EP4676645A1

EP4676645A1 - Fluidic device

Info

Publication number: EP4676645A1
Application number: EP24712565.1A
Authority: EP
Inventors: Liam CARR; Adriana TAVARES
Original assignee: University of Edinburgh
Current assignee: University of Edinburgh
Priority date: 2023-03-09
Filing date: 2024-03-06
Publication date: 2026-01-14
Also published as: WO2024184641A1; GB202303487D0; JP2026510780A; CN120882495A; KR20250155044A

Abstract

A fluidic apparatus 105 comprises an inlet 111; an outlet 112; and a first group of compartments 121 comprising two or more first compartments 121a, 121b, 121c, 121 d arranged in parallel between the inlet 111 and the outlet 112; wherein each of the first compartments 121a, 121b, 121c, 121 d is configured to receive a respective cell culture insert 150, and wherein a flow of fluid between the inlet 111 and the outlet 112 is evenly distributed and/or substantially identical between the first compartments 121a, 121b, 121c, 121d.

Description

Fluidic Device

Field of the Invention

The present invention relates to a fluidic device and associated methods for circulating a substance through compartments of a fluidic device. In particular, though not exclusively, the present invention relates to a fluidic device capable of mimicking an animal’s circulatory system in drug studies.

Background

Drug studies involve a number of stages. Typically, drug studies begin with individual cell work and other experiments in order to assess the potential efficacy of a drug. These initial stages are typically carried out in vitro. Promising candidates are then selected for subsequent in vivo animal studies (usually small rodents such as mice and rats). Overall, this approach leads to extremely low success rates of a drug candidate between the in vitro investigations and the in vivo animal studies.

It is desirable to improve the chances of success of in vivo studies and reduce unnecessary in vivo testing, for numerous reasons including time, cost, and ethical considerations.

In order to help bridge the gap between in vitro and in vivo drug studies, and help dismiss unsuitable candidates which might otherwise be studied in vivo, certain devices exist which aim to mimic drug distribution behaviour in an animal. A successful test using such devices increases the chance that the drug candidate(s) will translate successfully to human patients by having predictors of human in vivo success from the device.

An example of such a device is HU Ml MIC Chip4^TM by TissUse GmbH. This device is used in drug studies integrating up to four different organ models, for example intestine, liver, kidney and neuronal tissue. This device is aimed at investigating the toxic effects of a drug over time and uses separate and complex microfluidic circuits to model processes like excretion and reabsorption of various substances in the kidney model. The complexity and size of the device means that at least certain compartments are configured to house a 96-welll insert. Other compartments lack accessibility for sampling at regular intervals. The extremely small growth surface area of a 96-well insert is not compatible with the volume requirements for analysis of intracellular drug concentration using certain types of analytical techniques, such as high-performance liquid chromatography (HPLC) or liquid chromatography mass spectrometry (LC-MS), and would make normalising drug concentrations to cell numbers challenging. US 2005/142656 (Li et al) discloses a cell culture incubating apparatus comprising a culture plate sandwiched between a top plate and a bottom plate. In this arrangement, a flow of fluid is permitted via gravity/capillary action. None of the culture units is suitable to receive a cell culture insert, and the design does not allow sampling of any compartments. The flow is not evenly distributed between the compartments within each group of compartments arranged in parallel at least because the middle cell in each group of culture units is connected to both flow lines, while the outer culture units are only connected to one flow line. In addition, the fluid is configured to flow into, not through, each of the culture units.

CN1 15109699 (Ge et al) discloses an organ chip integrated with a microelectrode array. In this arrangement, a flow of fluid is permitted via gravity/capillary action. Although multiple cell culture wells are arranged in parallel, the flow of fluid is not evenly distributed between the cell culture wells because the two central wells are closer to the outlet flow path than the two outer cell culture wells. In addition, none of the cell culture wells are suitable to receive a cell culture insert, and the design does not allow sampling of any compartments as the wells are completely sealed.

It is an object of the invention to address and/or mitigate one or more problems associated with the prior art.

It is an object of the invention to provide a fluidic device capable of mimicking drug distribution behaviour in an animal and allowing for sampling and analysis, e.g. by HPLC or LC-MS analysis, of intracellular drug concentration in its compartments.

Summary

According to a first aspect, there is provided a fluidic apparatus comprising: an inlet; an outlet; and a first group of compartments comprising two or more first compartments arranged in parallel between the inlet and the outlet; wherein each of the first compartments is configured to receive a respective cell culture insert, and wherein a flow of fluid between the inlet and the outlet is evenly distributed and/or substantially identical between the first compartments.

The inlet may be in fluid communication with the first group of compartments.

The outlet may be in fluid communication with the first group of compartments. The fluidic apparatus may be configured to allow a flow of fluid between the inlet and the outlet through each of the first compartments separately.

Typically, the inlet may be configured to receive a flow of fluid, e.g. of biological fluid, into the fluidic apparatus. The outlet may be configured to allow the fluid to exit the fluidic apparatus.

The first compartments of the first group of compartments are arranged in parallel. In use, when a fluid flows between the inlet and the outlet, there may be no fluid flow between the first compartments of the first group of compartments.

The device may be configured such that the flow rate of a fluid between the inlet and the outlet is substantially identical and/or evenly distributed through each of the first compartments.

The fluidic apparatus may comprise a second group of compartments comprising one or more second compartments.

When the second group of compartments comprises a plurality of second compartments, e.g. two or more second compartments, the second compartments of the second group of compartments may be arranged in parallel, between the inlet and the outlet. In use, when a fluid flows between the inlet and the outlet, there may be no fluid flow between the second compartments of the second group of compartments.

The device may be configured such that the flow rate of a fluid between the inlet and the outlet is substantially identical through each of the second compartments.

The second group of compartments may be provided between the inlet and the first group of compartments.

The second group of compartments may be provided between the first group of compartments and the outlet.

The fluidic apparatus may comprise a third group of compartments comprising one or more third compartments arranged in parallel.

It will be appreciated that the fluidic device may comprise any number of further group(s) of compartments arranged in parallel, provided the flow of fluid between the inlet and the outlet is substantially identical through each compartment of a given group of compartments.

The inlet may be in direct fluid communication with a group of compartments, e.g. with the two or more first compartments of the first group of compartments, via respective inlet fluid channels.

1 The outlet may be in direct fluid communication with a group of compartments, e.g. with the two or more first compartments of the first group of compartments, via respective outlet fluid channels.

There may be provided two or more groups of compartments.

There may be provided a first group of compartments comprising two or more first compartments in direct fluid communication with the outlet via respective outlet fluid channels. There may be provided a second group of compartments comprising one or more second compartments in direct fluid communication with the inlet via (a) respective inlet fluid channel(s).

There may be provided a first group of compartments comprising two or more first compartments in direct fluid communication with the inlet via respective inlet fluid channels. There may be provided a second group of compartments comprising one or more second compartments in direct fluid communication with the outlet via (a) respective outlet fluid channel(s).

Each compartment of the second group of compartments may be in fluid communication, e.g. in direct fluid communication, with one or more first compartments of the first group of compartments via respective first fluid channels.

In an embodiment, the second group of compartments may comprise one compartment. This may be advantageous when the fluidic device aims to mimic systemic blood flow in an animal, as the second compartment may represent the heart of an animal. In such embodiment, the inlet may be in direct fluid communication with the second compartment via an inlet fluid channel.

The second compartment may be in direct fluid communication with each of the first compartments of the first group of compartments via respective first fluid channels.

Each of the first compartments of the first group of compartments may be in direct fluid communication with the outlet via respective outlet fluid channels.

In an embodiment, the second group of compartments may comprise two or more second compartments. In such embodiment, the inlet may be in direct fluid communication with each of the second compartments via respective inlet fluid channels.

Each of the second compartments may be in direct fluid communication with one or more of the first compartments of the first group of compartments via respective first fluid channels.

1 The number of first compartments of the first group of compartments may be less than, equal to, or more than, the number of second compartments of the second group of compartments.

The inlet may be in direct fluid communication with a group of compartments, e.g. with the two or more first compartments of the first group of compartments, or with the one more second compartments of the second group of compartments, nearest the inlet, via respective inlet fluid channels.

Each of the inlet fluid channel(s) may be substantially identical in dimension and/or may be symmetrically disposed relative to each other. This may allow a flow of fluid between the inlet and each of the compartments of a group of compartments nearest the inlet to be substantially identical and/or evenly distributed.

Each of the inlet fluid channel(s) may have a substantially identical length, width, height and/or diameter. When the inlet fluid channel(s) are substantially circular or semicircular in cross-section, the inlet fluid channel(s) may have a substantially identical length and diameter.

The outlet may be in direct fluid communication with a group of compartments, e.g. with the two or more first compartments of the first group of compartments, or with the one more second compartments of the second group of compartments, nearest the outlet, via respective outlet fluid channels.

Each of the outlet fluid channel(s) may be substantially identical in dimension and/or may be symmetrically disposed relative to each other. This may allow a flow of fluid between each of the compartments of a group of compartments nearest the outlet, and the outlet, to be substantially identical and/or evenly distributed.

Each of the outlet fluid channel(s) may have a substantially identical length, width, height and/or diameter. When the outlet fluid channel(s) are substantially circular or semi-circular in cross-section, the outlet fluid channel(s) may have a substantially identical length and diameter.

A group of compartments, e.g. the two or more first compartments of the first group of compartments, may be in direct fluid communication with an adjacent group of compartments, e.g. the one more second compartments of the second group of compartments, via respective fluid connection channels, e.g. first fluid channels.

Each of the fluid connection channel(s) between one group of compartments and an adjacent group of compartments may be substantially identical in dimension and/or

1 may symmetrically disposed relative to each other. This may allow a flow of fluid between one group of compartments and an adjacent group of compartments to be substantially identical and/or evenly distributed.

Each of the fluid connection channel(s) between one group of compartments and an adjacent group of compartments may have a substantially identical length, width, height and/or diameter. When the fluid connection channel(s) are substantially circular or semi-circular in cross-section, the fluid connection channel(s) may have a substantially identical length and diameter.

Advantageously, such a configuration of the fluidic device allows a flow of fluid between the inlet and the compartments of a group of compartments nearest the inlet, to be evenly distributed and/or substantially identical between the compartments of the group of compartments nearest the inlet. When one or more further groups of compartments are present, the configuration of the fluidic device allows a flow of fluid to be evenly distributed and/or substantially identical between the compartments of each group of compartments. This ensures that the flow rate of the fluid between the compartments of each group of compartments is substantially equal, which in turns may help conduct studies, e.g. drug studies, that aim to mimic the effects of a substance, e.g. a drug, in cells of one or more organs which are represented by one or more compartments of the fluidic device.

Preferably, the compartments of a given group of compartments or of each group of compartments may be substantially identical in size and/or configuration.

The two or more first compartments of the first group of compartments may be substantially identical in size and/or configuration.

The one or more second compartments of the second group of compartments may be substantially identical in size and/or configuration.

The one or more compartments or a further group of compartments may be substantially identical in size and/or configuration.

One or more compartments, e.g. the compartments, of the fluidic device, may each define a recess or cavity configured to receive a respective cell culture insert.

One or more compartments, e.g. the compartments, of the fluidic device, may be substantially spherical or hemispherical in shape. One or more compartments, e.g. the compartments, of the fluidic device, may be substantially cylindrical, cubic or frusto- conical in shape.

1 One or more compartments, e.g. the compartments, of the fluidic device, may comprise connection means for connecting with a respective cell culture insert. The connection means may comprise any conventional connecting mechanisms, for example one or more grooves configured to receive a corresponding engaging element of a respective cell culture insert.

The fluidic device may define a flowpath between the inlet and the outlet.

The flowpath may be defined by the channels and the compartments. The flowpath may be defined by the compartments and by the inlet fluid channel(s), the outlet fluid channel(s), and (when more than one group of compartments are present) the fluid connection channel(s) between the compartments of adjacent groups of compartments.

The fluidic apparatus may be made from any material compatible with flow of a fluid, e.g. biological fluid or pharmaceutical composition.

The fluidic apparatus may be made from a 3D-printed material. The material may be a polymeric material, e.g. an acrylic material such as VeroClear™. The flowpath may be made by 3D-printing the fluidic apparatus.

The fluidic apparatus may be made from a polymer, glass, silicon, or any other suitable material. The flowpath may be mechanically or chemically created into the material of the fluidic device, e.g. by etching or machining.

Typically, the size of the fluidic apparatus may be in the region of about 6cm x 8cm.

Typically, the fluid channels may have a width and/or diameter in the region of about 1 -5mm, e.g. about 2mm.

The compartments may have a dimension, e.g. width, diameter and/or depth, sized so as to receive a respective size of cell culture insert, e.g. a 96-well insert, a 48- well insert, a 24-well insert, a 12-well insert, or a 6-well insert. Preferably, the compartments may have a dimension, e.g. width, diameter and/or depth, sized so as to receive a respective size of cell culture insert selected from a 24-well insert, a 12-well insert, or a 6-well insert. By such provision, the apparatus may allow sampling of a volume of fluid sufficient for analysis of intracellular drug concentration using certain types of analytical techniques, such as high-performance liquid chromatography (HPLC) or liquid chromatography mass spectrometry (LC-MS).

Typically, the compartments may have a width and/or diameter in the region of about 14-37mm, e.g. about 16-35mm, e.g. about 20-25mm, e.g. about 22mm. Typically, when a compartment is configured to receive a 12-well insert, the compartment may have a width and/or diameter in the region of about 20-25mm, e.g. about 22mm. When

1 a compartment is configured to receive a 24-well insert, the compartment may have a width and/or diameter in the region of about 14-18mm, e.g. about 16mm. When a compartment is configured to receive a 6-well insert, the compartment may have a width and/or diameter in the region of about 33-37mm, e.g. about 35mm.

The compartments may have a depth in the region of about 15-20mm, e.g. about 17.9mm.

According to a second aspect, there is provided a fluidic apparatus comprising: an inlet; an outlet; a first group of compartments comprising two or more first compartments arranged in parallel between the inlet and the outlet; a second group of compartments comprising one or more second compartments arranged in parallel between the inlet and the outlet; and optionally one or more further group(s) of compartments arranged in parallel between the first group of compartments and the second group of compartments, wherein the inlet is in fluid communication with the first group of compartments or with the second group of compartments via respective inlet channel(s), wherein the outlet is in fluid communication with the second group of compartments or with the first group of compartments via respective outlet channel(s), wherein the compartments of each group of compartments are in fluid communication with one or more compartments of an adjacent group of compartments via respective fluid connection channels, wherein each compartment is configured to receive a respective cell culture insert, wherein each of the inlet fluid channel(s) are substantially identical in dimension and/or are symmetrically disposed relative to each other, wherein each of the outlet fluid channel(s) are substantially identical in dimension and/or are symmetrically disposed relative to each other, and wherein each of the fluid connection channels between the compartments of adjacent groups of compartment are substantially identical in dimension and/or are symmetrically disposed relative to each other.

Advantageously, by such provision, the fluidic apparatus is configured to allow a flow of fluid between the inlet and the outlet through each compartment, and the flow of

1 fluid may be evenly distributed and/or substantially identical between the compartments of each group of compartments.

In an embodiment, the fluidic apparatus may comprise: an inlet; an outlet; a first group of compartments comprising two or more first compartments arranged in parallel, wherein the outlet is in direct fluid communication with the two or more first compartments of the first group of compartments via respective outlet fluid channels, wherein each of the inlet fluid channel(s) are substantially identical in dimension and/or are symmetrically disposed relative to each other, a second group of compartments comprising one or more second compartments arranged in parallel, wherein the inlet is in direct fluid communication with the one or more second compartments of the second group of compartments via (a) respective inlet fluid channel(s), wherein each of the outlet fluid channel(s) are substantially identical in dimension and/or are symmetrically disposed relative to each other; and optionally one or more further group(s) of compartments arranged in parallel between the first group of compartments and the second group of compartments, wherein the compartments of each group of compartments are in fluid communication, e.g. direct fluid communication, with one or more compartments of an adjacent group of compartments via respective fluid connection channels, wherein each of the fluid connection channels between the compartments of adjacent groups of compartment are substantially identical in dimension and/or are symmetrically disposed relative to each other. wherein each compartment is configured to receive a respective cell culture insert.

The features described in relation to the device of the first aspect may equally apply to the device of the second aspect and, merely for brevity, are not repeated.

According to a third aspect, there is provided a fluidic system comprising: a fluidic apparatus according to the first aspect or second aspect; and a pump configured to pump a fluid through the fluidic apparatus.

1 The system may comprise an inlet conduit, e.g. cannula, configured to provide fluid communication between the pump and the inlet.

The system may comprise an outlet conduit, e.g. cannula, configured to provide fluid communication between the pump and the outlet.

The system may define a flowpath for circulating a fluid through the fluidic device.

The pump may be configured to circulate the fluid from the inlet to the outlet through the fluidic device. The pump may be configured to circulate the fluid and from the outlet to the inlet via the outlet conduit and the inlet conduit.

Advantageously, the pump may be configured to circulate the fluid from the inlet to the outlet through the fluidic device, and from the outlet to the inlet via the outlet conduit and the inlet conduit. The pump may be located between the outlet conduit and the inlet conduit.

The system may further comprise a fluid feed interface configured to allow the feeding of a fluid, e.g. a biological fluid or a pharmaceutical composition, into the system, e.g. into the flowpath.

The pump may be a peristaltic pump or roller pump.

The system may comprise at least one, typically a plurality of, cell culture inserts. The cell culture insert(s) may be configured to be provided in respective compartments of the fluidic device.

The cell culture inserts may comprise a 96-well insert, a 48-well insert, a 24-well insert, a 12-well insert, or a 6-well insert. Advantageously, the cell culture inserts may comprise a 24-well insert, a 12-well insert, or a 6-well insert. By such provision, the size of the inserts may allow the sampling of a volume of fluid sufficient to allow analysis thereof, e.g. by HPLC, LC-MS, or any other analytical technique. The size of the inserts may allow the sampling of a volume of fluid of about at least 1 OpL, e.g. at least 20pL, e.g. at least 50pL, e.g. at least 75pL. Advantageously, the cell culture inserts may be sized so as to permit sampling of an insert without requiring stopping the flow of fluid through the fluidic device. Typically, the cell culture inserts may comprise or may be 12- well inserts. 12-well inserts were identified as being sufficient large to allow accurate and reproducible quantification from the inserts by HPLC and/or LC-MS analysis, and sufficiently small to avoid the need for an excessive amount of cells to be grown to carry out an experiment.

The cell culture inserts may be seeded and/or populated with cells. The cells may be representative of a selected organ. One or more cell culture inserts may be seeded

1 and/or populated with cells selected from heart cells, lung cells, liver cells, kidney cells, brain cells, pancreas cells, spleen cells, etc.

In an embodiment, the second group of compartments may comprise one compartment, and the compartment may be configured to receive a cell culture insert seeded or populated with heart cells. This may be advantageous when the fluidic device aims to mimic systemic blood flow in an animal, as the second compartment may represent the heart of an animal. In such embodiment, the inlet may be in fluid communication with the second compartment via an inlet fluid channel.

The first compartments of the first group of compartments may be in fluid communication with the outlet via respective outlet fluid channels. Each of the first compartment may be configured to receive a cell culture insert seeded or populated with cells, e.g. with a different type of cells, selected from heart cells, lung cells, liver cells, kidney cells, brain cells, pancreas cells, spleen cells or the like.

One or more cell culture inserts, e.g. each cell culture insert, may have a semi- permeable portion. Typically, a bottom portion of a/the cell culture insert(s) may be configured to receive a semi-permeable membrane. The semi-permeable membrane may be configured to allow passage of small molecules, e.g. chemical substances such as drugs, but to prevent passage of larger molecules or substances such as cells.

An inside surface of the cell culture insert(s) may be populated with cells, e.g. with cells, selected from heart cells, lung cells, liver cells, kidney cells, brain cells, pancreas cells, spleen cells or the like. This may allow a drug which passes through the membrane into an insert to interact with the cells. This reaction can be assessed by analysing a sample taken from the insert.

An outer surface of the cell culture insert(s) may be populated with cells, e.g. with endothelial cells. Advantageously, this may provide a more realistic representation of the passage of a drug, e.g. a drug contained within the fluid, through a capillary wall (here represented by endothelial cells on the outer surface of the cell culture insert(s)) and into an organ (here represented by the cells provided on an inside surface of an insert).

According to a fourth aspect, there is provided a method of testing the effect a substance on one or more type of cells, the method comprising: providing a system according to the third aspect; providing a cell culture insert in one or more compartment of the fluidic apparatus; providing a fluid into a flowpath defined by the system and/or fluidic apparatus thereof;

1 circulating the fluid through the fluidic apparatus and/or system.

The method may comprise providing a cell culture insert in each compartment of the fluidic apparatus.

The method may comprise sampling one or more of the cell culture inserts, e.g. sampling each cell culture insert.

According to a fifth aspect there is provided a method of manufacturing a fluidic apparatus according to the first aspect or second aspect, the method comprising manufacturing the fluidic apparatus.

The method may comprise providing a starting structure made of a base material, and mechanically and/or chemically treating the base material, e.g. by etching or machining, so as to form the compartments and/or the channels.

The method may comprising manufacturing the fluidic apparatus by additive manufacturing, e.g. 3D printing.

Examples according to the disclosure may be formed using an additive manufacturing process. A common example of additive manufacturing is 3D printing; however, other methods of additive manufacturing are available. Rapid prototyping or rapid manufacturing are also terms which may be used to describe additive manufacturing processes.

As used herein, “additive manufacturing” refers generally to manufacturing processes wherein successive layers of material(s) are provided on each other to “buildup” layer-by-layer or “additively fabricate”, a three-dimensional component. This is compared to some subtractive manufacturing methods (such as milling or drilling), wherein material is successively removed to fabricate the part. The successive layers generally fuse together to form a monolithic component which may have a variety of integral sub-components. In particular, the manufacturing process may allow an example of the disclosure to be integrally formed and include a variety of features not possible when using prior manufacturing methods.

Additive manufacturing methods described herein enable manufacture to any suitable size and shape with various features which may not have been possible using prior manufacturing methods. Additive manufacturing can create complex geometries without the use of any sort of tools, molds or fixtures, and with little or no waste material. Instead of machining components from solid billets of plastic or metal, much of which is

1 cut away and discarded, the only material used in additive manufacturing is what is required to shape the part.

Suitable additive manufacturing techniques in accordance with the present disclosure include, for example, Fused Deposition Modeling (FDM), Selective Laser Sintering (SLS), 3D printing such as by inkjets and laserjets, Sterolithography (SLA), Direct Selective Laser Sintering (DSLS), Electron Beam Sintering (EBS), Electron Beam Melting (EBM), Laser Engineered Net Shaping (LENS), Electron Beam Additive Manufacturing (EBAM), Laser Net Shape Manufacturing (LNSM), Direct Metal Deposition (DMD), Digital Light Processing (DLP), Continuous Digital Light Processing (CDLP), Direct Selective Laser Melting (DSLM), Selective Laser Melting (SLM), Direct Metal Laser Melting (DMLM), Direct Metal Laser Sintering (DMLS), Material Jetting (MJ), NanoParticle Jetting (NPJ), Drop On Demand (DOD), Binder Jetting (BJ), Multi Jet Fusion (MJF), Laminated Object Manufacturing (LOM) and other known processes.

The additive manufacturing processes described herein may be used for forming components using any suitable material. For example, the material may be plastic, metal, composite, concrete, ceramic, polymer, epoxy, photopolymer resin, or any other suitable material that may be in solid, liquid, powder, sheet material, wire, or any other suitable form or combinations thereof. More specifically, according to exemplary embodiments of the present subject matter, the additively manufactured components described herein may be formed in part, in whole, or in some combination of materials including but not limited to pure metals, nickel alloys, chrome alloys, titanium, titanium alloys, magnesium, magnesium alloys, aluminum, aluminum alloys, iron, iron alloys, stainless steel, and nickel or cobalt based superalloys (e.g., those available under the name Inconel® available from Special Metals Corporation). These materials are examples of materials suitable for use in additive manufacturing processes which may be suitable for the fabrication of examples described herein.

Typically, the additive manufacturing processes described herein may use a polymeric material, e.g. an acrylic material such as VeroClear™.

As noted above, the additive manufacturing process disclosed herein allows a single component to be formed from multiple materials. Thus, the examples described herein may be formed from any suitable mixtures of the above materials. For example, a component may include multiple layers, segments, or parts that are formed using different materials, processes, and/or on different additive manufacturing machines. In this manner, components may be constructed which have different materials and material properties for meeting the demands of any particular application. In addition,

1 although the components described herein are constructed entirely by additive manufacturing processes, it should be appreciated that in alternate embodiments, all or a portion of these components may be formed via casting, machining, and/or any other suitable manufacturing process. Indeed, any suitable combination of materials and manufacturing methods may be used to form these components.

Additive manufacturing processes typically fabricate components based on three-dimensional (3D) information, for example a three-dimensional computer model (or design file), of the component.

Accordingly, examples described herein not only include products or components as described herein, but also methods of manufacturing such products or components via additive manufacturing and computer software, firmware or hardware for controlling the manufacture of such products via additive manufacturing.

The structure of one or more parts of the product may be represented digitally in the form of a design file. A design file, or computer aided design (CAD) file, is a configuration file that encodes one or more of the surface or volumetric configuration of the shape of the product. That is, a design file represents the geometrical arrangement or shape of the product.

Design files can take any now known or later developed file format. For example, design files may be in the Stereolithography or “Standard Tessellation Language” (.stl) format which was created for stereolithography CAD programs of 3D Systems, or the Additive Manufacturing File (.amf) format, which is an American Society of Mechanical Engineers (ASME) standard that is an extensible markup-language (XML) based format designed to allow any CAD software to describe the shape and composition of any three- dimensional object to be fabricated on any additive manufacturing printer.

Further examples of design file formats include AutoCAD (.dwg) files, Blender (.blend) files, Parasolid (.x_t) files, 3D Manufacturing Format (.3mf) files, Autodesk (3ds) files, Collada (.dae) files and Wavefront (.obj) files, although many other file formats exist.

Design files can be produced using modelling (e.g. CAD modelling) software and/or through scanning the surface of a product to measure the surface configuration of the product.

Once obtained, a design file may be converted into a set of computer executable instructions that, once executed by a processer, cause the processor to control an additive manufacturing apparatus to produce a product according to the geometrical arrangement specified in the design file. The conversion may convert the design file into slices or layers that are to be formed sequentially by the additive manufacturing

1 apparatus. The instructions (otherwise known as geometric code or “G-code”) may be calibrated to the specific additive manufacturing apparatus and may specify the precise location and amount of material that is to be formed at each stage in the manufacturing process. As discussed above, the formation may be through deposition, through sintering, or through any other form of additive manufacturing method.

The code or instructions may be translated between different formats, converted into a set of data signals and transmitted, received as a set of data signals and converted to code, stored, etc., as necessary. The instructions may be an input to the additive manufacturing system and may come from a part designer, an intellectual property (IP) provider, a design company, the operator or owner of the additive manufacturing system, or from other sources. An additive manufacturing system may execute the instructions to fabricate the product using any of the technologies or methods disclosed herein.

Design files or computer executable instructions may be stored in a (transitory or non-transitory) computer readable storage medium (e.g., memory, storage system, etc.) storing code, or computer readable instructions, representative of the product to be produced. As noted, the code or computer readable instructions defining the product that can be used to physically generate the object, upon execution of the code or instructions by an additive manufacturing system. For example, the instructions may include a precisely defined 3D model of the product and can be generated from any of a large variety of well-known computer aided design (CAD) software systems such as AutoCAD®, TurboCAD®, DesignCAD 3D Max, etc. Alternatively, a model or prototype of the component may be scanned to determine the three-dimensional information of the component.

Accordingly, by controlling an additive manufacturing apparatus according to the computer executable instructions, the additive manufacturing apparatus can be instructed to print out one or more parts of the product. These can be printed either in assembled or unassembled form. For instance, different sections of the product may be printed separately (as a kit of unassembled parts) and then subsequently assembled. Alternatively, the different parts may be printed in assembled form.

In light of the above, embodiments include methods of manufacture via additive manufacturing. This includes the steps of obtaining a design file representing the product and instructing an additive manufacturing apparatus to manufacture the fluidic device according to the design file. The additive manufacturing apparatus may include a processor that is configured to automatically convert the design file into computer executable instructions for controlling the manufacture of the fluidic device. In these

1 embodiments, the design file itself can automatically cause the production of the fluidic device once input into the additive manufacturing device. Accordingly, in this embodiment, the design file itself may be considered computer executable instructions that cause the additive manufacturing apparatus to manufacture the fluidic device. Alternatively, the design file may be converted into instructions by an external computing system, with the resulting computer executable instructions being provided to the additive manufacturing device.

Given the above, the design and manufacture of implementations of the subject matter and the operations described in this specification can be realized using digital electronic circuitry, or in computer software, firmware, or hardware, including the structures disclosed in this specification and their structural equivalents, or in combinations of one or more of them. For instance, hardware may include processors, microprocessors, electronic circuitry, electronic components, integrated circuits, etc. Implementations of the subject matter described in this specification can be realized using one or more computer programs, i.e., one or more modules of computer program instructions, encoded on computer storage medium for execution by, or to control the operation of, data processing apparatus. Alternatively or in addition, the program instructions can be encoded on an artificially-generated propagated signal, e.g., a machine-generated electrical, optical, or electromagnetic signal that is generated to encode information for transmission to suitable receiver apparatus for execution by a data processing apparatus. A computer storage medium can be, or be included in, a computer-readable storage device, a computer-readable storage substrate, a random or serial access memory array or device, or a combination of one or more of them. Moreover, while a computer storage medium is not a propagated signal, a computer storage medium can be a source or destination of computer program instructions encoded in an artificially-generated propagated signal. The computer storage medium can also be, or be included in, one or more separate physical components or media (e.g., multiple CDs, disks, or other storage devices).

Although additive manufacturing technology is described herein as enabling fabrication of complex objects by building objects point-by-point, layer-by-layer, typically in a vertical direction, other methods of fabrication are possible and within the scope of the present subject matter. For example, although the discussion herein refers to the addition of material to form successive layers, one skilled in the art will appreciate that the methods and structures disclosed herein may be practiced with any additive manufacturing technique or other manufacturing technology.

1 The features described in relation to any aspect of the invention may equally apply to any other aspect and, merely for brevity, are not repeated. For example, features described in relation to apparatus can apply in relation to methods, and vice versa.

Brief Description of Drawinas

Embodiments of the invention are described with reference to the accompanying drawings, in which:

Figure 1 shows a fluidic apparatus according to a first embodiment;

Figure 2 shows the fluidic apparatus of Figure 1 , fitted with cell culture inserts;

Figure 3 shows a cell culture insert for use with the apparatus device of Figure 1 ;

Figure 4 shows a fluidic system according to a second embodiment;

Figure 5 illustrates visual investigation of flow through the apparatus of Figure 1 using an aqueous food colouring medium;

Figure 6 shows maximum intensity projection images from PET/CT scans of the first apparatus of Figure 1 , tested using a flow rate of 1 .5mL/min;

Figure 7 shows an alternative fluidic apparatus according to a comparative example;

Figure 8 shows maximum intensity projection images from PET/CT scans of the apparatus of Figure 7, tested with a flow rate of 1 .5mL/min;

Figure 9 is a schematic view of the layout of the compartments of the apparatus of Figure 1 provided with populated cell inserts;

Figure 10 shows time-activity curves from the test of the apparatus of Figure 1 described in Figure 6;

Figures 11 to 14 show chromatograms illustrating measurable drug uptake and inter-compartment cross talk demonstrated by presence of hepatocyte metabolites in other compartments;

Figure 15 shows linear regressions of derived Ki from measured intracellular [¹⁸F]FDG in the inserts of a system of Figure 4, plotted against human in vivo SUV_mean, r²= 0.7966, p= 0.0416, n=1 , Pearson’s correlation.

Detailed

In the present disclosure, reference is made to a number of terms, which have the meanings provided below, unless a context indicates to the contrary.

1 The term “comprising” or variants thereof is to be understood herein to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term “consisting” or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

The term “about” herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified. For example, if a temperature is specified to be about 5 to about 13 ^qC, temperatures of 4.75 to 13.65 °C are included.

Reference to physical states of matter (such as liquid or solid) refer to the matter’s state at 25 °C and atmospheric pressure unless the context dictates otherwise.

The term “in direct fluid communication” herein means that there is no further compartment or group of compartments between the indicated parts.

As explained above, the present inventors have discovered that it is possible to provide a fluidic apparatus that allows an even flow of fluid between the inlet and the outlet through each compartment of the same group of compartments.

Figure 1 shows a fluidic apparatus 105 according to a first embodiment.

In this embodiment, the apparatus 105 has a body 1 10 which is 3D-printed from an acrylic polymer, namely VeroClear™. However, it will be appreciated that the fluidic apparatus 105 may alternatively be manufactured by other manufacturing techniques, for example chemical etching, mechanical machining, etc.

The fluidic apparatus 105 comprises an inlet 11 1 configured to receive a flow of fluid, e.g. biological fluid, into the fluidic apparatus, and an outlet 112 configured to allow the fluid to exit the fluidic apparatus.

In this embodiment, the fluidic apparatus comprises two groups of compartments.

The first group of compartments 121 which is disposed nearest the outlet 1 12, has four compartments 121 a, 121 b, 121 c, 121 d, disposed in parallel.

The second group of compartments 122, which is disposed nearest the inlet 1 11 , has a single second compartments 122.

The inlet 1 11 is in direct fluid communication with the second compartment 122 via an inlet channel 131.

1 The outlet 112 is in direct fluid communication with the first group 121 of compartments 121 a, 121 b, 121 c, 121 d via outlet channel 132.

Advantageously, the fluidic apparatus 105 is configured to allow a flow of fluid between the inlet 1 11 and the outlet 1 12 through each of the first compartments 121 a,121 b,121 c,121 d separately, and the flow of fluid is evenly distributed and/or substantially identical between the first compartments 121 a, 121 b, 121c, 121 d.

The second compartment 122 is in direct fluid communication with each of the first compartments 121 a, 121 b, 121c, 121d via respective fluid connection channels 133a,133b,133c, 133d.

Each of the fluid connection channels 133a, 133b, 133c, 133d between the second compartment 122 and the first compartments 121 a,121 b,121 c,121 d are substantially identical in dimension and are symmetrically disposed relative to each other, in this embodiment at an angle of ration of about 60°. Each of the fluid connection channels 133a, 133b, 133c, 133d has a substantially identical length of about 5.1 mm and diameter of about 2mm.

Advantageously, this arrangement allows a flow of fluid between the second compartment 122 and each of the first compartments 121 a, 121 b, 121c, 121 d of the first group of compartments 121 to be substantially identical and/or evenly distributed.

The outlet 1 12 is in direct fluid communication with each compartment 121 a,121 b,121 c,121 d of the first group 121 of compartments via respective outlet fluid channels 134a,134b,134c,134d.

Each of the outlet fluid channels 134a,134b,134c,134d are substantially identical in dimension and are symmetrically disposed relative to each other.

Each of the outlet fluid channels 134a,134b,134c,134d has a substantially identical length of about 12.4mm and diameter of about 2mm.

Advantageously, this allows a flow of fluid out of each of the between the each of the first compartments 121 a, 121 b, 121c, 121 d to be substantially identical and/or evenly distributed, as it may avoid any preferentially path of least resistance amongst the compartments of the same group of compartments, here amongst the first compartments 121 a, 121 b, 121 c, 121 d.

In this embodiment, the first and second outlet fluid channels 134a, 134b merge into a first auxiliary channel 135a in direct fluid communication with the outlet 132. Similarly, the third and fourth outlet fluid channels 134c,134d merge into a second auxiliary channel 135b in direct fluid communication with the outlet 132.

1 The first and second auxiliary channels 135a, 135b are substantially symmetrically disposed relative to the outlet, and have a substantially identical length and a substantially identical diameter of about 2mm. Again, this promotes even flow of a fluid through and away from each of the first compartments 121 a, 121 b, 121 c, 121 d, as it may avoid any preferentially path of least resistance amongst the compartments of the same group of compartments, here amongst the first compartments 121 a, 121 b, 121 c, 121d.

Preferably, the compartments of each group of compartments are substantially identical in size and/or configuration.

In this embodiment, the first compartments 121 a,121 b,121 c,121 d of the first group of compartments 121 are substantially identical in size and configuration. The first compartments 121 a, 121 b, 121c, 121 d of the first group of compartments 121 are substantially cylindrical, and have a diameter of about 22mm and a depth of about 17.9mm.. This may promote even flow of fluid through each of the first compartments 121 a,121 b,121 c,121 d of the first group of compartments 121.

Conveniently, in this embodiment, the second compartment 122 is also substantially cylindrical and has a diameter of about 22mm and a depth of about 17.9mm..

Conveniently, the size of the compartments was selected so as to receive a 12- well cell culture insert, for example a 12-well insert 150 as shown in Figure 3. 12-well inserts were identified as being sufficient large to allow accurate and reproducible quantification from the inserts by HPLC and/or LC-MS analysis, and sufficiently small to avoid the need for an excessive amount of cells to be grown to carry out an experiment. Without wishing to be bound by theory, it is believed that if the size of the inserts is too small, for example a 96-well insert, the amount of cells would be too low for there to be measurable concentrations of test compounds/metabolites, as the concentrations would be below the limit of detection of analytical techniques such as HPLC and/or LC-MS.

Each compartment has connection means in the form of recesses 125, arranged to receive a complementary engaging elements 151 of a respective cell culture insert 150. It will be appreciated that the particular shape and size of the recesses 125 may be configured to match or receive complementary engaging elements 151 of the respective cell culture insert 150 intended to be used with the apparatus 105. In some embodiments, there may be provided several sets of recesses 125, each set of recesses 125 configured to match or receive complementary engaging elements 151 of a

1 particular type of cell culture insert 150 intended to be used with the apparatus 105. This may improve the versatility of the apparatus 105.

Referring to Figure 3, the inserts 150 have a main body 154 defining a cavity 155 therein. The inserts 150 also have a flange 152 configure to engage with a rim of a respective compartment. The flange 152 may allow a bottom portion 153 of the insert 150 to be located at a predetermined depth or height within a respective compartment, so as to allow the bottom portion 153, typically equipped with a semi-permeable membrane, to be placed in the fluid flowpath of the fluidic device, in use. This may allow, in use, one or more substances within the fluid to permeate through the bottom portion 153, e.g. semi-permeable membrane, into the cavity 155. It will be appreciated that the semi-permeable membranes may have a different pore sizes depending on the desired migration between the fluid and the compartment cavity 155. For example, 3pM pore size may allow fluid and substrate to pass through, whilst larger pores (e.g. 12pM) may allow cell-cell interactions if the aim is for an “organ” compartment to be more complex and similar to in vivo, and smaller pores (e.g. 0.22pM) may be used if the goal is to prevent interactions between both layers etc.

Figure 2 shows the fluidic apparatus 105 of Figure 1 , with cell culture inserts 150 placed in their respective compartments 121 a, 121 b, 121c, 121 d, 122, with the engaging element 151 of the cell culture inserts 150 engaging corresponding recesses 125 of the compartments 121 a, 121 b, 121 c,121 d, 122.

Figure 4 shows a fluidic system 260 according to an embodiment. The system 260 includes the fluidic device 105 of Figure 1 .

The system 260 includes a pump, which in this embodiment is a peristaltic pump or roller pump 270. The pump 270 includes a cassette 274 which induces flow by creating pressure between the pump and the connected cannula 271. In this embodiment, the pump is equipped with two cassettes, but the pump may be equipped with a number of cassettes, for example four cassettes, and therefore multiple fluidic devices 105 could be operated by the pump 270 simultaneously.

The pump 270, in this embodiment the cassette 274, is connected to the inlet 1 11 via an inlet cannula 271 configured to provide fluid communication between the pump 270 and the inlet 11 1 , and to the outlet 112 via an outlet cannula configured to provide fluid communication between the pump 270 and the outlet 1 12.

1 Thus, the system 260 defines a flowpath for circulating a fluid through the fluidic device 105. The pump 270 is configured to circulate the fluid from the inlet 11 1 to the outlet 112 through the fluidic device 105, and from the outlet 1 12 to the inlet 1 11 via the outlet cannula 272, pump 270 and inlet cannula 271. Advantageously, this permits continuous circulation of fluid between the inlet 1 11 and the outlet 122 though the fluidic device 105, and between the outlet 1 12 and the inlet 1 11 through the pump 270, thus mimicking a mammal’s systemic circulation.

The system further includes a fluid feed interface 273 connected to the flowpath, here in communication with the outlet cannula 272, which allows the feeding of a fluid, e.g. a biological fluid, into the flowpath. In this embodiment, the system includes a syringe 275 containing the fluid, which is connected to the fluid feed interface 273. The arrangement shown in this embodiment may mimic a medical intravenous injection into the venous blood pool. It will be appreciated that other feed interface arrangements may be envisaged depending on the specific investigation being carried out. For example, in another embodiment, a separate inlet to an “intestine” compartment/insert may mimic the absorption of a drug through oral ingestion rather than injection.

Although not shown in Figure 4 for clarity, in use, it will be understood that cell culture inserts 150 will be provided in their respective compartments 121 a,121 b,121 c,121 d,122. Advantageously, the cell culture inserts are 12-well inserts. 12-well inserts were identified as being sufficient large to allow sampling a volume of fluid from the inserts which is suitable for HPLC analysis, and sufficiently small to avoid the need for an excessive amount of cells to be grown to carry out an experiment. This may allow the sampling of a volume of fluid of about at least 1 OpL, e.g. at least 20pL, e.g. at least 50pL, e.g. at least 75pL from the inserts 150. Advantageously, the cell culture inserts 150 are sized so as to permit sampling of an insert 150 without requiring stopping the flow of fluid through the fluidic device 105, in use.

Fluidic Device Optimisation

Basic visual test

In order to test a 3D-printed fluidic device for potential flaws, basic visual investigation of flow through the fluidic apparatus was carried out using an aqueous food colouring medium 380. An embodiment of an early iteration of the apparatus 305, as shown in Figure 5, was conducted, using the apparatus of Figure 4. The apparatus 305 of Figure 5 is generally similar to the apparatus 105 of Figure 1 , like parts denoted by

1 like numerals, incremented by ‘200’. Figure 5 shows that no fluid was passed through region 382 of the first auxiliary channel 335a, indicating the presence of a blockage. This simple method allows the testing of the various channels of the fluidic apparatus 305, before use in a substantial study, e.g. drug study.

Body-on-chip device optimisation

[¹⁸F]FDG or [¹⁸F]NaF from the Queen’s Medical Research Imaging (QMRI) Edinburgh Imaging Facility (University of Edinburgh, UK) was used in combination with a preclinical PET/CT small animal scanner (nanoPET/CT,Mediso) to test the perfusion performance of the fluidic device 105. This was carried out by visually inspecting the distribution of [¹⁸F]tracer using PMOD image analysis software (PMOD Technologies). The PET scan was obtained using a 1 :5 coincidence mode. Then, the CT scan was acquired (semi-circular full trajectory, maximum field of view, 480 projections, 35 kVp, 400 ms and 1 :4 binning) for structural overlay and attenuation correction. Tracer was circulated through the fluidic device using an MS-4/12 Reglo digital pump (ISMATECTM), 270. Once a seemingly even perfusion was achieved, volumes of interest were drawn using PMOD to extract time activity curves for each compartment, as well as the input function and the venous output.

Figure 6 shows maximum intensity projection images from PET/CT scans of the first apparatus of Figure 1 , tested using a flow rate of 1 .5mL/min.

As mentioned above, the flow was measured using [¹⁸F]FDG/[¹⁸F]NaF and a NanoScan MicroPET/CT system to overlay the gold standard flow measurements gained from PET, with the structural image gained from the CT scan. As shown in Figure 6, the resulting averaged image and time activity curves show that there is an even distribution of radioactivity throughout the organ compartments and within the interconnecting capillaries.

Comparative examples of an alternative design

Figure 7 shows an alternative fluidic apparatus 405 according to a comparative example. The apparatus 405 of Figure 7 is generally similar to the apparatus 105 of Figure 1 , like parts denoted by like numerals, incremented by ‘300’. However, in Figure 7, the fluid connection channels 433a, 433b, 433c, 433d between the second compartment 422 and the first compartments 421 a, 421 b, 421c, 421 d are not substantially identical in dimension and are not symmetrically disposed relative to each other. In addition, the outlet channels 432 were also not substantially identical in shape and

1 dimension, and not symmetrically disposed relative to each other. As a result, it was found that the flow of fluid between the second compartment 422 and each of the first compartments 421 a, 421 b, 421c, 421 d of the first group of compartments 421 was not identical and/or evenly distributed. This was evidenced by Figure 8 which shows maximum intensity projection images from PET/CT scans of the apparatus of Figure 7, tested with a flow rate of 1 .5mL/min

Examples and Experiments

Dual seeding of endothelial cells and “organ” cells on opposing sides of a cell culture insert

Three Corning 3pM polyethylene terephthalate 12-well inserts (inserts 150 of Figure 3) were turned upside down and placed into individual wells of a 6-well plate before being surrounded by 1 mL Dulbecco’s phosphate buffered saline (DBPS) in the well to aid in preventing evaporation. Human umbilical vein endothelial cells (HUVECs) (PromoCell™, Germany) were then seeded onto the underside of the transwell insert at a density of 25,000 cells/cm² in a total volume of 1 mL before being left for 4 hours to adhere to the membrane. After 4 hours, the inserts were turned back upright and carefully transferred from the DBPS plate to a new plate containing endothelial cell growth medium, before being left overnight to equilibrate and divide. The following day, the “organ” compartment specific cell types were seeded at their respective optimal seeding density into the inserts, and left for 24 hours to adhere to the membrane. The three cell types included were human dermal fibroblasts (HDFibro) that produce and deposit extracellular matrix components with an expected quantifiable drug uptake, HepG2 (ECCAC) hepatocarcinoma cell line to show that metabolites are produced, and the SH-SY5Y (ECCAC) neuroblast cell line was used as there should be very little drug uptake, but there is potential for metabolite uptake if there is compartment cross talk. On the day of the study, the inserts were inspected individually to confirm the presence of endothelial cells on the outer surface of the insert bottom portion 153 and of “organ” cells on the inside surface of the insert bottom portion 153.

Body-on-chip study with live cells

The chip was filled with 11 mL endothelial cell medium and 2.4mM docetaxel was made up in 500pL endothelial medium and dimethyl sulfoxide (76:24, v:v). The docetaxel dose was then mixed with 22.5 MBq [¹⁸F]FDG and made up to a final volume of 1 mL,

1 such that when injected into the chip, the final circulating concentration was 100pM docetaxel in 1% (v:v) DMSO. Five 12-well inserts were then placed into the chip as per Figure 9 , before the inlet and outlet points were cannulated, and the chip was then placed in the bed of the NanoScan PET/CT.

Figure 9 is a schematic view of the layout of the compartments of the apparatus of Figure 1 provided with populated cell inserts, where IO stands for inserts only, due to the two compartments 121 a, 122 requiring primary cells.

The peristaltic pump was set to a flow rate of 1.5mL/min before being switched on to allow flow through the chip. The radiotracer together with the drug were then injected through the inlet 1 11 and the PET acquisition was turned on for a 60-minute scan using 1 :5 scanning mode to confirm even distribution of flow through the compartments 122,121 a,121 b,121 c,121 d. As soon as the scan was terminated, a CT acquisition (semi-circular full trajectory, maximum field of view, 480 projections, 35 kVp, 400 ms and 1 :4 binningj.was immediately started to provide structural overlay and for attenuation correction of the PET data. Once finished, the chip was removed from the PET/CT and placed behind lead. The inserts were removed and placed into individual wells of a 6-well plate before removing the medium within the insert and washing the cells with DPBS. The cells were then lifted using 200pL Tryple (ThermoFisher Scientific), of which 20pL was taken for cell counting and the rest was transferred to a 500pL Eppendorf™. The Eppendorf™ tubes were then centrifuged at 1000x g for 5 minutes to form a pellet and the supernatant was removed. The cell pellet was then re-suspended in 75pL dH₂O to prevent clumping, before being lysed via the addition of 150pL acetonitrile. 75pL dH₂O was then added to make the final solution 50:50 dH₂0:acetonitrile (v:v) to match the mobile phase and docetaxel standards used for HPLC analysis. The Eppendorf™ tubes were then centrifuged, and the supernatant was removed for HPLC analysis of intracellular drug concentration.

Figure 10 shows time-activity curves from the test of the apparatus of Figure 1 described in Figure 6.

The input function refers to the inlet channel 131 leading to the heart compartment 122, and fluctuates due to the pulsing flow of the peristaltic pump 270. The heart compartment 122 peaks in activity soon after, before both the input function and heart decline as the radiotracer is then distributed from the heart to the other organs (compartments 121 a, 121b, 121 c, 121 d), reaching equilibrium around minute 4

1 Figures 11 to 14 show chromatograms illustrating measurable drug uptake and inter-compartment cross talk demonstrated by presence of hepatocyte metabolites in other compartments.

The chromatograms shown in Figures 1 1 -14 illustrate measurable drug uptake and inter-compartment cross talk demonstrated by presence of hepatocyte metabolites in other compartments:

Figure 1 1 : Chromatogram from an injection of 0.1 mg/mL docetaxel in mobile phase, with a retention time of 7.473 minutes.

Figure 12: Chromatogram from an injection of lysed hepatocytes 1 hour post docetaxel injection into the novel invention, demonstrating that the same metabolites are present;

Figure 13: Chromatogram from an injection of lysed SH-SY5Y cells used in the brain compartment, illustrating no drug uptake as expected, but presence of the metabolites produced in the liver compartment;

Figure 14: Chromatogram from an injection of lysed human dermal fibroblasts demonstrating measurable drug uptake as well as presence of the metabolites.

DDChip [¹⁸FjFDG kinetic study method

HUVEC barrier inserts were prepared (see above), where the respective organ cells were seeded 24 hours after the HUVECs (PromoCell™, Germany) were attached and transferred to a 12-well plate, allowing organ cells to adhere and grow for 48 hours prior to studies commencing. HUVECs were used between passages 4-5, SH-SY5Y (ECCAC, 94030304) were used and differentiated at passages 2-3, the SA7K (Sigma™, MA, USA) and HepG2 (ECACC, 8501 1430) cell lines were used at passage 5-6, and both HCM and HBEPCs (PromoCell, Germany) were used at passage 2. The device 105 (see Figure 4) was sterilised in 70% isopropanol inside a sterile hood for 30 minutes and left to dry before being stored in a sterile container for later use. During studies, the device 105 was connected to a MS-4/12 Reglo digital pump 270 (ISMATEC™, Wertheim, Germany) set to 1.5mL/min, using a closed circuit with a 3-way inlet to mimic an intravenous injection of docetaxel or [¹⁸F]FDG, illustrated in Figure 4.

The device was then filled with 11 mL endothelial growth medium pre-warmed to 37^eC such that injection of 1 mL brings the total volume to 12mL. Inserts 150 with an endothelial barrier and representing the heart, lung, liver, kidney, and brain were then

1 placed into the device 105 before placing it into the incubator ready for injection of [¹⁸F]FDG. [¹⁸F]FDG in saline was made up to a total of 1 mL with endothelial medium, with a concentration of 50.04 ± 1 1.96 MBq/mL, such that the circulating concentrations were 4.17 ± 1.00 MBq/mL (both expressed as mean ± SD, n=9) in the device 105. The flow was turned on and the dose of [¹⁸F]FDG was injected into the system 260, followed by clamping the inlet 1 11 to prevent backflow. At each respective timepoint the cassette 274 was removed from the system 260 to stop flow without interrupting other devices connected to the pump 260, after which the inserts 150 were processed to assess intracellular [¹⁸F]FDG concentration for kinetic modelling.

DDChip [¹⁸F1FDG kinetic study results

The cell lysate had measurable intracellular [¹⁸F]FDG from the earliest time point of 1 minute for all compartments. Patlak model derived Ki (Ki being the rate of influx for a model using irreversible binding) was highest for the brain compartment at 4.73x1 O'³ mL/cm³/min, followed by the kidney, heart, liver, then lung at 2.84x1 O'³, 2.43x1 O'³, 1 .01 x10³, and 1 .93x10'⁴ mL/cm³/min. These values were plotted against in vivo SUV_mean values (where SUV_mean is the average SUV (Standardised Uptake Value, calculated as concentration in tissue normalised to injected dose and body weight) across a tissue of interest) from healthy human volunteers for each organ compartment, as shown in Figure 15. Derived displayed a significant correlation with in vivo SUVmean (r²= 0.7966, p= 0.0416, n=1 , Pearson’s correlation). The significant linear correlation between the kinetics in the present system and human kinetics of [¹⁸F]FDG demonstrates the potential of the system 260 to be used in predicting small molecule drug kinetics for translation to human patients.

It will be understood that the present embodiments are provided by way of example only, and that various modifications can be made to the present embodiments without departing from the scope of the invention.

1

Claims

CLAIMS:

1 . A fluidic apparatus comprising: an inlet; an outlet; and a first group of compartments comprising two or more first compartments arranged in parallel between the inlet and the outlet; wherein each of the first compartments is configured to receive a respective cell culture insert, and wherein a flow of fluid between the inlet and the outlet is evenly distributed and/or substantially identical between the first compartments.

2. A fluidic apparatus according to claim 1 , wherein the inlet is in fluid communication with the first group of compartments and/or wherein the outlet is in fluid communication with the first group of compartments.

3. A fluidic apparatus according to claim 1 or claim 2, wherein the fluidic apparatus is configured to allow a flow of fluid between the inlet and the outlet through each of the first compartments separately.

4. A fluidic apparatus according to any preceding claim, wherein the flow rate of a fluid between the inlet and the outlet is substantially identical and/or evenly distributed through each of the first compartments.

5. A fluidic apparatus according to any preceding claim, wherein the fluidic apparatus comprises a second group of compartments comprising one or more second compartments arranged in parallel between the inlet and the outlet.

6. A fluidic apparatus according to claim 5, wherein the flow rate of a fluid between the inlet and the outlet is substantially identical through each of the second compartments.

7. A fluidic apparatus according to claim 5 or claim 6, wherein the second group of compartments is provided between the inlet and the first group of compartments, or between the first group of compartments and the outlet.

8. A fluidic apparatus according to any one of claims 5 to 7, wherein each compartment of the second group of compartments is in direct fluid communication with one or more first compartments of the first group of compartments via respective first fluid channels.

9. A fluidic apparatus according to any one of claims 5 to 8, wherein the two or more first compartments of the first group of compartments are in direct fluid communication with the outlet via respective outlet fluid channels, and wherein the one or more second compartments of the second group of compartments is/are in direct fluid communication with the inlet via (a) respective inlet fluid channel(s).

10. A fluidic apparatus according to any one of claims 5 to 8, wherein the two or more first compartments of the first group of compartments are in direct fluid communication with the inlet via respective inlet fluid channels, and wherein the one or more second compartments of the second group of compartments is/are in direct fluid communication with the outlet via (a) respective outlet fluid channel(s).

11. A fluidic apparatus according to any one of claims 5 to 10, wherein each of the inlet fluid channel(s) are substantially identical in dimension and/or are symmetrically disposed relative to each other.

12. A fluidic apparatus according to any one of claims 5 to 11 , wherein each of the outlet fluid channel(s) are substantially identical in dimension and/or are symmetrically disposed relative to each other.

13. A fluidic apparatus according to any one of claims 5 to 12, wherein each of the first fluid channel(s) are be substantially identical in dimension and/or are symmetrically disposed relative to each other.

14. A fluidic apparatus according to any preceding claim, wherein the compartments within each group of compartments are substantially identical in size and/or configuration.

15. A fluidic apparatus according to any preceding claim, wherein the fluidic apparatus defines a flowpath between the inlet and the outlet, wherein the flowpath is defined by the channels and the compartments.

16. A fluidic apparatus according to any preceding claim, wherein the compartments are configured to receive a cell culture insert selected from a 24-well insert, a 12-well insert, or a 6-well insert.

17. A fluidic apparatus comprising : an inlet; an outlet; a first group of compartments comprising two or more first compartments arranged in parallel between the inlet and the outlet; a second group of compartments comprising one or more second compartments arranged in parallel between the inlet and the outlet; and optionally one or more further group(s) of compartments arranged in parallel between the first group of compartments and the second group of compartments, wherein the inlet is in fluid communication with the first group of compartments or with the second group of compartments via respective inlet channel(s), wherein the outlet is in fluid communication with the second group of compartments or with the first group of compartments via respective outlet channel(s), wherein the compartments of each group of compartments are in fluid communication with one or more compartments of an adjacent group of compartments via respective fluid connection channels, wherein each compartment is configured to receive a respective cell culture insert, wherein each of the inlet fluid channel(s) are substantially identical in dimension and/or are symmetrically disposed relative to each other, wherein each of the outlet fluid channel(s) are substantially identical in dimension and/or are symmetrically disposed relative to each other, and wherein each of the fluid connection channels between the compartments of adjacent groups of compartment are substantially identical in dimension and/or are symmetrically disposed relative to each other.

18. A fluidic system comprising:

1 a fluidic apparatus according to any one of claims 1 to 17; and a pump configured to pump a fluid through the fluidic apparatus.

19. The system according to claim 18, further comprising an inlet conduit configured to provide fluid communication between the pump and the inlet; and an outlet conduit configured to provide fluid communication between the pump and the outlet.

20. The system according to claim 18 or claim 19, further comprising a fluid feed interface configured to allow the feeding of a fluid into a flowpath defined by the system.

21. The system according to any one of claims 18 to 20, further comprising cell culture inserts configured to be provided in respective compartments of the fluidic device.

22. A method of testing the effect a substance on one or more type of cells, the method comprising: providing a system according to any one of claims 18 to 21 ; providing a cell culture insert in one or more compartment of the fluidic apparatus; providing a fluid into a flowpath defined by the system and/or fluidic apparatus thereof; circulating the fluid through the fluidic apparatus and/or system.

23. A method of manufacturing a fluidic apparatus according to any one of claims 1 to 17, the method comprising manufacturing the fluidic apparatus by additive manufacturing, optionally by 3D printing.

24. A computer program comprising computer executable instructions that, when executed by a processor, cause the processor to control an additive manufacturing apparatus to manufacture the apparatus of any one of claims claim 1 to 17.

25. A method of manufacturing a device via additive manufacturing, the method comprising: obtaining an electronic file representing a geometry of a product wherein the product is an apparatus according to any one of claims claim 1 to 17; and

1 controlling an additive manufacturing apparatus to manufacture, over one or more additive manufacturing steps, the product according to the geometry specified in the electronic file.