Cell contact solution or gel thereof

Abstract

Provided is a cell contact solution or a gel thereof capable of improving the detectability of chemical substances such as odorants.　This cell contact solution or a gel thereof contains an unsaturated fatty acid.

Description

Cell contact solution or gel

[0001] The present invention relates to a solution for cell contact or a gel thereof.

[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, particularly the detectability of chemical substances after refrigerated storage of cells, differs depending on the type of component brought into contact with the cells.

[0006] Therefore, an object of the present disclosure is to provide a cell contact solution or gel thereof that can improve the detectability of chemical substances such as odorants.

[0007] As a result of intensive research conducted by the present inventors in view of the above-mentioned problems, they have found that the above-mentioned problems can be solved by a solution for cell contact or a gel thereof containing an unsaturated fatty acid. That is, the present disclosure includes the following aspects. That is, the present invention includes the following aspects.

[0008] Item 1. A solution for cell contact or a gel thereof containing an unsaturated fatty acid.

[0009] Item 2. The cell contact solution or gel thereof according to Item 1, wherein the concentration of the unsaturated fatty acid is 10 to 1000 μM.

[0010] Item 3. The cell contact solution or gel thereof according to Item 1, wherein the concentration of the unsaturated fatty acid is 50 μM or more.

[0011] Item 4. The cell contact solution or gel thereof according to Item 1, wherein the unsaturated fatty acid has a melting point of 0°C or lower.

[0012] Item 5. The cell contact solution or gel thereof according to Item 1, which contains a cell culture medium component.

[0013] Item 6. The cell contact solution or gel thereof according to Item 5, wherein the cell culture medium components include albumin.

[0014] Item 7. The solution for cell contact or a gel thereof according to any one of Items 1 to 6, which is a cell preservation solution, a cell treatment solution, or a cell culture medium.

[0015] Item 8. The cell contact solution or gel thereof according to Item 7, which is a cell maintenance medium.

[0016] Item 9. The cell contact solution or gel thereof according to any one of Items 1 to 6, which is used for contacting cells that are to be subjected to or have been subjected to refrigerated storage.

[0017] Item 10. The solution for cell contact or a gel thereof according to any one of Items 1 to 6, wherein the cells contain an exogenous polynucleotide comprising a coding sequence for a sensor protein.

[0018] Item 11. The cell contact solution or gel thereof according to Item 9, wherein the sensor protein is an olfactory receptor protein.

[0019] Item 12. A solution for cell contact or a gel thereof according to any one of Items 1 to 6, which is used for contacting cells to be used in measuring the activity of a sensor protein responding to a chemical substance.

[0020] Item 13. The solution for cell contact or a gel thereof according to any one of Items 1 to 6, wherein the cells are insect cells.

[0021] Item 14. A cell chip comprising a compartment containing cells and the cell contact solution or gel thereof according to any one of Items 1 to 6.

[0022] Item 15. A method for improving chemical response activity of a sensor protein in a cell, the method comprising contacting the cell with an unsaturated fatty acid.

[0023] Item 16. An agent for improving the chemical response activity of a cellular sensor protein, which contains an unsaturated fatty acid.

[0024] According to the present disclosure, it is possible to provide a cell contact solution or gel thereof that can improve the detectability of chemical substances such as odorants. In this specification, improving the detectability of a chemical substance not only means that the detectability of the chemical substance is higher than before contact with the cell contact solution or gel, but also includes suppressing the degree of decrease in detectability or maintaining the detectability when the detectability of the chemical substance tends to decrease.

[0025] Figure 1 shows the results of measuring olfactory receptor activity in Test Example 2. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the components added to the medium ((A) no addition (-), (B) 0.7% BSA (+BSA), (C) 0.7% BSA, 250 μM linoleic acid (+BSA + linoleic acid), (D) 0.7% BSA, 250 μM linolenic acid (+BSA + linolenic acid)). The legend indicates the concentration of compound a. Figure 1 shows the results of measuring olfactory receptor activity in Test Example 3. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the components added to the medium ((A) no addition (-), (B) 0.7% BSA, 250 μM linolenic acid (+linolenic acid)). The legend indicates the concentration of compound a (unit: μM). Figure 2 shows the results of measuring olfactory receptor activity in Test Example 4. The vertical axis indicates fluorescence intensity. The horizontal axis shows the components added to the medium ((A) none (-), (B) 0.7% BSA, 250 μM linoleic acid, 250 μM linolenic acid (+ fatty acid)) and the storage period. The legend shows the concentration of compound a (unit: μM).

[0026] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0027] In this specification, any combination of upper and lower limits of a certain numerical range is also directly and unambiguously disclosed.

[0028] In one aspect, the present disclosure relates to a solution for cell contact or a gel thereof (sometimes referred to herein as the "solution / gel of the present disclosure") containing an unsaturated fatty acid, which will be described below.

[0029] The solution / gel of the present disclosure is a liquid (the solution of the present disclosure) or a gel (the gel of the present disclosure). The gel of the present disclosure is obtained by gelling the solution of the present disclosure.

[0030] Unsaturated fatty acids are fatty acids with one or more unsaturated carbon bonds and have the general formula (1):

[0031]

[0032] [In the formula: R 1 represents a chain unsaturated hydrocarbon group.]

[0033] The number of carbon atoms in the chain unsaturated hydrocarbon group of the unsaturated fatty acid is not particularly limited and is, for example, 3 to 29. From the viewpoint of the effect of the unsaturated fatty acid in improving the detectability of chemical substances, the number of carbon atoms is preferably 7 or more, more preferably 11 or more, even more preferably 13 or more, still more preferably 15 or more, particularly preferably 16 or more, and is preferably 27 or less, more preferably 25 or less, even more preferably 23 or less, still more preferably 21 or less, particularly preferably 19 or less.

[0034] The chain unsaturated hydrocarbon group may be either a straight chain or a branched chain, but is preferably a straight chain from the viewpoint of the effect of improving the detectability of chemical substances by the unsaturated fatty acid.

[0035] From the viewpoint of improving the detectability of chemical substances, it is particularly preferable that the unsaturated fatty acid has a melting point of 0°C or lower and / or has two or more double bonds between carbon atoms. The melting point is preferably −1°C or lower, more preferably −2°C or lower, even more preferably −3°C or lower, even more preferably −4°C or lower, and particularly preferably −5°C or lower. The number of double bonds in the unsaturated fatty acid is preferably 2 to 6, more preferably 2 to 5, even more preferably 2 to 4, and even more preferably 2 to 3.

[0036] Specific examples of unsaturated fatty acids include the following substances:

[0037] Monounsaturated fatty acids: crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid; diunsaturated fatty acids: linoleic acid, eicosadienoic acid, docosadienoic acid; triunsaturated fatty acids: linolenic acid, pinolenic acid, eleostearic acid, mead acid, dihomo-γ-linolenic acid, eicosatrienoic acid; tetraunsaturated fatty acids: stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid; pentaunsaturated fatty acids: bosseopentaenoic acid, eicosapentaenoic acid, osbondoic acid, sardine acid, tetracosapentaenoic acid; hexaunsaturated fatty acids: docosahexaenoic acid, herring acid.

[0038] The concentration of the unsaturated fatty acid in the solution / gel of the present disclosure is not particularly limited and can be, for example, 10 μM or higher. From the viewpoint of the unsaturated fatty acid's effect of improving the detectability of chemical substances, the concentration is preferably 20 μM or higher, more preferably 30 μM or higher, even more preferably 50 μM or higher, even more preferably 70 μM or higher, particularly preferably 100 μM or higher, especially more preferably 150 μM or higher, especially more preferably 200 μM or higher, and especially even more preferably 220 μM or higher. The concentration is, for example, 2000 μM or lower, 1500 μM or lower, 1000 μM or lower, 500 μM or lower, 400 μM or lower, or 300 μM or lower.

[0039] The unsaturated fatty acids may be one type alone or two or more types in combination.

[0040] The solution / gel of the present disclosure preferably contains a cell culture medium component from the viewpoint of reducing damage to cells (particularly damage during storage). The cell culture medium component is not particularly limited as long as it is contained in a cell culture medium.

[0041] The medium usually contains a carbon source and inorganic salts that can be assimilated by the cells, and may also contain a nitrogen source.

[0042] Any carbon source that can be utilized by the cells may be used, for example, carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch, and starch hydrolysates; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.

[0043] Examples of nitrogen sources that can be used include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria, and digests thereof.

[0044] Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0045] More specific examples of media for culturing insect cells include TNM-FH medium (Pharmingen), Sf-900 III SFM medium (Life Technologies), ExCell400, ExCell405 (both JRH Biosciences), and Grace's Insect Medium (Nature, 195, 788 (1962)).

[0046] The solution / gel of the present disclosure preferably contains albumin to improve the solubility of unsaturated fatty acids. Examples of albumin include serum albumin, egg albumin, and milk albumin. A typical albumin used is bovine serum albumin (BSA).

[0047] The albumin concentration in the solution / gel of the present disclosure is preferably 0.01 to 5% by mass, more preferably 0.05 to 3% by mass, even more preferably 0.1 to 2% by mass, and even more preferably 0.2 to 1.5% by mass, from the viewpoint of the solubility of unsaturated fatty acids, etc.

[0048] The cell culture medium components may be one type alone or a combination of two or more types.

[0049] The solvent for the solution / gel of the present disclosure is not particularly limited as long as it does not significantly adversely affect cell viability. The solvent preferably contains water. The water content in the solvent is preferably 50% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, even more preferably 85% by mass or more, particularly preferably 90% by mass or more, and particularly preferably 95% by mass or more.

[0050] The gel of the present disclosure is preferably one that can be converted into a sol. By converting into a sol, high diffusibility of the detection target substance is ensured, which is thought to enable rapid and stable measurement. Furthermore, by converting into a sol, the detection target substance can be easily removed, and it can be replaced with a solution that has better diffusibility of the detection target substance.

[0051] The component that forms a gel that can be converted into a sol (gel-forming component: a component that forms a network by crosslinking) is not particularly limited, but examples include gelatin; polysaccharides such as agar, carrageenan, starch, and xanthan gum; and water-soluble synthetic polymers such as PVA and PEG. Among these, gelatin is particularly preferred because it is easy to develop the desired properties described above. Gelatin can be obtained by pretreating collagen raw materials such as cow bone, cowhide, pig bone, pig skin, and fish scales with acid or alkali, followed by washing with water, hot water extraction, purification, and concentration and drying. The sol and gel properties of gelatin vary depending on the type of collagen raw material, pretreatment, hot water extraction conditions, and other factors. To achieve the desired sol temperature, sol-gel transition temperature, sol viscosity, and gel modulus for the present invention, fish-derived gelatin (fish gelatin) is particularly preferred. By appropriately adjusting the pretreatment and hot water extraction conditions, the sol and gel properties suitable for the present invention can be obtained.

[0052] The gel-forming component may be one kind alone or a combination of two or more kinds.

[0053] The solution / gel of the present disclosure is used for contact with cells. Although not wishing to limit the interpretation, it is believed that the unsaturated fatty acid comes into contact with the sensor protein on the cell surface and / or is incorporated as a cell membrane component, thereby stabilizing the structure of the sensor protein and thereby improving the detectability of chemical substances.

[0054] The manner of contact is not particularly limited, and examples include a manner in which the solution / gel of the present disclosure is in contact with a cell layer, and a manner in which cells are dispersed in the solution / gel of the present disclosure.

[0055] More specifically, the solution / gel of the present disclosure can be, for example, a cell preservation solution, a cell treatment solution, or a cell culture medium.

[0056] In one embodiment, the cell preservation solution is for use in refrigerated storage. The temperature for refrigerated storage is, for example, 10°C or lower, or 6°C or lower. The period of refrigerated storage can be, for example, 5 days or more, 10 days or more, 15 days or more, 20 days or more, 25 days or more, 30 days or more, 40 days or more, 50 days or more, 60 days or more, or 70 days or more, and can be 200 days or less, 150 days or less, or 100 days or less. After refrigerated storage, the cells can be cultured at a predetermined temperature (e.g., 20°C or 25°C) or higher.

[0057] The cell treatment solution is a solution for transient treatment of cells, and is not particularly limited thereto. The treatment time can be, for example, 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, or 8 hours or more, and can be 16 hours or less, or 12 hours or less.

[0058] In one embodiment, the cell culture medium is a cell maintenance medium. The cell maintenance medium is not particularly limited as long as it does not require cell proliferation and is intended to keep cells alive.

[0059] In one embodiment, the solution / gel of the present disclosure is used for contacting cells that are subjected to or have been subjected to refrigerated storage. Refrigerated storage tends to reduce the detectability of chemical substances in cells, but contacting cells with the solution / gel of the present disclosure can suppress this reduction and can also increase the detectability of chemical substances compared to before refrigerated storage.

[0060] In one embodiment, the solution / gel of the present disclosure is used to contact cells that are to be used to measure the chemical response activity of a sensor protein.

[0061] 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 use in the solution / gel of the present disclosure and ease of management, such as the absence of CO2 or temperature management.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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).

[0071] 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).

[0072] 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.

[0073] 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.

[0074] 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.

[0075] The amino acid mutation is, for example, an amino acid substitution, insertion, addition, or deletion, preferably a substitution, and particularly preferably a conservative substitution.

[0076] 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.

[0077] 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, resulting in 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 fluorescence or luminescence from the cells is measured. The greater the measured amount of fluorescence or 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.

[0078] 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.

[0079] 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.

[0080] 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.).

[0081] 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.

[0082] 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).

[0083] 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).

[0084] In one aspect, the present invention relates to a cell chip (sometimes referred to herein as the "cell chip of the present disclosure") that includes compartments containing cells and the solution / gel of the present disclosure, which can improve the detectability of chemical substances of cells in the chip.

[0085] The shape of the compartment is not particularly limited as long as it is capable of retaining cells. From the viewpoint of cell retention, production efficiency, and chemical substance detection, the compartment is preferably in the form of a well.

[0086] The material of the compartment is not particularly limited as long as it can hold cells, and can be, for example, resin, metal, etc.

[0087] 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 mm 2 Below, 35mm 2 or less, or 15 mm 2 The following is the result.

[0088] From the viewpoint of detection sensitivity or production efficiency, the number of compartments 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.

[0089] 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.

[0090] The cell chip of the present disclosure may contain only cells of one type of sensor protein, but preferably contains 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 cells with different types of sensor proteins.

[0091] Chemical substances can be detected by measuring the chemical response 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 in 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 in 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 chemical response activity of the sensor protein can be measured, and chemical substances can be detected.

[0092] As described above, unsaturated fatty acids can improve the chemical response activity of a cellular sensor protein by contacting the cells with the unsaturated fatty acids. Therefore, in one aspect, the present invention relates to a method for improving the chemical response activity of a cellular sensor protein, which comprises contacting the cells with the unsaturated fatty acids; an agent for improving the chemical response activity of a cellular sensor protein, which contains the unsaturated fatty acids; and the like.

[0093] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0094] 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.

[0095] Test Example 2. Measurement of olfactory receptor activity 1 ORA cells were placed in a 96-well plate (CORNING 3903) at 5 x 10 4 Cells were seeded at 100 μL per well and placed in a 27°C incubator (without CO2 supply). Sf-900III SFM medium was used. 24 hours after seeding, the medium was removed from each well, and 100 μL of each of the following solutions (concentrations indicated are final concentrations): (A) Sf-900III SFM medium (-), or Sf-900III SFM medium supplemented with (B) 0.7% BSA (Fujifilm Wako 011-15144) (+BSA), (C) 0.7% BSA and 250 μM linoleic acid (+BSA, +linoleic acid), or (D) 0.7% BSA and 250 μM linolenic acid (+BSA, +linolenic acid) was added to each well. The cells were stored at 4°C for 29 days and then cultured at 27°C for 2 days. The medium was then removed from each well, and 80 μL of 0.1% BSA / 1× Hanks' buffer / 20 mM HEPES buffer was added to each well. Compound a (a substance to which ORA responds) solution or a solution identical to the compound a solution but without compound a was added to each well to final concentrations of 0, 0.1, 1, or 10 μM. The changes in fluorescence intensity before and after addition were measured using a microplate reader (FlexStation3, Molecular Devices).

[0096] The results are shown in Figure 1. Compared to the control groups (A) and (B), the odor response signals at each concentration were enhanced in the unsaturated fatty acid-added groups (C) and (D).

[0097] Test Example 3. Olfactory receptor activity measurement 2 ORA cells were plated in a 384-well plate (Greiner, 781098) at 3 × 10 4The cells were seeded at 40 μL per well and placed in a 27°C incubator (without CO2 supply). Sf-900III SFM medium was used. 24 hours after seeding, the medium was removed from each well, and 40 μL of (A) Sf-900III SFM medium containing 1% fish gelatin (-) or (B) the same medium supplemented with 0.7% BSA (Fujifilm Wako 011-15144) and 250 μM linolenic acid (+linolenic acid) (concentrations indicate final concentrations) was added to each well. The cells were stored at 4°C for 14 days and then cultured at 27°C for 3 days. The medium was then removed from each well, and 40 μL of 0.1% BSA / 1× Hanks' buffer / 20 mM HEPES buffer was added to each well. 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 give final concentrations 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).

[0098] The results are shown in Figure 2. Compared to the control group (A), the odor response signal was enhanced at each concentration in the unsaturated fatty acid-added group (B).

[0099] Test Example 4. Measurement of Olfactory Receptor Activity 3 ORA cells were cultured in a 96-well plate (CORNING 3903) at 5 x 10 4Cells were seeded at 100 μL per well and placed in a 27°C incubator (without CO2 supply). Sf-900III SFM medium was used. 24 hours after seeding, the medium was removed from each well, and 100 μL of either (A) Sf-900III SFM medium (-) or (B) Sf-900III SFM medium supplemented with 0.7% BSA (Fujifilm Wako 011-15144), 125 μM linoleic acid, and 125 μM linolenic acid (+ fatty acids) (concentrations indicated are final concentrations) was added to each well. After storage at 4°C for 27, 48, or 75 days, the cells were cultured at 27°C for 2 days. The medium was then removed from each well, and 80 μL of 0.1% BSA / 1× Hanks' buffer / 20 mM HEPES buffer was added to each well. Compound a (a substance to which ORA responds) was added to each well to final concentrations of 0, 0.1, 1, 10, and 100 μM, and the change in fluorescence intensity before and after the addition was measured using a microplate reader (FlexStation3, Molecular Devices).

[0100] The results are shown in Figure 3. Compared to the control group (A), the odor response signal was enhanced at each concentration in the unsaturated fatty acid-added group (B).

Claims

1. A solution or gel for cell contact containing unsaturated fatty acids.

2. The cell contact solution or gel thereof according to claim 1, wherein the concentration of the unsaturated fatty acid is 10 to 1000 μM.

3. The cell contact solution or gel thereof according to claim 1, wherein the concentration of the unsaturated fatty acid is 50 μM or more.

4. The cell contact solution or gel thereof according to claim 1, wherein the melting point of the unsaturated fatty acid is 0°C or lower.

5. The cell contact solution or gel thereof according to claim 1, which contains a cell culture medium component.

6. The cell contact solution or gel thereof according to claim 5, wherein the cell culture medium components include albumin.

7. The cell contact solution or gel thereof according to any one of claims 1 to 6, which is a cell preservation solution, a cell treatment solution, or a cell culture medium.

8. The cell contact solution or gel thereof according to claim 7, which is a cell maintenance medium.

9. A solution or gel thereof for cell contact according to any one of claims 1 to 6, which is used for contacting cells that are to be subjected to refrigerated storage or that have been subjected to refrigerated storage.

10. The solution or gel thereof for cell contact according to any one of claims 1 to 6, wherein the cells contain an exogenous polynucleotide comprising a coding sequence for a sensor protein.

11. The cell contact solution or gel thereof according to claim 9, wherein the sensor protein is an olfactory receptor protein.

12. A cell contact solution or gel thereof according to any one of claims 1 to 6, which is used for contacting cells to be subjected to measurement of chemical response activity of a sensor protein.

13. The solution or gel thereof for cell contact according to any one of claims 1 to 6, wherein the cells are insect cells.

14. A cell chip comprising a compartment containing cells and the cell contact solution or gel thereof according to any one of claims 1 to 6.

15. A method for improving a chemical response activity of a sensor protein in a cell, comprising contacting the cell with an unsaturated fatty acid.

16. An agent for enhancing the chemical response activity of a cellular sensor protein, which contains unsaturated fatty acids.

Images