Base material for cells

Abstract

Provided is a base material for cells, which has excellent detectability of chemical substances such as odorants. This base material for cells includes a compartment including a surface in which the molar ratio (O / C) of oxygen atoms with respect to carbon atoms is 0.150 or more.

Description

Substrate for cells

[0001] The present invention relates to a cell substrate and the like.

[0002] Groups of odorants that characterize specific human diseases and mental states have been identified, and because of their high utility as test markers, the development of various odor sensors targeting these has become active. Because biological olfactory receptors have superior properties in terms of diversity, sensitivity, selectivity, etc. that are not found in conventional odor sensor elements such as semiconductors, there are high expectations for the development of new odor sensors that use olfactory receptors as sensor elements.

[0003] Patent Document 1 discloses the use of cells expressing modified olfactory receptors or lipid bilayer membranes comprising modified olfactory receptors as odor sensors.

[0004] International Publication No. 2022 / 024902

[0005] Odor sensors that involve artificially preparing lipid bilayer membranes containing sensor proteins such as olfactory receptors do not necessarily have sufficient manufacturing efficiency, and further improvements in the manufacturing efficiency of odor sensors are therefore required. Therefore, the inventors focused on using cells that express sensor proteins. Through research based on this, the inventors discovered that the detectability of chemical substances such as odorants differs depending on the type of cell substrate.

[0006] Therefore, an object of the present disclosure is to provide a cell substrate that has excellent detectability for chemical substances such as odorous substances.

[0007] In view of the above-mentioned problems, the present inventors have conducted extensive research and have found that the above-mentioned problems can be solved by a cell substrate including a compartment having a surface in which the molar ratio of oxygen atoms to carbon atoms (O / C) is 0.150 or more.

[0008] Item 1. A cell substrate comprising a compartment having a surface in which the molar ratio of oxygen atoms to carbon atoms (O / C) is 0.150 or more.

[0009] Item 2. The cell substrate according to Item 1, wherein the molar ratio (O / C) of the surface is 0.195 or greater.

[0010] Item 3. The cell substrate according to Item 1, wherein the molar ratio (O / C) of the surface is 0.250 or less.

[0011] Item 4. The cell substrate according to Item 1, wherein the surface has a molar ratio of nitrogen atoms to carbon atoms (N / C) of 0.020 or more.

[0012] Item 5. The cell substrate according to Item 1, wherein the surface has a water contact angle of 60° or less.

[0013] Item 6. The cell substrate according to Item 1, wherein the surface area is 70% or more relative to 100% of the compartment area.

[0014] Item 7. The cell substrate according to Item 1, wherein the surface material is a resin.

[0015] Item 8. The area of ​​the compartment is 1 to 50 mm 2 Item 2. The cell substrate according to Item 1,

[0016] Item 9. The cell substrate according to any one of Items 1 to 8, which is used to measure the activity of a sensor protein with cells arranged on the surface.

[0017] Item 10. The cell substrate according to any one of Items 9, wherein the cells contain a polynucleotide comprising a coding sequence for a sensor protein.

[0018] Item 11. The cell substrate according to Item 9, wherein the sensor protein is an olfactory receptor protein.

[0019] Item 12. The cell substrate according to Item 9, which is used to measure the activity of the sensor protein after replacing or adding a solution with cells arranged on the surface.

[0020] Item 13. The cell substrate according to any one of Items 1 to 8, wherein the cells are insect cells.

[0021] Item 14. A method for measuring the activity of a sensor protein, comprising measuring the activity of the sensor protein of a cell placed on a surface having a molar ratio of oxygen atoms to carbon atoms (O / C) of 0.150 or more.

[0022] According to the present disclosure, a cell substrate with excellent detectability for chemical substances such as odorants can be provided.

[0023] This shows an image of cells observed in Test Example 3. The plate type at the top of the photograph and cell number 1 and cell number 2 on the left side of the photograph correspond to Table 1. This shows the results of measuring the activity of olfactory receptors in Test Example 3. The vertical axis indicates fluorescence intensity. The horizontal axis indicates plate type and cell number. Plate type corresponds to Table 1. The legend indicates the concentration of compound a.

[0024] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0025] In one aspect, the present disclosure relates to a cell substrate (sometimes referred to herein as the "substrate of the present disclosure") that includes a compartment having a surface with a molar ratio of oxygen atoms to carbon atoms (O / C) of 0.150 or more. This will be described below.

[0026] The substrate of the present disclosure is a cell substrate used for arranging (e.g., adhering) cells, and the form thereof is not particularly limited. The substrate of the present disclosure may be, for example, a cell plate, a cell chip, a cell device, a cell dish, a cell flask, a cell bag, or the like.

[0027] The substrate of the present disclosure includes a compartment. A compartment is an area where cells are arranged (e.g., where a cell layer is formed or where cells are adhered). The form of the compartment is not particularly limited as long as it is an embodiment where cells can be arranged (e.g., where a cell layer is formed or where cells are adhered). From the viewpoint of cell desiccation resistance, cell retention, production efficiency, or chemical substance detectability, the compartment is preferably in a form separated by walls (e.g., a well-like form and / or a form separated from the surrounding area by an outer wall). Furthermore, by making the surface outside the compartment a surface where cells cannot be arranged (e.g., where a cell layer is formed or where cells are adhered) (or where adhesion is significantly reduced), it is possible to distinguish it from a compartment where cells can be arranged (e.g., where a cell layer is formed or where cells are adhered).

[0028] The compartment includes a surface (sometimes referred to herein as "the surface of the present disclosure") having a molar ratio of oxygen atoms to carbon atoms (O / C) of 0.150 or greater.

[0029] The molar ratio (O / C) of the surface of the present disclosure is preferably 0.160 or more, more preferably 0.170 or more, and even more preferably 0.180 or more, from the viewpoint of chemical substance detectability (especially detectability of chemical substances at lower concentrations), and within the above range, the molar ratio (O / C) is particularly preferably 0.195 or more, and particularly preferably 0.205 or more, from the viewpoint of chemical substance detectability (especially detectability of chemical substances at lower concentrations). The upper limit of the molar ratio (O / C) is not particularly limited, but is preferably 0.300, more preferably 0.270, even more preferably 0.250, even more preferably 0.240, especially preferably 0.230, and especially preferably 0.220.

[0030] Although we do not wish to limit the interpretation, the molar ratio (O / C) represents, in one sense, the amount of oxidized carbon groups (C-O, O-C=O, C-O, C=O), and it is thought that a certain level of the amount of such groups or functional groups containing such groups affects the adhesiveness and adhesion mode of cells, etc., and contributes to improving cell reactivity in terms of the detectability of chemical substances.

[0031] From the viewpoint of chemical substance detectability, the surface of the present disclosure is preferably a surface having a nitrogen atom to carbon atom molar ratio (N / C) of 0.020 or more. The molar ratio (N / C) of the surface of the present disclosure is more preferably 0.030 or more, even more preferably 0.035 or more, and even more preferably 0.040 or more. The upper limit of the molar ratio (N / C) is not particularly limited, but is preferably 0.100, more preferably 0.080, even more preferably 0.070, even more preferably 0.060, and particularly preferably 0.050.

[0032] From the viewpoint of chemical substance detectability, the surface of the present disclosure is characterized by the ability to detect groups measured by C1s spectrum ((1) C-C, C-H; (2) C-O, C-N; (3) O-C=O; (4) CO3), groups measured by O1s spectrum (C-O, C=O), and groups measured by N1s spectrum ((1) C-N, C=N; (2) quaternary ammonium salts; (3) NO x The ratio of the sum of the state ratios of (2) to (4) in the C1s spectrum to the sum of (1) to (4) relative to the sum of 100 is preferably 0.15 or more, more preferably 0.16 or more, even more preferably 0.17 or more, still more preferably 0.18 or more, particularly preferably 0.19 or more, particularly more preferably 0.20 or more, particularly more preferably 0.21 or more, particularly still more preferably 0.22 or more, and particularly preferably 0.23 or more. The sum of the state ratios is not particularly limited, but is preferably 0.4 or less, more preferably 0.35 or less, and even more preferably 0.3 or less.

[0033] The surface molar ratio (O / C), molar ratio (N / C), and state ratio are values ​​measured by the method described in Test Example 4 below or a method equivalent thereto.

[0034] In one embodiment of the present disclosure, the water contact angle of the surface of the present disclosure is preferably 60° or less. The water contact angle is more preferably 55° or less, even more preferably 50° or less, and even more preferably 45° or less. The lower limit of the water contact angle is preferably 30°, more preferably 35°, and particularly preferably 40°.

[0035] The water contact angle of the surface is a value measured by the method described in Test Example 5 below or a method equivalent thereto.

[0036] The material constituting the surface of the present disclosure is not particularly limited as long as it is a material whose molar ratio (O / C) can be adjusted within a predetermined range. Examples of such materials include resins (e.g., polystyrene, polyethylene, polypropylene, poly(acrylic acid ester), poly(methacrylic acid ester), polyacrylamide, polyacrylonitrile, polyethylene terephthalate, poly(L-lactic acid), poly(glycolic acid), poly(ε-caprolactone), poly(ethylene glycol), and copolymers thereof), metals (gold, silver, copper, iron, zinc, aluminum, nickel, and alloys or oxides thereof), glass, silica, silicon, and composite materials. From the viewpoint of ease of adjusting the molar ratio (O / C) within a predetermined range, the material is preferably a resin.

[0037] The surface of the present disclosure can be obtained, for example, by performing a treatment on the resin surface to increase the molar ratio (O / C). Examples of such treatments include, but are not limited to, plasma treatment, corona treatment, and UV-ozone treatment. As another example, the surface of the present disclosure can also be obtained by using a resin obtained by a reaction to introduce oxygen-containing functional groups and / or by adding a resin containing a relatively large amount of oxygen-containing functional groups.

[0038] The objective effect of the present disclosure can be more effectively achieved by including more of the surface of the present disclosure in the compartment. From this viewpoint, the area of ​​the surface of the present disclosure relative to 100% of the compartment area (the area of ​​the region where cells are arranged (e.g., a cell layer is formed, or cells are adhered)) is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, still more preferably 95% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0039] The area of ​​the compartment is preferably 0.5 to 100 mm from the viewpoint of detection sensitivity or production efficiency. 2 , more preferably 1 to 50 mm 2 , more preferably 1.5 to 40 mm 2 , and even more preferably 2 to 40 mm 2 In a preferred embodiment of the present disclosure, the area is 50 mm2 Below, 35mm 2 or less, or 15 mm 2 The following is the result.

[0040] From the viewpoint of detection sensitivity or production efficiency, the number of compartments contained in the substrate of the present disclosure is preferably 1 to 2000, more preferably 4 to 1600, and even more preferably 8 to 400. In a preferred embodiment of the present disclosure, the number is 10 or more, 20 or more, or 50 or more.

[0041] The substrate and surface of the present disclosure can be used to measure the activity of a sensor protein in a cell. By measuring the activity of a sensor protein in a cell using the substrate and surface of the present disclosure, chemical substances can be detected with higher sensitivity. Therefore, in one aspect, the present disclosure relates to a method for measuring the activity of a sensor protein, the method comprising measuring the activity of the sensor protein in a cell containing a polynucleotide including a coding sequence for the sensor protein, which is arranged on a surface of the present disclosure.

[0042] When the substrate of the present disclosure is used to measure the activity of a cellular sensor protein, there are no particular limitations as long as cells are arranged on the surface of the present disclosure (e.g., a cell layer is formed, or cells are adhered). To prevent the cells from drying out, the compartment preferably contains a solution (e.g., a culture medium) or a gel (e.g., a gel containing culture medium components).

[0043] From the viewpoint of detection sensitivity, a compartment usually contains multiple cells. 2 The number of cells per 1000 mm is, for example, 50 to 20,000 cells / mm 2 From the viewpoint of detection sensitivity, cell viability, etc., the cell density is preferably 100 to 15,000 cells / mm 2 , more preferably 100 to 10,000 cells / mm 2 , and more preferably 200 to 10,000 cells / mm 2 , and even more preferably 500 to 7000 cells / mm 2 , particularly preferably 1000 to 5000 cells / mm 2 is.

[0044] In one aspect of the present disclosure, the substrate of the present disclosure can be used for solution replacement or addition while cells are placed on the surface of the present disclosure. In this case, for example, after solvent replacement or addition, the substrate can be used to measure the activity of the sensor protein. A solution (such as a culture medium) or gel suitable for holding cells may contain substances that adversely affect the detection of chemicals or may have physical properties that could adversely affect the detection of chemicals. Therefore, to reduce the risk of such adverse effects, it is preferable to replace or add the solution before activity measurement. In the case of a gel, the gel is converted to a liquid state as appropriate (for example, by increasing the temperature) before replacing or adding the solution. This allows the solution, such as a culture medium, to be replaced with a solution (such as a buffer solution) that can control the above-mentioned risk, or the above-mentioned risk can be reduced by dilution.

[0045] The substrate of the present disclosure may contain cells of only one type of sensor protein, but preferably contains cells of two or more (more preferably three or more, even more preferably four or more, even more preferably five or more, ten or more, fifteen or more, or twenty or more) types of different sensor proteins.

[0046] The cells are not particularly limited. From the viewpoint of suitability for detecting chemical substances, animal cells such as insect cells and mammalian cells are preferred, and insect cells are particularly preferred from the viewpoint of suitability for detecting chemical substances using the surface of the present invention and ease of management, such as no need for CO2 or temperature management.

[0047] Examples of insect cells include Sf cells, MG1 cells, and High Five cells. TM Examples of Sf cells that can be used include Sf9 cells (ATCC CRL1711) and Sf21 cells. Among insect cells, cells derived from insects of the family Arctiidae are particularly preferred.

[0048] The cells derived from an insect of the family Arctiidae are primary cultured cells or established cell lines of cells derived from an insect of the family Arctiidae that constitute the living body, and are not particularly limited as long as they are so.

[0049] Examples of the Arctiidae family include the Arctiinae subfamily, Lithosiinae subfamily, and Syntominae subfamily, with the Arctiinae being preferred among these. Examples of the Arctiinae subfamily include the genera Spilosoma, Spilarctia, and Rhagonis, with the Rhagonis genus being particularly preferred. The Rhagonis genus is not particularly limited, but the Rhagonis genus is particularly preferred.

[0050] Cells derived from an insect of the family Arctiidae can be obtained from a known biobank, or can be obtained by collecting and culturing from a living insect of the family Arctiidae according to or in accordance with a known method, or by establishing a line if necessary.

[0051] Examples of cells derived from Mulberry butterfly include FFPRI-SpIm-2AM-SF cells (MAFF number: 275052) and FFPRI-SpIm-2AM-IPL411 cells (MAFF number: 275053) from the National Agriculture and Food Research Organization Genebank.

[0052] The cells preferably contain an exogenous polynucleotide containing a coding sequence for a sensor protein, which allows the expression of any sensor protein and increases the expression level of the target sensor protein, thereby enhancing the detection sensitivity of the target chemical substance.

[0053] The exogenous polynucleotide is a polynucleotide containing a base sequence that is not derived from the genomic DNA (particularly chromosomal genomic DNA) of an insect cell, and is not particularly limited insofar as it is so.

[0054] The sensor protein may be selected from proteins capable of detecting the presence of a chemical substance, and may be, for example, a receptor protein that uses a chemical substance as a ligand. The sensor protein is preferably an olfactory receptor protein, and particularly preferably an insect olfactory receptor protein.

[0055] Insect olfactory receptor proteins are membrane proteins with seven transmembrane domains that function as odor sensors in living organisms. From the amino terminus (hereafter sometimes referred to as the "N-terminus") to the carboxyl terminus (hereafter sometimes referred to as the "C-terminus") of an olfactory receptor protein, they are composed of the N-terminal region (NT), the first transmembrane domain (TM1), the first extracellular loop (EC1), the second transmembrane domain (TM2), the first intracellular loop (IC1), the third transmembrane domain (TM3), the second extracellular loop (EC2), the fourth transmembrane domain (TM4), the second intracellular loop (IC2), the fifth transmembrane domain (TM5), the third extracellular loop (EC3), the sixth transmembrane domain (TM6), the third intracellular loop (IC3), the seventh transmembrane domain (TM7), and the C-terminal region (CT). In the present disclosure, each region is determined by structure prediction (default conditions) using TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membrane spanning protein segments, Biol. Chem. Hoppe-Seyler, 374 (1993), p. 166, https: / / embnet.vital-it.ch / software / TMPRED_form.html).

[0056] Insects from which insect olfactory receptor proteins are derived preferably include Diptera insects such as Culicidae and Drosophilidae, Lepidoptera insects such as Bombycidae, Hymenoptera insects such as Apidae, Orthoptera insects such as Acrididae, and Hemiptera insects such as Cimex, and more preferably Diptera insects such as Culicidae and Drosophilidae, Orthoptera insects such as Acrididae, and Hemiptera insects such as Cimex. Examples of Culicidae insects include Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. Examples of insects in the Drosophilidae family include Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila virillis. Examples of insects in the Bombycidae family include Bombyx mori, Bombyx mandarina, and Trilocha varians. Examples of insects in the Apidae family include Apis mellifera, Apis florea, Apis dorsata, and Bombus terrestris. Examples of insects belonging to the Acrididae family include the migratory locust (Locusta migratoria), and examples of insects belonging to the Cimex family include the bedbug (Cimex lectularius).

[0057] Specific examples of wild-type insect olfactory receptor proteins include AaOR1, AaOR2, AaOR4, AaOR5, AaOR6, AaOR8, AaOR9, AaOR10a, AaOR15, AaOR22, AaOR24, AaOR25, AaOR26, AaOR27, AaOR28, AaOR30, AaOR34, AaOR36, AaOR38, AaOR41a, AaOR41b, AaOR42, AaOR43, AaOR44, AaOR47, AaOR49, AaOR50, AaOR52, AaOR54, AaOR58, AaOR59, and AaOR60. , AaOR61, AaOR64, AaOR65, AaOR66, AaOR67a, AaOR69a, AaOR70, AaOR71, AaOR72a, AaOR73, AaOR74, AaOR75, AaOR77, AaOR78, AaOR79, AaOR81, AaOR83b, AaOR84, AaOR85, AaOR86, AaOR87, AaOR91, AaOR95, AaOR97, AaOR96, AaOR99, AaOR100, AaOR102, AaOR103, AaOR104a, AaOR105, AaOR107, AaOR108, AaOR1 09, AaOR110, AaOR112, AaOR114, AaOR116, AaOR117, AaOR118, AaOR122, AaOR125, AaOR128, AgOR1, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR8, AgOR9, A gOR10, AgOR11a, AgOR12a, AgOR12b, AgOR13, AgOR14, AgOR15, AgOR16a, AgOR17, AgOR18, AgOR20, AgOR21, AgOR23, AgOR25, AgOR26, AgOR27, AgOR28, AgO R30, AgOR34, AgOR36, AgOR37, AgOR38, AgOR39a, AgOR40, AgOR42, AgOR44, AgOR45, AgOR46, AgOR47, AgOR49, AgOR50, AgOR54, AgOR56a, AgOR57, AgOR60 , AgOR61, AgOR62, AgOR63, AgOR64, AgOR65, AgOR69, AgOR70, AgOR71, AgOR72, AgOR74, AgOR75, AgOR76a, AmOR1, AmOR3, AmOR9, AmOR10, AmOR13, AmOR41,AmOR51, AmOR52, AmOR55, AmOR71, AmOR73, AmOR78, AmOR85, AmOR89, AmOR90, AmOR114, AmOR115, AmOR118, AmOR120, AmOR121, AmOR161, BmOR1, BmOR2, BmOR3, BmOR4, BmOR5, BmOR 8, BmOR9, BmOR10, BmOR13, BmOR17, BmOR18, BmOR23, BmOR24, BmOR25, BmOR35, BmOR36, B mOR42, BmOR45, BmOR49, BmOR51, BmOR52, BmOR55, BmOR56, BmOR61, DmOR1a, DmOR9a, DmOR 19a, DmOR22a, DmOR22b, DmOR22c, DmOR24a, DmOR30a, DmOR33a, DmOR33b, DmOR33c, DmOR 35a, DmOR42b, DmOR43a, DmOR45a, DmOR45b, DmOR47a, DmOR49b, DmOR59b, DmOR65b, DmOR6 Examples include DmOR5c, DmOR67b, DmOR67c, DmOR69a, DmOR71a, DmOR74a, DmOR82a, DmOR83a, DmOR83c, DmOR85a, DmOR85c, DmOR85e, DmOR85f, DmOR88a, DmOR92a, DmOR94a, DmOR94b, and DmOR98b.

[0058] In this specification, OR indicates an olfactory receptor, Dm indicates that it is derived from Drosophila melanogaster, Bm indicates that it is derived from Bombyx mori, Ag indicates that it is derived from Anopheles gambiae, and Aa indicates that it is derived from Aedes aegypti. The amino acid sequences and coding sequences of various olfactory receptor proteins, including these, are publicly known or can be easily identified by sequence identity searches based on the publicly known sequences.

[0059] The sensor protein may contain amino acid mutations relative to the wild-type amino acid sequence, as long as the chemical response activity is not significantly reduced. "Not significantly reduced" means, for example, that the chemical response activity of the sensor protein containing the amino acid mutation is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more, relative to 100% of the chemical response activity of the wild-type sensor protein.

[0060] The amino acid mutation is, for example, an amino acid substitution, insertion, addition, or deletion, preferably a substitution, and particularly preferably a conservative substitution.

[0061] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0062] The sensor protein can include a wild-type amino acid sequence, or an amino acid sequence that has, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity to the wild-type amino acid sequence.

[0063] As used herein, "identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0064] As used herein, chemical response activity refers to the property of a sensor protein recognizing a chemical substance and exhibiting signal transduction activity (e.g., ion channel activity) either alone or in conjunction with other proteins. When the sensor protein is an olfactory receptor, it may be a G protein-coupled receptor or an ion channel receptor. In the case of an insect olfactory receptor, however, the term refers to the property of the olfactory receptor recognizing a chemical substance and activating an olfactory receptor complex formed by the olfactory receptor and an olfactory receptor co-receptor, thereby exhibiting ion channel activity. The chemical response activity of a sensor protein can be measured using as an indicator the signal transduction activity of the sensor protein in contact with a chemical substance (e.g., by quantifying and evaluating the amount of signal molecules). In the case of an insect olfactory receptor, the chemical response activity of the olfactory receptor can be measured using as an indicator the ion channel activity of the olfactory receptor complex formed by the olfactory receptor in contact with a chemical substance and an olfactory receptor co-receptor. For example, cells expressing proteins that emit fluorescence or luminescence in response to ions (such as calcium ions) that flow into the cells when (a) olfactory receptors, (b) olfactory receptor co-receptors, and (c) olfactory receptor complexes respond are contacted with a chemical substance, and the amount of luminescence from the cells is measured. The greater the measured amount of luminescence, the higher the response activity of the olfactory receptor to the chemical substance is determined to be. Specifically, measurements can be performed according to the method described in Patent Document 1.

[0065] The coding sequence of the sensor protein is not particularly limited as long as it is a base sequence that encodes the sensor protein. In one embodiment, the exogenous polynucleotide comprises an expression cassette for the sensor protein. The expression cassette is not particularly limited as long as it is a polynucleotide that can express the sensor protein in cells. A typical example of an expression cassette for a sensor protein is a polynucleotide that comprises a promoter and a coding sequence for the sensor protein placed under the control of the promoter.

[0066] When the sensor protein is an insect olfactory receptor, the exogenous polynucleotide preferably contains a coding sequence for an insect olfactory receptor co-receptor. Insect olfactory receptor co-receptors are membrane proteins with a seven-transmembrane structure, similar to olfactory receptors, and function by forming a heterocomplex with the olfactory receptor. The olfactory receptor complex, which is a heterocomplex composed of an olfactory receptor and an olfactory receptor co-receptor, has ion channel activity that is activated by odorants, and when activated, it transports sodium ions (Na + ), calcium ions (Ca 2+ ) into the cell.

[0067] The exogenous polynucleotide preferably contains a coding sequence for a protein that emits fluorescence or luminescence in response to ions (such as calcium ions) that flow into the cell in response to a sensor protein (particularly an olfactory receptor protein). Examples of such proteins include aequorin, Yellow Cameleon (YC), GCaMP, etc. Alternatively, the cells of the present disclosure preferably contain an ion-dependent fluorescent dye such as a calcium ion-dependent fluorescent dye (e.g., Fura-2, Fluo-3, Fluo-4, etc.).

[0068] Coding sequences such as those for insect olfactory receptor co-receptors, fluorescent or luminescent proteins, and drug resistance genes are preferably contained in the exogenous polynucleotide in the form of an expression cassette. The structure of the expression cassette is similar to that of the expression cassette for the sensor protein. The promoter of the expression cassette can be shared among multiple coding sequences.

[0069] The exogenous polynucleotide is preferably integrated into genomic DNA (particularly preferably chromosomal genomic DNA). This allows stable expression of the sensor protein, making it suitable for chemical detection. In this case, the polynucleotide can be a single contiguous region within the genomic DNA, or a combination of two or more contiguous regions (for example, a form in which the sensor protein coding sequence is included in contiguous region A and the drug resistance gene coding sequence is included in contiguous region B, which is a separate contiguous region from contiguous region A, or a form in which the sensor protein coding sequence is included in both contiguous region A and contiguous region B).

[0070] In another embodiment, the polynucleotide may be in a state where it is not integrated into genomic DNA. In this case, the exogenous polynucleotide may be in the form of, for example, a vector. In this case, the polynucleotide may be one type of polynucleotide molecule, or may be two or more types of polynucleotide molecules (for example, a form in which the sensor protein coding sequence is contained in polynucleotide molecule A and the drug resistance gene coding sequence is contained in polynucleotide molecule B, which is a molecule separate from polynucleotide molecule A, or a form in which the sensor protein coding sequence is contained in both polynucleotide molecule A and polynucleotide molecule B).

[0071] Chemical substances can be detected by measuring the activity of the sensor protein. The chemical substances can be chemical substances in a sample such as body fluids (e.g., urine, blood, saliva), air (e.g., indoor air, air inside a package), or water (e.g., river water, seawater, tap water, clean water, sewage). In this case, for example, by adding a sample to a compartment of the substrate of the present disclosure, the chemical substances in the sample can reach the cells and come into contact with the sensor protein of the cells. For example, by detecting ions flowing into the cells (e.g., detection by a protein that emits fluorescence or luminescence in response to ions), the activity of the sensor protein can be measured, and chemical substances can be detected.

[0072] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0073] Test Example 1. Preparation of Stably Expressing Cells A transposon vector was introduced into cells derived from the mulberry butterfly (Spilosoma imparilis) (SpIm cells), which contained an olfactory receptor protein (ORA) coding sequence, an olfactory receptor co-receptor coding sequence, a calcium sensor fluorescent protein coding sequence, and a puromycin resistance gene coding sequence under the control of a promoter sequence, with these sequences located between the 5'ITR (inverted repeat) and 3'ITR. Selection was then performed with puromycin to prepare SpIm cells stably expressing an olfactory receptor (hereinafter referred to as ORA cells), in which foreign DNA containing the coding sequence and promoter sequence was integrated into chromosomal genomic DNA. The olfactory receptor in question is an insect-derived olfactory receptor and is a receptor for compound a. ORA cells emit fluorescence in response to compound a.

[0074] Test Example 2: Cell adhesion to plates ORA cells were detached from a culture flask and mixed with medium (Sf-900 III, Gibco) to prepare a cell suspension. The cell density of the cell suspension was measured using a cell counter. When the cell density was 5 x 10 5 ~1×10 6 The cell density was adjusted to 100%. Then, using a dispenser (ASSIST PLUS, Integra), cells were seeded into three types of TC (Tissue Culture)-treated 384-well plates (resin, plates A to C) so that the same number of cells per well was used (40 μL / well). The number of cells seeded is shown in Table 1. After seeding, the plates were left to stand overnight at 27°C to allow cells to adhere. The plates were then stored at 4°C.

[0075]

[0076] Test Example 3. Measurement of Olfactory Receptor Activity 1 The plates stored at 4°C for 3 days in Test Example 2 were collected and left to stand at 27°C for 2 hours. After the cells were allowed to return to room temperature, the medium was replaced with assay buffer (Hanks' buffer containing 0.1% BSA and 20 mM HEPES) using a dispenser (40 μL / well). Cell adhesion was then observed under a microscope. The observed images are shown in Figure 1.

[0077] Compound a (a substance to which ORA responds) solution or a solution identical to compound a solution but without compound a was added to each well to a final concentration of 0, 0.1, 1, or 10 μM, and the change in fluorescence intensity before and after addition was measured using a microplate reader (FlexStation3, Molecular Devices).

[0078] The results are shown in Figure 2. Among plates A to C, when plates A and B (especially plate A) were used, higher fluorescence intensity was obtained than when plate C was used.

[0079] Test Example 4: Plate Surface Physical Property Measurement 1 The chemical composition and electronic state of elements present on the cell adhesion surface of the three types of plates (Plates A to C, unseeded) were nondestructively measured using an X-ray photoelectron spectrometer (Thermo Fisher Scientific, K-Alpha+). Details of the measurement method are described below. First, a cutter was inserted into the back of the plate to cut out the bottom of the plate along the shape of the wells, and the plate was then placed in a vacuum chamber to ensure a vacuum. Next, the measurement surface was inspected using the XPS camera to avoid analyzing contaminants such as cutting debris. Measurements were performed using an AlKα X-ray source with an X-ray spot diameter of 300 μm. To obtain more accurate measurement results, a neutralization gun was used to remove static electricity accumulated on the sample surface during measurement. Survey scan measurements were performed to identify the types of elements present in the sample. In addition, narrow scan measurements were performed to analyze the chemical bonding state of the elements detected during the survey scan.

[0080] The results are shown in Table 2. Survey scan measurements confirmed C1s, O1s, and N1s peaks on all plates. Narrow scan measurements performed waveform separation of the C1s and O1s spectra, revealing peaks presumably associated with CO, OC=O, and CO3, suggesting the presence of oxygen-containing functional groups. For N1s, the presence of CN and C=N was suggested on all plates. The proportions of different chemical states of each element were shown as state ratios (the total amount of each element is set to 100). To calculate the state ratios, the peak areas of each elemental state obtained from the narrow scan spectra were used to determine the proportion of each state. Plates A and B (especially Plate A) were found to have a higher molar ratio of oxygen atoms to carbon atoms (O / C) than Plate C.

[0081]

[0082] Test Example 5. Measurement of Plate Surface Properties 2 The cell adhesion surface of a plate (without cells seeded) was hollowed out using a biopsy trephine, placed horizontally on a contact angle meter (DMo-702, manufactured by Kyowa Interface Science), 0.2 μL of ultrapure water was dropped onto the surface, an image was taken, and the contact angle was calculated. Since the calculated value is expected to have an error of about 5°, measurements were taken on 3 to 4 plates of the same type and the average value was calculated. The results are shown in Table 3.

[0083]

[0084] Test Example 6. Olfactory Receptor Activity Measurement 2 A mixture of cells and culture medium was seeded onto a TC-treated plate, and the plate was left to stand overnight at 27°C to allow the cells to adhere. After that, the culture medium was aspirated using a dispenser, and 1-2% fish gelatin was added. The plate was then stored at 4°C. A cell adhesion plate was prepared in the same manner as in Test Example 2, except for the removal of the culture medium and the addition of fish gelatin. Using this cell adhesion plate, olfactory receptor activity was measured in the same manner as in Test Example 3. As a result, results showing the same trends as in Test Example 3 were obtained.

Claims

1. A cell substrate comprising a compartment having a surface with a molar ratio of oxygen atoms to carbon atoms (O / C) of 0.150 or more.

2. The cell substrate according to claim 1, wherein the molar ratio (O / C) of the surface is 0.195 or more.

3. The cell substrate according to claim 1, wherein the molar ratio (O / C) of the surface is 0.250 or less.

4. The cell substrate according to claim 1, wherein the surface has a molar ratio of nitrogen atoms to carbon atoms (N / C) of 0.020 or more.

5. The cell substrate according to claim 1, wherein the surface has a water contact angle of 60° or less.

6. The cell substrate according to claim 1, wherein the area of ​​said surface relative to 100% of said compartment area is 70% or more.

7. The cell substrate according to claim 1, wherein the surface material is a resin.

8. The area of ​​the compartment is 1 to 50 mm 2 The cell substrate according to claim 1 , 9. The cell substrate according to any one of claims 1 to 8, which is used to measure the activity of a sensor protein with cells disposed on the surface.

10. The cell substrate of any of claims 9, wherein the cells comprise a polynucleotide comprising a coding sequence for a sensor protein.

11. The cell substrate according to claim 9, wherein the sensor protein is an olfactory receptor protein.

12. The cell substrate according to claim 9, which is used to measure the activity of the sensor protein after solution replacement or addition with cells disposed on the surface.

13. The cell substrate according to any one of claims 1 to 8, wherein the cells are insect cells.

14. A method for measuring the activity of a sensor protein, comprising measuring the activity of the sensor protein of a cell disposed on a surface having a molar ratio of oxygen atoms to carbon atoms (O / C) of 0.150 or more.

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