WO2021168511A1

WO2021168511A1 - A microfluidic device for investigating interactions of substances with cells

Info

Publication number: WO2021168511A1
Application number: PCT/AU2021/050161
Authority: WO
Inventors: Nicolas Hans VOELCKER; Ziqiu Tong; Wing Yin TONG; Arianna ODDO; Helmut Thissen; Bo Peng
Original assignee: Commonwealth Scientific And Industrial Research Organisation; Monash University
Priority date: 2020-02-25
Filing date: 2021-02-25
Publication date: 2021-09-02

Abstract

The invention provides a microfluidic device for investigating an interaction of one or more substances with cells cultured therein, the microfluidic device comprising: one or more internal spaces including a vessel configured to contain a first fluid comprising the one or more substances; and a polymeric coating on walls of at least the vessel, wherein the polymeric coating comprises at least one cross-linked polymer chemically bonded to the walls and comprising a plurality of conjugating functional groups available for reaction, wherein polypeptide-containing macromolecules contacted with the polymeric coating in use are immobilised thereon by covalent bond formation with the conjugating functional groups, thereby adapting the walls for culturing cells.

Description

A microfluidic device for investigating interactions of substances with cells Technical Field

[1] The invention relates to a microfluidic device for investigating interactions of substances with cells cultured in the device, for example transport of substances across cellular barriers. The microfluidic device comprises one or more internal spaces including a vessel, typically in the form of a flow channel, and a cross-linked polymeric coating chemically bonded to walls of the vessel. In use, polypeptide-containing macromolecules are immobilised on the polymeric coating by covalent bond formation with conjugating functional groups present on the polymer, thereby adapting the walls for culturing cells. The invention also relates to a method of producing the microfluidic device and a use of the device to investigate an interaction of substances with cells.

Background of Invention

[2] Microfluidic devices are finding increasing application in many areas, one of the most promising being microfluidic cell culturing. To accurately simulate an in vivo biological system, it is important to mimic the biological microenvironment which includes exposure to soluble factors that regulate cell structure, function, behaviour, and growth. The use of microfluidics allows precise control over fluid flow in defined geometries and can facilitate simultaneous manipulation and analysis of substances important for cell culture in a micrometer and nanoliter scale. The use of spatially separate microchannel and microfluidic compartments also facilities high throughput screening of substances and reactions. Parallelization of experimental conditions allows for enhanced cell-based screening assays, such as immunophenotyping assays monitoring single cell cytokine production in response to external stimuli. The adoption of microfluidic devices to model complex diseases, including those which require soluble signal or therapeutic substances to cross tissue barriers, would thus seem promising.

[3] In one exemplary area of interest, effective therapies for neurodegeneration diseases and glioblastoma have yet to be developed. This is partly due to poor understanding of the neuropathology of these diseases, but also due to the inability of many potential therapeutics to reach their targets within the central nervous system (CNS). The blood-brain barrier (BBB) is a highly selective semi-permeable cellular barrier which blocks most therapeutic molecules from entering the brain site. For this reason, many promising therapeutics which demonstrate efficacy in vitro fail in pre- clinical trials.

[4] Animal models provide a relevant physiological representation of the human BBB, but the high cost, ethical concerns, low throughput and cross-species differences of these models hinder their application in drug development. Widely used in vitro models in which cells are cultured under static conditions over-simplify the biological microenvironment and the resultant lack of physiological relevance may cause inaccurate predictions of drug permeability.

[5] Microfluidic “organ-on-chip” systems provide potentially more physiologically relevant models than traditional well plate-based assays and have thus shown promise as drug screening platforms. Microfluidic blood brain barrier on chip devices (pBBBs) are proposed to more accurately model the blood brain barrier by providing control and flexibility with respect to parameters such as fluid shear stimulus, co-culture of multiple cell types, and the use of actual human BBB cells.

[6] Extracellular matrix (ECM) is a 3D network of extracellular macromolecules that structurally and biochemically supports surrounding cells. The ECM of the BBB, which includes the basement membrane, plays an important role in BBB formation and regulation. Providing a stable ECM substrate, under fluid shear stress conditions which mimic the environment of the in vivo human BBB, is essential for building a physiologically relevant in vitro BBB device.

[7] Previous mBBB models have utilized silanization or simply oxygen plasma to immobilize ECM proteins and thus support growth of endothelial cells (ECs). However, producing a protein-retentive surface in microfluidic channels via silanization involves multiple steps, and unwanted polymerization of the silane precursors may form surface layers having structural irregularities which lead to uneven ECM coatings. Physical absorption on plasma-activated surfaces is not suitable for longer-term cell culturing under shear stress. Previous approaches therefore do not immobilise the ECM macromolecules evenly or sufficiently robustly to withstand shear stresses which mimic those experienced in vivo at the human BBB. In other mBBB models, adherent collagen coatings have been used to culture cells representative of the BBB, but collagen alone does not adequately mimic the natural composition of the basement membrane which includes multiple functionally important proteins, including collagen IV, fibronectin and laminin.

[8] Apart from the critical role of the ECM coating itself, native BBB phenotypes depend strongly on the crosstalk between endothelium and several different types of brain cells. Therefore, faithfully replicating the interaction between multiple cell types may be critical to reliably forecast in vivo drug performance. The BBB is a dynamic and complex structure in which various different pathways rapidly regulate the transport of molecules and ions, including efflux, positive and negative transport, and receptor- mediated transcytosis. Many mBBB models have only demonstrated BBB junction tightness using fluorescence labelled macromolecules, and it has not been established that these systems representatively model other important BBB functional pathways including transcellular routes, for example those regulating the transport of small molecules.

[9] While the above discussion specifically relates to challenges with modelling the BBB, it will be appreciated that similar considerations apply for microfluidic models of other important cellular barriers, including but not limited to the blood-lymph barrier, the blood-testis barrier and the placental barrier, as well as blood tumour barriers. In each of these cases, the ability of the device’s in vitro cellular barrier to model a target in vivo cellular barrier, preferably containing multiple cell types and for a range of transport pathways, relies on initially forming a well-controlled and robust immobilised coating of polypeptide-containing macromolecules on which the cells can be cultured.

[10] Furthermore, a wide range of other biological systems involving interactions between cells and various substances remain challenging to model and improved microfluidic systems are needed. For example, the growth of CNS neurons (e.g. cortical, hippocampal and spinal cord neurons) involved in the pathology of most neurodegenerative diseases and injuries can also be modelled in microfluidic devices. In one study, microfluidic devices with discrete chambers for primary rat cortical and hippocampal neurons were investigated as an in vitro model of axonal injury, and the results following introduction of growth factors showed the ability to selectively lesion axons and biochemically analyze their somata for immediate early gene expression. [11 ] Other models have been explored looking at the interactions between tumor cells and epithelial cells, interactions that are important to study since most endocrine glands consist of clusters or cords of secretory cells surrounded by capillary networks permeating the tissues. So a relevant study looked at the growth of new microvessels from preexisting blood vessels on a microfluidic device. Endothelial cells were cultured in one microchannel in direct contact with hydrogel scaffolds. Angiogenesis was subsequently induced by applying a chemical or physical angiogenic stimulus [e.g., fluid shear stress, interstitial flow] and interaction with the macromolecule, the growth factor, VEGF, the latter being applied from the channel opposite to the cell monolayer to induce invasion into the scaffolds and thus promote further cell growth.

[12] For microfluidic cell culture models generally, the ability to form a well- controlled and robust immobilised coating of polypeptide-containing macromolecules on the internal surfaces of the microfluidic device, with precise attachment of soluble factors that regulate cell structure, function, behaviour, and growth, is critical to its relevance. This is particularly challenging in view of the microscale of the microfluidic device channels, the propensity for channel blockages and the requirement for accuracy and reproducibility.

[13] There is therefore an ongoing need for microfluidic devices for investigating interactions of substances with cells cultured in the device, e.g. the transport of substances across a cellular barrier, for methods of producing such devices, and for methods of using such devices to investigate interactions of a range of substances with cultured cells, which at least partially address one or more of the above-mentioned short-comings or provides a useful alternative.

[14] A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Summary of Invention

[15] In accordance with a first aspect the invention provides a microfluidic device for investigating an interaction of one or more substances with cells cultured therein, the microfluidic device comprising: one or more internal spaces including a vessel configured to contain a first fluid comprising the one or more substances; and a polymeric coating on walls of at least the vessel, wherein the polymeric coating comprises at least one cross-linked polymer chemically bonded to the walls and comprising a plurality of conjugating functional groups available for reaction, wherein polypeptide-containing macromolecules contacted with the polymeric coating in use are immobilised thereon by covalent bond formation with the conjugating functional groups, thereby adapting the walls for culturing cells.

[16] The microfluidic devices of the invention provide for facile immobilisation of a wide variety of polypeptide-containing macromolecules, including natural, synthetic and modified proteins, on the polymeric coating. The polymeric coating itself is robust, easy to produce as a thin yet continuous layer on the internal walls of the device in a single process step and with low risk of blockages, and immobilises proteins more effectively than prior approaches due to the conjugating functional groups being present on a cross-linked polymer. The conjugating functional groups may be configured to conjugate native proteins, so that a protein or a mixture of proteins can be immobilised without the need to synthetically functionalise the proteins for conjugation. Advantageously, a complex protein matrix which accurately models the ECM in biological tissue can thus be immobilised on the internal walls of the device.

[17] As a result of these advantages, the walls of the vessel can be conditioned for culturing cells in a manner which accurately models an in vivo biological system of interest. The relevance of the cellular model can be enhanced by co-culturing multiple different cell types present in the biological system and/or by culturing the cells under conditions of hydrodynamic shear flow. In an exemplary embodiment, the inventors have demonstrated that transport of a range of substances across an in vitro cellular barrier produced in microfluidic devices according to the invention is regulated in a manner which accurately models the complexity of the in vivo blood brain barrier.

[18] In some embodiments of the first aspect, the internal spaces of the microfluidic device further include: a chamber adjacent to the vessel; and at least one microchannel providing fluid communication between the vessel and the chamber, wherein the chamber is configured to contain a second fluid for receiving the one or more substances if transportable from the vessel through the at least one microchannel. In such embodiments, the microfluidic device may be a microfluidic device for investigating the transport of one or more substances across a cellular barrier cultured therein. The walls of the vessel can be conditioned for culturing a cellular barrier which separates the vessel and the chamber of the microfluidic device and which thus accurately models an in vivo cellular barrier of interest.

[19] In some such embodiments, the vessel and the chamber are elongated and in substantially parallel alignment. A plurality of spaced-apart microchannels may provide fluid communication between the chamber and the vessel.

[20] In some such embodiments, the microfluidic device further comprises: a compartment adjacent to the chamber, the compartment configured to contain a third fluid for supporting cell growth in the chamber in use; and at least one conduit providing fluid communication between the compartment and the chamber.

[21] In some embodiments of the first aspect, the vessel is a flow channel configured to convey a flow of fluid. When present, the chamber and the compartment may optionally also be flow channels configured to convey a flow of fluid.

[22] In some embodiments of the first aspect, the conjugating functional groups are configured for conjugation to a native protein, such as to an amine or thiol moiety of the native protein. The conjugating functional groups may be selected from active esters and epoxides, such as from N-hydroxy succinimide esters (NHS-esters) and terminal epoxides.

[23] In some embodiments of the first aspect, the conjugating functional groups are configured for conjugation to a modified protein via a click reaction, such as an alkyne-azide or a tetrazine-TCO click reaction.

[24] In some embodiments, the cross-linked polymer comprises a backbone selected from a poly(vinyl), a polyether and a carbohydrate. In some embodiments, the cross-linked polymer comprises a poly(vinyl) backbone.

[25] In some embodiments, the cross-linked polymer comprises photoactivated residues of a plurality of photoactivatable functional groups, wherein photoactivation has (i) cross-linked the polymer and (ii) chemically bonded the cross-linked polymer to the walls via covalent bonds. The photoactivatable functional groups may be selected from ketones, azides and azirines, such as a diaryl ketone, for example a benzoylphenyl group.

[26] In some embodiments, the cross-linked polymer is a photoactivated product of a poly(vinyl) copolymer comprising (i) polymerised units comprising photoactivatable functional groups and (ii) polymerised units comprising the conjugating functional groups. The poly(vinyl) copolymer may comprise the polymerised units comprising the photoactivatable functional groups in an amount of from 0.5 mol% to 25 mol%, such as from 1 mol% to 10 mol%, of the total polymerised units in the pol(vinyl) copolymer.

[27] In some embodiments, the polymerised units comprising the conjugating functional groups have a structure selected from Formula (3) or Formula (4):

Formula (3) Formula (4) wherein R¹ in Formula (3) and Formula (4) is independently hydrogen or methyl.

[28] In some embodiments, the pol(vinyl) copolymer has a structure according to Formula (5): wherein x, y and z are mole fractions of the respective polymerised units in the pol(vinyl) copolymer, wherein x is from 0.05 to 0.25, y is from 0.05 to 0.95, z is from 0 to 0.9 and x+y+z = 1 , wherein each R¹, R² and R³ is independently selected from hydrogen and methyl, wherein each R⁴ is independently selected from

wherein each R⁵ is independently selected from -O- and -NH-, and wherein each R⁶ is independently selected from -NH2, -OH, -0(Ci-C6 alkyl), -

(polyethylene glycol), -NH(CI-C6 alkyl) and -NH(2-hydroxypropyl).

[29] In some embodiments, the microfluidic device comprises a unitary body in which the one or more internal spaces are at least partly formed. The unitary body may comprise a transparent polymeric material, for example selected from polydimethylsiloxane, cyclic olefin copolymer and poly(methyl methacrylate), and the cross-linked polymer is chemically bonded to the transparent polymeric material.

[30] In accordance with a second aspect the invention provides a method of producing a microfluidic device for investigating an interaction of one or more substances with cells cultured therein, the method comprising: (a) providing a precursor microfluidic device comprising one or more internal spaces including a vessel configured to contain a first fluid comprising the one or more substances; and (b) forming a polymeric coating on walls of at least the vessel by: contacting the walls with a polymer comprising: (i) a plurality of photoactivatable functional groups and (ii) a plurality of conjugating functional groups, and activating the photoactivatable functional groups with light, wherein the photoactivated functional groups cross-link the polymer and chemically bond to the walls, thereby producing a cross-linked polymer comprising a plurality of the conjugating functional groups available for conjugation to polypeptide- containing macromolecules via covalent bond formation.

[31] In some embodiments of the second aspect, the internal spaces of the precursor microfluidic device further include: a chamber adjacent to the vessel; and at least one microchannel providing fluid communication between the vessel and the chamber. In use, the chamber will thus be configured to contain a second fluid for receiving the one or more substances if transportable from the vessel through the at least one microchannel.

[32] In some such embodiments, the vessel and the chamber are elongated and in substantially parallel alignment. A plurality of spaced-apart microchannels may provide fluid communication between the chamber and the vessel.

[33] In some embodiments of the second aspect, the vessel is a flow channel configured to convey a flow of fluid. When present, the chamber and the compartment may optionally also be flow channels configured to convey a flow of fluid.

[34] In some embodiments of the second aspect, the conjugating functional groups are configured for conjugation to a native protein, such as to an amine or thiol moiety of the native protein. The conjugating functional groups may be selected from active esters and epoxides, such as N-hydroxy succinimide esters (NHS-esters) and terminal epoxides.

[35] In some embodiments of the second aspect, the conjugating functional groups are configured for conjugation to a modified protein via a click reaction, such as an alkyne-azide and a tetrazine-TCO click reaction.

[36] In some embodiments, the polymer comprises a backbone selected from a poly(vinyl), a polyether and a carbohydrate. In some embodiments, the cross-linked polymer comprises a poly(vinyl) backbone. [37] In some embodiments, the photoactivatable functional groups are selected from ketones, azides and azirines, such as a diaryl ketone, for example a benzoylphenyl group.

[38] In some embodiments, the polymer is a poly(vinyl) copolymer comprising (i) polymerised units comprising the photoactivatable functional groups and (ii) polymerised units comprising the conjugating functional groups. The pol(vinyl) copolymer may comprise the polymerised units comprising the photoactivatable functional groups in an amount of from 0.5 mol% to 25 mol%, such as from 1 mol% to 10 mol%, of the total polymerised units in the pol(vinyl) copolymer.

[39] In some embodiments, the polymerised units comprising the conjugating functional groups have a structure selected from Formula (3) or Formula (4):

Formula (3) Formula (4) wherein R¹ in Formula(3) and Formula (4) is independently hydrogen or methyl.

[40] In some embodiments, the pol(vinyl) copolymer has a structure according to Formula (5): wherein x, y and z are mole fractions of the respective polymerised units in the pol(vinyl) copolymer, wherein x is from 0.05 to 0.25, y is from 0.05 to 0.95, z is from 0 to 0.9 and x+y+z = 1 , wherein each R¹, R² and R³ is independently selected from hydrogen and methyl, wherein each R⁴ is independently selected from

(polyethylene glycol), -NH(CI-C6 alkyl) and -NH(2-hydroxypropyl).

[41 ] In some embodiments, the precursor microfluidic device comprises a unitary body in which the one or more internal spaces are at least partly formed. The unitary body may comprises a transparent polymeric material, for example selected from polydimethylsiloxane, cyclic olefin copolymer and poly(methyl methacrylate), and the photoactivated functional groups chemically bond to the transparent polymeric material.

[42] In some embodiments, the method further comprises contacting polypeptide-containing macromolecules with the polymeric coating to immobilise the polypeptide-containing macromolecules thereon by covalent bond formation with the conjugating functional groups, thereby adapting the walls for culturing cells. [43] In accordance with a third aspect the invention provides a microfluidic device for investigating an interaction of one or more substances with cells cultured therein, produced by a method according to any of the embodiments disclosed herein.

[44] In accordance with a fourth aspect the invention provides use of a microfluidic device according to any of the embodiments disclosed herein to investigate an interaction of one or more substances with cells, the use comprising: contacting the polymeric coating with polypeptide-containing macromolecules, thereby immobilising the polypeptide-containing macromolecules thereon by covalent bond formation with the conjugating functional groups; culturing cells on the immobilised polypeptide- containing macromolecules; conveying a first fluid comprising the one or more substances into the vessel; and determining an interaction of the one or more substances with the cells.

[45] The culturing of cells may take place before, simultaneously with and/or after the conveying of the first fluid into the vessel.

[46] In some embodiments of the fourth aspect, the cells are cultured to form a cellular barrier on the walls of the vessel and determining an interaction of the one or more substances with the cells comprises measuring or observing transport of the one or more substances across the cellular barrier.

[47] In some embodiments of the fourth aspect, the cells are cultured to form a cellular barrier on the walls of the vessel, and determining an interaction of the one or more substances with the cells comprises (i) determining an amount of the one or more substances transported across the cellular barrier to another internal space of the microfluidic device; or (ii) observing an effect attributable to the one or more substances transported across the cellular barrier in another internal space of the microfluidic device.

[48] In some such embodiments, determining the amount of the one or more substances transported across the cellular barrier comprises measuring (i) an amount of the one or more substances present in a second fluid contained in the other internal space or (ii) an amount of the one or more substances remaining in the first fluid. [49] In some such embodiments, the effect attributable to the one or more substances is an effect on cells cultured in the other internal space, such as cancer cells.

[50] In some such embodiments, the cellular barrier is a simulated blood-tissue barrier, such as the blood brain barrier.

[51 ] In some embodiments of the fourth aspect, the conveying comprises flowing the first fluid through the vessel.

[52] In some embodiments of the fourth aspect, the cells are cultured on the immobilised polypeptide-containing macromolecules under conditions of hydrodynamic shear flow in the vessel.

[53] In some embodiments, the polypeptide-containing macromolecules comprise at least one native extracellular matrix (ECM) protein, such as a plurality of native extracellular matrix (ECM) proteins.

[54] In some embodiments, the polypeptide-containing macromolecules comprise at least one modified protein comprising functional groups configured for conjugation via a click reaction, for example selected from an alkyne-azide and a tetrazine-TCO click reaction.

[55] In some embodiments, the cells cultured on the immobilised polypeptide- containing macromolecules comprise endothelial cells. The cells cultured on the immobilised polypeptide-containing macromolecules may further comprise tumour cells. The cells cultured on the immobilised polypeptide-containing macromolecules may further comprise astrocytes and pericytes.

[56] In some embodiments, the one or more substances comprise at least one selected from the group consisting of pharmaceutical compounds, therapeutics, exosomes, nanomicelles, nanoparticles, toxins, small molecules, nucleic acids, oligonucleotides, oligopeptides, proteins, ribozymes, small interfering RNAs, microRNAs, short hairpin RNAs, aptamers, viruses, and antibodies or antigen binding parts thereof. [57] In accordance with a fifth aspect the invention provides a method of a investigating an interaction of one or more substances with cells, the method comprising: providing a microfluidic device according to any of the embodiments disclosed herein; contacting the polymeric coating with polypeptide-containing macromolecules, thereby immobilising the polypeptide-containing macromolecules thereon by covalent bond formation with the conjugating functional groups; culturing cells on the immobilised polypeptide-containing macromolecules; conveying a first fluid comprising the one or more substances into the vessel; and determining an interaction of the one or more substances with the cells.

[58] It will be appreciated that embodiments disclosed herein in the context of the fourth aspect are generally applicable also to the fifth aspect.

[59] In accordance with a sixth aspect, the invention provides a microfluidic device for culturing cells, produced by contacting the polymeric coating of a microfluidic device according to any of the embodiments of the first or third aspects with polypeptide-containing macromolecules, thereby immobilising the polypeptide- containing macromolecules thereon by covalent bond formation with the conjugating functional groups.

[60] Where the terms “comprise”, “comprises” and “comprising” are used in the specification (including the claims) they are to be interpreted as specifying the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.

[61] As used herein, the terms “first”, “second”, “third” etc in relation to various features of the disclosed devices are arbitrarily assigned and are merely intended to differentiate between two or more such features that the device may incorporate in various embodiments. The terms do not of themselves indicate any particular orientation or sequence. Moreover, it is to be understood that the presence of a “first” feature does not imply that a “second” feature is present, the presence of a “second” feature does not imply that a “first” feature is present, etc.

[62] Further aspects of the invention appear below in the detailed description of the invention. Brief Description of Drawings

[63] Embodiments of the invention will herein be illustrated by way of example only with reference to the accompanying drawings in which:

[64] Figure 1 schematically depicts in perspective view a precursor microfluidic device as used in some embodiments of the invention.

[65] Figure 2 schematically depicts the internal spaces of the precursor microfluidic device of Figure 1 , including the vessel, chamber and compartment channels, the microchannels connecting the vessel and chamber and the conduits connecting the chamber and compartment.

[66] Figure 3 schematically depicts in side view the precursor microfluidic device of Figure 1 .

[67] Figure 4 schematically depicts in side view a microfluidic device according to some embodiments of the invention, in which a polypeptide-containing macromolecule has been immobilised on the polymeric coating.

[68] Figure 5 is a graph of water contact angles for uncoated cyclic olefin copolymer (COC) substrates and COC substrates coated with a photoactivated polymer comprising benzoylphenyl groups and hydrophilic zwitterionic groups, as measured in Example 6.

[69] Figure 6 schematically depicts a method of fabricating a precursor microfluidic device used in some embodiments of the invention, as produced in Example 7.

[70] Figure 7 depicts the design and dimensions of an array of eight precursor microfluidic devices formed in a single unitary body, as produced in Example 7.

[71] Figure 8 is a graph of measured fluorescence intensities of the vessel walls of a series of microfluidic devices having different surface modifications, after treatment with Cy5-amine dye in Example 11 . [72] Figure 9 is a graph of measured fluorescence intensity of the vessel walls of a series of microfluidic devices having different photoactivated polymer coatings, after contact with Alex488 anti-rabbit antibody in Example 12.

[73] Figure 10 is a graph of the permeability coefficients across a cellular barrier for caffeine, nitrofurantoin, sucrose, dextran-3 kDa, alanine and glucose, as determined using a microfluidic device according to an embodiment of the invention in Example 14.

[74] Figure 11 is a graph which compares the permeability coefficients for caffeine, sucrose and dextran-3 kDa, as determined in Example 14, against previously reported values for the permeability of these compounds across the blood brain barrier.

[75] Figure 12 is a graph of the efflux coefficient across a cellular barrier for rhodamine 123, as determined using a microfluidic device according to an embodiment of the invention in Example 15, with comparison again a control microfluidic device in which the cellular barrier was inhibited with p-gp inhibitor Elacridar.

[76] Figure 13 is a graph of the uptake rates of transferrin- and bovine serum albumin-functionalised porous silicon nanoparticles into a cellular barrier, as determined using a microfluidic device according to an embodiment of the invention in Example 16.

[77] Figure 14 schematically depicts in perspective view a precursor microfluidic device as used in some embodiments of the invention.

Detailed Description

Method of producing a microfluidic device

[78] The present invention relates to a method of producing a microfluidic device for investigating an interaction of one or more substances with cells cultured within the microfluidic device. The method includes providing a precursor microfluidic device having internal spaces in which a polymeric coating can be formed. These internal spaces include a vessel configured to contain a first fluid comprising the one or more substances subject to investigation. The methods include forming a polymeric coating on walls of at least the vessel. The walls are contacted with a polymer comprising: (i) a plurality of photoactivatable functional groups and (ii) a plurality of conjugating functional groups. The photoactivatable functional groups are then photoactivated with light, so that the photoactivated functional groups cross-link the polymer and chemically bond to the walls of the vessel. The resultant polymeric coating on the walls comprises a cross-linked polymer with a plurality of the conjugating functional groups remaining available for conjugation to polypeptide-containing macromolecules via covalent bond formation.

Precursor microfluidic device

[79] The precursor microfluidic device includes one or more internal spaces including at least a vessel to receive a first fluid containing the one or more substances subject to investigation.

[80] In some embodiments, for example where the microfluidic device is ultimately intended for investigating the transport of substances across a cellular barrier, a chamber adjacent to the vessel is configured to receive a second fluid, and at least one microchannel connects the vessel and the chamber. The microchannels allow transport of the one or more substances from the first fluid in the vessel to the second fluid in the chamber, but have a cross-sectional area sufficiently small that a continuous cellular barrier separating the vessel from the chamber can be formed on the vessel walls while avoiding cell migration into the microchannels and the chamber. As used herein, a precursor microfluidic device refers to a device having the required structural configuration of the microfluidic devices according to the invention, but in which the photoactivated polymeric coating is yet to be formed.

[81 ] A precursor microfluidic device according to some embodiments is depicted in Figures 1 , 2 and 3. Precursor microfluidic device 100 comprises internal spaces including vessel 146, chamber 148 and compartment 150. The vessel, the chamber and the compartment are formed as recesses in solid unitary body 162 made of a transparent polymeric material such as polydimethylsiloxane (PDMS), and are enclosed by glass sheet 170 to which the unitary body is adhered. The vessel thus has walls 115 which are in part made of polymer (of the unitary body 162) and in part of glass (glass sheet 170).

[82] Vessel 146, chamber 148 and compartment 150 are each in the form of elongated channels, having linear portions arranged substantially in parallel and with the chamber intermediate the vessel and compartment. The linear portions of the channels may be about 2 cm in length, and the channels may have cross-sectional dimensions of about 500 pm in width and 100 pm in height (marked “w” and “h" in Figure 3). The distance between the channels may be about 80 pm.

[83] Precursor microfluidic device 100 further comprises a plurality of spaced apart microchannels 112 which provide fluid communication between vessel 146 and chamber 148. The microchannels are also formed as recesses in unitary body 162, and may have a height of about 3 pm and a width of about 3 pm. Similarly, a plurality of spaced apart conduits 113 provide fluid communication between chamber 148 and compartment 150. The conduits have a height of about 3 pm and a width of about 50 pm.

[84] Precursor microfluidic device 100 further comprises access holes 164, 166 formed through unitary body 162 and connected to each end of the elongated vessel 146, chamber 148 and compartment 150 channels. Holes 164 may have a diameter of about 1 mm, while hole 166 at one end of the chamber may have a diameter of about 2 mm diameter to provide an enlarged fluid reservoir. Each of the vessel, chamber and compartment is thus configured to receive fluid via one access hole to fill the internal space, or to receive a flow of fluid through the channel, flowing into one access hole and out of the other access hole.

[85] The structure of the precursor microfluidic device in this embodiment follows from the nature of the investigations to be conducted using the final, polymer-coated device. As will be described in greater detail hereafter, a cellular barrier will ultimately be assembled on polymer-coated walls 115 of vessel 146 by culturing cells in the vessel. Optionally, this may be done under conditions of flow through the vessel, thus mimicking the fluid shear stress conditions at the in vivo cellular barrier being modelled. A fluid comprising one or more substances subject to investigation can then be conveyed into vessel 146, either to fill the vessel statically or as a flow through the vessel. If the substance can traverse the cellular barrier, it will be transported from vessel 146, via microchannels 112, into a second fluid contained in chamber 148. The rate of permeation will depend on both the integrity of the cellular barrier and the inherent transmissibility of the substance through the cellular barrier. For some applications, it will be desirable to culture cells also in chamber 148, and compartment 150 may contain a third fluid to support growth of the cells in the chamber.

[86] While in some preferred embodiments the vessel and chamber are elongated channels arranged in substantially parallel alignment and connected by a plurality of microchannels, as described above, it will be apparent from the above discussion that other configurations of the precursor device may be adopted without departing from the scope of the invention. In particular, the chamber and the vessel need not be substantially elongated, and a single microchannel may be sufficient to provide fluid communication between these spaces. It will also be appreciated that the compartment and conduits are not required for all applications where cell transport across a cellular barrier will be investigated.

[87] In some embodiments, the distance between the vessel and the chamber along the connecting microchannels may be less than 200 pm, or less than 100 pm, such as from 20 to 100 pm. In some embodiments, the microchannels have a largest cross-sectional dimension of between 0.4 and 8 pm.

[88] In the methods of the invention, the precursor microfluidic device is internally coated by photoactivation of a polymer. Accordingly, at least a portion of the body of the device may be sufficiently transparent to light, preferably UV light, so that photoactivatable functional groups of the polymer can be photoactivated. Moreover, the walls of the vessel should generally be susceptible to covalent bond forming reactions with the photoactivated polymer. Suitable transparent materials meeting these requirements include a wide range of polymeric materials and glass. Suitable polymers may include PDMS, cyclic olefin copolymer, poly(methyl methacrylate) and the like.

[89] The precursor microfluidic devices can be fabricated by conventional microfabrication methodologies, and in particular by photolithography. Briefly, a positive mold of the internal spaces of the device is built up on a silicon substrate by applying a layer of photoresist, curing the photoresist by irradiation through a mask, baking, and then processing with developer to produce a microstructured layer. Multiple such layers can be assembled to provide all features of the mold. A curable polymer resin such as PDMS is then cast over the mold, cured, and peeled away to provide the unitary body of the device. The access holes may subsequently be punched into the cured polymer. Finally, the unitary body is adhered to a glass sheet to provide the precursor microfluidic device.

[90] The embodiment described with reference Figures 1 , 2 and 3 is a precursor microfluidic device configured for applications where transport of substances across a cellular barrier is to be investigated. It thus includes chamber 148 to receive the transported substances and microchannels 112 to provide fluid communication. It will be appreciated, however, that these features are not essential to all embodiments of the invention in its most general form.

[91 ] Depicted in Figure 14 is a precursor microfluidic device 400 suitable for other embodiments of the invention. While fabricated in the same manner as precursor microfluidic device 100, device 400 has only a single elongated flow channel in the form of vessel 446 connected to access holes 164 on each end. In the methods of the invention, the walls of vessel 446 can be internally coated by photoactivation of a polymer as will be described hereafter. The resultant microfluidic device is suitably adapted for investigating various interactions between cells cultured on the walls of vessel 446 and substances introduced into vessel 446 before, during or after the cell culturing.

Polymer

[92] The methods of the invention comprise forming a polymeric coating on internal walls of at least the vessel of the precursor microfluidic device by contacting the walls with a polymer (also referred to herein as a precursor polymer) and activating the polymer with light such that it cross-links and chemically bonds to the walls, typically by covalent bonds. This results in the formation of a cross-linked polymer which is anchored to the walls and includes conjugating functional groups available for reaction with polypeptide-containing macromolecules such as those present in an ECM.

[93] Accordingly, the precursor polymer has a molecular structure that includes both a plurality of photoactivatable functional groups and a plurality of conjugating functional groups. The photoactivatable functional groups are susceptible to activation by light such that they can cross-link the polymer and react with surface moieties on the walls of the vessel. At least a portion of the conjugating functional groups are unreacted in this process such that they remain available for conjugation.

[94] Apart from these requirements, the polymer structure is not considered to be particularly limited. Typically, the precursor polymer will be a linear polymer with the photoactivatable functional groups and conjugating functional groups presenting pendant to the backbone chain. In some embodiments, the polymer is a vinyl polymer, a polyether, a polyester, a polyurethane, a polysiloxane or a carbohydrate, or contains segments of such polymers. In some embodiments the precursor polymer comprises a backbone selected from a poly(vinyl), a polyether and a carbohydrate.

[95] In some embodiments, the precursor polymer is a poly(vinyl) polymer. As used herein, a poly(vinyl) polymer is an addition polymer of one or more ethylenically unsaturated monomers, thus comprising an extended saturated alkane (...-C-C-C-C- C-... ) backbone chain. Suitable ethylenically unsaturated monomers for forming a poly(vinyl) polymer may have vinyl (-CH=CH2) or vinylidene (-CR²=CH2 where R² = Ci- C6 alkyl) polymerisable moieties. Polymerisation of the ethylenically unsaturated monomers, for example by a free radical propagation mechanism, forms the extended backbone chain having the monomers incorporated therein as polymerised units (or residues). As used herein, polymerised units of a poly(vinyl) polymer encompass both as-polymerised forms of the ethylenically unsaturated monomers as well as post polymerisation chemically modified forms.

[96] In some embodiments, the polymer is a poly(vinyl) copolymer which includes a plurality of polymerised units comprising the photoactivatable functional groups and a plurality of different polymerised units comprising the conjugating functional groups.

[97] In some embodiments, the polymerised units comprising the photoactivatable functional groups are residues of ethylenically unsaturated monomers which contained the photoactivatable functional groups prior to polymerisation. Examples of such monomers are N-benzoylphenyl (meth)acrylamides such as N-(4- benzoylphenyl) acrylamide and benzoylphenyl (meth)acrylates such as 4- benzoylphenyl acrylate. In other embodiments, the polymerised units having the photoactivatable functional groups are derived by post-polymerisation chemical modification of the pol(vinyl) co-polymer. For example, as-polymerised units derived from an amine-containing monomer (such as N-(3-aminopropyl)methacrylamide) may be reacted with 4-benzoylbenzoyl chloride to provide pendant benzoylphenyl groups. Suitable photoactivatable functional groups for the precursor polymers used in the methods of the invention will be described more generally hereafter.

[98] In some embodiments, the polymerised units having the conjugating functional groups are residues of ethylenically unsaturated monomers which contained the conjugating functional groups prior to polymerisation. An example of such monomers is glycidyl (meth)acrylate; the corresponding polymerised unit in the copolymer thus comprises an epoxide group pendant to the backbone chain that can conjugate to amine or thiol moieties of a polypeptide. In other embodiments, the polymerised units having the conjugating functional groups are derived by post polymerisation chemical modification of the pol(vinyl) co-polymer. For example, as- polymerised units derived from acrylic acid or other acid-functionalised monomers may be converted to active esters, for example N-hydroxysuccinimide (NHS) esters. The active ester functional groups pendant to the backbone chain are then available for conjugation to amine moieties of a polypeptide. Suitable conjugating functional groups for the precursor polymers used in the methods of the invention will be described more generally hereafter.

[99] Optionally, the poly(vinyl) copolymer further comprises polymerised units of one or more further ethylenically unsaturated monomers of conventional type, these polymerised units generally lacking either photoactivatable functional groups or covalently conjugating functional groups. The further monomers may include one or more acrylic monomers, vinyl aromatics, vinyl esters, vinyl ethers, vinyl chloride, and the like. Examples of suitable acrylic monomers may include (meth)acrylic acid, (meth)acrylamide, alkyl (meth)acrylates such a methyl(meth)acrylate, ethyl(meth)acrylate, butyl(meth)acrylate, hexyl(meth)acrylate, and 2-ethylhexyl (meth)acrylate, substituted alkyl (meth)acrylates such hydroxyethyl methacrylate, zwitterionic (meth)acrylates, and acrylonitrile. As used herein, “(meth)acrylate” includes in the alternative both an acrylate and a methylacrylate. Examples of suitable vinyl aromatics may include styrene and substituted styrenes. Examples of suitable vinyl esters may include vinyl acetate. [100] The polymer generally comprises the photoactivatable functional groups and the conjugating functional groups in amounts sufficient to achieve their respective functionalities according to the principles disclosed herein. In the case where the polymer is a poly(vinyl) copolymer, the pol(vinyl) copolymer may comprise polymerised units comprising the photoactivated functional groups in an amount of from 0.5 mol% to 50 mol%, such as from 1 mol% to 25 mol%, for example from 1 mol% to 10 mol%, of the total polymerised units in the pol(vinyl) copolymer. Amounts of 4-10 mol% have been found adequate to produce a robust cross-linked polymeric coating anchored to polymeric substrates after photoactivation. The pol(vinyl) copolymer may comprise polymerised units comprising the conjugating functional groups in an amount of at least 5 mol%, or 10 mol%, or 20 mol%, or 30 mol%, or 40 mol%, or 50 mol%, of the total polymerised units in the pol(vinyl) copolymer.

[101] In some embodiments, the pol(vinyl) copolymer has a structure according to Formula (1 ) below, in which each R_p is independently an organyl substituent comprising a photoactivatable functional group as described herein, each R_c is independently an organyl substituent comprising a conjugating functional group as described herein, x, y and z are mole fractions of the respective polymerised units in the pol(vinyl) copolymer, wherein x is from 0.05 to 0.25, y is from 0.05 to 0.95, z is from 0 to 0.9 and x+y+z = 1 , each R¹, R² and R³ is independently selected from hydrogen and methyl, and each R⁶ is independently selected from -NH2, -OH, -0(Ci-C6 alkyl), -(polyethylene glycol), - NH(CI-C6 alkyl) and -NH(2-hydroxypropyl).

[102] As used herein,

refers generally to a point of connection to another portion of a molecular structure, for example the point of connection to the chain terminal groups or to a polymer segment of different composition (when denoting the end-points of a defined polymeric structure), or to an adjacent polymerised unit (when used to define the structure of an individual polymerised unit in a polymer). [103] The poly(vinyl) copolymers described herein may be prepared by conventional methods known to the skilled person, for example free radical polymerisation. Free-radical polymerisation of a mixture of ethylenically unsaturated monomers suitable to produce the required polymer structure can be initiated with a radical initiator, such as 2,2'-azobis(2-methylpropionitrile). Optionally, the molecular weight and polydispersity of the polymer can be controlled with living polymerisation methods, for example reversible addition-fragmentation chain transfer (RAFT) polymerization. In such cases, the poly(vinyl) copolymer will comprise terminal chain groups derived from the selected RAFT agent, for example 4-cyano-4- (ethylsulfanylthiocarbonylsulfanyl) pentanoic acid. Where the photoactivatable functional groups and/or the conjugating functional groups are to be introduced post polymerisation, this may be facilitated by including monomers having suitable target functionality for grafting. For example, pendant amine groups as grafting targets may be included via a monomer such as N-(3-aminopropyl)methacrylamide, while pendant carboxylic acid grafting target groups can be introduced via acrylic acid, with optional subsequent activation to active esters as disclosed herein.

Photoactivatable functional groups

[104] As described herein, the precursor polymer includes a plurality of photoactivatable functional groups, typically pendant from the polymer backbone. The photoactivatable functional groups are susceptible to excitation when irradiated with light of suitable wavelength, leading to formation of reactive species such as radicals. The photoactivated species are capable of covalent bond-forming reactions to cross link the polymer chain and to chemically bond the polymer to a substrate containing suitable target functionalities, for example C-FI bonds.

[105] In some embodiments, the photoactivatable functional groups are selected from ketones, azides and diazirines. Suitable ketones include aryl ketones and particularly diarylketones such as benzoylphenyl groups. Suitable azides include aryl azides such as phenyl azides.

[106] In some embodiments, the photoactivatable functional groups comprise benzoylphenyl groups (i.e. benzophenone derivatives). Without wishing to be bound by any theory, it is believed that the photoexcited state of the benzoylphenyl group can readily insert into carbon-hydrogen (or oxygen-hydrogen) bonds of many materials, thereby abstracting a hydrogen atom and forming a radical pair. Subsequent collapse of the radical pair leads to covalent bond formation between the carbon radical and the benzoylphenyl radical. Since carbon-hydrogen bonds at the surface of many polymeric substrates can be activated in this manner, photoactivation of pendant benzoylphenyl groups provides an effective means to anchor a polymeric coating to a polymeric substrate, as depicted in Scheme 1 (A) below. Furthermore, it is believed that coupling of two pendant benzoylphenyl radicals can cross-link the polymer as depicted in Scheme 1 (B). Accordingly, photoactivation of a polymer comprising a plurality of pendant benzoylphenyl groups, in the vicinity of a glass or polymeric substrate, provides a polymeric coating which is both cross-linked and covalently bonded to the substrate surface.

Scheme 1

[107] The benzoylphenyl groups may be connected to the polymer at any position, for example the 4-phenyl position. It is not excluded that the benzoylphenyl groups may be substituted at one or more of the other carbon atoms, for example with Ci-Ce alkyl groups.

[108] The photoactivatable functional group, such as a benzoylphenyl group, may be directly connected to the polymerised residue of the polymerisable functional group incorporated into the polymer chain. For example, the benzoylphenyl group may be incorporated into the polymer via a monomer such as a N-benzoylphenyl (meth)acrylamide, e.g. N-(4-benzoylphenyl) acrylamide or a benzoylphenyl (meth)acrylate, e.g. 4-benzoylphenyl acrylate. In other embodiments, the pendant photoactivatable functional group is separated from the backbone chain by a linker.

[109] In some embodiments, the benzoylphenyl group is present in a polymerised unit having the structure of Formula (2) below, in which R² is selected from hydrogen and methyl and R⁵ is selected from -O- and -NH-.

[110] Polymers according to the invention having a variety of photoactivatable functional groups may be prepared by previously disclosed methodologies. As another example, phenylazide photoactivatable groups may be grafted to amine target functionalities on a polymer chain via a commercially available reagent such as N- succinimidyl-5-azido-2-nitrobenzoate, as disclosed by Flook et al ( Biomacromolecules 2009, 10, 573-579). Diazirine groups can be incorporated by commercially available bifunctional reagents such as (S)-2-amino-4-(3/-/-diazirin-3-yl)pentanoic acid hydrochloride (7-/-L-photo-methionine HCI), which can be grafted to active ester functionalities on the polymer.

Conjugating functional groups

[111] As described herein, the precursor polymer also includes a plurality of conjugating functional groups, typically pendant from the polymer backbone. The role of the conjugating functional groups is to conjugate to polypeptide-containing macromolecules via covalent bond formation. Thus, when a cross-linked polymeric coating produced from the precursor polymer is contacted with polypeptide-containing macromolecules, the macromolecules are immobilised by covalent chemical bonds.

[112] Preferably, the conjugating functional groups are configured for facile and substantially irreversible conjugation reactions at ambient reaction conditions and without the need for chemical or photochemical activation. Moreover, since the precursor polymer is first subjected to a photoactivation step to form the polymer coating inside a microfluidic device, at least a portion of the conjugating functional groups must survive the activation and cross-linking process and remain available for conjugation reactions on the coating.

[113] In some embodiments, the conjugating functional groups are configured for conjugation to a native protein. For example, the conjugating functional groups may be selected to target amine or thiol moieties in native proteins. A wide range of conjugating functional groups are reportedly suitable for this purpose, and the polymers of the present invention may incorporate any such groups according to the principles disclosed herein. In some embodiments, the conjugating functional groups configured for conjugation to a native protein are selected from the group consisting of active esters, epoxides, isocyanates, isothiocyanates, sulfonyl chlorides, maleimides, anhydrides, chloroformates, aldehydes, ketones, carbodiimides and imidoesters.

[114] In some embodiments, the conjugating functional groups are selected from active esters and epoxides. We have demonstrated that photoactivated polymeric coatings comprising such conjugating functional groups are highly effective in immobilising native proteins, including ECM proteins and antibodies, under ambient conditions.

[115] As used herein, an active ester is an ester functional group which is highly susceptible to nucleophilic attack as a result of electronegative substituents. In particular, active esters provide enhanced rates of reaction with amines to form amide linkages. Non-limiting examples of suitable active esters may include N-hydroxy succinimide esters (NHS-esters), including sulfo-NHS esters (produced from N- hydroxysulfosuccinimide), fluorophenyl esters, and stabilised triazole esters (e.g. produced from HATU = 1 -[bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate) and HBTU = 3- [bis(dimethylamino)methyliumyl]-3/-/-benzotriazol-1 -oxide hexafluorophosphate). Epoxide groups are susceptible to ring opening conjugation reactions with amines or thiols to form beta-hydroxy secondary amine or thioether linkages respectively.

[116] In some embodiments, the conjugating functional groups are selected from N-hydroxy succinimide esters (NHS-esters) and terminal epoxides such as glycidyl groups.

[117] The conjugating functional group may be directly connected to the polymerised residue of the polymerisable functional group incorporated into the polymer chain or separated therefrom by no more than two atoms. In other embodiments, the conjugating functional group is separated from the backbone chain by a linker.

[118] In some embodiments, the conjugating functional group is present in a polymerised unit having the structure of Formula (3) or Formula (4) below, in which R¹ in either formula is independently hydrogen or methyl.

[119] In some embodiments, the conjugating functional groups are configured for conjugation to a modified protein via a click reaction. Click reactions refer generally to coupling reactions occurring between complementary pairs of functional groups which are thermodynamically predisposed to react with high selectivity, high conversion, a lack of complex by-products and under biological conditions. The resultant covalently bonded linking groups are chemically stable and biologically inert. Click chemistry is widely used for bioconjugation to biological macromolecules, for example to attach fluorophores or other reporter molecules. A particular advantage of click chemistry is that it allows selective bioconjugation of probe molecules to pre-functionalised target biological macromolecules in complex biological systems, also known as biorthogonal reactions. A wide range of click chemistries have been reported for bioconjugation reactions. These include [3+2] cycloaddition reactions, such as the azide-alkyne Huisgen cycloaddition between an azide functional group and an alkyne group to form a 1 ,2,3-triazole linker, inverse-demand Diels-Alder reactions between a tetrazine and a strained cycloalkene functional group, such as tetrazine and trans-cyclooctene (the tetrazene-TCO reaction), and the Staudinger ligation reaction between a tertiary phosphine and an azide functional group to form a phosphazo linker.

[120] The conjugating functional groups may thus comprise any of the functional groups known to participate in click reactions with a complementary functional group via these or other click reactions, according to the principles disclosed herein. In some embodiments, the conjugating functional groups are configured for conjugation to a modified protein via an alkyne-azide or a tetrazine-TCO click reaction.

[121] Polymers according to the invention having click conjugating groups can be produced by a variety of conventional synthetic methodologies for labelling macromolecules with these groups. As one example, the known alkyne-based click group dibenzo-bicyclo-octyne (DBCO), configured for copper-free alkyne-azide click coupling reactions, may be grafted to active ester and amine functionalities on the polymer via commercially available reagents such as dibenzocyclooctyne-amine and dibenzocyclooctyne-sulfo-A/-hydroxysuccinimidyl ester respectively. Similarly, the tetrazine click group, configured for tetrazine-TCO click couplings, can be introduced via commercially available ester-reactive and amine-reactive reagents such as tetrazine-amine and tetrazine-NHS ester respectively.

Forming the polymeric coating

[122] The polymeric coating is formed in the methods of the invention by contacting the walls of at least the vessel with a precursor polymer as described herein, and activating the photoactivatable functional groups with light. Optionally, the walls of other interior spaces of the microfluidic device, e.g. the microchannels and the chamber when present, may be coated in the same way.

[123] Typically, the precursor polymer is in solution when contacted with the walls and photoactivated. For example, the polymer may be dissolved in an aprotic organic solvent of suitable polarity and which is compatible with the materials of the device. The polymer may be dissolved at a concentration in the range of 0.1 to 50 wt%, such as from 1 to 10 wt%. For example, we have found that 2 wt% solutions of polymer in dimethylsulfoxide (DMSO) are suitable to provide a functionally acceptable polymeric coating on the internal walls of a microfluidic device, without blocking the microchannels. The vessel and optionally other interior spaces of the precursor microfluidic device may be filled with the polymer solution before photoactivation to ensure a consistent coating is formed on the walls.

[124] The polymer is then activated by irradiation with light of a suitable wavelength and intensity to activate the photoactivatable functional groups. It will be appreciated that the wavelengths of light suitable for photoactivation will depend on the nature of the photoactivatable functional groups. In some embodiments, the light is UV light. In the case of benzoylphenyl groups, a wavelength in the range of 200 to 400 nm is suitable. For example, we have found that irradiation for 20 seconds with broad spectrum UV light and an intensity of 34 J/cm² is sufficient to photoactivate benzoylphenyl-containing polymer precursors for coating formation. The body of the microfluidic precursor device is generally transparent to activating wavelengths of the light, such that the polymer solution in the internal spaces can be irradiated through the body.

[125] After the irradiation, the photoactivated functional groups cross-link the polymer and covalently bond to the walls of the vessel as described herein. In the case of benzoylphenyl functional groups, this is believed to take place via the mechanism shown in Scheme 1 . The resultant polymeric coating comprises a cross-linked polymer with a plurality of the conjugating functional groups remaining available for conjugation to polypeptide-containing macromolecules. The coating may have a thickness in the nanometer range, for example from 1 to 10 nm. We have found that even coatings of 3-5 nm in thickness are produced when 2 wt% solutions of the photoactivatable polymer in dimethylsulfoxide (DMSO) are photoactivated under UV irradiation.

[126] Referring again to Figure 1 , a solution of the precursor polymer may be conveyed into vessel 146 via access holes 164, and optionally also into chamber 148 and compartment 150. Once the internal spaces are filled, the solution is irradiated with light through the unitary body 162 which is formed of transparent polydimethylsiloxane (PDMS). Thus walls 115 are coated with a consistent polymeric coating of cross-linked polymer according to the principles disclosed herein. Similarly, with reference to Figure 14, a solution of the precursor polymer may be conveyed into vessel 446 via one access hole 464 and photoactivated therein to coat the walls of the vessel.

Microfluidic devices

[127] The invention also relates to a microfluidic device for investigating an interaction of one or more substances with cells cultured within the microfluidic device. The microfluidic device includes one or more internal spaces including a vessel configured to contain a first fluid comprising the one or more substances, and a polymeric coating on the walls of at least the vessel. The polymeric coating comprises at least one cross-linked polymer which is chemically bonded to the walls and a plurality of conjugating functional groups available for reaction. In use, polypeptide-containing macromolecules are contacted with the polymeric coating and thus immobilised on the polymeric coating by covalent bond formation with the conjugating functional groups. The walls of the vessel can thus be adapted for culturing cells.

[128] In some embodiments, and particularly for microfluidic devices to be used for investigating the transport of substances across a cellular barrier, the device further includes a chamber adjacent to the vessel, configured to contain a second fluid for receiving the one or more substances if transportable from the vessel, and at least one microchannel which provides fluid communication between the vessel and the chamber.

[129] The structural configuration of the precursor microfluidic device has already been described herein in the context of the methods of the invention. The microfluidic devices according to the invention generally comprise such a precursor microfluidic device, wherein a polymeric coating has been formed on walls of the vessel.

[130] The polymeric coating is disposed on the internal walls of the vessel, and optionally also on the walls of other internal spaces of the microfluidic device e.g. the microchannels and the chamber when present. The polymeric coating may have a thickness in the nanometer range (1 -100 nm), for example from 1 to 10 nm. Preferably, the coating is a continuous coating which covers the walls of the vessel. [131] In embodiments where the microfluidic device includes the chamber and connecting microchannels, the polymeric coating may be present as a continuous coating at least in regions proximate to the microchannels. Thus, the vessel and the chamber will be separated by a cellular barrier cultured on the coating such that any transmission of substances from fluid in the vessel to fluid in the chamber, through the microchannels, must occur through the cellular barrier.

[132] The polymeric coating comprises a cross-linked polymer which is chemically bonded to the internal walls of the vessel, typically by covalent bonds. Advantageously, the chemical bonding anchors the coating to the walls, thus providing a stable substrate of homogeneous thickness to which polypeptide-containing macromolecules can be attached and cells subsequently cultured. Chemical bonding of a polymeric coating to a substrate can thus be distinguished from coatings which rely on weaker dispersive adhesion (i.e. physisorption).

[133] In some embodiments, the chemical bonding is provided by photoactivation of a precursor polymer, as described herein in the context of the methods of the invention. The cross-linked polymer may thus comprise photoactivated residues of a plurality of photoactivatable functional groups, wherein the photoactivation has (i) cross-linked the polymer and (ii) chemically bonded the cross-linked polymer to the walls via covalent bonds. As disclosed herein, suitable photoactivatable functional groups may include ketones, azides and azirines. In some embodiments the photoactivatable functional groups comprise a diaryl ketone, for example a benzoylphenyl group.

[134] In some embodiments, the cross-linked polymer comprises a backbone selected from a poly(vinyl), a polyether and a carbohydrate. In some embodiments, the backbone is a poly(vinyl) backbone.

[135] The cross-linked polymer comprises a plurality of conjugating functional groups available for covalent bond-forming conjugation reactions with polypeptide- containing macromolecules. Preferably, the conjugating functional groups are configured for facile and substantially irreversible conjugation reactions at ambient reaction conditions and without the need for chemical or photochemical activation. In some embodiments, the conjugating functional groups are configured for conjugation to a native protein, for example to an amine or thiol moiety of the native protein. The conjugating functional groups may be selected from active esters and epoxides, for example N-hydroxy succinimide esters (NHS-esters) and terminal epoxides. In some embodiments, the conjugating functional groups are configured for conjugation to a modified protein via a click reaction, for example an alkyne-azide and a tetrazine-TCO click reaction.

[136] In some embodiments, the cross-linked polymer is a photoactivated product of a poly(vinyl) copolymer comprising (i) polymerised units comprising photoactivatable functional groups and (ii) polymerised units comprising the conjugating functional groups, as previously disclosed herein.

Uses of the microfluidic devices

[137] The invention also relates to the use of microfluidic devices as disclosed herein to investigate an interaction of one or more substances with cells. The use comprises contacting the polymeric coating on the walls of the vessel with polypeptide- containing macromolecules, such as those which form part of an extracellular matrix (ECM) in vivo, thereby immobilising the polypeptide-containing macromolecules by covalent bond formation with the conjugating functional groups of the cross-linked polymer. Cells are then cultured on the immobilised polypeptide-containing macromolecules. A fluid comprising the one or more substances is conveyed into the vessel (after, simultaneously with or before culturing the cells) and an interaction of the one or more substances with the cells is determined.

[138] In some embodiments, the use is to investigate the transport of one or more substances across a cellular barrier cultured on the walls of the vessel and the transportability of the substances is assessed by determining the amount of the substance transported across the cellular barrier or by observing an effect attributable to the presence of the one or more substances in another internal space of the microfluidic device. However, it is envisaged that other interactions of substance(s) with the cultured cells, for example promotion or inhibition of cell growth or cell differentiation by substances such as therapeutics, may also be investigated and thus fall within the scope of the invention.

Polypeptide-containing macromolecules [139] To adapt the microfluidic devices for cell culturing, polypeptide-containing macromolecules are first immobilised on the polymeric coating. As used herein, a polypeptide-containing macromolecule may be a natural, synthetic or modified protein. The purpose of the polypeptide-containing macromolecules is to contribute to a medium which supports the maintenance, viability, growth, and replication of a cell comprised therein. In this regard, the medium is typically a three-dimensional scaffold which mimics the ECM component of cellular environments found in vivo.

[140] The ECM surrounding a cell performs several critical functions. It provides a complex, nanoscale architecture of structural proteins such as collagen, fibronectin, laminin, elastin, and glycoproteins to create the mechanical properties inherent in the cellular microenvironment. Cells sense these mechanics through their cell surface integrins, and bind to specific adhesion motifs present on the ECM proteins. Cell adhesion in a three-dimensional system leads to and influences a series of subsequent cellular responses that are more physiologically relevant compared to cells grown on two-dimensional surfaces. Furthermore, the ECM is vital for sequestering soluble biomolecules and growth factors, and releasing these signaling molecules with spatial- temporal control to guide processes such as cell migration, matrix degradation and deposition. Therefore, the polypeptide-containing macromolecules encompassed herein contribute to a medium which exhibits the mechanical and biochemical properties of in vivo ECM, not only at the initial stage of cell seeding, but also, in a dynamic and tunable manner as the cells grow and develop.

[141] The three-dimensional scaffold, which includes the polypeptide-containing macromolecules may be in the form of a gel matrix which may contain a gel or gel-like material. For example, the gel matrix may be a hydrogel. Flydrogels are comprised of complex protein molecules of natural or synthetic origin. Due to their significant water content, hydrogels possess biophysical characteristics very similar to natural tissue, and serve as highly effective matrices for three-dimensional cell culture. Relevant hydrogels may include thermosensitive hydrogels, photosensitive hydrogels, ionic polymerisation hydrogels, irreversible gelling hydrogels, enzymatic, covalent, or noncovalent polymerisable hydrogels, or cross-linked hydrogels. Such hydrogels are known in the art with further examples summarized in the publications of Panwar A et al. 2016, Molecules, 21 : 685; and Aljohani W et al., 2018, International Journal of Biological Macromolecules, 107(Pt A): 261-275.

[142] Naturally derived hydrogels for cell culture are typically formed of proteins and ECM components (Corning Inc., Sigma Aldrich, Trevigen, Inc., NovaMatrix and Xylyx Bio), such as gelatin, collagen, laminin, fibrin (the combination of fibrinogen and thrombin - thrombin is used to rapidly polymerize fibrinogen to form fibrin - see Duong H et al., 2009, Tissue Engineering Part A, 15(7): 1865-1876), fibronectin, heparin sulfate proteoglycan, hyaluronic acid, chitosan, basement membrane extract (Cultrex^®), alginate, Matrigel, and silk. Derived from natural sources, these gels are inherently biocompatible and bioactive. They also promote many cellular functions due to the presence of various endogenous factors, which can be advantageous for supporting viability, proliferation, function, and development of many cell types.

[143] Matrigel from Corning Life Sciences is an ECM-based natural hydrogel that has been used extensively for three-dimensional cell culture in vitro and in vivo. This reconstituted basement membrane is extracted from Engelbreth-Holm-Swarm (EHS) mouse tumors and contains all the common ECM molecules found in the basement membrane (i.e. laminin, collagen IV, heparin sulfate proteoglycan, and nidogen/entac- tin).

[144] Collagen Type I is a common ECM molecule found in stromal compartments and bone. It can be isolated from various biological sources including bovine skin, rat tail tendon, and human placenta. Collagen I can also be electrospun into membranes, and can support 3D cell growth and differentiation.

[145] Other naturally derived hydrogels include cell-derived ECM hydrogels (TissueSpec^®) and decellularised matrix derived from tissue. These hydrogels are isolated from specific tissues or organs which preserve the biomolecules from original tissues (see Pati F etal., 2014, Nature Communications, 5: 3935; Choudhury D etal., 2018, Trends Biotechnol., 36(8): 787-805).

[146] In some embodiments, the polypeptide-containing macromolecules may comprise native proteins. As used herein, native proteins refer to proteins which have not been synthetically modified to include non-biological functional groups receptive to conjugation. Instead, the native proteins include one or more inherent functionalities suitable for conjugation, for example amine or thiol moieties. In such embodiments, the cross-linked polymer in the polymeric coating should comprise conjugating functional groups configured for covalent bond-forming conjugation to these target moieties.

[147] In some embodiments, the polypeptide-containing macromolecules comprise a plurality of different native proteins. In some embodiments, the polypeptide- containing macromolecules comprise a protein mixture able to model the ECM present in biological tissue, for example Matrigel. As indicated above, the protein mixture may comprise at least one, and preferably a plurality, of proteins selected from laminin, collagen (e.g. collagen type IV), fibrin, and entactin/nidogen.

[148] Since the polymeric coatings in some embodiments of the invention are configured to immobilise native proteins, a wide variety of naturally occurring proteins, mixtures thereof and other polypeptide-containing macromolecules can be immobilised on the devices without the need to synthetically predispose the macromolecules for conjugation. This property may be advantageous, for example, in high-throughput screening experiments. Moreover, multiple different polypeptide-containing macromolecules on the polymeric coating can simultaneously be immobilised on such coatings, thus creating a substrate for cell culturing which is representative of the modelled in vivo environment.

[149] In other embodiments, the polypeptide-containing macromolecules are functionalised for bioorthogonal conjugation reactions. For example, they may be configured for conjugation via a click reaction such as an alkyne-azide or a tetrazine- TCO click reaction. Polypeptides can readily be functionalised in this way using commercially available bifunctional reagents having both click groups and polypeptide conjugating groups, e.g. TCO-NHS ester, TCO-maleimide, tetrazine-NHS ester, azide- NHS ester, alkyne-NHS ester and the like. In such embodiments, the cross-linked polymer in the polymeric coating should comprise complementary click chemistry functional groups as disclosed herein. This approach advantageously allows selective immobilisation of labelled polypeptide-containing macromolecules in the presence of other biomolecules.

Immobilisation of polypeptide-containing macromolecules [150] The polypeptide-containing macromolecules are contacted with the polymeric coating so that covalent bond forming reactions occur between the conjugating functional groups on the cross-linked polymer and the target functionalities on the macromolecules. The microfluidic device is preferably sterilised as an initial step. The polypeptide-containing macromolecules are typically introduced to at least the vessel of the microfluidic device in a fluid, for example an aqueous fluid such as phosphate-buffered saline (PBS). The vessel is preferably filled to provide contact with all polymer-coated parts of the walls. The fluid may be incubated in the device at ambient conditions for a time sufficient to allow the conjugation reactions to take place.

[151] An embodiment of a microfluidic device with a configuration as generally disclosed in Figures 1 to 3, but now adapted for culturing of cells, is schematically depicted in Figure 4. Vessel 546, chamber 548 and compartment 550 are formed in unitary PDMS body 510 and enclosed by glass sheet 570. The vessel and chamber are connected by a plurality of microchannels 512, while the chamber and compartment are connected via conduits 514. The walls of the vessel are coated with a cross-linked polymeric coating 520 covalently bonded to the walls by photoactivated residues 521 of benzoylphenyl groups. Prior to contact with a protein mixture, the cross-linked polymer includes NFIS-ester functional groups 523 available for conjugation. Contact of the coating with a protein mixture, for example ECM-proteins, immobilises proteins 524 on coating 520. The coating and immobilised proteins are connected by amide linkages 525, formed by conjugation between the NFIS ester functionalities and amine moieties present in the proteins.

[152] It will be appreciated that the microfluidic device depicted in Figure 14, which includes only a single internal microchannel, can be similarly adapted for cell culturing.

Culturing cells

[153] Once the polypeptide-containing macromolecules have been immobilised on the polymeric coating, the macromolecules, as part of the three-dimensional scaffold which mimics the ECM, support the culture of cells, for example to produce a cellular barrier between the vessel and the chamber for transport investigations. It will be appreciated that the type of cells which are chosen to be cultured will depend on the nature of the in vivo biological system, e.g. cellular barrier, which is to be modelled in the device. In the case of the blood brain barrier, the cells may include endothelial cells, and optionally also astrocytes and/or pericytes. Such a model may also be useful to screen for substances which have efficacy in treating Alzheimer’s disease, Parkinson’s disease and other neurodegenerative diseases.

[154] In the case where the device will be used to study transport across a blood tumour barrier, the cells may include tumour cells, optionally together with endothelial cells, and further optionally also with astrocytes and/or pericytes. Endothelial cells are also required for the formation of blood-retina and blood-thymus barriers.

[155] Epithelial cells can be chosen to model cellular barriers of the skin, respiratory tract, and gastrointestinal tract, as well as modelling the blood-placenta and blood-testis barriers. Mesothelial cells can be chosen to model cellular barriers of the pleural cavity, peritoneal cavity, or pericardial cavity.

[156] The aforementioned cells can be obtained from commercial sources or extracted from relevant tissue, as would be known to those skilled in the art. Accordingly, cells suitable to simulate a wide variety of cellular barriers or other biological systems may thus be cultured, and the present invention is therefore not limited to those cell types and cellular barriers described above.

[157] The cells may be introduced to the vessel in a fluid such as cell culture medium, in one or more seeding stages, and the device may be positioned in several orientations to facilitate seeding of different portions of the walls. The cells may then be incubated in the device at a suitable temperature, such as about 37°C, for a time sufficient to culture the cells.

[158] The cell culture medium may be any medium which is capable of maintaining the viability and/or growth of a cell. Exemplary media include those available from commercial sources such as ThermoFisher Scientific (Gibco Cell Culture Media), HyClone™ Cell Culture Media, and Sigma Aldrich (such as DMEM - Dulbecco's Modified Eagle Medium, DMEM/F12, Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, Flam's F-10 Nutrient Mixture, Flam's F-12 Nutrient Mixture, Media 199, MEM, Minimum Essential Media RPMI Medium 1640, Advanced Media, Opti-MEM I Reduced Serum Media IMDM, Iscove's Modified Dulbecco's Medium, Gibco Cell Culture Bags, FluoroBrite DMEM Media). Suitable cell culture media is also available from STEMCELL™ Technologies (mTeSR™ Plus, mTeSR™, mTeSR™1 , MethoCult™ H4034 Optimum, BrainPhys™ Neuronal Medium, IntestiCult™ Organoid Growth Medium (Mouse), AggreWell™ EB Formation Medium, Agar Leukocyte Conditioned Medium), LONZA (media OGM™, Osteoblast Growth Medium SingleQuots™ Supplements and Growth Factors, EGM™ -2 MV, Microvascular Endothelial Cell Growth Medium-2 BulletKit™, EGM™ Endothelial Cell Growth Medium BulletKit™, EGM™-MV Microvascular Endothelial Cell Growth Medium BulletKit™, MEGM™ Mammary Epithelial Cell Growth Medium BulletKit™, MBM™-4 Melanocyte Growth Basal Medium-4, LGM-3™ Lymphocyte Growth Medium-3, FGM™ Fibroblast Growth Medium BulletKit™, FGM™-2 Fibroblast Growth Medium-2 BulletKit™, PGM- 2™ Preadipocyte Growth Medium-2 BulletKit™, KGM™ Gold Keratinocyte Growth Medium BulletKit™, tEGM™ Retinal Pigment Epithelial Cell Growth Medium BulletKit™, B-ALI™ Bronchial Air-Liquid Interface Medium BulletKit™, SAGM™ Small Airway Epithelial Cell Growth Medium BulletKit™, KGM™-CD Keratinocyte Growth Medium BulletKit™- Chemically Defined, SCGM™ Stromal Cell Growth Medium BulletKit™, KGM™-2 Keratinocyte Growth Medium-2 BulletKit™, Calcium Free, AGM™, Astrocyte Growth Medium BulletKit™, MsGM™ Mesangial Cell Growth Medium BulletKit™, ABM™ Astrocyte Basal Medium, SkGM™-2 Skeletal Muscle Cell Growth Medium-2 BulletKit™, BEGM™ Bronchial Epithelial Cell Growth Medium BulletKit™, PrEGM™ Prostate Epithelial Cell Growth Medium BulletKitT^M, SmGM™- 2 Smooth Muscle Cell Growth Medium -2 BulletKit™ etc) and PromoCell (Endothelial Cell Growth Medium, Fibroblast Growth Medium, Adipocyte Nutrition Medium, Airway Epithelial Cell Growth Medium, Chondrocyte Growth Medium, Keratinocyte Growth Medium, Mammary Epithelial Cell Growth Medium, Melanocyte Growth Medium, Osteoblast Growth Medium, Pericyte Growth Medium, Skeletal Muscle Cell Growth Medium, Smooth Muscle Cell Growth Medium, Small Airway Epithelial Cell Growth Medium, etc).

[159] In some embodiments, the cells are cultured under conditions of hydrodynamic shear flow. Cell culture medium may thus be flowed through the vessel to provide the required shear stress, for example for a time period of up to several days. It is believed that shear conditions representative of blood flow may facilitate the culturing of a cell barrier which accurately models an in vivo blood-tissue barrier. [160] The culturing of cells according to these methods may produce a cellular barrier which effectively separates the vessel and the chamber. Any transmission of substances from a fluid in the vessel to a fluid in the chamber, via the microchannels, must therefore occur through the cellular barrier. Referring again to the embodiment depicted in Figure 4, cells may be cultured on the immobilised ECM proteins 524 present on the walls of vessel 546. We have found that a co-culturing endothelium, astrocyte and pericyte cells on the immobilised ECM proteins in a flow of cell culture medium through the vessel produces a cellular barrier which accurately models the in vivo blood brain barrier with respect to its ability to regulate transport of a variety of substances across the barrier.

Investigating transport of substances across the cellular barrier

[161] As described herein, in some embodiments the use of the microfluidic device is to investigate transport of substance(s) across a cellular barrier, and in this case the microfluidic device may comprise a chamber to receive the substances transported from the vessel and microchannels between the vessel and chamber to provide fluid communication. Once the cellular barrier has been cultured, as described above, the microfluidic device is ready for use in such investigations. A fluid containing the substance or substances under investigation, typically at a known concentration, is thus conveyed into the vessel of the microfluidic device. The vessel may simply be filled with the fluid, or the fluid may be flowed through the vessel to maintain a constant concentration of the substance or to more accurately simulate an in vivo system, such as blood flowing along a blood-tissue barrier.

[162] In some embodiments, the transportation of the substance(s) across the cellular barrier is assessed by determining an amount of the one or more substances transported across the cellular barrier, typically after a predetermined time so that a rate of permeation can be calculated. This may be done by measuring the amount of the substance(s) present in a second fluid contained in the chamber, or the amount of the substance(s) remaining in the first fluid. The second fluid present in the chamber may be an aqueous fluid such as cell culture medium or HBSS.

[163] A wide variety of techniques may be used to determine the amount of the substances in either fluid. Such techniques include high-performance liquid chromatography (HPLC), fluorescence reader (for fluorescent substances), beta- scintillation counters (for radiolabelled substances) and confocal fluorescence microscopy. The fluid may be withdrawn for analysis after a predetermined time. Alternatively, the amounts can be determined in situ, for example with a fluorescence microscope, allowing the transportation of the substance(s) to be observed in real time.

[164] In some embodiments, the transportation of the substance(s) across the cellular barrier is assessed by observing an effect attributable to the one or more substances in the chamber of the microfluidic device. The effect may, for example, be the effect of the substance on cells cultured in the chamber, such as cancer cells. The effect on cells can be observed by in situ imaging using a number of approaches. For example, cell apoptosis/death or other cell fate can be monitored via cell staining techniques using specific fluorescent dyes, as disclosed for example by Peng et al ( ChemBioChem 2018, 19, 986 - 996). Thus, the effectiveness of therapeutics in treating diseases of the central nervous system or other organs can be directly investigated in a manner which accounts for potential difficulties in traversing the blood- tissue barrier.

[165] The use according to the invention may thus further include a step of culturing cells in the chamber of the microfluidic device. Cells such as glioblastoma cells may be cultured in the chamber in Matrigel, optionally using cell growth medium present in the compartment to support the cell growth. The walls of the chamber may be coated with the same polymeric coating as present in the vessel, so that cell culturing can also be promoted in the chamber according to the principles disclosed herein.

[166] It is known that tissue barriers such as the blood brain barrier (BBB) regulate the transport of different substances by a variety of mechanisms. The BBB acts as a physical barrier, with the complex tight junctions between endothelial cells forcing most molecular traffic to take a transcellular route across the BBB. The effectiveness of the tight junctions in the cultured cellular barrier to perform this function can be assessed by measuring the transportation of substances having a known permeability across the BBB. We have thus demonstrated the physical integrity of cellular barriers produced according to the invention using substances such as caffeine, sucrose and dextran. [167] Transcellular transport across the BBB includes both passive and positive mechanisms. The large surface area of the lipid membranes of the endothelium allows passive diffusion of lipid-soluble agents, such as caffeine. Transport proteins/carriers located on the surface of the endothelial cell membranes positively convey other substances, such as glucose, amino acids and nucleotides across the BBB. Other transport proteins/carriers will also efflux unwanted substrates back into the lumen, for example rhodamine. Receptor-mediated transcytosis is another positive transport route whereby certain peptides and proteins, such as transferrin, are actively transferred across the BBB. We have demonstrated the effectiveness of cellular barriers produced according to the invention in modelling the transport of substances known to be subject to these various mechanisms.

[168] The invention is thus considered useful for a wide range of substances of interest, including but not limited to pharmaceutical compounds or other therapeutics, exosomes, nanomicelles, nanoparticles, toxins, small molecules, nucleic acids (including nucleic acid vectors), oligonucleotides (including antisense oligonucleotides), oligopeptides, proteins, ribozymes, small interfering RNAs, microRNAs, short hairpin RNAs, aptamers, viruses, and antibodies or antigen binding parts thereof. These examples are non-limiting and other substances are contemplated.

[169] In some embodiments, the microfluidic devices are used to investigate the effect of various materials, such as toxins, pathogens (both virus and bacteria) or metastatic cancer cells, on the effectiveness of the cellular barrier. Accordingly, the use may comprise a step of exposing the cellular barrier to these materials before or while transporting a substance (for example a substance of known permeability in a healthy BBB) across the cellular barrier.

[170] One application of the invention is in high throughput screening studies, and accordingly arrays of similar microfluidic devices, optionally constructed in a single unitary body, may be used to simultaneously investigate transport of different substances or different cellular barriers.

Other uses

[171] In other embodiments the microfluidic device is used to conduct one or more of a wide range of investigations of interactions between internally cultured cells and one or more substances. Such interactions to be investigated may include, but are not limited to, an effect of the substance(s) on one or more of: cell attachment, cell growth, cell survival, cell differentiation, cell apoptosis/death and multicellular structure. The activity or interaction of substances on cell motility, cell migration, cell to cell interactions as well as cell-protein signal interactions are also important activities that can be studied using this microfluidic device.

[172] Substances of interest in such investigations may include, but are not limited to, pharmaceutical compounds or other therapeutics, exosomes, nanomicelles, nanoparticles, toxins, small molecules, nucleic acids (including nucleic acid vectors), oligonucleotides (including antisense oligonucleotides), oligopeptides, proteins, ribozymes, small interfering RNAs, microRNAs, short hairpin RNAs, aptamers, viruses, and antibodies or antigen binding parts thereof. These examples are non-limiting and other substances are contemplated.

[173] The effect on cells can be observed by in situ imaging using a number of approaches. For example, cell apoptosis/death or other cell fate can be monitored via cell staining techniques using specific fluorescent dyes, as disclosed for example by Peng et al ( ChemBioChem 2018, 19, 986 - 996). Thus, the effectiveness of therapeutics in treating diseases of the central nervous system or other organs can be directly investigated under conditions which accurately mimic the in vivo microenvironment.

EXAMPLES

[174] The present invention is described with reference to the following examples. It is to be understood that the examples are illustrative of and not limiting to the invention described herein.

Example 1. Synthesis of N-(4-benzophenyl)acrylamide and N-(4- benzophenvDacrylamide (BPAm) homopolymer (1)

[175] 4-Aminobenzophenone (5.0 g, 25.4 mmol) and triethylamine (4.25 mL, 30.5 mmol) were added to a round-bottom flask and dissolved in dichloromethane (40 mL) on ice. Acryloyl chloride (2.3 mL, 27.9 mmol) in dichloromethane (10 mL) was then added dropwise to the stirred solution. The reaction mixture was allowed to warm to room temperature and left to stir for 18 h in the dark. For the remainder of the work up, the fumehood light was turned off and only laboratory lighting was used. The reaction mixture was diluted with dichloromethane (50 ml_) and washed with 1 M HCI (3 x 120 ml_), deionized water (1 x 150 ml_) and saturated brine (1 c 150 ml_). The organic phase was dried over MgSC , filtered and the solvent removed under reduced pressure to afford an orange powder. The monomer was stored at 4 °C in the dark. Yield = 5.90 g (93%).

[176] The N-(4-benzophenyl)acrylamide product (BPAm) was characterised with 1 H NMR (400 MHz, CDCI3) d/ppm: 8.28 (1 H, br s, NH) 7.80 (2H, dt, J = 8.8 Hz, 2.1 Hz, Ar CH meta- to acrylamide), 7.77-7.72 (4H, m, Ar CH ortho- to acrylamide and Ar CH ortho- to carbonyl of benzophenone), 7.57 (1 H, tt, J = 6.9 Hz, 1 .4 Hz, 1 H, Ar CH para- to carbonyl of benzophenone), 7.47 (2H, tt, J = 7.7 Hz, 1 .5 Hz, Ar CH meta- to carbonyl of benzophenone), 6.47 (1 H, dd, J = 16.9 Hz, 1 .6 Hz, CH=CH2), 6.34 (1 H, dd, J = 16.8 Hz, 9.7 Hz, CH=CH2 cis to NHAr), 5.78 (1 H, dd, J = 10.1 Hz, 1 .4 Hz, CH=CH2 trans to NHAr).

[177] The sterically hindered homopolymer of BPAm was found difficult to produce by RAFT polymerisation, so a different approach was followed from the examples that follow. Working within a nitrogen filled glovebox, 100mg of BPA was dissolved in 2ml of nitrogen purged dioxane to which was added 2mg azobisisobutyronitrile (AIBN) dissolved in 1 ml of nitrogen purged dioxane. This was further purged for l Omins with nitrogen before sealing and heating at 70C for 24h, with the reaction vessel wrapped in aluminium foil. The contents were then diluted with further dioxane to allow transfer into dialysis tubing (molecular weight cutoff 3.5-5.0 kDa) and dialysed against dioxane before precipitation into diethyl ether. The product was obtained by removing the solvent under reduced pressure.

[178] Homopolymer (1 ) of BMAm having the structure shown below, was characterised with 1 H NMR (400 MHz, CDCI3) d/ppm: 8.32-6.17 (br m, Ar), 3.40-1.02 (CH2 and CH of backbone). Example 2. Synthesis of copolymer (2) containing 5 mol% N-(4- benzophenvDacrylamide and 95 mol% 2-(N-3-sulfopropyl-N,N-dimethyl ammoniurrOethyl methacrylate

[179] A copolymer was made from 5 mol% N-(4-benzophenyl)acrylamide and 95 mol% 2-(N-3-sulfopropyl-N,N-dimethyl ammonium)ethyl methacrylate using reversible addition-fragmentation chain-transfer (RAFT) polymerisation. Working within a glovebox providing a nitrogen atmosphere, 2-(N-3-sulfopropyl-N,N-dimethyl ammonium)ethyl methacrylate (3.0 g, 10.7 mmol), N-(4-benzophenyl)acrylamide (135 mg, 0.54 mmol), RAFT agent 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl) pentanoic acid (17 mg, 0.11 mmol) and initiator 4,4'-azobis(4-cyanovaleric acid) (3 mg, 0.011 mmol) were dissolved in 20 ml_ of a nitrogen purged solution containing 75% (v/v) DMF and 25% (v/v) water. The mixture was then further purged with nitrogen for 10 min, sealed and the reaction was stirred for 20 h at 70 °C. The product was purified by dialysis against 75% (v/v) DMF and 25% (v/v) water (molecular weight cutoff 3.5-5.0 kDa) and the soluble material recovered by lyophilisation to yield a yellow powder. The polymer was kept at 4 °C in the dark for storage. The reaction aimed to achieve a molecular weight of approx. 30 kDa.

[180] The polymer was characterised by 1 H NMR (400 MFIz, D20) d/ppm: 8.24- 7.69 (br m, Ar of BPAm), 4.41 (br s, OCH2CH2N of DMAPS), 3.71 (br s, OCH2CH2N of DMAPS), 3.49 (br s, N(CH3)2CH2CH2CH2S03 of DMAPS), 3.14 (N(CH3)2 of DMAPS), 2.88 (br s, N(CH3)2CH2CH2CH2S03 of DMAPS), 2.18 (br s, N(CH3)2CH2CH2CH2S03 of DMAPS), 2.07-1.42 (CH2 and CH of backbone), 1.42- 0.56 (CFI3 of backbone); and FT-IR (neat) v/ cm-1 : 2980 (C-FI stretch), 1700 (C=0 stretch), 1155 (S=0 stretch), 1035 (C-0 stretch). The structure of the synthesized polymer (2) is shown below:

Example 3. Synthesis of copolymer (3) containing 4 mol% N-(4- benzophenvDacrylamide and 96 mol% qlvcidyl methacrylate

[181] A copolymer was made from 5 mol% N-(4-benzophenyl)acrylamide and 95 mol% glycidyl (meth)acrylate following a RAFT-polymerisation method as described in Example 2. NMR analysis indicated approximately 4 mol% incorporation of N-(4- benzophenyl)acrylamide in the final polymer. The structure of the synthesized polymer (3) is shown below:

Example 4. Synthesis of copolymer (4) containing 10 mol% N-(4- benzophenvDacrylate and 90 mol% acrylic acid, and activated with N- hvdroxysuccinimide

[182] Working within a glovebox providing a nitrogen atmosphere, 4-benzophenyl acrylate (BPAc; commercially available) (1.75 g, 6.9 mmol), RAFT agent 4-cyano-4- (ethylsulfanylthiocarbonylsulfanyl) pentanoic acid (53 mg, 346 pmol) and initiator 4,4'- azobis(4-cyanovaleric acid) (10 mg, 35 pmol) were placed in a clean 50 ml flask to which 15 ml of nitrogen purged dioxane was added. To this mixture, distilled acrylic acid (AAc) (5.0 g, 69 mmol) was added using a further 5 ml of dioxane to complete the transfer. Following further nitrogen purging for 10 minutes, the flask was sealed and incubated at 70 °C for 24 h keeping the sample wrapped in aluminium foil. The contents where then diluted with further dioxane to allow transfer into dialysis tubing (molecular weight cutoff 3.5-5.0 kDa) and dialysed against dioxane before precipitation into diethyl ether. The product was obtained by removing the solvent under reduced pressure.

[183] The copolymer was characterised with 1 H NMR (400 MHz, DMSO) d/ppm: 12.91 -11 .46 (br,s OH of AAc), 7.90-7.13 (br m, Ar BPAc), 2.86-1 .17 (CH2 and CH of backbone). Peak integration indicates that the resultant BPAc/AAc ratio is similar to the feed ratio.

[184] To convert the carboxylic acid groups in the copolymer into activated ester groups, a round bottom flask containing the above dried copolymer (1.0 g) and dimethylformamide (DMF, 10 ml, dried over molecular sieve) was allowed to stir overnight to ensure complete dissolution of the copolymer whilst excluding moisture. Subsequently, 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 3.2 g, 1 .2 equiv.), N-hydroxy succinimide (NHS, 1.9 g, 1.2 equiv.) and 4-dimethylaminopyridine (DMAP, 0.1 equiv.) was added with a further 6 ml of dry DMF. The compounds developed a deep orange colour with overnight stirring. The NHS activated polymer was obtained by washing with anhydrous ethanol three times, precipitation into diethyl ether and removing the solvent under reduced pressure.

[185] The polymer was characterised by 1 H NMR (400 MHz, molecular sieve dried CD3CN) and it was found that the carboxylic acid peak was no longer present, indicating that full conversion had taken place. The structure of the synthesized copolymer (4) is shown below:

Example 5. Synthesis of terpolymer (5) containing 5 mol% N-(4- benzophenyhacrylamide, 20 mol% acrylic acid and 75 mol% acrylamide [186] Working within a nitrogen filled glovebox, N-(4-benzophenyl)acrylamide (1.04 g, 4.1 mmol), acrylamide (4.41 g, 61.3 mmol), RAFT agent 4-cyano-4- (ethylsulfanylthiocarbonylsulfanyl) pentanoic acid (53 mg, 346 pmol) and initiator 4,4'- azobis(4-cyanovaleric acid) (11 .6 mg, 41 pmol) were placed in a clean 100 ml flask to which 20 ml of nitrogen purged dioxane was added. After the solids were fully dissolved, distilled acrylic acid (1.18 g, 16.6 mmol) was added using a further 5 ml of dioxane to complete the transfer. Further purging for 10 minutes was carried out and the flask was sealed. Pleating of the contents at 70 °C was then done for 24 hours keeping the sample dark by wrapping in foil. The contents where then diluted with further dioxane to allow transfer into a dialysis tubing (molecular weight cutoff = 3.5-5 kDa) and dialysed against dioxane before precipitation into diethyl ether. The reaction aimed to achieve a molecular weight of approx. 19 kDa.

[187] The terpolymer was characterised with 1 FI NMR (400 MFIz, DMSO) d/ppm: 12.66-11 .53 (br,s OH of AAc), 10.40-9.78 (br, s, NH BPAm), 7.85-7.42 (br m, Ar BPAm), 7.42-6.37 (br, s, NH2 from AAm) 2.86-0.77 (CH2 and CH of backbone). Peak integration indicates that the resultant ratio of polymers is similar to the monomer feed ratio. The structure of the synthesized polymer (5) is shown below:

Example 6. Modification of polymer surfaces with solutions containing copolymer (2)

[188] Cyclic olefin copolymer (COC) samples were cut to size and subjected to cleaning in a surfactant solution (1% (v/v) RBS-35, Pierce, Rockford, IL, USA) with ultrasonication for 30 min, followed by thorough rinsing with Milli-Q™ water. A cavity was then produced by placing an 8 mm diameter O-ring onto a COC substrate. Quartz glass plates were then placed on top of this cavity and underneath the COC substrate. Subsequently, Milli-Q™ water or a polymer dissolved in Milli-Q™ water were injected using a syringe to completely fill the cavity. The polymer used in these experiments was copolymer (2). Polymer concentrations of either 5 mg/ml or 20 mg/ml in Milli-Q™ water were used. Samples were then exposed to a high power UV source (Fusion UV Systems) for 10 s. After exposure, the COC samples were washed over a minimum of 24 h in Milli-Q™ water to remove any non-covalently bound polymer. After drying the samples in air, the water contact angle was then measured on the modified COC substrate. The results are shown in Figure 5.

[189] The results demonstrate that surface immobilisation of benzophenone- based polymers can be achieved on polymer substrates using this method. Untreated COC polymer samples (88°) and surfactant cleaned COC samples (96°) became more hydrophilic after UV irradiation in the presence of solutions of copolymer (2). Moreover, the experiments demonstrate that the resulting contact angle is dependent on the polymer concentration in solution, with a concentration of 20 mg/ml of polymer providing a more hydrophilic surface (11 °) compared to 5 mg/ml (45°). Importantly, the contact angle obtained on the COC surface was not changed after being exposed to 20 mg/ml of the polymer solution without UV irradiation (95°).

Example 7. Fabrication of microfluidic device structure

[190] Microfluidic devices were fabricated via a conventional two-step photolithography technique, as schematically depicted in Figure 6. In the specific design used in subsequent Examples, the microfluidic device had the geometry depicted in Figure 7, including eight separately operable (i.e. unconnected) arrays of microchannel-connected blood / brain / medium channels in a single PDMS body, to facilitate high throughput experiments.

[191] Direct write lithography masks were designed using L-Edit software and fabricated. As seen in Figure 6, one mask 310 had two arrays of about 100 rectangular transparent features 312 of 500 pm length and feature separation of 50 pm. The features in one array 314 had a thickness of 50 pm and those in the other array 316 had a thickness of 3 pm. The second mask 320 had parallel main channel features 322, 324 and 326 to form the blood, brain and medium channels respectively.

[192] In a first fabrication step, permanent negative epoxy photoresist (SU-8 3005, Microchem) was spin-coated at 4,000 rpm onto a silicon wafer 330 to achieve a coating 332 with thickness of 3 pm. It was then exposed to UV light 334 (90 mJ/cm², EVG 6200 Mask Aligner) through the first mask 310, baked on a hot plate at 95°C for 3 min, and processed with developer to generate the first layer of photoresist 336. The arrays of features in this layer eventually formed the microchannels connecting the main channels. In a second fabrication step, the silicon wafer with crosslinked first layer of photoresist thereon was spin-coated with a thicker second layer 340 of photoresist at 1 ,000 rpm to 100 pm in height, and baked at 95°C for 45 min. The second mask 320 was then aligned perpendicularly to the array of features in the first layer. The wafer was exposed to UV light 342 (250 mJ/cm², EVG 6200 Mask Aligner, EV Group) through the mask, baked (3 min), and processed with developer to generate the second layer of photoresist 344. The resulting blood channel 346 and brain channel 348 features were connected by the 3 pm width microchannel features, while the brain channel and medium channel 350 features were connected by the 50 pm width microchannel features. The microstructured silicon wafer mold 352 was then further protected with silanization using trichloro(1 H,1 H,2H,2H-perfluorooctyl)silane (Sigma-Aldrich) under vacuum condition overnight.

[193] A poly(dimethyl siloxane) (PDMS, Dow Chemical) replica was obtained by casting a mixture (360) of PDMS prepolymer and curing agents (10:1 ) over the wafer mold 352 and baking at 80°C for 1 h. The cured PDMS replica 362 was then peeled from the mold. A 1 mm hole puncher (ProSciTech Pty. Inc.) was used to create flow ports 364 at each end of the blood and medium channels and one end of the brain channel, and a 2 mm hole puncher (ProSciTech Pty. Inc.) was used to create a larger reservoir 366 at the other end of the brain channel. The PDMS replica was then irreversibly sealed to a rectangular glass slide 370 (Deckglaser) of length and width of 24 mm x 50 mm via a brief treatment with oxygen plasma (40 s) in a plasma cleaner (Harrick Plasma) to form the precursor microfluidic blood-brain barrier (pBBB) device 380.

[194] Scanning electron microscopy (SEM) was used to characterize the dimensions of the pBBB device, including the microchannels. Images were captured with a Zeiss Sigma FESEM (Field Emission Scanning Electron Microscope). The images confirmed that the dimensions of the device were 3 and 50 pm width for the interconnecting microchannels, 80 pm for the distance between each main channel and 500 mίti for the width of each main channel, as seen in Figure 7. This demonstrated the successful fabrication of the mBBB chips.

Example 8. Preparation of cross-linked polymeric coatings on walls of the channels

[195] To produce the polymer-coated microfluidic device, a solution of the selected copolymer (2 wt%) in DMSO (10 pL) as produced in Examples 1 to 5 was injected inside each of the microfluidic main channels using a pipette. The injected copolymer was then crosslinked under broad spectrum UV irradiation through the PDMS body for 20 s (34 J/cm²). The microfluidic channels were then washed with DMSO and ethanol subsequently and dried with nitrogen. It was estimated on the basis of X-ray photoelectron spectroscopy (XPS) analysis that the thickness of the polymeric coatings was 3 to 5 nm.

[196] The polymer-coated devices thus produced are designated with the following device numbers: 8-1 having a coating of homopolymer (1 ) [containing N-(4- benzophenyl)acrylamide as the only monomer], 8-2 having a coating with copolymer (2) [containing 5 mol% N-(4-benzophenyl)acrylamide and 95 mol% 2-(N-3-sulfopropyl- N,N-dimethyl ammonium)ethyl methacrylate)], 8-3 having a coating with copolymer (3) [4 mol% N-(4-benzophenyl)acrylamide and 96 mol% glycidyl methacrylate], 8-4 having a coating with copolymer (4) [containing 10 mol% 4-benzophenyl acrylate and 90 mol% acrylic acid activated with N-hydroxysuccinimide)], and 8-5 having a coating with copolymer (5) [containing 5 mol% N-(4-benzophenyl)acrylamide, 20 mol% acrylic acid and 75 mol% acylamide].

Example 9. Small molecule functionalisation of walls of the channels

[197] A previously reported silane-based surface functionalisation method was investigated as an alternative to the method described in Example 8. A fabricated microfluidic device as prepared in Example 7 was treated with oxygen plasma (30 s). This was immediately followed by the injection of 10% (v/v) of either 3-aminopropyl- trimethoxysilane (APTMS, Sigma-Aldrich) or 3-aminopropyl-trimethoxysilane (APTES) in ethanol into each of the main channels. The solution was incubated in the microfluidic device for 15 min at room temperature. The microfluidic channels were then flushed with ethanol, washed with water and subsequently ethanol and dried in an oven at 80 °C for 2 h. The silanised surface was then further modified to provide aldehyde surface functionality by incubating with 2.5% glutaraldehyde (Merck). After incubating for 15 min, the channels were washed with water and ethanol and dried at 80 °C for 2 h. The aldehyde functional groups are reported to conjugate proteins by condensation reactions with amine moieties to form Schiff bases.

[198] The surface-modified devices thus produced are designated as device 9-s1 and 9-s2, having a small molecule surface modification in the channels via APTMS- glutaraldehyde and APTES-glutaraldehyde respectively.

Example 10 (comparative). Plasma activation of walls of the channels

[199] A previously reported plasma activation method of modifying the microchannel surfaces was investigated as an alternative to the method described in Example 8. The fabricated microfluidic chips were treated with oxygen plasma (30 s). This method is reported to provide activated sites on PDMS and glass surfaces capable of electrostatic interactions with proteins, thus physically absorbing proteins on the walls.

[200] The surface-modified device thus produced is designated as device 10-p, having a plasma-treated surface modification in the channels.

Example 11. Immobilisation of amine-containing ECM-proteins and dyes on the walls of the channels

[201] The protein immobilisation efficiency of the wall-modification methods in devices 8-1 , 8-4, 9-s1 , 9-s2 and 10-p was investigated using either cyanine 5 (Cy5) amine (Lumiprobe, 430C0) or Cy5 labeled collagen (Collagen-Cy5), prepared by labelling rat tail collagen I (Sigma-Aldrich) with Cy5 by adding 0.9 equiv. of Cy5 NHS ester (Lumiprobe, 43020) in PBS for 1 h at RT. The resulting Collagen-Cy5 was directly used without further purification.

[202] Cy5-amine (10 mM) in PBS or Collagen-Cy5 (25 pg/ml) was injected into the channel of the microfluidic devices and incubated under dark at RT for 1 h. This was followed by a thorough wash with DMSO (in the case of Cy5-amine) or PBST (in the case of Collagen-Cy5) three times. The treated devices were further washed with PBS once and examined under confocal microscope. The corresponding fluorescence intensities of each device were further analysed and quantified using ImageJ (NIH, Bethesda, MD) to explore the functionalisation and coating efficiency.

[203] On device 9-s1 (APTMS-glutaraldehyde modification), the Collagen-Cy5 was unevenly adhered to the channel walls and it was observed under high magnification that the microchannels connecting the blood channel and the brain channel were blocked. On device 10-p (plasma treatment modification) a thick and even coating of Collagen-Cy5 was formed on the PDMS surface, but a thin uneven coating was formed on the glass surface. On device 8-4 (photoactivated polymer coating functionalised with NHS-ester according to the invention), an even coating was formed on the walls of the blood channel, the microchannels and the brain channel, and it was apparent under high magnification that the microchannels were not blocked and thus continued to provide fluid communication between the two channels.

[204] The measured fluorescence intensity (in relative fluorescence units, RFU) after treatment with Cy5-amine dye is shown in Figure 8 for device 8-4 (photoactivated polymer coating functionalized with NFIS-ester), device 8-1 (photoactivated coating lacking protein-conjugating functional groups), 9-s1 (APTMS-glutaraldehyde modification), 9-s2 (APTES-glutaraldehyde modification) and 10-p (plasma treatment modification). Device 8-4 provided the best amine capture and immobilization capability. The effect of the amine-reactive NFIS-ester groups on the polymer coating is seen by comparing the results of devices 8-4 (polymer coating functionalized with NFIS-ester) and 8-1 (polymer coating lacking amine-reactive functionality). Device 8-4 also significantly outperformed devices 9-s1 and 9-s2 which had surfaces modified with small-molecule aldehyde functionality, and vastly exceeded the performance of device 10-p having a plasma-activated surface.

Example 12. Immobilisation of antibodies on the walls of the channels

[205] Further studies to simulate protein immobilisation were then conducted to investigate the effectiveness of different conjugating groups. Alex488 anti-rabbit antibody (ThermoFisher, 20 pg/ml) was injected into the channels of the microfluidic devices and incubated under dark at RT for 1 h. This was followed by a thorough wash with PBST (three times). The treated devices were further washed with PBS once and examined under confocal microscope. The corresponding fluorescence intensities were analysed and quantified using ImageJ (NIFH, Bethesda, MD). The measured fluorescence intensity is shown in Figure 9 for devices coated with different photoactivated polymeric coatings, namely device 8-4 (coating functionalized with NHS-ester conjugating groups), device 8-3 (coating functionalized with terminal epoxide groups), device 8-2 (coating functionalized with zwitterionic 2-(N-3-sulfopropyl- N,N-dimethyl ammonium)ethyl groups and device 8-5 (coating functionalised with acid functional groups).

[206] The polymeric coatings comprising functional groups configured for facile conjugation to native proteins via covalent bond formation with amines (i.e. NHS-ester and epoxy groups) provided significant improvements in their capacity to immobilise the antibodies, compared to coatings with functional groups capable only of electrostatic interactions. The best results were provided by the NHS-ester functional groups.

Example 13. Immobilisation of ECM-proteins and subsequent cell culture on the walls of the channels

[207] Prior to use in cell culture assays, surface-modified microfluidic devices 8-4 were sterilized within a laminar hood (ThermoFisher) built-in UV (l= 256 nm) for 1 h. After sterilization, the microfluidic devices were immediately incubated with ECM- proteins (MatriGel solution in PBS containing laminin as a major component, collagen type IV, heparin sulfate proteoglycan, entactin, and other minor components; 25 pg/ml, Sigma-Aldrich) for 1 h at room temperature.

[208] The resultant device, adapted for culturing of cells, is schematically depicted in Figure 4. The blood channel (546) and the brain channel (548) formed in the unitary PDMS body (510) and enclosed by the glass coverslip (570) are in fluid communication via a plurality of 3 gm-dimension microchannels (512), while the brain channel and medium channel (550) are in fluid communication via larger, 50 pm-width / 3 pm height, conduits (514). The walls of the blood channel are coated with a cross-linked polymeric coating (520) formed (as described in Example 8) by photoactivation of benzophenone groups on the precursor polymer and reaction of the resultant activated species with each other (to form cross-links) and with surface functionalities (522), believed to be C- H bonds, on the PDMS and glass walls. The ECM-proteins, when contacted with the polymeric coating, conjugate by reaction between the polymer’s NHS-ester functional groups and amine moieties on the proteins, thus forming covalent amide bonds which immobilise the proteins (524) on the walls of the blood channel. The blood channel is thus adapted for cell culturing to simulate the BBB on its walls.

[209] Immortalized human brain microvascular endothelial cells (hCMEC/D3, passages: 3-10; Merck) were maintained in Endothelial Cell Growth Basal Medium-2 (EBM-2, Lonza) with supplemented basic fibroblast growth factor (bFGF). Immortalized human astrocytes, fetal-hTERT (passages: 3-8; Applied Biological Materials) and human immortalized pericyte (passages: 3-8, Celther) were maintained in DMEM (Gibco, ThermoFisher) medium with N2 supplement (17502048, ThermoFisher) and DMEM medium only, respectively. All the cells were cultured on T25 tissue culture flasks coated with collagen type I (10 pg/ml, rat tail, Sigma-Aldrich).

[210] Before seeding cells, the surface functionalized microfluidic devices were equilibrated in cell culture medium for 1 h at 37°C. The astrocytes and pericytes were first detached from the cell culture flasks and resuspended in the culture medium at cell concentrations of 1 x 10⁶ and 5 x 10⁵ cells/ml, respectively. The cell mixture suspension was then injected into the blood channel of the devices. The loaded astrocytes and pericytes cells adhered to the upper wall surfaces (PDMS) of the blood channel when the chips were placed in an inverted position for 1 h in a cell culture incubator at 37°C supplied with 5% CO2.

[211] After overnight incubation, the microfluidic devices were then injected with endothelial cells (ECs). ECs were detached from the cell culture flask and resuspended in the culture medium at a concentration of 6 x 10⁶ cells/ml and injected into the blood channel. The injected ECs adhered to the upper wall surfaces (PDMS) of the blood channel when the chips were placed in an inverted position for 1 h in a cell culture incubator at 37°C supplied with 5% CO2. After the initial attachment to the upper surface, freshly isolated cells at the same concentration were gently reinjected and then attached to the lower (glass) surface at an upright position for another 1 h.

[212] After 24 h incubation, the blood channel was subjected to a prescribed physiological hydrodynamic shear flow of cell culture medium (EBM-2) for 72 h at 2 pl/min (shear stress: 38 mPa) to form a simulated blood-brain barrier (BBB). Thus, three types of BBB cells were co-cultured in the brain channel to better mimic the spatial configuration of cells and the resulting interactions which affect transport pathways across the BBB.

[213] The device was rinsed with PBS that has been warmed to 37 °C, and the cells were then fixed with formaldehyde (3.7 %) in PBS at room temperature for 10 min. The cells were then rinsed three times with PBS, followed by staining with TRITC- phalloidin (10 pg/ml) in PBS for 40 min at RT. The labelled cells inside the blood channel were rinsed three times with PBS prior to imaging with confocal microscopy. According to the confocal imaging, the cultured BBB cells formed a homogenous and 3D vessel-like structure along the blood channel, covering both the PDMS and glass portions of the walls. Thus, materials transported from the blood channel to the brain channel must cross the simulated BBB. Moreover, the astrocytes and pericytes were observed on top of the endothelium cells, replicating the crosstalk between different cells believed to be important to the function of the BBB.

[214] It is believed that the consistent shear stress during culturing enhanced the expression of tight junction proteins and formed tight boundaries between each cell. This was supported by contrasting the observed results against those for cells cultured in a similar device for the same period of time, but under static conditions.

Example 14. Permeability studies

[215] Prior to the permeability testing, cells were cultured for three to four days to form a simulated BBB on the walls of the blood channel in microfluidic devices 8-4 as described in Example 13.

[216] Test analytes for the permeability testing included fluorescein isothiocyanate (FITC)-dextrans (Sigma-Aldrich) of molecular weight of 3 and 10 kDa at 50 pg/mL; caffeine at 100 mM; nitrofurantoin at 100 pM, sucrose [¹⁴C] at 0.1 pCi/mL (PerkinElmer, 2812793), D-glucose [¹⁴C] at 0.1 pCi/mL (PerkinElmer, 2389266), alanine-L [³H] at 1 pCi/mL (PerkinElmer, 2390162). These analytes were either dissolved in cell culture medium (dextran, caffeine, nitrofurantoin, sucrose and glucose) or in HBSS buffer (alanine) prior to injection into the blood channel of a microfluidic device.

[217] The brain channel, the medium channel and their corresponding reservoirs were filled with cell culture medium or HBSS (alanine), then sealed with breathable polyurethane membranes (Sigma) to prevent evaporation. The selected analyte was then flowed through the blood channel at 2 pl/min for 3 h in a cell culture incubator at 37°C supplied with 5% CO2, using a programmable syringe pump (New Era Pump Systems, Inc). At the end of the assays, the samples from the brain channels were taken out and analyzed using a fluorescence microplate reader for FITC-Dextran, LC- MS for caffeine and nitrofurantoin, or a b-scintillation counter for glucose, alanine and sucrose.

[218] The BBB permeability of each analyte in the microfluidic devices was calculated as an apparent permeability (Papp) coefficient independent of flow rate and analyte size according to the following equation (as described by Y.l. Wanget al., Microfluidic blood-brain barrier model provides in vivo-like barrier properties for drug permeability screening. Biotechnol Bioeng 2017, 114 { 1 ), 184-194):

Valval V_ai app = -º -, when t «

AC_tt A - app

where V_ai (imL) and Cat (mol/mL) are the volume and concentration of analytes in the brain channel, respectively; A (cm²) is the contact area between blood and brain channels (the cross-sectional area of the microchannels); Ci (mol/mL) is the analyte concentration in the blood channel; and t (s) is the total perfusion time. Po (cm/s) represents the permeability from a blank device with ECM coating devoid of cells, and PBBB represents the BBB permeability of the target analyte (cm/s).

[219] The permeability {PBBB) of the various analytes through the simulated BBB created in microfluidic devices 8-4 is shown in Figure 10, denoting caffeine (13-1 ), nitrofurantoin (13-2), sucrose (13-3), dextran - 3 kDa (13-4), alanine (13-5) and glucose (13-6). The integrity of the cultured BBB was evaluated using dextran, nitrofurantoin and sucrose. As expected, dextran-FITC (3 kDa) has the lowest PBBB (1 .43 ^c 10⁶ cm/s) among the three analytes due to its relatively larger molecular weight and size. Nitrofurantoin, a small hydrophilic molecule that has very low BBB permeability in vivo [Friden et al, J Med Chem 52(20) (2009) 6233-43], exhibited similar PBBB (3.83 ^c 10⁶ cm/s) to that of sucrose (4.5 ^c 10⁶ cm/s). Importantly, these results closely resemble reported in vitro and vivo data [Franke et al, Brain Res 818(1 ) (1999) 65-71 ; Yaun et al, Microvasc Res 77(2) (2009) 166-73; Lippmann et al, Nat Biotechnol 30(8) (2012) 783-

91]·

[220] The permeability results for caffeine, sucrose and dextran (MW of 3kDa) are compared against the values reported in the literature in Figure 11 . The transcellular diffusion pathway was further verified with caffeine, a small lipophilic molecule that is known to rapidly pass through the BBB. The PBBB of caffeine obtained was 26.4 x 10⁶ cm/s, an approximately 18-fold increase compared to dextran.

Example 15. Efflux assay

[221] The functional activity of P-glycoprotein (P-gp) in the simulated BBB of microfluidic devices was examined using P-gp substrate rhodamine 123 (Rh123). Prior to the efflux assay, cells were cultured to form a simulated BBB on the walls of the blood channel in microfluidic devices 8-4 as described in Example 13.

[222] The blood channel of the resulting microfluidic device was then incubated with 1 mM Rh123 in cell culture medium. For the control experiment, the blood channel was first incubated with p-gp inhibitor Elacridar (10 pM, Sigma-Aldrich) for 1 h prior to the injection of Rh123. In each case, after incubating with Rh123 for 30 min, the channel was washed three times with PBS, refilled with fresh cell culture medium (EMB- 2), and incubated in a culturing incubator at 37°C supplied with 5% CO2.

[223] At different time points, the remaining Rh123 in the cells was analysed under an EVOS fluorescence microscope equipped with live-cell culture capability (ThermalFisher). In the microfluidic device with non-inhibited BBB, Rh123 was gradually pumped out by the endothelial cells. By contrast, the efflux activity was successfully blocked with the microfluidic device having inhibited BBB, and Rhd123 thus remained in the cells after 18 h. The relative efflux coefficients for the two systems was obtained with a similar calculation as for the permeability coefficient described in Example 13, and the comparative result can be seen in Figure 12.

Example 16. Nanoparticle cellular uptake

[224] Transferrin (Tf) functionalized porous silicon nanoparticles (Tf@pSiNPs) have previously been shown capable of crossing the BBB in conventional in vitro and in vivo models. Tf@pSiNPs were prepared according to a previously reported procedure (M Luo et al, Systematic Evaluation of Transferrin-Modified Porous Silicon Nanoparticles for Targeted-Delivery of Doxorubicin to Glioblastoma. ACS Appl Mater Interfaces 2019).

[225] A two-step EDC/NHS reaction was thus employed to conjugate Tf to undecylenic acid-decane functionalized pSiNPs (UnpSiNPs). Accordingly, 5 mg of UnpSiNPs dispersed in ethanol were centrifuged (20,000 ref, 15 min) and resuspended in 0.1 M MES buffer (pH=6). 1 -Ethyl-3-(3-dimethylamino) propyl carbodiimide, hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) were directly added to UnpSiNPs at final concentrations of 2.6 mM and 5 mM, respectively. The reaction components were mixed well and allowed to react at room temperature for 15 min. After the NHS ester activation, MES buffer was replaced by PBS buffer (pH =7.4). Then the suspension was added dropwise to a Tf solution in PBS buffer (10 mg/mL). The mixture was agitated for 2 h at room temperature. 50 mM Tris was added to quench the reaction for 15 min. The Tf@pSiNPs were washed with PBS three times to remove the free Tf by centrifugation.

[226] A similar method was used to conjugate bovine serum albumin (BSA) onto pSiNPs (BSA@pSiNPs) as a negative control due to the inert property of BSA.

[227] To visualize the cellular uptake of pSiNPs by the simulated BBB, Tf@pSiNPs and BSA@pSiNPs were first labelled with Cy5 fluorescent dye using NHS-Cy5 ester (Lumiprobe, 13020). This NHS-Cy5 ester conjugates to free amine moieties on Tf or BSA attached to the pSiNPs. Each type of labelled nanoparticles was then diluted in EBM-2 medium to a concentration of 10 pg/ml.

[228] Prior to the uptake experiments, cells were cultured to form a simulated BBB on the walls of the blood channel in microfluidic devices 8-4 as described in Example 13. The brain channel, the medium channel and their corresponding reservoirs were then filled with cell culture medium and sealed with breathable polyurethane membranes (Sigma) to prevent evaporation. The nanoparticle solutions were then flowed through the blood channel at 2 pl/min using a programmable syringe pump (Harvard Apparatus, Inc). The real time cellular uptake of nanoparticles in the blood channel was monitored under a confocal microscope (Nikon Ti-E and A1 R Confocal) at 5 min intervals for 5 h. The corresponding fluorescence intensity was analyzed with ImageJ (NIH, Bethesda, MD) and plotted in GraphPad Prism 7.

[229] The results for the uptake, as measured by the fluorescence intensity (in relative fluorescence units, RFU) of the nanoparticles is shown in Figure 13. The transferrin coated pSiNPs (Tf@pSiNP) exhibited significantly higher cellular uptake rates compared to bovine serum albumin functionalized pSiNPs (BSA@pSiNP).

[230] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope of the present invention.

Claims

1 . A microfluidic device for investigating an interaction of one or more substances with cells cultured therein, the microfluidic device comprising: one or more internal spaces including a vessel configured to contain a first fluid comprising the one or more substances; and a polymeric coating on walls of at least the vessel, wherein the polymeric coating comprises at least one cross-linked polymer chemically bonded to the walls and comprising a plurality of conjugating functional groups available for reaction, wherein polypeptide-containing macromolecules contacted with the polymeric coating in use are immobilised thereon by covalent bond formation with the conjugating functional groups, thereby adapting the walls for culturing cells.

2. The microfluidic device according to claim 1 , wherein the internal spaces include: a chamber adjacent to the vessel; and at least one microchannel providing fluid communication between the vessel and the chamber, wherein the chamber is configured to contain a second fluid for receiving the one or more substances if transportable from the vessel through the at least one microchannel.

3. The microfluidic device according to claim 2, wherein the vessel and the chamber are elongated and in substantially parallel alignment, and wherein a plurality of spaced-apart microchannels provide fluid communication between the chamber and the vessel.

4. The microfluidic device according to claim 2 or claim 3, further comprising: a compartment adjacent to the chamber, the compartment configured to contain a third fluid for supporting cell growth in the chamber in use; and at least one conduit providing fluid communication between the compartment and the chamber.

5. The microfluidic device according to any one of claims 1 to 4, wherein the vessel is a flow channel configured to convey a flow of fluid.

6. The microfluidic device according to any one of claims 1 to 5, wherein the conjugating functional groups are configured for conjugation to a native protein.

7. The microfluidic device according to any one of claims 1 to 6, wherein the conjugating functional groups are selected from active esters and epoxides.

8. The microfluidic device according to any one of claims 1 to 5, wherein the conjugating functional groups are configured for conjugation to a modified protein via a click reaction.

9. The microfluidic device according to any one of claims 1 to 8, wherein the cross- linked polymer comprises a backbone selected from a poly(vinyl), a polyether and a carbohydrate.

10. The microfluidic device according to any one of claims 1 to 9, wherein the cross- linked polymer comprises photoactivated residues of a plurality of photoactivatable functional groups, wherein photoactivation has (i) cross-linked the polymer and (ii) chemically bonded the cross-linked polymer to the walls via covalent bonds.

11.The microfluidic device according to claim 10, wherein the photoactivatable functional groups are selected from ketones, azides and azirines.

12. The microfluidic device according to claim 10 or claim 11 , wherein the cross-linked polymer is a photoactivated product of a poly(vinyl) copolymer comprising (i) polymerised units comprising photoactivatable functional groups and (ii) polymerised units comprising the conjugating functional groups.

13. The microfluidic device according to claim 12, wherein the poly(vinyl) copolymer comprises the polymerised units comprising the photoactivatable functional groups in an amount of from 0.5 mol% to 25 mol% of the total polymerised units in the pol(vinyl) copolymer.

14. The microfluidic device according to claim 12 or claim 13, wherein the polymerised units comprising the conjugating functional groups have a structure selected from Formula (3) or Formula (4):

15. The microfluidic device according to any one of claims 12 to 14, wherein the pol(vinyl) copolymer has a structure according to Formula (5):

Formula (5) wherein x, y and z are mole fractions of the respective polymerised units in the pol(vinyl) copolymer, wherein x is from 0.05 to 0.25, y is from 0.05 to 0.95, z is from 0 to 0.9 and x+y+z = 1 , wherein each R¹, R² and R³ is independently selected from hydrogen and methyl, wherein each R⁴ is independently selected from

wherein each R⁵ is independently selected from -O- and -NH-, and wherein each R⁶ is independently selected from -NH2, -OH, -0(Ci-C6 alkyl), -(polyethylene glycol), -NH(CI-C6 alkyl) and -NH(2-hydroxypropyl).

16. The microfluidic device according to any one of claims 1 to 15, comprising a unitary body in which the one or more internal spaces are at least partly formed.

17. The microfluidic device according to claim 16, wherein the unitary body comprises a transparent polymeric material and wherein the cross-linked polymer is chemically bonded to the transparent polymeric material.

18. A method of producing a microfluidic device for investigating an interaction of one or more substances with cells cultured therein, the method comprising:

(a) providing a precursor microfluidic device comprising one or more internal spaces including a vessel configured to contain a first fluid comprising the one or more substances; and

(b) forming a polymeric coating on walls of at least the vessel by: contacting the walls with a polymer comprising: (i) a plurality of photoactivatable functional groups and (ii) a plurality of conjugating functional groups, and activating the photoactivatable functional groups with light, wherein the photoactivated functional groups cross-link the polymer and chemically bond to the walls, thereby producing a cross-linked polymer comprising a plurality of the conjugating functional groups available for conjugation to polypeptide-containing macromolecules via covalent bond formation.

19. The method according to claim 18, wherein the internal spaces include: a chamber adjacent to the vessel; and at least one microchannel providing fluid communication between the vessel and the chamber.

20. The method according to claim 19, wherein the vessel and the chamber are elongated and in substantially parallel alignment, and wherein a plurality of spaced-apart microchannels provide fluid communication between the chamber and the vessel.

21. The microfluidic device according to any one of claims 18 to 20, wherein the vessel is a flow channel configured to convey a flow of fluid.

22. The method according to any one of claims 18 to 21 , wherein the conjugating functional groups are configured for conjugation to a native protein.

23. The method according to any one of claims 18 to 22, wherein the conjugating functional groups are selected from active esters and epoxides.

24. The method according to any one of claims 18 to 21 , wherein the conjugating functional groups are configured for conjugation to a modified protein via a click reaction.

25. The method according to any one of claims 18 to 24, wherein the polymer comprises a backbone selected from a poly(vinyl), a polyether and a carbohydrate.

26. The method according to any one of claims 18 to 25, wherein the photoactivatable functional groups are selected from ketones, azides and azirines.

27. The method according to any one of claims 18 to 26, wherein the polymer is a poly(vinyl) copolymer comprising (i) polymerised units comprising the photoactivatable functional groups and (ii) polymerised units comprising the conjugating functional groups.

28. The method according to claim 27, wherein the pol(vinyl) copolymer comprises the polymerised units comprising the photoactivatable functional groups in an amount of from 0.5 mol% to 25 mol% of the total polymerised units in the pol(vinyl) copolymer.

29. The method according to claim 27 or claim 28, wherein the polymerised units comprising the conjugating functional groups have a structure selected from Formula (3) or Formula (4):

30. The method according to any one of claims 27 to 29, wherein the pol(vinyl) copolymer has a structure according to Formula (5):

wherein x, y and z are mole fractions of the respective polymerised units in the pol(vinyl) copolymer, wherein x is from 0.05 to 0.25, y is from 0.05 to 0.95, z is from 0 to 0.9 and x+y+z = 1 , wherein each R¹, R² and R³ is independently selected from hydrogen and methyl, wherein each R⁴ is independently selected from

31. The method according to any one of claims 18 to 30, wherein the precursor microfluidic device comprises a unitary body in which the one or more internal spaces are at least partly formed.

32. The method according to claim 31, wherein the unitary body comprises a transparent polymeric material and wherein the photoactivated functional groups chemically bond to the transparent polymeric material.

33. The method according to any one of claims 18 to 32, further comprising contacting polypeptide-containing macromolecules with the polymeric coating to immobilise the polypeptide-containing macromolecules thereon by covalent bond formation with the conjugating functional groups, thereby adapting the walls for culturing cells.

34. A microfluidic device for investigating an interaction of one or more substances with cells cultured therein, produced according to the method of any one of claims 18 to 32.

35. Use of a microfluidic device according to any one of claims 1 to 17 or 34 to investigate an interaction of one or more substances with cells, the use comprising: contacting the polymeric coating with polypeptide-containing macromolecules, thereby immobilising the polypeptide-containing macromolecules thereon by covalent bond formation with the conjugating functional groups; culturing cells on the immobilised polypeptide-containing macromolecules; conveying a first fluid comprising the one or more substances into the vessel; and determining an interaction of the one or more substances with the cells.

36. Use according to claim 35, wherein the cells are cultured to form a cellular barrier on the walls of the vessel, and wherein determining an interaction of the one or more substances with the cells comprises (i) determining an amount of the one or more substances transported across the cellular barrier to another internal space of the microfluidic device; or (ii) observing an effect attributable to the one or more substances transported across the cellular barrier in another internal space of the microfluidic device.

37. Use according to claim 36, wherein determining the amount of the one or more substances transported across the cellular barrier comprises measuring (i) an amount of the one or more substances present in a second fluid contained in the other internal space or (ii) an amount of the one or more substances remaining in the first fluid.

38. Use according to claim 36, wherein the effect attributable to the one or more substances is an effect on cells cultured in the other internal space.

39. Use according to any one of claims 36 to 38, wherein the cellular barrier is a simulated blood-tissue barrier.

40. Use according to any one of claims 35 to 39, wherein the conveying comprises flowing the first fluid through the vessel.

41. Use according to any one of claims 35 to 40, wherein the cells are cultured on the immobilised polypeptide-containing macromolecules under conditions of hydrodynamic shear flow in the vessel.

42. Use according to any one of claims 35 to 41 , wherein the polypeptide-containing macromolecules comprise at least one native extracellular matrix (ECM) protein.

43. Use according to any one of claims 35 to 41 , wherein the polypeptide-containing macromolecules comprise at least one modified protein comprising functional groups configured for conjugation via a click reaction.

44. Use according to any one of claims 35 to 43, wherein the cells cultured on the immobilised polypeptide-containing macromolecules comprise endothelial cells.

45. Use according to claim 44, wherein the cells cultured on the immobilised polypeptide-containing macromolecules further comprise tumour cells.

46. Use according to claim 44 or claim 45, wherein the cells cultured on the immobilised polypeptide-containing macromolecules further comprise astrocytes and pericytes.

47. Use according to any one of claims 35 to 46, wherein the one or more substances comprise at least one selected from the group consisting of pharmaceutical compounds, therapeutics, exosomes, nanomicelles, nanoparticles, toxins, small molecules, nucleic acids, oligonucleotides, oligopeptides, proteins, ribozymes, small interfering RNAs, microRNAs, short hairpin RNAs, aptamers, viruses, and antibodies or antigen binding parts thereof.

48. A microfluidic device for culturing cells, produced by contacting the polymeric coating of a microfluidic device according to any one of claims 1 to 17 or 34 with polypeptide-containing macromolecules, thereby immobilising the polypeptide- containing macromolecules on the polymeric coating.