Cell culture materials

Abstract

A material for culturing cells is disclosed. The material comprises a bulk-modified elastomer having a Shore hardness (DIN EN ISO 868) in the range of Shore 00 20 to Shore A 80 and comprising a plurality of fatty acid moieties covalently bound to the elastomer bulk, wherein the carboxylic acid groups of the moieties are obtainable on the outer surface of the material to provide the binding, and wherein the bulk-modified elastomer is obtained by forming, in a mold having a polar inner surface, a composition comprising a vinyl-functionalized elastomer or a hydride-functionalized elastomer or at least one precursor thereof, a free or saponified unsaturated fatty acid in the range of 0.5 - 5% by weight based on the total weight of the vinyl-functionalized elastomer or hydride-functionalized elastomer or at least one precursor thereof, and a crosslinking catalyst; and subjecting the vinyl-functionalized elastomer or hydride-functionalized elastomer to bulk modification by covalently binding the free or saponified unsaturated fatty acid to the elastomer bulk through a crosslinking reaction between the vinyl groups or hydride groups of the elastomer and the unsaturated carbon-carbon bonds of the unsaturated fatty acid in the mold to obtain the material. Also disclosed are a fluid device module and a fluid device, a cell culture method, and a drug testing method.

Description

Technical Field

[0001] The present invention relates to a cell culture plate comprising an elastomeric material for culturing cells.

[0002] The present invention also relates to a fluid device module and a fluid device comprising such a cell culture plate.

[0003] The present invention also relates to a method for culturing cells.

[0004] The present invention also relates to a method for drug testing using a fluid device module comprising such an elastic material.

[0005] The present invention relates to the use of an elastomeric material for cell culture.

[0006] The present invention relates to the manufacture of a cell culture plate. Background Art

[0007] In vitro testing of mammalian cells or tissues is an important technique for obtaining clinically important information on mammalian materials in research. For example, such tests can be performed on biopsy mammalian cell or tissue materials to identify abnormalities or diseases in such mammalian materials or to expose diseased mammalian materials to drugs (e.g., experimental drugs) to monitor the response of the diseased mammalian materials to such exposure. For example, such methods are frequently used in oncology procedures. This can provide important insights into how diseases in individuals can be effectively treated without exposing the individual to a range of potentially effective drugs that may be undesirable for many reasons (e.g., drug toxicity). Additionally, the efficacy of experimental drugs against certain existing diseases for which no established satisfactory drug treatment has been obtained can be tested in this way. There are many other well-known reasons for deploying such in vitro tests.

[0008] A common method for such in vitro testing is to immobilize mammalian materials in a fluid device, which is sometimes referred to as an organ-on-chip. In this method, mammalian materials are typically immobilized on a membrane that separates two fluid channels of the fluid device, where the first channel is for feeding the mammalian materials and the second channel is for exposing the mammalian materials to a compound or chemical composition of interest (e.g., drug treatment) to, for example, test the efficacy and / or toxicity of a drug, as previously explained. The fluid device or at least its membrane can be made of an elastomer such that the fluid device can be cut into pieces or slices to obtain slices of the membrane comprising the mammalian materials for, for example, evaluation purposes, such as evaluating the response of the mammalian materials to exposure to a compound or chemical composition of interest.

[0009] The challenge in such in vitro assays is to ensure that mammalian material (e.g., cells or tissues) is properly stabilized on the membrane of a fluidic device and develops on that membrane such that the mammalian material remains viable for the duration of the assay process. This is particularly challenging in oncology processes where the experiments can be quite long. To this end, biocompatible materials such as fibronectin can be used to stabilize mammalian material, which ensures that mammalian cells remain proliferative by providing a chemical environment that mimics the natural tissue's chemical environment to the mammalian cells.

[0010] A commonly used method is to coat a micro-well cell plate with a polymer having carboxylic acid groups, since such groups are capable of (covalently) binding to fibronectin, thereby promoting the stabilization of mammalian cells on the membrane within the fluidic device as the polymer undergoes bio-linkage with fibronectin. However, this method is not without its drawbacks. First, the fluid flow through the fluidic device can partially remove the aforementioned polymer, which impairs the ability to evaluate the mammalian material at the desired time point (since at least some of this material may have been lost). In another method, the membrane is treated with UV or plasma to generate binding sites for fibronectin on the membrane. However, this method has the drawback that it is quite difficult to avoid non-uniform distribution of such binding sites, which again hampers the evaluation of the assay results.

[0011] Secondly, it is difficult to fabricate membranes incorporating such biocompatible coatings, which makes the fabrication of such fluidic devices quite expensive and cumbersome. For example, thin film technology (which is expensive) is typically required to fabricate such membranes, and integrating such membranes within the fluidic device without leakage is not straightforward. Additionally, in cases where the membrane itself is not made of a biocompatible material, it is usually necessary to provide the membrane with multiple cell-sized pores with small spacing to promote cell growth (which is also difficult to achieve).

[0012] Dongeun Huh et al. disclosed an example of the microfabrication of such artificial organ wafers based on thin film technology in Nature Protocols (Volume 8, Issue 11, 2013, pages 2135 - 2157). In this protocol, micro-engineering techniques were used to fabricate a multi-layer microfluidic device that includes two parallel elastomeric microchannels separated by a thin porous flexible membrane and two fully-height but evacuated chambers on either side, and the production of this device takes approximately three and a half days. The overall microfabrication process includes over 100 steps, where a large number of the steps are critical steps. This clearly demonstrates the complexity of such a fabrication process.

[0013] An improvement to this laborious method is disclosed in WO 2018 / 021906 A1, which discloses a silicon-based (PDMS) fluid device capable of using a coating such as collagen to render this PDMS fluid device biocompatible. Such a device can be fabricated in only a few manufacturing steps, but this device still has the drawback that it needs to be coated with a biocompatible material, which, as described above, can erode from the polymer surface when exposed to a fluid flow, resulting in an unwanted loss of cell material from the surface of the fluid device. The above problem is solved in WO 2018 / 021906 A1 by treating the surface of the device with plasma and then functionalizing the surface with 3-aminopropyltriethoxysilane and glutaraldehyde before coating the functionalized surface with collagen, and removing unbound collagen by flushing the microchannels of the fluid device with EGM-2 (Endothelial Cell Growth Medium-2 BulletKit, sold by Lonza). Therefore, a large number of processing steps are still required to provide a biocompatible fluid device.

[0014] The applicant has disclosed a solution to this problem in WO 2019 / 015988 A1. In this application, a material for binding to cell culture proteins is disclosed. The material contains a bulk-modified elastomer comprising a plurality of fatty acid moieties covalently bound to an elastomeric body, wherein the carboxylic acid groups of the moieties can be used to provide such binding on the surface of an object formed from the bulk-modified elastomer by bulk-modifying the elastomer with fatty acid moieties in a mold according to the shape of the object and forming a polar inner surface such that the hydrophilic carboxylic acid groups of the fatty acid moieties are attracted to the polar inner surface. It has been demonstrated in this prior application that biocompatible materials such as fibronectin can be covalently bound to the surface carboxylic acid groups of the bulk-modified elastomer while maintaining their cell culture properties.

[0015] There is a need for further improvement in cell culture plates and their manufacture. SUMMARY OF THE INVENTION

[0016] The present invention at least partially meets this need, and the present invention seeks to provide a plurality of aspects defined by the independent claims, each of which can use or provide an elastic material for culturing cells without covalently modifying the surface with cell culture proteins.

[0017] According to one aspect, there is provided a cell culture plate according to claim 1.

[0018] The term substrate or cell culture plate suitable for culturing cells means any device or surface intended for cell culture. The substrate defined in this way is intended herein to embody the use of an elastomeric material as defined in the field of cell culture. Cell culture means including, for example, keeping cells alive and / or proliferating the number of cells and / or differentiating cells. A residue is a part of a precursor molecule that carries an acidic group in either form and an olefinic bond capable of participating in a reaction to covalently bond the precursor to the elastomeric body. A residue is the part of the precursor remaining after such a reaction.

[0019] It has been found that when this surface is brought into direct contact with a cell culture medium in the presence of cells, such cells can be cultured without any further surface preparation. Thus, the step of covalently modifying the material surface with cell culture proteins can be omitted, thereby facilitating the use of such a surface. While not wishing to be bound by theory, it is believed that the acidic groups available at the surface are playing an important role in this cell culture function. After all, reference elastomers without bulk modification do not exhibit such cell culture properties. It has also been found that the acidic groups can exist at the surface in either free form or conjugate base form, where the free form means that the acidic group carries a proton capable of dissociating, and the conjugate base form means that in addition to the proton, there is also a positive counterion. While not wishing to be bound by theory, it is believed that in a cell culture medium (which is usually buffered at a pH of around 7.2), such acidic groups having a pKa value usually less than 5.5 are generally present to a large extent in dissociated form, regardless of the original form in which they are available at the surface.

[0020] The acidic groups preferably have a pKa less than 5, such that most of the groups are in dissociated form. In an alternative, the pKa is less than 4.5 or even less than 4. The pKa can be in the range of 5 to 1 or 5 to 2 or 5 to 3.

[0021] Importantly, a cell culture plate having any specific shape can now be effectively prepared in one step by mixing suitable precursors for forming a bulk-modified elastomer and reacting these precursors within a reaction vessel (e.g., the reaction vessel of an injection molding device) to form the cell culture plate and shape and prepare it such that it can be used for cell culture without further adjustment. This can also make it easier and more consistent to remanufacture a series of such devices.

[0022] In some embodiments, the one or more acidic groups are: phosphorous-based acidic groups, sulfur-based acidic groups, and carboxylic acid groups, or a mixture of two or more of these items. Phosphoric acid groups and their conjugate bases are preferred over sulfonic acid groups, and carboxylic acid groups are preferred over phosphoric acid groups. Residues carrying these groups are more easily used during bulk modification because, during such a modification method, their precursors have acidic groups in the form of conjugate bases with metal counterions, and such residues mix better with other components to form an elastomer. The acidic groups can be selected according to the desired (as described above) pKa.

[0023] In some preferred embodiments, the residue comprises or consists of the following aliphatic moiety, the aliphatic moiety comprising 3 or more and less than 50 carbon atoms, the one or more acidic groups being covalently bound to the aliphatic moiety, and the aliphatic moiety being covalently bound to the elastomer bulk. The aliphatic moiety can be linear or branched. It can include one or more carbon-carbon double bonds or carbon-carbon triple bonds or aryl units or phenyl units. The aliphatic moiety can include cyclic units, for example, cyclohexyl or cyclopentyl or other cyclic units. The aliphatic moiety is preferably a saturated hydrocarbon moiety. The aliphatic moiety preferably consists only of carbon atoms and hydrogen atoms.

[0024] In some embodiments, the aliphatic moiety is a linear chain coupled to the elastomer bulk at the end. In some embodiments, the aliphatic moiety also does not include carbon-carbon triple bonds to increase the flexibility of the residue.

[0025] In some embodiments, the residues within the bulk-modified elastomer can be different from each other, each residue being selected as defined herein. In some embodiments, at least a portion of the residues can be bound to the elastomer bulk via two covalent bonds. This can be caused by two residue precursors having olefinic bonds for participating in the modification reaction.

[0026] In some embodiments, the moiety includes at least 5 carbon atoms, and more preferably includes at least 10 carbon atoms. Preferably, the aliphatic moiety includes less than 40 or less than 30 carbon atoms. A linear moiety is preferred, but this is not essential per se. The number of carbon atoms in such a moiety is preferably between 5 and 30, more preferably between 5 and 20 or between 5 and 15. The moiety and / or the chain can have one or more aryl groups. One or more of the acidic groups can be directly attached to an aryl group (for example, a benzene ring).

[0027] In some embodiments, the residue is the remaining portion of an unsaturated fatty acid precursor covalently bound to the elastomer bulk via the reaction of one or more (if any) of its vinyl groups (carbon-carbon double bonds).

[0028] For example, the unsaturated fatty acid residue is one or more residues of a fatty acid selected from the group consisting of myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, octadecenoic acid, linoleic acid, elaidic linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

[0029] In some embodiments, the residue is the remainder of a precursor having a linear or branched alkyl chain carrying one or more acidic groups and at least one ethenyl group (carbon-carbon double bond). Preferably, the last such ethenyl group is the ethenyl group of the terminal chain. The terminal ethenyl group can provide enhanced reactivity as compared to non-terminal ethenyl groups during the formation of the elastomeric body during the manufacturing process. A straight chain having one terminal ethenyl group and one acidic group (e.g., a carboxyl group) is a preferred example. In this case, 5 to 15 carbon atoms can be present in the chain.

[0030] In some embodiments, the elastomeric body comprises a siloxane or polybutadiene backbone. Polybutadiene and polyisoprene are examples of polydienes. Polydimethylsiloxane is an example of a polyoxane (siloxane). Siloxanes have higher water permeability and are more transparent, thus allowing easier optical inspection of cell cultures.

[0031] In some embodiments, the residue is covalently bound to the elastomeric body due to the reaction between the unsaturated carbon-carbon bond of the precursor of the residue and the vinyl functional group or hydride functional group of the elastomeric body. In polydienes, this reaction is the ethenyl group, while in siloxanes, this binding typically occurs with the silyl hydride functional group.

[0032] The concentration of the residue in the body-modified elastomer or the concentration of the corresponding precursor for the residue in the mixture of precursors used to form the elastomer is preferably in the range of 2.10 -4 to 2.10 -2 mol / kg by weight in the total weight of the elastomer. It has been found that lower concentrations result in non-reproducible cell culture properties, while higher concentrations are difficult to achieve because incomplete mixing of the precursors and / or incomplete reaction between the precursors during the manufacture of the body-modified material can result in non-reproducible or degraded culture properties.

[0033] In some embodiments, the body-modified elastomer can be obtained or is obtained by: mixing a vinyl-functionalized elastomer or a hydride-functionalized elastomer or at least one of its precursors with a residue precursor having a concentration in the range of 2.10 -4 to 1.10 -2 mol / kg by weight in the total weight of the body-modified elastomer, and heating the mixture.

[0034] In some embodiments, the heating is carried out in a reaction vessel having a metal-metal oxide inner surface. It is believed that this causes acidic groups (in conjugate base form) to be at least partially associated with the oxide surface in the reaction vessel, such that during the polymerization reaction a modified bulk is formed and at least a portion of the acidic groups can be obtained at the surface of the material.

[0035] In a further aspect, there is provided a cell culture plate as described herein, wherein the substrate comprises a fluid device module, the fluid device module comprising flow channels extending on a membrane comprising the material.

[0036] In some embodiments, the cell culture plate comprises a first major surface and a second major surface, the first major surface comprising a first recessed structure defining a first flow channel, the second major surface being opposite the first major surface and comprising a second recessed structure defining a second flow channel; wherein the membrane separates the first flow channel from the second flow channel.

[0037] In some embodiments of the fluid device, the cell culture plate of the fluid device is a monolithic fluid device.

[0038] According to one aspect of the invention, there is provided a use of a material having an outer surface for culturing cells thereon, the material comprising a bulk-modified elastomer, the bulk-modified elastomer having an elastomer bulk and a plurality of residues, each residue comprising one or more acidic groups in free form and / or conjugate base form, the plurality of residues being covalently bound to the elastomer bulk such that a portion of the one or more acidic groups can be obtained on the outer surface, the use comprising contacting the outer surface with cells to be cultured and a cell growth medium. As explained for the cell culture plate embodying this use, this use was not known or apparent when such a material offered significant advantages.

[0039] According to yet another aspect, there is provided a method of culturing cells, comprising the steps of:

[0040] using a cell culture plate according to any one of the preceding claims, optionally, the substrate being a molded substrate; and

[0041] culturing cells directly on the exposed surface of the article.

[0042] In some embodiments of the method, the cell culture plate is a cell culture plate as defined herein.

[0043] In some embodiments, the cell culture method is part of a drug testing method, the drug testing method comprising the following additional steps:

[0044] Expose the cultured cells to the drug to be tested through another flow channel of the pair of flow channels of the fluid device; and

[0045] Monitor the response of the cultured cells to the drug to be tested.

[0046] Other non-limiting embodiments

[0047] In some embodiments, the bulk-modified elastomer is obtained by forming, in a mold having a polar inner surface, a composition comprising a vinyl-functionalized elastomer or a hydride-functionalized elastomer or at least one precursor thereof, a free or saponified unsaturated fatty acid in the range of 0.5-5% by weight based on the total weight of the vinyl-functionalized elastomer or hydride-functionalized elastomer or at least one precursor thereof, and a crosslinking catalyst; and subjecting the vinyl-functionalized elastomer or hydride-functionalized elastomer to bulk modification by covalently bonding the free or saponified unsaturated fatty acid to the elastomer body through a crosslinking reaction between the vinyl group or hydride group of the elastomer and the unsaturated carbon-carbon bond of the unsaturated fatty acid in the mold to obtain the material.

[0048] In some embodiments, there is provided a use of a material for culturing cells, the material comprising a bulk-modified elastomer having a Shore hardness (DIN EN ISO 868) in the range of Shore 00 20 to Shore A 80 and comprising a plurality of fatty acid moieties covalently bonded to the elastomer body, wherein the carboxylic acid groups of the moieties can be obtained on the outer surface of the material to provide the bonding, and wherein the bulk-modified elastomer is obtained by forming, in a mold having a polar inner surface, a composition comprising a vinyl-functionalized elastomer or a hydride-functionalized elastomer or at least one precursor thereof, a free or saponified unsaturated fatty acid in the range of 0.5-5% by weight based on the total weight of the vinyl-functionalized elastomer or hydride-functionalized elastomer or at least one precursor thereof, and a crosslinking catalyst; and subjecting the vinyl-functionalized elastomer or hydride-functionalized elastomer to bulk modification by covalently bonding the free or saponified unsaturated fatty acid to the elastomer body through a crosslinking reaction between the vinyl group or hydride group of the elastomer and the unsaturated carbon-carbon bond of the unsaturated fatty acid in the mold to obtain the material.

[0049] Surprisingly, a relatively soft elastomer has been discovered, i.e., an elastomer having a Shore hardness within a certain range, which ranges from Shore 00 20 to Shore A 80 (or Shore A 2 - 80). When the bulk modified with unsaturated fatty acids previously disclosed in WO 2019 / 015988 A1 can be used as a biocompatible material, cell materials can be directly cultured on such a compatible biomaterial without an extracellular matrix (e.g., fibronectin). At this time, the amount of unsaturated fatty acids is in the range of 0.5 - 5 (wt%) based on the weight in the total weight of the elastomer or at least one of its precursors. Without wishing to be bound by theory, it is believed that when the concentration of unsaturated fatty acids is lower than 0.5 wt%, the density of carboxylic acid groups or carboxylate groups in the case of saponified unsaturated fatty acids is too low to make the outer surface on which the cells of the elastomer are to proliferate hydrophilic enough to achieve the proliferation conditions. And when the concentration is higher than 5 wt%, some unreacted unsaturated fatty acids can leach out from the bulk modified elastomer and poison the cells adhered to the outer surface of the elastomer. Therefore, it is expected that the bulk modified elastomer has a covalent bonding fraction of free or saponified fatty acid groups in the range of 0.5 - 5 wt% based on the total weight of the elastomer, assuming that a complete conversion of the crosslinking reaction between the elastomer (or one or more of its precursors) and the unsaturated fatty acids in free or saponified form is expected within this range.

[0050] A bulk modified elastomer having a hardness within the range of Shore 00 20 to Shore A 80 is selected to ensure that the elastomer has sufficient flexibility to promote cell proliferation. Because, for example, it is well known per se that the softness of a cell carrier material is crucial for stem cell growth (and differentiation) because the tissues obtained from stem cell growth want to move, e.g., (heart) muscle tissue, lung tissue, intestinal tissue, and vascular tissue. Some organs need to grow in a movable carrier (such as cardiac muscle). Therefore, when the bulk modified elastomer material has a Shore A hardness exceeding 80, the material becomes insufficiently stretchable to promote the required cell proliferation directly on its functionalized surface. In this case, the use of a scaffold or extracellular matrix material will be required to provide the developed cell culture with the required flexibility.

[0051] In addition, the unsaturated fatty acids used in the bulk modification of the elastomer can be used in free form or saponified form (e.g., as sodium salt). The advantage of using the saponified form of unsaturated fatty acids is that the risk of catalyst poisoning by the protons of the unsaturated fatty acids is avoided. At the same time, it is surprisingly found that cell cultures will also proliferate on the bulk modified elastomer carrying a suitable counterion (e.g., Na + or K +) on the outer surface of the carboxylate group. Thus, this has the further advantage that saponification does not have to be reversed before using the bulk-modified elastomer for cell culture purposes.

[0052] Thus, in this way, it is possible to fabricate materials for cell culture in a small number of steps. For example, in some embodiments, materials for cell culture can be fabricated in a single step by combining an elastomer with a fatty acid in a coating (spin coating, dip coating, spray coating, dispensing) or an injection molding process (wherein crosslinking of the carbon-carbon double bonds of the elastomer with the fatty acid can be achieved without significant epoxidation of the carbon-carbon double bonds of the fatty acid due to limited exposure to environmental oxygen in the spin coating or injection molding process). Thus, a material is provided in which the elastomer is bulk-modified with a fatty acid moiety in which carboxylic acid groups or carboxylate groups of (saponified) fatty acids can be obtained for direct binding to harvested cells. Furthermore, by controlling the curing process and / or the nature of the mold in which the material is formed, such carboxylic acid groups or carboxylate groups are also present on the outer surface of the material, thereby providing a substantially uniform distribution of the carboxylic acid groups or carboxylate groups on such outer surface, which makes the material particularly suitable as a membrane material for fluid devices because each cross-section of the material will exhibit the same surface properties, in contrast to membrane materials onto which an anchor for a biocompatible material is to be grafted or otherwise formed as previously explained. Additionally, since only a certain fraction of the carbon-carbon double bonds of the elastomer are typically consumed in such crosslinking reactions, the materials of the present invention retain the elastomeric properties of the elastomer, which improves the suitability of the materials of the present invention for fluid devices and facilitates slicing or otherwise cutting the materials of the present invention for research purposes.

[0053] Preferably, each fatty acid moiety in the free or saponified fatty acid moiety is covalently bound to the elastomer bulk through a crosslinking reaction between the vinyl functional group or hydride functional group of the elastomer and the unsaturated carbon-carbon bond of the unsaturated fatty acid to ensure that the number of carboxylic acid groups or carboxylate groups available for binding to biocompatible materials can be optimized.

[0054] The unsaturated fatty acid can be any suitable unsaturated fatty acid. In an exemplary embodiment, the unsaturated fatty acid is selected from the following: myristoleic acid, palmitoleic acid, petroselinic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, undecylenic acid, and docosahexaenoic acid. Linoleic acid is specifically mentioned.

[0055] Similarly, the elastomer can be any suitable elastomer. In an exemplary embodiment, the elastomer comprises a polybutadiene backbone or a siloxane backbone. In the case where the elastomer includes a siloxane backbone, prior to crosslinking between the unsaturated fatty acid molecules and the elastomer molecules, at least a fraction of the carboxylic acid groups within the siloxane backbone can be saponified to saponified products of the carboxylic acid groups, and the elastomer molecules can be required to prevent the crosslinking catalyst from being deactivated by the protons of the carboxylic acid groups of the unsaturated fatty acid molecules. Similarly, polybutadiene can be crosslinked with the saponified unsaturated fatty acid because, as previously explained, the saponified product of the unsaturated fatty acid does not significantly affect the cell culture properties of the bulk-modified material.

[0056] In a set of embodiments, the composition comprises the free or saponified unsaturated fatty acid in the range of 0.5 - 2% by weight based on the total weight of the vinyl-functionalized elastomer or hydride-functionalized elastomer or at least one of its precursors, because it has been found that when the amount of the free or saponified unsaturated fatty acid added to the bulk of the elastomer is within this range, particularly good cell proliferation is achieved. For example, when the composition comprises the free or saponified unsaturated fatty acid in an amount of approximately 1% by weight based on the total weight of the vinyl-functionalized elastomer or hydride-functionalized elastomer or at least one of its precursors, excellent cell proliferation is achieved directly on the outer surface of the bulk-modified elastomer.

[0057] According to yet another aspect, there is provided a fluid device module, which includes a flow channel extending on a membrane, and the membrane comprises the material of the present invention according to any one of the embodiments described herein. Such a fluid device module can include a first major surface and a second major surface, the first major surface includes a first recessed structure defining a first flow channel, the second major surface is opposite to the first major surface and includes a second recessed structure defining a second flow channel; wherein, the membrane separates the first flow channel from the second flow channel. In a preferred embodiment, the fluid device module is a monolithic fluid device module, which has the advantage that the device module can be manufactured with a small number of processing steps (e.g., a single processing step by injection molding).

[0058] The membrane can include a plurality of pores or grooves extending through the membrane. Such pores or grooves are preferably cell-sized to prevent cells from passing through the membrane.

[0059] In an embodiment, the first flow channel and the second flow channel can be accessed through corresponding diaphragms, such that individual flow channels can be accessed separately without the risk of cross-contamination.

[0060] According to yet another aspect, there is provided a fluid device, the fluid device comprising: a fluid device module of any one of the embodiments described herein; and a pair of cover plates for fluid-sealing the fluid device module. The cover plates may be arranged such that one of the cover plates covers the first major surface, thereby sealing the first fluid passage, and the other cover plate of the cover plates covers the second major surface, thereby sealing the second fluid passage. Such a fluid device can be manufactured in a simple manner because the fluid device module can be formed in a small number of processing steps as previously explained, and due to the flexibility of the fluid device module, such a fluid device module is robust against leakage.

[0061] According to yet another aspect, there is provided a method of culturing cells, the method comprising: providing a molded article formed of an elastomeric material modified by the body of any one of the embodiments described herein, the article including an outer surface of the material as one of the exposed surfaces of the material; and culturing the cells directly on the exposed surface. According to this method, the cells can proliferate directly on the exposed surface of such an article (e.g., the fluid device module as described above), without attaching an extracellular matrix (e.g., fibronectin, etc.) to the exposed surface of the article, thereby significantly simplifying the cell culture process.

[0062] According to yet another aspect, there is provided a method of drug testing, the method comprising providing a fluid device module according to an embodiment; directly applying harvested cells to the surface of the membrane of the fluid device module; culturing the harvested cells on the surface; forming a fluid device with the prepared fluid device module; feeding the cultured cells through one of a pair of flow channels of the fluid device; exposing the cultured cells to a drug to be tested through the other of the pair of flow channels of the fluid device; and monitoring the response of the cultured cells to the drug to be tested. Such a method of drug testing can be deployed in a simple and straightforward manner, particularly in terms of preparing the fluid device to be used in such a method of drug testing, thereby providing a significant simplification of the existing methods of drug testing (wherein preparing such a fluid device is usually very cumbersome).

[0063] Monitoring the response of the cultured cells to the drug to be tested can include: stabilizing and fixing the cultured cells within the fluid device, and sectioning the fluid device module to obtain sections for microscopic evaluation, the sections including at least a portion of the stabilized and fixed cultured cells. According to the teachings of the present invention, since the cultured cells are uniformly distributed on the membrane surface of the fluid device module (due to the bulk modification of the elastomer with free or saponified unsaturated fatty acids as previously explained), generating such sections for microscopic evaluation is no longer critical or subject to variable results.

[0064] Alternatively, in a preferred embodiment, a confocal microscope can be used to monitor the response of the cultured cells within the assembled fluid device. This facilitates real-time monitoring of such response. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Embodiments of the present invention are described in more detail with reference to the accompanying schematic drawings, which are not drawn to scale, in which:

[0066] Figure 1 Depicts the bulk modification of an elastomer with an unsaturated fatty acid according to an embodiment;

[0067] Figure 2 Depicts a perspective view of a fluid device module according to an embodiment;

[0068] Figure 3 Depicts another perspective view of a fluid device module according to an embodiment;

[0069] Figure 4 Depicts a perspective view of a fluid device module according to another embodiment;

[0070] Figure 5 Depicts an exploded perspective view of a fluid device according to an embodiment;

[0071] Figure 6 Depicts an exploded perspective view of a fluid device according to another embodiment;

[0072] Figure 7 Depicts an exploded perspective view of a fluid device according to yet another embodiment;

[0073] Figure 8 Depicts an exploded perspective view of a fluid device according to yet another embodiment;

[0074] Figure 9 Is a microscopic image of a cell culture developed on a bulk-modified elastomer according to an embodiment;

[0075] Figure 10is a microscopic image of a cell culture developed on an elastomer without such bulk modification;

[0076] Figure 11 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 1 day of development;

[0077] Figure 12 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 2 days of development;

[0078] Figure 13 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 7 days of development;

[0079] Figure 14 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 4 days of development;

[0080] Figure 15 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 4 days of development;

[0081] Figure 16 is a graph showing the measured contact angles for substrates containing siloxane modified with 1 wt% linoleic acid. The vertical axis is the contact angle (in degrees). Four sets (each set including two bars) represent the average contact angles for 4 batches of substrates. The left bar of each set represents the contact angle at pH = 4, and the right bar of each set represents the contact angle at pH = 10. The set of bars on the far left represents unmodified siloxane, while the other bars represent the modified substrates used in cell culture examples 1, 2, and 3, respectively. Detailed Description

[0082] It should be understood that the same reference numerals are used throughout the drawings to indicate the same or similar parts.

[0083] Figure 1 Depicts the reaction between the double bond of the precursor of residue 20 and the double bond of the polymer backbone 10 of the bulk-modified elastomeric material for making some embodiments of the present invention. In this example, the reaction occurs between the fatty acid precursor residue linolenic acid and a vinyl-functionalized elastomer 10 in the form of polybutadiene (hereinafter simply referred to as elastomer). Such a reaction can be catalyzed with a suitable catalyst (e.g., peroxide). Although in this Figure 1 example, polybutadiene 10 is shown reacting with saponified linoleic acid (wherein R is, for example, an alkali metal, such as lithium, sodium, or potassium ions), it is apparent that other double bond precursor residues and other double bond backbone polymers can also be used in this type of reaction.

[0084] It has been found that such a reaction can provide a bulk-modified elastomer, wherein at least a certain fraction of the carboxylic acid groups of the saturated fatty acid molecules 20 participating in the reaction with the elastomer 10 are present on the outer surface of the bulk-modified elastomer, such that these carboxylic acid groups can be used to directly immobilize a cell culture on the outer surface of the bulk-modified elastomer 10. Accordingly, there is no longer a need for the step of binding a protein to such carboxylic acid groups to facilitate cell immobilization, and this saves a significant amount of time and effort and opens up new opportunities for defining cell culture plates incorporating the material. For example, by using a suitable reaction vessel, elastomer formation and elastomer shaping can now be accomplished in one step. In a preferred example, such a reaction vessel is a mold, e.g., a mold of an injection molding device. It should be noted that Figure 1 The example of

[0085] Preferably, the elastomer 10 is soft or flexible to facilitate cell-initiated movement. For this reason, the elastomer 10 can generally have a certain range of Shore hardness determined according to the DIN EN ISO 868 standard, which ranges from Shore 00 20 (i.e., Shore A 2) to Shore A 80, i.e., a Shore A hardness of 2 - 80. Suitable elastomers include polyenes, e.g., polybutadiene and siloxanes.

[0086] For example, the unsaturated fatty acid 20 can be a free unsaturated fatty acid (in which case, R = H) or can be a saponified fatty acid (in which case, R = Na + or K + )(and the COO group is COO− or a carboxylate anion group). Such a saponification reaction can be achieved in any suitable manner (e.g., using a concentrated NaOH or KOH solution). Since such a saponification reaction is well known per se, this will not be further explained in detail here for the sake of brevity.

[0087] Importantly, there is no need to reverse the saponification of the unsaturated fatty acid residues in the bulk-modified elastomer, since it has surprisingly been found that cell proliferation can also occur on the surface when the outer surface of the bulk-modified elastomer 10 is functionalized with saponified carboxylic acid groups (i.e., carboxylate groups having a counterion such as Na + or K + as a counterion), as will be further explained in detail below.

[0088] It may be considered surprising that carboxylic acid groups or carboxylate groups are present on the outer surface of the bulk-modified elastomer 10, since the elastomer 10 can be non-polar in nature, especially when the elastomer 10 is dissolved in a non-polar solvent. In such a non-polar environment, the unsaturated fatty acid molecules 20 tend to form micelles in which the carboxylic acid moieties of these molecules rotate inwards within such micelles, which typically causes such carboxylic acid moieties to end up within the bulk of the modified elastomer. Additionally, it is well known per se that the carbon-carbon double bonds of such unsaturated fatty acids 20 are prone to rapid epoxidation in the presence of oxygen, which reduces the ability of these compounds to react in a crosslinking reaction with the elastomer 10.

[0089] Embodiments of the present invention are based on the insight that if a reaction mixture comprising the elastomer 10 and the unsaturated fatty acid 20 in saponified form is brought into contact with a polar surface, this promotes the orientation of the carboxylic acid groups of the fatty acid molecules along such polar surface, such that the outer surface of the bulk-modified elastomeric material exhibits a high density and uniform distribution of these carboxylic acid groups. Additionally, it has been found that when the elastomer 10 is bulk-modified in this manner during an injection molding process at elevated temperatures (e.g., at a temperature in the range of 120 - 240 °C), the epoxidation of the carbon-carbon double bonds of the unsaturated fatty acid molecules 20 does not significantly interfere with the bulk modification of the elastomer 10, i.e., does not significantly inhibit the reaction between the unsaturated fatty acid molecules 20 and the elastomer 10. Without wishing to be bound by theory, it is believed that during such an injection molding process, molecular oxygen is substantially absent in the reaction mixture within the injection molding apparatus, thus inhibiting the epoxidation of these unwanted carbon-carbon double bonds.

[0090] To obtain an elastomer 10 on which cells can be directly cultured, use is made of 2.10 by weight in the total weight of the elastomer 10 or by weight in the total weight of one or more precursors of the elastomer 10 -4 to 1.10 -2The elastomer 10 is bulk-modified with an amount of unsaturated fatty acid 20 in free or saponified form in the range of from

[0091] While Figure 1 Examples are described for poly-diene type bulk elastomers, but other elastomers can also be used. Preferably, such elastomers also have the desired Shore hardness.

[0092] Thus, elastomers that can be used are, for example, homopolymers, copolymers, block copolymers, terpolymers, block terpolymers, etc. Particularly suitable elastomers can be selected from polyenes, for example, polybutadiene or isoprene or chloroprene. In the case of using polyenes, any suitable polymerization process can be used to manufacture the polyene, after which, as referenced Figure 1The coupling described above. Particularly preferred is polybutadiene formed in a Nd- or Li-catalyzed polymerization reaction, as it is well-known per se. This polybutadiene has a low degree of branching (usually below 5%) and a low polydispersity (Mw / Mn) of approximately 2. Since Li-catalyzed polybutadiene has a high degree of 1,2-vinyl content (about 11%), Li-catalyzed polybutadiene is particularly preferred. However, other types of polybutadiene (e.g., polybutadiene obtained from Co-, Ni- or Ti-catalyzed polymerization reactions) can alternatively be used. In the case of using a polyene such as polybutadiene as elastomer 10, a peroxide catalyst can be used to catalyze the crosslinking reaction between elastomer 10 and unsaturated fatty acid 20. In another advantageous embodiment, Li-catalyzed polybutadiene is used, which has a certain degree of branching in the 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 unsaturated fatty acid 20 is increased. It should be noted that, for the sake of completeness, how to control the degree of branching in the synthesis of polybutadiene is well-known per se, such that this will not be further explained merely for the sake of brevity.

[0093] Another class of desired elastomers are siloxanes (polysiloxanes), as they are suitable for injection molding processes. Such siloxanes can include a polydimethylsiloxane (PDMS) backbone (which includes vinyl moieties) to facilitate crosslinking via a Pt-catalyzed (with (polymethyl)hydrosiloxane) addition reaction. As an alternative or supplement to this crosslinking with (polymethyl)hydrosiloxane, the vinyl-functionalized PDMS backbone can react with unsaturated fatty acid 20.

[0094] Such elastomers in the form of siloxanes can be formed by the following crosslinking reactions: crosslinking reactions between vinyl-functionalized linear or branched siloxane monomers or oligomers (e.g., T-branched or Q-branched siloxane monomers or oligomers) and linear hydride-functionalized siloxane monomers or oligomers, crosslinking reactions between hydride-functionalized linear or branched siloxane monomers or oligomers (T-branched or Q-branched siloxane monomers or oligomers) and linear vinyl-functionalized siloxane monomers or oligomers or mixtures of vinyl-functionalized and hydride-functionalized linear or branched siloxane monomers or oligomers (e.g., mixtures of T-branched or Q-branched siloxane monomers or oligomers and / or linear hydride-functionalized and vinyl-functionalized siloxane monomers or oligomers).

[0095] For example, a two-component siloxane can be formed by a crosslinking reaction between linear component 1 and linear component 2, and each linear component can correspond to the following general formula:

[0096]

[0097] In Component 1, R1 and R2 are each independently selected from C1-C3 alkyl groups, and R3-R8 are each independently selected from C1-C3 alkyl groups and vinyl groups, provided that at least one of R3-R5 and at least one of R6-R8 are vinyl groups. Preferably, at least three of R3-R8 are vinyl groups. In all embodiments, n can range from 100 to 200,000. In a particular embodiment of Component 1, each of the groups R1-R8 that is an alkyl group is a methyl group, i.e., Component 1 is a vinyl-functionalized PDMS with terminal vinyl groups.

[0098] In Component 2, R1 is hydrogen, R2 = C1-C3 alkyl, and R2-R8 are each independently selected from C1-C3 alkyl groups or hydrogen, provided that at most one of R3-R5 and at most one of R6-R8 are hydrogen, and n can have any suitable value, e.g., n = 3 - 1000 or more specifically n = 3 - 10. In a particular embodiment of Component 2, none of the groups R3-R8 are hydrogen. In another particular embodiment of Component 2, each of R2-R8 is a methyl group.

[0099] Component 2 is generally used as a crosslinker for Component 1. This crosslinking reaction, which is typically catalyzed by Pt, is well-known per se, see for example WO 2009 / 147602 A2, and thus will not be explained in detail for the sake of brevity.

[0100] Bulk modification of such siloxanes can be accomplished by forming an elastomer in the presence of unsaturated fatty acid 20, and a covalent bond between the siloxane and the unsaturated fatty acid 20 can be formed through the reaction between the hydride functional groups of the (crosslinked) siloxane and the unsaturated fatty acid 20 as suggested by the following reaction mechanism:

[0101]

[0102] To prevent catalyst inhibition by the protons of the carboxylic acid groups of the unsaturated fatty acid 20 during this crosslinking reaction, the unsaturated fatty acid can be used in its saponified form (e.g., as the sodium salt of such unsaturated fatty acid) for the crosslinking reaction with the siloxane elastomer. In some embodiments, if desired, the reaction product (the bulk-modified elastomer) can subsequently be treated with a protonic acid (e.g., HCl, etc.) in order to restore the carboxylic acid groups on the surface of the bulk-modified elastomer, but this is not necessary in cases where cell culture is to be directly performed on the surface of the bulk-modified elastomer, since it has been shown that cells also proliferate when the polar surface groups are carboxylate anions (i.e., saponified carboxylic acid groups). Any siloxane elastomer having free silyl hydride groups available for reaction with the olefinic bonds of the residue precursor can be used for this elastomer bulk modification.

[0103] It should be noted that elastomer formation need not be initiated prior to the modification reaction. This can be done in some embodiments, but preferably, the modification is carried out at least in part during elastomer formation by mixing appropriate elastomer-forming components with the modification precursor residues.

[0104] Alternatively, a siloxane backbone such as a (polymethyl)hydrogensiloxane backbone can be crosslinked with a rubbery polymer such as polybutadiene and polyisoprene, where unsaturated fatty acids are incorporated into the crosslinked product. This can be done, for example, to tune the water permeability of the final material, since siloxanes generally have a rather open structure while such rubbery polymers have a rather closed structure, such that the openness (i.e., water permeability) of the crosslinked product can be tuned by the ratio of siloxane to rubbery polymer therein. In this way, the properties of the material according to this embodiment can be optimized for cell / protein interactions to construct a good scaffold. For example, in the case where the elastomer is a silicone-based (e.g., PDMS-based) elastomer, a material with high permeability to water and other compounds (e.g., drugs) is obtained, which promotes the maximized perfusion of these compounds into the cells on the membrane of the fluid device module 100. However, in cases where real-time monitoring of the cell consumption of drugs is required, a polyene-based material (e.g., a polybutadiene-based material) that is not permeable to water and drugs can preferably be used, since the perfusion rate of these compounds to the cells can be controlled by the density of the pores through the membrane.

[0105] In an embodiment, the polysiloxane can be formed from a multi-component starting material as previously explained to prevent premature crosslinking of the polysiloxane. Such multi-component starting materials are well known per se; for example, a kit of such multi-component starting materials is sold by Wacker Chemie AG of Munich, Germany (trade name Elastosil).

[0106] Linoleic acid has been used as a precursor for acidic groups carrying residues, and the bulk-modified elastomer is modified using this precursor. It should be understood that this is only a non-limiting example, since other acidic groups carrying residues can also be used, which will be described below. Unless otherwise stated, the residues and their precursors below will also achieve the advantages of linoleic acid described above.

[0107] Thus, for example with respect to unsaturated fatty acid 20, any suitable unsaturated fatty acid can be used for the purpose of reacting it with elastomer 10. For example, unsaturated fatty acid 20 can be selected from myristoleic acid, palmitoleic acid, petroselinic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, undecylenic acid, and docosahexaenoic acid.

[0108] More generally, in the context of the present application, in the case of referring to examples of Figure 1 unsaturated fatty acid 20, it should be understood that this is intended to refer to any organic compound that includes one or more acidic groups in free form or conjugate base form and includes at least one carbon-carbon double bond.

[0109] Typically, the residues in the bulk-modified elastomer are obtained from the reaction of a residue precursor (for modifying the bulk-modified elastomer) with the elastomer bulk or its precursor. In this sense, fatty acids are just one type of such residue precursor. There are also other types and other types can also be used, as long as they have at least one carbon-carbon double bond and one or more acidic groups, and when used in combination with silicone chemistry, such acidic groups in conjugate base form are required. In polyene chemistry, such conjugate base form is not required per se.

[0110] In some preferred embodiments, the residue precursor comprises or consists of the following aliphatic moiety, which aliphatic moiety includes 3 or more and less than 50 carbon atoms and at least one carbon-carbon double bond, wherein one or more acidic groups are covalently bonded to the aliphatic moiety. The aliphatic moiety can be linear or branched. It can include one or more (e.g., 2, 3, or 4) carbon-carbon double bonds or carbon-carbon triple bonds (preferably only double bonds) or aryl units or phenyl units. The aliphatic moiety can include cyclic units, such as cyclohexyl or cyclopentyl, etc. The aliphatic moiety preferably includes only carbon atoms and hydrogen atoms. The aliphatic moiety can be a saturated hydrocarbon moiety. In some embodiments, the aliphatic moiety also does not include carbon-carbon triple bonds to improve the flexibility of the residue.

[0111] In some embodiments, the aliphatic moiety includes at least one terminal carbon-carbon double bond (H2C=CH-R, wherein R is the remainder of the aliphatic moiety), preferably, this moiety is completely linear and has at least one (e.g., only one) acidic group attached to the other terminal of this moiety.

[0112] In some embodiments, a mixture of precursor residues can be used to achieve a bulk-modified elastomer, at least part of whose residues are mutually different. Each precursor residue in the precursor residues can be selected as defined herein. In some embodiments, at least part of the aliphatic moiety of the residue precursor includes at least 2 carbon-carbon double bonds. In this way, the residues in the bulk-modified elastomer can be covalently bonded to the elastomer bulk via two or more bonds. Thus, the polymer chains of the elastomer bulk can be crosslinked via the residues.

[0113] In some embodiments, the portion of the precursor residue comprises at least 5 carbon atoms, more preferably comprises at least 10 carbon atoms. Preferably, the aliphatic portion comprises fewer than 40 or fewer than 30 carbon atoms. The number of carbon atoms in such a portion is preferably between 5 and 30, more preferably between 5 and 20 or between 5 and 15. The portion and / or chain may have one or more aryl groups. One or more of the acidic groups in the acidic group may be directly attached to an aryl group (e.g., a benzene ring).

[0114] In some embodiments, the residue precursor is an unsaturated fatty acid and the residue is the remaining portion of such an unsaturated fatty acid precursor covalently bound to the remainder of the elastomer via the reaction of one or more (if any) of its vinyl groups (carbon-carbon double bonds).

[0115] For example, the unsaturated fatty acid residue is one or more residues of a fatty acid selected from the group consisting of myristoleic acid, palmitoleic acid, petroselinic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

[0116] In some embodiments, the residue is the remaining portion of the precursor having a linear or branched alkyl chain carrying one or more acidic groups and at least one olefinic bond (carbon-carbon double bond). Preferably, the last such vinyl group is a terminal chain vinyl group. The terminal vinyl group can provide enhanced reactivity compared to non-terminal vinyl groups during the formation of the elastomer body during the manufacturing process. A straight chain having one terminal vinyl group and one acidic group (e.g., a carboxyl group) is a preferred example. In such a case, 5 to 15 carbon atoms may be present in the chain.

[0117] Thus, the residue may have an aliphatic portion comprising at least 3 carbon atoms to which one or more acidic groups are covalently bound. The corresponding precursor for such a residue comprises at least one carbon-carbon double bond in its aliphatic portion. Preferably, the aliphatic portion comprises 5 or more, 7 or more, 9 or more, 11 or more carbon atoms. Preferably, the aliphatic portion comprises fewer than 30 or fewer than 20 or fewer than 15 carbon atoms. The aliphatic portion of the residue or precursor may be linear or branched, with linear being preferred. It may have a cyclic structure. The aliphatic tail may terminate in a non-aromatic ring structure, e.g., a 5- 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 for unsaturated fatty acid 20 is retinoic acid.

[0118] The aliphatic moiety may have one, two, three or four double bonds. Preferably, the number of double bonds is less than 3. More preferably, there is only 1 double bond. At least one double bond is substituted by at least two hydrogen atoms. More preferably, at least one double bond is substituted by three hydrogen atoms. Such double bonds generally exhibit increased reactivity in the reactions described herein, which is higher for the latter.

[0119] The acidic group carried by the aliphatic moiety is preferably attached to a saturated carbon atom of the aliphatic component. The acidic group may be a carboxylic acid group and its carboxylate group (conjugate base group), phosphorus including acidic groups (e.g., phosphoric acid group) and corresponding phosphates and hydrogen phosphates, phosphoric acid group and corresponding conjugate base, and / or sulfur including acidic groups (e.g., sulfuric acid group) and corresponding sulfates.

[0120] The phosphoric acid group is a phosphorus oxyacid in which each phosphorus atom is in the +5 oxidation state and is bonded to four oxygen atoms. Two or more of these PO4 units (tetrahedra) can be linked by oxygen atoms with shared single bonds, thereby forming a linear or branched chain with at least one of the oxygen atoms bonded to the remainder of the residue. The unshared single-bonded oxygen atoms are completed with acidic hydrogen atoms or are capable of accepting protons. The general formula of the phosphoric acid group is H n+1-2x P n O 3n+1-x , where n is the number of phosphorus atoms and x is the number of basic cycles in the molecular structure, the number of which is between 0 and (n + 2) / 2. One bond is reserved for bonding to the residue (e.g., the aliphatic moiety of such a residue). Preferably, the acidic group includes an orthophosphoric acid group or a polyphosphoric acid group R-O(O=PoHO) n -H, where for orthophosphoric acid, n = 1, and for polyphosphoric acid, n > 2, and R represents the remainder of the residue to which the group is bonded. For example, phosphoric acid groups well known in the art may be present and will not be described in further detail herein. Further definitions can be found in many textbooks on organic chemistry.

[0121] Any of the conjugate bases of the acidic groups (e.g., carboxylate, phosphate, hydrogen phosphate, bisulfate, etc.) is preferably associated with or saponified with a metal counterion (e.g., counterions of alkali metals (e.g., lithium, sodium and potassium) and counterions of alkaline earth metals (e.g., barium and calcium)).

[0122] Those skilled in the art will immediately think of many other compounds other than the exemplary compounds specifically mentioned in this application.

[0123] Each residue or precursor of such a residue may have an acidic group. In this case, the molar concentration of the residue in the bulk-modified elastomer or in the precursor used to prepare such a bulk-modified elastomer also reflects the molar concentration of the acidic group present in the bulk-modified elastomer. To calculate the final molar concentration of the acidic group based on the molar concentration of the residue, it is necessary to multiply the molar concentration of the residue by the number of acidic groups per residue. It can be beneficial for each residue to have more than one acidic group when: the molar concentration of such groups in the required bulk-modified elastomer is greater than 1.10 -2 mol residue / kg of bulk-modified elastomer, and is substantially not troubled by problems associated with amounts of residues above this range limit, as explained previously.

[0124] As disclosed previously, the concentration of the residue in the bulk-modified elastomer or the concentration of the corresponding precursor for the residue in the mixture of precursors used to form the elastomer is preferably in the range of 2.10 -4 to 2.10 -2 mol / kg, based on the total weight of the elastomer or the total weight of one or more precursors of the elastomer. For siloxane-type elastomers, the residue concentration range is preferably 2.10 -4 to 1.10 -2 mol / kg. This largely avoids rinsing the substrate before use to remove unreacted precursors. For low concentrations of acidic groups, the residue molar concentration range can be 2.10 -4 to 2.10 -3 mol / kg. For high concentrations of acidic groups, the residue molar concentration range can be 1.10 -3 to 5.10 -3 mol / kg. Cell proliferation for different cell lines may require different concentrations within the range.

[0125] When using well-known calculations to account for the molar weight of the residue or the precursor for the residue, the molar concentrations disclosed herein can be converted to weight percentages.

[0126] It should be noted that when using hydrosilylation on the olefinic chemical used to form a silicone-based elastomer, a residue precursor with an acidic group in the form of a conjugate base is used. For example, they are saponified. For this reason, they are added as salts of alkali metals or alkaline earth metals. This avoids contamination of the Pt catalyst used for the aforementioned chemical. However, this is not required for polyene-type modifications, as acid-insensitive chemical catalysts are used.

[0127] The following gives an example synthesis process for manufacturing a bulk-modified elastomer from polyenes such as polybutadiene (Synthesis Example 1).

[0128] Synthesis Example 1

[0129] Insert a polybutadiene block obtained from Lanxess AG in Cologne, Germany, into a compounding or coextrusion extruder. The block includes a peroxide (e.g., dicumyl peroxide) as a catalyst. Knead at a temperature in the range of 110 - 150 °C to mix an amount of linoleic acid (concentrated 60 - 74% obtained from Aldrich Chemical) in the range of 1 - 5 wt% based on the weight of the polybutadiene block into the polybutadiene block. Inject the resulting extruded mixture into a stainless-steel mold that defines a fluid device and has an oxidized inner surface that contacts the extruded mixture at a temperature in the range of 150 - 200 °C for 3 - 10 minutes to produce a single-piece fluid device module of polybutadiene rubber crosslinked with linoleic acid.

[0130] The following gives an example synthesis process for manufacturing a bulk-modified elastomer from silicone rubber (Synthesis Example 2).

[0131] Synthesis Example 2

[0132] Mix 4.848 g of a sodium linoleate solution in a solution of water and isopropanol (solvent weight ratio 1:1) (weight fraction of sodium linoleate in the solution is 30% (by weight)) into the silicone component A (240 g) of Elastosil LR3040 obtained from Wacker Chemie AG, and stir vigorously under vacuum conditions at room temperature to evaporate water from the composition. Mix the resulting mixture with component B (240 g) of Elastosil LR 3040 obtained from Wacker Chemie AG (including a Pt catalyst), and feed it into an injection molding device, where the resulting mixture is injected into a stainless-steel mold that has an oxidized inner surface that contacts the resulting mixture and defines a fluid device module and is molded at a temperature in the range of 150 - 200 °C for 10 - 60 seconds to produce a single-piece fluid device module of silicone rubber crosslinked with sodium linoleate, where sodium linoleate is present in the polymer matrix, and the amount of sodium linoleate is 1% based on its weight relative to the weight of the polymer matrix (based on the starting material weight). Optionally, the molded product can subsequently be placed 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.

[0133] Synthesis Example 3

[0134] Same as Example 2, but using an appropriate amount of sodium undecanoate to achieve the same molar concentration as that of sodium linoleate in Example 3.

[0135] To further demonstrate the bulk modification of the elastomers in Synthesis Examples 1 and 2, after the reaction with linoleic acid as described above, each elastomer block was molded in a polar mold, and then sliced at -40 °C with a microtome to obtain internal slices of the modified elastomer, i.e., slices where neither of the major surfaces of the elastomer was the outer surface of the molded elastomer block.

[0136] To show that a portion of the acidic groups of the modified residues can actually be obtained on the surface of the molded elastomer, these internal slices were then brought into contact with 2 μL water droplets 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 the unmodified elastomer to compare the contact angles of the internal slices of the linoleic acid-modified elastomer measured with the contact angles of the corresponding internal slices of the unmodified elastomer measured. For example, the results for the silicone-based elastomer are shown in the graph in Figure 16 The contact angles of the unmodified silicone are very insensitive to the pH of the droplet. Without wishing to be bound by theory, the small increase in the contact angle measured for the unfunctionalized silicone with increasing pH can be the result of the pH-dependence of the hydrogen bond between water molecules and the silanol moieties in the silicone. For the modified silicone batches used for culturing different cell types described below, the contact angle at pH = 4 is much lower than the contact angle at pH = 10.

[0137] The behavior observed for the modified silicone (not shown) is the same regardless of the form of the carboxylic acid groups available on the surface (free form or conjugate base form).

[0138] In addition, it can be seen that the bulk modification is evident from observing the same behavior at the cut surface prepared by cutting a batch of substrates in half.

[0139] Therefore, both the internal slices and the outer surface of the polybutadiene crosslinked with linoleic acid (obtained after cutting) show a strong pH-dependence of the measured contact angles, with the measured contact angles decreasing as the pH increases.

[0140] Although not shown here, a similar contact angle behavior was observed for the modified polybutadiene, while the contact angles measured for the unmodified polybutadiene were largely independent of pH.

[0141]

[0142] ​The strong pH-dependence of the measured contact angles of the bulk and surface of the functionalized elastomers clearly demonstrates the presence of carboxylic acid groups both in the bulk and on the surface of the modified elastomers. This can be understood as follows. At lower pH, such carboxylic acid groups remain mostly protonated, thus minimizing the surface charge of the inner slices of the elastomer. As the pH increases, this surface charge increases due to the increased deprotonation of the carboxylic acid groups, which decreases the contact angle of the water droplets with the surface of these inner slices. On the other hand, in the absence of such carboxyl groups on the surface exposed to the water droplets (e.g., in the unmodified elastomer), changes in the pH of the water droplets do not cause changes in the surface charge, making the measured contact angles of water droplets at different pHs with such a surface highly insensitive to these pH changes.

[0143] The pH of the droplets used in the method for discerning the contact angle depends on the pKa value of the acidic groups used in the substrate. In the case of linoleic acid groups, the pKa is approximately 4.7. Thus, the low pH droplets should have a pH below 4.7, while for the high pH droplets, the pH is about 2 pH units higher than this pKa, thus causing complete dissociation in the droplets. A person skilled in the art will be able to select the correct conditions for a specific acidic group with a specific pKa.

[0144] Another method for checking for surface acidic groups (carboxylic acid groups) is carried out. For this, all thick slices or sections are treated with HCl as described herein to reverse the conjugate base of any available acidic groups into its acid form. The NH2 groups of cadaverine are reacted with the acidic groups by using the well-known coupling chemicals of EDC / NHS, and the resulting dried thick slices are coupled to fluorescently labeled cadaverine. The comparison of the treatment of the modified elastomer and the unmodified elastomer clearly shows an increase in fluorescence, with the latter having a rather uniform spatial gray scale and the unmodified elastomer lacking fluorescence. This staining test can be used to show the presence of surface-available acidic groups (e.g., carboxylic acid groups).

[0145] IR measurements are used to study the bulk presence of acidic groups in reflection mode and transmission mode as well as in elemental analysis.

[0146] After the polymerization reaction for forming the modified elastomer has been completed or carried out, IR and Raman spectroscopy are used to determine the conversion rate and state. For example, this is achieved by looking at the reduction in the vibrational absorption of the double bond of the precursor of the modified residue.

[0147] The bulk-modified elastomer can be molded in any suitable form to produce an article or a cell culture plate on which cells can be directly cultured. These articles can be in any suitable shape. In a particular embodiment, the mold used in such an injection molding process can be shaped in the form of a fluid device module such that a single-piece fluid device module can be formed in a single-step injection molding process. Since the carboxylic acid groups or carboxylate groups have a high density and a uniform distribution on the (one or more) outer surfaces of such a single-piece fluid device module, such a device module can be directly used as a cell culture plate. The mold can be formed of or have an inner surface coated with any suitable polar material (e.g., stainless steel, metal, or metal oxide (e.g., alumina)) so as to enable the carboxylic acid groups or carboxylate groups of the bulk-modified elastomer to have a desired orientation on at least one of its outer surfaces. In Figure 2 and Figure 3 Typical examples of the fluid device module 100 are schematically depicted, where perspective views of the opposite major surfaces of the fluid device module 100 are schematically depicted. The fluid device module 100 can include a membrane 110 on which at least one flow channel 115 extends. Preferably, the fluid device module 100 includes opposite flow channels spatially separated by the membrane 110. At least the membrane 110 is made of the bulk-modified elastomer 30, but in a preferred embodiment, the entire fluid device module 100 is made of the bulk-modified elastomer 30, thereby producing a single-piece fluid device 100.

[0148] The fluid device module 100 can include one or more septa 120 through which access to such flow channels 115 can be obtained. Preferably, each flow channel 115 can be accessed through a dedicated set of septa 120 such that cross-contamination between multiple flow channels 115 is avoided. As explained above, the exposed surface (e.g., the membrane 110) of the fluid device module 100 can be used to directly bind cell cultures such that cells can be cultured (proliferated) on a biocompatible surface.

[0149] The membrane 110 can include a plurality of pores or grooves such that liquid nutrients and solutions having compounds such as potential drugs can reach both sides of the membrane and the cells / tissues, and the pores or grooves can be formed in any suitable manner (e.g., by laser cutting). Such pores or grooves in the membrane 110 of the fluid device module 100 preferably have an order of magnitude similar to the typical diameter of the cells to be stabilized on the membrane 110, and the spacing between such pores or grooves is not particularly critical.

[0150] The fluid device module 100 can be formed into a Figure 4The fluid device 200 schematically depicted therein, the cover plate fluid-tightly seals the fluid device module 100 such that the fluid passing through one or more flow channels 115 of the fluid device module 100 cannot leak from the fluid device 200.

[0151] In an embodiment, the cover plates 210, 220 are arranged such that: the cover plate 210 covers the first main surface of the fluid device module 100, thereby sealing the first fluid channel 115 of the fluid device module; and the other cover plate 220 covers the second main surface of the fluid device module 100, thereby sealing the second fluid channel 115 of the fluid device module. The cover plates 210 and 220 can be made of any suitable material (e.g., glass or plastic material). Preferably, the cover plate is stretchable and flexible to ensure a good fit with the fluid device module 100 while maintaining the flexibility of the fluid device module 100 in the fluid device 200. Such a fluid device 200 can be deployed as an organ-on-a-chip as will be readily understood by those skilled in the art.

[0152] Figure 5Schematically depicts an exemplary embodiment of a fluid device 200, wherein the device includes a fluid device module 100 according to the present invention, and the fluid device module 100 is between two rigid cover plates 210 and 220. The upper plate 210 includes a first inlet 231 and a first outlet 232, and the first inlet 231 and the first outlet 232 are in fluid communication with an invisible lower channel (not shown) below the membrane 110 in the fluid device module 100, such that the membrane 110 is in fluid contact with the fluid passing through the lower channel. The upper plate 210 further includes a second inlet 233 and a second outlet 234, and the second inlet 233 and the second outlet 234 are in fluid communication with an upper channel 115 above the membrane 110, such that the membrane 110 is in fluid contact with additional fluid passing through the upper channel 115. For example, the fluid passing through the lower channel may include a drug to be tested on a cell culture attached to the membrane 110 as explained in more detail below. Since the membrane 110 is made porous (e.g., by laser drilling or grooving in the membrane 110), the drug can pass through the membrane 110 to reach the cell culture. Alternatively, when forming the fluid device module 100 in a polar mold, such pores or grooves can be formed in the membrane 110. This has the advantage that a regular pattern of such fluid channels (pores) can be formed through the membrane 110. The additional fluid passing through the upper channel 115 can be in direct contact with such cell culture and provide nutrients to such cell culture. In some embodiments, the inlets 231, 233 and the outlets 232, 234 are diaphragms through which (e.g., by pressing the diaphragm) fluid can be hydraulically pumped onto the membrane 110 through the aforementioned channels. Alternatively, the inlets 231, 233 and the outlets 232, 234 can be ferrules to which pipes can be attached (e.g., clamped), which is very useful, for example, when the fluid device 200 is used as a well plate or the like in a laboratory on a chip system.

[0153] In Figure 6 the embodiment schematically depicted, for example when molding the fluid device module 100, the upper channel and the lower channel 115 are incorporated in the (monolithic) fluid device module 100 (only one channel is shown). Alternatively, at least one of the upper channel and the lower channel can be formed in one of the cover plates. Figure 7 Schematically depicts a top perspective view of an exemplary embodiment of the fluid device 200, and Figure 8 schematically depicts a bottom perspective view of an exemplary embodiment of the fluid device 200, wherein the upper channel 115 is formed in the upper cover plate 210 while the lower channel 115' is formed in the fluid device module 100. Those skilled in the art will readily understand that it is of course equally feasible to form the lower channel 115' in the bottom cover plate 220. The fluid device 200 (e.g., in Figure 7 and Figure 8The device shown in (e.g., can be used as a micro-well in a laboratory for a chip device, where multiple such micro-wells can be clamped or otherwise fixed to facilitate parallel testing of multiple different samples in a single laboratory on the chip device, where each micro-well typically includes one of these samples.)

[0154] When stabilizing the cell culture 50 in such a fluid device 200 and proliferating the cell culture, the fluid device 200 can be used in a method in which the stabilized and proliferated cell culture 50 is exposed to a fluid comprising a compound of interest, which can flow through the flow channels 115 of the fluid device 200 so as to expose the stabilized cell culture 50 to the compound of interest in the fluid and monitor the response of the stabilized cell culture 50 to such exposure. Such a method can be deployed, for example, in an oncology setting, in which the stabilized cell culture 50 can be exposed to a drug (e.g., a chemotherapeutic drug) in order to monitor the response of the stabilized cell culture 50 to such a drug. To this end, the stabilized cell culture 50 can include tumor cells in order to test the efficacy of the drug and / or can include healthy cells in order to test the toxicity of the drug on healthy tissue.)

[0155] This method of drug testing typically involves providing a fluid device module 100 according to one or more embodiments of the present invention. For example, providing a monolithic fluid device module 100 and applying a harvested cell culture to at least the membrane 110, on which the cell culture can directly bind to carboxylic acid groups or carboxylate groups on the exposed surface to obtain a prepared fluid device module 100. According to this fluid device module 100, a fluid device 200 (such as an organ-on-a-chip) is formed as previously explained. The cell culture within the fluid device 200 can be fed through one of a pair of flow channels 115 of the fluid device 200 to keep the cell culture alive, while the cell culture can be exposed to a drug that can be tested through the other of the pair of flow channels 115 of the fluid device 200. Wherein, the drug testing method is generally completed by monitoring the response of the cell culture to the drug to be tested. This can be achieved, for example, by the following operations: disassembling the fluid device 200 by removing the cover plates 210, 220, and cutting the fluid device module 100 such that a section of the module including the cell culture can be obtained, and the section can be studied under a microscope or the like to study the effect of the drug being tested on the cell culture. In such a study, the cell culture is generally stained, treated with formalin, and fixed in a fixative (such as paraffin) to facilitate such microscopic study. This staining, treatment, and fixation of the cell culture can be deployed inside the fluid device 200, for example, by passing a suitable chemical reagent through the fluid channel 115 of the fluid device 200 where the cell culture is exposed.

[0156] In an alternative embodiment, such disassembly of the fluid device 200 can be avoided, and a confocal microscope can be used to monitor in real time the response of the cell culture to one or more compounds (such as the drug to be tested). In particular, in an embodiment where the fluid device 200 includes transparent cover plates 210, 220 (such as glass plates or polymer plates), a confocal microscope can be used to monitor the cell culture within the assembled fluid device 200. This can be achieved by the fact that at least the bottom cover plate 220 can be kept thin, for example, having a thickness in the range of 150 - 300 μm, wherein the distance between the membrane 110 of the fluid device module 100 carrying the cell culture and the bottom cover plate 220 is less than 200 μm, such that the focal length of the confocal microscope is limited (usually about 500 μm from the object to be studied to the objective lens). For this purpose, the membrane 110 can have a thickness in the range of 10 - 100 μm, for example, a thickness of 20 - 30 μm. The thickness of the upper cover plate 210 is not important; any suitable thickness can be expected, for example, a thickness in the range of 300 μm - 3 mm.

[0157] It is particularly suitable to evaluate cell cultures within the fluid device 200 using a confocal microscope, where the diameter of the cell spheroids is less than 500 μm (e.g., approximately 200 μm), in order to ensure that the overall focal length of the optical path contributes to capturing sharp images using the confocal microscope. For larger spheroids, it may be necessary to disassemble the fluid device 200 such that the membrane 110 including the spheroid can be pressed onto a glass coverslip for positioning within the optical objective of the confocal microscope.

[0158] Additionally, it has also been noted that the aforementioned dimensions of the fluid device 200 are typically applied during real-time monitoring of cell cultures within the fluid device 200. In embodiments where the fluid device 200 is sectioned as previously explained (e.g., for digital pathology), the dimensions of the fluid device 200 are not as critical as long as formalin application and paraffin fixation can be performed on the fluid device 200.

[0159] The cell culture substrates disclosed above are merely examples. There are other such substrates. For one substrate, such a substrate can be a microplate. The substrate can be formed for use with a microplate, e.g., thick slices of the substrate can be arranged to have an adjusted shape and size such that they can be inserted into the standard-sized wells of a microplate. Alternatively, a microplate with open microwells can be used with a portion of the material that closes the bottom of the closed microplate. Such an assembly or arrangement can be clamped.

[0160] As will be readily understood by those skilled in the art, such drug testing methods can vary significantly in terms of protocol (e.g., drug dosage, dosing frequency, etc.). It should be understood that the drug testing methods of the present invention are not limited to a specific protocol as long as these methods can be deployed using the fluid device 200 according to embodiments of the present invention, in which cells are cultured directly on the exposed surface of the device (e.g., membrane 110).

[0161] At this point, proof of the concept of culturing mammalian cells directly on the body-modified elastomers of the present invention will be provided in the form of the following experimental evidence.

[0162] General procedure:

[0163] The sample was cleaned by rinsing it in isopropanol for 5 minutes. After drying, the sample was placed in a 24-well plate. A cell culture of immortalized human prostate fibroblasts (WPMY-1 cells, ATCC CRL-2854) was provided in DMEM (Dulbecco's Modified Eagle Medium, Thermofisher) with 10% FBS (fetal bovine serum), 1% penicillin / streptomycin, and 1% Glutamax. The total amount of 2.0×10 5Cells were seeded on all samples and placed in an incubator overnight at 37 °C in an atmosphere of 5% CO2. Images of the resulting cell cultures were taken using a Leica inverted bright-field microscope at 100x magnification.

[0164] Example 1: On a modified silicone elastomer body according to the teachings of the present invention, a WPMY-1 cell culture was developed with 0.5 wt% linoleic acid according to the general procedure described above. For this purpose, Synthesis Example 2 was modified by replacing 4.84 g of sodium linoleate with 2.41 g of linoleic acid to obtain the body-modified silicone elastomer used in this example.

[0165] Comparative Example 1: On a silicone elastomer identical to that of Example 1 but without body modification, a WPMY-1 cell culture was developed according to the general procedure described above.

[0166] Example 2: On a body-modified silicone elastomer prepared according to Synthesis Example 2 (i.e., containing 1.0% by weight of sodium linoleate), a WPMY-1 cell culture was developed according to the general procedure described above.

[0167] Figure 9 The cell culture resulting from Example 1 after overnight incubation is shown. It can be clearly seen in this image that the cells show good adhesion to the surface of the linoleic acid-functionalized silicone.

[0168] Figure 10 The cell culture resulting from Example 1 after overnight incubation is shown. It can be clearly seen in this image that the cells show minimal adhesion to the surface of the unfunctionalized silicone elastomer.

[0169] Figure 11 The cell culture resulting from Example 2 after overnight incubation is shown. It can be clearly seen in this image that the cells show good adhesion to the surface of the sodium linoleate-functionalized silicone. Figure 12 The cell culture of Example 2 after 2 days of incubation is shown, and Figure 13 The cell culture of Example 2 after 7 days of incubation is shown. Continuous cell proliferation can be clearly identified from these images, demonstrating that mammalian cell material can be directly cultured and proliferated on the body-functionalized elastomers of the present invention.

[0170] By using other cell types on the substrate and performing a preparation and treatment similar to that of Figure 2 but using 1 wt% linoleic acid, similar results were obtained.

[0171] Example 3: On the bulk-modified silicone elastomer prepared according to Synthesis Example 2 (i.e., containing 1.0% by weight of sodium linoleate), the endothelial cell Ea.hy926 cell culture was developed according to the general procedure described above.

[0172] Example 4: On the bulk-modified silicone elastomer prepared according to Synthesis Example 2 (i.e., containing 1.0% by weight of sodium linoleate), the epithelial cell Caco-2 cell culture was developed according to the general procedure described above.

[0173] Figure 14 and Figure 15 respectively show cell proliferation after, for example, 3 days and 4 days. Thus, all cell lines showed good proliferation on the substrate after several days.

[0174] List of non-limiting examples.

[0175] Example 1. A material for culturing cells, the material comprising a bulk-modified elastomer having a Shore hardness (DIN EN ISO 868) in the range of Shore 00 20 to Shore A 80 and comprising a plurality of fatty acid moieties covalently bound to the elastomer body, wherein the carboxylic acid groups of the moieties are accessible on the outer surface of the material to provide the binding, and wherein the bulk-modified elastomer is obtained by:

[0176] forming in a mold having a polar inner surface a composition comprising a vinyl-functionalized elastomer or a hydride-functionalized elastomer or at least one precursor thereof, a free or saponified unsaturated fatty acid in the range of 0.5 - 5% by weight based on the total weight of the vinyl-functionalized elastomer or hydride-functionalized elastomer or at least one precursor thereof, and a crosslinking catalyst; and

[0177] bulk-modifying the vinyl-functionalized elastomer or hydride-functionalized elastomer by a crosslinking reaction between the vinyl groups or hydride groups of the elastomer and the unsaturated carbon-carbon bonds of the unsaturated fatty acid in the mold to covalently bind the free or saponified unsaturated fatty acid to the elastomer body to obtain the material.

[0178] Example 2. The material according to Example 1, wherein each fatty acid moiety in the fatty acid moieties is covalently bound to the elastomer body by a crosslinking reaction between the vinyl groups or hydride groups of the elastomer and the unsaturated carbon-carbon bonds of the unsaturated fatty acid.

[0179] Example 3. Based on the material of Example 2, the unsaturated fatty acids are selected from the following: myristoleic acid, palmitoleic acid, petroselinic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

[0180] Example 4. Based on the material of any one of Examples 1-3, wherein the elastomer comprises a polybutadiene backbone or a siloxane backbone.

[0181] Example 5. Based on the material of any one of Examples 1-4, wherein the composition comprises the free or saponified unsaturated fatty acids in the range of 0.5-2% by weight based on the total weight of the vinyl-functionalized elastomer or hydride-functionalized elastomer or at least one of its precursors.

[0182] Example 6. Based on the material of any one of Examples 1-5, wherein the polar inner surface of the mold is a metal oxide inner surface.

[0183] Example 7. A fluid device module including a flow channel that extends over a membrane comprising the material of any one of Examples 1-6.

[0184] Example 8. The fluid device module according to Example 7, including a first main surface and a second main surface, the first main surface including a first recessed structure defining a first flow channel, the second main surface being opposite to the first main surface and including a second recessed structure defining a second flow channel; wherein the membrane separates the first flow channel from the second flow channel.

[0185] Example 9. The fluid device module according to Example 7 or 8, wherein the fluid device is a monolithic fluid device.

[0186] Example 10. A fluid device including the fluid device module according to any one of Examples 7-9 and a pair of cover plates for fluid-sealing the fluid device module.

[0187] Example 11. The fluid device according to Example 10, wherein the cover plates are arranged such that one of the cover plates covers the first main surface to seal the first fluid channel, and the other cover plate covers the second main surface to seal the second fluid channel.

[0188] Example 12. A method for culturing cells, including:

[0189] Provide a molded article formed from the material according to any one of Embodiments 1-5, the article including an outer surface of the material as one of the exposed surfaces of the article; and

[0190] Cultivate the cells directly on the exposed surface of the article.

[0191] Embodiment 13. The method according to Embodiment 12, wherein the article includes a fluid device module according to any one of Embodiments 7-9.

[0192] Embodiment 14. A drug testing method, comprising:

[0193] Provide a fluid device module according to Embodiment 8;

[0194] Apply the harvested cells to the surface of the membrane of the fluid device module;

[0195] Cultivate the harvested cells on the surface;

[0196] Form a fluid device with the prepared fluid device module;

[0197] Feed the cultivated cells through one of the pair of flow channels of the fluid device;

[0198] Expose the cultivated cells to the drug to be tested through the other of the pair of flow channels of the fluid device; and

[0199] Monitor the response of the cultivated cells to the drug to be tested.

[0200] Embodiment 15. The drug testing method according to Embodiment 14, wherein monitoring the response of the proliferating cell culture to the drug to be tested includes:

[0201] Stabilize and fix the cultivated cells in the fluid device, and

[0202] Section the fluid device module to obtain sections for microscopic evaluation, the sections including at least a portion of the stabilized and fixed proliferating cell culture; or

[0203] Use a confocal microscope to monitor the cultivated cells in the fluid device.

[0204] It should be noted that the above embodiments illustrate rather than limit the present invention, and those skilled in the art will be able to design many alternative embodiments without departing from the scope of the claims. In the claims, any reference signs in parentheses shall not be construed as limiting the claims. The word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The present invention can be implemented by means of hardware comprising several different elements. In device claims enumerating several units, several of these units can be embodied by the same item of hardware. The fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used advantageously.

Claims

1. A cell culture plate comprising a material having an outer surface for culturing cells thereon, the material comprising a bulk-modified elastomer having an elastomer bulk and a plurality of residues, each residue comprising one or more acidic groups in free form and / or conjugate base form, the plurality of residues being covalently bound to the elastomer bulk such that a portion of the one or more acidic groups is obtainable on the outer surface, wherein the residue concentration in the bulk-modified elastomer is in the range of 2.10 -4 to 2.10 -2 mol / kg; and wherein the residues are covalently bound to the elastomer bulk due to a reaction between an unsaturated carbon-carbon bond of a precursor of the residues and a vinyl functional group or a hydride functional group of the elastomer bulk.

2. The cell culture plate according to claim 1, wherein the one or more acidic groups are selected from the group consisting of phosphoacidic groups, sulfoacidic groups, and carboxylic acid groups, or a mixture of two or more of these items.

3. The cell culture plate according to claim 1, wherein the residue comprises or consists of the following aliphatic moiety, the aliphatic moiety comprising 3 or more and less than 50 carbon atoms, the one or more acidic groups being covalently bound to the aliphatic moiety, and the aliphatic moiety being covalently bound to the elastomeric body.

4. The cell culture plate according to claim 1, wherein the residue is an unsaturated fatty acid residue.

5. The cell culture plate according to claim 4, wherein the unsaturated fatty acid residue is one or more residues of a fatty acid selected from the group consisting of myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, octadecenoic acid, linoleic acid, linolelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

6. The cell culture plate according to claim 1, wherein the elastomeric body comprises a silicone or polybutadiene backbone.

7. The cell culture plate according to any one of claims 1 to 6, wherein the bulk-modified elastomer is obtained or can be obtained by the following operation: mixing a vinyl-functionalized elastomer and / or a hydride-functionalized elastomer or at least one of their precursors with a residue precursor having a concentration in the range of 2.10 -4 to 2.10 -2 mol / kg, based on the weight of the total bulk-modified elastomer, and heating the mixture, wherein the residue precursor contains one or more acidic groups in free form and / or conjugate base form and unsaturated carbon-carbon bonds.

8. The cell culture plate according to claim 7, wherein when the elastomer is a silicone-based elastomer, the concentration range of the residue precursor is 2.10 -4 to 1.10 -2 mol / kg.

9. The cell culture plate according to claim 7, wherein the heating is carried out in a reaction vessel having a metal-metal oxide inner surface.

10. The cell culture plate according to any one of claims 1 to 6, wherein the substrate comprises a fluid device module, the fluid device module comprising flow channels extending on a membrane comprising the material.

11. The cell culture plate according to claim 10, comprising a first major surface and a second major surface, the first major surface comprising a first recessed structure defining a first flow channel, the second major surface being opposite to the first major surface and comprising a second recessed structure defining a second flow channel; wherein the membrane separates the first flow channel from the second flow channel.

12. The cell culture plate according to claim 10, wherein the fluid device is a monolithic fluid device.

13. Use of a material having an outer surface for directly culturing cells thereon, said material comprising a bulk-modified elastomer having an elastomer body and a plurality of residues, each residue comprising one or more acidic groups in free form and / or conjugate base form, said plurality of residues being covalently bound to the elastomer body such that a portion of said one or more acidic groups is obtainable on said outer surface, wherein the residue concentration in the bulk-modified elastomer is in the range of 2.10 -4 to 2.10 -2 mol / kg, said use comprising bringing said outer surface into contact with cells to be cultured and a cell growth medium; and wherein said residues are covalently bound to said elastomer body due to a reaction between the unsaturated carbon-carbon bonds of the precursors of said residues and the vinyl or hydride functional groups of the elastomer body.

14. A method of culturing cells, comprising the steps of: using the cell culture plate according to any one of claims 1 to 12; and culturing cells directly on the outer surface of the cell culture plate.

15. The method according to claim 14, wherein the cell culture plate is the cell culture plate according to any one of claims 10 to 12.

16. The method according to claim 14, wherein the substrate is a molded substrate.

17. A method of drug testing, comprising the method according to claim 15 and further comprising the steps of: exposing the cultured cells to a drug to be tested through another flow channel of a pair of flow channels of the fluid device; and monitoring the response of the cultured cells to the drug to be tested.

18. A method of manufacturing a cell culture plate, wherein the cell culture plate comprises a material having an outer surface for culturing cells thereon, the material comprising a bulk-modified elastomer having an elastomer body and a plurality of residues, the method comprising: Heating a vinyl-functionalized elastomer and / or a hydride-functionalized elastomer or at least one of its precursors in a mold with a mixture of residue precursors at a concentration in the range of 2.10 -4 to 2.10 -2 mol / kg, and heating the mixture, wherein the residue precursors comprise one or more acidic groups in free form and / or conjugate base form and unsaturated carbon-carbon bonds.

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