Fluidic bioculture system

Abstract

There are provided fluidic bioculture devices and methods of culturing cells using the devices. One such fluidic bioculture device comprises a device body comprising: a first layer; and a second layer comprising a recess, the recess defining a chamber well and configured to receive one or more scaffolds for cell culturing, wherein the first layer is disposed on top of the second layer. The fluidic bioculture device further comprises a scaffold for culturing cells suspended in media in the chamber well, wherein the scaffold comprises a substantially cylindrical structure having a lumen. The device comprises a first fluid feed channel comprising: a first fluid entry point for receiving a fluid; and a first fluid exit point for allowing egress of the fluid from the fluidic bioculture device. The first fluid entry point and first fluid exit point define a first fluid flowpath through the lumen of the scaffold.

Description

[0001] FLUIDIC BIOCULTURE SYSTEM

[0002] Field

[0003] The present invention relates to a fluidic bioculture system, in particular to a fluidic device for supporting and screening cell cultures.

[0004] Background

[0005] There is a demand for high throughput screening of therapeutic treatments. However, these screenings are limited to expensive and highly restrictive animal models, with poor clinical translation, ex-vivo or basic in-vitro testing. The use of certain animal models can raise ethical concerns and regulatory considerations.

[0006] Cell culture conditions often simplify the complex in vivo environment. Cells are removed from their natural three-dimensional context, and the absence of interactions with neighbouring cells, extracellular matrix, and physiological gradients can affect their behaviour. The dynamic responses of cells, such as response to mechanical forces or fluid flow, are challenging to replicate in static cell culture conditions.

[0007] Conventional in-vitro testing systems involve using glass or plastic cell multi-well plates and tissue culture plates to grow the cells and tissues to be studied. These systems require cell culture media to be monitored and replaced by a researcher several times over the course of a week (dependent on the cell type being studied). Further, any dynamic movement or agitation of the fluids and cell matrix, which can be especially necessary with the study of certain cell types, requires the use of external rotators or shakers and as such are restricted to specific space considerations, timetables or routines. These cell culture set ups are not representative of the natural physiological continual systemic flow (e.g., blood circulation processes and plant vascular systems). These laboratory devices and processes are also typically not fully sealed and so are partially or fully open to the environment, and thereby susceptible to contamination (e.g. bacteria and fungus).

[0008] These shortcomings in existing technologies are particularly evident in the fields of research concerned with, among others, Alzheimer’s disease, Dementia and Parkinson’s (ADP). The prior art systems are particularly insufficient when attempting to produce a viable and representative blood brain barrier (BBB) model for extended periods of time. Existing BBB models typically use a permeable trans-well insert or variations thereof. The process involves growing endothelial cells on the upper and lower sides of a semi-permeable membrane (also referred to as apical and basolateral sides). This model can be complemented and enhanced by the addition of other brain cell types (i.e astrocytes, pericytes, neurons, microglia) on the other end of the membrane. Tight junctions between endothelial are then expected to form, and a communication between the two layers through the pores is required to reflect a viable model. However, the cellular growth in these models is restricted to the flat 2D surfaces of the membrane. These surfaces do not fully replicate the mechanical and biochemical cues that cells experience in their natural micro-environments, and can lead to some cell lines undergoing spontaneous immortalisation or transformation. This can lead to abnormal growth patterns and behaviours and thereby compromising the relevance of the cell culture model. In nature cells grow in all directions, thereby forming 3D physiological structures (i.e., organs, blood vessels) and without cells being physically separated by a non-biological membrane.

[0009] 3D cell culture growth is an emerging field. One example of such a growth / culture method is the use of nano / micro polymer fibres as scaffolds. These fibres are typically produced using melt blown or electrospinning techniques to produce 3D scaffolds for cell culture. These 3D scaffolds are generally limited to disk like geometers and are used as inserts in plastic cell multi-well plates and tissue culture plates. These systems, while improved by virtue of the use of 3D scaffolds, still have the drawbacks discussed previously regarding the static nature of the media, and risk of contamination (e.g., residual solvents and impurities). Many cell culture models consist of a homogeneous population of cells, whereas tissues in vivo are often heterogeneous. This lack of heterogeneity may not fully capture the complexity of cell interactions in tissues.

[0010] Tissues in vivo are highly organized and composed of various cell types interacting in a dynamic manner (e.g. blood brain barrier). Cell culture models often do not fully replicate this complexity, limiting their ability to model certain physiological processes accurately (e.g. the highly selective and semipermeable blood brain barrier that separates the circulating blood from the brain's extracellular fluid).

[0011] Continuous monitoring and regular assessment are crucial for maintaining healthy in vitro cell cultures, ensuring reproducibility, and obtaining reliable experimental results. Adjustments in culture conditions, media changes, and subculturing can be performed based on the observations made during monitoring. Beyond basic microscopy, these methods typically involve removing the cells from the multi-well plates and tissue culture plates for further analysis (e.g. real-time polymerase chain reaction, PCR).

[0012] The present invention aims to at least partially address some of the above-mentioned problems and needs.

[0013] Summary

[0014] According to a first aspect of the present invention, there is provided a fluidic bioculture device. The fluidic bioculture device comprises a device body comprising a first layer; and a second layer. The second layer comprises a recess, the recess defining a chamber well and configured to receive one or more scaffolds for cell culturing. The first layer is disposed on top of the second layer. The fluidic bioculture device further comprises a scaffold for culturing cells suspended in media in the chamber well, wherein the scaffold comprises a substantially cylindrical structure having a lumen. The fluidic bioculture device further comprises a first fluid feed channel comprising a first fluid entry point for receiving a fluid, and a first fluid exit point for allowing egress of the fluid from the fluidic bioculture device. The first fluid entry point and first fluid exit point define a first fluid flowpath through the lumen of the scaffold. Advantageously, the fluidic bioculture device comprising a first fluid flowpath through the lumen of the scaffold allows a dynamic fluid flow through the scaffold during the culturing of the cells. This provides an in-vitro environment more closely aligned to the experience of the cells in their natural environment. The potentially continuous, dynamic fluid flow provided by the first fluid flowpath is more representative of, for example, natural blood circulation than the use of agitators and shakers required in the prior art systems.

[0015] Advantageously, a substantially cylindrical scaffold can be used to more closely replicate blood vessels, for example. The substantially cylindrical geometry naturally promotes the alignment of endothelial cells along the curvature of the structure, closely mimicking the arrangement of brain capillaries and other tubular biological structures, like bronchiole and alveoli. This spatial orientation is important for maintaining physiological functions such as barrier integrity, selective permeability, and intercellular communication.

[0016] The substantially cylindrical scaffold allows cells to interact with surrounding extracellular matrix components in all directions. This enhances the physiological relevance of in vitro models, promoting more natural cellular behaviour, including polarization, differentiation, and cytoskeletal organization.

[0017] Further advantageously, by introducing fluid flow through the inner lumen of the scaffold, shear stress can be generated. This is a critical mechanical cue for endothelial cell function. Shear stress regulates endothelial cell alignment, tight junction formation, and permeability, which are essential factors for accurately modelling vascularized tissues. In prior art static cultures, endothelial cells lack these biomechanical stimuli, resulting in non-physiological behaviour. The present invention allows for precise control over flow rate within the lumen of the scaffold, enabling the recreation of physiological shear stress levels, which is crucial for studying endothelial responses in vascular models, including BBB functionality and drug transport studies. Yet further advantageously, the device being configured to allow continuous flow within the scaffold ensures efficient mass transport of nutrients, oxygen, and signalling molecules, supporting long-term cell viability. Simultaneously, waste metabolites are effectively removed, preventing toxic buildup that can occur in prior art static cultures.

[0018] The scaffold may comprise a permeable or non-permeable textured surface. Textured surfaces can feature a variety of topographical cues, such as grooves, ridges, pits, pillars, or nanopatterns, with dimensions ranging from micrometres to nanometres. These features can be engineered to mimic the structural organization of native tissues or to create specific microenvironments for cells. 3D textured surfaces can influence various cellular processes, including adhesion, spreading, migration, proliferation, differentiation, and gene expression. Cells may align along surface features, change their morphology, or exhibit altered behaviour compared to cells cultured on smooth surfaces. Textured surfaces can exert mechanical forces on cells through cellsubstrate interactions, leading to mechanotransduction. By understanding the mechanisms underlying mechanotransduction, the system allows a user to gain insights into tissue physiology, disease mechanisms, and potential therapeutic interventions.

[0019] Advantageously, the scaffold’s suspension within the chamber well allows for continuous exposure to culture media from all directions. This dynamic interaction with the surrounding medium supports cell survival, differentiation, and maturation by providing an optimal supply of oxygen, nutrients, and signalling molecules while efficiently removing metabolic waste.

[0020] The fluidic bioculture device may be described as a laminate fluidic bioculture device.

[0021] The first fluid entry point and the first fluid exit point may be interchangeable, such that fluid flow through the chamber well via the first flowpath can be reversed by a user.

[0022] The fluid may be or comprise cell culture media. Examples of the fluid may include mammalian cell culture media (e.g., Dulbecco’s Modified Eagle Medium), cell buffers (e.g., phosphate buffered saline), biofluids (e.g., blood, plasma, cerebrospinal fluid, urine, saliva, etc.), pharmaceutical fluids (e.g., medicinal drugs, nutraceuticals, biologies, pharmaceutical excipients), and biomarkers.

[0023] The scaffold may comprise a cylindrical nanofiber tube. Advantageously, the suspended nanofiber scaffold allows for the regulation and measurement of molecular transport across the barrier. The nanofiber scaffold can be engineered with specific pore sizes and surface coatings to model tissue-specific permeability, such as the selective filtration properties of the blood-brain barrier. This feature is particularly useful for testing drug candidates, as it enables differentiation between compounds that can or cannot cross the barrier.

[0024] The first fluid entry point and first fluid exit points may comprise apertures formed in the second layer of the device body. Advantageously, this allows an end user to identify the entry and exit points easily, both visually and in a tactile manner if desirable

[0025] The scaffold may be mounted in the first fluid feed channel and be suspended across the recess.

[0026] The fluidic bioculture device may comprise a second fluid feed channel, comprising a second fluid entry point for receiving a fluid comprising cell culture media and a second fluid exit point for allowing egress of the fluid from the fluidic bioculture device. The second fluid entry points and second fluid exit points may define a second fluid flowpath into the chamber well and surrounding the scaffold.

[0027] Advantageously, the provision of a first and second fluid flowpath allows for various fluid characteristics to be altered and provided separately based on the needs of each cell type on either end of an embedded scaffold, such as continuous perfusion, different flow rates, fluid compositions (isocratic and gradient) and concentrations, viscosity, temperature among others. This allows for an end user to provide a variety of environments to cells being cultured in the fluidic bioculture device and allows for variations to be provided with the cells in situ. This also allows for an end user to more closely replicate the dynamic environment being mimicked.

[0028] A substantially cylindrical scaffold further allows independent fluid flow both in the interior and exterior of the scaffold, generating more naturalistic cell growth. In a similar vein, the present invention allows for the provision and maintaining of two distinct culture media: one inside the tubular scaffold and another in the surrounding chamber well. This setup ensures that different cell types, such as endothelial cells lining the inner scaffold and astrocytes / pericytes on the outer surface, receive their respective optimal nutrients, growth factors, and signalling molecules. This is particularly beneficial for complex co-culture systems, such as those modelling the blood-brain barrier (BBB), where endothelial cells require a different biochemical environment than the surrounding glial cells. The system could also allow to pass air / gasses within the tube in contact with pneumocytes and keep the external surface in medium, mimicking a lung alveoli.

[0029] The cylindrical scaffold in combination with the distinct fluid flowpaths allows for the controlled co-culture of multiple cell types in a physiologically relevant arrangement. For example, endothelial cells can grow along the inner surface of the scaffold, while astrocytes, pericytes, or other supporting cells can attach to the outer surface. This spatial distribution accurately recreates the cell-cell interactions found in vivo, particularly in complex tissue structures like the blood-brain barrier (BBB). The close proximity of different cell types facilitates essential signalling mechanisms, including paracrine communication, extracellular matrix remodelling, and biochemical crosstalk, which are critical for barrier function and tissue homeostasis.

[0030] The combination of inner and outer media flow also enables the establishment of physiologically relevant nutrient gradients, further enhancing tissue-like functionality.

[0031] The second fluid entry point and the second fluid exit point may be interchangeable, such that fluid flow through the chamber well via the second flowpath can be reversed by a user. Providing the first fluid flowpath and second fluid flowpath through the lumen of the scaffold and externally surrounding the scaffold respectively means that the scaffold (and so the cells being cultured) can be supplied with independent fluid flow internally and externally, allowing adjustments to be made depending on the physiological requirements of the cultured cells on each side. This enhances cell-cell and cell- extracellular matrix interactions, by controlling cell positioning and spatial organisation within the culture environment, which can be particularly beneficial in providing a more natural environment for in-vitro research such as BBB research and generation, and in tissue development, morphogenesis, and disease progression.

[0032] The second fluid entry point and second fluid exit points may comprise apertures formed in the second layer of the device body.

[0033] The apertures and channels defining the fluid entry and exit points and feed channels may be micrometre to millimetre in diameter, to represent natural biological systemic flow (e.g., capillaries, veins, arteries, xylem, phloem). The application of millimetre diameter fluid flow reduces the risk of bubble formation and accumulation in the fluidic bioculture device.

[0034] The fluidic bioculture device may comprise a third layer, disposed underneath the second layer such that the first and third layer act to enclose the second layer.

[0035] At least a portion of the second layer may comprise a flexible material configured to seal the second layer to any adjacent layers on the application of pressure.

[0036] According to a second aspect of the present invention, there is provided a fluidic bioculture device. The fluidic bioculture device comprises a device body, comprising a first plate comprising a first recess and a second plate comprising a second recess. The first and second recesses correspond to form a chamber well when the device is assembled. The fluidic bioculture device further comprises an intermediate layer disposed between the first plate and second plate. The intermediate layer is configured to receive one or more scaffolds for cell culturing. The fluidic bioculture device further comprises a first fluid feed channel, the first fluid feed channel comprising: a first fluid entry point for receiving a fluid (such as cell culture media); and a first fluid exit point for allowing egress of the fluid from the fluidic bioculture device. The first fluid entry point and first fluid exit point define a first fluid flowpath through the chamber well.

[0037] Advantageously, the fluidic bioculture device comprising a first fluid flowpath through the chamber well allows a dynamic fluid flow across the scaffold during the culturing of the cells. This provides an in-vivo environment more closely aligned to the experience of the cells in their natural environment. The potentially continuous, dynamic fluid flow provided by the first fluid flowpath is more representative of, for example, natural blood circulation than the use of agitators and shakers required in the prior art systems.

[0038] The fluidic bioculture device may be described as a laminate fluidic bioculture device. Each of the first plate, second plate, and intermediate layer may each be referred to as layers of the fluidic bioculture device.

[0039] The first fluid entry point and the first fluid exit point may be interchangeable, such that fluid flow through the chamber well via the first flowpath can be reversed by a user.

[0040] The fluid may be or comprise cell culture media. Examples of the fluid may include mammalian cell culture media (e.g., Dulbecco’s Modified Eagle Medium), cell buffers (e.g., phosphate buffered saline), biofluids (e.g., blood, plasma, cerebrospinal fluid, urine, saliva, etc.), pharmaceutical fluids (e.g., medicinal drugs, nutraceuticals, biologies, pharmaceutical excipients), and biomarkers.

[0041] As noted, the intermediate layer is configured to receive a scaffold for growing cells. The intermediate layer may be permeable. Alternatively, the intermediate layer may be non-permeable. This scaffold may be 2D or 3D. The chamber well provided by the recesses advantageously allows a 3D scaffold to be suspended in a variety of orientations. The first fluid entry point and exit point may be in fluid communication with the first recess, such that the first fluid flowpath flows above the intermediate layer. Additionally, or alternatively, the first fluid entry point and exit point may be in fluid communication with the second recess, such that the first fluid flowpath flows below the intermediate layer.

[0042] Advantageously, this provides flexibility of fluid provision. When the intermediate layer is non-permeable, the first and second fluid flows can be independent (parallel) or connected (series), thereby enabling independent cell cultures and the study of one interacting with the other via cell-cell and cell-extracellular matrix interactions (e.g. mimicking two discrete organ tissues). The fluid flow may be restricted to just the first or the second recess (i.e. just above or below the intermediate layer and scaffold) to allow cell cultures on either end of the scaffold with more static needs to be examined, while still providing flow at the other end of the scaffold for those cultures requiring flow. These ‘static’ culture may include tumours and neurons. Those cultures benefitting from fluid flow may include endothelial and epithelial cells.

[0043] The first plate may comprise a first pair of apertures defining the first fluid entry point and first fluid exit point respectively. Advantageously, this allows an end user to identify the entry and exit points easily, both visually and in a tactile manner if desirable. The first plate may comprise additional apertures to facilitate additional input and / or output channels. The apertures defining the fluid entry and exit points may be situated on the top surface of the plate. Alternatively, they may be disposed on the end or side of the plate.

[0044] The fluidic bioculture device may comprise a second fluid feed channel. The second fluid feed channel comprises a second fluid entry point for receiving a fluid; and a second fluid exit point for allowing egress of the fluid from the fluidic bioculture device. The second fluid entry point and second fluid exit point define a second fluid flowpath through the chamber well. The second fluid entry point may receive a fluid feed that can be the same or different to the fluid received by the first fluid entry point. Advantageously, the provision of a first and second fluid flowpath allows for various fluid characteristics to be altered and provided separately based on the needs of each cell type on either end of an embedded scaffold, such as continuous perfusion, different flow rates, fluid compositions (isocratic and gradient) and concentrations, viscosity, temperature among others. This allows for an end user to provide a variety of environments to cells being cultured in the fluidic bioculture device, and allows for variations to be provided with the cells in situ. This also allows for an end user to more closely replicate the dynamic environment being mimicked.

[0045] The second fluid entry point and the second fluid exit point may be interchangeable, such that fluid flow through the chamber well via the second flowpath can be reversed by a user.

[0046] The second fluid entry point and exit point may be in fluid communication with the second recess, such that the second fluid flowpath flows below the intermediate layer.

[0047] Advantageously, providing the second fluid flowpath below the intermediate layer and the first fluid flowpath above the intermediate layer means that the scaffold (and so the cells being cultured) can be supplied with independent fluid flow on each side of the scaffold, allowing adjustments to be made depending on the physiological requirements of the cultured cells on each side. This enhances cell-cell and cell- extracellular matrix interactions, by controlling cell positioning and spatial organisation within the culture environment, which can be particularly beneficial in providing a more natural environment for in-vitro research such as BBB research and generation, and in tissue development, morphogenesis, and disease progression.

[0048] The first plate may comprise a second pair of apertures, said apertures defining the second fluid entry point and second fluid exit point respectively; and the second plate may comprise a pair of channels corresponding to the second pair of apertures and in fluid communication with the second recess. In contrast with the first pair of apertures, the second pair of apertures may be through holes connecting the channels in the second plate to a fluid supply. Providing both pairs of apertures for the fluid supply in the first plate advantageously improves accessibility for the second fluid flowpath. Instead of needing entry into the second recess directly through the second plate, all operations can be performed from above the fluidic bioculture device. The corresponding apertures and channels advantageously provide a visual and tactile guide when assembling the fluidic bioculture device.

[0049] The apertures and channels in the first plate and / or second plate may be micrometre to millimetre in diameter, to represent natural biological systemic flow (e.g., capillaries, veins, arteries, xylem, phloem). The application of millimetre diameter fluid flow reduces the risk of bubble formation and accumulation in the fluidic bioculture device.

[0050] At least a portion of the intermediate layer may comprise a membrane configured to receive one or more scaffolds. The membrane may be semi-permeable, which may incorporate fibres, porous membranes or gels. Advantageously, use of a membrane may allow fluid flow in and around the scaffold from both above and below as it passes through the membrane. A defined portion of the intermediate layer may comprise the membrane. Advantageously, this guides a user to where the scaffold should be embedded, and can help to ensure that the scaffold is situated correctly within the chamber well on assembly of the fluidic bioculture device.

[0051] The membrane may comprise biological tissue. The membrane may comprise ex-vivo skin or membranes of the gastrointestinal tract. Advantageously, this may allow for the fluidic bioculture device be utilised as a confined flow diffusion cell, to study (among others) the permeability of substances through a biological membrane.

[0052] At least a portion of the intermediate layer may comprise a flexible material (e.g., polydimethylsiloxane) configured to seal the first and second plate to the intermediate layer on the application of pressure. Advantageously, this ‘self-seal’ upon application of pressure enables a user to easily seal the fluidic bioculture device after the scaffold has been embedded and facilitate removal of the scaffold after cell adhesion. Alternatively, the fluidic bioculture device may be sealed with a separate adhesive. Alternatively, sealing the fluidic bioculture device may involve a heat activated mechanism (e.g., an ultrasonic weld).

[0053] Sealing may be facilitated via polymerisation of the first and second plates to the intermediate layer. This polymerisation may be facilitated using one or more of ultraviolet, infrared or catalytic curing.

[0054] The components of the fluidic bioculture device, in particular the first plate or second plate, may be formed through injection moulding. Alternatively, they may be formed by additive manufacturing (including, for example, 3D printing techniques). Alternatively, they may be produced by machine milling.

[0055] The fluidic bioculture device may comprise multiple chamber wells. The first plate may comprise at least a first auxiliary recess; the second plate may comprise at least a second auxiliary recess. The first auxiliary recess and second auxiliary recess may correspond to form an auxiliary chamber well. The device may comprise a first auxiliary fluid feed channel comprising: a first auxiliary fluid entry point for receiving a fluid; and a first auxiliary fluid exit point for allowing egress of the fluid from the fluidic bioculture device. The first auxiliary fluid entry point and first auxiliary fluid exit point may define a first auxiliary fluid flowpath through the auxiliary chamber well.

[0056] As such, the device may comprise multiple sites for providing a scaffold and culturing cells. These sites may be independent. The sites may be configured to be interconnected to run in either series or parallel, either with additional channels within the fluidic bioculture device, or by connecting the fluid flowpaths with external channels. The provision of multiple sites can allow for simultaneous culturing of multiple cell types, with connections between these sites providing opportunities for interactions between different cells more representative of a biological system than isolated instances. Multiple sites allow for multi-organ / tissue modelling. For example, this could observe a user to observe interactions caused by fluid flow from a liver into a kidney.

[0057] The fluidic bioculture device may comprise a scaffold for culturing cells, wherein the scaffold is embedded in the intermediate layer in the chamber well of the fluidic bioculture device.

[0058] The scaffold may comprise a 3D scaffold. As noted previously, 3D scaffolds provide a more realistic replication of the 3D growth seen in physiological structures. The 3D scaffold allows for a better representation of the natural environment for cell growth, and maintaining homeostasis and physiological cell functions including cell division, gene expression and morphology for longer periods of time compared to cell culturing using 2D scaffolds. An example 3D scaffold may include disc-like geometries comprising polymer fibres. The fibres of the example 3D scaffold may be electro-spun or melt-blown onto a static or moving surface (such as a rotating cylinder) and may be made up of a single or combination of biocompatible materials (e.g., polyacrylonitrile, polyetheramines, poly(glycolic acid), poly(lactic-co-glycolic) acid, poly(caprolactone), poly(l-lactic acid), poly(vinyl alcohol), hyaluronan / hyaluronic acid, polyethylene terephthalate, chitosan, gelatine, chitin, dextran, alginate etc) in varying diameters, concentrations and hardness. The fibres can be combined with extracellular matrix protein (e.g., collagen, laminin, fibronectin, perlecan, nidogen) to impove bootability and cell growth.

[0059] The 3D scaffold may comprise a substantially cylindrical scaffold. As discussed with reference to the first aspect, advantageously a substantially cylindrical scaffold can be used to more closely replicate blood vessels, for example. A substantially cylindrical scaffold further allows independent fluid flow both in the interior and exterior of the scaffold, generating more naturalistic growth of the cells.

[0060] The 3D scaffold may comprise a permeable or non-permeable textured surface. Textured surfaces can feature a variety of topographical cues, such as grooves, ridges, pits, pillars, or nanopatterns, with dimensions ranging from micrometres to nanometres. These features can be engineered to mimic the structural organization of native tissues or to create specific microenvironments for cells. 3D textured surfaces can influence various cellular processes, including adhesion, spreading, migration, proliferation, differentiation, and gene expression. Cells may align along surface features, change their morphology, or exhibit altered behaviour compared to cells cultured on smooth surfaces. Textured surfaces can exert mechanical forces on cells through cell-substrate interactions, leading to mechanotransduction. By understanding the mechanisms underlying mechanotransduction, the system allows a user to gain insights into tissue physiology, disease mechanisms, and potential therapeutic interventions.

[0061] The application of a flexible intermediate layer in the fluidic bioculture device enables mechanical movement of the cell culture scaffold via the application of pulsed fluid flow into the second recess, enabling mechanical stimulation of the cell culture.

[0062] In both the first and second aspects of the present invention, the fluidic bioculture device may comprise a biofilter to protect the cultured cells from environmental contamination and / or to reduce the risk of the cells contaminating the environment. The biofilter may be gas-permeable. The biofilter may be comprise a 0.1 to 0.2 urn membrane, and as such act to be anti-pathogenic whilst still permitting the cell media to enter and exit the fluidic bioculture device through the fluid entry and exit points. Example biofilters may include pleated polypropylene high efficiency particulate air (HEPA) filters. Advantageously, the use of a biofilter allows the fluidic bioculture device to be a sealed system to safely study pathogens without the need for a containment hood, and conversely to reduce the risk of the cells being cultured becoming contaminated. The biofilter may be integrated into the fluidic bioculture device. Alternatively, a third party biofilter may be utilised to protect the cells from contamination (such as a Luer lock or a millipore syringe filter).

[0063] Biofilters may be positioned before all the fluid entry points to the fluidic bioculture device as well as any potential air access points at or by the recesses, ensuring sterile containment of the device. Filters may be placed both before all the fluid entry and after all or selected exit points to fluidic bioculture device upon user and experimental requirements. For example, in the case of microorganism use it may be required that the microorganism is maintained solely within the fluidic bioculture device and not allowed to exit and potentially contaminate the surrounding environment.

[0064] The fluidic bioculture device may comprise at least one sensor configured to measure conditions within the chamber well. The at least one sensor may comprise one or more of a temperature sensor or a pH sensor. The at least one sensor may comprise a pair of sensors, each one of said pair being disposed in the first and second recess respectively, so as to monitor conditions either side of the intermediate layer. Advantageously, the provision of sensors as part of the fluidic bioculture device provides inline monitoring of the cells and surroundings during culturing, allowing for non-intrusive measurement.

[0065] The fluidic bioculture device may comprise at least one pair of electrodes. The electrodes may be disposed either side of the intermediate layer, or as in the first aspect, disposed in the interior lumen and outside the scaffold, and configured to measure electrical resistance across the scaffold in use.

[0066] The fluidic bioculture device may comprise two pairs of electrodes. The first pair of electrodes may be disposed in the first recess; and the second pair of electrodes may be disposed in the second recess, such that the electrode pairs provide a 4-point probe to measure electrical resistance across the scaffold in use. Alternatively, the first pair of electrodes may be disposed in the first feed channel, or the lumen of the scaffold in the first aspect and the second pair of electrodes disposed in the chamber well. Advantageously, this allows for trans-epithelial electrical resistance (TEER) measurements to be made, to determine cell confluence and the integrity of cellular growth. Such a measurement can be useful in modelling BBBs because the TEER measurements can be used to measure barrier integrity and tightness, in addition to cell confluency. The device body may be shaped such that the fluidic bioculture device has the form factor of at least one of:

[0067] (i) a microscope slide; and / or

[0068] (ii) a cell culture plate; and / or

[0069] (iii) a trans well; and / or

[0070] (iv) a cylindrical insert; and / or

[0071] (v) a third party insert, such as an Emulate® Pod Portable Module.

[0072] Advantageously, this allows for the integration of the fluidic bioculture device with existing analytical systems such as plate readers, microscopes and multi-axis stages.

[0073] In a similar vein, the fluidic bioculture device may be configured to be compatible with temperature control systems. In particular, the fluidic bioculture device could be temperature-controlled with an incubator, by coupling with a heat block (e.g. an aluminium heat block), or using the second recess, in conjunction with a non- permeable intermediate layer, with temperature controlled recirculating fluid, to provide an external heat or cooling source. In this instance, a thermocouple could be utilised to monitor and prevent the temperature of the fluidic bioculture device exceeding a desired limit. Similarly, biomarkers and stains can be incorporated into any one of the fluidic bioculture device components, the scaffold or the cells themselves in order to identify features such as the junction of the cells with optical microscopy. Utilisation of the second recess as a tease source enables rapid temperature changes compared to a heat plate or incubator.

[0074] The device body of the fluidic bioculture device may be formed of a biocompatible material. This biocompatible material may be the same material across each component, or may differ for each. For example, the first plate, intermediate layer or second plate may be formed of one or more of polystyrene, polycarbonate, silicone, glass, or variations and combinations thereof. Other examples of materials that could be used for the production of the first and second plates include BioMed Durable Resin and BioMed Clear Resin. Advantageously, the biocompatible materials reduce the risk of the cells or fluid being damaged by the fluidic bioculture device, or vice-versa. Further advantageously, the materials listed may allow for the fluidic bioculture device to be used in applications in medical scanners (e.g., MRI and X-ray scanners). This may allow for further modelling and analysis to be performed without requiring for the cells being grown to be removed from the device.

[0075] The fluidic bioculture device may comprise one or more non-return valves. The one or more non-return valves may be incorporated into one or more of the fluid flow channels in the fluidic bioculture device. Non-return, or one-way valves, may advantageously act to reduce or prevent backward flow of fluid and enable pressurisation of the fluidic bioculture device. One such means of pressurisation may comprise narrowing of one or more of the fluid flow channels, or provision of fluid flow via a larger channel (e.g., millimetre diameter) into a micrometre diameter channel. Pressurisation of the fluidic bioculture device may advantageously enable the study of systolic and diastolic pressure effects on cell and tissue behaviour.

[0076] The scaffold may comprise a biodegradable polymer. The scaffold may be treated with collagen. Advantageously, a biodegradable polymer reduces waste, and could allow for applications where the cells cultured using the system of the second aspect are used in tissue-replacement therapies in addition to medical research. Further advantageously, treating the scaffold with collagen may act to encourage cell migration and adhesion.

[0077] The scaffold may be pre-seeded with cells and frozen for storage. Advantageously, this may allow for end users to obtain the system (i.e. the fluidic bioculture device and seeded scaffold) in a Teady-to-go’ form. This reduces the work required at the point of the end user and can enable straightforward scaling of the research and / or medical applications.

[0078] The scaffold may comprise a biological membrane (e.g., ex-vivo skin or membranes of the gastrointestinal tract). This may allow for the fluidic bioculture device be utilised as a confined flow diffusion cell, to study the permeability of substances through a biological membrane.

[0079] According to a third aspect of the present invention, there is provided a system for culturing cells. The system comprises two or more fluidic bioculture devices as defined in the first or second aspect comprising a scaffold. The fluidic bioculture devices are arranged in an array.

[0080] The two or more fluidic bioculture devices may be arranged in series, such that the cells cultured on the scaffolds of each fluidic bioculture device can be linked. This may comprise arranging the fluid feed channels in series, for example, to enable flow of cell culture media from one fluidic bioculture device to another. Fluidic bioculture devices can be linked either with external tubing / connection or embedded channels within the individual device.

[0081] The system may further comprise a mount configured to house the two or more fluidic bioculture devices, wherein the mount has a form factor corresponding to conventional cell culture plates. Advantageously, this allows for the system to be integrated into standard laboratory imaging devices (such as a microplate reader, or optical microscope).

[0082] According to a fourth aspect of the present invention, there is provided a method of culturing cells using the fluidic bioculture device of any embodiment of the second aspect of the invention, the method comprising the steps of: disposing the intermediate layer of the fluidic bioculture device on top of the second plate; seeding a scaffold with cells to be grown; embedding the scaffold in the membrane of the intermediate layer, positioned such that the scaffold sits within the boundaries of the second recess; disposing the first plate on top of the intermediate layer, such that the first and second recess define a chamber well in which the scaffold is retained; sealing the first and second plates and intermediate layer together. The steps of seeding and embedding the scaffold may be performed in any order.

[0083] Embedding the scaffold may comprise using an adhesive to attach it to the intermediate layer. Alternatively, embedding the scaffold may comprise using semicured PDMS for the first and / or second plates, and applying the scaffold before fully curing the PDMS. Alternatively, embedding the scaffold may comprise the use of ultrasound to weld the fibres of the scaffold to the intermediate layer.

[0084] The flexible material (e.g., polydimethylsiloxane) of the intermediate layer may act to seal the fluidic bioculture device upon the application of pressure such that the step of sealing the first and second plates and intermediate layer together comprises applying pressure to at least one of the first and second plates.

[0085] Advantageously, and as discussed with reference to the first aspect, ‘self-sealing’ enables an end user to easily complete assembly of the fluidic bioculture device from its component parts. This advantageously avoids the need to introduce additional components, such as adhesive, which could contaminate the cell culture. Further, it avoids the need to introduce further environmental conditions such as heat or moisture, which could affect the growth of the cells being cultured.

[0086] The method may further comprise the step of freezing the fluidic bioculture device after sealing to enable transport and / or storage of the seeded scaffold. As noted with respect to the third aspect of the present invention, this can enable an end user to scale up production of tissue, or simply store spare fluidic bioculture devices until the cell culture is required for further experimentation.

[0087] The method may further comprise the steps of: providing fluid flow to the chamber well via the first fluid feed channel; and / or providing fluid flow to the chamber well via the second fluid feed channel. Providing fluid flow to the chamber well may comprise feeding the fluid via a peristaltic or syringe pump or other pressured system. As noted with respect to the first aspect of the invention, providing fluid flow via both the first and second fluid feed channel can allow a user to vary the fluid characteristics on either side of a permeable scaffold, and replicate fluid flow more closely representative of a natural environment.

[0088] According to fifth aspect of the present invention, there is provided a method of culturing cells using the fluidic bioculture device of any embodiment of the first aspect of the invention, the method comprising the steps of: seeding the scaffold with cell to be grown; suspending the scaffold in media in the chamber well of the second layer; sealing the first layer and second layer together; providing fluid flow through the lumen of the scaffold via the first fluid feed channel.

[0089] The method may further comprise providing fluid flow to the chamber well via the second fluid feed channel.

[0090] Providing fluid flow to the chamber well may comprise feeding the fluid via a peristaltic or syringe pump or other pressured system. As noted with respect to the first aspect of the invention, providing fluid flow via both the first and second fluid feed can allow a user to vary the fluid characteristics on either side of a permeable scaffold, and replicate fluid flow more closely representative of a natural environment.

[0091] The flexible material (e.g., polydimethylsiloxane) of the second layer may act to seal the fluidic bioculture device upon the application of pressure such that the step of sealing the first and second layers together comprises applying pressure to at least one of the first and second layers.

[0092] Advantageously, and as discussed with reference to the first aspect, ‘self-sealing’ enables an end user to easily complete assembly of the fluidic bioculture device from its component parts. This advantageously avoids the need to introduce additional components, such as adhesive, which could contaminate the cell culture. Further, it avoids the need to introduce further environmental conditions such as heat or moisture, which could affect the growth of the cells being cultured.

[0093] The method may further comprise the step of freezing the fluidic bioculture device after sealing to enable transport and / or storage of the seeded scaffold. As noted with respect to the third aspect of the present invention, this can enable an end user to scale up production of tissue, or simply store spare fluidic bioculture devices until the cell culture is required for further experimentation.

[0094] According to a sixth aspect of the present invention, there is provided tissue cultured according to the method of the third aspect, for use in at least one of medical research; and / or tissue replacement therapies. As noted previously, such tissue could include a more natural BBB model than that produced by conventional techniques. The tissue cultured could be considered ‘organ on a chip’, with other diagnostic applications including those related to lungs and respiration.

[0095] These and other aspects of the invention will be apparent from, and elucidated with reference to, the embodiments described hereinafter.

[0096] Brief Description of Drawings

[0097] Embodiments will be described, by way of example only, with reference to the drawings, in which

[0098] Figure 1 a illustrates a perspective diagrammatic view of a fluidic bioculture device according to an example embodiment of the present invention;

[0099] Figure 1 b illustrates a plan view of the fluidic bioculture device of Figure 1 a;

[0100] Figure 1 c illustrates a side view of the fluidic bioculture device of Figure 1a;

[0101] Figure 2 illustrates a perspective view of a fluidic bioculture device according to an alternative example embodiment of the present invention;

[0102] Figure 3 illustrates a perspective view of a fluidic bioculture device according to an alternative example embodiment of the present invention ; Figure 4 illustrates a perspective view of a tissue culture plate housing a pair of fluidic bioculture devices of Figure 3;

[0103] Figure 5 illustrates a method of culturing cells according to an example embodiment of the present invention;

[0104] Figure 6a illustrates a diagrammatic plan view of a fluidic bioculture device according to an alternative example embodiment of the present invention;

[0105] Figure 6b illustrates a side view of the fluidic bioculture device of Figure 6a;

[0106] Figure 6c illustrates an additional side view of the fluidic bioculture device of Figure 6a;

[0107] Figure 7a illustrates a diagrammatic plan view of a fluidic bioculture device according to an alternative example embodiment of the present invention;

[0108] Figure 7b illustrates a side view of the fluidic bioculture device of Figure 7a;

[0109] Figure 8a illustrates a diagrammatic plan view of a fluidic bioculture device according to an alternative example embodiment of the present invention;

[0110] Figure 8b illustrates a side view of the fluidic bioculture device of Figure 8a; and

[0111] Figure 9 illustrates a method of culturing cells according to an example embodiment of the present invention;

[0112] Figure 10a illustrates a diagrammatic plan view of a fluidic bioculture device according to an alternative example embodiment of the present invention;

[0113] Figure 10b illustrates a side view of the fluidic bioculture device of Figure 10a;

[0114] Figure 11 a illustrates a partial side and cross-sectional view of a fluidic bioculture device according to an alternative example embodiment of the present invention; andFigure 11 b illustrates a partial side view of a fluidic bioculture device according to an alternative example embodiment of the present invention.

[0115] It should be noted that the Figures are diagrammatic and not drawn to scale. Relative dimensions and proportions of parts of these Figures have been shown exaggerated or reduced in size, for the sake of clarity and convenience in the drawings. The same reference signs are generally used to refer to corresponding or similar feature in modified and different embodiments.

[0116] Detailed Description Figures 1 a, 1 b and 1 c illustrate a fluidic bioculture device 100. Figure 1 a illustrates a perspective view of the fluidic bioculture device 100. Figure 1 b illustrates a plan view of the fluidic bioculture device 100. Figure 1 c illustrates a side view of the fluidic bioculture device 100.

[0117] Fluidic bioculture device 100 comprises a first plate 110, a second plate 130 and an intermediate layer 120 disposed between the first plate 110 and second plate 130. The intermediate layer 120 is configured to receive a scaffold (not pictured) for growing one or more cell cultures.

[0118] In the illustrated embodiment, the first plate 110 and second plate 130 are shaped such that the fluidic bioculture device 100 takes the form factor of a microscope slide, in order to aid compatibility with analysis techniques. For ease of assembly, the intermediate layer 120 is shaped to share edges with the first plate 110 and second plate 130, allowing for straightforward alignment of the components.

[0119] The first plate 110 and second plate 130 comprise a first recess 142 and second recess 144 respectively. The first recess 142 and second recess 144 have a depth smaller than the depth of the first plate 110 and second plate 130, such that the recesses 142, 144 do not pass fully through the first plate 110 and second plate 130. The first and second recesses 142, 144 are disposed on the internal surfaces of the first plate 110 and second plate 130 in use, such that they define a chamber well 140. The intermediate layer 120 comprises a membrane in which the scaffold can be received, such that the scaffold can be suspended inside the chamber well 140 in use. The first recess 142 will sit above the intermediate layer 120 in use, whereas the second recess 144 will sit below the intermediate layer 120 in use.

[0120] The fluidic bioculture device 100 further comprises a first fluid feed channel 150, comprising a first fluid entry point 151 and a first fluid exit point 155. The first fluid entry point 151 and first fluid exit point 155 are in fluid communication with the chamber well 140 to define a first flowpath through the chamber well 140. In the illustrated embodiment, the first fluid feed channel 150 is in fluid communication with the first recess 142. As can be seen most clearly in Figure 1 b, the first fluid entry point 151 comprises an aperture 112 in the first plate 110, and a channel 113 in the first plate 110 fluidly connecting the aperture 112 to the chamber well 140, more particularly to the first recess 142. The first fluid exit point 155 comprises an aperture 116 in the first plate 110, and a channel 115 in the first plate 110 fluidly connecting the chamber well 140 (more particularly the first recess 142) to the aperture 116. The apertures 112, 116 may be defined as a first pair of apertures 112, 116. In the illustrated embodiment, the first pair of apertures are diametrically opposite.

[0121] The fluidic bioculture device 100 further comprises a second fluid feed channel 160, comprising a second fluid entry point 161 and a second fluid exit point 165. The second fluid entry point 161 and second fluid exit point 165 are in fluid communication with the chamber well 140 to define a second flowpath through the chamber well 140. In the illustrated embodiment, the second fluid feed channel 160 is in fluid communication with the second recess 144. As can be seen most clearly in Figure 1 b, the second fluid entry point 151 comprises an aperture 114 in the first plate 110, and a channel 133 in the second plate 130 fluidly connecting the aperture 114 to the chamber well 140, more particularly to the second recess 144. The second fluid exit point 165 comprises an aperture 118 in the first plate 110, and a channel 135 in the second plate 130 fluidly connecting the chamber well 140 (more particularly the second recess 144) to the aperture 118. The apertures 114, 118 may be defined as a second pair of apertures 114, 118. In the illustrated embodiment, the second pair of apertures are diametrically opposite. The second pair of apertures 114, 118 are through-holes in the first plate 110, in order for fluid to access the channels 133, 135 in the second plate 130.

[0122] In the illustrated embodiment, both pairs of apertures 112, 114, 116, 118 are shaped as funnels configured to guide the fluid flow into / out of the respective channels, or to receive nozzles of an external device such as a peristaltic pump. In the illustrated embodiment, the first fluid entry point 151 and first fluid exit point 155 may be interchangeable, such that fluid flow through the chamber well 140 may be reversed. In the illustrated embodiment, the second fluid entry point 161 and first fluid exit point 165 may be interchangeable, such that fluid flow through the chamber well 140 may be reversed. As such, the first pair of apertures 112, 116 may be identical. The second pair of apertures 114, 118 may be identical. It is envisaged that other aperture shapings or channel shapes and sizes may be incorporated.

[0123] In the illustrated embodiment, it is envisaged that the first plate 110 and second plate 130 are formed of a biocompatible material. For example, the first plate 110 and second plate 130 may be formed of one or more of polystyrene, polycarbonate, silicone, glass, or variations and combinations thereof. Other examples of materials that could be used for the production of the first and second plates include BioMed Durable Resin and BioMed Clear Resin. It is envisaged that the intermediate layer 120 is formed of a flexible material, such as silicone.

[0124] Figure 2 illustrates a fluidic bioculture device 200. Similar to the fluidic bioculture device 100 of Figures 1 a-c, the fluidic bioculture device 200 comprises a first plate 210, a second plate 230 and an intermediate layer 220 disposed between the first plate 210 and second plate 230. The first plate 210 and second plate 230 comprise recesses 242, 244, forming a chamber well 240 similar to the fluidic bioculture device 100 of Figures 1 a-c. In contrast to the fluidic bioculture device 100 of Figures 1 a-c, the first and second fluid entry points 251 , 261 and the first and second fluid exit points 255, 265 are formed in the ends of the fluidic bioculture device 200. Rather than passing vertically through the first and second plates 210, 230, the channels 233, 235 defining the fluid flowpaths run parallel to the top and bottom surfaces of the fluidic bioculture device 200. This arrangement may act to reduce the effect of gravity on fluid flow and provide a constant flow pressure. This arrangement may improve accessibility for a user to change fluid provision, or insert a micro-organism or similar.

[0125] The fluidic bioculture device 200 also comprises additional apertures 271 , 272 and corresponding channels 273, 274. These are arranged to join the first and second fluid flowpath respectively. These additional apertures 271 , 272 may improve accessibility for a user to change fluid provision, or insert a micro-organism or similar In addition to the features present in the fluidic bioculture device 100 of Figures 1a-c, fluidic bioculture device 200 comprises two pairs of electrodes 281 , 282. The first pair of electrodes 281 is disposed in the first recess 242 in the first plate 210. The second pair of electrodes is disposed in the second recess 244, in the second plate 230. The electrode pairs 281 , 282 provide a 4-point probe to measure electrical resistance across the scaffold (not pictured) when the fluidic bioculture device 200 is in use.

[0126] Figure 3 illustrates an alternative fluidic bioculture device 300. In contrast with the fluidic bioculture device 100 of Figures 1 a-c, fluidic bioculture device 300 comprises a plurality of recesses 342 in the first plate 310 and corresponding recesses 344 in the second plate 330. These recesses 342, 344 form a first, second and third chamber well 340 respectively, and each have associated first and second fluid entry and exit points 351 , 361 , 355, 365 and channels 333, 335 defining the first and second fluid flowpaths. The provision of a plurality of chamber wells 340 advantageously allows for parallel analysis of multiple cell cultures, potentially under different conditions. In the illustrated embodiment, each chamber well 340 and associated fluid is separate. It is envisaged that auxiliary channels (not pictured) could be provided to enable fluid flow between chamber wells 340. These auxiliary channels could be integrated in the fluidic bioculture device 300, or may be external channels couplable to the fluid entry and exit points 351 , 361 , 355, 365.

[0127] Figure 4 illustrates a plurality of fluidic bioculture devices 300 as illustrated in Figure 3. These fluidic bioculture devices 300 form an array of devices, housed by a tissue culture plate 400. The provision of tissue culture plate 400 enables a plurality of fluidic bioculture devices 300 and associated chamber wells 340 to be assessed simultaneously, and improves the scalability of research on these cell cultures. In the illustrated embodiment tissue culture plate 400 takes the form factor of a standard tissue culture plate, with dimensions of 85.40 x 127.60 x 20.20mm.

[0128] Tissue culture plate 400 comprises a base plate 410. In the illustrated embodiment the base plate 410 comprises three recessed portions 420, each shaped to receive a fluidic bioculture device 300. In the illustrated embodiment, each recessed portion 420 comprises a window 422 along its length to allow for optical analysis of the cells being cultured when the fluidic bioculture device 300 is housed in the tissue culture plate 400. Each recessed portion 420 further comprises a plurality of protrusions 424 disposed on the surface, positioned to correspond to the fluid entry and exit points 351 , 361 , 355, 365 of the fluidic bioculture devices 300. These protrusions 424 may act to seal the fluid entry and exit points 351 , 361 , 355, 365 to provide a closed system for analysis. Alternatively, the protrusions 424 may comprise apertures in order for fluid provision to be continued during analysis of the cells.

[0129] Tissue culture plate 400 further comprises three retaining caps 430, configured to be placed over each fluidic bioculture device 300. Each retaining cap comprises a window 432 along its length, similar to the recessed portions 420, in order to allow optical analysis of the cells. Each retaining cap 430 further comprises a plurality of through- holes 434 positioned to correspond with an aperture 414 in the base plate 410. It is envisaged that a retaining means can be used to couple the retaining cap 430 to the base plate 410 using the through-holes 434 and apertures 414 (e.g. with a shaped clamp, or nut and bolt).

[0130] Figure 5 illustrates a method 5000 of culturing cells according to an example embodiment of the present invention, using the fluidic bioculture device 100 of Figures 1a-c.

[0131] Method 5000 comprises the steps of:

[0132] Step 5100: Disposing the intermediate layer 120 of the fluidic bioculture device 100 on top of the second plate 130 of the fluidic bioculture device 100.

[0133] Step 5200: Seeding a scaffold with cells to be grown using the fluidic bioculture device 100.

[0134] The scaffold may comprise a 2D scaffold for receiving cells, or a 3D scaffold, as discussed previously. Step 5300: Embedding the scaffold in the membrane of the intermediate layer 120, positioned such that the scaffold sits within the boundaries of the second recess 144 in the second plate 130.

[0135] Positioning the scaffold within the boundaries of the second recess 144 in the second plate 130 ensures that the scaffold will be suspended within the chamber well 140 of the fluidic bioculture device 100 upon assembly of the fluidic bioculture device 100.

[0136] It can be appreciated that steps 5200 and 5300 may be performed in any order.

[0137] Step 5400: Disposing the first plate 110 on top of the intermediate layer 120, such that the first and second recesses 142, 144 define a chamber well 140 in which the scaffold is retained.

[0138] Step 5500: Sealing the first and second plates 110, 130 and intermediate layer 120 together.

[0139] As discussed previously, the step of sealing the plates 110, 130 and intermediate layer 120 may comprise applying pressure to one of the first or second plates 110, 130. Alternatively, sealing 5500 may comprise heating the fluidic bioculture device to activate a sealing mechanism in the intermediate layer 120. The heat-activated sealing may be reversible. Alternatively, sealing 5500 may comprise using a separate adhesive to adhere the layer 110, 120, 130 together.

[0140] Step 5550: Freezing the fluidic bioculture device 100 after sealing to enable transport and / or storage of the seeded scaffold.

[0141] Step 5555: Thawing the frozen fluidic bioculture device 100 to allow for the seeded cells to be grown. It can be appreciated that step 5550 and step 5555 are only performed when it is desirable to store the seeded cells for later use.

[0142] Step 5600: Providing fluid flow to the chamber well 140 via one or more of the first fluid feed channel 150 and / or the second fluid feed channel 160.

[0143] Providing fluid flow to the chamber well 140 may comprise feeding the fluid one or more of the first fluid feed channel 150 and / or the second fluid feed channel 160 via a peristaltic or syringe pump. Providing fluid flow via the first fluid feed channel 150 provides a flowpath of the fluid above the intermediate layer 120 (and so by extension the embedded scaffold). Providing fluid flow via the second fluid feed channel 160 provides a flowpath of the fluid above the intermediate layer 120 (and so by extension the embedded scaffold). As discussed previously, fluid flow via both the first fluid feed channel 150 and second fluid feed channel 160 can allow a user to vary the fluid characteristics above and below the scaffold independently of the other flowpath, allowing for a more dynamic environment.

[0144] Figures 6a to 6c illustrate a fluidic bioculture device 600. Figure 6a illustrates a plan view of the fluidic bioculture device 600. Figure 6b illustrates a side view of the fluidic bioculture device 600. Figure 6c illustrates an additional side view of the fluidic bioculture device 600, magnified relative to Figure 6b.

[0145] Similar to the fluidic bioculture device 100 of Figures 1a-c, fluidic bioculture device 600 comprises a first fluid feed channel 650. In contrast with fluidic bioculture device 100, only two layers 610 and 620 are provided to form fluidic bioculture device 600. The second layer 620 may be analogous to the second plate 130 of fluidic bioculture device 100, with the exception that the chamber well 640 is formed from a recess in the second layer 620 itself, across which a scaffold 605 is suspended. The first layer 610 may be a flat plate, such as a glass slide or cover slip. A second fluid feed channel 660 provides fluid flow into the chamber well 640. In the illustrated example of Figures 6a-c, fluid feed channels 650 and 660 have an alternate configuration (i.e., the inlet and outlet of second fluid feed channel 650 are disposed on opposite sides of the scaffold 605). This may act to promote a more dynamic fluid flow around the exterior of the scaffold 605.

[0146] In the illustrated example of Figures 6a-c, the first fluid feed channel is formed within the second layer 620. The scaffold 605 is mounted within the first fluid feed channel 650 such that fluid flow via the first fluid feed channel flows through the lumen of the scaffold 605. The second fluid feed channel 660, in contrast, flows directly into the chamber well 640.

[0147] The scaffold 605 of fluidic bioculture device 600 comprises a substantially cylindrical nanofiber tube. As discussed previously, this has multiple advantages. It can be used to more closely replicate blood vessels, for example. Introducing fluid flow through the inner lumen of the scaffold 605 via the first fluid channel 650 can generate shear stress can be generated, which is beneficial for cell culturing. It also allows for continuous flow within the scaffold 605, ensuring efficient mass transport of nutrients, oxygen, and signalling molecules, supporting long-term cell viability. The scaffold 605 is suspended in media in the chamber well and so can have multi-axial interactions with the media. A nanofiber scaffold 605 further advantageously allows for the regulation and measurement of molecular transport across the barrier. The nanofiber scaffold 605 can be engineered with specific pore sizes and surface coatings to model tissuespecific permeability, such as the selective filtration properties of the blood-brain barrier.

[0148] In Figure 6c, a schematic example can be seen of how a 3D tubular nanofiber scaffold could be loaded with cells to mimic the blood brain barrier (BBB). In the illustrated example, endothelial cells 606 are plated inside to form a capillary-like structure, and astrocytes 607 are plated on the outside surface of the scaffold 605. As discussed throughout this specification, the present invention provides an in vitro environment closer to that of the in vivo environment being mimicked.

[0149] Figures 7a and 7b illustrate an alternative fluidic bioculture device 700. It is similar in structure to the fluidic bioculture device 600 of Figures 6a-c, comprising first and second layers 710, 720, with the chamber well 740 formed within the recess of the second layer 720 and having the scaffold 705 suspended in media and across the chamber well 740. In the example fluidic bioculture device 700, however, the fluid feed channels 750, 760 have a different positioning within the second layer 720. The two channels 750, 760 are parallel, such that either or both of the channels 750, 760 could receive scaffold 705 (as demonstrated in Figure 7b). This provides a flexible set up, and alternatively allows for parallel assessment of different cell cultures in the same environment, or different test setups across the same environment, within a single device.

[0150] Figures 8a and 8b illustrate an alternative fluidic bioculture device 800. It is similar in structure to the fluidic bioculture device 600 of Figures 6a-c, comprising first and second layers 810, 820, with the chamber well 840 formed within the recess of the second layer 820 and having the scaffold 805 suspended in media and across the chamber well 840. In the example fluidic bioculture device 800, however, a single fluid feed channel 850 is provided, with a Y-shape set up (i.e. the channel 850 has two inlets 851 , 852 and a single outlet 853). The scaffold 805 may also have the same shape, such that it sits in each sub-channel of the channel 850, and all are fluidly connected such that fluid flows through the lumen of the scaffold, allowing for different fluid flows to be provided into the scaffold 805 simultaneously. This allows for a heterogenous set-up, which may assist in mimicking the in vivo environment, where cell cultures are not discretely separated.

[0151] Additionally, microstructures 808 are shown growing within the chamber well 840. These are illustrative only, but can demonstrate that both the interior and the exterior surfaces of the scaffold 705 can be utilised for cell culturing. This allows for a variety of variables that can be controlled and / or measured with a single device set up (e.g. flow rate, positioning, fluid media used).

[0152] Figure 9 illustrates a method 9000 of culturing cells according to an example embodiment of the present invention, using the fluidic bioculture device 600 of Figures 6a-c. It can be appreciated that method 9000 may equally apply when using the fluidic bioculture device 700 of Figure 7 or fluidic bioculture device 800 of Figure 8.

[0153] Method 9000 comprises the steps of:

[0154] Step 9100: Seeding a scaffold 605 with cells to be grown using the fluidic bioculture device 600.

[0155] The fluidic bioculture device 600 comprises a 3D nanofiber scaffold 605, having a form factor of a cylindrical tube. The step 9100 of seeding the scaffold may comprise seeding the exterior surface of the scaffold 605 and / or the interior surface of the scaffold 605.

[0156] Step 9200: Embedding the scaffold 605 within the second layer 620, positioned such that the scaffold sits within the boundaries of the chamber well 640 formed by the recess in the second layer 620.

[0157] Positioning the scaffold within the boundaries of the chamber 640 in the second layer 620 ensures that the scaffold will be suspended within the media in the chamber well 640 of the fluidic bioculture device 600 upon assembly of the fluidic bioculture device 700.

[0158] It can be appreciated that steps 9100 and 9200 may be performed in any order.

[0159] Step 9300: Disposing the first plate 610 on top of the second layer 620, such that the chamber well 640 in which the scaffold 605 is suspended is covered.

[0160] Step 9400: Sealing the first and second layers 610, 620 together.

[0161] As discussed previously, the step of sealing the layers 610, 620 may comprise applying pressure to one of the first or second layers 7610, 620. Alternatively, sealing 9400 may comprise heating the fluidic bioculture device to activate a sealing mechanism in the second layer 720. The heat-activated sealing may be reversible. Alternatively, sealing 9400 may comprise using a separate adhesive to adhere the layers 610, 620 together.

[0162] Step 9450: Freezing the fluidic bioculture device 600 after sealing to enable transport and / or storage of the seeded scaffold.

[0163] Step 9455: Thawing the frozen fluidic bioculture device 600 to allow for the seeded cells to be grown.

[0164] It can be appreciated that step 9450 and step 9455 are only performed when it is desirable to store the seeded cells for later use.

[0165] Step 9500: Providing fluid flow to at least one of the scaffold 605 and chamber well 640 via one or more of the first fluid feed channel 650 and / or the second fluid feed channel 660.

[0166] Providing fluid flow to the chamber well 640 may comprise feeding the fluid one or more of the first fluid feed channel 650 and / or the second fluid feed channel 660 via a peristaltic or syringe pump. Providing fluid flow via the first fluid feed channel 650 provides a flowpath of the fluid through the lumen of the cylindrical scaffold 605. Providing fluid flow via the second fluid feed channel 660 provides a flowpath of the fluid into the chamber well 640 directly, and the external surface of the scaffold 605. As discussed previously, fluid flow via both the first fluid feed channel 650 and second fluid feed channel 760 can allow a user to vary the fluid characteristics through and surrounding the scaffold independently of the other flowpath, allowing for a more dynamic environment.

[0167] Figures 10a and 10b illustrate an alternative fluidic bioculture device 1000. It is similar in structure to the fluidic bioculture device 1000 of Figures 6a-c, comprising first and second layers 1010, 1020, with the chamber well 1040 formed within the recess of the second layer 1020 and having the scaffold 1005 suspended in media and across the chamber well 1040. In the example fluidic bioculture device 1000, however, the fluid feed channels 1050, 1060 have a different orientation within the second layer 1020. The two channels 1050, 1060 are disposed perpendicularly, such that the scaffold 1005 is suspended in the chamber well1040 perpendicularly to the second fluid feed channel 1060. This provides an alternative fluid flow profile, wherein the velocity of fluid flowing through the second fluid feed channel 1060 may be greater when passing the exterior surface of the scaffold 1005.

[0168] Figures 11 a and 11 b each illustrate a partial side and cross-sectional view of fluidic bioculture device 1100. Figure 11 a illustrates the chamber well 1140 of the fluidic bioculture device 1100, whereas Figure 11 b illustrates the device with a plurality of chamber wells 1140. In contrast with the fluidic bioculture device 600 of Figure 6a, only a first fluid feed channel 1150 is present in the example fluidic bioculture device 1100 of Figures 11 a and 11 b. The first fluid feed channel similarly flows through the lumen of the scaffold 1105. In the illustrated example, the scaffold 1105 is suspended in the media substantially vertically in the chamber well 1140, such that a supplied fluid would exit the scaffold 1105 and surround the exterior of the scaffold 1105 within the chamber well. This may lead to an alternative environment for cell culturing compared to other illustrated examples, and allow for dynamic flow through and around the scaffold 1105 without the requirement of multiple fluid feed channels. As can be seem in Figure 11 b, this arrangement can be scaled to provide fluid to multiple chamber wells 1140 (and hence scaffolds 1105) with a single fluid feed channel 1150.

[0169] Although the appended claims are directed to particular combinations of features, it should be understood that the scope of the disclosure of the present invention also includes any novel feature or any novel combination of features disclosed herein either explicitly or implicitly or any generalisation thereof, whether or not it relates to the same invention as presently claimed in any claim and whether or not it mitigates any or all of the same technical problems as does the present invention. Features which are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. The applicant hereby gives notice that new claims may be formulated to such features and / or combinations of such features during the prosecution of the present application or of any further application derived therefrom.

[0170] For the sake of completeness, it is also stated that the term "comprising" does not exclude other elements or steps, the term "a" or "an" does not exclude a plurality, and reference signs in the claims shall not be construed as limiting the scope of the claims.

Claims

CLAIMS1 . A fluidic bioculture device, comprising: a device body comprising: a first layer; and a second layer comprising a recess, the recess defining a chamber well and configured to receive one or more scaffolds for cell culturing, wherein the first layer is disposed on top of the second layer; a scaffold for culturing cells suspended in media in the chamber well, wherein the scaffold comprises a substantially cylindrical structure having a lumen; and a first fluid feed channel comprising: a first fluid entry point for receiving a fluid; and a first fluid exit point for allowing egress of the fluid from the fluidic bioculture device; wherein the first fluid entry point and first fluid exit point define a first fluid flowpath through the lumen of the scaffold.

2. The fluidic bioculture device of claim 1 , wherein the scaffold comprises a cylindrical nanofiber tube.

3. The fluidic bioculture device of claim 1 or claim 2, wherein the first fluid entry point and first fluid exit points comprise apertures formed in the second layer of the device body.

4. The fluidic bioculture device of any of claims 1 to 3, wherein the scaffold is mounted in the first fluid feed channel and is suspended across the recess.

5. The fluidic bioculture device of any of claims 1 to 4, further comprising: a second fluid feed channel comprising: a second fluid entry point for receiving a fluid comprising cell culture media; anda second fluid exit point for allowing egress of the fluid from the fluidic bioculture device; wherein the second fluid entry points and second fluid exit points define a second fluid flowpath into the chamber well and surrounding the scaffold.

6. The fluidic bioculture device of claim 5, wherein the second fluid entry point and second fluid exit points comprise apertures formed in the second layer of the device body.

7. The fluidic bioculture device of any of claims 1 to 6, further comprising: a third layer, disposed underneath the second layer such that the first and third layer act to enclose the second layer.

8. The fluidic bioculture device of any preceding claim, wherein at least a portion of the second layer comprises a flexible material configured to seal the second layer to any adjacent layers on the application of pressure.

9. A fluidic bioculture device, comprising: a device body comprising: a first plate comprising a first recess; a second plate comprising a second recess, wherein the first and second recess correspond to form a chamber well; and an intermediate layer disposed between the first plate and second plate and configured to receive one or more scaffolds for cell culturing; and a first fluid feed channel comprising: a first fluid entry point for receiving a fluid; and a first fluid exit point for allowing egress of the fluid from the fluidic bioculture device; wherein the first fluid entry point and first fluid exit point define a first fluid flowpath through the chamber well.

10. The fluidic bioculture device of claim 9, wherein the first fluid entry point and exit point are in fluid communication with the first recess, such that the first fluid flowpath flows above the intermediate layer.

11. The fluidic bioculture device of claim 9 or claim 10, wherein the first plate comprises a first pair of apertures defining the first fluid entry point and first fluid exit point respectively.

12. The fluidic bioculture device of any of claims 9 to 11 , wherein the first fluid entry point and exit point are in fluid communication with the second recess, such that the first fluid flowpath flows below the intermediate layer.

13. The fluidic bioculture device of any of claims 9 to 12, further comprising: a second fluid feed channel comprising: a second fluid entry point for receiving a fluid comprising cell culture media; and a second fluid exit point for allowing egress of the fluid from the fluidic bioculture device; wherein the second fluid entry points and second fluid exit points define a second fluid flowpath through the chamber well.

14. The fluidic bioculture device of claim 13, wherein the second fluid entry point and exit point are in fluid communication with the second recess, such that the second fluid flowpath flows below the intermediate layer.

15. The fluidic bioculture device of any of claims 11 to 13, wherein: the first plate comprises a second pair of apertures, said apertures defining the second fluid entry point and second fluid exit point respectively; and the second plate comprises a pair of channels corresponding to the second pair of apertures and in fluid communication with the second recess.

16. The fluidic bioculture device of any of claims 9 to 15, wherein at least a portion of the intermediate layer comprises a membrane configured to receive one or more scaffolds.

17. The fluidic bioculture device of any of claims 9 to 16, wherein at least a portion of the intermediate layer comprises a flexible material configured to seal the first and second plate to the intermediate layer on the application of pressure.

18. The fluidic bioculture device of claim any of claims 9 to 17, wherein: the fluidic bioculture device comprises two pairs of electrodes; and the first pair of electrodes is disposed in the first recess; and the second pair of electrodes is disposed in the second recess, such that the electrode pairs provide a 4-point probe to measure electrical resistance across the scaffold in use.

19. The fluidic bioculture device of any of claims 9 to 18, wherein: the first plate comprises at least a first auxiliary recess; the second plate comprises at least a second auxiliary recess, wherein the first auxiliary recess and second auxiliary recess correspond to form a auxiliary chamber well; the device comprises a first auxiliary fluid feed channel comprising: a first auxiliary fluid entry point for receiving a fluid; and a first auxiliary fluid exit point for allowing egress of the fluid from the fluidic bioculture device; wherein the first auxiliary fluid entry point and first auxiliary fluid exit point define a first auxiliary fluid flowpath through the auxiliary chamber well.

20. The fluidic bioculture device of any of claims 9 to 19, further comprising: a scaffold for culturing cells, wherein the scaffold is embedded in the intermediate layer in the chamber well.

21. The fluidic bioculture device of claim 20, wherein the scaffold comprises a 3D scaffold.

22. The fluidic bioculture of claim 21 , wherein the 3D scaffold comprises a substantially cylindrical scaffold.

23. The fluidic bioculture device of any preceding claim, wherein the scaffold comprises a biodegradable polymer, and optionally wherein the scaffold is treated with a biocompatible coating (e.g. collagen).

24. The fluidic bioculture device of any preceding claim, wherein the scaffold is preseeded with cells and frozen for storage.

25. The fluidic bioculture device of any preceding claim, wherein the fluidic bioculture device comprises a biofilter to protect the cultured cells from environmental contamination and / or to reduce the risk of the cells contaminating the environment.

26. The fluidic bioculture device of any preceding claim, wherein the device body is shaped such that the fluidic bioculture device has the form factor of at least one of:(i) a microscope slide; and / or(ii) a cell culture plate;(iii) a transwell insert; and / or(iv) a cylindrical insert; and / or(v) a third party insert, such as an Emulate® Pod Portable Module27. The fluidic bioculture device of any preceding claim, wherein device body is formed of a biocompatible material.

28. A system for culturing cells, comprising: two or more fluidic bioculture devices as defined in any of claims 1 to 7 or 19 to 26 as dependent on 1 to 7 or 19 to 23, wherein the fluidic bioculture devices are arranged in an array.

29. The system of claim 28, wherein the two or more fluidic bioculture devices are arranged in series, such that the cells cultured on the scaffolds of each fluidic bioculture device can be linked.

30. The system of claim 28 or claim 29, further comprising: a mount configured to house the two or more fluidic bioculture devices, wherein the mount has a form factor corresponding to conventional cell culture plates.31 . A method of culturing cells using the fluidic bioculture device of any of claims 9 to 22 or claims 23 to 27 as dependent on any of claims 9 to 22, the method comprising the steps of: disposing the intermediate layer of the fluidic bioculture device on top of the second plate; seeding a scaffold with cell to be grown; embedding the seeded scaffold in the membrane of the intermediate layer, positioned such that the scaffold sits within the boundaries of the second recess; disposing the first plate on top of the intermediate layer, such that the first and second recess define a chamber well in which the scaffold is retained; sealing the first and second plates and intermediate layer together.

32. The method of claim 31 , wherein the flexible material of the intermediate layer acts to seal the fluidic bioculture device upon the application of pressure such that the step of sealing the first and second plates and intermediate layer together comprises applying pressure to at least one of the first and second plates.

33. The method of claim 31 or claim 32, further comprising: freezing the fluidic bioculture device after sealing to enable transport and / or storage of the seeded scaffold.

34. The method of any of claims 31 to claim 33, further comprising: providing fluid flow to the chamber well via the first fluid feed channel; and / orproviding fluid flow to the chamber well via the second fluid feed channel.

35. A method of culturing cells using the fluidic bioculture device of any of claims 1 to 8 or 23 to 27 as dependent on any of claims 1 to 8, the method comprising the steps of: seeding the scaffold with cell to be grown; suspending the scaffold in media in the chamber well of the second layer; sealing the first layer and second layer together; providing fluid flow through the lumen of the scaffold via the first fluid feed channel.

36. Tissue cultured according to the method of any of claims 31 to 35 for use in at least one of:(i) medical research; and / or (ii) tissue replacement

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