Cell culture substrate and application thereof

Abstract

The present invention relates to a cell culture substrate comprising an elastomeric material for culturing cells and the use thereof, a fluidic device module and a fluidic device comprising such a cell culture substrate, and a cell culture method and a pharmaceutical test method using a fluidic device module comprising such an elastomeric material.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080067111.8, filed on September 24, 2020, with the invention name “Cell Culture Material”. Technical Field

[0002] The present invention relates to a cell culture substrate comprising an elastomeric material for culturing cells. The present invention also relates to a fluidic device module and a fluidic device comprising such a cell culture substrate. The present invention also relates to a method for culturing cells. The present invention also relates to a pharmaceutical testing method utilizing a fluidic device module comprising such an elastomeric material. The present invention also relates to the use of an elastomeric material for cell culture. The present invention also relates to the manufacture of a cell culture substrate. Background Art

[0003] In vitro testing of mammalian cells or tissues is an important technique for obtaining clinically important information about mammalian materials in research. For example, such testing can be performed on biopsied mammalian cells or tissue material to determine an abnormality or disease in such mammalian material or to expose the sick mammalian material to a drug (e.g., an experimental drug) to monitor the response of the sick mammalian material to such exposure. For example, such methods are frequently used in oncology procedures. This can provide important insights into how to effectively treat a disease in an individual without having to expose the individual to a range of potentially effective drugs that may be undesirable for many reasons (e.g., drug toxicity). In addition, the efficacy of experimental drugs for certain existing diseases can be tested in this way for which no established satisfactory drug treatment has yet to be obtained. There are many other well-known reasons for deploying such in vitro testing.

[0004] A common approach to such in vitro testing is to immobilize mammalian material in a fluidic device, sometimes referred to as an organ-on-chip. In this approach, the mammalian material is typically immobilized on a membrane that separates two fluidic channels of the fluidic device, wherein the first channel is used to feed the mammalian material and the second channel is used to expose the mammalian material to a compound or chemical of interest (e.g., a drug treatment), for example, to test the efficacy and / or toxicity of the drug, as previously explained. The fluidic device, or at least its membrane, can be made of an elastomer so that the fluidic device can be cut into pieces or slices in order to obtain slices of the membrane comprising the mammalian material, for example, for evaluation purposes, for example, to evaluate the response of the mammalian material to exposure to the compound or chemical of interest.

[0005] The challenge in such in vitro experiments is to ensure that the mammalian material (e.g., cells or tissue) is properly stabilized and developed on the membrane of the fluidic device so that the mammalian material remains viable for the duration of the experimental procedure. This is particularly challenging in oncology procedures, where experiments can be quite lengthy. To this end, biocompatible materials such as fibronectin can be used to stabilize the mammalian material, ensuring that the mammalian cells remain proliferating by providing them with a chemical environment that mimics that of native tissue.

[0006] A common method is to coat microporous cell plates with a polymer having a carboxylic acid group, because this group can be (covalently) bound to fibronectin, thereby promoting the stabilization of mammalian cells on the membrane in the fluidic device as fibronectin makes the polymer bioconnected. However, this method is not without its shortcomings. First, the aforementioned polymer can be partially removed by the fluid flow of the fluidic device, which impairs the ability to evaluate mammalian materials at the required time point (due to at least some of this material may have been lost). In another method, the membrane is disposed of with UV or blood plasma to generate a binding site for fibronectin on the membrane. However, this method has the following shortcomings: it is quite difficult to avoid the uneven distribution of this binding site, which again hinders the evaluation of the test results.

[0007] Secondly, it is difficult to manufacture membranes that include such biocompatible coatings, which makes manufacturing such fluidic devices quite expensive and cumbersome. For example, such membranes typically need to be manufactured using thin-film technology (which is expensive), and such membranes need to be integrated into the fluidic device without leakage (which is not straightforward). In addition, if the membrane itself is not made of a biocompatible material, it is typically necessary to provide the membrane with multiple cell-sized pores with a small spacing to promote cell growth (which is also difficult to achieve).

[0008] Dongeun Huh et al. disclose an example of microfabrication of artificial organ chips based on thin film technology in Nature Protocols (Volume 8, Issue 11, 2013, Pages 2135-2157). In this scheme, micro-engineering technology is used to manufacture a multi-layer microfluidic device, which contains two parallel elastomeric microchannels separated by a thin porous flexible membrane and two fully height but vacuum chambers on either side. The production of this device takes about 3 and a half days. The total microfabrication process includes more than 100 steps, of which a large number are key steps. This clearly shows the complexity of this manufacturing process.

[0009] An improvement to this laborious approach is disclosed in WO 2018 / 021906 A1, which discloses a silicon-based (PDMS) fluidic device that can be made biocompatible using a coating such as collagen. Such a device can be manufactured with only a small number of manufacturing steps, but still suffers from the disadvantage that it needs to be coated with a biocompatible material, which, as described above, can erode the biocompatible material from the polymer surface when exposed to fluid flow, resulting in an unwanted loss of cellular material from the surface of the fluidic device. This problem is addressed 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 fluidic device with EGM-2 (Endothelial Cell Growth Medium-2 Bullet Kit, sold by Lonza). Therefore, a large number of processing steps are still required to provide a biocompatible fluidic device.

[0010] The applicant 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 the elastomer bulk, wherein the carboxylic acid groups of the moieties can be used to provide the binding on the surface of an object formed by the bulk-modified elastomer by the following operation: the elastomer is bulk-modified with fatty acid moieties in a mold according to the shape of the object and a polar inner surface is formed, so that the hydrophilic carboxylic acid groups of the fatty acid moieties are attracted to the polar inner surface. In this prior application, it has been demonstrated that biocompatible materials such as fibronectin can be covalently bound to the surface carboxylic acid groups of bulk-modified elastomers while maintaining their cell culture properties.

[0011] There is a need for further improvements in cell culture substrates and their manufacture. Summary of the Invention

[0012] This need is at least partially met by the present invention which seeks to provide, through the various aspects defined herein, each of which allows for the use or provision of elastic materials for culturing cells without the need for covalently modifying the surface with cell culture proteins.

[0013] According to one aspect, the present application provides a cell culture substrate comprising a material having an outer surface for culturing cells thereon, the material comprising a bulk-modified elastomer, the bulk-modified elastomer having an elastomeric body 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 elastomeric body such that a portion of the one or more acidic groups is available on the outer surface.

[0014] The term substrate or cell culture substrate suitable for culturing cells is meant to include any device or surface intended for cell culture. Substrates defined in this manner are intended to embody the purposes of the elastic material defined in the field of cell culture in this article. Cell culture means, for example, to keep cells alive and / or proliferate the number of cells and / or differentiate cells. Residue is the part of the precursor molecule that carries an acidic group in any form and can participate in the reaction to covalently bind the precursor to the olefinic bond of the elastomer body. Residue is the remaining part of the precursor after this reaction.

[0015] It has been found that when this surface is directly contacted with cell culture medium in the presence of cells, it is possible to cultivate this cell without any further surface preparation. Therefore, it is possible to save the step of covalently modifying the material surface with cell culture proteins, thereby promoting the use of this surface. Although it is not desirable 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, this cell culture property has not been demonstrated with reference to the elastomer of non-bulk modification. It has also been found that acidic groups can be present at the surface in free form or conjugate base form, and free form means that the acidic group carries the proton that can dissociate, and the conjugate base form means that in addition to the proton, there is also a positive counter ion. It is not desirable to be bound by theory, it is believed that in cell culture medium (which is usually buffered when pH is about 7.2), this acidic group with a pKa value usually less than 5.5 usually exists in a dissociated form to a great extent, and no matter how their original form can be obtained at the surface.

[0016] The acidic groups preferably have a pKa of less than 5 so that most of the groups are in dissociated form. In alternative embodiments, the pKa is less than 4.5 or even less than 4. The pKa may be in the range of 5 to 1, or 5 to 2, or 5 to 3.

[0017] Importantly, cell culture substrates of any specific shape can now be efficiently prepared in a single step by mixing appropriate precursors for forming bulk-modified elastomers and reacting these precursors within a reaction vessel (e.g., that of an injection molding apparatus) to form the cell culture substrate and shape and prepare it for cell culture without further adjustments. This also makes remanufacturing a series of such devices easier and more consistent.

[0018] In some embodiments, the one or more acidic groups are: phosphorus-based acidic groups, sulfenyl acidic groups and carboxylic acid groups, or two or more mixtures in these items. Phosphoric acid group and their conjugate base are better than sulfonic acid group, and carboxylic acid group is better than phosphoric acid group. During the bulk modification, it is easier to use the residue that carries these groups, because during this modification method, their precursor has the acidic group in the form of the conjugate base with metal counterion, and this residue is better mixed with other parts to form elastomer. Can select acidic group according to required (as previously mentioned) pKa.

[0019] In some preferred embodiments, the residue comprises or consists of an aliphatic moiety comprising 3 or more and less than 50 carbon atoms, to which the one or more acidic groups are covalently bonded, and to which the aliphatic moiety is covalently bonded. The aliphatic moiety can be linear or branched. It can comprise one or more carbon-carbon double bonds or carbon-carbon triple bonds or aromatic units or phenyl units. The aliphatic moiety can comprise cyclic units, for example, cyclohexyl or cyclopentyl or other cyclic units. The aliphatic moiety is preferably a saturated hydrocarbon moiety. The aliphatic moiety preferably comprises only carbon and hydrogen atoms.

[0020] In some embodiments, the aliphatic portion is a linear chain that is coupled to the elastomeric bulk at its terminus. In some embodiments, the aliphatic portion also does not include a carbon-carbon triple bond to increase the flexibility of the residue.

[0021] In some embodiments, the residues in the bulk-modified elastomer can be different from one another, each residue being selected as defined herein. In some embodiments, at least a portion of the residues may be bound to the elastomer bulk via two covalent bonds. This may be caused by two residue precursors having olefinic bonds that participate in the modification reaction.

[0022] In some embodiments, the part comprises at least 5 carbon atoms, and more preferably comprises at least 10 carbon atoms. Preferably, the aliphatic part comprises less than 40 or less than 30 carbon atoms. Linear part is preferred, but this is not necessary per se. The number of carbon atoms in this part is preferably between 5 and 30, more preferably between 5 and 20 or 5 and 15. This part and / or chain can have one or more aryl groups. One or more in the acidic group can be directly attached to the aryl group (for example, a phenyl ring).

[0023] In some embodiments, the residue is the remaining portion of an unsaturated fatty acid precursor covalently bound to the elastomeric bulk via reaction of one or more of its vinyl groups (carbon-carbon double bonds), if present.

[0024] For example, the unsaturated fatty acid residues are one or more residues of fatty acids selected from the group consisting of myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, octadecenoic acid, linoleic acid, elaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid and docosahexaenoic acid.

[0025] In some embodiments, the residue is the remainder of a precursor of a linear or branched alkyl chain having one or more acidic groups and at least one vinyl group (carbon-carbon double bond). Preferably, the last such vinyl group is a vinyl group of a terminal chain. Compared with the non-terminal vinyl groups during the formation of the elastomer bulk in the manufacturing process, the terminal vinyl group can provide the reactivity of improvement. A straight chain with a terminal vinyl group and an 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.

[0026] 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, thereby allowing easier optical inspection of cell cultures.

[0027] In some embodiments, the residue is covalently bonded to the elastomeric bulk due to a reaction between an unsaturated carbon-carbon bond of a precursor of the residue and a vinyl or hydride functional group of the elastomeric bulk. In polydienes, this reaction is with a vinyl group, while in siloxanes, this bonding typically occurs with a silyl hydride functional group.

[0028] 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 between 2.10 and 1.5%, calculated as a weight relative to the total weight of the elastomer. -4 to 2.10-2 It has been found that lower concentrations result in infertile cell culture properties, while higher concentrations are difficult to achieve because incomplete mixing of precursors and / or incomplete reactions between precursors that occur during the manufacture of bulk-modified materials can lead to infertile or degraded culture properties.

[0029] In some embodiments, the bulk modified elastomer is or is obtainable by combining a vinyl functionalized elastomer or a hydride functionalized elastomer or at least one precursor thereof with a vinyl functionalized elastomer at a concentration of 2.10% by weight based on the total weight of the bulk modified elastomer. -4 to 1.10 -2 mol / kg range of the residue precursors are mixed, and the mixture is heated.

[0030] In some embodiments, the heating is carried out in a reaction vessel having an inner surface of a metal oxide. It is believed that this causes the acidic groups (in the form of a conjugate base) to at least partially associate with the oxide surface in the reaction vessel, so that during the polymerization reaction, a modified body is formed, at least a portion of the acidic groups are available at the surface of the material.

[0031] In a further aspect, there is provided a cell culture substrate as described herein, wherein the substrate comprises a fluidic device module comprising a flow channel extending over a membrane comprising the material.

[0032] In some embodiments, the cell culture substrate 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.

[0033] In some embodiments of the fluidic device, the cell culture substrate of the fluidic device is a monolithic fluidic device.

[0034] According to one aspect of the present invention, there is provided a use of a material having an outer surface for culturing cells thereon, the material comprising a bulk-modified elastomer having an elastomeric bulk and a plurality of residues, each residue comprising one or more acidic groups in free form and / or in conjugate base form, the plurality of residues being covalently bound to the elastomeric bulk such that a portion of the one or more acidic groups is available on the outer surface, the use comprising contacting the outer surface with cells to be cultured and a cell growth medium. As explained with respect to a cell culture substrate embodying this use, such a use is not known or apparent while such a material offers significant advantages.

[0035] According to another aspect, there is provided a method for culturing cells, comprising the steps of:

[0036] Using the cell culture substrate according to the aforementioned present invention, optionally, the substrate is a molded substrate; and

[0037] Cells are cultured directly on the exposed surface of the article.

[0038] In some embodiments of the method, the cell culture substrate is a cell culture substrate as defined herein.

[0039] In some embodiments, the cell culture method is part of a drug testing method comprising the following additional steps:

[0040] exposing the cultured cells to a drug to be tested through the other of the pair of flow channels of the fluidic device; and

[0041] The cultured cells are monitored for their response to the drug being tested.

[0042] Other non-limiting embodiments

[0043] In some embodiments, the bulk-modified elastomer is obtained by forming 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 the hydride-functionalized elastomer or at least one precursor thereof, and a cross-linking catalyst in a mold having a polar inner surface; and bulk-modifying the vinyl-functionalized elastomer or the hydride-functionalized elastomer by covalently bonding the free or saponified unsaturated fatty acid to the elastomer bulk through a cross-linking 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.

[0044] 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 (DINEN ISO 9001) in the range of Shore 00 20 to Shore A80. 868) and comprising a plurality of fatty acid moieties covalently bonded to said elastomer bulk, wherein carboxylic acid groups of said moieties are available on an outer surface of said material to provide said bonding, and wherein said bulk-modified elastomer is obtained by forming a composition comprising a vinyl-functionalized elastomer or a hydride-functionalized elastomer or at least one precursor thereof, 0.5-5% by weight of free or saponified unsaturated fatty acids based on the total weight of said vinyl-functionalized elastomer or hydride-functionalized elastomer or at least one precursor thereof, and a cross-linking catalyst in a mold having a polar inner surface; and bulk-modifying said vinyl-functionalized elastomer or hydride-functionalized elastomer by covalently bonding said free or saponified unsaturated fatty acids to said elastomer bulk by a cross-linking reaction between the vinyl or hydride groups of said elastomer and the unsaturated carbon-carbon bonds of said unsaturated fatty acids in said mold to obtain said material.

[0045] Surprisingly, it was found that a relatively soft elastomer, i.e. an elastomer having a Shore hardness in a range of Shore 00 20 to Shore A 80 (or Shore A2-80), when modified with unsaturated fatty acids as previously disclosed in WO 2019 / 015988A1, can be used as a biocompatible material, and cell material can be cultured directly on this compatible biomaterial without the need for an extracellular matrix (e.g., fibronectin), when the amount of unsaturated fatty acids is in the range of 0.5-5 (wt%) by weight based on the total weight of the elastomer or at least one precursor thereof. Without wishing to be bound by theory, it is believed that when the concentration of unsaturated fatty acids is below 0.5 wt%, the density of carboxylic acid groups or carboxylate groups in the case of saponified unsaturated fatty acids is too low to render the outer surface of the elastomer on which cells are to proliferate sufficiently hydrophilic to achieve the desired proliferative conditions, whereas when the concentration is above 5 wt%, some unreacted unsaturated fatty acids may leach from the bulk-modified elastomer and poison cells attached to the outer surface of the elastomer. Therefore, bulk-modified elastomers are expected to have a covalently bound 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 within this range complete conversion of the cross-linking reaction between the elastomer (or one or more precursors thereof) and the unsaturated fatty acids in free or saponified form is expected to occur.

[0046] The bulk modified elastomer is selected to have a hardness in the range of Shore A 20 to Shore A 80 to ensure that the elastomer has sufficient flexibility to promote cell proliferation, because, for example, it is well known that the softness of the cell carrier material is crucial for stem cell growth (and differentiation) because the tissues grown from stem cells want to move, for example, (cardiac) muscle tissue, lung tissue, intestinal tissue and vascular tissue. Some organs need to be grown in a carrier that can move (such as the myocardium). Therefore, when the bulk modified elastomeric material has a Shore A hardness of more than 80, the material becomes insufficiently stretchable and cannot promote the desired cell proliferation directly on its functionalized surface. In this case, it will be required to use a scaffold or extracellular matrix material to provide a cell culture with its desired flexibility.

[0047] Furthermore, the unsaturated fatty acids used in bulk modification of elastomers can be used in free form or in saponified form (e.g., as sodium salts). The advantages of using saponified unsaturated fatty acids are that the risk of catalyst poisoning by the unsaturated fatty acid protons is avoided, and surprisingly, cell culture also propagates to bulk-modified elastomers carrying suitable counterions (e.g., Na + or K + ) on the outer surface of the carboxylate groups of the elastomer. This therefore has the further advantage that the saponification does not have to be reversed before using the bulk-modified elastomer for cell culture purposes.

[0048] Thus, in this way, materials for cell culture can be manufactured in a small number of steps, for example, in some embodiments in a single step, by combining an elastomer with a fatty acid in a coating (spin coating, dip coating, spray coating, dispensing) or injection molding process (wherein cross-linking of the carbon-carbon double bonds of the elastomer and the fatty acid can be achieved without significant epoxidation of the carbon-carbon double bonds of the fatty acid due to limited exposure to ambient oxygen during 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 the carboxylic acid group or carboxylate group of the (saponified) fatty acid is available for direct binding to harvested cells. Furthermore, by controlling the curing process and / or the properties 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 carboxylic acid groups or carboxylate groups on such outer surface, which makes the material particularly suitable for use as a membrane material for fluidic devices, as each cross-section of the material will exhibit the same surface properties, in contrast to membrane materials to which anchors for biocompatible materials are to be grafted or otherwise formed as previously explained. Additionally, since typically only a fraction of the carbon-carbon double bonds of the elastomer are consumed in such a cross-linking reaction, the material of the present invention retains the elastomeric properties of the elastomer, which improves the suitability of the material of the present invention for use in fluidic devices and facilitates slicing or other cutting of the material of the present invention for research purposes.

[0049] Preferably, each of the free or saponified fatty acid moieties is covalently bonded to the elastomer bulk by a cross-linking reaction between the vinyl or hydride functional groups of the elastomer and the unsaturated carbon-carbon bonds of the unsaturated fatty acid to ensure that the number of carboxylic acid groups or carboxylate groups available for binding to the biocompatible material can be optimized.

[0050] Unsaturated fatty acids can be any suitable unsaturated fatty acids. In an exemplary embodiment, the unsaturated fatty acids are selected from the following: myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, octadecenoic acid, linoleic acid, elaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, undecanoic acid and docosahexaenoic acid. Linoleic acid is particularly mentioned.

[0051] Similarly, elastomer can be any suitable elastomer. In exemplary embodiments, elastomer comprises polybutadiene backbone or siloxane backbone. When elastomer comprises siloxane backbone, before the crosslinking between unsaturated fatty acid molecule and the elastomer molecule, the carboxylic acid group of at least a certain fraction in the siloxane backbone can be saponified into the saponified product of carboxylic acid group, and the elastomer molecule can be required to prevent the crosslinking catalyst from being deactivated by the proton of the carboxylic acid group of the unsaturated fatty acid molecule. Equally, polybutadiene can be crosslinked with the unsaturated fatty acids of saponification, because as previously explained, the saponified product of unsaturated fatty acid can not significantly affect the cell culture properties of the material of body modification.

[0052] In one set of embodiments, the composition includes the free or saponified unsaturated fatty acid in an amount 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 precursor thereof, as it has been found that particularly good cell proliferation is achieved when the amount of free or saponified unsaturated fatty acid added to the bulk of the elastomer is within this range. For example, excellent cell proliferation directly on the outer surface of the bulk-modified elastomer is achieved when the composition includes the free or saponified unsaturated fatty acid in an amount of about 1% by weight based on the total weight of the vinyl-functionalized elastomer or hydride-functionalized elastomer or at least one precursor thereof.

[0053] According to another aspect, a fluid device module is provided, comprising a flow channel extending on a membrane, the membrane comprising a material of the present invention according to any of the embodiments described herein. Such a fluid device module may comprise 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. 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, such as a single processing step performed by injection molding.

[0054] The membrane may comprise a plurality of holes or grooves extending through the membrane. Such holes or grooves are preferably cell-sized to prevent cells from passing through the membrane.

[0055] In embodiments, the first flow channel and the second flow channel are accessible through respective septa, such that separate flow channels can be individually accessed without risk of cross contamination.

[0056] According to another aspect, a fluidic device is provided, comprising: a fluidic device module according to any of the embodiments described herein; and a pair of cover plates for fluidically sealing the fluidic device module. The cover plates can be arranged so that one of the cover plates covers the first major surface, thereby sealing the first fluid channel, and the other of the cover plates covers the second major surface, thereby sealing the second fluid channel. Such a fluidic device can be manufactured in a simple manner because the fluidic device module can be formed in a small number of processing steps as previously explained, and due to the flexibility of the fluidic device module, the fluidic device module is robust against leakage.

[0057] According to another aspect, a method for culturing cells is provided, the method comprising: providing a molded article formed from the body-modified elastomeric material of any of the embodiments described herein, the article comprising 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, cells can be directly proliferated on the exposed surface of such an article (e.g., a fluidic device module as described above) without the need to attach an extracellular matrix (e.g., fibronectin, etc.) to the exposed surface of the article, thereby significantly simplifying the cell culture process.

[0058] According to another aspect, a drug testing method is provided, the method comprising providing a fluidic device module according to an embodiment; applying harvested cells directly to the surface of a membrane of the fluidic device module; culturing the harvested cells on the surface; forming a fluidic device with the prepared fluidic device module; feeding the cultured cells through one of a pair of flow channels of the fluidic device; exposing the cultured cells to a drug to be tested through the other of the pair of flow channels of the fluidic device; and monitoring the response of the cultured cells to the drug to be tested. This drug testing method can be deployed in a simple and straightforward manner, particularly in terms of preparing the fluidic device to be used in the drug testing method, thereby providing a significant simplification of existing drug testing methods (in which preparing such fluidic devices is typically very cumbersome).

[0059] Monitoring the response of cultured cells to a drug being tested can include stabilizing and fixing the cultured cells within the fluidic device and slicing the fluidic device module to obtain a slice for microscopic evaluation, the slice comprising at least a portion of the stabilized and fixed cultured cells. According to the teachings of the present invention, since the cultured cells are evenly distributed on the membrane surface of the fluidic device module (due to bulk modification of the elastomer with free or saponified unsaturated fatty acids, as previously explained), generating such a slice for microscopic evaluation is no longer critical or subject to variable results.

[0060] Alternatively, in a preferred embodiment, monitoring of the response of the cultured cells can be performed within the assembled fluidic device using confocal microscopy. This facilitates real-time monitoring of such responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Embodiments of the invention are described in more detail by way of non-limiting example with reference to the accompanying schematic drawings, which are not drawn to scale, in which:

[0062] Figure 1 depicts bulk modification of elastomers with unsaturated fatty acids according to embodiments;

[0063] Figure 2 depicts a perspective view of a fluidic device module according to an embodiment;

[0064] Figure 3 depicts another perspective view of a fluidic device module according to an embodiment;

[0065] Figure 4 depicts a perspective view of a fluidic device module according to another embodiment;

[0066] Figure 5 depicts an exploded perspective view of a fluidic device according to an embodiment;

[0067] Figure 6 depicts an exploded perspective view of a fluidic device according to another embodiment;

[0068] Figure 7 depicts an exploded perspective view of a fluidic device according to yet another embodiment;

[0069] Figure 8 depicts an exploded perspective view of a fluidic device according to yet another embodiment;

[0070] Figure 9 is a microscopic image of a cell culture grown on a bulk-modified elastomer according to an embodiment;

[0071] Figure 10is a microscopic image of a cell culture developed on an elastomer without such bulk modification;

[0072] Figure 11 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 1 day of development;

[0073] Figure 12 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 2 days of development;

[0074] Figure 13 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 7 days of development;

[0075] Figure 14 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 4 days of development;

[0076] Figure 15 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 4 days of development;

[0077] Figure 16 : is a graph showing the measured contact angles for substrates comprising siloxane modified with 1 wt % linoleic acid. The vertical axis is the contact angle (in degrees). Four sets (each set comprising two bars) represent the average contact angles for four 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 modified substrates used in cell culture examples 1, 2, and 3, respectively. DETAILED DESCRIPTION

[0078] It should be understood that the same reference numerals are used throughout the drawings to indicate the same or like parts.

[0079] Figure 1 Depicted is a reaction between the olefinic bonds of a precursor of residue 20 and the olefinic bonds of a polymer backbone 10 used to make a bulk-modified elastomeric material according to some embodiments of the present invention. In this embodiment, the reaction occurs between the fatty acid precursor residue linolenic acid and a vinyl functionalized elastomer 10 in the form of polybutadiene (hereafter simply referred to as elastomer). This reaction can be catalyzed by a suitable catalyst (e.g., peroxide). Although in this embodiment, the reaction between the olefinic bonds of a precursor of residue 20 and the olefinic bonds of a polymer backbone 10 used to make a bulk-modified elastomeric material according to some embodiments of the present invention is depicted. In this embodiment, the reaction occurs between the fatty acid precursor residue linolenic acid and a vinyl functionalized elastomer 10 in the form of polybutadiene (hereafter simply referred to as elastomer). Figure 1 In FIG, polybutadiene 10 is shown reacted with a saponified form of linoleic acid (wherein R is, for example, an alkali metal, e.g., lithium, sodium, or potassium ion), but it is clear that other olefinic precursor residues and other olefinic backbone polymers can also be used in this type of reaction.

[0080] It has been found that such a reaction can provide a bulk-modified elastomer in which the carboxylic acid groups of at least a certain fraction of the saturated fatty acid molecules 20 that participate in the reaction with the elastomer 10 are present on the outer surface of the bulk-modified elastomer, so that these carboxylic acid groups can be used to stabilize cell cultures directly on the outer surface of the bulk-modified elastomer 10. Therefore, there is no longer a step of binding proteins to such carboxylic acid groups to promote cell stabilization, and this saves considerable time and effort and opens up new opportunities for defining cell culture substrates comprising such materials. For example, by using an appropriate reaction vessel, elastomer formation and elastomer shaping can now be accomplished in one step. In a preferred embodiment, such a reaction vessel is a mold, for example a mold of an injection molding device. It should be noted that Figure 1 is merely an example of an olefinic chemistry that can be used in this process. Reactions based on the addition of silane hydrides to olefinic bonds in the presence of a catalyst, as described below, can also be used, as explained in more detail below.

[0081] Preferably, the elastomer 10 is soft or flexible to facilitate cell-induced migration. For this reason, the elastomer 10 may generally have a Shore hardness range of from Shore 0020 (i.e., Shore A2) to Shore A 80, i.e., a Shore A hardness of 2-80, as determined in accordance with DIN EN ISO 868. Suitable elastomers include polyenes, such as polybutadiene and silicones.

[0082] For example, the unsaturated fatty acid 20 may be a free unsaturated fatty acid (in which case R = H) or may be a saponified fatty acid (in which case R = Na + or K + ) (and the COO group is a COO- or a carboxylic acid anion group). This saponification reaction can be achieved in any suitable manner (e.g. using concentrated NaOH or KOH solution). Since this saponification reaction is well known per se, this will not be explained in further detail for the sake of brevity.

[0083] Importantly, there is no need to reverse the saponification of the unsaturated fatty acid residues in the bulk-modified elastomer, as it has been surprisingly found that when the outer surface of the bulk-modified elastomer 10 is saponified with carboxylic acid groups (i.e., with a moiety such as Na + or K + Cell proliferation can also occur on the surface when the surface is functionalized with a carboxylate group of a counterion such as a counterion, as will be explained in further detail below.

[0084] The presence of carboxylic acid groups or carboxylate groups on the outer surface of the bulk-modified elastomer 10 can be surprising, as the elastomer 10 can be non-polar in nature, particularly when 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 inward within the micelle, which generally results in the carboxylic acid moieties ending up within the bulk of the modified elastomer. Furthermore, it is well known that the carbon-carbon double bonds of such unsaturated fatty acids 20 are susceptible to rapid epoxidation in the presence of oxygen, which reduces the ability of these compounds to undergo a crosslinking reaction with the elastomer 10.

[0085] Embodiments of the present invention are based on the insight that if a reaction mixture comprising an elastomer 10 and an unsaturated fatty acid 20 in saponified form is contacted with a polar surface, this promotes the orientation of the carboxylic acid groups of the fatty acid molecules along such a polar surface, so that the outer surface of the bulk-modified elastomeric material exhibits a high density and uniform distribution of these carboxylic acid groups. Furthermore, 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., a temperature in the range of 120-240° C.), 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, the substantial absence of molecular oxygen in the reaction mixture within the injection molding apparatus inhibits epoxidation of these unwanted carbon-carbon double bonds.

[0086] In order to obtain an elastomer 10 on which cells can be cultured directly, a 2.10% cellulose acetate copolymer is used, which is calculated 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 using an amount of the unsaturated fatty acids 20 in free or saponified form within the range of 10 mol / kg. If a lower amount of the unsaturated fatty acids 20 in free or saponified form is used, it has been found that cells do not proliferate significantly on the bulk-modified elastomer 10, possibly because the surface of the bulk-modified elastomer 10 on which the cells are to be seeded is too hydrophobic in nature. On the other hand, if a higher amount of the unsaturated fatty acids 20 in free or saponified form is used, cells also largely fail to proliferate on the surface of the bulk-modified elastomer 10, and / or the proliferation results vary greatly between experiments, making them irreproducible. This may be caused by unreacted unsaturated fatty acids 20 in free or saponified form leaching from the bulk-modified polymer and poisoning the cell culture. Furthermore, it has been found that when the amount of the unsaturated fatty acids 20 in free or saponified form is within the above-mentioned range, the flexibility or hardness of the unmodified elastomer 10 is not significantly altered by bulk modification with the unsaturated fatty acids 20 in free or saponified form. Furthermore, the transparency of the elastomer to at least optical radiation (e.g., visible light) decreases, for example, due to increased scattering caused by opacity when higher concentrations are used. This can impair the use of cell culture substrates for optical monitoring of cell proliferation or culture.

[0087] Although Figure 1 The examples are described for bulk elastomers of the polydiene type, but other elastomers may also be used. Preferably, such elastomers also have the desired Shore hardness.

[0088] 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 produce the polyene, after which the polymerization can be carried out as described in reference to Figure 1The coupling is described. Particularly preferred is polybutadiene formed in Nd- or Li-catalyzed polymerization reactions, as it is well known per se that such polybutadiene has a low degree of branching (typically less than 5%) and a low polydispersity (Mw / Mn) of approximately 2. Li-catalyzed polybutadiene is particularly preferred because it has a high 1,2-vinyl content (approximately 11%). However, other types of polybutadiene (e.g., polybutadiene obtained from Co-, Ni-, or Ti-catalyzed polymerization reactions) may also be used instead. In the case of using a polyene such as polybutadiene as the elastomer 10, a peroxide catalyst may be used to catalyze the crosslinking reaction between the elastomer 10 and the unsaturated fatty acid 20. In another advantageous embodiment, Li-catalyzed polybutadiene is used that 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 within this range, the reactivity of the polybutadiene with the unsaturated fatty acid 20 is improved. It should be noted for the sake of completeness that how to control the degree of branching when synthesizing polybutadiene is well known per se, so that this will not be explained further merely for the sake of brevity.

[0089] Another class of desirable elastomers is siloxanes (polysiloxanes) because they are suitable for injection molding. Such siloxanes can include a polydimethylsiloxane (PDMS) backbone (which includes vinyl moieties) to promote crosslinking via a Pt-catalyzed addition reaction with (polymethyl)hydrogensiloxane. As an alternative or in addition to crosslinking with (polymethyl)hydrogensiloxane, the vinyl-functionalized PDMS backbone can be reacted with unsaturated fatty acids.

[0090] Such elastomers in the form of siloxanes can be formed by 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).

[0091] For example, a two-component siloxane can be formed by a crosslinking reaction between a linear component 1 and a linear component 2, each of which can correspond to the following general formula:

[0092]

[0093] In component 1, R1 and R2 are independently selected from C1-C3 alkyl groups, and R3-R8 are 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 may be in the range of 100-200,000. In a specific embodiment of component 1, each of the alkyl groups R1-R8 is a methyl group, i.e., component 1 is a vinyl-functionalized PDMS having a terminal vinyl group.

[0094] In component 2, R1 is hydrogen, R2 = C1-C3 alkyl, and R2-R8 are individually selected from C1-C3 alkyl or hydrogen, with the proviso that at most one of R3-R5 and at most one of R6-R8 is hydrogen, and n can have any suitable value, for example, n = 3-1000 or more specifically n = 3-10. In a specific embodiment of component 2, none of the groups R3-R8 is hydrogen. In another specific embodiment of component 2, each of R2-R8 is methyl.

[0095] Component 2 generally acts as a crosslinker for component 1. Such crosslinking reactions, which are generally catalyzed by Pt, are well known per se, see for example WO 2009 / 147602 A2, and therefore will not be explained in detail for the sake of brevity.

[0096] Bulk modification of such siloxanes can be accomplished by forming an elastomer in the presence of unsaturated fatty acids 20, with covalent bonds between the siloxane and the unsaturated fatty acids 20 being formed by reaction between the hydride functional groups of the (crosslinked) siloxane and the unsaturated fatty acids 20 as suggested by the following reaction mechanism:

[0097]

[0098] To prevent catalyst inhibition during this crosslinking reaction by the protons of the carboxylic acid groups of the unsaturated fatty acid 20, the unsaturated fatty acid can be used in a saponified form (e.g., as a sodium salt of the unsaturated fatty acid) for the crosslinking reaction with the silicone elastomer. In some embodiments, if desired, the reaction product (bulk-modified elastomer) can then be treated with a protonic acid (e.g., HCl, etc.) to restore the carboxylic acid groups on the surface of the bulk-modified elastomer, but this is not necessary when cell culture is to be performed directly on the surface of the bulk-modified elastomer, as it has been demonstrated that cells can also proliferate when the polar surface groups are carboxylate anions (i.e., saponified carboxylic acid groups). Any silicone elastomer having free silicon hydride groups for reaction with the olefinic bonds of the residue-bearing precursor can be used for this elastomer bulk modification.

[0099] It should be noted that elastomer formation need not be initiated prior to carrying out the modification reaction. This may be accomplished in some embodiments, but preferably, modification is at least partially performed during elastomer formation by mixing appropriate amounts of elastomer-forming components with the modified precursor residue.

[0100] Alternatively, the siloxane backbone such as (polymethyl) hydrogen siloxane backbone can be cross-linked with the rubbery polymer such as polybutadiene and polyisoprene, and wherein, unsaturated fatty acid is incorporated in this cross-linked product.For example, this operation can be carried out to tune the water permeability of final material, because siloxane has quite open structure usually, and this rubbery polymer has quite closed structure, makes the openness (that is, water permeability) of cross-linked product can be tuned by the ratio of siloxane and rubbery polymer wherein.In this way, can optimize the character of the material according to this embodiment to build good support for cell / protein interaction.For example, when elastomer is (for example based on PDMS) elastomer based on siloxane, obtain and have highly permeable material for water and other compounds (for example, medicine), this material promotes the maximization perfusion in the cell on the film of these compounds to fluid device module 100. However, in cases where real-time monitoring of drug consumption by cells is desired, the use of polyene-based materials (e.g., polybutadiene-based materials) that are impermeable to water and drugs may be preferred because the density of pores through the membrane can be used to control the rate of perfusion of these compounds to the cells.

[0101] In an embodiment, the polysiloxane can be formed from a multi-component starting material as explained above to prevent premature crosslinking of the polysiloxane. Such multi-component starting materials are well known per se; for example, such a multi-component starting material kit is sold by Wacker Chemie AG of Munich, Germany under the trade name Elastosil. However, other siloxanes can also form elastomers and can also react with olefinic bonds.

[0102] Linoleic acid has been used as a precursor for the acidic groups carrying residues with which bulk-modified elastomers are modified. It should be understood that this is by way of non-limiting example only, as other acidic groups carrying residues may also be used, as will be described below. Unless otherwise indicated, the residues and their precursors described below will also achieve the advantages of linoleic acid described above.

[0103] Thus, for example, with respect to the unsaturated fatty acid 20, any suitable unsaturated fatty acid may be used for the purpose of reacting it with the elastomer 10. For example, the unsaturated fatty acid 20 may be selected from myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, octadecenoic acid, linoleic acid, elaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, undecanoic acid, and docosahexaenoic acid.

[0104] More generally, in the context of this application, reference is made to Figure 1 In the case of the exemplary unsaturated fatty acid 20, it should be understood that this is intended to refer to any organic compound comprising one or more acidic groups in free form or in conjugate base form and comprising at least one carbon-carbon double bond.

[0105] Typically, the residue in a bulk-modified elastomer is derived from the reaction of a residue precursor (used to modify the bulk-modified elastomer) with the elastomer bulk or its precursor. In this sense, fatty acids are just one type of such residue precursor. Other types exist and can be used, as long as they possess at least one carbon-carbon double bond and one or more acidic groups, which, when used in conjunction with siloxane chemistries, require a conjugate base form. In polydiene chemistries, this conjugate base form is not inherently required.

[0106] In some preferred embodiments, the residue precursor comprises or consists of an aliphatic portion comprising 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 bound to the aliphatic portion. The aliphatic portion can be linear or branched. It can comprise one or more (e.g., 2, 3, or 4) carbon-carbon double bonds or carbon-carbon triple bonds (preferably only double bonds) or an aromatic unit or a phenyl unit. The aliphatic portion can comprise a cyclic unit, such as a cyclohexyl or cyclopentyl group. The aliphatic portion preferably comprises only carbon atoms and hydrogen atoms. The aliphatic portion can be a saturated hydrocarbon portion. In some embodiments, the aliphatic portion further comprises no carbon-carbon triple bond to improve the flexibility of the residue.

[0107] In some embodiments, the aliphatic moiety includes at least one terminal carbon-carbon double bond (H2C=CH-R, where R is the remainder of the aliphatic moiety), and preferably, the moiety is completely linear, having at least one (e.g., only one) acidic group attached to the other end of the moiety.

[0108] In certain embodiments, the mixture of precursor residues can be used to realize the elastomer of body modification, and at least the part of its residue is different from each other.Can choose each precursor residue in the precursor residue as defined herein.In certain embodiments, at least the part of the aliphatic part of the residue precursor comprises at least 2 carbon-carbon double bonds.In this way, the residue in the elastomer of body modification can be covalently bonded to the elastomer body with two or more keys.Therefore, the polymer chain of elastomer body can become crosslinked via residue.

[0109] In certain embodiments, the part of the precursor residue includes at least 5 carbon atoms, more preferably includes at least 10 carbon atoms. Preferably, the aliphatic part includes less than 40 or less than 30 carbon atoms. The number of carbon atoms in this part is preferably between 5 and 30, more preferably between 5 and 20 or between 5 and 15. This part and / or chain can have one or more aryl groups. One or more acidic groups in the acidic group can be directly attached to the aryl group (for example, a phenyl ring).

[0110] In some embodiments, the residue precursor is an unsaturated fatty acid, and the residue is covalently bonded to the remainder of the elastomer via reaction of one or more of its vinyl groups (carbon-carbon double bonds), if present.

[0111] For example, the unsaturated fatty acid residues are one or more residues of fatty acids selected from the group comprising myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, octadecenoic acid, linoleic acid, elaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid and docosahexaenoic acid.

[0112] In certain embodiments, the residue is the remainder of a linear or branched alkyl chain of a precursor having 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. Compared with the non-terminal vinyl group during the formation of the elastomer bulk in the manufacturing process, the terminal vinyl group can provide the reactivity of improvement. The straight chain with a terminal vinyl group and an acidic group (e.g., carboxyl group) is a preferred example. In this case, 5 to 15 carbon atoms can be present in the chain.

[0113] 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 includes 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 less than 30, less than 20, or less 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, for example, a 5-membered or 6-membered cyclopentyl or cyclohexyl group, which itself may contain at least one double bond. A non-limiting example of an organic compound intended to fall within the above definition for unsaturated fatty acids 20 is retinoic acid.

[0114] The aliphatic moiety may have one, two, three, or four olefinic bonds. Preferably, the number of olefinic bonds is less than 3. More preferably, there is only one olefinic bond. At least one olefinic bond is substituted with at least two hydrogen atoms. More preferably, at least one olefinic bond is substituted with three hydrogen atoms. Such olefinic bonds generally exhibit increased reactivity in the reactions described herein, with the reactivity being higher for the latter.

[0115] The acidic group carried by the aliphatic portion is preferably attached to a saturated carbon atom of the aliphatic component. The acidic group can be a carboxylic acid group and a carboxylate group thereof (a conjugate base group), a phosphorus comprising an acidic group (e.g., a phosphate group) and corresponding phosphates and hydrogen phosphates, a phosphate group and corresponding conjugate bases, and / or a sulfur comprising an acidic group (e.g., a sulfate group) and corresponding sulfates.

[0116] The phosphate group is a phosphoric acid in which each phosphorus atom is in the oxidation state +5 and is bound to four oxygen atoms. Two or more of these PO4 units (tetrahedrons) can be linked by a shared single-bonded oxygen, thereby forming a linear or branched chain with at least one of the oxygen atoms bound to the rest 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 phosphate 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, which number is between 0 and (n+2) / 2. One bond remains for binding to the residue (e.g., the aliphatic portion of such residue). Preferably, the acidic group comprises an orthophosphate group or a polyphosphate group RO (O=PoHO) n -H, where n=1 for orthophosphoric acid and n>2 for polyphosphoric acid, and R represents the remainder of the residue to which the group is attached. For example, a phosphate group, well known in the art, may be present and will not be described in further detail. Further definitions can be found in many textbooks on organic chemistry.

[0117] Any of the conjugate bases of the acidic group (e.g., carboxylates, phosphates, hydrogen phosphates, hydrogen sulfates, etc.) preferably associates with or saponifies with metal counterions (e.g., counterions of alkali metals (e.g., lithium, sodium, and potassium) and counterions of alkaline earth metals (e.g., barium and calcium)).

[0118] Many other compounds besides the exemplified compounds explicitly mentioned in this application will immediately occur to those skilled in the art.

[0119] Each residue or precursor of such a residue may have one 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 groups present in the bulk-modified elastomer. To calculate the final molar concentration of acidic groups based on the molar concentration of the residues, it is necessary to multiply the molar concentration of the residues by the number of acidic groups per residue. Having more than one acidic group per residue may be beneficial when the desired molar concentration of such groups in the bulk-modified elastomer is greater than 1.10 -2 The present invention relates to an amount of at least one mole of residue per kg of bulk-modified elastomer, without being substantially plagued by the problems associated with amounts of residues above the limits of this range, as explained hereinbefore.

[0120] As disclosed above, 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 between 2.10 and 3.5, calculated as a weight relative to the total weight of the elastomer or the total weight of one or more precursors of the elastomer. -4 to 2.10 -2 For silicone elastomers, the residue concentration range is preferably 2.10 -4 to 1.10 -2 mol / kg. This largely avoids the need to rinse the substrate before use to remove unreacted precursors. For low concentrations of acidic groups, the residual molar concentration can be in the range of 2.10 -4 to 2.10 -3 mol / kg. For high concentrations of acidic groups, the residue molar concentration can be in the range of 1.10 -3 to 5.10 -3 Cell proliferation for different cell lines may require different concentrations within a range.

[0121] The molar concentrations disclosed herein can be converted to weight percents when taking into account the molar weight of the residue or precursor to the residue using well-known calculations.

[0122] It should be noted that when using silyl hydride addition on olefinic chemistries used to form siloxane-based elastomers, residue precursors with acidic groups in the form of conjugate bases are used. For example, they are saponified. For this reason, they are added as salts of alkali or alkaline earth metals. This avoids contamination of the Pt catalyst used with the aforementioned chemistries. However, this is not necessary for polyene-based modifications, as acid-insensitive chemical catalysts are used.

[0123] An exemplary synthetic scheme for making bulk-modified elastomers based on polyenes such as polybutadiene is given below (Synthesis Example 1).

[0124] Synthesis Example 1

[0125] A polybutadiene block obtained from Lanxess AG of Cologne, Germany, was inserted into a compound or mixing extruder. The block included a peroxide (e.g., dicumyl peroxide) as a catalyst. An amount of linoleic acid (60-74% concentrated from Aldrich Chemical) in the range of 1-5 wt% based on the weight of the polybutadiene block was mixed into the polybutadiene block by kneading at a temperature in the range of 110-150° C. The resulting extruded mixture was injected into a stainless steel mold that defined the fluidic device and had an oxidized inner surface that was contacted with the extruded mixture for 3-10 minutes at a temperature in the range of 150-200° C. to produce a monolithic fluidic device module of polybutadiene rubber cross-linked with linoleic acid.

[0126] An exemplary synthetic scheme for making a bulk-modified elastomer from silicone rubber is given below (Synthesis Example 2).

[0127] Synthesis Example 2

[0128] 4.848 g of a sodium linoleate solution in a solution of water and isopropyl alcohol (solvent weight ratio 1:1) (the weight fraction of sodium linoleate in the solution is 30% by weight) was mixed into 240 g of the silicone component A of Elastosil LR3040 obtained from Wacker Chemie AG and stirred vigorously under vacuum at room temperature to evaporate water from the composition. The resulting mixture was mixed with component B (240 g) of Elastosil LR 3040 obtained from Wacker Chemie AG (including a Pt catalyst) and fed into an injection molding apparatus where the resulting mixture was injected into a stainless steel mold having an oxidized inner surface that contacted the resulting mixture and defined a fluidic device module and molded at a temperature in the range of 150-200° C. for 10-60 seconds to produce a monolithic fluidic device module of silicone rubber crosslinked with sodium linoleate, wherein the sodium linoleate was present in the polymer matrix in an amount of 1% by weight relative to the weight of the polymer matrix (based on the weight of the starting material). Optionally, the molded product can then be placed in an aqueous solution of HCl (pH 2-4) to convert at least the sodium carbonate groups at the surface of the molded product into free carboxylic acid groups.

[0129] Synthesis Example 3

[0130] Same as Example 2, but using an appropriate amount of sodium undecanoate to achieve the same molar concentration as the sodium linoleate of Example 3.

[0131] To further demonstrate the bulk modification of the elastomers in Synthesis Examples 1 and 2, blocks of each elastomer were molded in a polar mold after reaction with linoleic acid as described above and then sliced ​​with a microtome at -40°C to obtain internal slices of the modified elastomer, i.e., slices in which none of the major surfaces of the elastomer were the outer surfaces of the molded elastomer block.

[0132] In order to demonstrate that a portion of the acidic groups of the modified residues were actually available on the surface of the molded elastomers, these internal sections were subsequently contacted with 2 μL of water drops at different pH values ​​(pH 4, pH 7, and pH 10) for 3 minutes, after which the contact angles of the water drops with these internal sections were measured. The same procedure was repeated with the unmodified elastomer to compare the contact angles measured for the internal sections of the linoleic acid-modified elastomers with the contact angles measured for the corresponding internal sections of the unmodified elastomers. For example, in Figure 16 The results for silicone-based elastomers are shown in the graphs in .

[0133] As can be seen from these graphs, the contact angle of the unmodified siloxane is very insensitive to the pH of the droplet. Without wishing to be bound by theory, the small increase in contact angle measured for the unfunctionalized siloxane with increasing pH may be a result of the pH dependence of hydrogen bonding between water molecules and the silanol moieties in the siloxane. For the modified siloxane batches used for culturing different cell types described below, the contact angle at pH = 4 was significantly lower than the contact angle at pH = 10.

[0134] The behavior observed for the modified siloxanes (not shown) is the same regardless of the form of the surface-available carboxylic acid groups (free or conjugated base form).

[0135] In addition, bulk modification is evident as can be seen from observing the same behavior at cut surfaces prepared by cutting a batch of substrates in half.

[0136] Thus, both the inner slice and the outer surface of polybutadiene cross-linked with linoleic acid (obtained after cutting) show a strong pH dependence of the measured contact angle, which decreases with increasing pH.

[0137] Although not shown here, similar contact angle behavior was observed for the modified polybutadiene, while the measured contact angle for unmodified polybutadiene was largely independent of pH.

[0138] The strong pH dependence of the measured contact angles for the bulk and surface of the functionalized elastomer clearly demonstrates the presence of carboxylic acid groups in the bulk and on the surface of the modified elastomer. This can be understood as follows. At lower pH, these carboxylic acid groups remain mostly protonated, thereby minimizing the surface charge of the internal sections of the elastomer. As pH rises, this surface charge increases due to the increase in deprotonation of the carboxylic acid groups, which reduces the contact angle of the water droplet with the surface of these internal sections. On the other hand, in the absence of such carboxyl groups at the surface exposed to the water droplet (for example, in unmodified elastomers), changes in the pH of the water droplet do not cause changes in the surface charge, making the measured contact angles of the water droplet with this surface at different pH values ​​extremely insensitive to these pH changes.

[0139] The pH of the droplet used for contact angle discrimination depends on the pKa value of the acidic group used in the substrate. In the case of linoleic acid groups, the pKa is approximately 4.7, so low-pH droplets should have a pH below 4.7, while high-pH droplets should have a pH approximately 2 pH units higher than the pKa to induce complete dissociation in the droplet. Those skilled in the art will be able to select the correct conditions for a specific acidic group with a specific pKa.

[0140] An alternative method for examining surface acidic groups (carboxylic acid groups) was performed. To this end, all slabs or sections were treated with HCl as described herein to reverse the conjugate base of any available acidic group to its acidic form. The NH2 group of cadaverine was reacted with the acidic group using the well-known EDC / NHS coupling chemical, and the resulting dry slab was coupled to fluorescently labeled cadaverine. Comparison of the treatment of the modified and unmodified elastomers clearly showed an increase in fluorescence, with the latter having a fairly uniform spatial grayscale, and the unmodified elastomer lacking fluorescence. This staining test can be used to illustrate the presence of available acidic groups (e.g., carboxylic acid groups) on the surface.

[0141] The bulk presence of acidic groups was investigated using IR measurements in reflection mode and transmission mode as well as in elemental analysis.

[0142] After the polymerization reaction to form the modified elastomer is completed or carried out, IR and Raman spectroscopy are used to determine the conversion and state, for example, by observing a decrease in vibrational absorption of the olefinic bonds of the precursor to the modified residue.

[0143] The bulk modified elastomer can be molded in any suitable form to produce an article or cell culture substrate on which cells can be cultured directly. These articles can take any suitable shape. In a particular embodiment, the mold used in this injection molding process can be shaped in the form of a fluid device module so that a monolithic fluid device module can be formed in a single-step injection molding process. Because the carboxylic acid groups or carboxylate groups have a high density and uniform distribution on the (one or more) outer surfaces of this monolithic fluid device module, it is possible to use this device module directly as a cell culture substrate. The mold can be formed by or have an inner surface coated with any suitable polar material (e.g., stainless steel, metal or metal oxide (e.g., alumina)) so that the carboxylic acid groups or carboxylate groups of the bulk modified elastomer have a desired orientation on at least one of its outer surfaces. In Figure 2 and Figure 3 An exemplary embodiment of a fluidic device module 100 is schematically depicted in FIG, wherein perspective views of opposing major surfaces of the fluidic device module 100 are schematically depicted. The fluidic device module 100 may include a membrane 110 on which at least one flow channel 115 extends. Preferably, the fluidic device module 100 includes opposing flow channels that are spatially separated by the membrane 110. At least the membrane 110 is made of a bulk-modified elastomer 30, but in a preferred embodiment, the entire fluidic device module 100 is made of a bulk-modified elastomer 30, thereby creating a monolithic fluidic device 100.

[0144] The fluidic device module 100 may include one or more membranes 120 through which such flow channels 115 are accessible. Preferably, each flow channel 115 is accessible through a dedicated set of membranes 120, so that cross contamination between multiple flow channels 115 is avoided. As explained above, the exposed surface of the fluidic device module 100 (e.g., membrane 110) can be used to directly bind cell culture, so that cells can be cultured (proliferated) on a biocompatible surface.

[0145] The membrane 110 may include a plurality of holes or grooves so that liquid nutrients and solutions with compounds such as potential drugs can reach both sides of the membrane and the cells / tissue. The holes or grooves may be formed in any suitable manner (e.g., by laser cutting). Such holes or grooves in the membrane 110 of the fluidic device module 100 are preferably of the same order of magnitude as the typical diameter of the cells to be stabilized on the membrane 110. The spacing between such holes or grooves is not particularly critical.

[0146] The fluid device module 100 can be formed by providing a pair of cover plates 210, 220. Figure 4As schematically depicted in the fluidic device 200 , the cover plate fluidically seals the fluidic device module 100 , such that fluid passing through one or more flow channels 115 of the fluidic device module 100 cannot leak from the fluidic device 200 .

[0147] In an embodiment, the cover plates 210, 220 are arranged such that: the cover plate 210 covers the first major surface of the fluidic device module 100, thereby sealing the first fluid channel 115 of the fluidic device module; and the other cover plate 220 covers the second major surface of the fluidic device module 100, thereby sealing the second fluid channel 115 of the fluidic device module. The cover plates 210 and 220 can be made of any suitable material (e.g., glass or plastic material). Preferably, the cover plates are retractable and flexible to ensure a good fit with the fluidic device module 100 while maintaining the flexibility of the fluidic device module 100 in the fluidic device 200. Such a fluidic device 200 can be deployed as an organ-on-a-chip as will be readily understood by those skilled in the art.

[0148] Figure 5An exemplary embodiment of a fluidic device 200 is schematically depicted, wherein the device comprises a fluidic device module 100 according to the present invention, the fluidic device module 100 being between two rigid cover plates 210 and 220. The upper plate 210 comprises a first inlet 231 and a first outlet 232, the first inlet 231 and the first outlet 232 being in fluid communication with a lower channel (not visible) below the membrane 110 in the fluidic device module 100, such that the membrane 110 is in fluid contact with a fluid passing through the lower channel. The upper plate 210 further comprises a second inlet 233 and a second outlet 234, the second inlet 233 and the second outlet 234 being in fluid communication with an upper channel 115 above the membrane 110, such that the membrane 110 is in fluid contact with another fluid passing through the upper channel 115. For example, the fluid through lower passage can comprise the medicine that will be tested on the cell culture being attached to film 110 as explained in more detail below, because film 110 (for example, by laser drilling or groove in film 110) is made into porous, therefore this medicine can reach cell culture by film 110.Alternatively, when forming fluid device module 100 in polarity mould, can form this hole or groove in film 110.This has the following advantages: the regular pattern of this fluid channel (hole) can be formed by film 110.Can directly contact with this cell culture and provide nutrients to this cell culture through the other fluid of upper passage 115.In certain embodiments, inlet 231,233 and outlet 232,234 are diaphragms, by this diaphragm (for example, by pressing this diaphragm), fluid can be pumped to on film 110 by aforementioned passage hydraulically. Alternatively, the inlets 231 , 233 and outlets 232, 234 may be ferrules to which tubing may be attached (eg, clamped), which is useful, for example, when the fluidic device 200 is used as a well plate or the like in a lab-on-a-chip system.

[0149] exist Figure 6 In the embodiment schematically depicted in FIG, the upper channel and the lower channel 115 are incorporated into the (monolithic) fluidic device module 100 (only one channel is shown), for example, when the fluidic device module 100 is molded. 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 example embodiment of a fluidic device 200, and Figure 8 Schematically depicts a bottom perspective view of an exemplary embodiment of a fluidic device 200, wherein the upper channel 115 is formed in the upper cover plate 210, while the lower channel 115' is formed in the fluidic device module 100. A skilled person will readily appreciate that it is equally feasible to form the lower channel 115' in the bottom cover plate 220. The fluidic device 200 (e.g., in Figure 7 and Figure 8The device shown in ) can, for example, be used as a microwell in a lab on a chip device, wherein a plurality of such microwells can be clamped or otherwise secured to facilitate parallel testing of a plurality of different samples within a single lab on a chip device, wherein each microwell typically includes one of these samples.

[0150] When a cell culture 50 is stabilized and propagated in such a fluidic device 200, the fluidic device 200 can be used in a method in which the stabilized, propagating cell culture 50 is exposed to a fluid comprising a compound of interest, which can be passed through the flow channel 115 of the fluidic device 200 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 this exposure. This method can be deployed, for example, in an oncology setting, where the stabilized cell culture 50 can be exposed to a drug (e.g., a chemotherapeutic drug) to monitor the response of the stabilized cell culture 50 to the drug. To this end, the stabilized cell culture 50 can include tumor cells to test the efficacy of the drug and / or can include healthy cells to test the toxicity of the drug on healthy tissue.

[0151] Such a drug testing method generally involves providing a fluidic device module 100 according to one or more embodiments of the present invention, for example, providing a monolithic fluidic device module 100 and applying a harvested cell culture to at least a membrane 110, on which the cell culture can directly bind to the carboxylic acid groups or carboxylate groups on the exposed surface, to obtain a prepared fluidic device module 100, from which a fluidic device 200 (e.g., an organ-on-a-chip) is formed as previously explained. The cell culture within the fluidic device 200 can be fed through one of a pair of flow channels 115 of the fluidic 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 fluidic 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: by removing the cover plate 210, 220 to disassemble the fluidic device 200, and cutting the fluidic device module 100 so that a slice of the part including the cell culture of the module can be obtained, the slice can be studied under a microscope, etc., so as to study the effect of the drug being tested on the cell culture. In such research, the cell culture is usually dyed, the cell culture is disposed of with formalin and the cell culture is fixed in a fixative (e.g., paraffin) to facilitate such microscopic research. This dyeing, disposal and fixation of the cell culture can be deployed in the fluidic device 200, for example, by making appropriate chemical reagents be exposed to the fluid channel 115 of the fluidic device 200 therein to achieve this.

[0152] In an alternative embodiment, such disassembly of the fluidic device 200 can be avoided, and confocal microscopy can be used to monitor the response of the cell culture to one or more compounds (e.g., a drug to be tested) in real time. In particular, in embodiments where the fluidic device 200 includes transparent cover plates 210, 220 (e.g., glass plates or polymer plates), a confocal microscope can be used to monitor the cell culture within the assembled fluidic device 200. This can be achieved by the fact that at least the bottom cover plate 220 can be kept very thin, for example, with a thickness in the range of 150-300 μm, wherein the distance between the membrane 110 of the fluidic device module 100 carrying the cell culture and the bottom cover plate 220 is less than 200 μm, so that the focal length of the confocal microscope is limited (typically about 500 μm from the object to be studied to the objective lens). To this end, 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 critical; any suitable thickness is contemplated, for example, a thickness of 300 μm-3 mm.

[0153] Confocal microscopy is particularly suitable for evaluating cell cultures within the fluidic device 200, where the diameter of the cell spheroids is less than 500 μm (e.g., approximately 200 μm) to ensure that the overall focal length of the optical path facilitates the capture of sharp images using the confocal microscope. For larger spheroids, it may be necessary to dismantle the fluidic device 200 so that the membrane 110 containing the spheroids can be pressed onto a glass coverslip for positioning within the optical objective of the confocal microscope.

[0154] Additionally, it is noted that the aforementioned dimensions of the fluidic device 200 are typically applied during real-time monitoring of cell cultures within the fluidic device 200. In embodiments where the fluidic device 200 is sectioned (e.g., for digital pathology) as previously explained, the dimensions of the fluidic device 200 are less critical as long as formalin application and paraffin fixation can be performed on the fluidic device 200.

[0155] The cell culture substrates disclosed above are merely examples. Other such substrates exist. Regarding one substrate, such a substrate may be a microporous cell plate. The substrate may be formed for use with a microporous cell plate. For example, thick sheets of substrate may be arranged with a shape and size adjusted to allow insertion into the standard-sized wells of a microporous cell plate. Alternatively, a microporous cell plate with open microwells may be used with a portion of material that closes the bottom of the microporous cell plate. Such an assembly or arrangement may be clamped.

[0156] As will be readily appreciated by those skilled in the art, such drug testing methods can vary significantly in 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 particular protocol, so long as such methods can be deployed using a fluidic device 200 according to an embodiment of the present invention in which cells are cultured directly on an exposed surface of the device (e.g., membrane 110).

[0157] At this point, proof of concept for culturing mammalian cells directly on the bulk-modified elastomers of the present invention will be provided in the form of the following experimental evidence.

[0158] General process:

[0159] The samples were cleaned by rinsing them in isopropanol for 5 minutes. After drying, the samples were placed in 24-well microplates. Cell cultures of immortalized human prostate fibroblasts (WPMY-1 cells, ATCC CRL-2854) were maintained in DMEM (Dulbecco's Modified Eagle Medium, Thermofisher) with 10% FBS (fetal bovine serum), 1% penicillin / streptomycin, and 1% Glutamax. A total of 2.0×10 5Cells were seeded on all samples and placed in an incubator overnight at 37° C. in an atmosphere of 5% CO 2 . Images of the cell cultures thus generated were taken at 100× magnification using a Leica inverted bright-field microscope.

[0160] Example 1: WPMY-1 cell culture was grown on a bulk-modified silicone elastomer according to the teachings of the present invention using 0.5 wt% linoleic acid according to the general protocol described above. To this end, Synthesis Example 2 was modified by replacing 4.84 g of sodium linoleate with 2.41 g of linoleic acid to obtain the bulk-modified silicone elastomer used in this example.

[0161] Comparative Example 1: WPMY-1 cell culture was grown on the same silicone elastomer as in Example 1 but without bulk modification according to the general protocol described above.

[0162] Example 2: WPMY-1 cell cultures were grown on a bulk-modified silicone elastomer prepared according to Synthesis Example 2 (ie, containing 1.0% by weight of sodium linoleate) according to the general protocol described above.

[0163] Figure 9 Shown is the cell culture obtained as a result of Example 1 after overnight cultivation. It can be clearly seen in this image that the cells show good adhesion to the surface of the linoleic acid functionalized silicone.

[0164] Figure 10 Shown is the cell culture obtained as a result of Example 1 after overnight incubation. It can be clearly seen in this image that the cells show minimal adhesion to the surface of the unfunctionalized silicone elastomer.

[0165] Figure 11 Shown is the cell culture obtained as a result of Example 2 after overnight incubation. 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 shows the cell culture of Example 2 after 2 days of culture, and Figure 13 The cell culture of Example 2 is shown after 7 days of culture. Continued cell proliferation can be clearly identified in these images, demonstrating that mammalian cell material can be cultured and propagated directly on the bulk functionalized elastomers of the present invention.

[0166] By using other cell types on the substrate Figure 2 Similar results were obtained by similar preparation and treatment of the case of (but using 1 wt% linoleic acid).

[0167] Example 3: Endothelial Ea.hy926 cell cultures were grown on the bulk-modified silicone elastomer prepared according to Synthesis Example 2 (ie, containing 1.0% by weight of sodium linoleate) according to the general protocol described above.

[0168] Example 4: Epithelial Caco-2 cell cultures were grown on a bulk-modified silicone elastomer prepared according to Synthesis Example 2 (ie, containing 1.0% by weight of sodium linoleate) according to the general protocol described above.

[0169] Figure 14 and Figure 15 Cell proliferation is shown, for example, after 3 and 4 days, respectively. Thus, all cell lines showed good proliferation on the substrate after several days.

[0170] List of non-limiting embodiments.

[0171] Embodiment 1. A material for culturing cells, comprising a bulk-modified elastomer having a Shore hardness (DIN EN ISO 868) in the range of Shore 00 20 to Shore A80 and comprising a plurality of fatty acid moieties covalently bonded to the bulk of the elastomer, wherein the carboxylic acid groups of the moieties are available on the outer surface of the material to provide the bonding, and wherein the bulk-modified elastomer is obtained by:

[0172] forming a composition comprising a vinyl-functionalized elastomer or a hydride-functionalized elastomer or at least one precursor thereof, in the range of 0.5-5% by weight of a free or saponified unsaturated fatty acid based on the total weight of the vinyl-functionalized elastomer or the hydride-functionalized elastomer or at least one precursor thereof, and a cross-linking catalyst in a mold having a polar inner surface; and

[0173] The material is obtained by bulk modification of the vinyl functionalized elastomer or hydride functionalized elastomer by covalently bonding the free or saponified unsaturated fatty acid to the elastomer bulk through a cross-linking 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.

[0174] Embodiment 2. A material according to embodiment 1, wherein each of the fatty acid moieties is covalently bonded to the elastomer body via a cross-linking reaction between the vinyl group or hydride group of the elastomer and the unsaturated carbon-carbon bond of the unsaturated fatty acid.

[0175] Embodiment 3. A material according to embodiment 2, wherein the unsaturated fatty acids are selected from the group consisting of myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, elaidic acid, octadecenoic acid, linoleic acid, elaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

[0176] Embodiment 4. A material according to any one of embodiments 1-3, wherein the elastomer comprises a polybutadiene backbone or a siloxane backbone.

[0177] Embodiment 5. A material according to any of embodiments 1 to 4, wherein 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 precursor thereof.

[0178] Embodiment 6. The material according to any one of embodiments 1-5, wherein the polar inner surface of the mold is a metal oxide inner surface.

[0179] Embodiment 7. A fluidic device module comprising a flow channel extending across a membrane comprising the material according to any one of embodiments 1-6.

[0180] Embodiment 8. A fluid equipment module according to embodiment 7, comprising a first main surface and a second main surface, wherein the first main surface comprises a first recessed structure defining a first flow channel, and the second main surface is opposite to the first main surface and comprises a second recessed structure defining a second flow channel; wherein the membrane separates the first flow channel from the second flow channel.

[0181] Embodiment 9. A fluidic device module according to embodiment 7 or 8, wherein the fluidic device is a monolithic fluidic device.

[0182] Embodiment 10: A fluidic device comprising the fluidic device module according to any one of embodiments 7 to 9 and a pair of cover plates for fluidically sealing the fluidic device module.

[0183] Embodiment 11. A fluidic device according to embodiment 10, wherein the cover plates are arranged such that one of the cover plates covers the first major surface, thereby sealing the first fluid channel, and the other of the cover plates covers the second major surface, thereby sealing the second fluid channel.

[0184] Embodiment 12: A method for culturing cells, comprising:

[0185] providing a molded article formed from the material according to any one of embodiments 1 to 5, the article comprising an outer surface of the material as one of the exposed surfaces of the article; and

[0186] The cells are cultured directly on the exposed surface of the article.

[0187] Embodiment 13. The method of embodiment 12, wherein the article comprises a fluidic device module according to any one of embodiments 7-9.

[0188] Embodiment 14: A drug testing method comprising:

[0189] Providing a fluidic device module according to embodiment 8;

[0190] applying the harvested cells to the surface of the membrane of the fluidic device module;

[0191] culturing the harvested cells on the surface;

[0192] forming a fluidic device using the prepared fluidic device module;

[0193] feeding the cultured cells through one of the pair of flow channels of the fluidic device;

[0194] exposing the cultured cells to a drug to be tested through the other flow channel of the pair of flow channels of the fluidic device; and

[0195] The cultured cells are monitored for their response to the drug being tested.

[0196] 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 comprises:

[0197] Stabilizing and fixing the cultured cells in the fluidic device, and

[0198] Slicing the fluidic device module to obtain a slice for microscopic evaluation, the slice comprising at least a portion of the stabilized and fixed proliferating cell culture; or

[0199] Confocal microscopy was used to monitor the cultured cells within the fluidic device.

[0200] It should be noted that the above-described embodiments illustrate rather than limit the invention, and that a person skilled in the art will be able to devise many alternative embodiments without departing from the scope of the claims. In the claims, any figure signs in brackets 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 a device-type claim 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 to advantage.

Claims

1. A cell culture substrate comprising a material having an outer surface for culturing cells thereon, the material comprising a bulk-modified elastomer, the 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 in conjugate base form, the plurality of residues being covalently bound to the elastomer body such that a portion of the one or more acidic groups is available on the outer surface, wherein the concentration of the residues in the bulk-modified elastomer or the concentration of the corresponding precursors for the residues in a mixture of precursors used to form the elastomer is between 2.10 and 3.0 by weight based on the total weight of the elastomer. -4 to 1.10 -2 mol / kg range. 2 . The cell culture substrate according to claim 1 , wherein the one or more acidic groups are selected from the group consisting of a phosphorus-based acidic group, a sulfur-based acidic group and a carboxylic acid group, or a mixture of two or more of these.

3. The cell culture substrate according to claim 1 or 2, wherein the residue comprises or consists of an aliphatic moiety comprising 3 or more and less than 50 carbon atoms, the one or more acidic groups being covalently bonded to the aliphatic moiety, and the aliphatic moiety being covalently bonded to the elastomeric bulk.

4. The cell culture substrate according to any one of claims 1 to 3, wherein the residue is an unsaturated fatty acid residue.

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

6. The cell culture substrate according to any one of claims 1 to 5, wherein the elastomeric body comprises a silicone or polybutadiene backbone.

7. The cell culture substrate of claim 6, wherein the residue is covalently bound to the elastomeric body due to a reaction between an unsaturated carbon-carbon bond of a precursor of the residue and a vinyl functional group or a hydride functional group of the elastomeric body.

8. The cell culture substrate according to any one of claims 1 to 7, wherein the bulk modified elastomer is or is obtainable by combining a vinyl functionalized elastomer and / or a hydride functionalized elastomer or at least one precursor thereof with a vinyl functionalized elastomer as defined herein at a concentration of 2.10% by weight based on the total weight of the bulk modified elastomer. -4 to 2.10 -2 mol / kg, and heating the mixture. Preferably, when the elastomer is a silicon-based elastomer, the range is 2.10 -4 to 1.10 -2 mol / kg.

9. The cell culture substrate of claim 8, wherein the heating is performed in a reaction vessel having an inner surface of a metal oxide.

10. The cell culture substrate according to any one of claims 1 to 9, wherein the substrate comprises a fluidic device module comprising a flow channel extending over a membrane comprising the material.

11. The cell culture substrate 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 substrate according to claim 10 or 11, wherein the fluidic device is a monolithic fluidic device.

13. Use of a material having an outer surface for culturing cells thereon, said 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 in conjugate base form, said plurality of residues being covalently bound to said elastomer bulk such that a portion of said one or more acidic groups is available on said outer surface, wherein the concentration of said residues in said bulk-modified elastomer or the concentration of the corresponding precursors for said residues in a mixture of precursors used to form said elastomer is between 2.10 and 30% by weight based on the total weight of said elastomer. -4 to 1.10 -2 mol / kg, and the use comprises contacting the outer surface with cells to be cultured and a cell growth medium.

14. A method for culturing cells, comprising the following steps: Use of the cell culture substrate according to any one of claims 1 to 12, optionally wherein the substrate is a molded substrate; and Cells are cultured directly on the exposed surface of the article.

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

16. A pharmaceutical testing method comprising the method according to claim 15 and the further steps of: exposing the cultured cells to a drug to be tested through the other of the pair of flow channels of the fluidic device; and The cultured cells are monitored for their response to the drug being tested.

17. A method for manufacturing a cell culture substrate, comprising: The vinyl functionalized elastomer and / or hydride functionalized elastomer or at least one precursor thereof is heated in a mold with a mixture of 2.10% by weight based on the total weight of the bulk modified elastomer and a mixture of 2.10% by weight based on the total weight of the bulk modified elastomer. -4 to 1.10 - 2 mol / kg, and heating the mixture.

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