Cell culture materials

Abstract

To provide improved cell culture substrates and a method for producing the same.SOLUTION: Cell culture materials are disclosed that include a bulk-modified elastomer having covalently bonded fatty acid moieties. The bulk-modified elastomer is obtained by forming a composition comprising a vinyl-functionalized or hydride-functionalized elastomer or at least one precursor thereof, a free or saponified unsaturated fatty acid in a range of 0.5 to 5 wt% of a total weight of the vinyl-functionalized or hydride-functionalized elastomer or at least one precursor thereof, and a crosslinking catalyst in a mold having a polar inner surface, and bulk modifying the vinyl-functionalized or hydride-functionalized elastomer by covalently bonding the free or saponified unsaturated fatty acid to the elastomer bulk in the mold by a cross-linking reaction between a vinyl or hydride group of the elastomer and an unsaturated carbon-carbon bond of the unsaturated fatty acid to obtain the material.SELECTED DRAWING: Figure 1

Description

[Technical Field]

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

[0002] The present invention further relates to fluidic devices and fluidic device modules comprising such cell culture substrates.

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

[0004] The present invention further relates to a method of drug testing using a fluidic device module having such an elastomeric material.

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

[0006] The present invention relates to the manufacture of cell culture substrates. [Background technology]

[0007] In vitro testing of mammalian cells or tissues is an important technique for obtaining clinically relevant information about the mammalian material under investigation. For example, biopsied mammalian cell or tissue material can be subjected to such testing to determine abnormalities or diseases in such mammalian material, or to expose diseased mammalian material to drugs (e.g., experimental drugs) and monitor the diseased mammalian material's response to such exposure. Such approaches are frequently used, for example, in oncology procedures. This approach can provide important insights into how to effectively treat an individual's disease without exposing the individual to a range of potentially effective drugs that may be undesirable for a number of reasons, including drug toxicity. Furthermore, the effectiveness of experimental drugs for existing diseases for which no established, satisfactory drug treatment is yet available can be tested in this manner. There are many other reasons for deploying such in vitro testing.

[0008] A common approach to such in vitro testing is to immobilize mammalian material on a fluidic device, sometimes referred to as an organ-on-chip. In such an approach, the mammalian material is typically immobilized on a membrane that separates two channels of the fluidic device, one channel used to deliver the mammalian material and the second channel used to expose the mammalian material to a compound or composition of interest, such as a pharmaceutical treatment (e.g., to test the efficacy and / or toxicity of the drug, as described above). The fluidic device, or at least its membrane, can be formed from an elastomer so that the fluidic device can be diced or sliced ​​to obtain slices of membrane containing the mammalian material for evaluation purposes, such as to evaluate the mammalian material's response to exposure to a chemical compound or composition of interest.

[0009] A challenge in such in vitro studies is ensuring that mammalian material, e.g., cells or tissue, stabilized on the membrane of the fluidic device will develop normally and survive for the duration of the test procedure. This is particularly difficult in oncology procedures, where experiments can be quite lengthy. To this end, mammalian material can be stabilized using a biocompatible material, such as fibronectin, which ensures that the mammalian cells continue to proliferate by providing them with a chemical environment that mimics that of natural tissue.

[0010] A common approach is to coat well cell plates with a polymer containing carboxylic acid groups. Such groups can (covalently) bind to fibronectin, making the polymer biocompatible and facilitating the stabilization of mammalian cells on a membrane within a fluidic device. However, this approach has drawbacks. First, fluid flow through the fluidic device can partially remove the polymer, which impairs the ability to evaluate the mammalian material at a desired time point due to the fact that at least a portion of the material may be lost. In other approaches, the membrane is treated with UV or plasma to generate fibronectin binding sites on the membrane. However, this approach has the drawback that it is quite difficult to avoid uneven distribution of such binding sites, which hinders the evaluation of test results.

[0011] Second, membranes containing such biocompatible coatings are extremely difficult to manufacture, which makes the manufacture of such fluidic devices quite expensive and cumbersome. For example, such membranes usually need to be manufactured using expensive thin-film techniques and need to be leak-proof and integrated into the fluidic device, which is not easy. Furthermore, if the membrane itself is not made of a biocompatible material, it usually needs to have multiple cell-sized pores at a small pitch to promote cell growth, which is also difficult to achieve.

[0012] An example of such a human organ-on-a-chip microfabrication based on thin-film technology is described by Dongeun Huh et al. in Nature Protocols, Vol. 8, No. 11, 2013, pp. 2135–2157. In this protocol, microengineering is used to fabricate a multilayer microfluidic device containing two parallel elastomeric microchannels separated by a thin, porous, flexible membrane and two full-height, vacuum chambers on either side. This device takes approximately three and a half days to fabricate. The entire microfabrication procedure involves over 100 steps, of which a significant number are critical. This demonstrates the complexity of such a fabrication procedure.

[0013] An improvement over this laborious approach is disclosed in WO 2018 / 021960 A1, which describes a silicon-based (PDMS) fluidic device that can be made biocompatible using, for example, a collagen coating. While such devices can be produced in only a few manufacturing steps, they require a coating of biocompatible material, which, as mentioned above, suffers from the disadvantage that exposure to fluid flow erodes the polymer surface, leading to the undesired loss of cellular material from the fluidic device surface. This is addressed in WO 2018 / 021906 A1 by plasma-treating the device surface and then functionalizing it with 3-aminopropyltriethoxysilane and glutaraldehyde before coating the functionalized surface with collagen, while flushing the microchannels of the fluidic device with EGM-2 (Endothelial Cell Growth Medium-2 Bullet Kit, sold by Lonza). Therefore, a significant number of processing steps are still required to provide a biocompatible fluidic device.

[0014] A solution to this problem has been disclosed by the applicant in document WO 2019 / 015988 A1. In this application, a material for binding to cell culture proteins is disclosed. The material comprises a bulk-modified elastomer having a plurality of fatty acid moieties covalently bonded to the elastomer bulk, the carboxylic acid groups of which are available for binding on the surface of an object formed from the bulk-modified elastomer by bulk-modifying the elastomer with fatty acid moieties in a mold having the shape of the object and polar interior surfaces to which the hydrophilic carboxylic acid groups of the fatty acid moieties are attracted. This prior application also demonstrates that biocompatible materials such as fibronectin can be covalently bonded to the surface carboxylic acid groups of the bulk-modified elastomer while retaining its cell culture properties. Summary of the Invention [Problem to be solved by the invention]

[0015] There is a need for further improvements in cell culture substrates and their manufacture. [Means for solving the problem]

[0016] This need is at least partially met by the present invention, which seeks to provide several aspects defined by the independent claims that utilize or provide elastomeric materials for culturing cells without the need to covalently modify the surface with cell culture proteins.

[0017] According to one aspect, a cell culture substrate is provided having a material with an exterior surface for culturing cells, the material comprising an elastomer bulk and a bulk-modified elastomer having a plurality of residues each comprising one or more acidic groups in free and / or conjugate base form, the plurality of residues being covalently bonded to the elastomer bulk such that a portion of the one or more acidic groups are available on the exterior surface.

[0018] The term substrate adapted for culturing cells or cell culture substrate (matrix) is meant to include any device or surface intended for use in cell culture. Substrates thus defined are intended to embody the use of the elastomeric material defined herein in the field of cell culture. Cell culture may, for example, involve keeping cells alive, expanding the number of cells, and / or differentiating cells. Residues are portions of precursor molecules that possess the acidic group in some form and have an ethylene bond capable of participating in a reaction that covalently bonds the precursor to the elastomer bulk. Residues are portions of the precursor that remain after such a reaction.

[0019] It has been found that when such surfaces are placed in direct contact with cell culture medium in the presence of cells, such cells can be cultured without any further surface treatment. Therefore, the step of covalently modifying the material surface with cell culture proteins can be omitted, facilitating use. Without wishing to be bound by theory, it is believed that the acidic groups available on the surface play an important role for this cell culture function. Consequently, the bulk unmodified elastomers used as references did not exhibit such cell culture properties. It has also been found that the acidic groups can be present on the surface either in a free form, meaning that they carry a dissociable proton, or in a conjugate base form, with a positive counterion other than a proton. Without wishing to be bound by theory, it is believed that in the cell culture medium, which is typically buffered at a pH of about 7.2, such acidic groups, which typically have a pKa value less than 5.5, are present largely in a dissociated form, regardless of their original surface-available form.

[0020] The acidic groups preferably have a pKa of less than 5 so that most of these groups are in dissociated form. In other approaches, the pKa is less than 4.5 or even less than 4. The pKa can range from 5 to 1, 5 to 2, or 5 to 3.

[0021] Importantly, cell culture substrates with any particular shape can be effectively prepared in one step by mixing appropriate precursors to form bulk-modified elastomers and reacting such precursors in a reaction vessel, such as that of an injection molding apparatus, to form, shape, and prepare the surface of the cell culture substrate for use in cell culture without further adaptation, which may also make serial replication of such devices easier and more consistent.

[0022] In some embodiments, the one or more acidic groups are phosphorus-based (phosphorus-based) acidic groups, sulfur-based acidic groups, and carboxylic acid groups, or a mixture of two or more of these. Phosphoric acid groups and their conjugate bases are preferred over sulfonic acid groups, and carboxylic acid groups are preferred over phosphoric acid groups. Residues bearing such groups are easier to use during the bulk modification process, because their precursors and such residues having acidic groups in the conjugate base form with a metal counterion mix better with other components to form elastomers during such modification processes. The acidic group can be selected according to the required pKa (as specified earlier in this specification).

[0023] In some preferred embodiments, the residue comprises or consists of an aliphatic moiety containing at least 3 but 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 elastomer bulk. The aliphatic moiety can be linear or branched. The moiety can have one or more carbon-carbon double or triple bonds, or aryl or benzene units. The aliphatic moiety can contain cyclic units such as cyclohexyl, cyclopentyl, or others. The aliphatic moiety is preferably a saturated hydrocarbon moiety. The aliphatic moiety preferably contains only carbon and hydrogen atoms.

[0024] In some embodiments, the aliphatic moiety is a linear chain terminally attached to the elastomer bulk, and in some embodiments, the aliphatic moiety further lacks a carbon-carbon triple bond to enhance flexibility of the residue.

[0025] In some embodiments, the residues within a bulk-modified elastomer may be different from one another, each selected as defined herein. In some embodiments, at least a portion of the residues may be attached to the elastomer bulk via two covalent bonds. This may result, for example, from a residue precursor having two ethylene bonds for participation in the modification reaction.

[0026] In some embodiments, the moiety has at least 5 carbon atoms, more preferably at least 10 carbon atoms. Preferably, the aliphatic moiety has fewer than 40 or fewer than 30 carbon atoms. Linear moieties are preferred, but this is not required. The number of carbon atoms in such moieties is preferably between 5 and 30, more preferably between 5 and 20 or between 5 and 15. The moiety and / or chain may have one or more aryl groups. One or more of the acidic groups may be directly attached to an aryl group, such as a benzene ring.

[0027] In some embodiments, the residues are remnants of unsaturated fatty acid precursors covalently bonded to the elastomer bulk via reaction of one or more (if present) of their ethylene groups (carbon-carbon double bonds).

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

[0029] In some embodiments, the residue is the remainder of a precursor having a linear or branched alkyl chain bearing one or more acidic groups and at least one ethylene group (carbon-carbon double bond). Preferably, at least one such ethylene group is a terminal chain ethylene group. Terminal ethylene groups can enhance reactivity compared to non-terminal ethylene groups during the formation of the elastomer bulk in the manufacturing process. A linear chain with one terminal ethylene group and one acidic group (e.g., carboxyl group) is a preferred example. In such cases, the chain may have 5 to 15 carbon atoms.

[0030] In some embodiments, the elastomer bulk comprises a silicone or polydiene backbone. Polybutadiene and polyisopropylen are examples of polydienes. Polydimethylsiloxane is an example of a polythioxane (silicone). Silicones have higher water permeability and are more transparent, facilitating optical inspection of cell cultures.

[0031] In some embodiments, the residue is covalently bonded to the elastomer bulk as a result of a reaction between an unsaturated carbon-carbon bond of the residue's precursor and a vinyl or hydride functionality of the elastomer bulk. In polydienes, such reaction is with a vinyl group, while in silicones, such bonding is usually with a silylhydride functionality.

[0032] In some embodiments, the residue concentration in the bulk modified elastomer or corresponding precursor is, relative to the residue in the mixture of precursors from which the elastomer is formed, preferably less than 2.10% by weight of the total elastomer. -4 ~1.10 -2 mol / kg (molal concentration). Lower concentrations result in irreproducible cell culture characteristics, while higher concentrations have proven difficult to produce in that incomplete mixing of the precursors and / or incomplete reaction between the precursors occurs during the preparation of the bulk modified material, resulting in irreproducible or deteriorated culture characteristics.

[0033] In some embodiments, the bulk-modified elastomer comprises a vinyl-functionalized or hydride-functionalized elastomer or at least one precursor thereof, and 2.10% by weight of the total weight of the bulk-modified elastomer. -4 ~1.10 -2 The modified bulk may be obtained by mixing the modified bulk with a residue precursor at a concentration in the range of 1000 mol / kg and heating the mixture. In some embodiments, the heating is carried out in a reaction vessel having an inner surface of a metal oxide. This is believed to cause the acidic groups (in conjugate base form) to at least partially bond with the oxide surface while in the reaction vessel, making at least some of the acidic groups available on the surface of the material during the polymerization reaction to form the modified bulk.

[0034] In another aspect, there is provided a cell culture substrate as claimed, the substrate having a fluidic device module including a flow channel extending over a membrane having the material.

[0035] In some embodiments, the cell culture substrate has a first major surface with a first recessed structure defining a first flow path, and a second major surface opposite the first major surface with a second recessed structure defining a second flow path, the membrane separating the first flow path from the second flow path.

[0036] In some embodiments of the fluidic device, the cell culture substrate of the fluidic device is a monolithic fluidic device.

[0037] According to one aspect of the present invention, there is provided a use of a material having an exterior surface for culturing cells, the material comprising an elastomer bulk and a bulk-modified elastomer having a plurality of residues, each residue comprising one or more acidic groups in free and / or conjugated base form, the plurality of residues being covalently bonded to the elastomer bulk such that a portion of the one or more acidic groups are available on the exterior surface, the use comprising contacting the exterior surface with cells to be cultured and cell growth medium. As described for the cell culture substrate embodying this use, such a use offers significant advantages, but was not known or obvious.

[0038] According to yet another aspect, - using a cell culture substrate according to any of the preceding paragraphs, optionally a molded substrate; and - culturing cells on the exposed surface of the article; A method for culturing cells having

[0039] In some embodiments of the method, the cell culture substrate is as defined herein.

[0040] In some embodiments, the cell culture method is part of a drug testing method, the latter further comprising: - exposing the cultured cells to the agent to be tested through the other of the pair of flow paths of the fluidic device; and - monitoring the response of the cultured cells to the agent to be tested; It has.

[0041] Particularly useful concentration ranges for modification are described below. Other Non-Limiting Embodiments

[0042] In some embodiments, the bulk-modified elastomer is obtained by forming a composition comprising a vinyl- or hydride-functionalized elastomer or at least one precursor thereof, a free or saponified unsaturated fatty acid in the range of 0.5 to 5 wt % based on the total weight of the vinyl- or hydride-functionalized elastomer or at least one precursor thereof, and a crosslinking catalyst in a mold having a polar inner surface; and bulk-modifying the vinyl- or hydride-functionalized elastomer by covalently bonding the free or saponified unsaturated fatty acid to the elastomer bulk in the mold via a crosslinking reaction between the vinyl or hydride groups of the elastomer and an unsaturated carbon-carbon bond of the unsaturated fatty acid to obtain the material.

[0043] In some embodiments, there is provided a use of a material for culturing cells, the material having a Shore hardness (DIN EN ISO 14021) ranging from Shore 0020 to Shore A80. 868), and having a plurality of fatty acid moieties covalently bonded to the elastomer bulk, the carboxylic acid groups of the moieties being available on the exterior surface of the material to effect said bonding, wherein the bulk-modified elastomer is obtainable by forming, in a mold having a polar interior surface, a composition comprising a vinyl- or hydride-functionalized elastomer or at least one precursor thereof, a free or saponified unsaturated fatty acid in the range of 0.5 to 5 weight percent based on the total weight of the vinyl- or hydride-functionalized elastomer or at least one precursor thereof, and a crosslinking catalyst; and bulk-modifying the vinyl- or hydride-functionalized elastomer by covalently bonding the free or saponified unsaturated fatty acid to the elastomer bulk in the mold by a crosslinking reaction between the vinyl or hydride groups of the elastomer and the unsaturated carbon-carbon bonds of the unsaturated fatty acid to obtain the material.

[0044] Surprisingly, it has been found that relatively soft elastomers, i.e., elastomers having a Shore hardness in the range of Shore 0020 to Shore A 80 (or Shore A 2-80), determined using the measurement method specified in DIN EN ISO 868, when bulk-modified with unsaturated fatty acids as previously disclosed in document WO 2019 / 015988 A1, can function as biocompatible materials on which cellular material can be directly cultured without the need for an extracellular matrix such as fibronectin, provided that the amount of unsaturated fatty acids is in the range of 0.5 to 5 weight percent (wt%), based on the total weight of the elastomer or at least one precursor thereof. While not wishing to be bound by theory, it is believed that when the concentration of unsaturated fatty acids is less than 0.5 wt%, the density of carboxylic acid or carboxylate groups in the case of saponified unsaturated fatty acids is too low to render the exterior surface of the elastomer hydrophilic enough to achieve suitable growth conditions for cells to grow on, while when the concentration exceeds 5 wt%, some unreacted unsaturated fatty acids may leach out of the bulk-modified elastomer and harm cells attached to its exterior surface. Therefore, it is predicted that bulk-modified elastomers will have a covalently bound fraction of free or saponified fatty acid groups in the range of 0.5 to 5 wt%, based on the total weight of the elastomer, assuming that the total conversion of crosslinking reactions between the elastomer (or one or more of its precursors) and the unsaturated fatty acids in free or saponified form is predicted within this range.

[0045] The bulk-modified elastomer is selected to have a hardness in the range of Shore 00 20 to Shore A 80 to ensure that the elastomer is sufficiently flexible to promote cell growth. For example, it is well known that the flexibility of the cell support material is essential for stem cell growth (and differentiation) due to the fact that tissues grown from stem cells, such as (cardiac) muscle, lung, intestinal, and vascular tissue, desire movement. Some organs, such as cardiac muscle, require growth within a movable support. Therefore, if the bulk-modified elastomer material has a Shore A hardness greater than 80, the material will be insufficiently stretchable to promote the desired cell growth directly on its functionalized surface, necessitating the use of a scaffold or extracellular matrix material to impart the desired flexibility to the developing cell culture.

[0046] Furthermore, the unsaturated fatty acids used for bulk modification of the elastomer can be used in free or saponified form, e.g., as sodium salts. The use of saponified unsaturated fatty acids has the advantage that the risk of catalyst poisoning by unsaturated fatty acid protons is avoided, while surprisingly, cell cultures do not tolerate Na + or K + It has been found that bacteria can also grow on the exterior surface of bulk-modified elastomers bearing carboxylate groups with suitable counterions such as PEG, PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-30, PEG-31, PEG-32, PEG-33, PEG-34, PEG-35, PEG-36, PEG-37, PEG-38, PEG-39, PEG-40, PEG-41, PEG-42, PEG-43, PEG-44, PEG-45, PEG-46, PEG-47, PEG-48, PEG-49 ...

[0047] Thus, in this manner, cell culture materials can be fabricated in a few steps, and in some embodiments, in a single step, by combining the elastomer and fatty acid in a coating (spin coating, dip coating, spray coating, dispensing) or injection molding process, where crosslinking of the elastomer with the fatty acid carbon-carbon double bonds can be achieved without significant epoxidation of the fatty acid carbon-carbon double bonds due to limited exposure to ambient oxygen in the spin coating or injection molding process. As a result, a material is provided in which the elastomer is bulk-modified with fatty acid moieties, making the carboxylic acid or carboxylate groups of the (saponified) fatty acids 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 or carboxylate groups are also present on the outer surface of the material, thereby resulting in a substantially uniform distribution of carboxylic acid or carboxylate groups on such outer surface, making the material particularly suitable for use as a membrane material in fluidic devices. This is because each cross-section of the material exhibits the same surface characteristics, as opposed to membrane materials, which, as previously described, require implantation or otherwise formation of anchors for biocompatible materials. Furthermore, because such cross-linking reactions typically consume only a portion of the elastomer carbon-carbon double bonds, the materials of the present invention retain the elastomeric properties of the elastomer, which increases their suitability for use in fluidic devices and facilitates slicing or otherwise cutting the materials of the present invention for research purposes.

[0048] Preferably, each of the free or saponified fatty acid moieties is covalently bonded to the elastomer bulk via a cross-linking reaction between a vinyl or hydride functional group of the elastomer and an unsaturated carbon-carbon bond of the unsaturated fatty acid, ensuring that the number of carboxylic acid or carboxylate groups available for binding to biocompatible materials can be optimized.

[0049] The unsaturated fatty acid can be any suitable unsaturated fatty acid.In exemplary embodiments, the unsaturated fatty acid is selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosperenteinoic acid, erucic acid, undodecanoic acid and docosahexaenoic acid.Particularly mentioned is linoleic acid.

[0050] Similarly, the elastomer can be any suitable elastomer. In exemplary embodiments, the elastomer has a polybutadiene backbone or a silicone backbone. When the elastomer includes a silicone backbone, at least a portion of the carboxylic acid groups within the silicone backbone can be saponified. This is because saponification of the carboxylic acid groups prior to crosslinking between unsaturated fatty acid molecules and elastomer molecules may be required to prevent the crosslinking catalyst from being inactivated by protons from the carboxylic acid groups of the unsaturated fatty acid molecules. Similarly, the polybutadiene can be crosslinked with saponified unsaturated fatty acids. This is because the cell culture properties of the bulk modified material are not significantly affected by saponification of the unsaturated fatty acids, as previously explained.

[0051] In one set of embodiments, the composition comprises free or saponified unsaturated fatty acids in the range of 0.5 to 2 wt. % based on the total weight of the vinyl- or hydride-functionalized elastomer or at least one precursor thereof. Particularly good cell growth has been found to be achieved when the amount of free or saponified unsaturated fatty acids incorporated into the bulk of the elastomer is within this range. For example, excellent cell growth is achieved directly on the exterior surface of the bulk-modified elastomer when the composition has about 1 wt. % free or saponified unsaturated fatty acids based on the total weight of the vinyl- or hydride-functionalized elastomer or at least one precursor thereof.

[0052] According to yet another aspect, there is provided a fluidic device module including flow channels extending through a membrane, the membrane comprising a material of the invention according to any of the embodiments described herein. Such a fluidic device module may have a first major surface with a first recessed structure defining a first flow channel; and a second major surface opposite the first major surface with a second recessed structure defining a second flow channel, the membrane separating the first flow channel from the second flow channel. In a preferred embodiment, the fluidic device module is a monolithic fluidic device module, which has the advantage that the device module can be manufactured in a few processing steps, for example, in a single processing step by injection molding.

[0053] The membrane may have a plurality of holes or channels extending therethrough, such holes or channels preferably having cell-sized dimensions to prevent cells from passing through the membrane.

[0054] In one embodiment, the first and second flow paths are accessible through respective septa (separator membranes), allowing the separate flow paths to be accessed independently without risk of cross-contamination.

[0055] According to yet another aspect, there is provided a fluidic device 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 flow path, and the other of the cover plates covers the second major surface, thereby sealing the second flow path. Such a fluidic device can be manufactured in a simple manner, as the fluidic device module can be formed in a small number of processing steps as described above, and is robust to leaks due to the flexible nature of the fluidic device module.

[0056] According to yet another aspect, there is provided a method of culturing cells, the method comprising the steps of: providing a molded article formed from any of the bulk-modified elastomeric materials of the embodiments described herein, the article having an exterior surface of the material as one of its exposed surfaces; and culturing the cells directly on the exposed surface. This method allows cells to grow directly on the exposed surface of such an article without the need to attach an extracellular matrix, such as fibronectin, to the exposed surface of the article, thereby significantly simplifying the cell culture process.

[0057] According to yet another aspect, a drug testing method is provided, the drug testing method comprising the steps of: preparing a fluidic device module according to one embodiment; supplying (applying) collected cells to a surface of the membrane of the fluidic device module; culturing the collected cells on the surface; forming a fluidic device using the prepared fluidic device module; supplying 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 reaction of the cultured cells to the drug to be tested. Such a drug testing method can be performed in a simple and easy manner, particularly in terms of preparation of the fluidic device to be used in such a drug testing method, thereby significantly simplifying existing drug testing methods in which preparing such a fluidic device is usually very tedious.

[0058] Monitoring the response of the cultured cells to the agent to be tested can include immobilizing and fixing the cultured cells in the fluidic device, and slicing the fluidic device module to obtain slices for microscopic evaluation, the slices comprising at least a portion of the immobilized and fixed cultured cells. According to the teachings of the present invention, the generation of such slices for microscopic evaluation is no longer stringent or subject to variable results due to the fact that the cultured cells are uniformly distributed over the membrane surface of the fluidic device module due to the bulk modification of the elastomer with free or saponified unsaturated fatty acids as described above.

[0059] As another example, in preferred embodiments, monitoring of the responses of the cultured cells can also be performed within the assembled fluidic device using confocal microscopy, which facilitates real-time monitoring of such responses. [Brief explanation of the drawings]

[0060] [Figure 1] FIG. 1 illustrates the bulk modification of an elastomer with an unsaturated fatty acid according to one embodiment. [Figure 2] FIG. 2 shows a perspective view of a fluidic device module according to one embodiment. [Figure 3] FIG. 3 shows another perspective view of a fluidic device module according to one embodiment. [Figure 4] FIG. 4 shows a perspective view of a fluidic device module according to another embodiment. [Figure 5] FIG. 5 shows an exploded perspective view of a fluidic device according to one embodiment. [Figure 6] FIG. 6 shows an exploded perspective view of a fluidic device according to another embodiment. [Figure 7] FIG. 7 shows an exploded perspective view of a fluidic device according to yet another embodiment. [Figure 8] FIG. 8 shows an exploded perspective view of a fluidic device according to yet another embodiment. [Figure 9] FIG. 9 is a microscopic image of a cell culture grown on a bulk-modified elastomer according to one embodiment. [Figure 10] FIG. 10 is a microscopic image of a cell culture grown on an elastomer without such bulk modification. [Figure 11] FIG. 11 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after one day of growth. [Figure 12] FIG. 12 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after two days of growth. [Figure 13] FIG. 13 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 7 days of growth. [Figure 14] FIG. 14 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 4 days of growth. [Figure 15] FIG. 15 is a microscopic image of a cell culture on a bulk-modified elastomer according to another embodiment after 4 days of growth. [Figure 16] FIG. 16 is a graph showing contact angles measured for substrates with silicone having a 1 wt % linoleic acid modification, where the vertical axis is contact angle in degrees, and four sets of two bars each represent the average contact angle for four batches of substrates, with the left bar in each set representing the contact angle at pH 4 and the right bar in each set representing the contact angle at pH 10, with the leftmost set of bars representing unmodified silicone while the other bars represent the modified substrates used in Cell Culture Examples 1, 2, and 3, respectively. DETAILED DESCRIPTION OF THE INVENTION

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

[0062] It should be understood that the same reference numerals are used throughout the figures to indicate the same or similar parts.

[0063] FIG. 1 illustrates the reaction between an ethylene bond of a precursor residue 20 and an ethylene bond of a polymer backbone 10 to produce a bulk-modified elastomeric material according to some embodiments of the present invention. In this example, the reaction is between a fatty acid precursor residue, linoleic acid, and a vinyl-functionalized elastomer (hereafter simply referred to as elastomer) 10 in the form of polybutadiene. Such reactions can be catalyzed with a suitable catalyst, such as peroxide. While this illustration shows polybutadiene 10 reacting with the saponified form of linoleic acid, where R is an alkali metal, such as lithium, sodium, or potassium ion, it will be apparent that other ethylene-linked precursor residues and other ethylene-linked backbone polymers can be used in such types of reactions.

[0064] It has been found that such a reaction can result in a bulk-modified elastomer such that at least some of the carboxylic acid groups of the unsaturated fatty acid molecules 20 that participated in the reaction with the elastomer 10 are present on the exterior surface of the bulk-modified elastomer, making these carboxylic acid groups available for stabilizing cell culture directly on the exterior surface of the bulk-modified elastomer 10. Thus, the step of attaching proteins to such carboxylic acid groups to facilitate cell stabilization is no longer required, which saves considerable time and effort and opens up new opportunities for defining cell culture substrates comprising the material. For example, the formation of the elastomer and molding of the elastomer can now be carried out in one step using a suitable reaction vessel. In a preferred example, such a reaction vessel is, for example, a mold in an injection molding machine. Note that the example in FIG. 1 is just one type of ethylene bond chemistry that can be used in such a process. As described below, reactions based on the addition of a silyl hydride to an ethylene bond in the presence of a catalyst can also be used, as explained in more detail below.

[0065] Preferably, elastomer 10 is soft or flexible to facilitate cell-guided movement. For this reason, elastomer 10 may typically have a Shore hardness, determined according to the DIN EN ISO 868 standard, ranging from Shore 00 20 (i.e., Shore A 2) to Shore A 80, i.e., a Shore A hardness of 2 to 80. Suitable elastomers include polyenes such as polybutadiene and silicone.

[0066] The unsaturated fatty acid 20 can be a free unsaturated fatty acid, in which case R=H, or a saponified fatty acid, in which case R=Na, for example. + Or K + (and COO group is COO - or carboxylate anion groups). Such saponification can be achieved in any suitable manner, for example using concentrated NaOH or KOH solutions. Such saponification reactions are well known per se and will not be described in further detail, for the sake of brevity only.

[0067] Importantly, the saponification of the unsaturated fatty acid residues in the bulk-modified elastomer need not be reversed, as it has surprisingly been found that cell growth on the exterior surface of bulk-modified elastomer 10 occurs even when the surface is functionalized with saponified carboxylic acid groups, i.e., carboxylate groups having counterions such as Na or K counterions, as explained in more detail below.

[0068] The presence of carboxylic acid or carboxylate groups on the exterior surface of bulk-modified elastomer 10 may be considered surprising given that elastomer 10 can be nonpolar, especially when elastomer 10 is dissolved in a nonpolar solvent. In such a nonpolar environment, unsaturated fatty acid molecules 20 tend to form micelles, where the carboxylic acid moieties of these molecules are rotated inward within such micelles, leading to such carboxylic acid moieties terminating 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 epoxidization in the presence of oxygen, which reduces the ability of such compounds to participate in crosslinking reactions with elastomer 10.

[0069] Embodiments of the present invention are based on the insight that when a reaction mixture containing elastomer 10 and saponified unsaturated fatty acid 20 is contacted with a polar surface, this promotes the orientation of the carboxylic acid groups of the fatty acid molecules along such polar surface, such that the exterior surface of the bulk-modified elastomeric material exhibits a dense and uniform distribution of these carboxylic acid groups. Furthermore, it has been found that when elastomer 10 is bulk-modified in such a manner in an injection molding process at high temperatures, e.g., temperatures in the range of 120-240°C, epoxidation of the carbon-carbon double bonds of unsaturated fatty acid molecules 20 does not significantly interfere with the bulk modification of elastomer 10, i.e., does not significantly inhibit the reaction between unsaturated fatty acid molecules 20 and elastomer 10. Without wishing to be bound by theory, it is believed that in such an injection molding process, molecular oxygen is substantially absent from the reaction mixture within the injection molding apparatus, thereby suppressing undesired epoxidation of these carbon-carbon double bonds.

[0070] To obtain an elastomer 10 on which cells can be directly cultured, the elastomer 10 is preferably 2.10 times the total weight of the elastomer 10 or the total weight of one or more precursors of the elastomer 10. -4 ~1.10 -2The bulk-modified elastomer 10 is bulk-modified with a certain amount of unsaturated fatty acid 20 in free or saponified form within the range of mol / kg (molar weight concentration). It has been found that when lower amounts of unsaturated fatty acid 20 in free or saponified form are used, cells do not substantially grow on the bulk-modified elastomer 10, which may be due to the surface of the bulk-modified elastomer 10 on which the cells are seeded being excessively hydrophobic in nature. On the other hand, when higher amounts of unsaturated fatty acid 20 in free or saponified form are used, cells again grow poorly on the surface of the bulk-modified elastomer 10 and / or growth results vary irreproducibly from experiment to experiment, which may be due to unreacted unsaturated fatty acid 20 in free or saponified form leaching out of the bulk-modified polymer and harming the cell culture. Furthermore, it has been found that when the amount of unsaturated fatty acid 20 in free or saponified form is within the aforementioned range, the flexibility or hardness of the unmodified elastomer 10 is not significantly altered by bulk modification with unsaturated fatty acid 20 in free or saponified form. Furthermore, the transparency of the elastomer to at least optical radiation, e.g., visible light, decreases due to, for example, increased scattering due to opacity when higher concentrations are used, which can impair the use of the cell culture substrate for optical inspection of cell growth or culture.

[0071] Although the example of Figure 1 is described with respect to a polydiene-type bulk elastomer, other elastomers may be used, preferably such elastomers also having the desired Shore hardness.

[0072] Thus, usable elastomers include, for example, homopolymers, copolymers, block copolymers, terpolymers, block terpolymers, etc. Particularly suitable elastomers can be selected from polyenes, such as polybutadiene, or isoprene or neoprene. When polyenes are used, they can be formed using any suitable polymerization process, followed by bonding as described with reference to FIG. 1. Particularly preferred are polybutadienes formed via Nd- or Li-catalyzed polymerization reactions. Such polybutadienes are well known for their low branching degree (typically less than 5%) and low polydispersity (Mw / Mn) of about 2. Li-catalyzed polybutadienes are particularly preferred due to their high 1,2-vinyl content (about 11%). However, other types of polybutadienes, such as those obtained from Co-, Ni-, or Ti-catalyzed polymerization reactions, can also be used instead. When using polyenes, such as polybutadiene, as elastomer 10, the crosslinking reaction between elastomer 10 and unsaturated fatty acid 20 can be catalyzed using a peroxide catalyst. In another advantageous embodiment, Li-catalyzed polybutadiene having a branching degree in the range of 5-15% is used, since it has been found that when the branching degree of polybutadiene is within this range, the reactivity of polybutadiene with unsaturated fatty acids 20 is improved. For completeness, it is noted that how to control the degree of branching in polybutadiene synthesis is well known and will not be further described for the sake of brevity.

[0073] Another class of desirable elastomers are silicones (polysiloxanes) because they are suitable for injection molding processes. Such silicones may have a polydimethylsiloxane (PDMS) backbone containing vinyl moieties to facilitate crosslinking via a Pt-catalyzed addition reaction with, for example, (polymethyl)hydrogensiloxane. Alternatively, or in addition to such crosslinking with (polymethyl)hydrogensiloxane, the vinyl-functionalized PDMS backbone may be reacted with an unsaturated fatty acid 20.

[0074] Such elastomers in the form of silicones may be formed by crosslinking reactions between vinyl-functionalized linear or branched silicone monomers or oligomers, such as T- or Q-branched silicone monomers or oligomers, and linear hydride-functionalized silicone monomers or oligomers, between hydride-functionalized linear or branched silicone monomers or oligomers, such as T- or Q-branched silicone monomers or oligomers, and linear vinyl-functionalized silicone monomers or oligomers or mixtures of vinyl-functionalized and hydride-functionalized linear or branched silicone monomers or oligomers, such as mixtures of T- or Q-branched silicone monomers or oligomers and / or linear hydride-functionalized and vinyl-functionalized silicone monomers or oligomers.

[0075] For example, the two-component silicone can be formed by a crosslinking reaction between linear component 1 and linear component 2, each of which can correspond to the following general formula: [ka]

[0076] In Component 1, R1 and R2 are independently selected from C1-C3 alkyl, and R3-R8 are independently selected from C1-C3 alkyl and vinyl, where at least one of R3-R5 and at least one of R6-R8 is vinyl. Preferably, at least three of R3-R8 are vinyl. In all embodiments, n can be in the range of 100-2,000,000. In certain embodiments of Component 1, each of the alkyl groups R1-R8 is a methyl group. That is, Component 1 is a vinyl-functionalized PDMS having terminal vinyl groups.

[0077] In component 2, R1 is hydrogen, R2=C1-C3 alkyl, R2-R8 are independently selected from C1-C3 alkyl or hydrogen, where at most one of R3-R5 and at most one of R6-R8 is hydrogen, and n can have any suitable value, such as n=3-1,000, or more specifically n=3-10. In particular embodiments of component 2, none of the R3-R8 groups are hydrogen. In other particular embodiments of component 2, each of R2-R8 is methyl.

[0078] Component 2 typically acts as a crosslinker for component 1. Such crosslinking reactions are usually Pt-catalyzed and are well known per se (see, for example, WO2009 / 147602 A2), and therefore, for the sake of brevity, will not be described in further detail.

[0079] Such bulk modification of silicones can be achieved by forming the elastomer in the presence of unsaturated fatty acids 20, and a covalent bond between the silicone and unsaturated fatty acids 20 can be formed by reaction between the hydride functional groups of the (crosslinked) silicone and the unsaturated fatty acids 20, as suggested by the following reaction mechanism: [ka]

[0080] To prevent catalytic inhibition of the carboxylic acid groups of the unsaturated fatty acid 20 by protons during such crosslinking reactions, the unsaturated fatty acid may be used in a saponified form, e.g., as the sodium salt of such an unsaturated fatty acid, in the crosslinking reaction with the silicone elastomer. In some embodiments, if desired, the reaction product (bulk-modified elastomer) can then be treated with a protic acid, such as HCl, to restore the carboxylic acid groups on the surface of the bulk-modified elastomer; however, this is not necessary, given that when the bulk-modified elastomer is used to culture cells directly on its surface, the cells have been demonstrated to grow even when the polar surface groups are carboxylate anions, i.e., saponified carboxylic acid groups. Any silicone elastomer with free silyl hydride groups present to react with residual precursors bearing ethylene bonds can be used for bulk-modifying such elastomers.

[0081] It should be noted that elastomer formation need not be initiated prior to carrying out the modification reaction, and while this may be done in some embodiments, preferably the formation is carried out at least in part during elastomer formation by mixing appropriate amounts of elastomer-forming components and modification precursor residues.

[0082] As another example, a silicone backbone, such as a (polymethyl)hydrogensiloxane backbone, can be crosslinked with a rubbery polymer, such as polybutadiene or polyisoprene, and unsaturated fatty acids can be incorporated into such crosslinked products. This can be done, for example, to tailor the water permeability of the final material. Silicones typically have a fairly open structure, while such rubbery polymers have a fairly closed structure. Thus, the openness, and therefore water permeability, of the crosslinked product can be tailored by the ratio of silicone to rubbery polymer in the product. In this way, the properties of the material according to this embodiment can be optimized with respect to cell / protein interactions to build a good scaffold. For example, if the elastomer is silicone-based, e.g., PDMS-based, the resulting material is highly permeable to water and other compounds (e.g., drugs), facilitating maximum perfusion of these compounds to cells on the membrane of the fluidic device module 100. However, if real-time monitoring of drug consumption by cells is desired, a polyene-based material, such as a polybutadiene-based material, which is impermeable to water and drugs, may be preferred. The rate of perfusion of these compounds into the cells can be controlled using the density of openings through the membrane.

[0083] In one embodiment, the polysiloxane can be formed from a multi-component starting material, as described 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 under the trade name Elastosil by Wacker Chemi AG, Munich, Germany. However, other silicones that can form elastomers and react with ethylene bonds are also possible.

[0084] Linoleic acid has been used as a precursor for the acidic group-bearing residue to which the bulk-modified elastomer is modified. This is by way of non-limiting example only, and it should be understood that other acidic group-bearing residues may also be used, as described herein below. The aforementioned benefits obtained with linoleic acid will also be achieved with the following other residues and their precursors, unless otherwise specified.

[0085] Thus, for example, with respect to unsaturated fatty acid 20, any suitable unsaturated fatty acid can be used for reacting with elastomer 10. For example, unsaturated fatty acid 20 can be selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosperentanoic acid, erucic acid, undodecanoic acid, and docosahexaenoic acid.

[0086] In the context of the present application, where reference is made to unsaturated fatty acid 20 in connection with the example of Figure 1, it should be understood that this is intended to refer more generally to any organic compound containing one or more acidic groups, either in free or conjugate base form, and containing at least one carbon-carbon double bond.

[0087] Generally, the residues in the bulk-modified elastomer result 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. Others exist and can be used as long as they have at least one carbon-carbon double bond and one or more acidic groups, which, when used in the context of silicone chemistry, require the conjugate base form. In polydiene chemistry, such a conjugate base form itself is not required.

[0088] In some preferred embodiments, the residue precursor comprises or consists of an aliphatic moiety containing 3 or more carbon atoms and fewer than 50 carbon atoms, and at least one carbon-carbon double bond when the one or more acidic groups are covalently bonded to the aliphatic moiety. The aliphatic moiety can be linear or branched. The moiety can contain one or more (e.g., 2, 3, or 4) carbon-carbon double or triple bonds (preferably only double bonds) or an aryl or benzene unit. The aliphatic moiety can contain a cyclic unit such as cyclohexyl, cyclopentyl, or others. The aliphatic moiety preferably contains only carbon and hydrogen atoms. The aliphatic moiety can be a saturated hydrocarbon moiety. In some embodiments, the aliphatic moiety further does not contain a carbon-carbon triple bond to enhance the flexibility of the residue.

[0089] In some embodiments, the aliphatic moiety has at least one terminal carbon-carbon double bond (HC=CH-R, where R is the remainder of the aliphatic moiety), and preferably the moiety is entirely linear with at least one (e.g., only one) acidic group attached to the other end of the moiety.

[0090] In some embodiments, a mixture of precursor residues can be used to arrive at a bulk-modified elastomer in which at least some of the residues differ from one another. Each of the precursor residues can be selected as defined herein. In some embodiments, at least some of the aliphatic moieties of the residue precursors have at least two carbon-carbon double bonds. In this manner, the residues in the bulk-modified elastomer can be covalently bonded to the elastomer bulk by more than one bond. As a result, polymer chains in the elastomer bulk can be crosslinked via the residues.

[0091] In some embodiments, the moiety of the precursor residue comprises at least 5 carbon atoms, more preferably at least 10 carbon atoms. Preferably, the aliphatic moiety comprises fewer than 40 or fewer than 30 carbon atoms. The number of carbon atoms in such moieties is preferably between 5 and 30, more preferably between 5 and 20 or between 5 and 15. The moiety and / or chain may have one or more aryl groups. One or more of the acidic groups may be directly attached to an aryl group, such as a benzene ring.

[0092] In some embodiments, the residue precursor is the remnant (carbon-carbon double bond) of such an unsaturated fatty acid precursor covalently bonded to the elastomer bulk via reaction of one or more (if present) of the ethylene groups of the unsaturated fatty acid and the residue.

[0093] For example, the unsaturated fatty acid residue is one or more residues of a fatty acid selected from the group consisting of myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicospermaceous acid, erucic acid, and docosahexaenoic acid.

[0094] In some embodiments, the residue is the remainder of a precursor having a linear or branched alkyl chain bearing one or more acidic groups and at least one ethylene group (carbon-carbon double bond). Preferably, at least one such ethylene group is a terminal chain ethylene group. Terminal ethylene groups may provide enhanced reactivity compared to non-terminal ethylene groups during the formation of the elastomer bulk in the manufacturing process. A linear chain with one terminal ethylene group and one acidic group, such as a carboxyl group, is a preferred example. In such cases, the chain may have 5 to 15 carbon atoms.

[0095] Thus, the residue may have an aliphatic portion containing at least three carbon atoms to which one or more acidic groups are covalently bonded. The corresponding precursors for such residues contain at least one carbon-carbon double bond in their aliphatic portions. Preferably, the aliphatic portion contains 5 or more, 7 or more, 9 or more, or 11 or more carbon atoms. Preferably, the aliphatic portion contains fewer than 30, fewer than 20, or fewer than 15 carbon atoms. The aliphatic portion of the residue or precursor may be linear or branched, with linear being preferred. It may also have a cyclic structure. The aliphatic tail may terminate in a non-aromatic ring structure, such as a 5- 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 be included within the above definition of unsaturated fatty acid 20 is retinoic acid.

[0096] The aliphatic moiety may have one, two, three, or four ethylene bonds. Preferably, there are fewer than three. More preferably, there is only one. At least one ethylene bond is replaced with at least two hydrogen atoms. More preferably, at least one ethylene bond is replaced with three hydrogen atoms. Such ethylene bonds typically exhibit increased reactivity in the reactions described herein, exhibiting higher reactivity relative to the latter.

[0097] The acidic groups carried by the aliphatic moiety are preferably attached to saturated carbon atoms of the aliphatic moiety and may be phosphorus-containing acidic groups such as carboxylic acid groups and their carboxylate groups (conjugate base groups), phosphate groups and the corresponding phosphates and hydrogen phosphates, phosphite groups and the corresponding conjugate bases, and / or sulfur-containing acidic groups such as sulfonic acid groups and the corresponding sulfonates.

[0098] The phosphate group is a phosphorus oxoacid in which each phosphorus atom is in the oxidation state +5 and is bonded to four oxygen atoms. Two or more of these PO4 units (tetrahedra) are connected by shared, single-bonded oxygen atoms to form a linear or branched chain in which at least one of the oxygen atoms is bonded to the rest of the residue. The non-shared, single-bonded oxygen atoms can be completed with an acidic hydrogen atom or can accept a proton. The general formula for a 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 (0 to (n+2) / 2). There is one bond reserved for attachment to the residue, e.g., the aliphatic portion of such a residue. Preferably, the acidic group is an orthophosphate group or a polyphosphate group RO (O=POHO). n -H, n is 1 for orthophosphate and n>2 for polyphosphate, and R represents the remainder of the residue to which the group is attached. For example, phosphate groups are well known in the art and will not be described in further detail. Further definitions can be found in many textbooks on organic chemistry.

[0099] For example, any of the conjugate bases of the acidic groups, such as carboxylate, phosphate, hydrogen phosphate sulfonate, etc., are preferably combined with or saponified by metal counterions, such as those of alkali metals (e.g., lithium, sodium, and potassium) and alkaline earth (e.g., barium and calcium) metals.

[0100] In addition to the exemplary compounds explicitly mentioned in this application, many other compounds will be apparent to those skilled in the art.

[0101] There may be one acidic group per residue or precursor of such residue. In such cases, the molar concentration of the residue in the bulk-modified elastomer or of the precursor used to prepare such bulk-modified elastomer also reflects the molar concentration of acidic groups present in the bulk-modified elastomer. To calculate the final molar concentration of acidic groups from the molar concentration of the residue, the latter must be multiplied by the number of acidic groups per residue. If the molar concentration of such groups in the bulk-modified elastomer is 1.10, then the molar concentration of the acidic groups in the bulk-modified elastomer is 1.10. -2 If higher mol residues / kg bulk modified elastomer are required without substantially encountering the problems associated with residue amounts higher than this range limit, as previously discussed, it can be beneficial to have two or more acidic groups per residue.

[0102] As previously disclosed herein, the residue concentration in the bulk modified elastomer or corresponding precursor, relative to the residue in the mixture of precursors for forming the elastomer, is preferably less than 2.10% by weight of the total weight of the elastomer or of one or more precursors of the elastomer. -4 From 2.10 -2 In the case of silicone type elastomers, this residue concentration range is preferably 2.10 -4 From 1.10 -2 mol / kg. This largely avoids washing the substrate to remove unreacted precursors before use. For low concentrations of acidic groups, the residue molality ranges from 2.10 -4 ~2.10 -3 mol / kg. If the concentration of acidic groups is high, the range is 1.10 -3 From 5.10 -3 Cell growth of different cell lines may require different concentrations within the above ranges.

[0103] The molar concentrations disclosed herein can be converted to weight percent when considering the molar weight of the residue or precursor of the residue using commonly known calculations.

[0104] It should be noted that when using silyl hydride addition over vinyl bond chemistry to form silicone-based elastomers, the residual precursors are used with the acid groups in the form of their conjugate bases. For example, they are saponified. They are added as such, either as alkali or alkaline earth metal salts. This avoids contamination of the Pt catalyst used with the aforementioned chemistry. However, this is not required for polydiene-type modifications, since the chemistry uses an acid-insensitive catalyst.

[0105] An exemplary synthetic procedure for producing such bulk-modified elastomers from polyenes such as polybutadiene is provided below (Synthetic Example 1). [Synthesis Example 1]

[0106] Polybutadiene chunks obtained from Lanxess AG, Cologne, Germany, are inserted into a compounder or mixing extruder. The chunks contain a peroxide, such as dicumyl peroxide, as a catalyst. Linoleic acid (60-74% concentrated, obtained from Aldrich Chemistry) in an amount ranging from 1 to 5% by weight of the polybutadiene chunks is mixed into the polybutadiene bulk by kneading at a temperature ranging from 110 to 150°C. The resulting extruded mixture is injected into a stainless steel mold, defining a fluidic device and having an oxidized inner surface in contact with the extruded mixture, for 3 to 10 minutes at a temperature ranging from 150 to 200°C to obtain a monolithic fluidic device module of the linoleic acid-crosslinked polybutadiene rubber.

[0107] An exemplary synthetic procedure for producing such bulk-modified elastomers from silicone rubber is provided below (Synthetic Example 2). [Synthesis Example 2]

[0108] 4.848 g of a solution of sodium linoleate in a solution of water and isopropyl alcohol (solvent weight ratio 1:1) such that the weight fraction of sodium linoleate in the solution was 30 wt % was mixed with 240 g of silicone component A of Elastosil LR3040 from Wacker Chemie AG and vigorously stirred at room temperature under vacuum conditions to evaporate water from the composition. The resulting mixture was mixed with 240 g of Elastosil LR3040 (Wacker Chemie AG) component B (containing a Pt catalyst) and fed to an injection molding apparatus, where it was injected into a stainless steel mold having an oxidized inner surface in contact with the resulting mixture and defining a fluidic device module, and molded at a temperature ranging from 150 to 200°C for 10 to 60 seconds to obtain 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 wt% relative to the weight of the polymer matrix (based on the weight of the starting materials). Optionally, the molded product can then be immersed in an aqueous HCl solution (pH 2 to 4) to convert at least the sodium carbonate groups on the surface of the molded product to free carboxylic acid groups. [Synthesis Example 3]

[0109] Same as Example 2, but use the appropriate amount of sodium undecanoate to reach the same molar concentration with respect to sodium linoleate in Example 3.

[0110] To further demonstrate the bulk modification of the elastomers in Synthesis Examples 1 and 2, blocks of each elastomer after reaction with linoleic acid were molded in a polar mold as described above and then sliced ​​at −40° C. using a microtome to obtain internal slices of the modified elastomer, i.e., slices where none of the major surfaces were on the exterior surface of the molded elastomer block.

[0111] To demonstrate that some of the modified residual acidic groups were indeed available on the surface of the molded elastomer, these internal slices were then contacted with 2 μl water droplets of different pHs (pH 4, pH 7, and pH 10) for 3 minutes, after which the contact angles of the water droplets with these internal slices were measured. The same procedure was repeated for unmodified elastomer to compare the measured contact angles of the internal slices of the linoleic acid-modified elastomer with those of the corresponding unmodified elastomer. By way of example, results for a silicone-based elastomer are shown in the graph in FIG. 16.

[0112] As can be seen from these graphs, the contact angle of unmodified silicone is nearly insensitive to the pH of the droplet. Without wishing to be bound by theory, the small increase in contact angle measured for unfunctionalized silicone with increasing pH may be the result of the pH dependence of hydrogen bonding between water molecules and silanol moieties in the silicone. For the modified silicone batches used for culturing different cell types described below, the contact angle at pH=4 is significantly lower than the contact angle at pH=10.

[0113] The observed behavior for the modified silicones (not shown) is the same regardless of the form (free or conjugated base) of the carboxylic acid groups available on the surface.

[0114] Furthermore, the bulk modification is evident from the identical behavior observed in cut surfaces prepared by cutting a batch of substrates into two halves.

[0115] Thus, both the internal slice (obtained after cutting) and the external surface of polybutadiene cross-linked with linoleic acid showed a strong pH dependence of the measured contact angle, with decreasing contact angles measured with increasing pH.

[0116] Although not shown here, similar contact angle behavior is observed for modified polybutadiene, whereas the measured contact angle for unmodified polybutadiene is nearly pH independent.

[0117] The strong pH dependence of the measured contact angles for the bulk and surface of the functionalized elastomer clearly indicates 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 largely protonated, thereby minimizing the surface charge of the interior slices of the elastomer. As the pH increases, this surface charge increases due to increased deprotonation of the carboxylic acid groups, which reduces the contact angle of the water droplet with the surface of these interior slices. On the other hand, if such carboxylic acid groups are not present on the surface exposed to the water droplet, as in unmodified elastomers, changes in the pH of the water droplet do not cause a change in surface charge, and as a result, the measured contact angles of water droplets of different pH with such surfaces are much less sensitive to these pH changes.

[0118] The pH of the droplets to be used in the contact angle discrimination method depends on the pKa value of the acidic group used on the substrate. For linoleic acid groups, the pKa is about 4.7, so low pH droplets should have a pH below 4.7, and high pH droplets should have a pH about 2 pH units above the pKa to cause complete dissociation within the droplet. Those skilled in the art will be able to select the correct conditions for a particular acidic group with a particular pKa.

[0119] Another method was implemented to check the availability of surface acidic groups (carboxylic acid groups). To this end, all slabs or slices were treated with HCl as described herein to convert any accessible conjugate bases of the acidic groups back to their acidic form. The resulting dried slabs were then coupled to fluorescently labeled cadaverine by reacting the NH groups of the cadaverine with the acidic groups using the well-known coupling chemistry of EDC / NHS. Comparison of the modified and unmodified elastomers after treatment clearly shows the increased fluorescence of the latter with a very uniform spatial gray scale and the absence of fluorescence in the unmodified elastomer. Such a staining test can be used to indicate the presence of surface-accessible acidic groups, such as carboxylic acid groups.

[0120] The bulk presence of acidic groups was investigated using IR measurements in both reflection and transmission modes and elemental analysis.

[0121] The completion or progress of the polymerization reaction to form the modified elastomer was followed using IR and Raman spectroscopy to determine the rate and state of conversion, for example, by observing the decrease in absorption of the ethylene bond vibrations of the precursor to the modifying residue.

[0122] The bulk-modified elastomer can be molded into any suitable form to produce an article or cell culture substrate on which cells can be directly cultured. Such articles can take any suitable shape. In certain embodiments, the mold used in such an injection molding process can be molded into the shape of a fluidic device module, such that a monolithic fluidic device module can be formed in a single-step injection molding process. The high density and uniform distribution of carboxylic acid or carboxylate groups on the exterior surface(s) of such a monolithic fluidic device module allows such device modules to be used in a simple manner as cell culture substrates. The mold can be formed from any suitable polar material, such as stainless steel, a metal, or a metal oxide such as aluminum oxide, or can have an interior surface coated with such a material, to achieve the desired orientation of carboxylic acid or carboxylate groups on at least one of the exterior surfaces of the bulk-modified elastomer. A typical example of a fluidic device module 100 is shown schematically in FIGS. 2 and 3, which depict a perspective view of the opposite major surface of the fluidic device module 100. The fluidic device module 100 may have a membrane 110 through which at least one flow path 115 extends. Preferably, the fluidic device module 100 has opposing flow channels that are spatially separated by the membrane 110. While at least the membrane 110 is formed from the bulk modified elastomer 30, in a preferred embodiment, the entire fluidic device module 100 is formed from the bulk modified elastomer 30, thereby resulting in a monolithic fluidic device module 100.

[0123] The fluidic device module 100 may have one or more septa 120 to provide access to the flow channels 115 as described above. Each flow channel 115 is preferably accessible through a dedicated set of septa 120, avoiding cross-contamination between multiple flow channels 115. As previously mentioned, the exposed surfaces of the fluidic device module 100, such as the membrane 110, can be used to directly attach cell cultures, allowing cells to be cultured (grown) on the biocompatible surface.

[0124] The membrane 110 may have a plurality of holes or grooves to allow liquid nutrients and solutions, along with potential chemical compounds such as drugs, to reach both sides of the membrane and the cells / tissues, and these holes or grooves may be formed in any suitable manner, such as, for example, via laser cutting. Such holes or grooves in the membrane 110 of the fluidic device module 100 are preferably on the same order of magnitude as the typical diameter of the cells to be immobilized on the membrane 110, and the pitch between such holes or grooves is not particularly critical.

[0125] The fluidic device 200 shown schematically in Figure 4 can be formed from a fluidic device module 100 as described above by providing a pair of cover plates 210, 220 which fluidically seal the fluidic device module 100 so that fluid passing through one or more flow paths 115 of the fluidic device module 100 cannot leak from the fluidic device 200.

[0126] In one embodiment, the cover plates 210, 220 are arranged such that the cover plate 210 covers a first major surface of the fluidic device module 100, thereby sealing the first flow path 115 of the fluidic device module, while the other cover plate 220 covers a second major surface of the fluidic device module 100, thereby sealing the second flow path 115 of the fluidic device module. The cover plates 210 and 220 can be formed from any suitable material, such as glass or a plastic material. Preferably, the cover plates are stretchable and flexible to ensure a good fit with the fluidic device module 100 while maintaining the flexibility of the fluidic device module 100 within the fluidic device 200. Such a fluidic device 200 can be deployed as a biofunctional chip, as will be readily understood by those skilled in the art.

[0127] 5 shows a schematic representation of an exemplary embodiment of a fluidic device 200, comprising a fluidic device module 100 according to the present invention between two rigid cover plates 210 and 220. The upper plate 210 has a first inlet 231 and a first outlet 232 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 fluid passing through the lower channel. The upper plate 210 also has a second inlet 233 and a second outlet 234 in fluid communication with an upper channel 115 on the membrane 110, such that the membrane 110 is in fluid contact with other fluids passing through the upper channel 115. For example, fluid passing through the lower flow channel may carry a chemical to be tested (tested) on a cell culture attached to the membrane 110, as described in more detail below, and the chemical can reach the cell culture through the membrane 110 due to the fact that the membrane 110 is made porous, e.g., by drilling holes or grooves in the membrane 110 with a laser, as described above. As another example, such holes or grooves can be formed in the membrane 110 when the fluidic device module 100 is formed in a polar mold. This approach has the advantage that a regular pattern of such fluid passages (pores) can be formed through the membrane 110. Other fluids passing through the upper flow channel 115 can come into direct contact with such cell cultures and provide nutrients to such cell cultures. In some embodiments, the inlets 231, 233 and outlets 232, 234 are diaphragms through which fluids can be hydraulically pumped through the grooves on the membrane 110, e.g., by pressing on the diaphragm. As another example, the inlets 231, 233 and outlets 232, 234 may be ferrules to which tubing can be attached, e.g., 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.

[0128] In the embodiment shown schematically in FIG. 6, for example, upper and lower flow channels 115 are incorporated into the (monolithic) fluidic device module 100 when the fluidic device module 100 is molded (only one of these channels is shown). Alternatively, at least one of the upper and lower flow channels can be formed in one of the cover plates. FIG. 7 schematically shows a perspective top view of an exemplary embodiment of a fluidic device 200, while FIG. 8 schematically shows a perspective bottom view, in which the upper flow channels 115 are formed in the top cover plate 210, while the lower flow channels 115' are formed in the fluidic device module 100. As will be readily apparent to those skilled in the art, it is equally possible to form the lower flow channels 115' in the bottom cover plate 220. For example, a fluidic device 200 such as the device shown in Figures 7 and 8 can be used as a microwell in a lab-on-a-chip device, in which case multiple such microwells can be clamped or otherwise secured to facilitate parallel testing of multiple different samples within a single lab-on-a-chip device, with each microwell typically containing one of these samples.

[0129] Upon immobilizing and growing a cell culture 50 within such a fluidic device 200, the fluidic device 200 can be used in a method in which the immobilized, growing cell culture 50 is exposed to a fluid containing a compound of interest, and the fluid is passed through the flow channels 115 of the fluidic device 200, exposing the immobilized cell culture 50 to the compound of interest therein, and the response of the immobilized cell culture 50 to such exposure can be monitored. Such a method can be performed in an oncology setting, in which the immobilized cell culture 50 is exposed to a drug, such as a chemotherapy drug, and the response of the immobilized cell culture 50 to such drug can be monitored. To this end, the immobilized cell culture 50 can contain tumor cells to test the efficacy of the drug and / or healthy cells to test the toxicity of the drug to healthy tissue.

[0130] Such a drug testing method typically includes the steps of preparing a fluidic device module 100 according to one or more embodiments of the present invention, e.g., preparing a monolithic fluidic device module 100, and supplying (applying) a harvested cell culture to at least the membrane 110, where the cell culture can directly bind to carboxylic acid or carboxylate groups on the exposed surface of the membrane, and obtaining the prepared fluidic device module 100 from which a fluidic device 200, e.g., a biofunctional chip, is formed, as described above. The cell culture in the fluidic device 200 can be supplied through one of the pair of channels 115 of the fluidic device 200 to keep the cell culture alive, while the cell culture can be exposed to a drug to be tested through the other of the pair of channels 115 of the fluidic device 200, and the drug testing method is typically completed by monitoring the response of the cell culture to the drug being tested. This can be accomplished, for example, by disassembling the fluidic device 200 by removing the cover plates 210, 220 and slicing the fluidic device module 100 to obtain slices containing a portion of the cell culture of the module, which can be examined under a microscope or the like to investigate the effect of the drug under test on the cell culture. During such examination, the cell culture is typically stained, treated with formalin, and fixed with a fixative such as paraffin to facilitate such microscopic examination. Staining, treating, and fixing of such cell cultures can be performed within the fluidic device 200 by passing appropriate chemicals through the channels 115 of the fluidic device 200 to which the cell culture will be exposed.

[0131] In other embodiments, disassembly of such fluidic device 200 can be avoided, and monitoring of the cell culture's response to one or more compounds, such as drugs, can be achieved in real time using confocal microscopy. In particular, when embodiments of fluidic device 200 include transparent cover plates 210, 220, e.g., glass or polymer plates, confocal microscopy can be used to monitor the cell culture within the assembled fluidic device 200. This is made possible by the fact that at least the bottom cover plate 220 can be kept thin, e.g., in the range of 150-300 μm, such that 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, within the focal length limits of the confocal microscope (typically about 500 μm from the object to be investigated to the objective lens). To this end, the membrane 110 can have a thickness in the range of 10-100 μm, e.g., 20-30 μm. The thickness of the upper cover plate 210 is not critical and any suitable thickness is contemplated, such as a thickness between 300 μm and 3 mm.

[0132] Evaluation of cell cultures within fluidic device 200 using a confocal microscope is particularly suitable when the cell spheroids have diameters of less than 500 μm, e.g., about 200 μm, to ensure that the total focal length of the optical path facilitates capture of clear images by the confocal microscope. For larger spheroids, fluidic device 200 must be disassembled so that membrane 110 containing the spheroids can be pressed against a glass coverslip for positioning in the optical objective of the confocal microscope.

[0133] Furthermore, it should be noted that the above-described dimensions of the fluidic device 200 generally apply during real-time monitoring of cell cultures within the fluidic device 200. In embodiments in which the fluidic device 200 is sliced ​​as described above, e.g., for digital pathology, the dimensions of the fluidic device 200 are less critical as long as formalin and paraffin fixation can be performed.

[0134] The cell culture substrates described above are merely examples. Other such substrates exist. As an example, such a substrate may be a well cell plate. Such a substrate may be configured for use with a well cell plate, e.g., a slab of the substrate may be configured with a shape and size adapted to be inserted into a standard-sized well of a well cell plate. As another example, a well cell plate with open wells may be used, with a portion of the material closing the bottom of the well cell plate. Such an ensemble or device may be clamped.

[0135] As will be readily understood by those skilled in the art, such drug testing methods can vary widely in terms of protocols, such as drug dosage, frequency of administration, etc. It should be understood that drug testing methods according to the present invention are not limited to any particular protocol, so long as such methods can be performed using fluidic device 200 according to embodiments of the present invention in which cells are cultured directly on an exposed surface of the device, such as the membrane 10.

[0136] Proof of concept for mammalian cell culture directly on bulk-modified elastomers of the present invention will now be provided in the form of the following experimental evidence. [General Procedure]

[0137] The samples were cleaned by washing them in isopropanol for 5 minutes. After drying, the samples were placed in a 24-well plate. Immortalized human prostate fibroblast (WPMY-1 cells, ATCC CRL-2854) cell culture was provided in DMEM (Dulbecco's Modified Eagle's Medium, Thermo Fisher Scientific) containing 10% FBS (fetal bovine serum), 1% penicillin / streptomycin, and 1% Glutamax. 2.0x10 5 The total amount of cells was seeded into all samples and placed in an incubator overnight at 37°C and 5% CO2. Images of the cell cultures thus generated were made at 100x magnification using a Leica inverted brightfield microscope.

[0138] Example 1: WPMY-1 cell cultures were grown on silicone elastomer bulk-modified with 0.5 wt. % linoleic acid according to the teachings of the present invention, following the general procedure described above. To this end, Synthesis Example 2 was modified by substituting 48.488 g of sodium linoleate with 2.41 g of linoleic acid to obtain the bulk-modified silicone elastomer used in this example.

[0139] Comparative Example 1: WPMY-1 cell cultures were grown on the same silicone elastomer as in Example 1, but without bulk modification, according to the general procedure described above.

[0140] Example 2: WPMY-1 cell cultures were grown on bulk-modified silicone elastomer (i.e., containing 1.0 wt. % sodium linoleate) prepared according to Synthesis Example 2, following the general procedure described above.

[0141] Figure 9 shows the resulting cell culture of Example 1 after overnight incubation. In this image, it can be clearly seen that the cells exhibit good attachment to the linoleic acid functionalized silicone surface.

[0142] Figure 10 shows the resulting cell culture of Comparative Example 1 after overnight incubation. In this image, it is clearly seen that the cells show minimal attachment to the surface of the non-functionalized silicone elastomer.

[0143] Figure 11 shows the resulting cell culture of Example 2 after overnight incubation. The image clearly shows good cell attachment to the sodium linoleate-functionalized silicone surface. Figure 12 shows the cell culture of Example 2 after two days of incubation, and Figure 13 shows the cell culture of Example 2 after seven days of incubation. Ongoing cell growth is clearly visible in these images, demonstrating that mammalian cell material can be cultured and grown directly on the bulk-functionalized elastomers of the present invention.

[0144] Similar results were obtained using other cell types on substrates prepared and treated as for Example 2 but with 1% by weight linoleic acid.

[0145] Example 3: Endothelial Ea.hy926 cell cultures were grown on bulk-modified silicone elastomer (i.e., containing 1.0 wt. % sodium linoleate) prepared according to Synthesis Example 2, following the general procedure described above.

[0146] Example 4: Epithelial Caco-2 cell cultures were grown on bulk-modified silicone elastomer (i.e., containing 1.0 wt. % sodium linoleate) prepared according to Synthesis Example 2, following the general procedure described above.

[0147] Figures 14 and 15 show the cell growth after 4 days for Examples 3 and 4, respectively. Thus, all cell lines show good growth on the substrate after several days. Non-limiting list of examples

[0148] Example 1: A material for culturing cells, the material comprising a bulk-modified elastomer having a Shore hardness (DIN EN ISO 868) in the range of Shore 00 20 to Shore A 80 and having a plurality of fatty acid moieties covalently bonded to the elastomer bulk, the carboxylic acid groups of the moieties being available on the exterior surface of the material to effect said bonding, the bulk-modified elastomer comprising: forming a composition having a vinyl-functionalized or hydride-functionalized elastomer or at least one precursor thereof, a free or saponified unsaturated fatty acid in the range of 0.5 to 5 wt. % of the total weight of the vinyl-functionalized or hydride-functionalized elastomer or at least one precursor thereof, and a crosslinking catalyst in a mold having a polar inner surface; and bulk modifying the vinyl- or hydride-functionalized elastomer by covalently bonding the free or saponified unsaturated fatty acid to the bulk of the elastomer in the mold via a crosslinking reaction between the vinyl or hydride groups of the elastomer and the unsaturated carbon-carbon bond of the unsaturated fatty acid to obtain the material; This is obtained by:

[0149] Example 2: The material of Example 1, wherein each of the fatty acid moieties is covalently bonded to the elastomer bulk via a crosslinking reaction between a vinyl or hydride functional group of the elastomer and an unsaturated carbon-carbon bond of the unsaturated fatty acid.

[0150] Example 3: The material of example 2, wherein the unsaturated fatty acids are selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosperanoic acid, erucic acid, and docosahexaenoic acid.

[0151] Example 4: Materials of Examples 1-3, wherein the elastomer has a polybutadiene backbone or a silicone backbone.

[0152] Example 5: The materials of Examples 1-4, wherein the composition has free or saponified unsaturated fatty acids in the range of 0.5-2 wt. % of the total weight of the vinyl- or hydride-functionalized elastomer or at least one precursor thereof.

[0153] Example 6: Materials of Examples 1-5, where the polar inner surface of the mold is a metal oxide inner surface.

[0154] Example 7: Fluidic device module including a flow channel extending over a membrane having the material of any of Examples 1-6.

[0155] Example 8: A fluidic device module of Example 7, having a first main surface having a first concave structure defining a first flow path, and a second main surface opposite the first main surface having a second concave structure defining a second flow path, the membrane separating the first flow path from the second flow path.

[0156] Example 9: The fluidic device module of Example 7 or 8, wherein the fluidic device is a monolithic fluidic device.

[0157] Example 10: A fluidic device comprising the fluidic device module of any one of Examples 7 to 9, and a pair of cover plates for fluidically sealing the fluidic device module.

[0158] Example 11: The fluidic device of Example 10, wherein the cover plates are arranged such that one of the cover plates covers a first main surface, thereby sealing a first flow path, and the other of the cover plates covers a second main surface, thereby sealing a second flow path.

[0159] Example 12: A method of culturing cells, comprising: providing a molded article formed from the material of any of Examples 1-5, the article having an exterior surface of the material as one of its exposed surfaces; and culturing the cells directly on the exposed surface of the article; It has.

[0160] Example 13: The method of example 12, wherein the article comprises the fluidic device module of any of examples 7-9.

[0161] Example 14: A method for testing a drug, the method comprising: Preparing a fluidic device module according to Example 8; providing the collected cells onto a surface of a membrane of the fluidic device module; culturing the harvested cells on the surface; forming a fluidic device using the prepared fluidic device module; supplying the cultured cells through one of a pair of channels of the fluidic device; exposing the cultured cells to a chemical to be tested through the other of the pair of channels of the fluidic device; monitoring the response of the cultured cells to the agent to be tested; It has.

[0162] Example 15: The drug testing method of Example 14, wherein monitoring the response of the expanded cell culture to the drug to be tested comprises: Immobilizing and fixing the cultured cells within the fluidic device and slicing the fluidic device module to obtain slices for microscopic evaluation, the slices having at least a portion of the immobilized and fixed expanded cell culture; or monitoring the cultured cells in the fluidic device using a confocal microscope; It has.

[0163] It should be noted that the above-described embodiments illustrate rather than limit the present invention, and that those skilled in the art can design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. The word "comprises" does not exclude the presence of elements or steps other than those stated in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The invention can be implemented by means of hardware comprising several distinct elements. In a device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain means are recited in mutually different dependent claims does not indicate that a combination of these means cannot be used to advantage.

Claims

1. 1. A cell culture substrate comprising a material having an exterior surface for culturing cells, the material comprising a bulk-modified elastomer having an elastomer bulk and a plurality of residues, each residue comprising one or more acidic groups in free and / or conjugate base form, the plurality of residues being covalently bonded to the elastomer bulk such that a portion of the one or more acidic groups are available on the exterior surface.

2. 10. The cell culture substrate of claim 1, wherein the one or more acidic groups are selected from the group consisting of phosphorus-based acidic groups, sulfur-based acidic groups, and carboxylic acid groups, or a mixture of two or more thereof.

3. 3. The cell culture substrate of claim 1 or claim 2, wherein the residue comprises or consists of an aliphatic moiety containing at least 3 but less than 50 carbon atoms, the one or more acidic groups are covalently bonded to the aliphatic moiety, and the aliphatic moiety is covalently bonded to the elastomer bulk.

4. 4. The cell culture substrate of claim 1, wherein the residue is an unsaturated fatty acid residue.

5. 5. The cell culture substrate of claim 4, wherein the unsaturated fatty acid residue is one or more residues of a fatty acid selected from the group consisting of myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicospartan acid, erucic acid, and docosahexaenoic acid.

6. 6. The cell culture substrate of claim 1, wherein the elastomer bulk has a silicone or polybutadiene backbone.

7. 7. The cell culture substrate of claim 6, wherein the residue is covalently bonded to the elastomer bulk as a result of a reaction between an unsaturated carbon-carbon bond of a precursor of the residue and a vinyl or hydride functional group of the elastomer bulk.

8. The residue concentration in the bulk modified elastomer or corresponding precursor is preferably less than 2.10% by weight of the total elastomer, relative to the residue in the mixture of precursors for forming the elastomer. -４ ~2.10 -２ 8. The cell culture substrate of claim 1, wherein the concentration of the soluble solids in the cell culture medium is in the range of 1000 mol / kg (molal concentration).

9. The bulk-modified elastomer comprises a vinyl-functionalized and / or hydride-functionalized elastomer or at least one precursor thereof, and 2.10% by weight of the total weight of the bulk-modified elastomer. -４ ~2.10 -２ and heating the mixture, and preferably, when the elastomer is a silicone-based elastomer, the range is 2.10 -４ ~1.10 -２ 9. The cell culture substrate according to claim 1, wherein the cell culture substrate is denatured in an amount of 0.1 mol / kg.

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

11. The cell culture substrate of claim 1 , wherein the substrate has a fluidic device module including a flow path extending onto a membrane having the material.

12. 12. The cell culture substrate of claim 11, having a first main surface having a first recessed structure defining a first flow path, and a second main surface opposite the first main surface having a second recessed structure defining a second flow path, the membrane separating the first flow path from the second flow path.

13. The cell culture substrate according to claim 11 or claim 12, wherein the fluidic device is a monolithic fluidic device.

14. 1. A method of using a material having an exterior surface for culturing cells, the material comprising an elastomer bulk and a bulk-modified elastomer having a plurality of residues each comprising one or more acidic groups in free and / or conjugated base form, the plurality of residues being covalently bonded to the elastomer bulk such that a portion of the one or more acidic groups are available on the exterior surface, the method comprising contacting the exterior surface with cells to be cultured and a cell growth medium.

15. 1. A method of culturing cells, the method comprising: Using the cell culture substrate according to any one of claims 1 to 13, optionally a molded substrate; and Culturing cells on the exposed surface of the substrate. A method for culturing cells comprising:

16. 16. The method for culturing cells according to claim 15, wherein the cell culture substrate is a cell culture substrate according to any one of claims 11 to 13.

17. A drug testing method including the cell culturing method according to claim 15, exposing the cultured cells to a chemical to be tested through the other of the pair of channels of the fluidic device; and monitoring the response of the cultured cells to the agent to be tested; The drug inspection method further comprises:

18. 1. A method for producing a cell culture substrate, comprising: In a mold, a mixture of a vinyl-functionalized and / or hydride-functionalized elastomer or at least one precursor thereof and a bulk-modified elastomer in an amount of 2.10% by weight of the total weight of the bulk-modified elastomer is mixed. -４ ~2.10 -２ and a residue precursor defined in a concentration range of 2.10 mol / kg (molal concentration), and heating the mixture, preferably when the elastomer is a silicone-based elastomer, said range being 2.10 mol / kg (molal concentration). -４ ~1.10 -２ mol / kg, A method for producing a cell culture substrate comprising:

Images