Multicellular vascularized tumor sphere 3D chip, its application and usage method

Abstract

The invention relates to a microfluidics device comprising a lower layer comprising a stroma compartment having a central zone having a tumour microenvironment medium input channel and a tumour microenvironment (TME) medium output channel, and a periphery having at least one infusion input channel and at least one infusion output channel. The central zone and the periphery being demarked by a plurality of interspersed pillars which partially separate the central zone from the periphery, wherein the pillars are arranged to retain a tumour microenvironment medium in the central zone and to permit an infusion fluid to flow from the infusion input channel into the central zone. A middle layer comprising a tumour compartment which is collocated with the central zone; the tumour compartment having a stroma medium input channel and a stroma medium output channel. A tumour retaining portion to position a tumour in a desired location.

Description

[0001] DEVICE

[0002] Field of the Invention

[0003] The present invention relates to a microfluidics device for use in creating a tumour microenvironment (TME) or stromal milieu, to a medium for use in the creation of a TME or stromal milieu, to methods of creating a TME or stromal milieu and methods of testing therapeutic candidates using the device, medium and milieu of the disclosure.

[0004] Background to the Invention

[0005] Overall approval rates for oncology drugs from Phase I clinical trials is only 3.4% [1], Although immunotherapy has emerged as a revolutionary approach, showing high success rates and improved survival rates, its effectiveness is limited in solid cancers by the surrounding tissue, which creates an immunosuppressive tumour microenvironment (TME)[l-3], Because the TME promotes immune evasion and hampers the antitumour immune response [4,5], the tumour's response to immunotherapies is dependent on the characteristics of the particular TME.

[0006] For example, non-small cell lung carcinoma (which often shows high mutational burden) is often susceptible to treatment with immunotherapies like checkpoint inhibitors [6], However, only around 40% of patients with solid tumours are eligible for these immunotherapies and, among those, only 12% show a successful response [7], Many complex mechanisms contribute to this situation, such as cancer- associated fibroblasts' (CAFs) aberrant modification of the extracellular matrix (ECM) [8] or the release of immunosuppressive cytokines by tumour-associated macrophages (TAMs) [9] and other immunosuppressive cells, which render usual checkpoint inhibitors ineffective.

[0007] Currently there are no approved immunotherapies for the treatment of pancreatic cancer

[0010] , with only a few clinical trials undergoing

[0011] , Pancreatic tumours are particularly difficult to access because, in addition to the presence of immunosuppressive cells, they often have a fibrotic stroma, which further limits the infiltration of immune cells, leads to their exhaustion, and limits the penetration of immunotherapies

[0012] ,

[0008] Patients that do not qualify for immunotherapy find themselves with chemotherapy and radiotherapy as their only therapeutic options. Both of which have debilitating side effects and poorer outcomes. Ongoing research aims to optimise and expand the application of immunotherapies, providing new hope for cancer patients worldwide

[0013] , As a result, a better understanding of the features of cancer that prevent the application of immunotherapies is crucial for identifying novel targets and in extending the benefits of immuno-oncology to a larger number of patients.

[0009] At present, the assessment of immuno-oncology candidates relies on inaccurate models such as patient- derived xenografts (PDX) implanted onto mouse models. Engraftment success in PDX mice can be as low as 30% and the fidelity of the tumour is lost [2], Furthermore, the process of serial passaging in immunocompromised mice leads to heterogeneity loss, selection bias, and TME changes [3], The TME changes are particularly significant when testing immunotherapies because the journey of immune cells from the bloodstream through the stroma and into the tumour is not replicated in most animal studies. This leads to false positives that go into clinical trials and it is only in Phase II clinical trials that efficacy is found to be deficient. Thus, there is a need to provide a better screening method for immune-oncology candidates that more accurately mimics the TME.

[0010] Current efforts to improve our understanding of the mechanisms of disease, drug efficacy, and drug toxicity in the preclinical stage include using organoids. Organoids are a mixed population of cells that self-organise to form a complex tissue that is reminiscent of organs [14,15], For example, liver organoids composed of hepatic stem cells, hepatocytes, fibroblasts, and endothelial cells self-organise to form a structure that is similar to a liver

[0016] , These 3D cultures have significant changes in pharmacokinetic and metabolic processing of drugs that is more similar to a human system. However, drug response in organoids is still not proportional to a human physiological response

[0017] because aspects of the architecture and the microenvironment are not represented in organoids. Furthermore, typical drug toxicity occurs systematically. Therefore, although organoid systems are helpful for assessing a drug's toxicity and eliminating highly toxic drug candidates, these results do not necessarily transfer to a human system.

[0011] Microphysiological systems are another recent development and typically use microfluidic devices designed to mimic cell-cell interactions and tissue organisation. The most common systems are the chips from Emulate® and Mimetas ®.

[0012] Emulate® chips rely on a polydimethylsiloxane (PDMS) membrane sandwiched between an upper and lower channel (US11773359B2, US9725687B2). Cell-cell interactions between cell types can then be studied using this chip. For example, Emulate® has a liver-on-a-chip system wherein the top channel is coated in an extracellular matrix protein solution that allows the growth of epithelial hepatocytes and the bottom channel contains hepatic stellate cells and endothelial cells that organise to mimic blood vessels. This system mimics drug infiltration through blood vessels and its eventual processing by hepatocytes. Due to the separation between the two channels, released factors such as cytokines and enzymes can be easily quantified between the two channels. However, PDMS is not completely inert and could bind growth factors or even the drugs flowing into the system. This could lead to inaccuracy in dosing. Furthermore, as mentioned above, toxicity is typically systemic, which requires multiple organs to be modelled together.

[0013] Mimetas® chips typically contain 3 channels with a "phase guide" between, where a hydrogel or ECM can be restricted in order to study barrier integrity and migration of cells (US11629319B2). The use of phase guides avoids the use of a PDMS membrane that potentially absorbs drug compounds. To feed the cells, flow is induced by using differential static pressures between the two sides of their device and a rocking system that lets the media flow from one side to the other back and forth. Mimetas® have also adapted a transepithelial electrical resistance (TEER) measurement device for their chips to monitor barrier integrity (US20180196035A1, USD898216S1, US20200393397A1).

[0014] Although existing solutions go some way to improving our ability to study tumours in a more physiologically accurate environment, these systems are missing biochemical and biophysical gradients that lead to tumour heterogeneity and TME heterogeneity. For example, CAFs could have several phenotypes (myofibroblast, inflammatory, vascular) and their phenotype is correlated with its spatial distribution with the tumour [8,18], Furthermore, there is no system that allows for continuous monitoring of the tumour microenvironment and how it changes over time. It is against this backdrop that we have developed a microfluidic device that contains a novel gel formulation where biochemical and biophysical gradients can be generated to reproduce the tumour microenvironment. Furthermore, we have developed a system that monitors the TME and the tumour independently in a high throughput manner. Finally, we provide a means of testing new immuno-oncology approaches and monitoring how the tumour and the TME responds to these treatments.

[0015] Summary of the Invention

[0016] According to a first aspect there is provided a microfluidics device comprising: a lower layer comprising a stroma compartment having: o a central zone having a tumour microenvironment medium input channel and a tumour microenvironment medium output channel, and o a periphery having at least one infusion input channel and at least one infusion output channel, the central zone and the periphery being demarked by a plurality of interspersed pillars which partially separate the central zone from the periphery, wherein the pillars are arranged to retain a tumour microenvironment medium in the central zone and to permit an infusion fluid to flow from the infusion input channel into the central zone; a middle layer comprising a tumour compartment which is collocated with the central zone; the tumour compartment having a stroma medium input channel and a stroma medium output channel; and a tumour retaining portion to position a tumour in a desired location.

[0017] The device of may further comprising an upper layer comprising one or more ports providing access to one or more channel selected from the group consisting of tumour microenvironment medium input channel, tumour microenvironment medium output channel, infusion input channel, infusion output channel, stroma medium input channel, stroma medium output channel, tumour medium input channel, and tumour medium output channel.

[0018] The device periphery may have two infusion input channels and two infusion output channels.

[0019] The device tumour microenvironment medium input channel and the tumour microenvironment medium output channel may be located on opposite sides of the central zone.

[0020] The pillars may be cylindrical.

[0021] The pillars may have a wall between them.

[0022] The wall or walls may be shorter than the pillars.

[0023] The stroma medium input channel may be serpentine.

[0024] The stroma medium output channel may be serpentine.

[0025] The serpentine channel may have at least two switchbacks.

[0026] The tumour retaining portion may be a hoop.

[0027] The tumour retaining portion may have a lower surface which, in use, is in contact with the tumour microenvironment medium in the central zone. The tumour retaining portion may be a cylinder with solid walls having a cylinder diameter and a bottom with an aperture of smaller diameter than the cylinder diameter.

[0028] The tumour retaining portion may be integral in the upper layer.

[0029] The one or more ports may comprise attachment means.

[0030] The device may further comprise one or more sensors.

[0031] The device may have at least two sensors and at least one sensor is configured to monitor the stroma and at least one sensor is configured to monitor the tumour.

[0032] In a second aspect there is provided a method of creating a tumour microenvironment within a device according to any preceding claim, comprising the steps: a. introducing a suitable tumour microenvironment medium and cells to the central zone to create a stroma, b. flowing stroma medium over the surface of the stroma by adding stroma medium via the stroma medium input channel and removing stroma medium via the stroma medium output channel, c. placing a tumour in the tumour retaining device, and d. allowing a period of time to elapse.

[0033] In a third aspect there is provided a method of screening an oncology therapeutic candidate comprising setting up a tumour microenvironment as disclosed herein and introducing an infusion comprising the oncology therapeutic candidate via the infusion input channel.

[0034] In a fourth aspect there is provided a tumour microenvironment medium comprising a hydrogel, one or more extracellular matrix proteins, and transglutaminase.

[0035] In a fifth aspect there is provided a stromal milieu comprising a hydrogel, one or more extracellular matrix proteins, transglutaminase, and stromal cells.

[0036] The one or more extracellular matrix proteins may be selected from collagen, fibronectin and elastin.

[0037] The one or more extracellular matrix proteins may comprise collagen-l in a concentration in the range 2mg / ml to 5mg / ml.

[0038] The transglutaminase may have a concentration in the range O.lmg / ml to lOmg / ml.

[0039] The tumour microenvironment medium or stromal milieu may further comprise biochemical factors.

[0040] In a sixth aspect there is provided a kit of parts comprising a microfluidics device as disclosed herein and a tumour microenvironment medium or ingredients to make the TME medium as described herein.

[0041] Brief Description of the Drawings

[0042] For a better understanding of the invention and to show how the same may be carried into effect, there will now be described by way of example only, specific embodiments, methods and processes according to the present invention with reference to the accompanying drawings in which:

[0043] Figure 1 shows the microfluidic design (A / C) Diagram and schematics show the different parts of the microfluidic device where each component of the TME will be placed. Bottom layer contains a fibrotic gel with CAFs in the centre channel; the middle layer serves as a feeding system for the fibrotic gel; and the top one houses the tumour and its feeding system. The dimensions of the device are 24x24x1.5mm. (B) 3D-printed microfluidic chip showing the different compartments and channels. 1 - upper layer, 2 - middle layer, 3 - lower layer, 4 -TME medium and tumour-associated cells, 5 - tumour compartment, 6 - medium channels, 7 - infusion channels, 8 - central zone, 9 tumour-retaining portion.

[0044] Figure 2 shows design embodiments to integrate biocompatible electrodes and high throughput compatibility. (A) Redesign of the device to properly fit a 384 well plate (B). This allows for the device to be compatible with automated liquid handling and scale up the assays developed with the TM Ernie system.

[0045] Figure 3 shows the fabrication of collagen gels hydrogel. (A) SHG Microscopy images showing collagen I fibres on different collagen concentration hydrogels. The single optical sections show representative images of self-assembled collagen gels. Gels were polymerised at 37°C. Scale bar, 100 pm. (B) Mean fibre length of the collagen gels after polymerization. (C) Image shows separation of the collagen I hydrogel and the drug channels through circular micropillars in the microfluidic device.

[0046] Figure 4 shows tuneable hydrogel characterisation - Pseudo-coloured images obtained via SHG microscopy of collagen hydrogels of different concentrations with and without cross-linking.

[0047] Figure 5 shows more tuneable hydrogel characterisations - (A) Integrated density of collagen signal measure for Collagen I hydrogels of different concentration with and without cross-linking. (B) Quantification of collagen fibre width in non cross-linked and cross-linked samples. Mean ±SD; N = 3; (*, P < 0.05; P, **P < 0.01). Black, without cross-linker; Grey, with cross-linker. (C) Quantification of collagen fibre length in non cross-linked and cross-linked samples. Mean ±SD; N = 3; (*, P<0.05).

[0048] Figure 6 shows the stiffness tunability. (Graph shows Storage modulus (G1) and loss modulus (G") as a function of time for non-crosslinked and crosslinked 2 mg / ml collagen hydrogels, measured at 37 °C. Quantification shows the comparison of the shear storage modulus of 2mg / ml collagen hydrogels without and with the addition of crosslinking agent measured by rheology. Mean ± SD; N = 3; ****, P < 0.0001.

[0049] Figure 7 shows remodelling of hydrogels by MRC5-SV2 cells. (A) Pseudo-coloured images obtained via SHG microscopy of 2mg / ml collagen hydrogels with and without crosslinking. Upper images show structure of native collagen gels, bottom images show collagen network architecture after culturing MRC5-SV2 cells for 7 days. (B) images show structure of native collagen gels, bottom images show collagen network architecture after culturing MRC5-SV2 cells for 7 days.

[0050] Figure 8 shows fibronectin deposition by MRC5-SV2 cells on hydrogels. Representative multiphoton images of MRC5-SV2 cells growing for 7 days on 2mg / ml Collagen I hydrogels without cross-linker (top), with O.lmg / ml TG (middle), and with 5mg / ml TG (bottom). Scale bar, 50 pm. Boxed regions are shown at a higher magnification.

[0051] Figure 9 shows TGFP MRC5-SV2 cells on hydrogels. Representative multiphoton images of MRC5-SV2 cells growing for 7 days on 2mg / ml Collagen I hydrogels without cross-linker immunostained to visualise TGFP (Green). Scale bar, 20 pm. Figure 10 shows culturing PANC-1 spheroids on collagen hydrogels and co-culture with MRC5-SV2 induce metastatic-like phenotypes. Confocal images show PANC-1 spheroids grown for 7 days in U- shaped ultralow attachment plates (upper panels) or on 2mg / ml Collagen I hydrogels (lower panels). Coculture with MRC5-SV2 cells at a 9:1 ratio was performed (second and forth rows). Scale bar, 200 pm. Dotted lines indicate shape of the spheroid.

[0052] Figure 11. Monitoring polymerisation of hydrogels with impedance measurements. Impedance spectroscopy bode plots of the hydrogel, pre-polymerisation and post-polymerisation, water, and after swelling for 24hr. Distinct profiles could be seen between the different conditions.

[0053] Figure 12 Nyquist plots of different hydrogels with different collagen concentrations that have polymerised within a microfluidic device. (A) Without crosslinker. (B) With crosslinker. Distinct profiles could be observed for different concentrations of collagen. Evidently, adding the crosslinker led to increasing resistance especially when compared to hydrogels with the same collagen concentration (n=3).

[0054] Figure 13. Detecting a spheroid within a microfluidic device with an impedance analyser. (A) Differently sized spheroids after 7 days with different number of cells seeded. These spheroids were generated by spinning down the cells in an ultra-low attachment U-bottom plate. (B) Impedance spectroscopy bode plot of the microfluidic device with and without a spheroid within the microfluidic device. Increasing impedance is measured across frequencies when there is a spheroid within the microfluidic device.

[0055] Detailed Description

[0056] The present disclosure provides a microfluidics device comprising: a lower layer comprising a stroma compartment having: o a central zone having a tumour microenvironment medium input channel and a tumour microenvironment medium output channel, and o a periphery having at least one infusion input channel and at least one infusion output channel, o the central zone and the periphery being demarked by a plurality of interspersed pillars which partially separate the central zone from the periphery, wherein the pillars are arranged to retain a tumour microenvironment medium in the central zone and to permit an infusion fluid to flow from the infusion input channel into the central zone; a middle layer comprising a tumour compartment which is collocated with the stroma compartment; the tumour compartment having a medium input channel and a medium output channel; and a tumour retaining portion to position a tumour in a desired location.

[0057] Advantageously, the medium input and medium output channels help confine the TME medium in the lower layer.

[0058] Microfluidics Device

[0059] Microfluidics device as employed herein refers to a system that manipulates a small amount of fluids (10“9to 10“18litres) using small channels with sizes ten to hundreds micrometres. Microfluidics is a multidisciplinary field that involves molecular analysis, molecular biology, and microelectronics. It has practical applications in the design of systems that process low volumes of fluids to achieve multiplexing, automation, and high-throughput screening, all of which are relevant to the present device.

[0060] Lower layer as employed herein refers to the layer which is lower than the middle layer in use.

[0061] Stroma compartment as employed herein refers to the entirety of the channels and compartments of the lower layer. The channels and compartments are in fluid communication, therefore at least in principle, fluids could move in all areas of the stroma compartment. However, various features of the media used, in combination with physical features of the device, constrain the flow of the different media used to controlled zones.

[0062] The stroma compartment can be considered to be a well within the lower layer. In some embodiments, the stroma compartment is a central region (e.g. a central zone and periphery) with a number of arms (channels) extending therefrom.

[0063] Stroma as employed herein refers to the tumour stroma. In cancer biology, the stroma is defined as the non-malignant cells found in the supportive tissue surrounding tumours. These cells include fibroblasts, immune cells, endothelial cells, and various other cell types.

[0064] Stromal cells within the tumour microenvironment represent an important cellular component in cancer development, influencing tumour metabolism, growth, metastasis, immune evasion, and resistance to chemotherapy. These cells can originate from neighbouring non-cancerous stromal cells or undergo transdifferentiation from tumour cells.

[0065] Stromal cells contribute to tumour initiation, progression and drug resistance and the stroma is known to evolve as the tumour develops. Understanding the interactions between cancer cells and stromal cells is essential for developing effective cancer treatments. Alterations in the stroma, including the activation of fibroblasts into carcinoma-associated fibroblasts (CAFs) and remodelling of the extracellular matrix (ECM), are recognised as important in cancer progression and potential targets for therapy and diagnosis.

[0066] Central zone as employed herein refers to the middle of the lower layer as depicted in Figure 1C. The central zone 8 is defined the area inside the pillars and does not necessarily extend to include the TME medium input and output channels. In the examples, the central zone is shown as a circle but it will be appreciated that other shapes may be suitable.

[0067] In some embodiments the central zone is circular with a diameter of approximately 5.2 mm.

[0068] In some embodiments the central zone is approximately 500 pm deep.

[0069] "The central zone and the periphery being demarked by a plurality or interspersed pillars" as employed herein refers to the semi-permeable boundary created by the pillars. That is, the pillars partially separate the central zone from the periphery. The pillars do not fully block flow of liquids between the central zone and the periphery but they do act to retain TME medium in the central zone (at least in part due to the viscosity of the TME medium) such that it does not enter the periphery, whilst permitting infusion to enter the central zone from the periphery.

[0070] Pillars as employed herein refers to a plurality of interspersed posts that hinder the flow of TME medium into the periphery. The pillars may be spaced at regular intervals and at a uniform distance from the edge of the periphery, the edge being the outer wall of the stroma compartment. The pillars may be any suitable cross section and may be solid or hollow. The pillars may be full height, such that, in use, the top of the pillars contacts the lower surface of the middle layer.

[0071] Air pockets can be created within the device when the inlets and outlets, except for the TME medium input / output channels, are plugged. These air pockets can occur in the periphery and the middle layer of the device. The TME medium flow is then guided from the TME medium inlet channel to the outlet channel and restricted from going into the periphery and middle layer as the TME medium cannot overcome the fluidic resistance presented by the air pockets.

[0072] In some instances, the TME medium is prepared into a pre-stromal milieu by combining additional stromal components, such as cells, with the TME medium prior to adding it to the device.

[0073] As employed herein stromal milieu refers to TME medium and additional ingredients, such as cells, that together form the stroma. Pre-stromal milieu refers to the ingredients with before culturing / incubation, or after initial culture but before settling in the central zone of the device to form the stroma.

[0074] It will be appreciated that TME medium as employed herein may be substituted with stromal milieu.

[0075] As employed herein, stroma and stromal milieu may be used interchangeably.

[0076] As employed herein reference to TME medium may be TME alone or TME medium plus cells, otherwise referred to as stromal milieu or pre-stromal milieu.

[0077] Once the TME medium or stromal milieu is in situ, access to the channels may be closed by closing ports or by inserting sensors into the ports.

[0078] In one embodiment the pillars are cylindrical, such as a circular cross section.

[0079] In some embodiments the pillars are approximately 250 pm diameter / wide. For example, in the range approximately 200 to 300 pm.

[0080] In some embodiments the pillars are approximately 500 pm high. For example, in the range approximately 200 to 600 pm.

[0081] In some embodiments the spaces between the pillars may have walls of a lesser height than the pillars. The walls may fully span the gap between pillars, or partially span the gap between pillars.

[0082] In some embodiments the spaces are approximately 500 pm between pillars. For example, in the range approximately 400 to 600 pm.

[0083] In some embodiments the pillars are approximately 800 pm from the edge of the periphery. For example, in the range approximately 700 to 900 pm. In some embodiments all spaces have a wall. Where all spaces have a wall, the wall may not span the entrances to the TME medium input and output channels.

[0084] In some embodiments not all spaces have a wall.

[0085] In some embodiments the wall is approximately 350 pm high. For example, in the range approximately 150 to 400 pm.

[0086] Retain a tumour microenvironment medium as employed herein refers to the property by which the pillars constrain flow of TME medium from the central zone into the periphery. As noted above, this is in part due to the viscosity of the TME medium and in part due to air pockets that form or may form in the side channels, in the middle layers, and between the pillars.

[0087] It will be appreciated that TME medium may also refer to pre-stromal milieu or stromal milieu.

[0088] Tumour microenvironment medium input channel as employed herein refers to the channel through which TME medium flows and enters the central zone. The input channel and central zone do not have pillars or obstructions that would interfere with the flow of TME medium from the channel into the central zone. As such, the TME medium input channel is continuous with the central zone.

[0089] In some embodiments the TME medium input channel is approximately 1 mm wide. For example, in the range approximately 0.75 to 1.25mm.

[0090] In some embodiments the TME medium input channel is approximately 500 pm high. For example, in the range approximately 400 to 600 pm.

[0091] In some embodiments the TME medium input channel is approximately 6.79 mm long. For example, in the range approximately 5 to 8 mm.

[0092] Tumour microenvironment medium output channel as employed herein refers to the channel through which TME medium flows from the central zone. The output channel and central zone do not have pillars or obstructions that would interfere with the flow of TME medium from the central zone into the output channel. As such, the TME medium output channel is continuous with the central zone.

[0093] In some embodiments the TME medium output channel is approximately 1 mm wide. For example, in the range approximately 0.75 to 1.25mm.

[0094] In some embodiments the TME medium output channel is approximately 500 pm high. For example, in the range approximately 400 to 600 pm.

[0095] In some embodiments the TME medium output channel is approximately 6.79 mm long. For example, in the range approximately 5 to 8 mm.

[0096] Tumour microenvironment medium input and output channels together make up the tumour microenvironment medium channel. They are each considered to be arms or branches off the central zone and are in fluid communication with the central zone. In use, TME medium enters the central zone via the TME medium input channel and exits the central zone via the TME medium output channel. Once loaded with TME medium, the channels may be closed off or sealed. TME medium does not usually continue to flow through the channel in normal use because it is intended that the central zone will form a stroma.

[0097] Periphery as employed herein refers to the outer boundary of the stroma compartment. The periphery is defined and demarked by the pillars, being that the periphery is the area outside of the pillars. The periphery does not necessarily extend to the infusion input or output channels.

[0098] In some embodiments the periphery is approximately 800 pm wide when measured from the outer edge of the pillar to the edge of the periphery. For example, the periphery may be in the range approximately 700 to 900 pm wide.

[0099] Infusion as employed herein refers to a liquid composition of ingredients which may include a drug or pharmaceutical. In general, the infusion enters the microfluidics device via the infusion input channel, flows around the periphery (some of it entering the central zone via the spaces between the pillars), and exits via the infusion output channel.

[0100] Infusion input channel as employed herein refers to the channel through which the infusion enters the periphery.

[0101] In some embodiments the infusion input channel is approximately 1 mm wide. For example, in the range approximately 0.75 to 1.25mm.

[0102] In some embodiments the infusion input channel is approximately 500 pm high. For example, in the range approximately 400 to 600 pm.

[0103] In some embodiments the infusion input channel is approximately 9.5 mm long. For example, in the range approximately 8 to 11 mm.

[0104] Infusion output channel as employed herein refers to the channel through which the infusion exits the periphery.

[0105] In some embodiments the infusion output channel is approximately 1 mm wide. For example, in the range approximately 0.75 to 1.25mm.

[0106] In some embodiments the infusion output channel is approximately 500 pm high. For example, in the range approximately 400 to 600 pm.

[0107] In some embodiments the infusion output channel is approximately 9.5 mm long. For example, in the range approximately 8 to 11 mm.

[0108] Infusion input and output channels together make up one infusion channel. They are in fluid communication with the periphery and infusion can flow from the input channel, to the periphery, to the output channel unhindered. Infusion may also enter the central zone where it contacts the TME medium and may penetrate into the stroma, in use. In Figure IB infusion 7 can be seen entering the central zone 4.

[0109] In one embodiment the lower layer comprises two infusion input channels and two infusion output channels. In general, one infusion input channel and one infusion output channel form a continuous channel. In general, where there are two infusion input and two infusion output channels, they will be located with one infusion input channel and one infusion output channel on one side of the tumour microenvironment medium input channel and a tumour microenvironment medium output channel. The other infusion input channel and infusion output channel will be located on the other side of the tumour microenvironment medium input channel and a tumour microenvironment medium output channel.

[0110] In some examples, the three sets of channels (considering an input channel and an output channel to be a "set") run alongside each other.

[0111] In some embodiments the sets of channels are substantially parallel.

[0112] Middle layer as employed herein refers to the layer that sits above the lower layer, in use.

[0113] Tumour compartment as employed herein refers to the region of the middle layer in which a tumour could be placed. The tumour compartment is open to, or in communication with, the stroma in use. Therefore, the tumour compartment is positioned in, at least, an overlapping location with the stroma. Since the stroma is positioned in the central zone of the lower layer, the tumour compartment is positioned to at least overlap with the central zone. Thus, the tumour compartment is collocated with the central zone of the stroma compartment.

[0114] Collocated as employed herein means that the tumour compartment and the central zone are placed together. In this case, the tumour compartment is positioned, at least partially, on top of, or above, the central zone.

[0115] It will be appreciated that the tumour compartment may be any suitable size and shape. In its simplest form, the tumour compartment is a hole through the middle layer.

[0116] In some embodiments the tumour compartment is a circle.

[0117] In some embodiments the tumour compartment has a width or diameter of approximately 5mm. For example, in the range approximately 3 to 10 mm. Conceptually, the tumour compartment is required to be smaller than the central zone to ensure any flow of liquids (stroma or infusion) into tumour retaining portion must necessarily have travelled through the stroma.

[0118] Stroma medium input channel as employed herein refers to the channel through which the medium enters the tumour compartment.

[0119] In some embodiments the stroma medium input channel is approximately 200 pm wide. For example, in the range approximately 150 to 250 pm.

[0120] In some embodiments the stroma medium input channel is approximately 200 pm high. For example, in the range approximately 150 to 250 pm.

[0121] In some embodiments the stroma medium input channel is approximately 39 mm long. For example, in the range approximately 30 to 50mm. Stroma medium output channel as employed herein refers to the channel through which the medium exits the tumour compartment.

[0122] In some embodiments the stroma medium output channel is approximately 200 pm wide. For example, in the range approximately 150 to 250 pm.

[0123] In some embodiments the stroma medium output channel is approximately 200 pm high. For example, in the range approximately 150 to 250 pm.

[0124] In some embodiments the stroma medium output channel is approximately 39 mm long. For example, in the range approximately 30 to 50mm.

[0125] In some embodiments the stroma medium input and output channels are connected to each other with a channel, conceptually the tumour compartment, that is smaller than the central zone of the lower layer but larger than the tumour retaining portion.

[0126] Stroma medium input and output channels together make up a stroma medium channel. They are each in fluid communication with the central zone and stroma medium can flow from the stroma medium input channel, through the tumour compartment (over the surface of the stroma), to the output channel unhindered. Stroma medium may also enter the central zone where it contacts the TME medium and may penetrate into the stroma, in use. In Figure IB medium 6 can be seen entering the central zone 4.

[0127] In some embodiments the stroma medium input channel and stroma medium output channel are each independently serpentine.

[0128] In some embodiments the stroma medium input channel is serpentine.

[0129] In some embodiments the stroma medium output channel is serpentine.

[0130] Serpentine as employed herein means curved, or winding. That is, not in a straight line. Serpentine may include acute curves or switchbacks, such as 180° curves that create loops, hairpins or switchbacks.

[0131] In some embodiments the serpentine channel has at least two switchbacks. Such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 switchbacks.

[0132] In some embodiments the serpentine channel has 5 or more switchbacks.

[0133] Switchback as employed herein means a path (channel) that forms very sharp bends from one direction to (almost) the opposite.

[0134] Tumour retaining portion as employed herein refers to a device that physically constrains movement of a tumour positioned above or on top of the stroma. Many configurations to tumour retaining portion could be conceived however, one of the simpler options would be a simple ring or hoop that is fixed in position and provides a small aperture in which to place the tumour. In addition to retaining the tumour in position, the tumour retaining portion also prevents the flow of stroma medium and / or infusion fluid into the tumour directly. In order to contact the tumour, such fluids must travel through the stroma. The tumour retaining portion may have its own medium that is flowed in separately through a microfluidic fitting.

[0135] In some embodiments the tumour retaining portion is a hoop.

[0136] Hoop as employed herein refers can be any shape, including round. The hoop may be substantially continuous and unbroken. The hoop forms a barrier to protect the tumour from the flow of fluids that may cause the tumour to migrate from the desired location. The barrier formed may be a thin barrier or a thick one. The aperture in the hoop may be smaller in diameter than the diameter of the inner surface of the barrier such that the hoop has a "floor" with an aperture formed therein.

[0137] In one embodiment the tumour retaining portion is approximately 1.5 mm wide or in diameter. For example, in the range approximately 1 to 2 mm.

[0138] In one embodiment the tumour aperture is approximately 1000 pm wide or in diameter. For example, in the range approximately 500 to 1500 pm.

[0139] In some embodiments the tumour retaining portion has a lower surface which, in use, is in contact with the tumour microenvironment medium in the central zone of the lower layer stroma compartment.

[0140] In some embodiments the tumour retaining portion is a cylinder with solid walls having a cylinder diameter and a bottom with an aperture of smaller diameter than the cylinder diameter.

[0141] It will be appreciated that the shape of the cylinder is exemplary and not limited to a circular cross section. For example, the cross section could be substituted for a "tube" with any suitable cross section, such as oval, square, triangular etc.

[0142] In some embodiments the tumour retaining portion is integral in the upper layer.

[0143] Upper layer as employed herein refers to a layer that, in use, is positioned above the middle layer.

[0144] In one embodiment the microfluidics device further comprises an upper layer comprising one or more ports providing access to one or more channel selected from the group consisting of tumour microenvironment medium input channel, tumour microenvironment medium output channel, infusion input channel, infusion output channel, medium input channel and medium output channel.

[0145] Port as employed herein refers to access points that are formed in the top layer and permit direct access to the channels of the microfluidics device. Where the channel is not in the neighbouring layer, it will be appreciated that the port will necessarily require access through any intermediate layers. Ports may access input or output channels.

[0146] In some embodiments the port comprises attachment means.

[0147] Attachment means as employed herein refers to any suitable means of attaching tubing, ducting, piping, sensors or other attachment to the port. For example, push fit, screw fit, friction fit, luers, mini-luers etc.

[0148] In one embodiment the attachment means is a luer or mini-luer.

[0149] In some embodiments the microfluidics device comprises one or more sensors. Suitable sensors include electrical impedance sensors, O2 sensors, CO2 sensors, pH sensors, absorptionbased sensors, and other suitable sensors for a parameter to be measured.

[0150] Tumour Microenvironment (TME) Medium

[0151] A key aspect contributing to the immune escape of tumours is the ECM

[0020] , Particularly, a dense ECM creating a fibrotic stroma is a distinctive feature observed in various types of immune-excluded tumours, which creates a physical barrier that impedes the infiltration of immune cells such as cytotoxic T cells [21,22],

[0152] This dense network of ECM components, including collagen and fibronectin, not only physically obstructs the movement of immune cells but hinders effective immune surveillance and response within the TME [12,23], The fibrotic stroma is often associated with an altered biochemical environment that contributes to immunosuppression. The presence of specific cells (CAFs, TRegs, and TAMs), signalling molecules, and cytokines within the fibrotic matrix can modulate immune cell function, promoting an environment that is conducive to immune evasion by the tumour [24-26], This phenomenon not only limits the efficacy of immunotherapies, that rely on robust immune infiltration, but poses a considerable challenge for the development of therapeutic interventions aimed at overcoming immune escape. Modelling this environment in vitro is key for the development of an efficient screening platform.

[0153] Creating a fibrotic stroma involves replicating the ECM composition and architecture, and incorporating key components that contribute to fibrosis. The presently disclosed ECM medium, and the stromal milieu created by addition of cells etc to the ECM medium, mimics the 3D architecture and viscoelastic properties of a fibrotic stroma using components present in the physiological TME. Advantageously, the composition can be fine-tuned to control porosity and biophysical characteristics, to tailor them to the unique features of the TME across various types of cancers.

[0154] The present disclosure provides a stromal milieu comprising a hydrogel, one or more extracellular matrix proteins, transglutaminase, immune cells, and ECM modifying cells.

[0155] The present disclosure provides a TME medium comprising a hydrogel, one or more extracellular matrix proteins and transglutaminase.

[0156] The complex stromal milieu includes biochemical and biophysical gradients that lead to different phenotypes among different cell types. This includes fibroblasts that can be classified as myofibroblasts (myCAF), inflammatory fibroblasts (iCAFs), or other phenotypes

[0019] , To recreate this gradient, the presently disclosed microfluidic device allows for the interaction of the tumour stroma with the tumour. Potentially, endothelial cells can be seeded on the infusion channels to act as miniaturised blood vessels. For this purpose, infusion channels could be coated with relevant ECM proteins. ECM coating allows for the correct polarisation of endothelial cells in the apicobasal axis to generate functional endothelial tubular structures.

[0157] TME medium as employed herein refers to a medium suitable for growing or creating a tumour microenvironment, such as fibrotic stroma.

[0158] Hydrogel as employed herein is a biphasic material, a mixture of porous, permeable solids and at least 10% by weight or volume of interstitial fluid composed completely, or mainly, of water. In hydrogels the porous permeable solid is a water insoluble three-dimensional network of natural or synthetic polymers and a fluid, having absorbed a large amount of water or biological fluids. Extracellular matrix (ECM) protein as employed herein refers to proteins typically found in the extracellular matrix. The ECM is a network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide structural and biochemical support to surrounding cells.

[0159] In some embodiments the ECM protein is selected from collagen, fibronectin, laminin, and elastin. More than one ECM protein may be included in the TME medium to create the desired TME.

[0160] In one embodiment the ECM medium comprises collagen-l at a concentration in the range 0.5mg / ml to 6.0 mg / ml. For example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0 mg / ml.

[0161] In one embodiment the ECM medium comprises collagen-l at a concentration in the range 2mg / ml to 5mg / ml.

[0162] Given that the main ECM component of the TME is collagen I [27-29], collagen l-based hydrogels can be used to accurately mimic the tumour matrix. Second Harmonic Generation (SHG) microscopy has been used to image collagen fibres (Figure 3A), showing architectural differences among different ECM concentrations. Higher collagen concentrations (such as those in the range 3.5 to 5mg / ml) reflect a more physiological tumour tissue with longer fibres, while lower concentrations show a more compact architecture but with thinner fibres (Figure 3A). Gels have been assessed using the presently disclosed microfluidic device to assess polymerisation within the platform.

[0163] Cell migration through the ECM is influenced by the alignment, density, and porosity of collagen fibres [30,31], The width of collagen fibres particularly affects spacing and porosity within the gel, creating paths of greater and lesser resistance [32,33], To further tune the collagen gel microarchitecture within the TME medium, a crosslinking agent (such as transglutaminase) can be used with different collagen concentrations (Figure 4)

[0034] ,

[0164] Transglutaminase catalyses the formation of isopeptide bonds between y-carboxamide groups of glutamine residue side chains and the e-amino groups of lysine residue side chains between collagen fibres. Importantly, bonds formed by transglutaminase exhibit high resistance to proteolytic degradation. Thus, advantageously, use of transglutaminase provides a number of well-controlled gel morphologies with different mechanical properties and local topography (Figure 4 and 5A).

[0165] Crosslinking significantly increases fibre width and influences local stiffness (Figure 5B). Since collagen fibres serve as anchoring points for cell adhesion receptors such as integrins, the width of collagen fibres determines the available surface area for cell adhesion. Consequently, cell spreading and migration will be different on fibres of varying widths, leading to variations in cell morphology, cytoskeletal organisation, and signalling pathway activation [35,36], As mentioned above, an increased fibre width also results in a reduced pore size of the hydrogel network. Fibre length change was not statistically significant at lower Collagen I concentrations when using crosslinking (Figure 5C).

[0166] In some embodiments the transglutaminase has a concentration in the range O.lmg / ml to lOmg / ml. For example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0 mg / ml.

[0167] A change in the bulk mechanical properties after crosslinking is also evident from the rheological properties of the hydrogels (Figure 6). Bulk stiffness 2 mg / ml collagen gels were significantly increased from 246.3 ± 67.4 Pa to 469.7 ± 84.2 Pa after crosslinking. This is particularly important since being able to modulate stiffness and tailor it to the specific tissue being tested is key to accurately mirror the response to therapeutic candidates.

[0168] Among other cells in the TME, CAFs contribute significantly to ECM deposition and remodelling within the TME, to create a dense and fibrotic stroma that supports tumour progression and therapeutic resistance. To recreate a desmoplastic TME in our system, we cultured MRC5-SV2 lung-derived fibroblast cells on our proprietary TME medium and analysed remodelling of the Collagen I network via SHG (Figure 7).

[0169] It is known that fibroblasts growing on collagen hydrogels induce contraction of the gel, and that such contraction is regulated by a balance between contractile forces generated by the cells and the resistance provided by the collagen matrix

[0038] , We observed that MRC5 cells induced hydrogel contraction differently depending on the hydrogel formulation. A higher contraction was observed in 2 mg / ml Collagen hydrogels compared to 5 mg / ml collagen hydrogels, while the addition of cross-linkers reduced hydrogel contraction (Figure 7B). This further confirms that a higher concentration of collagen and cross-linkers results in a stiffer matrix that is more challenging for fibroblasts to deform. Higher contraction was also observed at 14 days compared to 7 days of growth in 2 mg / ml collagen matrices without transglutaminase and with 0.1 and 1 mg / ml transglutaminase (Figure 7B).

[0170] Since fibrosis induced by CAFs presents a significant challenge in the context of immunotherapies, matrix remodelling and deposition by fibroblasts in the presently disclosed system was tested. In contrast to the initial collagen hydrogels, the collagen matrix observed after culturing fibroblasts exhibits a distinct fibrous structure, reminiscent of physiological collagen matrices. These remodelled structures showed denser collagen fibres, which can be translated into stiffer substrates and smaller pores. Consistently, the addition of fibroblasts induces the aggregation and stiffening of the collagen networks in all conditions (Figure 7A).

[0171] Fibroblasts can be established fibroblast cell lines from different organ origins, modified to become immortalised, or primary fibroblast lines derived from biopsies. These fibroblasts are detached from their culture vessel and mixed with the TME medium. Other stromal cells may be added to the TME medium as well at this stage.

[0172] The inlets and outlets of the device are plugged except for the TME medium inlet and outlet. The TME medium (comprising cells, referred to as pre-stromal milieu herein) is then introduced into the inlet and allowed to grow, for example, for approximately 4 to 14 days thereby forming the stromal milieu, or stroma.

[0173] Tumour cells can be cell lines or primary cells derived from tumour biopsies. If from primary cells, they may contain tissue resident immune cells as well. The tumour cells are counted and seeded onto ultralow attachment U-bottom plates and centrifuged at 100-300 RCF and can be grown for up to 14 days.

[0174] The tumour spheroids are then added onto the tumour compartment after 24 hours or up to 7 days.

[0175] Fibronectin is a further ECM protein (glycoprotein) which is relevant to the formation of the TME. The upregulation of fibronectin has been associated with a wide range of human cancers

[0040] , CAFs play a crucial role in the deposition of fibronectin, promoting tumour progression through mechanisms such as invasion, angiogenesis, and therapy resistance [41,42], The deposition of fibronectin in the TME is particularly relevant in the shaping of the stromal ECM network since it acts as both a scaffold for the deposition of other matrix proteins such as collagen, and a binding site for functionalisation by soluble factors

[0043] , The secretion and assembly of fibronectin by fibroblasts in the present TME medium was considered. Immunostaining showed the assembly of networks of fibronectin within the collagen matrix (Figure 8). More consistent bundles of nanofibers, with a clearer meshwork structure, were observed in cross-linked hydrogel relative to non-crosslinked. Fibronectin staining was also evident in regions devoid of cells, suggesting its involvement, not only in pericellular matrix formation, but also in shaping an interconnected microenvironment at the micro-to-mesoscale level.

[0176] Stromal cells as employed herein refers to cells such as carcinoma-associated fibroblasts (CAFs), including myofibroblasts (myCAF) and inflammatory fibroblasts (iCAF), tumour-associated macrophages (TAMs), and other cells present in the cellular microenvironment.

[0177] In some embodiments the fibroblasts have a concentration in the range of approximately 20,000 cells / mL to 500,000 cells / mL

[0178] In some embodiments the TAM concentration is in the range of approximately 20,000 cells / mL to 500,00 cells / mL.

[0179] In some embodiments the ECM proteins comprise collagen, fibronectin, laminin, hyaluronic acid, and elastin.

[0180] In some embodiment transglutaminase has a concentration in the range of approximately 0.01 mg / mL to 10 mg / mL.

[0181] In some embodiments the TME medium or stromal milieu comprises biochemical factors in the range of approximately 1 ng / mL to 10,000 ng / mL.

[0182] In some embodiments there are 2 or more biochemical factors, each with individual concentrations in the range of approximately 1 ng / mL to 10,000 ng / mL.

[0183] In some embodiments the TME medium comprises transglutaminase at a concentration in the range of approximately 0.01 mg / mL to 10 mg / mL.

[0184] CAFs are also known to secrete and respond to various cytokines that play crucial roles in modulating the TME and influencing the efficacy of immunotherapies. TGF-P is relevant in the TME is, exerting a wide range of effects [44-46], among which the generation of a pro-fibrotic phenotype is particularly relevant to the presently disclosed system [47-49], TGF-P is stored in the matrix in a latent form and, once activated, regulates ECM formation and remodelling in fibrosis. In cancer, TGF-P contributes to the generation of CAFs

[0050] , which promote tumour progression and chronic tumour fibrosis via TGF-P signalling

[0051] , The production of TGF-P by the fibroblasts present in our system was explored and expression of this cytokine intracellularly was observed via immunostaining (Figure 9).

[0185] In some embodiments the TME medium or stromal milieu further comprises additional biochemical factors.

[0186] Biochemical factors as employed herein refers to any protein-based molecule that can alter cell states, including but not limited to growth factors and cytokines.

[0187] Growth factors may include TGF-P, FGF, PDGF, VEGF, and other suitable growth factors.

[0188] In one embodiment the growth factors are selected from the group consisting of TGF-P, FGF, PDGF, and VEGF.

[0189] Cytokines may include IL-2, IL-6, IL-8, IL-10, IL-12, IL-13, 1 Fy, TNFa, CXCL12 and other suitable cytokines.

[0190] In one embodiment the cytokines are selected from the group consisting of IL-2, IL-6, and I Fy.

[0191] Where employed, the biochemical factors may be embedded into the TME medium, for example, with transglutaminase. The addition of transglutaminase modifies the time-dependent presentation and degradation of biochemical factors which influences the reaction of stromal cells, infiltrating drugs, and the tumour.

[0192] System, Method and Use

[0193] The present disclosure also provides a method of creating a tumour microenvironment within the microfluidics device according to the disclosure, comprising the steps: a. introducing a suitable tumour microenvironment medium and cells to the central zone to create a stroma, b. flowing stroma medium over the surface of the stroma by adding stroma medium via the stroma medium input channel and removing stroma medium via the stroma medium output channel, c. placing a tumour in the tumour retaining device, and d. allowing a period of time to elapse.

[0194] Suitable TME media are disclosed herein. It will be appreciated that the device and the TME medium disclosed herein function in synergy as a system and are particularly beneficial when used together.

[0195] As such, there is also provided a kit of parts comprising the microfluidics device of the disclosure and a TME medium, or ingredients to make a TME medium, according to the disclosure.

[0196] The disclosure also provides a method of screening an oncology therapeutic candidate comprising setting up a tumour microenvironment according to the disclosure and introducing an infusion comprising the oncology therapeutic candidate via the infusion input channel. Oncology therapeutic candidate as employed herein refers to a pre-clinical or clinical therapeutic, which may be a biological or chemical entity (small or large molecule) or combinations or hybrids thereof, with potential activity against a cancer or its surrounding tissue.

[0197] Working as a system, the present disclosure functions as follows, in use:

[0198] The device is assembled with a lower layer and middle layer, and optionally an upper layer.

[0199] TME medium is added to the lower layer via the TME medium input channel, such that it enters the central zone and resides there, without entering the periphery (because it is restricted by the pillars). The TME medium may be filled such that it touches the lower side of the tumour retaining portion. The TME medium input and output channels (collectively, the TME medium channel) are then closed or sealed off. Cells and ECM develop in the central zone to produce a fibrotic stroma which mimics the TME of a physiological tumour after a period of time. Exemplary periods include approximately 4 to 14 days of culture.

[0200] For example, the fibrotic stroma, or stromal milieu is allowed to form for a minimum of 4 days and up to 14 days in the central zone of the lower layer. The tumour is grown separately for a minimum of 3 days then incorporated into the device (in the tumour retaining portion) and allowed to interact with the fibrotic stroma for a minimum of 1 day typically up to 7 days, before drug infusion is initiated via the infusion input channel.

[0201] In the present example, there are two infusion input channels and two infusion output channels (collectively, two infusion channels), as shown in Figure 1. The infusion channels run either side of the TME medium channel.

[0202] In the example shown in Figure 1C the pillars are not present where the TME medium channel meets the central zone, so that flow of the TME medium into the central zone is not hindered. However, the pillars (shown as cylindrical in Figure 1C) are positioned around the edge of the central zone and demark the central zone and the periphery. In Figure 1C the pillars are partially connected by a series of walls of lesser height than the pillars. This configuration works in combination with serpentine channels in the middle layer to retain the TME medium in the central zone.

[0203] The infusion medium, which may comprise an oncology therapeutic candidate, is added to the device via the infusion input channel(s) and flows around the periphery, exiting via the infusion output channel. The infusion medium is able to flow between the pillars and infuse the TME medium or stromal milieu in the central zone. Oncology therapeutic candidates that can penetrate the TME have a greater likelihood of reaching a tumour and working against the tumour.

[0204] The TME medium and cells (stroma or stromal milieu) in the central zone are fed by stroma medium provided via the stroma medium input channel of the middle layer. Stroma medium flows over the top of the TME medium / stromal milieu, partially diffusing into it and creating a gradient within the stroma. Some of the medium flows away via the medium output channel.

[0205] The middle layer of Figure 1C has serpentine channels and a tumour compartment, which in the example is a hole in the centre of the middle layer. The tumour compartment sits directly above the stroma in the central zone. Medium is added to the device via the medium input channel and flows directly out of the input channel onto the top surface of the stroma so that medium can be fed to the stroma. Excess medium flows out of the other side of the tumour compartment and along the medium output channel. Separately, medium feeding the tumour in the tumour retaining portion can flow into the tumour compartment. Some tumour medium and / or stroma medium may flow out via the infusion output channel(s) having passed through the TME medium. Some mixing with the drug infusion is possible.

[0206] The tumour is positioned in the centre of the tumour compartment (hole), in the centre of, and on top of, the stroma. The tumour is retained in position by the tumour retaining portion, which allows control of the gradient within the stroma that the tumour is in contact with. Tumour may directly sit on stroma or may be in ECM or different medium, for example. The tumour retaining portion creates a wall around the tumour which shelters it from the flow of medium and prevents the tumour migrating across the top of the stroma.

[0207] In the example of Figures 1 and 2, the top layer provides the tumour retaining device in the form of a cylinder with solid walls and an aperture in the bottom which has a smaller diameter than the cylinder. The tumour is placed in this smaller aperture. The shape and dimensions of the aperture may vary. Some flow of stroma medium from the stroma medium input channel will flow through the TME medium / stromal milieu and into the tumour via the aperture.

[0208] The top layer also has a series of ports for accessing the channels of the various layers. The ports are shown with a luer or mini-luer fixing but any suitable attachment means could be used.

[0209] The device may also comprise various sensors, such as electrical impedance sensors.

[0210] Functional Example of the System

[0211] There are currently no immunotherapies approved for the treatment of pancreatic cancer, the primary reasons for which are fibrosis and the effect of the immunosuppressive TME in pancreatic tumours [12,22,52], Consequently, understanding the complex interactions within the TME, particularly the role of CAFs and ECM components, is essential for improving outcomes for patients with this disease.

[0212] With this in mind, pancreatic cancer and its TME was modelled within our system using PANC-1 cells to generate a simplified pancreatic tumour model that could be monitored. PANC-1 spheroids have been widely used as a relevant model for the study of various aspects of pancreatic cancer biology and to evaluate potential therapeutic interventions [55,56], PANC-1 spheroids, like many other 3D culture models, reproduce tumour heterogeneity more closely than traditional monolayer cultures. This heterogeneity includes differences in cell morphology, gene expression, and response to therapy, mirroring the complexity observed in vivo tumours. PANCI- spheroids were generated by culturing them on ultra-low attachment plates. Since a fibrotic stroma is a hallmark feature of pancreatic cancer that significantly influences disease progression and therapeutic response, culturing pancreatic cancer cells within suitable matrices is key to generate a physiologically relevant model. After 5 days in culture, cells were either kept on the plates for an additional 7 days or transferred to collagen matrices and grown for 7 days. The inclusion of PANC-1 spheroids on collagen matrices resulted in a less spherical shape, inducing migration and spreading of the outer cell layers of the spheroid in the collagen matrix (Figure 10). To model crosstalk between pancreatic cancer cells and relevant stromal components such as CAFs, PANC-1 cells were co-cultured with MRC5 fibroblast. The secretion of fibronectin by PANC-1 spheroids and co-culture spheroids were analysed and an increased amount of fibronectin deposition on coculture spheroids, compared to PANCI- spheroids was observed (Figure 10). This difference is more pronounced in spheroids grown on collagen matrices, where fibronectin deposition and assembly on coculture spheroids create a dense network surrounding the structure (Figure 10). Fibronectin, which is abundant in the pancreatic cancer stroma but not in normal tissues, supports tumour progression by promoting several enabling hallmarks of cancer, such as cell invasion

[0042] .

[0213] An increased migration of cells outside the borders of the co-culture spheroids compared to PANC-1 spheroids was also observed (Figure 10). This migratory behaviour is particularly enhanced in spheroids included on collagen TME medium, where there is increased migration from the spheroid towards the hydrogels (Figure 10). Since both a fibrotic stroma and invasive behaviour are hallmarks of pancreatic cancer, a system like the one developed here, is relevant for creating a more advanced in vitro TME model.

[0214] In the context of this specification "comprising" is to be interpreted as "including".

[0215] Aspects of the invention comprising certain elements are also intended to extend to alternative embodiments "consisting" or "consisting essentially" of the relevant elements.

[0216] Where technically appropriate, embodiments of the invention may be combined.

[0217] Embodiments are described herein as comprising certain features / elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features / elements.

[0218] Approximately as employed herein is intended to mean ±10%.

[0219] Technical references such as patents and applications are incorporated herein by reference.

[0220] Any embodiments specifically and explicitly recited herein may form the basis of a disclaimer either alone or in combination with one or more further embodiments.

[0221] Examples

[0222] Design and fabrication of microwells and microfluidic devices.

[0223] Computer Assisted Design (CAD) software such as Fusion360 and OnShape were used to design the microfluidic devices and microwells. Microwells were printed using Anycubic Photon Mono 2 printer with Liqcreate Biomed Clear resin. The print was then washed and cured further based on manufacturer's recommendations. Copper tape was then attached on the sides of the microwells. The microfluidic devices were also printed with Liqcreate Biomed Clear resin by a supplier.

[0224] Hydrogel formation

[0225] Collagen hydrogel solutions were prepared using acid-soluble Collagen I from rat tail tendon (Corning) at variable concentrations following manufacturer's instructions. Briefly, collagen I stock solution was combined with concentrated 10X PBS, water, and neutralisation agent (IN sodium hydroxide (NaOH)) at pH 7.4 Collagen solutions were mixed and pipetted on ice and subsequently polymerized at 37°C for 1 hour. When transglutaminase cross-linked gels were formed, fresh transglutaminase solutions were prepared in 10X PBS.

[0226] Cell Culture

[0227] MRC5-SV2 cells were purchased from ECACC and cultured in MEM (Sigma-Aldrich) medium supplemented with with 10% foetal bovine serum (Biowest), 2mM L-glutamine (Gibco), and 1% Non- Essential Amino Acids (Gibco) following manufacturer's instructions. PANC-1 cells were purchased from ECACC and cultured in Dulbecco's modified Eagle medium (Sigma-Aldrich) supplemented with with 10% foetal bovine serum (Biowest), 2mM L-glutamine (Gibco), and 1% Sodium pyruvate (Gibco) following manufacturer's instructions. Cells between passages 3 and 10 were used for the experiments. Cells were maintained at 379C in a humidified atmosphere with 5%CO2. Medium was replaced with fresh medium every two-three days. For cell passaging and seeding, cells were harvested using TrypLE (Gibco) and centrifuged at (200xg) for 5 min.

[0228] Spheroid formation

[0229] Homogenous spheroids were formed with PANC-1 cells. Heterogeneous cell suspensions for the formation of co-culture spheroid were obtained by mixing PANC-1 with MRC5 at a 9:1 ratio. The seeding number of PANC-1 cells was between 612 and 10,000 cells for the impedance measurement experiment. For immunostaining experiments, cell numbers were optimised to achieve a radius of around 250 pm and a viability above 90% at day 7. Complete DM EM medium was used for both homogeneous and co-culture spheroids. Spheroids were formed using ultra-low attachment plates, which were centrifuged to facilitate cell aggregation and spheroid formation. Spheroids were grown at 379C in a humidified atmosphere with 5%CO2 for several days depending on the experimental conditions. For the analysis of invasion and fibronectin deposition, half of the spheroids grown for 5 days in ultra-low attachment plates were included in 2mg / ml Collagen I hydrogels, while the rest were kept in the plate, and cultured for an additional 7 days.

[0230] Fluorescence microscopy

[0231] Leica SP8 DIVE multiphoton microscope and Nikon CSU-W1 Spinning disk microscope with Two Photometries Prime BSI sCMOS were used for multiphoton and confocal imaging of immunostained samples. Objectives used were u25x / 1.0 W IR Apo objective on the Leica SP8 Dive and CFI Plan Apochromat VC 20X Air in the Nikon CSU-W1 were CFI Plan Apochromat VC 20X Air and CFI Apochromat TIRF 60XC Oil. Image and video analyses, z-stack projections and 3D deconvolution were done with ImageJ software.

[0232] SHG microscopy

[0233] The SHG microscopy for detecting a signal generated by the material itself, such as collagen structures, was performed on a Leica SP8 DIVE multiphoton microscope equipped with a tunable 680-1300 nm IR Laser Spectra Physics InSight DS+ DUAL. Images were taken using a 25x / 1.0 W IR Apo objective motCORR™. IR laser was used at 950 nm to excite the sample, generating a signal at half the wavelength (475 nm) detectable by a high-sensitivity detector. Impedance measurements

[0234] Impedance measurements were performed using Digilent Analog Discovery 2. The software Waveform was used to control the compensation, and amplitude / frequency of the alternating current. Briefly, copper wires are connected to the positive and negative terminals of the Digilent Analog. The wires were then connected to each other to short the circuit and measure the compensation values. The wires were directly interfaced with the outlets of the microfluidic device where the gel or cells could be measured.

[0235] Quality assurance and monitoring of fibrotic tissue with electrodes

[0236] Manufacturing a fibrotic tissue consistently for drug testing requires quality control and quality assurance measures. Transepithelial electrical resistance has been measured as a method of assessing barrier integrity in transwells and recently on microfluidic devices. The amplitude and frequency of the alternating current was controlled with the Digilent Analog Discovery 2 multifunctional with integrated impedance analyzer. A 3D-printed microwell was fabricated with copper tape attached to its sides. The copper tape is then connected to the Digilent Analog Discovery 2. A collagen gel (TME medium) was then formed on the microwell and its impedance profile was measured pre-polymerisation and postpolymerisation (Figure 11). Varying frequencies of the alternating current provided a distinct impedance spectroscopy bode plot profile between pre-polymerisation and post-polymerisation, indicating that impedance measurements could be used to monitor the fabrication of the hydrogels.

[0237] Impedance sensing is a powerful tool to monitor the changes within hydrogels. By monitoring the hydrogels, we can assure consistency in the fabricated hydrogels and minimise data noise when performing any assays with them. In a microfluidic device, hydrogels with different concentrations of collagen with and without cross-linker were formed. After 1 hour of incubation at 37°C, the impedance was measured with copper wires attached to the inlets of the microfluidic device. The Nyquist plots (reactance vs resistance) of the different setups had different profiles (Figure 12). Consistently, when a crosslinker is added, the resistance increases significantly (Figure 12B). As previously shown, fibroblasts start modifying the ECM within the collagen gels.

[0238] Furthermore, it was observed that when there is a bubble within the device, this could also be detected with the impedance analyser as the profile changes significantly - further highlighting the utility of the impedance analyser as a monitoring tool for the fabrication of the hydrogels.

[0239] Impedance sensing could also be used to detect spheroids. Different sizes of spheroids (Figure 13A) were flowed into a microfluidic chip. Copper wires attached to the Digilent Analog Discovery 2 were then placed onto the outlets to detect the spheroids. While the difference between just media and the spheroid (Figure 13B) is easily distinguishable, between the differently sized spheroids cannot be detected.

[0240] Finally, the microfluidic chips have been redesigned (Figure 2A) to fit a 384 well plate cartridge (Figure 2B). This ensures compatibility with automated liquid handling and increases the throughput of the assays.

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Claims

Claims1. A microfluidics device comprising: a lower layer comprising a stroma compartment having: o a central zone having a tumour microenvironment medium input channel and a tumour microenvironment medium output channel, and o a periphery having at least one infusion input channel and at least one infusion output channel, the central zone and the periphery being demarked by a plurality of interspersed pillars which partially separate the central zone from the periphery, wherein the pillars are arranged to retain a tumour microenvironment medium in the central zone and to permit an infusion fluid to flow from the infusion input channel into the central zone; a middle layer comprising a tumour compartment which is collocated with the central zone; the tumour compartment having a stroma medium input channel and a stroma medium output channel; and a tumour retaining portion to position a tumour in a desired location.

2. The device of claim 1 further comprising an upper layer comprising one or more ports providing access to one or more channel selected from the group consisting of tumour microenvironment medium input channel, tumour microenvironment medium output channel, infusion input channel, infusion output channel, stroma medium input channel, stroma medium output channel, tumour medium input channel, and tumour medium output channel.

3. The device of claim 1 or claim 2 wherein the periphery has two infusion input channels and two infusion output channels.

4. The device of any preceding claim wherein the tumour microenvironment medium input channel and the tumour microenvironment medium output channel are located on opposite sides of the central zone.

5. The device of any preceding claims wherein the pillars are cylindrical.

6. The device of any preceding claim wherein at least some of the pillars have a wall between them.

7. The device of claim 6 wherein the wall or walls are shorter than the pillars.

8. The device of any preceding claim wherein the stroma medium input channel is serpentine.

9. The device of any preceding claim wherein the stroma medium output channel is serpentine.

10. The device of claim 8 or 9 wherein the serpentine channel has at least two switchbacks.

11. The device according to any preceding claim wherein the tumour retaining portion is a hoop.

12. The device according any preceding claim wherein the tumour retaining portion has a lower surface which, in use, is in contact with the tumour microenvironment medium in the central zone.

13. The device of any preceding claim wherein the tumour retaining portion is a cylinder with solid walls having a cylinder diameter and a bottom with an aperture of smaller diameter than the cylinder diameter.

14. The device according to any one of claims 2 to 13 wherein the tumour retaining portion is integral in the upper layer.

15. The device of claim 2 wherein the one or more ports comprises attachment means.

16. The device of any preceding claim further comprising one or more sensors.

17. The device of claim 16 wherein there are at least two sensors and wherein at least one sensor is configured to monitor the stroma and at least one sensor is configured to monitor the tumour.

18. A method of creating a tumour microenvironment within a device according to any preceding claim, comprising the steps: a. introducing a suitable tumour microenvironment medium and cells to the central zone to create a stroma, b. flowing stroma medium over the surface of the stroma by adding stroma medium via the stroma medium input channel and removing stroma medium via the stroma medium output channel, c. placing a tumour in the tumour retaining device, and d. allowing a period of time to elapse.

19. A method of screening an oncology therapeutic candidate comprising setting up a tumour microenvironment according to claim 18 and introducing an infusion comprising the oncology therapeutic candidate via the infusion input channel.

20. A tumour microenvironment medium comprising a hydrogel, one or more extracellular matrix proteins, and transglutaminase.

21. A stromal milieu comprising a hydrogel, one or more extracellular matrix proteins, transglutaminase, and stromal cells.

22. The tumour microenvironment medium according to claim 20 or the stromal milieu according to claim 21 wherein the one or more extracellular matrix proteins are selected from collagen, fibronectin and elastin.

23. The tumour microenvironment medium or stromal milieu of claim 22 wherein one or more extracellular matrix proteins comprises collagen-l in a concentration in the range 2mg / ml to 5mg / ml.

24. The tumour microenvironment medium or stromal milieu of any one of claims 20 to 23 wherein the transglutaminase has a concentration in the range O.lmg / ml to lOmg / ml.

25. The tumour microenvironment medium or stromal milieu of any one of claims 20 to 24 further comprising biochemical factors.

26. A kit of parts comprising a microfluidics device according to any one of claims 1 to 17 and a tumour microenvironment medium according to any one of claims 20 to 25.

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