Patent ID: 12241082

DETAILED DESCRIPTION OF THE EMBODIMENTS

It should be understood that the Figures are merely schematic and are not drawn to scale. It should also be understood that the same reference numerals are used throughout the Figures to indicate the same or similar parts.

FIG.1schematically depicts the cross-linking reaction for producing the bulk-modified elastomer material according to embodiments of the present invention. In this cross-linking reaction, a vinyl-functionalized elastomer10(from here on simply referred to as elastomer) is cross-linked to an unsaturated fatty acid20by a cross-linking reaction between one of the carbon-carbon double bonds of the elastomer10and one of the carbon-carbon double bonds of the unsaturated fatty acid20, which cross-linking reaction may be catalyzed using an appropriate cross-linking catalyst. It has surprisingly been found by the present inventors that such a cross-linking reaction can provide a bulk-modified elastomer in which the carboxylic acid groups of at least a fraction of the unsaturated fatty acid molecules20having engaged in the cross-linking reaction with the elastomer10are presented on an external surface of the bulk-modified elastomer, such that these carboxylic acid groups are available for binding reactions with cell-stabilizing (i.e. cell-culturing) proteins such as fibronectin, collagen or elastin. This is surprising given that the elastomer10can be non-polar in character, in particular when the elastomer10is dissolved in a non-polar solvent. In such a non-polar environment, the unsaturated fatty acid molecules20tend to form micelles in which the carboxylic acid moieties of these molecules are turned inwardly within such micelles, which typically leads to such carboxylic acid moieties ending up within the bulk of the modified elastomer. Alternatively, the cross-linking reaction between the elastomer10and the unsaturated fatty acid20may involve the formation of a covalent bond by a reaction between a hydride functional group of the elastomer10and the carbon-carbon double bond of the unsaturated fatty acid20, such as for example in the case of hydride-functionalized silicones, as will be explained in further detail below.

In addition, it is well-known per se that the carbon-carbon double bonds of such unsaturated fatty acids20are prone to rapid epoxidation in the presence of oxygen, which reduces the ability of such compounds to engage in cross-linking reactions with the elastomer10.

Embodiments of the present invention are based on the insight that if a reaction mixture including the elastomer10and the unsaturated fatty acid20is brought into contact with a polar surface, this promotes orientation of the carboxylic acid groups of the fatty acid molecules along such a polar surface such that an external surface of the bulk-modified elastomer material exhibits a high density and homogeneous distribution of these carboxylic acid groups. Moreover, it has been found that when bulk modifying the elastomer10in this manner in an injection molding process at elevated temperatures, e.g. temperatures in a range of 120-240° C., epoxidation of the carbon-carbon double bonds of the unsaturated fatty acid molecules20does not significantly interfere with the bulk modification of the elastomer10, i.e. does not significantly inhibit the cross-linking reaction between the unsaturated fatty acid molecules20and the elastomer10. Without wishing to be bound by theory, it is believed that in such injection molding processes, molecular oxygen is largely absent from the reaction mixture within the injection molding apparatus, thereby suppressing the unwanted epoxidation of these carbon-carbon double bonds.

The mold used in such an injection molding process may be shaped in the form of a fluidic device module such that a monolithic fluidic device module may be formed in a single step injection molding process. Due to the high density and homogeneous distribution of carboxylic acid groups on the external surface(s) of such a monolithic fluidic device module, such a device module can be made biocompatible in a straightforward manner by binding a cell-culturing protein such as fibronectin, collagen or elastin to the exposed carboxylic acid groups on the external surface of the bulk-modified elastomer. The mold may be made of or have an inner surface coated with any suitable polar material, such as stainless steel, a metal or a metal oxide such as aluminium oxide in order to achieve the desired orientation of the carboxylic acid groups of the bulk-modified elastomer on at least one of its external surfaces.

Any suitable elastomer may be used for this purpose. For example, the elastomer may be a homopolymer, a copolymer, a block copolymer, a terpolymer, a block terpolymer and so on. A particularly suitable elastomer may be selected from polyenes such as polybutadiene. Where polybutadiene is used, the polybutadiene may be made using any suitable polymerization process. Particularly preferred is a polybutadiene formed in a Nd or Li-catalyzed polymerization reaction, as it is well-known per se that such polybutadiene has a low degree of branching (typically below 5%) and a low degree of polydispersity (Mw/Mn) of around 2. Li-catalyzed polybutadiene is particularly preferred due to its high degree of 1,2-vinyl content (about 11%). However, other types of polybutadiene, e.g. polybutadiene obtained from a Co, Ni or Ti-catalyzed polymerization reaction may be used instead. Where a polyene such as polybutadiene is used as the elastomer10, the cross-linking reaction between the elastomer10and the unsaturated fatty acid20may be catalyzed using a peroxide catalyst. In another advantageous embodiment, a Li-catalyzed polybutadiene is used having a degree of branching in a range of 5-15% as it has been found that when the degree of branching of the polybutadiene is in this range, the reactivity of the polybutadiene with the unsaturated fatty acid20is improved. It is noted for the sake of completeness that it is well-known per se how to control the degree of branching when synthesizing polybutadiene such that this will not be further explained for the sake of brevity only.

Another class of elastomer is that are specifically mentioned is silicones (polysiloxanes) for injection molding. Such silicones may comprise a PDMS backbone including vinyl moieties to facilitate cross-linking through Pt-catalyzed addition reactions, e.g. with (poly methyl) hydrogen siloxanes. Instead of or in addition to such cross-linking with (poly methyl) hydrogen siloxanes, the vinyl-functionalized PDMS backbone may be cross-linked with unsaturated fatty acids20. Such silicones may be formed by a cross-linking reaction between a vinyl-functionalized linear or branched silicone monomer or oligomer, e.g. a T-branched or Q-branched silicone monomer or oligomer and a linear hydride-functionalized silicone monomer or oligomer, between a hydride-functionalized linear or branched silicone monomer or oligomer, e.g. a T-branched or Q-branched silicone monomer or oligomer and a linear vinyl-functionalized silicone monomer or oligomer or mixtures of vinyl-functionalized and hydride-functionalized linear or branched silicone monomers or oligomers, e.g. a T-branched or Q-branched silicone monomer or oligomer and/or a mixture of linear hydride-functionalized and vinyl-functionalized silicone monomer or oligomer.

For example, the two-component silicone may be formed by a cross-linking reaction between a linear component1and a linear component2, which each may correspond to the general formula below:

In component1, R1and R2are individually selected from C1-C3alkyl, R3-R8are individually selected from C1-C3alkyl and vinyl, with the proviso that at least one of R3-R5and at least one of R6-R8is vinyl. Preferably, at least three of R3-R8are vinyl. In all embodiments, n may be in the range of 100-200,000. In a specific embodiment of component1, each of the groups R1-R8that is an alkyl group is a methyl group, i.e. the component1is vinyl-functionalized PDMS having terminal vinyl groups.

In component2, R1is hydrogen, R2=C1-C3alkyl and R2-R8are individually selected from C1-C3alkyl or hydrogen, with the proviso that at most one of R3-R5and at most one of R6-R8is hydrogen, and and n may have any suitable value, such as n=3-1,000 or more specifically n=3-10. In a specific embodiment of component2, none of the groups R3-R8are hydrogen. In another specific embodiment of component2, each of R2-R8are methyl.

Component2typically acts as a cross-linking agent for component1. Such cross-linking reactions, which are typically Pt-catalyzed, are well-known per se, see for example WO 2009/147602 A2 and are therefore not explained in further detail for the sake of brevity. Any suitable silicone elastomer may be used. It should be understood that when cross-linking such silicones in the presence of an unsaturated fatty acid20, the covalent bond between the silicone and the unsaturated fatty acid20alternatively may be formed by a reaction between a hydride functional group of the (cross-linked) silicone and the unsaturated fatty acid20as suggested in the below reaction mechanism:

In order to prevent catalyst inhibition by the protons of the carboxylic acid groups of the unsaturated fatty acids20during such cross-linking reactions, the unsaturated fatty acid may be used in a saponified form in the cross-linking reaction with a silicone elastomer, e.g. as the sodium salt of such an unsaturated fatty acid, with the cross-linking reaction product subsequently being treated with a strong protic acid, e.g. HCl or the like, in order to reinstate the carboxylic acid groups on the surface of the bulk-modified elastomer. In this embodiment, a fraction of the fatty acid moieties within the bulk of the bulk-modified elastomer may remain saponified due to the fact that the protic acid may be unable to reach the bulk due to the length of the diffusion channels between the surface of the bulk-modified elastomer and its bulk. As will be readily understood, this is not a problem given that the bulk-modified elastomer can still be made biocompatible due to the availability of the carboxylic acid groups on one or more of its surfaces. Any suitable type of polysiloxane may be used for this purpose.

Alternatively, a silicone backbone such as a (poly methyl) hydrogen siloxane backbone may be crosslinked with rubber-like polymers such as polybutadiene and polyisoprene, with the unsaturated fatty acid being incorporated in such a crosslinked product. This for example may be done to tune the water permeability of the final material as silicones typically have a rather open structure whilst such rubber-like polymers have a rather closed structure, such that the openness, i.e. the water permeability, of the crosslinked product can be tuned by the ratio of silicone and rubber-like polymer therein. In this manner, the properties of the material according to this embodiment may be optimized for cell/protein interaction to build good scaffolds. For example, where the elastomer is a silicone-based, e.g. PDMS-based, a material that is highly permeable to water and other compounds (e.g. drugs) is obtained that facilitates maximized perfusion of these compounds into the cells on the membrane of the fluidic device module100. However, where real-time monitoring of drug consumption by cells is desirable, a polyene-based material such as a polybutadiene-based material that is non-permeable to water and drugs may be preferable, as the rate of perfusion of these compounds to the cells can be controlled using the density of apertures through the membrane.

In an embodiment, the polysiloxane may be formed from a multi-component starting material as previously explained in order to prevent premature cross-linking of the polysiloxane. Such multi-component starting materials are well-known per se; for example, such multi-component starting material kits are marketed by Wacker Chemie AG from Munich, Germany under the tradename Elastosil.

Regarding to unsaturated fatty acid20, any suitable unsaturated fatty acid may be used for the purpose of cross-linking it with the elastomer10. For example, the unsaturated fatty acid20may be selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoeladic acid, α-linolenic acid, arachidonic acid, eicospaentaenoic acid, erucic acid and docosahexaenoic acid. Linoleic acid has been used in the examples of the present invention but it should be understood that this is by way of non-limiting example only and that other unsaturated fatty acids may be used instead. In the context of the present application, where reference is made to an unsaturated fatty acid20, it is to be understood that this is intended to refer to any organic compound comprising a carboxylic acid head group covalently bound to an aliphatic tail comprising in between 10-30 carbon atoms and at least one carbon-carbon double bond in the aliphatic tail. The aliphatic tail may terminate in a non-aromatic ring structure, e.g. a five-membered or 6-membered cyclopentyl or cyclohexyl group, which may itself contain at least one double bond. A non-limiting example of an organic compound intended to fall within the above definition of an unsaturated fatty acid20is retinoic acid. Many other compounds in addition to the example compounds explicitly mentioned in the present application will be immediately apparent to the skilled person.

The elastomer10and the unsaturated fatty acid20may be mixed in any suitable ratio in a composition to form the bulk-modified elastomer. Preferably, unsaturated fatty acid20is present in such a composition in a range of 0.05-35% by weight of the elastomer10such that upon bulk modification of the elastomer10the bulk-modified elastomer retains its flexibility to the remaining presence of carbon-carbon double bonds in the elastomer bulk.

An example synthesis procedure for producing the bulk-modified elastomer from a polyene such as polybutadiene is given below (synthesis example 1).

Synthesis Example 1

Polybutadiene chunks as obtained from Lanxess AG, Cologne, Germany are inserted into a compounder or mix extruder. The chunks contain a peroxide such as Dicumylperoxide as a catalyst. An amount of linoleic acid (60-74% concentrated as obtained from Aldrich Chemistry) in a range of 1-30 wt % by weight of the polybutadiene chunks is mixed into the polybutadiene bulk by kneading at a temperature in the range of 110-150° C. The resulting extruded mixture is injected into a stainless steel mold defining a fluidic device and having an oxidized inner surface contacting the extruded mixture for 3-10 minutes at a temperature in a range of 150-200° C. to yield a monolithic fluidic device module of a polybutadiene rubber cross-linked with the linoleic acid.

An example synthesis procedure for producing the bulk-modified elastomer from a silicone rubber is given below (synthesis example 2).

Synthesis Example 2

A 121 g sodium linoleate solution in water (30 wt %) is mixed into silicone component A (240 g) of Elastosil LR 3040 from Wacker Chemie AG and vigorously stirred under vacuum conditions at room temperature to evaporate the water from the composition. The resultant mixture is mixed with component B (277 g) of Elastosil LR 3040 from Wacker Chemie AG (including Pt-catalyst) and fed into an injection molding apparatus where the resultant mixture is injected into a stainless steel mold having an oxidized inner surface contacting the resultant mixture and defining a fluidic device module and molded for 10-60 seconds at a temperature in a range of 150-200° C. to yield a monolithic fluidic device module of a silicone rubber cross-linked with the sodium linoleate. At higher concentrations of sodium linoleate extra cross linker (polymethylhydrosiloxane) may be added to the reaction mixture to prevent an excess of double bonds of the unsaturated fatty acids decreasing the conversion rate. The molded product is subsequently dropped in an aqueous HCl solution (pH 2-4) to convert at least the sodium carbonate groups at the surface of the molded product into free carboxylic acid groups.

The thus bulk-modified elastomer may be made biocompatible by reacting the free carboxylic acid groups on its surface with a cell-culturing protein such as fibronectin, collagen or elastin. This is schematically depicted inFIG.2in which a cell-culturing protein40can be bound to the free carboxylic acid groups of the fatty acid tails33extending from a surface31of the bulk-modified elastomer30to form a cell culturing scaffold. The cell-culturing protein40of such a cell culturing scaffold can stabilize mammalian cells or tissue50, e.g. human tissue, such that the cells50remain alive and multiply normally over a prolonged period of time, as the cell-culturing protein40mimics the natural environment of such cells, as is well-known per se. A well-documented binding protocol for covalently binding fibronectin to surface carboxylic acid groups is given below:

A description of this protocol can be found in: L. H. H. Olde Daminnk, P. J. Dijkstra, M. J. A. Luyn, P. B. v. Wachem, P. Nieuwenhuis, and J. Feijen, “Cross-linking of dermal sheep collagen using a water-soluble carbodiimide”, Biomaterials, 17(8) pp. 765-774 (1996).

In this protocol, the carboxylic acid groups (compound I) may be reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, compound II) followed by a reaction with N-hydroxysuccinimide (NHS, compound IV), which can react with the amine groups of fibronectin (compound VII) to covalently bind the fibronectin to the carboxylic acid groups of the bulk-modified elastomer30. In this manner, the fibronectin can form a scaffold for carrying and culturing mammalian cells. As is well-known per se, the integrin receptors of such cells can recognize specific amino acid sequences of the fibronectin, e.g. a sequence such as an Arg-Gly-Asp-Ser sequence, which triggers the cells to accept the new environment and stay alive. As previously explained, such a scaffold can be formed with high uniformity in terms of coverage of the surface of the bulk-modified elastomer30, e.g. the surface of a membrane of a fluidic device module due to the high density homogeneous distribution of the carboxylic acid groups across such a surface.

A typical example of a fluidic device module100is schematically depicted inFIG.3andFIG.4, in which perspective views of opposing major surfaces of the fluidic device module100are schematically depicted. The fluidic device module100may comprise a membrane110over which at least one flow channel115extends. Preferably, the fluidic device module100comprises opposing flow channels spatially separated by the membrane110. At least the membrane110is made of the bulk-modified elastomer30although in a preferred embodiment the entire fluidic device module100is made of the bulk-modified elastomer30, thereby yielding a monolithic fluidic device module100.

The fluidic device module100may comprise one or more septums120through which such flow channels115may be accessed. Each flow channel115preferably is accessible through a dedicated set of septums120, such that cross-contamination between multiple flow channels115is avoided. As explained above, the fluidic device module100may be made bio-compatible by reacting the carboxylic acid groups on the membrane110as explained above to covalently bind a cell culturing protein, e.g. fibronectin, collagen or elastin, to the membrane110, such that cells50may be cultured on the thus formed cell culturing scaffold of the biocompatible membrane110. As such cell immobilization procedures on biocompatible membranes including such cell-culturing proteins are well-known per se, this will not be explained in further detail for the sake of brevity only. It suffices to say that any suitable protocol may be used for this purpose.

The membrane110may comprise a plurality of holes or grooves such that liquid nutrition and solutions with compounds such as potential drugs may reach both sides of the membrane and cells/tissue, which holes or grooves may be formed in any suitable manner, e.g. through laser cutting. Because the membrane110can be made biocompatible as explained above, the size and pitch of these holes or grooves is less critical than in prior art devices in which the holes or grooves used to immobilize and culture cellular material. In other words, such holes or grooves in the membrane110of the fluidic device module100may be substantially larger than a typical diameter of the cells to be immobilized on the membrane110, with the pitch between such holes or grooves not being particularly critical.

A fluidic device200as schematically depicted inFIG.5may be formed from such a fluidic device module100by the provision of a pair of cover plates210,220, which cover plates fluidly seal the fluidic device module100such that fluids passed through the one or more flow channels115of the fluidic device module100cannot leak out of the fluidic device200.

In an embodiment, the cover plates210,220are arranged such that the cover plate210covers a first major surface of the fluidic device module100, thereby sealing a first fluidic channel115of the fluidic device module and the other cover plate220covers a second major surface of the fluidic device module100, thereby sealing a second fluidic channel115of the fluidic device module. The cover plates210and220may be made of any suitable material, e.g. glass or a plastic material. Preferably, the cover plates are stretchable and flexible to ensure a good fit with the fluidic device module100whilst retaining the flexibility of the fluidic device module100in the fluidic device200. Such a fluidic device200may be deployed as an organ on chip as will be readily understood by the skilled person.

FIG.6schematically depicts an example embodiment of a fluidic device200in which the device comprises a fluidic device module100according to the present invention in between two rigid cover plates210and220. The upper plate210comprises a first inlet231and a first outlet232in fluid connection with a lower channel (not visible) underneath the membrane110in the fluidic device module100such that the membrane110is in fluid contact with a fluid passing through this lower channel. The upper plate210further comprises a second inlet233and a second outlet234in fluid connection with an upper channel115over the membrane110such that the membrane110is in fluid contact with a further fluid passing through this upper channel115. For example, the fluid passing through the lower channel may comprise drugs to be tested on the cell cultures attached to the membrane110as explained in more detail below, which can reach the cell cultures through the membrane110owing to the fact that the membrane110is made porous, e.g. by laser drilling holes or grooves in the membrane110as previously explained. Alternatively, such holes or grooves may be formed in the membrane110when forming the fluidic device module100in the polar mold. This has the advantage that a regular pattern of such fluid passages (pores) may be formed through the membrane110. The further fluid passing through the upper channel115may be in direct contact with such cell cultures and provide such cell cultures with nutrients. The inlets231,233and the outlets232,234in some embodiments are septa through which fluids may be hydraulically pumped over the membrane110through the aforementioned channels, e.g. by pressing the septa. Alternatively, the inlets231,233and the outlets232,234may be ferrules to which tubing may be attached, e.g. clamped, which for instance is useful when the fluidic device200is used as a well plate or the like in a lab on chip system.

In an embodiment, the upper and lower channels are incorporated in the (monolithic) fluidic device module100, e.g. when molding the fluidic device module100. Alternatively, at least one of the upper and lower channels may be formed in one of the cover plates.FIG.7schematically depicts a perspective top view andFIG.8schematically depicts a perspective bottom view of an example embodiment of a fluidic device200in which the upper channel115is formed in the upper cover plate210whilst the lower channel115′ is formed in the fluidic device module100. It is of course equally feasible to form the lower channel115′ in the bottom cover plate220as will be readily understood by the skilled person. The fluidic device200such as the device shown inFIG.7andFIG.8for example may be used as a microwell in a lab on chip device in which a plurality of such microwells may be clamped or otherwise secured to facilitate parallel testing of a plurality of different samples within a single lab on chip device, in which each microwell typically comprises one of these samples.

Upon immobilizing a cell culture50in such a fluidic device200, typically after the membrane110has been made biocompatible as explained above, the fluidic device200may be used in methods in which the immobilized cell culture50is exposed to a fluid comprising a compound of interest, which fluid may be passed through a flow channel115of the fluidic device200in order to expose the immobilized cell culture50to the compound of interest in the fluid and monitor the reaction of the immobilized cell culture50to such exposure. Such a method for example may be deployed in an oncology setting where the immobilized cell culture50may be exposed to a drug, e.g. a chemotherapy drug, in order to monitor the response of the immobilized cell culture50to such a drug. To this end, the immobilized cell culture50may include tumor cells in order to test the efficacy of the drug and/or may include healthy cells in order to test the toxicity of the drug on healthy tissue.

Such a drug testing method typically involves providing a fluidic device module100in accordance with one or more embodiments of the present invention, e.g. providing a monolithic fluidic device module100and binding at least the membrane110of the fluidic device module100to a cell culturing protein40, e.g. fibronectin, to obtain a cell culturing scaffold. Next, a harvested cell culture50is applied to the cell culturing scaffold to obtain a prepared fluidic device module100from which a fluidic device200, e.g. an organ on chip, is formed as previously explained. The cell culture within the fluid device200may be fed through one of a pair of flow channels115of the fluidic device200in order to keep the cell culture alive, whilst the cell culture may be exposed to a drug to be tested through the other of the pair of flow channels115of the fluidic device200, with the drug testing method typically being completed by monitoring the response of the cell culture50to the drug to be tested. This for example may be achieved by disassembling the fluidic device200by removing the cover plates210,220and slicing the fluid device module100such that a slice of this module including a portion of the cell culture50is obtained, which slice may be investigated under a microscope or the like in order to investigate the effects of the drug under test on the cell culture50. During such investigations, the cell culture50is typically stained, treated with formalin and fixated in a fixating agent such as paraffin to facilitate such microscopic investigation. Such staining, treating and fixating of the cell culture50may be deployed within the fluidic device200, e.g. by passing the appropriate chemical agents through a fluidic channel115of the fluidic device200in which the cell culture50is exposed.

In an alternative embodiment, such disassembly of the fluidic device200can be avoided and the monitoring of the response of the cell culture50to one or more compounds such as a drug to be tested can be achieved in real time using confocal microscopy. In particular, where embodiments of the fluidic device200comprise transparent cover plates210,220, e.g. glass plates or polymer plates, confocal microscopy may be used to monitor the cell culture50within an assembled fluidic device200. This is made possible through the fact that at least the bottom cover plate220can be kept thin, for example having a thickness in a range of 150-300 μm, with the distance between the membrane110of the fluidic device module100carrying the cell culture50and the bottom cover plate220being less than 200 μm such that the focal length limits of confocal microscopy (typically around 500 μm from the object to be investigated to the lens objective). To this end, the membrane110may have a thickness in a range of 10-100 μm, such as a thickness of 20-30 μm. The thickness of the upper cover plate210is not critical; any suitable thickness may be contemplated, such as a thickness of 300 μm-3 mm.

The evaluation of the cell culture50within the fluidic device200using confocal microscopy is particularly suitable where the cell spheroids have a diameter of less than 500 μm, e.g. about 200 μm in order to ensure that the overall focal length of the optical path facilitates the capture of a sharp image with the confocal microscope. For larger spheroids, the fluidic device200may have to be dismantled such that the membrane110including the spheroids can be pressed onto a glass cover slip for positioning in the optical objective of the confocal microscope.

It is furthermore noted that the aforementioned dimensions of the fluidic device200typically apply during the real-time monitoring of the cell culture50within the fluidic device200. In embodiments in which the fluidic device200is sliced as previously explained, e.g. for digital pathology, the dimensions of the fluidic device200are less critical as long as the formalin and paraffin fixation can be performed thereon.

As will be readily understood by the skilled person, such drug testing methods may significantly differ in terms of protocol, e.g. drug dosages, administration frequencies, and so on. It should be understood that the drug testing method of the present invention is not limited to a particular protocol as long as such methods may be deployed using a fluidic device200according to an embodiment of the present invention in which at least the membrane110is made biocompatible by converting it into a cell culturing scaffold by reacting its carboxylic acid groups with a cell culturing protein as previously explained.

At this point, supplementary evidence is provided for the cross-linking reaction between the elastomer10and the carbon-carbon double bonds of the unsaturated fatty acid20, as well as for the successful binding of fibronectin to (the carboxylic acid groups of) such a bulk-modified elastomer.

In the following, FT-IR spectra were recorded using a Thermo iS50 FTIR bench in combination with a heatable Golden Gate ATR module. An average of 64 scans was used for all measurements. In the measurements, the resolution was set to 4 cm−1using a spectral range from 4000-650 cm−1. To allow for such a resolution to be used, the internal spectrometer aperture was set to 50%.

Raman spectra were recorded using a Renishaw InVia Raman microscope combined with a laser excitation wavelength of 514 nm provided by a Coherent Innova 70C Ar+laser. A 50 mW excitation power was used in combination with a 63× glass correction objective. The spectra were recorded through a glass top cover using a grating consisting of 1200 lines/mm and with a 2 s integration time over a spectral range of 662-3,000 cm−1using an average of 300 spectra such that each spectrum took 600 s to be recorded.

FIG.9depicts part of an FT-IR spectrum of sodium linoleate. The solid arrow indicates a C—H stretch vibration at approximately 3,050 cm−1, which has been assigned to the C—C double bonds of the sodium linoleate, whereas the dashed arrow indicates the C═O stretch vibration at approximately 1600 cm−1, which has been assigned to the carboxylate groups of the sodium linoleate.FIG.10depicts a ratio between the intensity of this C—H stretch vibration and C═O stretch vibration as a function of time during monitoring of the progress of a reaction according to aforementioned synthesis example 2. The samples were recorded in between two quartz plates whilst heating the samples to 200° C. to induce the cross-linking reaction. As can be clearly seen fromFIG.10, a reduction in C—C double bonds in the sodium linoleate can be seen during the cross-linking reaction whilst the carboxylate stretch vibration intensity remains unaltered, which provides a clear indication that the C—C double bonds are being consumed in the cross-linking reaction, thereby clearly suggesting cross-linking between the sodium linoleate and the silicone elastomer.

To further corroborate this, the cross-linking reaction was further monitored with Raman spectroscopy, using the same sample setup as used for the recordal of the FT-IR spectra in order to minimize the impact of environmental oxygen on the crosslinking reaction. In the Raman spectrum, the C═O stretch vibration was inactive whilst the C═C stretch vibration showed clear activity as depicted inFIG.11.

Upon initial thermal stabilization of the sample up to t=250 s (during which the sample is thermally equilibrated with the laser used in the Raman spectroscopy), the Raman spectrum shows a steady decrease in the C═C stretch vibration of the sodium linoleate, thereby clearly indicating that these bonds are active in the cross-linking with the silicone elastomer.

To further demonstrate the bulk modification of the elastomers in synthesis examples 1 and 2, a block of each elastomer after reaction with the linoleic acid was molded in a polar mold as previously explained and subsequently sliced with a microtome at −40° C. to obtain an internal slice of the modified elastomer, i.e. a slice in which none of its major surfaces were external surfaces of the molded elastomer block. These internal slices were subsequently brought into contact with a 2 μl droplet of water of different pH (pH 4, pH 7 and pH 10) for 3 minutes after which the contact angles of the water droplets with these internal slices were measured. The same procedure was repeated with unmodified elastomers to compare the measured contact angles of an internal slice of the linoleic acid modified elastomers against the measured contact angles of an internal slice of the corresponding unmodified elastomer. The results for the silicone-based elastomer are shown in the graph inFIG.12and the results for the polybutadiene-based elastomer are shown in the graph inFIG.13. For the silicone-based elastomer, a functionalized elastomer formed using 6.6 wt % linoleic acid was used for both the bulk and surface contact angle measurements, whereas for the polybutadiene elastomer a functionalized elastomer formed using 6 wt % linoleic acid was used for the surface contact angle measurements and a functionalized elastomer formed using 1.5 wt % linoleic acid was used for the bulk contact angle measurements. This difference in concentration of the linoleic acid between surface and bulk contact angle measurements for the polybutadiene elastomer merely is the result of availability of samples at the time of measurement.

As can be seen from these graphs, the internal slices of the polybutadiene crosslinked with linoleic acid and the silicone crosslinked with (saponified) linoleic acid both showed a strong pH dependency of the measured contact angle with decreasing contact angles being measured with increasing pH, whereas the measured contact angles of the unmodified polybutadiene and silicone were largely pH independent. Without wishing to be bound by theory, the small increase in contact angle measured for the unfunctionalized silicone with increasing pH may be result of a pH dependence on the hydrogen bonding between the water molecules and silanol moieties in the silicone.

The strong pH dependency of the measured contact angle for the bulk of the functionalized elastomers clearly demonstrates the presence of carboxylic acid groups within the bulk of the elastomers crosslinked with an unsaturated fatty acid such as linoleic acid. This can be understood as follows. At lower pH, such carboxylic acid groups remain largely protonated, thereby minimizing the surface charge of the internal slices of the elastomers. With increasing pH, this surface charge increases due to increased deprotonation of the carboxylic acid groups, which decreases the contact angle of the water droplets with the surface of these internal slices. On the other hand, where such carboxylic groups are not present at the surface exposed to the water droplets, such as in the unmodified elastomers, the change in pH of the water droplets does not cause a change in the surface charge, such that the measured contact angle of the water droplets of different pH with such a surface is far less sensitive to these pH changes.

To further demonstrate the ability of the material of the present invention to bind a cell culturing protein such as fibronectin, example embodiments of the material were bound to fibronectin, and surface plasmon resonance (SPR) spectra were recorded to demonstrate this binding reaction.

SPR experiments were performed on amine SPR chips (High capacity amine chip SPR affinity Sensors, Sierra Sensors, Hamburg, Germany) and on bare gold SPR chips (Bare gold 12 pk. SPR affinity Sensors, Sierra Sensors, Hamburg, Germany) spin coated with a solution of polybutadiene chunks and linoleic acid in n-heptane using a SPR2 spectrometer as provided by Sierra Sensors, Hamburg, Germany. The solution was prepared by dissolving the polybutadiene chunks in n-heptane in a glass bottle at 70° C. over a period of 72 hours whilst shaking the bottle, after which the linoleic acid was added and stirred into the mixture. After flow was observed and system was stabilized by flushing and degassing, an angle scan was performed to check the correlation between all channels. The angle scan data was saved and preceded with the essay for fibronectin cross-linking.

The chip surface was pre-conditioned by injecting 3 phases of 2 solutions over the surface. The first injection consisted of 60 μl of 100 mM Hydrochloric acid (HCl) at a flow rate of 10 μl/min and a dissociation time set to 1 second. The second injection consisted of 60 μl Elution buffer consisting of 1 M sodium chloride (NaCl) and 100 mM sodium hydroxide (NaOH) at a flow rate of 10 μl/min and dissociation time set to 1 second. The first phase consisted of injecting the solutions over channels1and2, the second phase consisted of injecting the solutions over channels3and4, whilst the third and final phase consisted of injecting the solutions over channels1-4.

After the pre-conditioning all channels were cleaned and activated with EDC/NHS. This was done by mixing 100 μl EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, Sigma-Aldrich, Saint Louis, USA) and 100 μl NHS (N-hydroxysuccinimide, Sigma-Aldrich, Saint Louis, USA) of 9.6 mg/ml in MilliQ stocks together, after which 60 μl of the mixture was injected onto the surface on all four channels at a flow rate of 10 μl min with a 1 s dissociation.

After all channels were pre-conditioned and activated with EDC/NHS, the channels were injected with 60 μl Fibronectin from bovine plasma powder (Bio Reagent, suitable for cell culture Sigma Aldrich, Saint Louis, MO, USA) dissolved in a sodium acetate buffer with pH 5.5 (Biacore, Diegem, Belgium) at a flow rate of 10 μl/min. Channel 2 was injected with 1 μg/ml Fibronectin with a dissociation time of 1 s, channel 3 was injected with 5 μg/ml Fibronectin with a dissociation time of is and channel 4 was injected with 10 μg/ml Fibronectin with a dissociation time of 1 s (channel 1 was used as a reference).

All four channels of the system subsequently were flushed with 60 μl running buffer PBS 0.05% Tween at a flow rate of 10 μl/min and a dissociation time of is to determine, whether fibronectin had truly bonded to the material of the present invention. Spectral data was saved and analyzed with analyzer program (Analyzer R2 Sierra Sensors Sierra Sensors, Hamburg, Germany).

FIG.14depicts a surface plasmon resonance (SPR) spectrum of a fluidic device200comprising a fluidic device module100made of polybutadiene cross-linked with linoleic acid, which modified polybutadiene was reacted with EDC and NHS as described above and the resultant structure was reacted with fibronectin. As can be clearly seen from the SPR spectrum, the fibronectin is bound to the surface of the modified polybutadiene, thereby indicating that such a bulk-modified elastomer can be successfully converted into a cell-culturing scaffold.FIG.15shows the part of the SPR spectrum ofFIG.14in which this surface modification is clearly shown. As can be seen the SPR baseline is shifted by approximately 2,000 RU, thus clearly indicating the binding of the fibronectin to the linoleic acid-modified polybutadiene. What is more, the SPR spectrum remained stable under the fluid streams (flushing) to which the fluidic device200was exposed, thereby further demonstrating that the linoleic acid is (covalently) bound to the polybutadiene, as no linoleic acid was washed away during this procedure. As expected, strongest covalent binding between the fibronectin and the material of the present invention was observed at the highest concentration of fibronectin (i.e. the chip in channel 4).

Next, binding of cell lines MDA-MB231 and MCF-7 to the chip of channel 4 was investigated to demonstrate the ability of the covalently bound fibronectin to bind to such cell lines. To this end, the above described SPR procedure was repeated with fresh chips in channels2-4(channel 1 being used as a reference again), over which 60 μl of 10 μg/ml Fibronectin at a flow rate of 10 μl/min at a dissociation time of 1 s was run to coat the surfaces of the fresh chips with fibronectin. After the surfaces were recoated with fibronectin cells, 2*106cells/ml of MDA-MB231 were flowed across channel 2 in 4 injection steps of 200 μl, at a flow rate of 10 μl/min and a dissociation time set to the maximum of 1800 s to give the cells enough time to bind to the fibronectin. The same was done for channel 4 with 1.3*106cells/ml of the cell line MCF-7 in 4 injection steps of 200 μl. In total, cells were given almost 2 hours to bind to the chip surface. The cells subsequently were exposed to 200 μl running buffer PBS with 0.05% Tween that was injected over channels1-4at a flow rate of 10 μl/min and the maximum dissociation time to flush away any cells not bonded to the chip surfaces. Bonded cells were removed from the chip surfaces with an injection of 200 μl 0.25% trypsin 0.25% over channel 1, 2, 3 and 4 at a flow rate of 10 μl/min and the maximum dissociation time.

After each experiment, the SPR machine was cleaned by flushing the system with 70% ethanol, sanitized with 1-2% hypochlorite and desorbed with 0.5% SDS, 50 mM glycine-NaOH (pH 9.5) to reduce contaminants or other build-up of dirt in the system.

FIG.16depicts a SPR spectrum of the fibronectin-bound linoleic acid-modified polybutadiene-based fluidic device200after seeding the device with cells from MDA-MB 231 and MCF-7 cell lines. It can be seen that these cell lines attached to the fibronectin bound to the linoleic acid-modified polybutadiene. The cells remained attached to the fluidic device200whilst flushing the device with a running buffer but could be cleaved off by the trypsin solution. This therefore clearly demonstrates that the cells effectively bind to the device membrane, with the fibronectin being covalently bound to the membrane surface as the fibronectin (and bound cells) are not washed away by the running buffer.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. The word “comprising” does not exclude the presence of elements or steps other than those listed in a claim. The word “a” or “an” preceding an element does not exclude the presence of a plurality of such elements. The invention can be implemented by means of hardware comprising several distinct elements. In the device claim enumerating several means, several of these means can be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.