Cell contact solution or gel thereof

Abstract

Provided is a cell contact solution or a gel thereof capable of improving the detection of a chemical substance such as an odor substance. The osmotic pressure of the cell contact solution or the gel thereof is 340 mOsm / kg or less.

Description

Technical Field

[0001] This invention relates to solutions or gels for cell contact. Background Technology

[0002] A group of odor substances characterizing specific human diseases and mental states has been identified. Due to their high value as diagnostic markers, the development of various odor sensors targeting these substances has become prevalent. Because biological olfactory receptors possess superior characteristics in terms of diversity, sensitivity, and selectivity compared to previous odor sensor elements such as semiconductors, there is a promising prospect for developing new odor sensors that utilize olfactory receptors as sensor elements.

[0003] Patent document 1 discloses the use of cells that express altered olfactory receptors and lipid double membranes that possess altered olfactory receptors as odor sensors.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: International Publication No. 2022 / 024902 Summary of the Invention

[0007] The problem that the invention aims to solve

[0008] Odor sensors that involve the artificial preparation of lipid double membranes containing sensor proteins such as olfactory receptors may not be sufficiently efficient, necessitating further improvements in preparation efficiency. Therefore, the focus is on utilizing cells expressing sensor proteins. Based on this, the inventors discovered that the detectability of odor substances and other chemical substances varies depending on the physical properties of the liquid in contact with the cells, particularly the detectability of chemical substances after cold storage of the cells.

[0009] Therefore, the subject of this disclosure is to provide a cell contact solution or gel thereof that can improve the detectability of chemical substances such as odorants.

[0010] Methods for solving problems

[0011] The inventors, through in-depth research into the aforementioned problems, discovered that the problems can be solved by using a cell contact solution or gel with an osmotic pressure of 340 mOsm / kg or less. That is, the present invention comprises the following methods.

[0012] Project 1. A solution or gel for cell contact, with an osmotic pressure of less than 340 mOsm / kg.

[0013] Item 2. The cell contact solution or gel described in Item 1, wherein the osmotic pressure is below 300 mOsm / kg.

[0014] Item 3. The cell contact solution or gel described in Item 1, wherein the osmotic pressure is 210–300 mOsm / kg.

[0015] Item 4. The cell contact solution or gel described in Item 1, wherein the aforementioned cells are non-mammalian cells.

[0016] Item 5. The cell contact solution or gel described in Item 1, wherein the aforementioned cells are insect cells.

[0017] Item 6. The cell contact solution or gel described in Item 1, which contains cell culture medium components.

[0018] Item 7. The cell contact solution or gel described in any of Items 1 to 6, which is a cell preservation solution, cell treatment solution or cell culture medium.

[0019] Item 8. The cell contact solution or gel described in Item 7, which is a cell maintenance culture medium or a cell proliferation culture medium.

[0020] Item 9. The cell contact solution or gel described in any of Items 1 to 6, for contacting cells intended for or already intended for cold storage.

[0021] Item 10. The cell contact solution or gel described in any one of Items 1 to 6, wherein the cells contain an exogenous polynucleotide encoding a sensor protein sequence.

[0022] Item 11. The cell contact solution or gel described in Item 10, wherein the aforementioned sensor protein is an olfactory receptor protein.

[0023] Item 12. The cell contact solution or gel thereof described in any one of Items 1 to 6, used for contacting cells for measuring the chemical response activity of sensor proteins.

[0024] Item 13. A cell chip comprising a partition containing cells and a cell contact solution or gel thereof as described in any one of Items 1 to 6.

[0025] Project 14. A method for enhancing the chemical response activity of sensor proteins in cells, comprising contacting the aforementioned cells with a cell contacting solution or gel thereof as described in any one of Projects 1 to 6.

[0026] Invention Effects

[0027] According to this disclosure, a cell contact solution or gel thereof can be provided that can improve the detectability of chemical substances such as odorants. In this specification, the improvement in the detectability of the chemical substance includes not only an increase in the detectability of the chemical substance compared to before contact with the cell contact solution or gel, but also the suppression of the decrease in detectability or the maintenance of the detectability when the detectability of the chemical substance tends to decrease. Attached Figure Description

[0028] [ Figure 1 The results of the cell proliferation assay in Example 2 are shown. The vertical axis shows the absorbance representing the number of viable cells. The horizontal axis shows the concentration of the culture medium (100% for undiluted medium) and the osmotic pressure of the diluted liquid (in mOsm / kg).

[0029] [ Figure 2 The graph shows the results of the olfactory receptor activity assay for Example 3. The vertical axis shows fluorescence intensity. The horizontal axis shows the concentration of the culture medium (100% for undiluted medium) and the osmotic pressure of the diluted liquid. The legend shows the concentration of compound a (in μM).

[0030] [ Figure 3 The graph shows the results of the olfactory receptor activity assay for Example 4. The vertical axis shows fluorescence intensity. The horizontal axis shows the concentration of the culture medium (100% for undiluted medium), the liquid used in the dilution, the osmotic pressure of the diluted liquid (mOsm / kg), storage, and incubation days. The legend shows the concentration of compound a (μM). Detailed Implementation

[0031] In this specification, the expressions “containing” and “comprising” include the concepts of “containing”, “comprising”, “substantially composed of” and “composed of only”.

[0032] Furthermore, in this specification, the range of values ​​formed by any combination of the upper and lower limits is also directly and uniquely disclosed.

[0033] In one embodiment, this disclosure relates to cell contact solutions or gels thereof with an osmotic pressure of less than 340 mOsm / kg (sometimes referred to in this specification as "solutions / gels of this disclosure"). The following describes such solutions / gels.

[0034] The solution / gel of this disclosure is a liquid (the solution of this disclosure) or a gel (the gel of this disclosure). The gel of this disclosure is formed by gelling the solution of this disclosure.

[0035] From the viewpoint of improving the detectability of chemical substances, the osmotic pressure is preferably 330 mOsm / kg or less, more preferably 320 mOsm / kg or less, even more preferably 310 mOsm / kg or less, even more preferably 300 mOsm / kg or less, particularly preferably 290 mOsm / kg or less, and even more preferably 210 mOsm / kg or more, more preferably 220 mOsm / kg or more, even more preferably 230 mOsm / kg or more, and even more preferably 240 mOsm / kg or more.

[0036] Osmotic pressure, for example, if the solution / gel of this disclosure is prepared by diluting it with a solvent in a solution with a known osmotic pressure, can be calculated by multiplying the osmotic pressure before dilution by the composition ratio and adding it to each of the components individually. Furthermore, if the osmotic pressure of the solution / gel of this disclosure cannot be calculated, it can be determined using a general method for measuring the osmotic pressure of cell culture media.

[0037] From the viewpoint of mitigating damage to cells (especially damage during preservation), and further from the viewpoint of adjusting the osmotic pressure to a more appropriate range, the solutions / gels of this disclosure preferably contain cell culture medium components. There are no particular limitations as long as the cell culture medium components are present in the cell culture medium.

[0038] Culture media typically contain carbon sources and inorganic salts that are available to cells. In addition, culture media may contain nitrogen sources.

[0039] As a carbon source, anything that is available to the cell can be used. For example, carbohydrates such as glucose, fructose, sucrose and molasses containing them, starch and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used.

[0040] As nitrogen sources, for example, ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn extract, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermented microorganisms and their digests can be used.

[0041] As inorganic salts, for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

[0042] As more specific examples of culture media, in the case of insect culture media, examples include TNM-FH medium (manufactured by Pharmingen), Sf-900 III SFM medium (manufactured by Life Technologies), ExCell400, ExCell405 (both manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195, 788(1962)), etc.

[0043] Cell culture medium can be a single component or a combination of two or more components.

[0044] The solvent used in the solutions / gels disclosed herein is not particularly limited as long as it does not significantly adversely affect cell survival. 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, further preferably 80% by mass or more, even more preferably 85% by mass or more, particularly preferably 90% by mass or more, and especially preferably 95% by mass or more.

[0045] The solution disclosed herein is preferably a diluted culture medium prepared by diluting cell culture medium. The liquid or solvent used in the dilution may be the solvent described above. In this case, the cell culture medium concentration of the diluted culture medium (=(cell culture medium volume / diluted culture medium volume)×100) is preferably 95% or less, more preferably 90% or less, further preferably 85% or less, and even more preferably 50% or more, more preferably 60% or more, and even more preferably 65% ​​or more.

[0046] In one embodiment, the solution disclosed herein may also be a cell culture medium prepared by adding osmotic pressure adjusting components (such as salts), a cell culture medium prepared by reducing the amount of osmotic pressure adjusting components (such as salts), or a cell culture medium prepared by reducing the amount of solvent.

[0047] The gel disclosed herein is preferably sol-gelled. Sol-gelling is believed to ensure high diffusivity of the target substance, enabling rapid and stable determination. Furthermore, since sol-gelling allows for easy removal and replacement with a solution exhibiting superior diffusivity of the target substance, it is possible to achieve this.

[0048] The components that form the sol-gel (gel-forming components: components that cross-link to form a network) are not particularly limited. 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 from the viewpoint of easily exhibiting the desired properties described above. Gelatin is obtained by pretreating collagen raw materials such as bovine bone, bovine hide, pig bone, pig hide, and fish scales with acid or alkali, washing with water, extracting with warm water, purifying, concentrating, and drying. The sol and gel physical properties of gelatin vary depending on the type of collagen raw material, pretreatment, and warm water extraction conditions. In order to achieve the desired sol temperature, sol-gel transition temperature, sol viscosity, and gel elastic modulus as described above in this invention, fish-derived gelatin (fish gelatin) is particularly preferred. By appropriately adjusting the pretreatment and warm water extraction conditions, sol and gel physical properties suitable for this invention can be obtained.

[0049] The gel-forming component can be a single component or a combination of two or more components.

[0050] The solutions / gels disclosed herein are used for contact with cells.

[0051] There are no particular limitations on the mode of contact. For example, the modes of contact between the solution / gel of this disclosure and the cell layer, and the modes of dispersion of cells in the solution / gel of this disclosure are listed.

[0052] More specifically, the solutions / gels disclosed herein may be, for example, cell preservation solutions, cell treatment solutions, or cell culture media.

[0053] In one method, the cell preservation solution is used for cryopreservation. The cryopreservation temperature is, for example, below 10°C or below 6°C. The cryopreservation period can be, for example, more than 5 days, more than 10 days, more than 15 days, more than 20 days, more than 25 days, more than 30 days, more than 40 days, more than 50 days, more than 60 days, or more than 70 days, and can also be less than 200 days, less than 150 days, or less than 100 days. After cryopreservation, it can be provided for culture at a specified temperature (e.g., 20°C or 25°C).

[0054] Cell treatment solution is a liquid used for temporary treatment of cells, and there are no particular limitations in this definition. The treatment time may be, for example, more than 30 minutes, more than 1 hour, more than 2 hours, more than 4 hours, or more than 8 hours, or less than 16 hours or less than 12 hours.

[0055] In one embodiment, the cell culture medium is a cell proliferation medium. There are no particular limitations on the cell proliferation medium as long as its purpose is cell proliferation. The solution / gel of this disclosure can increase the rate of cell proliferation and is therefore particularly suitable for use as a cell proliferation medium.

[0056] In another approach, the cell culture medium is a cell maintenance medium. Cell maintenance media do not require cell proliferation; there are no particular restrictions as long as the cells can be kept alive.

[0057] In one embodiment, the solution / gel of this disclosure is used to contact cells intended for or already refrigerated. Refrigeration tends to decrease the detectability of chemical substances in cells, but by contacting the cells with the solution / gel of this disclosure, this decrease can be suppressed, and the detectability of chemical substances can be improved compared to before refrigeration.

[0058] In one embodiment, the solution / gel of this disclosure is used to contact cells for measuring the chemical response activity of sensor proteins.

[0059] There are no particular restrictions on the cells used. From the viewpoint of suitability for detecting chemical substances, animal cells such as mammalian cells and non-mammalian cells are preferred. From the viewpoint of being more suitable for use with the solutions / gels of this disclosure, non-mammalian cells are particularly preferred due to their ease of management, such as the absence of CO2 and strict temperature control. Examples of non-mammalian cells include, for example, insect cells, fish cells, amphibian cells, and reptile cells.

[0060] As insect cells, for example, Sf cells, MG1 cells, High Five cells are used. TM Cells, BmN cells, etc. For example, Sf9 cells (ATCC CRL1711) and Sf21 cells are used as Sf cells. Among insect cells, cells derived from insects of the family Arctiidae are particularly preferred.

[0061] Cells derived from insects of the family Triplophysa are primary cultured cells or cell lined cells of organisms that constitute cells derived from insects of the family Triplophysa, without any particular restriction in this definition.

[0062] As for the family Arctiinae, examples include the subfamilies Arctiinae, Lithosiinae, and Syntominae, with Arctiinae being the preferred subfamily. Within the Arctiinae subfamily, genera such as *Spilosoma*, *Spilarctia*, and *Rhagonis* are preferred, with *Spilosoma* being particularly preferred. Within the *Spilosoma* genus, there are no particular restrictions, but *Spilosoma imparilis* is particularly preferred.

[0063] Cells derived from insects of the family Lithopodidae can also be obtained from well-known biobanks, and can be collected / cultured from Lithopodidae organisms using or based on well-known methods, and can be lined as needed.

[0064] Cells used as sources of the mulberry leafminer moth include, for example, FFPRI-SpIm-2AM-SF cells (MAFF number: 275052) and FFPRI-SpIm-2AM-IPL411 cells (MAFF number: 275053) from the Agricultural Biological Resources Gene Bank.

[0065] The cells preferably contain exogenous polynucleotides encoding sensor proteins. This allows for the expression of any sensor protein, and further enables the increase of expression levels of the target sensor protein and the improvement of detection sensitivity for the target chemical substance.

[0066] Exogenous polynucleotides are polynucleotides that contain a base sequence that does not originate from the genomic DNA (specifically, chromosomal genomic DNA) of insect cells, and there are no particular restrictions in this definition.

[0067] Sensor proteins can be selected from proteins that can detect the presence of chemical substances, such as receptor proteins that use chemical substances as ligands. Olfactory receptor proteins are preferred, and insect olfactory receptor proteins are particularly preferred.

[0068] Insect olfactory receptor proteins are membrane proteins with a seven-transmembrane structure that function as odor sensors in organisms. They are formed by the following sequence from the amino terminus (hereinafter sometimes called the "N-terminus") to the carboxyl terminus (hereinafter sometimes called the "C-terminus"): N-terminal region (NT), first transmembrane domain (TM1), first extracellular loop (EC1), second transmembrane domain (TM2), first intracellular loop (IC1), third transmembrane domain (TM3), second extracellular loop (EC2), fourth transmembrane domain (TM4), second intracellular loop (IC2), fifth transmembrane domain (TM5), third extracellular loop (EC3), sixth transmembrane domain (TM6), third intracellular loop (IC3), seventh transmembrane domain (TM7), and C-terminal region (CT). In this disclosure, each region was determined by applying structural predictions (with default conditions) to TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membranespanning protein segments, Biol. Chem. Hoppe-Seyler, 374 (1993), p. 166, https: / / embnet.vital-it.ch / software / TMPRED_form.html).

[0069] Preferred insects that serve as sources of olfactory receptor proteins include: Diptera such as mosquitoes and fruit flies; Lepidoptera such as silkworm moths; Hymenoptera such as honeybees; Orthoptera such as grasshoppers; and Hemiptera such as bedbugs. Further preferred examples include: Diptera such as mosquitoes and fruit flies; Orthoptera such as grasshoppers; and Hemiptera such as bedbugs. Examples of mosquitoes include *Anopheles gambiae*, *Aedes aegypti*, and *Culex quinquefasciatus*. Examples of fruit flies include *Drosophila melanogaster*, *Drosophila pseudoobscura*, and *Drosophila virillis*. Examples of insects belonging to the family Bombyx mori include the domestic silkworm (Bombyx mori), the wild mulberry silkworm (Bombyx mandarina), and the trilocha varians. Examples of insects belonging to the family Apidae include the Western honeybee (Apis mellifera), the honey honeybee (Apis florea), the giant honeybee (Apis dorsata), and the ground bumblebee (Bombus terrestris). Examples of insects belonging to the family Locustae include the Asian migratory locust (Locusta migratoria). Examples of insects belonging to the family Cimexidae include the temperate bedbug (Cimex lectularius).

[0070] Specifically, wild-type insect olfactory receptor proteins include, for example, 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, AaOR60, and AaOR66. 1. 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, AaOR109, AaO R110, AaOR112, AaOR114, AaOR116, AaOR117, AaOR118, AaOR122, AaOR125, AaOR128, AgOR1, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR8, AgOR9, AgOR10, AgOR11a, AgOR12a, AgOR12b, AgOR13, AgOR14, AgOR15, AgOR16a, AgOR17, AgOR18, AgOR20, AgOR21, AgOR23, AgOR25, AgOR26, AgOR27, AgOR28, AgOR30, Ag OR34, 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, BmOR8, Bm OR9, BmOR10, BmOR13, BmOR17, BmOR18, BmOR23, BmOR24, BmOR25, BmOR35, BmOR36, BmOR 42. BmOR45, BmOR49, BmOR51, BmOR52, BmOR55, BmOR56, BmOR61, DmOR1a, DmOR9a, DmOR1 9a, DmOR22a, DmOR22b, DmOR22c, DmOR24a, DmOR30a, DmOR33a, DmOR33b, DmOR33c, DmOR 35a, DmOR42b, DmOR43a, DmOR45a, DmOR45b, DmOR47a, DmOR49b, DmOR59b, DmOR65b, DmO R65c, DmOR67b, DmOR67c, DmOR69a, DmOR71a, DmOR74a, DmOR82a, DmOR83a, DmOR83c, Dm OR85a, DmOR85c, DmOR85e, DmOR85f, DmOR88a, DmOR92a, DmOR94a, DmOR94b, DmOR98b, etc. ,

[0071] In this specification, OR represents the olfactory receptor, Dm represents the Drosophila melanogaster source, Bm represents the silkworm source, Ag represents the Anopheles gambiae source, and Aa represents the Aedes aegypti source. The amino acid sequences and coding sequences of the various olfactory receptor proteins containing them are well-known or can be easily identified by sequence identity searches based on well-known sequences.

[0072] Sensor proteins may contain amino acid mutations in the wild-type amino acid sequence, provided that 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, further 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.

[0073] Amino acid mutations, such as substitutions, insertions, additions, or deletions of amino acids, are preferred, with substitutions being particularly preferred, especially conservative substitutions.

[0074] The sensor protein may contain: a wild-type amino acid sequence, and an amino acid sequence having, for example, 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more of the same amino acid sequence as the wild-type amino acid sequence.

[0075] In this specification, chemical response activity refers to the property of a sensor protein to recognize a chemical substance, and the sensor protein, alone or in combination with other proteins, to exhibit signal transduction activity (e.g., ion channel activity). In the case of an olfactory receptor, the sensor protein may be a G protein-coupled receptor or an ion channel receptor; however, in the case of insect olfactory receptors, it refers to the property of the olfactory receptor recognizing a chemical substance, and the activation of the olfactory receptor complex formed by the olfactory receptor and its co-receptor to exhibit ion channel activity. The chemical response activity of a sensor protein can be measured as an indicator (e.g., quantifying / evaluating the amount of signaling molecules) of the sensor protein in contact with the chemical substance. In the case of insect olfactory receptors, the chemical response activity of the olfactory receptor can be measured as an indicator of the ion channel activity of the olfactory receptor complex formed by the olfactory receptor and its co-receptor in contact with the chemical substance. For example, a cell expressing a protein that fluoresces or emits light through ions (such as calcium ions) flowing into the cell in response to (a) the olfactory receptor, (b) the co-receptor, and (c) the olfactory receptor complex is contacted with a chemical substance, and the amount of fluorescence or luminescence in the cell is measured. The greater the amount of fluorescence or luminescence measured, the higher the responsiveness of the chemical substance to the olfactory receptor. Specifically, it can be measured according to the method described in Patent Document 1.

[0076] There are no particular restrictions on the coding sequence of a sensor protein, as long as it is a base sequence encoding a sensor protein. In one embodiment, an exogenous polynucleotide contains an expression cassette for the sensor protein. There are no particular restrictions on the expression cassette, as long as it is a polynucleotide capable of expressing a sensor protein within the cell. Typical examples of sensor protein expression cassettes include a promoter and a polynucleotide containing a sensor protein coding sequence configured under the control of that promoter.

[0077] When the sensor protein is an insect olfactory receptor, the exogenous polynucleotide preferably contains the coding sequence of the insect olfactory receptor co-receptor. The insect olfactory receptor co-receptor is a membrane protein with a seven-transmembrane structure, similar to the olfactory receptor, and functions by forming a heterogeneous complex with the olfactory receptor. The olfactory receptor complex, a heterogeneous complex composed of the olfactory receptor and the olfactory receptor co-receptor, possesses ion channel activity activated by odor substances, releasing sodium ions (Na+) upon activation. + ), calcium ions (Ca 2+ Cations such as ions flow into the cells.

[0078] The exogenous polynucleotide preferably contains the coding sequence of a protein that, in response to a sensor protein (particularly an insect olfactory receptor protein), emits fluorescence or light through ions (such as calcium ions) flowing into the cell. Examples of such proteins include Aequorin, Yellow Cameleon (YC), and GCaMP. Alternatively, the cells disclosed herein preferably contain ion-dependent fluorescent dyes such as calcium ion-dependent fluorescent dyes (e.g., Fura-2, Fluo-3, Fluo-4, etc.).

[0079] The coding sequences for insect olfactory receptor co-receptors, fluorescent or luminescent proteins, and antibiotic resistance genes are preferably contained in exogenous polynucleotides in the form of expression cassettes. The structure of the expression cassette is the same as that of sensor protein expression cassettes. The promoter of the expression cassette can be shared among multiple coding sequences.

[0080] Exogenous polynucleotides are preferably integrated into genomic DNA (particularly chromosomal genomic DNA). This allows for the stable expression of sensor proteins, making them suitable for chemical substance detection. In this case, the polynucleotide can be a single contiguous region within the genomic DNA, or it can be a combination of two or more contiguous regions (e.g., the sensor protein coding sequence is contained in contiguous region A, the drug resistance gene coding sequence is contained in contiguous region B, which is a different contiguous region from contiguous region A, or the sensor protein coding sequence is contained in both contiguous regions A and B).

[0081] In another approach, the polynucleotide can be in a state where it is not integrated into the genomic DNA. In this case, the exogenous polynucleotide can be, for example, in the form of a vector. In this case, the polynucleotide can be a single polynucleotide molecule, or more than two polynucleotide molecules (e.g., the sensor protein coding sequence is contained in polynucleotide molecule A, the drug resistance gene coding sequence is contained in polynucleotide molecule B which is a molecule different from polynucleotide molecule A, or the sensor protein coding sequence is contained in both polynucleotide molecule A and polynucleotide molecule B).

[0082] In one embodiment, the present invention relates to a cell chip (sometimes referred to in this specification as "cell chip of the present disclosure") comprising partitions containing cells and a solution / gel of the present disclosure. This allows for improved detectability of chemical substances in the cells within the chip.

[0083] The form of the partitions is not particularly limited as long as it preserves the cells. From the viewpoints of cell preservation, preparation efficiency, or chemical detection, pore-shaped partitions are preferred.

[0084] There are no particular restrictions on the material used for partitioning, as long as it can preserve the cells. Materials can include, for example, resin, metal, etc.

[0085] From the perspective of detection sensitivity or preparation efficiency, the area of ​​the partition is preferably 0.5–100 mm. 2 More preferably 1 to 50 mm 2 Further optimization is preferred, with a diameter of 1.5–40 mm. 2 Further preferred size: 2-40mm 2 In a preferred embodiment of this disclosure, the area is 50 mm². 2 Below, 35mm 2 Below or 15mm 2 the following.

[0086] From the viewpoint of detection sensitivity or preparation efficiency, the number of partitions is preferably 1 to 2000, more preferably 4 to 1600, and even more preferably 8 to 400. In a preferred embodiment of this disclosure, the number is 10 or more, 20 or more, or 50 or more.

[0087] From a detection sensitivity perspective, partitions typically contain multiple cells. The area per mm² of a partition... 2 The cell count (cells) is, for example, 50 to 20,000 cells / mm². 2 From the perspectives of detection sensitivity and cell viability, a concentration of 100–15,000 cells / mm² is preferred. 2 More preferably, 100 to 10,000 cells / mm 2 Further optimization is needed to select cells ranging from 200 to 10,000 per mm. 2 Further optimization yields 500–7000 cells / mm². 2 Especially preferred is 1000-5000 cells / mm². 2 .

[0088] The cell chip disclosed herein may contain only one type of sensor protein, but preferably contains two or more (more preferably three or more, further preferably four or more, even more preferably five or more, ten or more, fifteen or more, or twenty or more) different types of sensor proteins.

[0089] Chemical substances can be detected by measuring the chemical response activity of sensor proteins. These chemicals can be, for example, substances in samples such as bodily fluids (e.g., urine, blood, saliva), air (e.g., indoor air, air inside packaging), and water (e.g., river water, seawater, tap water, purified water, sewage). In this case, for example, by adding the sample to a partition of the substrate of this disclosure, the chemicals in the sample can reach the cells and contact the sensor proteins of the cells. For example, the chemical response activity of the sensor proteins can be determined by detecting ions flowing into the cells (e.g., by detection by proteins that emit fluorescence or light from ions), thus detecting the chemical substances.

[0090] Example

[0091] The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0092] Experiment 1. Preparation of cells with stable expression

[0093] In SpIm cells (derived from the mulberry snow moth *Spilosoma imparilis*), transposon vectors were introduced, containing the coding sequences for the olfactory receptor protein (ORA), olfactory receptor co-receptor, calcium sensor fluorescent protein, and puromycin resistance gene, configured under promoter control and positioned between the 5' ITR (inverted repeat sequence) and 3' ITR. These vectors were then selected with puromycin to prepare stably expressing olfactory receptor SpIm cells (hereinafter referred to as ORA cells) by integrating exogenous DNA containing the aforementioned coding sequences and promoter sequences into the chromosomal genomic DNA. This olfactory receptor is insect-derived and is a receptor for compound a. ORA cells fluoresce in response to compound a.

[0094] Experimental Example 2. Cell Proliferation Assay

[0095] ORA cells were cultured in 96-well plates (CORNING 3903) at a rate of 1×10⁻⁶. 4 Inoculate with 100 μL per well and incubate at 27°C (without CO2 supply). Use Sf-900III SFM medium. 24 hours after inoculation, remove the culture medium from each well. Add 100 μL of Sf-900III SFM medium or a 70-90% diluted medium (using ultrapure water) to each well and incubate at 27°C for 6 days. Add MTT reagent (11465007001 Roche) to each well, incubate at 4°C for 4 hours, add MTT dissolving reagent, and incubate overnight at 37°C. Measure the absorbance at 560 nm and a reference absorbance at 690 nm using an Infinite 200 or TECAN microplate reader.

[0096] The results are shown in Figure 1 Compared with a 100% culture medium concentration, the number of viable cells increased in the hypotonic group diluted with ultrapure water.

[0097] Experimental Example 3. Determination of Olfactory Receptor Activity 1

[0098] ORA cells were cultured in 384-well plates (Greiner_781098) at a rate of 3 × 10⁻⁶ cells / well. 4 Inoculate with 40 μL per well and incubate at 27°C (without CO2 supply). Use Sf-900III SFM medium. 24 hours after inoculation, remove the medium from each well and add 40 μL of Sf-900III SFM medium or a liquid containing 1% fish gelatin diluted to 63-91% with ultrapure water to each well. Incubate at 4°C in a gelled state for 31 days, then incubate at 27°C in a sol-gelled state for 3 days. Afterward, remove the medium from each well and add 40 μL of 0.1% BSA / 1×Hanks' buffer / 20mM HEPES buffer to each well. Add compound a (ORA-responsive substance) to each well at final concentrations of 0, 0.1, 1, and 10 μM, and measure the change in fluorescence intensity before and after addition using a microplate reader (FlexStation3, MolecularDevices).

[0099] The results are shown in Figure 2 Compared with a 100% culture medium concentration, the odor response signals were enhanced at all concentrations in the hypotonic group diluted with ultrapure water.

[0100] Experimental Example 4. Determination of Olfactory Receptor Activity 2

[0101] ORA cells were cultured in 96-well plates (CORNING 3903) at a rate of 5 × 10⁶ cells / well. 4Inoculate with 100 μL per well and incubate at 27°C (without CO2 supply). Use Sf-900III SFM medium. 24 hours after inoculation, remove the medium from each well and add 100 μL of Sf-900III SFM medium, or a liquid containing 1% fish gelatin diluted to 83% with ultrapure water or PBS (Nichisui Pharmaceutical), to each well. Incubate at 4°C in a gelled state for 26 or 54 days, then incubate at 27°C in a sol-gelled state for 2 days. Afterward, remove the medium from each well and add 80 μL of 0.1% BSA / 1×Hanks' buffer / 20 mM HEPES buffer to each well. Compound a (the substance that responds to ORA) was added to each well at final concentrations of 0, 0.1, 1, and 10 μM. The changes in fluorescence intensity before and after addition were measured using a microplate reader (FlexStation3, Molecular Devices).

[0102] The results are shown in Figure 3 Regarding the storage periods of 26 days and 54 days at 4°C, compared with 100% culture medium concentration, the odor response signal was enhanced in the hypotonic group diluted with ultrapure water at all concentrations. On the other hand, no enhancement was observed in the group with higher osmotic pressure due to dilution with PBS than in the group diluted with water.

Claims

1. A cell contact solution or gel thereof, having an osmotic pressure of less than 340 mOsm / kg.

2. The cell contact solution or gel of claim 1, wherein, The osmotic pressure is below 300 mOsm / kg.

3. The cell contact solution or gel of claim 1, wherein, The osmotic pressure is 210–300 mOsm / kg.

4. The cell contact solution or gel of claim 1, wherein, The cells in question are non-mammalian cells.

5. The cell contact solution or gel of claim 1, wherein, The cells in question are insect cells.

6. The cell contact solution or gel of claim 1, wherein the solution contains cell culture medium components.

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

8. The cell contact solution or gel of claim 7, wherein the solution is a cell maintenance culture medium or a cell proliferation culture medium.

9. The cell contact solution or gel according to any one of claims 1 to 6, for contacting cells intended for or already intended for cold storage.

10. The cell contact solution or gel according to any one of claims 1 to 6, wherein, The cells contain exogenous polynucleotides that encode sensor proteins.

11. The cell contact solution or gel of claim 10, wherein, The sensor protein is an olfactory receptor protein.

12. The cell contact solution or gel of any one of claims 1 to 6, used for contacting cells for measuring the chemical response activity of sensor proteins.

13. A cell chip comprising a partition containing cells and a cell contact solution or gel according to any one of claims 1 to 6.

14. A method for enhancing the chemical response activity of sensor proteins in cells, comprising contacting the cells with a cell contacting solution or gel according to any one of claims 1 to 6.

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