patient derived tissues / cells. This would enable testing of treatment strategies on the target as well as relevant organ systems. It may therefore be valuable to provide cell culture chambers that do not have a corresponding membrane or mould, but that are still part of the overall device / system.

[0058] In some embodiments, a cell culture chamber is provided on both sides of the membrane. In such embodiments, the system may comprise: a base portion comprising a first cell culture chamber; a body portion comprising a second cell culture chamber; wherein the first and / or second cell culture chamber comprises a mould; and a deformable membrane separating the first cell culture chamber(s) from the second cell culture chamber(s).

[0059] A system which enables cells to be cultured on both sides of the membrane advantageously enables the modelling of more complex biological systems. For example, in a system that is configured to mimic the lungs, vascular endothelial cells may be cultured on one side of the membrane to mimic blood vessels. Cell culture on both sides of the membrane may also be useful for modelling immune cell invasion into tissue cultured on the membrane. In some embodiments, the system comprises a plurality of wells and a single membrane, wherein a portion of the membrane is positioned over each well (i.e. instead of having a separate membrane for every well).

[0060] In some embodiments, the or each cell culture chamber comprises a further mould. In some embodiments the deformable membrane separates the mould from the further mould.

[0061] The base portion may further comprise securing means. It may be that the securing means is for securing a membrane to the base portion. In some embodiments, the base portion may comprise one or more holes for receiving screws for attaching the base portion to the body portion. In some embodiments, screw holes are located at the four corners of the base portion. This allows a secure connection between the body portion and the base portion.

[0062] The base portion may be fabricated by any suitable means. Conveniently, the base portion may be fabricated using 3D printing. Alternatively, the base portion may be fabricated using injection moulding.

[0063] The membrane provides a substrate on which cells are cultured. Preferably, the membrane is biocompatible. In some embodiments, the membrane is formed from a biocompatible polymer. The polymer may be natural or synthetic. Suitable biocompatible polymers include polysiloxanes (e.g. polydimethylsiloxane (PDMS)), polycarbonate (PC), polyethylene terephthalate (PET), poly-lactic acid (PLA), poly(lactic-coglycolic acid) (PLGA), or mixtures thereof. In some embodiments, the polymer may be a mixture of PDMS and one or more other polymers, such as, polyethylene glycol (PEG).

[0064] In some embodiments the membrane is formed from a naturally occurring protein, such as collagen, or polysaccharide, such as chitosan. Other suitable membrane materials are described by Corral-Najera et al. (2023) 9:107 Microsystems & Nanoengineering.

[0065] In some embodiments, the membrane comprises a surface modification. The surface modification may be present on one side (surface) of the membrane (e.g. the side / surface facing the cell-culture chamber), or it may be present on both sides (surfaces) of the membrane. The surface modification may be present on the whole of the surface, or it may be present on only a part (e.g. selected regions) of the surface of the membrane.

[0066] The surface modification may comprise a coating, a surface functionalisation of the membrane, or a combination of both. The term “coating” will be understood as a surface layer that is added to the membrane but which is distinct from the membrane in that it does not chemically bind to or otherwise change the chemical structure of the membrane itself (analogous to a coat of paint). The term “functionalisation” (or “functionalised”) refers to the addition of a component to the membrane which results in chemical modification of the molecules at the surface of the membrane, in order to alter the physical, chemical and / or biological properties of the membrane surface.

[0067] In some embodiments, the membrane is wholly or partially coated. In some embodiments, the membrane is wholly or partially functionalised. In some embodiments, the membrane is both surface functionalised and coated (wholly or partially). For example, at least part of the membrane surface may be functionalized with a chemically bound protein, and the chemically bound protein may be coated with growth factors.

[0068] The coating and / or surface functionalisation can be selected by the skilled person according to the desired function. It may be that the coating and / or surface functionalisation comprises a material that promotes or prevents cell adhesion to the membrane, prevents permeation through the membrane and / or instructs cell behaviour (e.g. in terms of morphology, proliferation, migration and / or differentiation). It may be that the coating and / or surface functionalisation comprises a component that mimics one or more aspects of the extracellular matrix. For example, a cell-adhesive membrane material may be coated, in-part, by a nonadhesive (or anti-fouling) coating. This advantageously provides an additional level of control over protein or cell patterning as it enables regions of the membrane to be blocked or masked.

[0069] The coating and / or the surface functionalisation may comprise an extracellular matrix component. In some embodiments the coating or the surface functionalisation comprises a peptide (e.g. a cell-penetrating peptide, an adhesion peptide, a growth-factor mimicking peptide, and / or a signalling peptide), a protein (such as collagen, fibronectin, laminin, a growth factor and / or a cytokine), a proteoglycan (e.g. aggrecan), a glycosaminoglycan (e.g. hyaluronic acid, chondroitin sulphate), an inorganic compound (e.g. hydroxyapatite), a synthetic, semi-synthetic, or naturally-occurring hydrogel (e.g. Matrigel, polyethylene glycol (PEG), poloxamer (e.g. Pluronic F-127)), a chemically inert material (e.g. parylene), or any combination thereof.

[0070] It will be appreciated that some of the components above (e.g. chemically inert materials such as parylene) may be more suited to coating a membrane, while others will be more suited for use in surface functionalisation of the membrane. In some cases, the component may be applied to the membrane either as a coating or as a surface functionalisation. For example, fibronectin (and other cell binding proteins) can be used to functionalize the surface, e.g. using APTES and glutaraldehyde to covalently bind the protein to the membrane’s surface. Equally, the fibronectin could alternatively be physisorbed onto the membrane surface as a coating.

[0071] The coating and / or the surface functionalisation may be permanent or it may be temporary / transient. For example, it may be that the coating and / or the surface functionalisation can be disrupted or removed from the surface of the membrane in response to certain stimuli (e.g. the addition of a solution, changes in pH, or changes in temperature).

[0072] The membrane may be fabricated using any suitable method known to those skilled in the art, such as the methods described herein.

[0073] In some embodiments, the membrane is fabricated using spin-coating. Spin-coating processes will be known to those skilled in the art, and are described hereinbelow.

[0074] Briefly, uncured (i.e. liquid) polymer is placed onto a substrate, which is loaded into a spincoater device. The substrate is then rotated (e.g. >500 RPM) for a period of time (e.g. 5 seconds to 30 minutes), allowing a layer of uncured polymer to coat the substrate, with the excess being removed by centrifugal force. The membrane thickness can be tuned by varying the speed (RPM) and time, achieving thicknesses from tens to hundreds of micrometres.

[0075] After the spin-coating process, the polymer is cured. This can be done at room temperature (e.g. on the benchtop), or at elevated temperatures (e.g. 60 to 120 °C), for example on a hotplate or inside an oven. The specific curing temperature is largely dependent on the thermostability of the substrate material. After curing (and optionally cooling), the membrane is removed from the substrate. The membrane may be stored in a dust-free environment or container until use.

[0076] Optionally, the substrate can be pre-treated to form a sacrificial layer. The purpose of this layer is to aid removal of the membrane from the substrate after curing. The sacrificial layer can be applied to the substrate by any suitable method, such as spraying, painting or dipping. In some embodiments the sacrificial layer is formed by dipping the substrate in a liquid (e.g. a Polyvinyl alcohol (PVA) solution). The substrate may then be dried (e.g. allowed to air-dry) prior to spincoating with. The sacrificial layer can later be removed, e.g. by dissolving the sacrificial layer in a solvent, such as water. The membrane is deformable. By “deformable”, it will be understood that the shape of the membrane can be changed by the application of force.

[0077] In some embodiments, the membrane is reversibly deformable. Thus, upon removal of a deforming force, the membrane will revert to its original shape. In other words, the membrane may undergo elastic deformation upon application of a force. The use of a reversibly deformable material allows the membrane to undergo controlled deformations in a reversible manner. This facilitates the accurate conformation of the membrane to complex geometries as well as cyclic deformation / relaxation of the membrane.

[0078] In other embodiments, the membrane is irreversibly deformable. Thus, upon removal of a deforming force, the membrane remains in its deformed shape. In otherwords, the membrane may undergo plastic deformation upon application of a force.

[0079] It may be that the membrane lies in a plane and the system is configured such that operation of the actuator causes out-of-plane deformation of the membrane. Thus, prior to actuation, the membrane may be substantially planar.

[0080] The membrane may be smooth (i.e. non-patterned) or it may be patterned. In embodiments wherein the membrane is fabricated by spin-coating, the substrate used during the spincoating process can be used to control the surface finish of the membrane. For example, a smooth substrate, e.g. glass or silica, can be used to prepare a smooth membrane, while a patterned substrate (e.g. a micropatterned or microstructured) substrate can be used to prepare a patterned membrane. By controlling the design of the substrates during membrane fabrication, user-defined membrane patterns with known features (extruded or embossed, or pores) and dimensions can be reproducibly created.

[0081] In some embodiments, the membrane is patterned. Advantageously, patterning can be used to provide cues to the residing cells. The patterning may be configured to influence cell patterning, differentiation, orientation, migration, and / or tissue maturation. Additionally, a patterned membrane may be configured to support enhanced cellular integration. This may help to enable membrane deformation without the growing cells and developing tissue detaching. The patterning may be regular or irregular (e.g. random).

[0082] Patterning of the membrane may be achieved by any suitable process known to those skilled in the art. It may be that the membrane is patterned by chemical and / or physical patterning. In some embodiments, the membrane is chemically patterned. The membrane may be chemically patterned by selectively coating or functionalising regions of the membrane with an additional component, as described above.

[0083] In some embodiments, the membrane is physically patterned. In other words, the membrane may be textured. In some embodiments, the membrane comprises a physical pattern or texture formed from embossed regions (i.e. protrusions, bumps, ridges), recessed regions (e.g. hollows, channels), cut-out regions and / or pores. Physical patterning may be achieved using a laser or a chemical-based etching process.

[0084] In some embodiments, the membrane is both chemically and physically patterned. It may be that the chemical patterning is correlated with the physical patterning. For example, a first protein may be applied to the concavities of the physical micropattern / microstructure of the membrane, and a second protein may be applied to the convexities of the physical micropattern / microstructure of the membrane.

[0085] The pattern may be formed on a macro (i.e. mm), micro (i.e. pm) or nano (i.e. nm) scale. In some embodiments, the membrane is micropatterned (i.e. the pattern is formed on a micro scale).

[0086] In some embodiments, the membrane is porous. Creating porosity can be advantageous because it enables the membrane to better mimic physiological interfaces or barrier functions, such as epithelium and endothelium function in the intestine, kidneys, lung, skin, blood vessels, and the blood-brain barrier. Porous membranes also facilitate the study or modelling of mass transport across barriers, e.g. mimicking liver function for toxicity testing. The use of a porous membrane can also allow products released from cells to diffuse to another cell type that are not in direct contact. For example, injured or diseased cells (including cancer cells) release soluble inflammatory signals that impact another cell type such as immune cells.

[0087] The membrane may comprise pores which are formed as through-holes (holes which extend through the membrane from one side to the other). Alternatively, the membrane may have a sponge- or foam-like porous structure. Methods of forming porous membranes are described by Quiros-Solano et al., Nature Scientific Reports volume 8, Article number: 13524 (2018).

[0088] The pores may have an average diameter of from 2 to 10 pm, from 3 to 8 pm or from 4 to 6 pm. Pore spacing can be tuned to change the % porosity of the membrane. The membrane may have a porosity of from 2 to 80%, from 3 to 70%, from 4 to 65%, from 5 to 60%, from 6 to 50%, from 8 to 40%, from 9 to 30% or from 10 to 20%.

[0089] The properties of the membrane, such as the thickness, surface area and / or mechanical stiffness of the membrane can be selected according to the desired deformation and consequently the known mechanical strain on any resident cells or tissue.

[0090] The or each membrane may have a thickness of from 10 to 1000 pm, from 25 to 800 pm, from 50 to 700 pm, from 100 to 500 pm, from 150 to 400 pm or from 200 to 300 pm.

[0091] The size and / or shape of the membrane can also be selected by the skilled person as required, for example depending on whether a single- or multi-well configuration is used, and whether each cell culture chamber has a corresponding membrane or whether a single membrane is provided for multiple cell culture chambers. In some embodiments, the or each membrane is square or rectangular. In some embodiments, the or each membrane is circular.

[0092] It will be appreciated that the stiffness of the membrane may depend on a number of factors including the membrane thickness, the membrane material and the preparation process. For PDMS membranes the stiffness will depend on the mixing ratio of the pre-polymer resin with the cross linker, as well as the curing temperature and time. By way of example, for a PDMS membrane (Sylgard 184 10:1 mix) having a thickness of 25 pm, 150 pm or 400 pm, the corresponding stiffness (Young’s modulus, E) is 2.836 MPa, 1.464 MPa or 1.019MPa, respectively (calculated using the methods described by Gao et al., Journal of Microelectromechanical Systems ( Volume: 24, Issue: 6, 2015).

[0093] The strain distributions that cells are exposed to during deformation may be simulated using commercial software packages, such as Abaqus, Ansys, COMSOL, or open-source packages such as FEBio.

[0094] In use, the body portion may form a top or upper component of the system. For example, it may be that the body portion serves as a lid which is placed over one or more cell culture chamber(s) of the base portion.

[0095] In another example, it may be that the body portion contains one or more cell culture chambers When present in the body portion, the cell culture chamber(s) will be located above the membrane. The one or more cell culture chambers in the body portion may be open or closed. An open cell culture chamber advantageously enables the addition of cells, nutrients, media and / or a cell-laden hydrogel into the cell culture chamber.

[0096] The body portion may be fabricated by any suitable means. Conveniently, the body portion may be fabricated using 3D printing. Alternatively, the body portion may be fabricated using injection moulding.

[0097] The body portion may be removably attachable to the base portion. For example, the body portion may comprise one or more holes for receiving screws for attaching the body portion to the base portion. In some embodiments, screw holes are located at the four corners of the body portion. This allows a secure connection between the body portion and the base portion.

[0098] In some embodiments, the system further comprises a lid. The lid may be removable. For example, it may be that in embodiments wherein the body portion comprises one or more cell culture chambers, a removeable lid may be provided for closing the cell culture chamber(s). The lid may be configured to fit onto or over the cell culture chambers. It may be that the lid is considered to comprise a part of the body portion.

[0099] In some embodiments, the lid comprises one or more channels (e.g. microfluidic channels) for delivering fluids into and / or out of the or each cell culture chamber.

[0100] In some embodiments of the assembled system, the membrane will be positioned between the body portion and the base portion such that it separates the cell culture chamber(s) located in one of the base portion and the body portion from the mould(s) located in the other of the base portion and the body portion. It may be that attachment of the body portion to the base portion holds the membrane in place.

[0101] In some embodiments, the membrane is positioned between the body portion and the base portion such that it separates a first cell culture chamber located in the base portion from a second cell culture chamber located in the body portion.

[0102] The microphysiological system comprises an actuator configured to cause the deformable membrane to confirm to the shape of a mould. This may conveniently enable a pre-defined 2D-to-3D shape-change during cell culture, either discrete or cyclical, as well as enabling multi-axial stimulation during cell culture. The actuator may use any suitable means for applying force to the membrane. In some embodiments, the actuator is configured to apply a physical force to the membrane, e.g. by changing the relative positions of the membrane and the mould. Physical displacement may be achieved, for example, by pulling the membrane over the mould, or by pushing the mould into the membrane. Such physical displacement of the membrane and / or the mould may be achieved using a mechanical actuator (e.g. a sliding mechanism) or an electrical linear actuator. Conveniently, an electrical linear actuator could be used to induce cyclic (i.e. repeated) deformation of the membrane.

[0103] In some alternative embodiments, the actuator is configured to apply a hydraulic force (i.e. a force applied using a liquid) or a pneumatic force (i.e. a force applied using a gas to the membrane. The actuator may be configured to apply positive or negative pressure to the membrane. Conveniently, in use, the application of positive or negative pressure causes deformation of the membrane, thereby causing local mechanical stimuli to cells or a user- defined transition from 2D-to-3D culture through conformation of the membrane to the mould. Employing hydraulic or pneumatic actuation to conform the membrane to the mould may enable higher resolution and fidelity membrane geometries compared to application of a physical force to the membrane. In particular, the application of negative pressure can enable more complex membrane geometries to be achieved.

[0104] In some embodiments, the actuator comprises an actuation chamber. The actuation chamber may be a hydraulic actuation chamber or a pneumatic actuation chamber. The actuation chamber may be disposed within the body portion and / or the base portion. In some embodiments, the actuation chamber is disposed within the body portion. In other embodiments, the actuation chamber is disposed within the base portion.

[0105] The actuation chamber may comprise or be constituted by a space around the mould, e.g. between the mould and the membrane, from which a fluid (e.g. a gas) can be removed, causing the membrane to be deformed over the mould. Thus, in some embodiments the mould is located in the actuation chamber (e.g. in a pneumatic actuation chamber).

[0106] Alternatively, the mould may be located outside of the actuation chamber. For example, in some embodiments the mould is separated from the actuation chamber by a wall or barrier. The wall or barrier may be perforated so as to allow fluid communication between the actuation chamber and a space surrounding the mould. In some embodiments, a conduit is provided between the actuation chamber and a surface of the base or body portion in which the actuation chamber is disposed. The conduit enables the application of force to the chamber. For example, the conduit may be configured to receive tubing through which a vacuum (i.e. negative pressure) can be applied.

[0107] In some embodiments, the cell culture chamber serves as an actuation chamber or a further actuation chamber. For example, increasing the volume of liquid in the cell culture chamber may be used to apply hydraulic pressure to the membrane, such that it conforms to the shape of the mould. In some embodiments, the body portion comprises a first actuation chamber (e.g. a pneumatic actuation chamber), and the cell culture chamber in the base portion serves as a second actuation chamber (e.g. a hydraulic actuation chamber). In other embodiments, the base portion comprises a first actuation chamber (e.g. a pneumatic actuation chamber), and the cell culture chamber in the body portion serves as a second actuation chamber (e.g. a hydraulic actuation chamber).

[0108] In other embodiments, the actuation chamber is external to the cell culture chamber. Separating the actuation chamber from the cell culture chamber advantageously means that the cells being cultured are not subjected to the pressure change.

[0109] In some embodiments, the system further comprises a seal, such as an O-ring, a washer, or a gasket, between the body portion and the base portion. In some embodiments, the system further comprises an O-ring. The body portion or the base portion may comprise an annular groove for receiving the seal, e.g. the O-ring or gasket. The annular groove may be located so as to provide a tight seal of the pneumatic actuation chamber when all of the components of the system are assembled.

[0110] The mould is a solid, rationally-designed three-dimensional shape to which the membrane can be conformed by the actuator. Thus, in some embodiments, the system enables the membrane to undergo a change between a substantially planar configuration and a three- dimensional configuration. The use of a mould to which the membrane can be conformed enables the generation of a more complex MPS / OoC platform than has previously been possible via the provision of multiaxial geometry changes. The invention therefore enables the study of complex biological tissues and processes that involve static or dynamic changes, such as respiratory, cardiac or vascular tissues, the digestive tract, embryonic development and certain diseases. In some embodiments, the body portion and / or the base portion comprises a plurality of moulds.

[0111] It may be that each mould is associated with a corresponding cell culture chamber. For example, it may be that the system comprises a base portion comprising a cell culture chamber, and a corresponding mould located within the body portion, on the other side of the membrane.

[0112] Alternatively, it may be that the body portion comprises the cell culture chamber, and a corresponding mould is located within the base portion, on the other side of the membrane.

[0113] In a further example, it may be that the base portion comprises a first cell culture chamber, and the body portion comprises a second cell culture chamber. A mould may be located in one or both of the first and second cell culture chambers.

[0114] In some embodiments, at least two moulds are associated with each cell culture chamber. For example, it may be that the system comprises a first mould located within the cell culture chamber in the base portion and a second (or further) mould located within the body portion. Alternatively, it may be that a first mould is located within the cell culture chamber in the base portion, and a second (or further) mould is located within the body portion. Thus, in such embodiments a mould is provided on either side of the membrane.

[0115] It may be that the body portion comprises at least 2, 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 500, 800 or at least 1000 moulds, or further moulds.

[0116] It may be that the base portion comprises at least 2, 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 500, 800 or at least 1000 moulds, or further moulds.

[0117] The mould may be formed from any suitable material. The material may be sufficiently rigid so as to not undergo significant deformation when brought into contact with the membrane. The material may be selected from a metal, a ceramic, or a polymer, such as a hydrogel. In some embodiments, the mould is formed from a resin, such as a photocurable resin (e.g. Formlabs BioMed Clear resin).

[0118] In some embodiments, the mould is transparent. In embodiments wherein a mould is provided on either side of the membrane (i.e. two moulds per cell culture chamber), it may be that at least one of the moulds is transparent. This allows the membrane to be visualised. The mould may be formed by any suitable method. Conveniently, the mould may be fabricated using additive manufacturing, 3D printing, micromachining, sintering, fused deposition or injection moulding, two-photon polymerization, or selective laser melting / sintering. Alternatively, replica moulding may be used to create the mould (at scale) from previously fabricated master moulds.

[0119] In some embodiments, the mould is formed by 3D printing. Using 3D printing for fabrication of the mould facilitates the generation of a multitude of user-defined, complex geometries which would otherwise be challenging to realise using traditional fabrication strategies.

[0120] In some embodiments, the mould is formed as a discrete component which is attached (e.g. glued) to the body portion. For example, the mould may be glued to the pneumatic actuation chamber of the body portion. In alternative embodiments, the mould can be fabricated as a part of the body portion.

[0121] The mould may have a first (e.g. a “bottom”) surface for attachment to the body or the base portion.

[0122] The mould may have a second (e.g. a “top”) surface. In use, the second surface will come into contact with the membrane upon the application of force to the membrane by the actuator (e.g. through the application of air or liquid pressure (i.e. a pneumatic or hydraulic pressure change), or due to physical movement of the membrane onto the mould). In some embodiments, the second surface of the mould is concave or convex.

[0123] In some embodiments, the mould is annular. In some embodiments wherein a mould is provided on either side of the membrane (i.e. two moulds per cell culture chamber), it may be that at least one of the moulds is annular.

[0124] The geometry of the second surface of the mould may be determined by the desired deformation of the membrane. It may be that the geometry of the second surface can be described through an explicit surface representation (or “height field”) in the form of z = f(x,y), where x and y represent values along two orthogonal directions in the plane, z is the elevation of the surface in the third perpendicular direction to the plane defined by x and y, and f is a function of x and y. However, deviations to this pattern are possible (i.e. surface geometries may be used that are not described by this representation). Rationally designed and bespoke geometries enable localised functionalisation and localised mechanical stimulation of cells during culture. The geometry of the solid mould can be designed using any suitable method, such as 3D CAD software. Preferably, the method used to design the mould geometry results in a 3D triangle mesh file (custom-made, open-source, or commercial). The mesh file may be prepared for 3D printing (“sliced”) using the manufacturer’s software.

[0125] The geometry of the solid mould can be designed to mimic the shape of a biological surface. In some embodiments the biological surface may be that of an intestine, a lung, a heart, a muscle, a brain, a nerve, a joint, skin, a liver, an eye, a bladder, or a tendon.

[0126] In some embodiments, a surface of the (or each) mould comprises one or more recesses and / or projections. For example, a surface of the mould may comprise a plurality of projections and / or recesses. Each projection or recess may be less than 10 mm, less than 5 mm or less than 1 mm in diameter, for example from 10 pm to 500 pm, from 50 pm to 400 pm or from 100 pm to 300 pm in diameter.

[0127] In use, the mould may be surrounded by a fluid, e.g. a liquid or a gas. In some cases, it may be that in use the mould is surrounded by a liquid. It may be that the liquid is used to supply soluble factors (e.g. nutrients, drugs, and / or bioactives) to the cells or tissue cultured on the membrane. In some cases, it may be that in use the mould is surrounded by a gas. The gas composition, humidity, pressure and / or temperature may be selected to simulate certain conditions or environments. For example, the gas may be selected to simulate atmospheric or ambient conditions. In another example, the gas may comprise an additional component e.g. an aerosol, a particulate or a pollutant. In some embodiments, the gas is air, for example to replicate lung tissue which is exposed to air.

[0128] In some embodiments, the system comprises a further mould. The further mould may be located in the cell culture chamber. This arrangement provides a mould on each side of the membrane. This can be particularly useful for modelling biological interfaces, such as the airliquid interface found in the lungs. In such embodiments, the actuator is configured to cause the deformable membrane to conform to the shape of either the mould in the body portion, or the mould in the base portion.

[0129] In some embodiments, the microphysiological system comprises a control unit. The control unit may be for controlling the actuator(s). Additionally or alternatively, the control unit may be configured to control the supply and / or removal of fluids, cells, media and / or reagents to the cell culture chamber(s). It will be appreciated that any embodiment of the components of the microphysiological system, including the body portion, the base portion, the cell culture chamber(s), the mould(s), the control unit and / or the membrane(s) described in relation to the first aspect of the invention is applicable to the second, third and fourth aspects of the invention, unless otherwise stated.

[0130] The microphysiological system of the invention may be configured in multiple different ways.

[0131] In some embodiments, the system comprises: a single base portion comprising a single cell culture chamber; a single body portion located on the base portion, the body portion comprising a single mould; a single deformable membrane separating the cell culture chamber from the mould; and an actuator configured to cause the deformable membrane to conform to the shape of the mould. Multiple systems could also be fluidly coupled together in series or in parallel (i.e. a modular system).

[0132] In some embodiments, the system comprises: a single base portion comprising a single mould; a single body portion located on the base portion, the body portion comprising a single cell culture chamber; a single deformable membrane separating the cell culture chamber from the mould; and an actuator configured to cause the deformable membrane to conform to the shape of the mould. Multiple systems could also be fluidly coupled together in series or in parallel (i.e. a modular system).

[0133] In some embodiments, the system comprises: a base portion comprising n cell culture chambers; a body portion located on the base portion, the body portion comprising n moulds; one or more deformable membranes separating the cell culture chambers from the moulds; and an actuator configured to cause the or each deformable membrane to conform to the shape of the moulds, wherein n is at least two. In some embodiments, the system comprises: a base portion comprising n moulds; a body portion located on the base portion, the body portion comprising n cell culture chambers; one or more deformable membranes separating the cell culture chambers from the moulds; and an actuator configured to cause the or each deformable membrane to conform to the shape of the moulds, wherein n is at least two. It may be that n is at least 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 800 or at least 1000. It may be that n is no greater than 2000, 1600, 1400, 1200, 1000, 800, 600, 400, 200, 100, 50, 40, 30, 20 or 10. In such embodiments, each cell culture chamber has a corresponding mould. Optionally, the system comprises a single membrane which separates each cell culture chamber from its corresponding mould. Alternatively, a separate membrane is provided for each cell culture chamber, i.e. the system comprises n membranes. In some embodiments, the system comprises: a base portion comprising n cell culture chambers; n body portions located on the base portion, each body portion comprising a mould; one or more deformable membranes separating the cell culture chambers from the moulds; and an actuator configured to cause the or each deformable membrane to conform to the shape of the moulds, wherein n is at least two. In some embodiments, the system comprises: a base portion comprising n moulds; n body portions located on the base portion, each body portion comprising a cell culture chamber; one or more deformable membranes separating the cell culture chambers from the moulds; and an actuator configured to cause the or each deformable membrane to conform to the shape of the moulds, wherein n is at least two. It may be that n is at least 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 800 or at least 1000. It may be that n is no greater than 2000, 1600, 1400, 1200, 1000, 800, 600, 400, 200, 100, 50, 40, 30, 20 or 10. In such embodiments, each cell culture chamber has a corresponding body portion and mould. Optionally, the system comprises a single membrane which separates each cell culture chamber from its corresponding mould. Alternatively, a separate membrane is provided for each cell culture chamber, i.e. the system comprises n membranes.

[0134] In some embodiments, the system comprises: a base portion comprising x cell culture chambers; a body portion located on the base portion, the body portion comprising y moulds; one or more deformable membranes separating the cell culture chambers from the moulds; and an actuator configured to cause the or each deformable membrane to conform to the shape of the moulds, wherein x is greater than y. In some embodiments, the system comprises: a base portion comprising y moulds; a body portion located on the base portion, the body portion comprising x cell culture chambers; one or more deformable membranes separating the cell culture chambers from the moulds; and an actuator configured to cause the or each deformable membrane to conform to the shape of the moulds, wherein x is greater than y. It may be that the system comprises a first set of cell culture chambers, wherein each cell culture chamber of the first set has a corresponding membrane and / or mould, and a second set of cell culture chambers, wherein none of the cell culture chambers has a corresponding membrane and / or mould. It may be that one or more cell culture chambers of the first set is connected to at least one cell culture chamber of the second set.

[0135] In some embodiments, the system comprises: a base portion comprising a cell culture chamber, the cell culture chamber comprising a first mould; a body portion located on the base portion, the body portion comprising a second mould; a deformable membrane separating the first mould in the cell culture chamber from the second mould; and an actuator configured to cause the deformable membrane to conform to the shape of either the first mould or the second mould.

[0136] In some embodiments, the system comprises: a base portion comprising a first mould; a body portion located on the base portion, the body portion comprising cell culture chamber comprising a second mould; a deformable membrane separating the first mould in the cell culture chamber from the second mould; and an actuator configured to cause the deformable membrane to conform to the shape of either the first mould or the second mould.

[0137] In some embodiments, the system comprises: a base portion comprising a first cell culture chamber; a body portion located on the base portion, the body portion comprising a second cell culture chamber; a deformable membrane separating the first cell culture chamber from the second cell culture chamber; a mould located in the first and / or the second culture chamber; and an actuator configured to cause the deformable membrane to conform to the shape of the mould(s).

[0138] Any embodiment of the microphysiological system described herein may conveniently be configured as a modular building block that can be assembled with one or more further building blocks, for example, to model the influence of different tissues on each other. This may involve the microfluidic coupling of cell culture chambers to enable cellular cross-talk.

[0139] Thus, in some embodiments, the microphysiological system comprises a first base portion which is connectable to a second base portion such that a first cell culture chamber within the first base portion is fluidly coupled to a second cell culture chamber within the second base portion. In some embodiments, the microphysiological system comprises a first body portion which is connectable to a second body portion such that a first cell culture chamber within the first body portion is fluidly coupled to a second cell culture chamber within the second body portion.

[0140] Additionally or alternatively, the system may comprise a first body portion which is connectable to a second body portion such that a first actuation chamber within the first body portion is fluidly coupled to a second actuation chamber within the second body portion. In some embodiments, the system may comprise a first base portion which is connectable to a second base portion such that a first actuation chamber within the first base portion is fluidly coupled to a second actuation chamber within the second base portion. Additionally or alternatively, in embodiments wherein the base portion or the body portion comprises two or more cell culture chambers, it may be that a first cell culture chamber is fluidly coupled to a second cell culture chamber, wherein the first and second cell culture chambers are within the same base or body portion.

[0141] In some embodiments, the microphysiological system comprises two or more modules, wherein each module comprises: a base portion and; a body portion located on the base portion, wherein one of the base portion and the body portion comprises a cell culture chamber, and the other of the base portion and the body portion comprises a mould; a deformable membrane separating the cell culture chamber from the mould; and an actuator configured to cause the deformable membrane to conform to the shape of the mould.

[0142] The cell culture chamber of at least one of the modules may be fluidly connected to a cell culture chamber of another module.

[0143] In some embodiments, the microphysiological system comprises a plurality of modules. For example, the microphysiological system may comprise at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 30, 40 or 50 modules. It may be that the actuators of the modules are simultaneously or individually operable, for example by a central control unit. Thus, it may be that the actuators within the modules can be operated simultaneously, or individually.

[0144] The membrane, the actuator and the body portion (e.g. comprising the mould) may be provided as a single unit for fitting onto or inserting into a base portion (e.g. comprising a cell culture chamber).

[0145] Thus, according to a second aspect of the invention, there is provided a device for a microphysiological system, the device comprising: a body portion for locating on or in a base portion, the body portion comprising a cell culture chamber or a mould; a deformable membrane; and an actuator configured to cause the deformable membrane to conform to the shape of the mould. In some embodiments, the body portion comprises a mould. In some embodiments, the body portion comprises a cell culture chamber. In some embodiments, each of the body portion and the base portion comprises a cell culture chamber.

[0146] The device will be configured such that, in use, when the body portion is located on or in the base portion, the deformable membrane will separate the cell culture chamber from the mould. In some embodiments, the deformable membrane will separate a first cell culture chamber in the base portion from a second cell culture chamber in the body portion.

[0147] In some embodiments, the deformable membrane is located in a holder. In use, the holder may form a bottom of the device which contacts the base portion.

[0148] The device may be configured for use with standard cell culture devices, such as standard multi-well plates. For example, the device may be configured to be inserted into an individual well of a standard multi-well plate (e.g. a 6- or 12-well plate). It may be that a device is placed into or onto each well of the multi-well plate. In such embodiments, it may be that a modified multi-well plate lid is provided which enables pneumatic actuation of the membrane(s).

[0149] According to a third aspect of the invention, there is provided a kit for assembling a microphysiological system, the kit comprising: a body portion or a base portion comprising a mould; an actuator configured to cause a deformable membrane (e.g. when present and positioned adjacent the mould) to conform to the shape of the mould.

[0150] In some embodiments, the kit further comprises a deformable membrane.

[0151] In some embodiments, the kit comprises a body portion comprising a mould, and further comprises a base portion comprising a cell culture chamber. Alternatively, the kit may comprise a base portion comprising a mould, and further comprises a body portion comprising a cell culture chamber. In some embodiments, the kit comprises a base portion comprising a first cell culture chamber and a body portion comprising a second cell culture chamber, wherein a mould is provided in the first and / or second cell culture chamber. The body portion may be configured to be received on or in the base portion such that a deformable membrane, when present, may be positioned between the cell culture chamber and the mould.

[0152] In some embodiments, the kit comprises a holder for holding a deformable membrane. The holder may be configured for receiving the body portion thereon. The kit may therefore comprise some or all of the components required for assembling a device for a cell culturing chamber, such as the device described herein.

[0153] According to a fourth aspect of the invention, there is provided a method of cell and / or tissue culture, the method comprising:

[0154] - culturing cells or tissue on a deformable membrane; and

[0155] - conforming the membrane, and the cells or tissue thereon, to the shape of a mould.

[0156] The method of cell and / or tissue culture may be for (patho-)physiological organ modelling.

[0157] Culturing the cells or tissue on the membrane can be achieved by methods known to those skilled in the art. In some embodiments, culturing the cells or tissue on the membrane comprises contacting the membrane with cells, such that cells adhere to the membrane, and allowing the cells to replicate on the membrane. The membrane may be contacted with a cellcontaining fluid, such as a culture medium. The fluid (e.g. culture medium) containing the cells may be flowed over the membrane. In some embodiments, the fluid containing the cells is introduced into a cell culture chamber where it contacts the membrane.

[0158] During culturing, the membrane may be maintained in contact with a culture medium. This provides nutrients to the growing cell or tissue layer. During culturing, the membrane and the cells or tissue thereon may be maintained under conditions which are suitable or optimized for growth of the cells. It will be appreciated that the conditions (e.g. temperature, pressure and / or humidity) will depend on the type of cell or tissue. In some embodiments, the cells or tissue are cultured under physiological conditions. For human cells or tissue these conditions may be from 36 to 38 °C.

[0159] The cells or tissue may be animal, plant, fungal or bacterial. In some embodiments, the cells or tissue are animal cells or tissue (e.g. human cells or tissue). The cells or tissue may be intestinal, cancer, lung, heart, muscle, brain, nerve, skin, liver, kidney, pancreatic, eye, bladder, embryonic, stomach, immune, lymphatic, fat, bone, cartilage, tendon, ligament, thyroid or oesophageal cells or tissue. In some embodiments the cells are stem cells, progenitor cells or precursor cells. The stem cells may be totipotent, pluripotent or multipotent.

[0160] In some embodiments the cells are stem cells. In such embodiments, the step of culturing the cells on the membrane may comprise allowing the stem cells to differentiate (i.e. mature). The cells or tissue may, at least initially, form a homogeneous layer (e.g. a monolayer) on the membrane. Thus, in some embodiments the method comprises culturing a layer of cells or tissue on the membrane. However, it will be appreciated that the arrangement of the cells or tissue on the membrane may depend on a number of factors, such as the initial cell density, and / or whether there are any non-adherent regions of the membrane. It will further be appreciated that, over time, the cells or tissue may rearrange in response to various stimuli (such as the shape change of the membrane) resulting in more or less populated regions. Furthermore, some cell types may stack upon one another to form multilayered structures.

[0161] The deformable membrane may be substantially planar, prior to the step of conforming the membrane to the shape of the mould. The mould may have a three-dimensional shape. Therefore, in some embodiments, conforming the membrane to the shape of the mould causes the membrane, and the cells or tissue thereon, to change from a substantially planar configuration to a three-dimensional configuration.

[0162] In some embodiments, the method further comprises reverting the membrane to a substantially planar configuration. During this step, the cells or tissue may also revert to a substantially planar or layer configuration.

[0163] In some embodiments, the method comprises repeatedly cycling the membrane between the substantially planar configuration and the three-dimensional configuration. Advantageously, subjecting the cell or tissue layer(s) to repeated cycling between planar and three-dimensional configurations is useful for mimicking many types of cyclic physiological deformation of cells or tissues in vivo, such as those encountered in the lungs during breathing, in the gut during peristalsis, or in the cardiovascular system as a consequence of pulsatile blood flow.

[0164] Alternatively, the method may comprise causing a single (i.e. discrete) conformational change of the membrane from the substantially planar configuration to the three-dimensional configuration. This could, for example, be used to model tissue folding as seen during embryonic development or to study wound healing after shape-changing-induced injury.

[0165] The change in membrane shape could be used to detach matured tissues from the substrate. Thus, in some embodiments reverting the membrane to the substantially planar configuration causes the cells or tissue, in the three-dimensional configuration, to detach from the membrane. For example, the membrane could initially be deformed into an array of small hemispherical wells suitable for the growth of spheroids or organoids, which could be harvested from the wells by a simple application of positive pressure to bring the membrane back to its original planar shape.

[0166] In some embodiments, the method comprises detaching the cells or tissue from the membrane. In some embodiments the cells or tissue are detached by reverting the membrane to a substantially planar configuration.

[0167] If it is desired to retain the cells / tissue on the membrane during reversion of the membrane to its original shape following deformation, one or more strategies can optionally be used to ensure that they remain attached. For example, the membrane may be provided with a surface modification such as adhesive extracellular matrix (ECM) components to ensure robust adhesion between the cells and the membrane. Additionally, or alternatively, the membrane can be physically micropatterned to increase cellular attachment to the membrane. Additionally, or alternatively, the deformation rate of the membrane can be modulated to ensure that the cell-membrane or cell-ECM / ECM-membrane interactions have time to account for the changes in shape and accompanying strains without compromising the integrity of the attachment.

[0168] In some embodiments the cells or tissue are damaged or detached using a greater magnitude or rate of deformation than is used during a previous step of conforming the membrane to the shape of the mould and reverting the membrane to a substantially planar configuration. The intentional damage of cells or tissue may be useful for modelling wound healing.

[0169] In some embodiments, the cells or tissue are detached from the membrane by exposing the membrane to an elevated temperature or a solvent. Such methods can be used for removing coatings applied to the membrane.

[0170] Additionally or alternatively, in some embodiments, the cells or tissue are detached from the membrane by applying positive pressure to the membrane. This can create a bulging of the membrane to help “pop” out the cells or tissues.

[0171] The membrane may be conformed to the shape of the mould by any suitable means. In some embodiments, the method may comprise applying a physical force to the membrane and / or to the mould, e.g. by changing the relative positions of the membrane and the mould. Physical displacement may be achieved, for example, by pulling the membrane over the mould, or by pushing the mould into the membrane. Such physical displacement of the membrane and / or the mould may be achieved using a mechanical actuator (e.g. a sliding mechanism) or an electrical linear actuator. Alternatively, the method may comprise applying a pneumatic force (i.e. positive or negative pressure) to the membrane. Thus, in some embodiments, conforming the membrane to the shape of the mould is carried out by pneumatic actuation of the membrane.

[0172] In some embodiments, the method is carried out using a microphysiological system as described herein.

[0173] In some embodiments, the method comprises culturing multiple layers of cells or tissues on the membrane. The ability to alter substrate shape after an initial period of planar cell / tissue culture offers interesting opportunities for building more complex tissues (e.g. biomimetic blood vessels comprising three cell layers and three distinct ECM profiles). For example, several layers of different cells could first be seeded in a layer-by-layer fashion on the membrane, after which it is deformed into a predefined configuration and allowed to mature. This way, geometrically complex, multi-layered tissues can be established that could not otherwise be constructed without the 2D-to-3D shape change, such as undulating crypt / villi- mimicking tissues that model the gut system.

[0174] In some embodiments, the method comprises culturing cells or tissue on both sides of the membrane. For example, cells or tissue may be cultured on a first side of the membrane, as described above. Cells or tissue may then be cultured on a second side of the membrane. This can be useful for modelling barrier tissues.

[0175] The systems, devices, kits and methods of the invention may therefore be used for modelling embryogenesis, tissue morphogenesis, tumour growth, and dynamic tissue movements in the lung, gut, respiratory or cardiovascular system.

[0176] The systems, kits and methods of the invention thus advantageously allows the study complex dynamic biological systems across a broad length scale, from mechanistic cellular studies to scalable tissue / organ systems. As such, the invention provides a tool to better mimic the dynamic and shape-changing nature of biology. Consequently, the model tissues created on the MPS / OoC platform of the invention will be more representative of native human tissues, and therefore improve the accuracy and reliability of OoC drug screening and development leading to higher confidence, reduced animal testing, faster screening, and lower costs.

[0177] The use of a mould to which the membrane can be conformed enables the generation of a more complex MPS / OoC platform than has previously been possible via the provision of multiaxial geometry changes. The invention therefore enables the study of complex biological tissues and processes that involve static or dynamic changes, such as respiratory, cardiac or vascular tissues, the digestive tract, embryonic development and certain diseases.

[0178] Thus, in a further aspect the invention provides the use of the system according to the first aspect of the invention, the device according to the second aspect of the invention, the kit according to the third aspect of the invention and / or the method according to the fourth aspect of the invention to model a biological process or a disease, or to generate a cell aggregate, a biological tissue, a spheroid, an organoid or an organ.

[0179] The process may be tissue growth, digestion, tissue or organ function, wound healing, embryonic development or disease progression.

[0180] As is known in the art, a spheroid is a 3D aggregate of cells. Spheroids can be used to mimic cellular interactions of native tissue, and thus find utility in research, drug screening and tissue regeneration.

[0181] It will be appreciated that the system may be used to model any biological process or disease, or to generate any type of cell aggregate, tissue, spheroid, organoid or organ. The cell aggregate, tissue, spheroid, organoid or organ may be selected from, but not limited to: cardiovascular (e.g. heart or blood vessels), respiratory (e.g. lung or trachea), digestive (e.g. oesophageal, stomach or intestinal), musculoskeletal (e.g. bone, bone marrow, cartilage, ligament, muscle, tendon or connective tissues), skin, mucous membrane, metabolic (e.g. liver, gall bladder), urinary (e.g. kidney, bladder), nervous system (e.g. brain, spinal cord or nerves) lymphatic, endocrine (e.g. pancreas, spleen), reproductive (e.g. ovarian, uterine, testicular), optical (e.g. eye or optic nerve), or embryonic.

[0182] In some embodiments, the system, device, kit and / or method is used for modelling multiple biological processes or diseases (e.g. simultaneously), or for generating multiple different types of cell aggregates, biological tissues, spheroids, organoids or organs. For example, the system, device, kit and / or method of the invention may be used to model a partial or whole organism (e.g. an animal- or human-on-a-chip).

[0183] The disease may be cancer, inflammatory bowel disease (e.g. colitis, Crohn’s, irritable bowel syndrome or coeliac disease), coronary heart disease, or respiratory disease (e.g. asthma or COPD). The cell aggregate, biological tissue, spheroid, organoid or organ may be generated for research purposes, or for transplantation. The invention therefore provides a platform for engineering personalized tissue or organ grafts for transplantation, such as intestinal or vascular grafts.

[0184] In some embodiments, the method comprises:

[0185] - culturing a layer of cells or tissue on a deformable membrane;

[0186] - conforming the membrane, and the layer of cells or tissue thereon, to the shape of a mould, thereby causing the membrane, and the layer of cells or tissue thereon, to change from a substantially planar configuration to a three-dimensional configuration; and

[0187] - detaching the cells or issue in the three-dimensional configuration from the membrane.

[0188] In some embodiments, the method further comprises implanting the cell aggregate, biological tissue, spheroid, organoid or organ into a recipient. The recipient may be an animal, such as a mouse, a rat, a rabbit, a dog, a cat, a pig, a goat, a horse, a cow, a guinea pig, a non-human primate (e.g. a monkey, a chimpanzee) or a human. In some embodiments the recipient is a human.

[0189] By enabling the transition from 2D-to-3D culture on-demand, the invention enables the engineering of complex tissue architectures or grafts that are otherwise challenging to realise using traditional tissue engineering strategies.

[0190] Figures 1 to 3 show exploded views of a system (1) according to the invention.

[0191] The system (1) comprises a base portion (10), formed from a substantially rectangular plate, and a cuboid body portion (30). The system (1) also comprises a deformable membrane (50). The base portion (10), the body portion (30), and the deformable membrane (50) are assembled such that the membrane (50) is held between a bottom face (35) of the body portion (30) and a top face (13) of the base portion (10).

[0192] The system (1) further comprises a solid mould (40). When the system (1) is assembled, the mould (40) is located between the body portion (30) and the deformable membrane (50). The mould (40) has an upper face (41) which may be substantially flat and a lower face (42) which is shaped or textured. The lower face (42) can interact with the deformable membrane (50).

[0193] The top face (13) of the base portion (10) comprises a depression (14). The depression (14) is located substantially in the centre of the base portion (10) such that, in the assembled system, the depression (14) receives with a corresponding protruding region (36) located substantially in the centre of the body portion (30).

[0194] The base portion (10) has a circular hole (21) therethrough, located within the depression (14). The hole (21) in the depression (14) in the base portion (10) partially defines a cell culture chamber (20). The hole (21) has an annular ledge (24) located towards the bottom face (14) of the base portion (10) for receiving a cover glass (11). In the assembled system, the cover glass (11) forms a bottom of the cell culture chamber (20).

[0195] The cell culture chamber (20) is fluidly connected to fluidic channels (22a, 22b) which extend through the base portion (10). The fluidic channels (22) are further connected to an inlet port (21a) and an outlet port (21b). The inlet and outlet ports (21a, 21b) are located on the top face (13) of the base portion (10). In the assembled system, the inlet and outlet ports (21a, 21 b) are received in corresponding port holes (32a, 32b) which extend through the body portion (30). The channels (22a, 22b) fluidly connect the inlet and outlet ports (21a, 21 b) to the cell culture chamber (20) within the base portion (10).

[0196] The base portion (10) comprises four screw holes (12a, 12b, 12c, 12d) which extend from the bottom face (14) to the top face (13) of the base portion (10). The screw holes (12a, 12b, 12c, 12d) align with corresponding screw holes (34a, 34b, 34c, 34d) in the body portion (30).

[0197] The body portion (30) comprises a pneumatic actuation chamber (60) configured to cause the deformable membrane (50) to conform to the shape of the mould (40). The pneumatic actuation chamber (60) is connected to a pneumatic inlet / outlet (31). The pneumatic actuation chamber (60) comprises a first section (63) and a second section (62) separated by a perforated barrier (61). The second section (62) contains the solid mould (40) and is flared outwardly towards the bottom face of the body portion (30). The perforated barrier (61) prevents the solid mould (40) from being moved into the first section (63) upon actuation. A circumferential groove (33) is located along the outer edge of the pneumatic actuation chamber (60) and is configured to hold an O-ring (70). In the assembled system, the O-ring (70) rests both in the circumferential groove (33) and on the deformable membrane (50), thereby providing a seal between the body portion (30) and the base portion (10).

[0198] The deformable membrane (50) is configured to be received with the depression (14) in the base portion (10). When assembled, the base portion (10) is fitted with a cover glass (11) which forms the bottom of the cell culture chamber (20). The deformable membrane (50) sits within the depression (14) of the base portion (10) and over the top of the cell culture chamber (20). The protruding region (36) of the body portion (30) is received within the depression (14) in the base portion (10) and the O-ring (70) forms a seal. The solid mould (40) is located above the deformable membrane (50), within the pneumatic actuation chamber (60). The inlet and outlet ports (21a, 21 b) extend though the port holes (32a, 32b) and screws (not shown) are fed into the screw holes (12a, 12b, 12c, 12d) of the base portion (10) and the body portion (30), securing the components of the system (1) together.

[0199] During use, a cell growth fluid comprising cells can be flowed through the inlet port (21a), through the fluidic channel (22a) and into the cell culture chamber (20) where it contacts the deformable membrane (50), enabling cells to adhere to the membrane. The cell growth fluid will then flow out the cell culture chamber (20), through a further fluidic channel (22b), and out of the outlet port (21b). After a layer of cells has been cultured on the membrane (50), a vacuum or negative pressure is applied to the pneumatic actuation chamber (60) using the pneumatic inlet / outlet (31). This causes the deformable membrane (50) to conform to the shape of the solid mould (40). The pressure can then be removed allowing the membrane to revert to its original planar configuration. This action of applying and releasing pressure can be repeated. Optionally, a positive pressure can be applied to cause the membrane to bulge, for example to remove the layer of cells.

[0200] Figures 4A, 4B, and 4C show schematic views of an alternative embodiment of a system according to the invention.

[0201] The system (101) comprises an annular base portion (110) and a body portion (130).

[0202] The device (101) also comprises a deformable membrane (150). The base portion (110), the body portion (130), and the deformable membrane (150) are assembled such that the membrane (150) is held between a bottom face (135) of the body portion (130) and a top face (113) of the base portion (110).

[0203] Housed within the body portion (130) is a lead screw (160). The screw (160) has a proximal end (164), to which is attached a mould (140), and a distal end (161). The mould (140) comprises multiple projections (142). Attached to the body portion (130) are an inlet tube (121a) and an outlet tube (121 b). The inlet tube (121a) is connected to a first fluid channel (122a) located within the body portion (130) and the outlet tube (121b) is connected to a second fluid channel (122b), also located within the body portion (130).

[0204] The base portion (110) defines a circular hole (121) which partially defines a cell culture chamber (120). The base portion (110) also comprises connecting fluid channels (123a, 123b). When assembled, the connecting fluid channels (123a, 123b) in the base portion (110) align with the first and second fluid channels (122a, 122b) in the body portion (130). A bottom face (114) of the base portion (110) is configured to receive a cover glass (111). In the assembled system, the cover glass (111) forms a lower surface of the cell culture chamber (120).

[0205] In this embodiment, the membrane is deformed via physical (mechanical actuation) rather than pneumatic actuation. The lead screw (160) can be rotated so as to press the projections (142) of the mould (140) into the deformable membrane (150), thus deforming the membrane.

[0206] Figures 5A, 5B, and 5C show schematic views of a device according to an embodiment of the invention.

[0207] The device (201) comprises a tubular holding portion (210) which is configured to receive a substantially cylindrical body portion (230) therein. The device (201) also comprises a deformable membrane (250).

[0208] The holding portion (210) comprises an annular top face (213), an annular bottom face (214), an inner surface (215) and an outer surface (216). The inner surface (215) of the holding portion (210) comprises a screw thread (295) which is configured to engage with a corresponding thread (290) located on an outer surface (291) of the body portion (230). Inwardly of the annular bottom face (214) is an annular sloped surface (222) which defines a central hole (221). The holding portion (210) further comprises a fluid inlet channel (221a) and a fluid outlet channel (221 b) which run through the length of the holding portion (210) from the top face (213) to the bottom face (214).

[0209] The body portion (230) comprises a vacuum chamber (260) that is connected to a pneumatic inlet / outlet (231) located on a top face (237) of the body portion (230) and to pneumatic ports (261) located substantially in the centre of a bottom face (235) of the body portion (230). The bottom face (235) of the body portion (230) also comprises multiple projections (242) which together provide a mould for shaping the deformable membrane (250). An annular groove (233) is located in the bottom face (235) of the body portion (230) and is configured to hold an O-ring (270). In the assembled system, the O-ring (270) rests both in the groove (233) and on the deformable membrane (250), thereby providing a seal between the body portion (230) and the holding portion (210).

[0210] The holding portion (210), the body portion (230), and the deformable membrane (250) are assembled such that the membrane (250) is held between the bottom face (235) of the body portion (230) and the central hole (221) of the holding portion (210), below the projections (242) on the bottom face (235) of the body portion (230). When a vacuum is applied to the pneumatic actuation chamber (260) using the pneumatic inlet / outlet (231), the deformable membrane (250) is caused to conform to the shape of the projections (242). The assembled device is configured to be inserted into a well of a standard multi-well plate, wherein the well serves as a culture chamber (see Figure 7).

[0211] Figures 6A, 6B, and 6C show schematic views of an alternative device according to the invention.

[0212] The device of Figures 6A, 6B, and 6C works in substantially the same way as the device shown in Figures 5A, 5B, and 5C with the exception that there are no screw threads (290, 295). Instead, the body portion may be held in place by the application of downward pressure or the body portion (330) and the holding portion (310) may be designed to enable a press-fit configuration where the body portion (330) and the holding portion (310) are held together by friction alone. Corresponding features between Figures 5 and 6 are denoted with the same reference numeral starting with ‘3’ instead of ‘2’.

[0213] Figures 7A, 7B, 7C, and 7D show a system comprising a base portion (402) in the form of a multi-well plate. A device (401) according to the invention (such as that shown in Figure 5 or Figure 6) is placed into each well (403). The devices (401) are configured such that they do not contact the bottom of the wells. This leaves a space between the bottom of the device (404) and the bottom of the well (405), this space defining a cell growth chamber (420).

[0214] Figure 7D shows four devices (401) arranged in series. Connectors (406) fluidly connect each device with the neighboring device. The connectors (406) attach to fluid inlet and outlet channels (e.g. channels 221a, 221 b of Figure 5 or 321a, 321b of Figure 6) such that a fluid outlet channel of a first device is connected to a fluid inlet channel (221a, 321a) of the next device. Cell growth medium may be supplied to each individual device as desired.

[0215] Figure 8A is a schematic representation, viewed from above and from the side, of a cell (500) on a deformable membrane (550) in an OoC system known from the prior art. The left-hand image shows the membrane (550) and the cell (500) thereon in a static (non-actuated) state, while the right-hand image shows the membrane (550) and the cell (500) thereon in a deformed (actuated) state. It can be seen that in such prior art systems, both the membrane (550) and the cell (500) thereon are only deformed in a single 2D plane upon actuation of the system. Thus, this ‘in-plane’ deformation is limited to simple cell models.

[0216] With reference to Figure 8B, there is provided a schematic representation, viewed from above and from the side, of cells (500) on a deformable membrane (550) in an embodiment of the microphysiological system of the present invention, in which deformation of the membrane (550) is enabled by conforming the flexible membrane to the shape of a mould. The left-hand image shows the membrane (550) and the cell (500) thereon in a static (non-actuated) state, while the right-hand image shows the membrane (550) and the cells (500) thereon in a deformed (actuated) state. It can be seen that in the actuated state the cells have been deformed in both 2D and 3D planes. This ‘out-of-plane’ deformation enables the cell layer to more accurately mimic complex cell structures and systems, thereby providing a more realistic representation of in vivo systems.

[0217] Figures 10A and 10B show a schematic representation of a cross section of a system (600) according to an embodiment of the invention. The system (600) comprises a base portion (610), a body portion (630) comprising a mould (640), and a deformable membrane (650). The system (600) is depicted in an un-actuated state (Figure 10A) and in an actuated state wherein the membrane (650) is deformed over the mould (640) (Figure 10B). In the embodiment shown the pneumatic actuation chamber (660) is constituted by the space surrounding the mould (640), as opposed to a distinct chamber or space within the body portion which is separated from the mould. Two pneumatic inlet / outlets (631a, 631b) extend through an upper surface (637) of the body portion (630).

[0218] Figures 11A and 11 B show a system similar to that shown in Figures 10A and 10B, wherein two pneumatic inlet / outlets (731 a, 731b) are located in side walls (738, 739) of the body portion (730).

[0219] Figure 12A and 12B show a system according to a further embodiment of the invention. The system comprises a deformable membrane (850) held between a body portion (830) and a base portion (810). A first solid mould (840a) is located in the body portion (830), and a second solid mould (840b) is located in the base portion (810), within a cell culture chamber (820). Both the first and second solid moulds (840a, 840b) are concave in shape. Figure 13 shows a variant of the system of Figure 12. In the embodiment shown, the second mould (840b) in the base portion (810) is annular, to enable visualisation of the cells / tissues cultured on the membrane (850) through the base portion (810).

[0220] The system of Figure 12 or Figure 13 can be used to model barrier or interfacial physiological systems in which there are two or more cell types separated by a membrane. For example, Figure 14 shows the system of Figure 13 in use as a lung MPS. The cell culture chamber (820) contains a liquid media (821) while the body portion (830) contains air (822) in the space surrounding the first mould (840a).

[0221] A porous membrane (850) provides an air-liquid barrier (Figure 14C). A layer of cells is cultured on both sides of the membrane (850): on the side which contacts the liquid media, endothelial cells (851) are grown, while on the side of the membrane (850) which contacts the air, epithelial cells (852) are grown.

[0222] The layer of endothelial cells remain submerged in liquid cell culture medium throughout the cultivation period. To achieve a layer of epithelial cells on the air side of the membrane, lung epithelial cells can be initially seeded onto the membrane in a liquid cell culture medium and allowed to grow to confluence in submerged culture. Once they have reached confluency and formed an effective / functional epithelial barrier, the liquid medium can be removed from their side of the device, exposing the apical side of the epithelium to air. During this time cell culture medium may remain in the endothelial side of the system, and via the endothelial cells and pores within the membrane, nutrient / waste exchange occurs with the basolateral side of the epithelium.

[0223] The cyclic motion of breathing is mimicked by the bidirectional deformation of the membrane (850). As air moves out of the body portion (830) and liquid moves into f the base portion (810), as in exhalation (Figure 14A), the membrane (850) conforms to the shape of the first mould (840a) in the cell culture chamber. As liquid moves out of the base portion (810) and air moves into the body portion (830), as in inhalation (Figure 14B), the membrane (850) conforms to the shape of the second mould (840b) in the body portion (830). The membrane (850) can be actuated by applying a hydraulic force (e.g. by forcing liquid media into and out of the cell culture chamber), by applying a pneumatic force (e.g. by drawing air into or out of the body portion (830)), or by a combination of both hydraulic and pneumatic actions.

[0224] Thus, the system of this embodiment may comprise both a pneumatic actuation chamber within the body portion, and / or a hydraulic actuation chamber (i.e. the cell culture chamber) within the base portion. In the system of this embodiment, the body portion also serves as a further cell culture chamber in which epithelial cells are cultured.

[0225] Figures 15A, 15B, 15C, 15D, 15E, 15F, 15G, and 15H show exploded, side, plan, and front views of a system (901) according to a further embodiment of the invention. The system (901) comprises a primary body portion (930’) and a secondary body portion (930”). When assembled, the primary body portion (930’) and the secondary body portion (930”) combine to provide a unified body portion (930). The embodiments of Figure 15 are considered to be in a “slide format”.

[0226] The system (901) further comprises a base portion (910). Solid moulds (940a, 940b, 940c) are located on the base portion (910).

[0227] The system (910) further comprises deformable membranes (950a, 950b, 950c) and gaskets (970a, 970b, 970c) held between the secondary body portion (930”) and the base portion (910). The deformable membranes (950a, 950b, 950c) are located directly over the solid moulds (940a, 940b, 940c).

[0228] The primary body portion (930’) includes cell culture chambers (920a, 920b, 920c). Circular holes (92T, 921”, and 92T”) pass though the secondary body portion (930”) to allow the cell culture chamber to extend from the primary body portion (930’) through to the membrane (950a-c). Two rows of inlet and outlet ports (921a, 921b) are provided on the secondary body portion (930”). When the slide is assembled, the inlet and outlet ports (921a, 921b) are received in corresponding port holes (932a, 932b) that are cut out of the primary body portion (930’).

[0229] During use, a cell growth fluid will fill the cell culture chambers (920a, 920b, 920c) and be separated from the solid moulds (940a, 940b, 940c) by the deformable membranes (950a, 950b, 950c).

[0230] With reference to Figures 15E and 15F, the system may further include lids (971a, 971b, 971c) for closing the cell culture chambers (920a, 920b, 920c). Optionally, the lids can contain one or more microfluidic channels (922) which enable the delivery of liquid / air to the culture chambers. The fluidic channels can also connect the ports on the lid to the body portion.

[0231] Figures 16A, 16B, and 16C show the system of Figures 15A-H scaled up to form an array. This may also be referred to as a microtitre (or well) plate format. In the system of Figures 16A-C, the secondary body portion (930”) is located above (on top of) the primary body portion (930’), which comprises an array of cell culture chambers (920), while the secondary body portion (930”) comprises a series of fluidic channels and connectors. The primary body portion (930’) and the secondary body portion (930”) are held within a container (980) which sits on top of the base portion (910) and which comprises further fluidic channels and connectors. A series of deformable membranes (950) are held by gaskets (970) between the primary body portion (930’) and the base portion (910). The system also comprises a removable lid (971) which is adapted to fit over the container (980).

[0232] Figures 17A, B, C, D, E, F, G, H, I, J, K, L, and M show schematic views of further embodiments of the invention in which the system is used upright, as opposed to inverted, meaning the cell culture chamber (1020) is located in a body portion (1030) above the membrane (1050), with the solid mould (1040) located below the membrane in a base portion (1010). These embodiments enable ‘open culture’, that is the top portion can be cultured without a permanent enclosure as shown in previous embodiments of cells (1044). The horizontal arrows in Figures 17A-M indicate flow paths for liquid media (1045, 1045’) and air (1046, 1046’) in and out of the culture chamber (1020) and the base portion (1010).

[0233] Figure 17A shows the system in an actuated state, in which the membrane (1050) and a layer of cells or tissue thereon (1044) has been conformed to the shape of the mould (1040).

[0234] In the embodiment of Figures 17B and 17C, the cell culture chamber contains a first portion of liquid media (1045), while a second portion of liquid media (1045’) surrounds the mould (1040), on the underside of the membrane (1050). This arrangement enables the layer of cells or tissue (1044) to be supplied with soluble factors such as nutrients, bioactives and / or drugs from both sides of the membrane (1050), which is porous in this embodiment. The second portion of liquid media (1045’) can optionally be used to hydraulically actuate the membrane.

[0235] The system can be used to generate liquid-liquid, gas-liquid, and gas-gas interfaces. The gas composition, humidity, pressure and / or temperature may be selected to simulate certain conditions or environments. For example, the gas may be selected to simulate atmospheric or ambient conditions. In another example, the gas may comprise an additional component e.g. an aerosol, a particulate or a pollutant. In some embodiments, the gas is air.

[0236] Figure 17C shows an “gas-liquid” system in which a gas (1046) is present in the cell culture chamber (1020), in contact with the layer of cells or tissue (1044), and a liquid media (1045’) is provided in the base portion (1010). In this configuration the liquid media (1045’) flows over the solid mould (1040).

[0237] In an alternative embodiment (Figure17D) a “liquid-gas” system may comprise a liquid media (1045) in the cell culture chamber (1020) and a gas (1046’) surrounding the mould in the base portion (1010). In this configuration the liquid media (1045) flows directly over the layer of cells or tissue (1044).

[0238] In a further embodiment (Figure 17E) a “gas-gas” system is provided, in which both the cell culture chamber (1020) and the base portion (1010) are filled with a gas (1046, 1046’), such as air.

[0239] The systems of Figures 17F-I further include a removable lid (1071). The lids in Figures 17G and 171 include holes (1072a, 1072b) to allow fluids to enter and exit the cell culture chamber.

[0240] Figures 17J and 17K show a further embodiment which includes a solid mould (1040) that has an alternative shape. Figure 17K in particular shows the layer of cells (1044) being deformed over the mould (1040) and taking on a corresponding shape.

[0241] Figure 17L shows cell-containing media (1044’) being poured from a pipet (1144) onto a membrane (1050) in an open culture cell chamber (1020). The cell-containing media (1044’) will then form a cell layer (1044) on the membrane (1050).

[0242] Figure 17M (i, ii,. iii, and iv) shows exploded views of cells (1044, 1044b, 1044b’) on different types of membrane (1050, 1050b, 1050c). In Figure 17Mi, a layer of cells (1044) is formed on a non-permeable membrane (1050). Figure 17Mii shows a layer of cells being formed held on a textured membrane (1050b). Figure 17Miii shows layers of cells (1044, 1044b) on both sides of a permeable membrane (1050c). Figure 17Miv shows a similar configuration to that of Figure 17Miii, except that the pore size is large enough to allow cells (1044b’) to translocate from one side of the membrane (1050c) to the other. Thus, in the embodiments of Figures 17Miii and 17Miv, a cell culture chamber is provided on each side of the membrane.

[0243] Example 1

[0244] Introduction

[0245] The inventors have developed a cell culture system which is a microphysiological system (MPS) or organ-on-a-chip (OoC) platform that offers the ability to study biological processes in more physiologically relevant, dynamic, and three-dimensional (3D) environments. The system enables the dynamic actuation of a membrane (which itself can be patterned) as a means of creating controllable, on-demand 2D-to-3D changes in living systems. This platform technology enables the study of numerous dynamic biological events, such as those occurring during embryonic development (or in the context of disease) and / or the development of more physiologically relevant tissue / organ models. Specifically, this platform enables the precise recapitulation of dynamic in vivo (bio)mechanical microenvironments by coupling multiscale and / or multimodal cues topography with on-demand multiaxial actuation.

[0246] Materials and methods

[0247] Fabrication of a planar (un-patterned) membrane

[0248] A glass slide substrate(76 x 52 mm) was pretreated by coating with polyvinylalcohol (PVA). A PVA solution (5 g PVA in 100 mL miliQ water) was stirred at 70 °C overnight. The coating was applied by dipping the substrate in the PVA solution, and allowing the substrate to air-dry.

[0249] Membranes were fabricated using the commercial silicone elastomer polydimethylsiloxane (PDMS, product name: Sylgard 184). A standard ratio of 10 parts base to 1 part curing agent was used (w / w), as recommended by the manufacturer. Throughout the fabrication process, environmental conditions were carefully controlled to avoid contaminating the uncured PDMS with solid fragments (dust, hair, etc.). The mixture was stirred vigorously for 3-5 minutes, and degassed first through centrifugation and then using a vacuum pump.

[0250] The membranes were fabricated using a WS-650SZ-6NPP / Lite spin-coater. 2-5 mL of uncured (liquid) PDMS was added onto the coated substrate, which was then loaded into the spincoater. The substrate was then rotated at 500 RPM for 30 seconds at room temperature, allowing a thin layer of uncured PDMS to form on the substrate, with the excess being removed by centrifugal force. Generally, around 2 mL PDMS is sufficient for generating a membrane thickness of 20 - 150 pm. A membrane thickness of 130 pm was achieved using 500 RPM. This can be varied by adjusting the RPM (e.g. 1 ,000 RPM for a thickness of 60 pm and 1 ,500 RPM for a thickness of 40 pm).

[0251] After the spin-coating process, the PDMS was cured. This was done using a hotplate set at 120 °C for 10 minutes. After curing (and cooling), the PDMS membrane was removed from the substrate by soaking the substrate in warm water (40 °C) until the membrane was liberated from the substrate by dissolving the PVA coating.

[0252] The membrane was labelled to enable fluorescent imaging of the deformation. A 1 mg / ml stock solution of difluoro{2-[1-(3,5-dimethyl-2H-pyrrol-2-ylidene-N)ethyl]-3,5-dimethyl-1 H-pyrrolato- NJboron (Merck 790389) was prepared in dichloromethane. 80 pL of stock solution was added to 40 mL of uncured PDMS solution, mixed by hand for 5 minutes, degassed and stored in a freezer (-20 °C) until usage.

[0253] Fabrication of a physically patterned membrane

[0254] For membranes with a micropattern, PDMS was prepared as before. 2-5 mL was added to a microfabricated substrate (see below) and loaded into the spin coater. The substrate was rotated at 500 RPM for 30 seconds at room temperature. After the spin coating process, the PDMS was cured at 60 °C for 4 hours. Once cooled, the membrane was liberated from the micropatterned substrate by rinsing with isopropanol and air-dried.

[0255] Fabrication of the mould

[0256] The solid mould was designed using a custom Matlab script. First the surface (z) of a double sinusoid height field was defined as z = a / 2*(cos(2*pi*x / p)+cos(2*pi*y / p)) with a the amplitude (0.5 mm) and p the period (1 mm) of the double sine wave. Next, the surface was converted to a solid by adding a solid base beneath the height field. The entire mesh was triangulated and exported in STL file format.

[0257] The solid mould was fabricated from Orange touch resin by 3D printing using a Prusa SL1S printer. The printer settings were: 0.025 mm layer height, 1.8 seconds curing time per layer. After 3D printing, the moulds were removed from the printer build platform, and washed in water until the mould were visibly clear of uncured resin (approx. 5 minutes), followed by washing for 2 minutes in isopropanol. The moulds were then dried at 45 °C for 10 minutes. Printed parts were visually inspected to ensure there was no residual solvent, liquid resin, or particles before proceeding. If necessary, the parts were re-washed or left to dry for longer than the described time until clean and dry. The moulds were then subjected to UV post-curing using a Form Cure UV curing station, for 30 minutes at 60 °C. Next, the moulds were washed again extensively with isopropanol and air-dried.

[0258] Base and body portions

[0259] The base and body portions were designed using 3D CAD design software (Solidworks, Dassault Systemes). Part files were converted to a .stl file format in preparation for printing. Pneumatic actuation chambers and supporting bases were fabricated using a Biomed Clear resin and a Formlabs Form 2 printer. The printer layer height was set to 0.100 mm. After printing, the parts were first washed in isopropanol until microfluidic channels were clear of uncured resin (approximately 15 minutes) and then washed for a further 5 minutes in isopropanol. The parts were then air dried for at least 30 minutes. The parts were visually inspected to ensure that there was no residual solvent, liquid resin, or particles remaining before proceeding. If necessary, parts were re-washed or left to dry for longer than the described time until clean and dry. The parts were then cured using a Form Cure UV curing station for 60 minutes at 60 °C.

[0260] Preparation for cell culture

[0261] Prior to cell culture, the components of the microphysiological system were sterilised by ethanol soaking, and / or UV exposure.

[0262] Confocal microscopy

[0263] Confocal microscopy was carried out using the following set-up:

[0264] • Leica SP5 confocal microscope

[0265] • Laser: 458 nm (14% power), Alexa 488 detection set (or FITC)

[0266] Objective: HCX PL S-APO, 10.0x0.30 DRY

[0267] • Voxel size: 0.7572x0.7572x1.8883 micronA3

[0268] • Resolution: 1.3206 pixels per micron

[0269] • Postprocessing of microscopy data: Imaris software (version 9).

[0270] Mechanical actuation

[0271] To assemble the device, an O-ring was inserted into the groove within the body portion. If the mould was fabricated separately to the body portion, the mould was glued to the centre of the body portion. The deformable membrane was cut to size to fit the base portion (depending on the configuration used this was either 25 mm x 25 mm or 14 mm). For the single-well configuration a cover-glass (19 mm) was glued to the underside of the base portion. Once the adhesive had dried, the body portion was assembled onto / into the base portion. For variants where screws were used, 4 x M2 screws were inserted into the base and used to fasten the base and body parts together. PTFE Tubing (I.D 1.6 mm) was used to connect the pneumatic port on the body piece to a 5 mL syringe. Actuation was achieved by drawing a syringe to a known volume. For the single-well device, this was 2 mL and resulted in the deformation of the membrane over the solid mould. The pressure at full actuation was calculated to be 0.236 Bar. The syringe was held at 2 mL (or 0.236 Bar) for 5 minutes to allow for imaging of the deformed membrane. When the syringe was returned to 0 mL the pressure within the body portion returned to atmospheric (1.013 bar) and the membrane reverted to planar.

[0272] Results and discussion Confocal microscopy was used to image the change in shape of the membrane upon pneumatic actuation. As shown in Figure 9, upon the application of negative pressure, the membrane deformed from a substantially planar configuration (static, 2D) to a 3D configuration (full actuation, 3D) in which the membrane substantially conformed to the shape of the mould. This demonstrates for the first time that out-of-plane deformation of a membrane in an OoC system can be achieved using a mould to dictate the shape adopted by the membrane. As such, this invention overcomes the limitations of current actively actuated MPSs / OoCs which only offer simple in-plane deformation and a reductionist representation of human biology. We present a novel device that represents a significant technical development over existing platforms, enabling the recapitulation of the complex geometric changes that occur in human biology in a reliable and reproducible fashion. Furthermore, this invention demonstrates the capacity to deliver multiscale and / or multimodal biologically relevant cues via membrane patterning (physical or chemical) and coinciding user-defined shape changes. This device is a platform for engineering advanced in vitro models of human cells, tissues, organs, and systems, which better mimic the native (micro)environment (healthy and / or diseased), and thus hold great potential for disease modelling as well as drug and therapeutic development.

[0273] Example 2

[0274] A microphysiological system was fabricated using the methods described in Example 1. A cell layer may be grown on the membrane by first inverting the device and perfusing a cell suspension (single cells homogenously distributed in a cell culture medium) through the microfluidic channels into the cell culture chamber. Delivering a known volume (380 pL) of cell suspension to fill the chamber ensures a homogeneous seeding of the cells onto the membrane. Keeping the device inverted, place it into an incubator for 30 minutes to 2 hours to allow for cell attachment. After this settling time, cell adhesion to the membrane can be confirmed microscopically and the device can be connected via the relevant ports to media reservoirs and a pneumatic control system for the remainder of the culture time. Examples of target MPSs, cell types used, the associated media and culture conditions are provided below, although this is not an exhaustive list.

[0275] Bone MPS: Murine preosteoblasts (MC3T3-E1 subclone 4, Sigma-Aldrich, St. Louis, MO, US) were expanded for 7 days in minimum essential medium (a-MEM, Sigma-Aldrich) with the addition of 10% fetal bovine serum and 1% penicillin-streptomycin (Thermo Fischer Scientific, Waltham, MA, USA). The medium was refreshed every 2-3 days. Cells were seeded on the fibronectin-coated PDMS membrane in culture medium, which were then cultured at 37 °C and 5% CO2 with the medium being refreshed every 2-3 days. To induce osteogenic differentiation, the culture medium was supplemented with 4 mM P-glycerophosphate and 50 pg / ml ascorbic acid (both from Sigma-Aldrich), starting from day 3.

[0276] Muscle MPS: Murine myoblasts (C2C12, ATCC, CRL-1772) were expanded at an initial density of 5.0 x 103viable cells / cm2in expansion medium composed of Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich) with the addition of 10% fetal bovine serum and 1 % penicillin-streptomycin (Thermo Fischer Scientific, Waltham, MA, USA), cultures were not allowed to become confluent as this depleted the myoblastic population in the culture. For experiments, cells were seeded onto a fibrin and / or Matrigel coated PDMS membrane in expansion medium. Cells were allowed to proliferate for 4 days, and became visually confluent, before initiating differentiation by supplementing DMEM with 10% horse serum (Thermo Fischer Scientific) and 50 ng / mL insulin-like growth factor 1 (IGF-1 , PeproTech). All culturing took place at 37°C and 5% CO2.

[0277] Cartilage MPS: Mesenchymal stem cells (MSCs) and / or chondrocytes (CCs) can be used to engineer various cartilage MPSs. Stable cartilage can be formed by seeding a 3:1 ratio of MSCs:CCs and culturing in Chondrogenic Differentiation Medium (CDM) created by supplementing hgDMEM GlutaMAX with 100 U / mL penicillin, 100 pg / mL streptomycin (both Gibco), 100 pg / mL sodium pyruvate, 40 pg / mL L-proline, 50 pg / mL L-ascorbic acid-2- phosphate, 4.7 pg / mL linoleic acid, 1.5 mg / mL bovine serum albumin, 1 x insulin-transferrin- selenium (ITS), 100 nM dexamethasone (all from Sigma-Aldrich), 2.5 pg / mL amphotericin B and 10 ng / mL of human transforming growth factor- p3 (TGF-P) (Peprotech, UK). To create hypertrophic cartilage, MSCs are first cultured in CDM followed by Hypertrophic Medium (HYP) which was composed of hgDMEM GlutaMAX supplemented with 100 U / ml penicillin, 100 pg / mL streptomycin (both Gibco), 1 x ITS, 4.7 pg / mL linoleic acid, 50 nM thyroxine, 100 nM dexamethasone, 250 pM ascorbic acid, 7 mM P-glycerophosphate and 2.5 pg / mL amphotericin B (all from Sigma). All culturing takes place under physioxic conditions (37 °C in a humidified atmosphere with 5% CO2 and 5% O2) for improved chondrogenic differentiation.

Claims

Claims1. A microphysiological system comprising: a base portion and a body portion, wherein one of the base portion and the body portion comprises a cell culture chamber and the other of the base portion and the body portion comprises a mould; a deformable membrane separating the cell culture chamber from the mould; and an actuator configured to cause the deformable membrane to conform to the shape of the mould.

2. The microphysiological system of claim 1 , wherein the base portion comprises the cell culture chamber and the body portion comprises the mould.

3. The microphysiological system of claim 1 , wherein the base portion comprises the mould and the body portion comprises the cell culture chamber.

4. The microphysiological system of any one of claims 1-3, wherein the membrane lies in a plane and the system is configured such that operation of the actuator causes out-of-plane deformation of the membrane.

5. The microphysiological system of any preceding claim, wherein the base portion and / or the body portion further comprises one or more channels (e.g. microfluidic channels) for delivering fluids to the membrane, optionally wherein both the base portion and the body portion comprises one or more channels for delivering fluids to the membrane.

6. The microphysiological system of any preceding claim, wherein the system comprises a further mould, located within the cell culture chamber.

7. A device for a microphysiological system, the device comprising: a body portion for locating on or in a base portion, the body portion comprising a cell culture chamber or a mould; a deformable membrane; and an actuator configured to cause the deformable membrane to conform to the shape of the mould.

8. The device of claim 7, wherein the body portion comprises a mould.

9. The device of claim 7, wherein the body portion comprises a cell culture chamber.

10. The microphysiological system of any one of claims 1 to 6, or the device of any one of claims 7 to 9, wherein the actuator comprises a pneumatic or hydraulic actuation chamber disposed within the body portion.11 . The microphysiological system of any one of claims 1 to 6 or 10, or the device of any one of claims 7 to 10, wherein the membrane is biocompatible, optionally wherein the membrane is formed from a biocompatible polymer (e.g. a polysiloxane, such as polydimethylsiloxane (PDMS)).

12. The microphysiological system of any one of claims 1 to 6, 10 or 11 , or the device of any one of claims 7 to 11 , wherein the membrane and / or the mould is patterned.

13. The microphysiological system of any one of claims 1 to 6 or 10 to 12, or the device of any one of claims 7 to 12, wherein the membrane comprises a surface modification, optionally wherein the surface modification comprises a coating, surface functionalisation of the membrane, or a combination thereof.

14. A kit for assembling a microphysiological system, the kit comprising: a body portion or a base portion comprising a mould; and an actuator configured to cause a deformable membrane to conform to the shape of the mould.

15. The kit of claim 14, further comprising a deformable membrane.

16. The kit of claim 14 or claim 15, further comprising a base portion or a body portion comprising a cell culture chamber.

17. The microphysiological system of any one of claims 1 to 6 or 10 to 12, the device of any one of claims 7 to 12, or the kit of any one of claims 14 to 16, further comprising a removable lid, optionally wherein the lid forms a part of the body portion.

18. A method of cell and / or tissue culture, the method comprising:- culturing cells or tissue on a deformable membrane; and- conforming the membrane, and the cells or tissue thereon, to the shape of a mould.

19. The method of claim 18, wherein conforming the membrane to the shape of the mould causes the membrane, and the cells or tissue thereon, to change from a substantially planar configuration to a three-dimensional configuration.

20. The method of claim 19, further comprising reverting the membrane to a substantially planar configuration.

21. The method of claim 18, comprising repeatedly cycling the membrane between the substantially planar configuration and the three-dimensional configuration.

22. The method of any one of claims 18 to 21 , wherein conforming the membrane to the shape of the mould is carried out by:(i) applying a physical force to the membrane and / or to the mould; or(ii) applying a pneumatic or hydraulic force to the membrane.

23. The method of any one of claims 18 to 22, wherein the method comprises culturing multiple layers of cells or tissues on the membrane.

24. The method of any one of claims 18 to 23, further comprising detaching the cells or tissue from the membrane.

25. The use of the system of any one of claims 1 to 6, 10 to 13 or 17, the device of any one of claims 7 to 13 or 17, the kit of any one of claims 14 to 17, or the method of any one of claims 18 to 24, to model a biological process or a disease, or to generate a cell aggregate, a biological tissue, a spheroid, an organoid or an organ.

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