Cell chip

Abstract

Provided is a technique capable of improving the detectability of a chemical substance such as a smell substance when cells are stored under refrigeration.　Specifically provided is a cell chip for use in storage under refrigeration associated with recovery culture, the cell chip containing cells.

Description

Cell chip

[0001] The present invention relates to a cell chip 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, which involve the artificial preparation of lipid bilayer membranes containing sensor proteins such as olfactory receptors, are not necessarily manufactured efficiently, and further improvements in the manufacturing efficiency of odor sensors are needed. Therefore, we focused on using cells that express sensor proteins.

[0006] When using cells as chemical sensors such as odor sensors, from the viewpoint of ease of use, it is desirable to prepare cells in advance in a container or the like and use them when needed, rather than culturing and preparing the cells each time. In the latter case, the cells must be stored for a certain period of time, and it is particularly important to store them in a refrigerator to prevent the cells from drying out. However, as the inventors continued their research, they found that storing cells in a refrigerator reduces their ability to detect chemical substances such as odorants.

[0007] Therefore, an object of the present disclosure is to provide a technology that can improve the detectability of chemical substances such as odorous substances when cells are stored in a refrigerator.

[0008] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that refrigerated storage of cells with recovery culture improves the detectability of chemical substances such as odorants compared to refrigerated storage without recovery culture. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0009] Item 1. A cell chip for use in refrigerated storage with recovery culture, comprising a compartment containing cells.

[0010] Item 2. The cell chip according to Item 1, wherein the refrigerated storage temperature is 10°C or lower.

[0011] Item 3. The cell chip according to Item 1, wherein the temperature of the recovery culture is 12°C or higher.

[0012] Item 4. The cell chip according to Item 1, wherein the continuous period of refrigerated storage is 3 days or more.

[0013] Item 5. The cell chip according to Item 1, wherein the continuous period of the recovery culture is from 1 day to 14 days.

[0014] Item 6. The cell chip according to Item 1, wherein the recovery culture is carried out after completion of refrigerated storage and / or during refrigerated storage.

[0015] Item 7. The cell chip according to Item 6, wherein the recovery culture is performed only after completion of refrigerated storage, and the continuous period of refrigerated storage is 3 days or more and 100 days or less, and / or the recovery culture is performed during refrigerated storage and after completion of refrigerated storage, and the continuous period of refrigerated storage before and after the interruption of refrigerated storage by the recovery culture during refrigerated storage is 3 days or more and 100 days or less.

[0016] Item 8. The cell chip according to any one of Items 1 to 7, wherein the cells contain an exogenous polynucleotide comprising a coding sequence for a sensor protein.

[0017] Item 9. The cell chip according to Item 8, wherein the sensor protein is an olfactory receptor protein.

[0018] Item 10. The cell chip according to Item 8, which is used for measuring the chemical substance response activity of the sensor protein.

[0019] Item 11. The cell chip according to any one of Items 1 to 7, wherein the cells are insect cells.

[0020] Item 12. A composition containing cells for use in refrigerated storage with recovery culture.

[0021] Item 13. A method for preserving cells, comprising subjecting the cells to refrigerated preservation followed by recovery culture.

[0022] Item 14. A method for measuring the chemical response activity of a sensor protein, comprising subjecting cells containing an exogenous polynucleotide comprising a coding sequence for the sensor protein to refrigerated storage followed by recovery culture.

[0023] According to the present disclosure, it is possible to provide a technology that can improve the detectability of chemical substances such as odorants when cells are stored in a refrigerator. Specifically, it is possible to provide a cell chip, a cell composition, a cell storage method, a method for measuring the chemical response activity of a sensor protein, etc.

[0024] 1 shows the results of measuring olfactory receptor activity in Test Example 2. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the storage period at 4°C, and if recovery culture is performed at 27°C after storage, this period is also indicated. The legend indicates the concentration of compound a. 1 shows the results of measuring olfactory receptor activity in Test Example 3. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the period of recovery culture at 27°C after 56 days of storage at 4°C. The legend indicates the concentration of compound a. 1 shows the results of measuring olfactory receptor activity in Test Example 4. The vertical axis indicates fluorescence intensity. The legend indicates the concentration of compound a. 1 shows the results of measuring olfactory receptor activity in Test Example 5, where refrigerated storage was performed for 49 days. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the temperature and period of recovery culture. The legend indicates the concentration of compound a. 1 shows the results of measuring olfactory receptor activity in Test Example 5, where refrigerated storage was performed for 56 days. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the temperature and period of recovery culture. The legend indicates the concentration of compound a.

[0025] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0026] In this specification, any combination of upper and lower limits of a certain numerical range is also directly and unambiguously disclosed.

[0027] In one aspect, the present disclosure relates to a cell chip containing cells for use in refrigerated storage with recovery culture (also referred to herein as the "cell chip of the present invention"). This will be described below.

[0028] Refrigerated storage is storage at a low temperature at which the cells or a composition containing the cells do not freeze, and is not particularly limited as long as this temperature is maintained. The temperature for refrigerated storage is, for example, 10°C or lower, and from the viewpoint of storage efficiency, is preferably 9°C or lower, more preferably 8°C or lower, even more preferably 7°C or lower, even more preferably 6°C or lower, and particularly preferably 5°C or lower. The temperature for refrigerated storage is, for example, 0°C or higher, 1°C or higher, or 2°C or higher.

[0029] As described below, recovery culture may be performed during the refrigerated storage period. In this case, the refrigerated storage period is interrupted midway. In this specification, the period of refrigerated storage that is not interrupted by recovery culture is referred to as the "continuous period," and the sum of the continuous periods before and after the middle period when the period is interrupted by recovery culture is referred to as the "total period."

[0030] The continuous period of refrigerated storage is not particularly limited and is, for example, one day or more. Even if the period of refrigerated storage is short enough that a decrease in detectability of chemical substances cannot be confirmed, it is possible to improve the detectability of chemical substances by performing recovery culture. From the viewpoint that the decrease in detectability of chemical substances due to refrigerated storage becomes more significant and the need for improving the detectability of chemical substances by recovery culture becomes greater, the continuous period of refrigerated storage is preferably three days or more, more preferably four days or more, even more preferably five days or more, still more preferably ten days or more, particularly preferably twenty days or more, particularly more preferably thirty days or more, and particularly more preferably forty days or more. From the viewpoint of the effect of improving the detectability of chemical substances by recovery culture, the continuous period of refrigerated storage is preferably 100 days or less, more preferably 80 days or less, even more preferably 65 days or less, and even more preferably 50 days or less.

[0031] The lower limit of the total refrigerated storage period is the same as the lower limit of the continuous period. On the other hand, by performing recovery culture during refrigerated storage (i.e., by keeping the continuous refrigerated storage period relatively short), refrigerated storage for a longer period is possible. From this perspective, the total refrigerated storage period is, for example, 400 days or less, preferably 300 days or less, more preferably 200 days or less, even more preferably 150 days or less, and even more preferably 100 days or less.

[0032] The method of refrigerated storage is not particularly limited, and the cells can be stored in a state where the temperature of the cells is adjusted to be equal to or lower than a predetermined temperature. Specifically, for example, the cells can be stored in a refrigerator where the temperature inside the refrigerator is adjusted to be equal to or lower than a predetermined temperature.

[0033] Recovery culture is culture at a temperature above refrigeration temperature, and is not particularly limited thereto. The temperature of recovery culture is, for example, 12°C or higher. The temperature of recovery culture can be adjusted depending on the optimal culture temperature for the cells, and is, for example, −15°C or higher, preferably −13°C or higher, more preferably −11°C or higher, even more preferably −9°C or higher, even more preferably −7°C or higher, particularly preferably −5°C or higher, especially more preferably −3°C or higher, especially even more preferably −2°C or higher from the optimal culture temperature. It is also, for example, +5°C or lower, +3°C or lower, or +2°C or lower from the optimal culture temperature. Furthermore, particularly when insect cells are used, the temperature of recovery culture is preferably 14°C or higher, more preferably 16°C or higher, even more preferably 18°C ​​or higher, even more preferably 20°C or higher, especially preferably 22°C or higher, especially more preferably 24°C or higher, especially even more preferably 25°C or higher, or, for example, 32°C or lower, 30°C or lower, or 29°C or lower. From the viewpoint of being more suitable for improving the detectability of chemical substances by recovery culture (particularly by recovery culture for a relatively short period), it is preferable to set the temperature of recovery culture higher and closer to the optimum culture temperature.

[0034] On the other hand, in one embodiment of the present invention, from the viewpoint of being more suitable for improving the detectability of chemical substances through a relatively long recovery culture, it is preferable to lower the temperature of the recovery culture. In this case, the temperature of the recovery culture is preferably −3°C or lower, more preferably −5°C or lower, even more preferably −7°C or lower, even more preferably −9°C or lower, and particularly preferably −11°C or lower from the optimal culture temperature. Similarly, particularly when insect cells are used, the temperature of the recovery culture is preferably 24°C or lower, more preferably 22°C or lower, even more preferably 20°C or lower, even more preferably 18°C ​​or lower, and particularly preferably 16°C or lower.

[0035] The timing of recovery culture may be any timing after the start of refrigerated storage. That is, recovery culture is performed after the end of refrigerated storage and / or during refrigerated storage. From the viewpoint of being able to provide for chemical substance detection in a state where the effect of recovery culture in improving the detectability of chemical substances is not impaired, recovery culture is preferably performed at least after the end of refrigerated storage, and can optionally also be performed during refrigerated storage.

[0036] The number of times of recovery culture is not particularly limited as long as it is one or more times, and is, for example, 10 times or less, 5 times or less, 3 times or less, or 2 times or less.

[0037] The duration of each recovery culture is not particularly limited as long as it is a period that can improve the detectability of chemical substances, and can be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more, and can also be, for example, 14 days or less, 12 days or less, 10 days or less, 8 days or less, 6 days or less, 5 days or less, 4 days or less, or 3 days or less. When the temperature of the recovery culture is increased and closer to the optimum culture temperature, the recovery culture period is preferably 1 day or more and 6 days or less, more preferably 1 day or more and 5 days or less, even more preferably 1 day or more and 4 days or less, even more preferably 1 day or more and 3 days or less, and particularly preferably 2 days or more and 3 days or less. On the other hand, when the temperature of the recovery culture is decreased, the recovery culture period is preferably 2 days or more and 14 days or less, more preferably 3 days or more and 14 days or less, even more preferably 4 days or more and 12 days or less, even more preferably 5 days or more and 12 days or less, particularly preferably 6 days or more and 10 days or less, and especially preferably 6 days or more and 8 days or less.

[0038] The recovery culture method is not particularly limited and can be carried out by incubating in an appropriate medium at a predetermined temperature. Specifically, for example, the culture can be carried out by incubating in a thermostatic device whose interior is adjusted to a predetermined temperature.

[0039] In one aspect of the present invention, from the viewpoint of more effectively improving the detectability of chemical substances, it is preferable that the recovery culture is performed only after the end of refrigerated storage, and that the continuous period of refrigerated storage is 3 days or more and 100 days or less, and / or that the recovery culture is performed during refrigerated storage and after the end of refrigerated storage, and that the continuous period of refrigerated storage before and after the interruption of refrigerated storage by the recovery culture during refrigerated storage is 3 days or more and 100 days or less.

[0040] The compartments of the cell chip of the present invention preferably contain a culture medium together with cells. This can further reduce damage to cells during refrigerated storage, and the transition from refrigerated storage to recovery culture and from recovery culture to refrigerated storage can be achieved simply by changing the temperature (either within the same chamber or by transferring to another chamber with a different temperature). When a compartment contains a culture medium, the compartments may be in a state where the cells are dispersed in the culture medium, or a cell layer is formed in the culture medium, for example.

[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 include, in the case of insect culture media, 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 medium solvent 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 (especially 100% by mass).

[0047] The medium may be in liquid or gel form.

[0048] The gel 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, allowing it to be replaced with a solution that has better diffusibility of the detection target substance.

[0049] 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.

[0050] The gel-forming component may be one kind alone or a combination of two or more kinds.

[0051] 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 refrigerated storage involving recovery culture and ease of management, such as the absence of CO2 or temperature control.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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).

[0061] 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).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The amino acid mutation is, for example, an amino acid substitution, insertion, addition, or deletion, preferably a substitution, and particularly preferably a conservative substitution.

[0066] 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.

[0067] 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 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.

[0068] 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.

[0069] 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.

[0070] 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.).

[0071] 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.

[0072] 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).

[0073] 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).

[0074] 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.

[0075] The material of the compartment is not particularly limited as long as it can hold cells, and can be, for example, resin, metal, etc.

[0076] 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, 35mm2 or less, or 15 mm 2 The following is the result.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] As described above, it has been found that subjecting cells to refrigerated storage with recovery culture improves the detectability of chemical substances such as odorants compared to refrigerated storage without recovery culture. Therefore, the present disclosure also relates to the following aspects as other aspects: (A) A composition for use in refrigerated storage with recovery culture, comprising cells; (B) A method for preserving cells, comprising subjecting cells to refrigerated storage with recovery culture; and (C) A method for measuring the chemical response activity of a sensor protein, comprising subjecting cells containing an exogenous polynucleotide including a coding sequence for the sensor protein to refrigerated storage with recovery culture.

[0082] For embodiments (A) to (C), the description regarding the cell chip of the present disclosure can be cited.

[0083] In embodiment (A), the composition is preferably contained in a container. The container is not particularly limited as long as it is suitable for culturing or storing cells, in addition to the cell chip described above.

[0084] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0085] 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.

[0086] Test Example 2. Olfactory receptor activity measurement 1 ORA cells were placed in a 96-well plate CORNING3903 at 5 x 10 4 The plates were seeded at 100 μL / well and placed in a 27°C incubator (without CO2 supply). Sf-900III SFM medium was used. 24 hours after seeding, the plates were transferred to 4°C and subjected to one of the following treatments (1) to (8): (1) storage at 4°C for 3 hours, (2) storage at 4°C for 2 days, (3) storage at 4°C for 5 days, (4) storage at 4°C for 12 days, (5) storage at 4°C for 14 days, (6) storage at 4°C for 12 days followed by incubation at 27°C for 2 days, (7) storage at 4°C for 31 days, or (8) storage at 4°C for 29 days followed by incubation at 27°C for 2 days.

[0087] After the treatments (1) to (8) above, the solution was 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 change in fluorescence intensity before and after addition was measured using a microplate reader (FlexStation3, Molecular Devices).

[0088] The results are shown in Figure 1. It was found that refrigerated storage reduced the detectability of chemical substances, and that recovery culture after refrigerated storage improved the detectability of chemical substances.

[0089] Test Example 3. Olfactory receptor activity measurement 2 ORA cells were placed in a 96-well plate CORNING3903 at 5 x 10 4 The plates were seeded at 100 μL / well and placed in a 27°C incubator (without CO2 supply). Sf-900III SFM medium was used. 24 hours after seeding, the plates were transferred to 4°C and subjected to one of the following treatments (1) to (3): (1) storage at 4°C for 56 days followed by culture at 27°C for 2 days, (2) storage at 4°C for 56 days followed by culture at 27°C for 3 days, or (3) storage at 4°C for 56 days followed by culture at 27°C for 8 days.

[0090] After the treatments (1) to (3) above, the solution was 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 change in fluorescence intensity before and after addition was measured using a microplate reader (FlexStation3, Molecular Devices).

[0091] The results are shown in Figure 2. Comparing this with the data for 31 days of refrigerated storage in Figure 1, it was found that the effects of recovery culture were exhibited even after long-term refrigerated storage.

[0092] Test Example 4. Olfactory receptor activity measurement 3 ORA cells were placed in a 96-well plate CORNING3903 at 5 x 10 4 The plates were seeded at 100 μL / well and placed in a 27°C incubator (without CO2 supply). Sf-900III SFM medium was used. 24 hours after seeding, the plates were transferred to 4°C and treated as follows: (1) stored at 4°C for 28 days, cultured at 27°C for 2 days, stored at 4°C for 42 days, and cultured at 27°C for 2 days.

[0093] After the treatment in (1) above, the solution was 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).

[0094] The results are shown in Figure 3. Compared with the data for 31 days of refrigerated storage in Figure 1, it was found that the effects of recovery culture were demonstrated even in the case of a very long refrigerated storage period of 70 days in total. It is believed that longer-term refrigerated storage is possible by performing recovery culture during the period.

[0095] Test Example 5. Measurement of Olfactory Receptor Activity 4 ORA cells were placed in a 96-well plate CORNING3903 at 5 x 10 4 The plates were seeded at 100 μL / well and placed in a 27°C incubator (without CO2 supply). Sf-900III SFM medium was used. 24 hours after seeding, the plates were transferred to 4°C and treated with the following steps (1) to (15). (1) Stored at 4°C for 49 days, (2) Stored at 4°C for 49 days and then cultured at 27°C for 2 days, (3) Stored at 4°C for 49 days and then cultured at 27°C for 3 days, (4) Stored at 4°C for 49 days and then cultured at 15°C for 4 days, (5) Stored at 4°C for 49 days and then cultured at 15°C for 5 days, (6) Stored at 4°C for 49 days and then cultured at 15°C for 6 days, (7) Stored at 4°C for 49 days and then cultured at 15°C for 7 days, (8) Stored at 4°C for 56 days, (9) Stored at 4°C for 56 days and then cultured at 27°C for 2 days, (10) Stored at 4°C for 56 days and then cultured at 27°C for 3 days, (11) Stored at 4°C for 56 days and then cultured at 20°C for 2 days, (12) Stored at 4°C for 56 days and then cultured at 20°C for 3 days, (13) Store at 4°C for 56 days and then culture at 20°C for 4 days, (14) Store at 4°C for 56 days and then culture at 20°C for 6 days, or (15) Store at 4°C for 56 days and then culture at 20°C for 7 days.

[0096] After the treatments (1) to (15) above, the solution was 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).

[0097] The results for treatments (1) to (7) are shown in Figure 4, and the results for treatments (8) to (15) are shown in Figure 5. It was found that the effect of recovery culture was exerted even at temperatures lower than the optimal culture temperature.

Claims

1. A cell chip for use in refrigerated storage with recovery culture, comprising a cell-containing compartment.

2. The cell chip according to claim 1, wherein the refrigerated storage temperature is 10°C or lower.

3. The cell chip according to claim 1, wherein the temperature of the recovery culture is 12°C or higher.

4. The cell chip of claim 1, wherein the continuous period of refrigerated storage is 3 days or longer.

5. The cell chip according to claim 1, wherein the continuous period of the recovery culture is from 1 day to 14 days.

6. The cell chip according to claim 1, wherein the recovery culture is carried out after completion of refrigerated storage and / or during refrigerated storage.

7. The cell chip according to claim 6, wherein the recovery culture is carried out only after the end of refrigerated storage, and the continuous period of the refrigerated storage is from 3 days to 100 days, and / or the recovery culture is carried out during and after the end of refrigerated storage, and the continuous period of refrigerated storage before and after the interruption of the recovery culture during refrigerated storage is from 3 days to 100 days.

8. The cell chip according to any one of claims 1 to 7, wherein the cells contain an exogenous polynucleotide comprising a coding sequence for a sensor protein.

9. The cell chip according to claim 8, wherein the sensor protein is an olfactory receptor protein.

10. The cell chip according to claim 8, for use in measuring the chemical response activity of the sensor protein.

11. The cell chip according to any one of claims 1 to 7, wherein the cells are insect cells.

12. A composition comprising cells for use in refrigerated storage with recovery culture.

13. A method for preserving cells, comprising subjecting the cells to refrigerated preservation with recovery culture.

14. A method for measuring a chemical response activity of a sensor protein, comprising subjecting cells containing an exogenous polynucleotide comprising a coding sequence for the sensor protein to refrigerated storage with recovery culture.

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