Cell chip

Abstract

Provided is a cell chip having excellent cell dryness resistance, cell retention, and detectability of chemical substances such as odor substances. A cell chip comprises a partition comprising cells and a gel that can be solated.

Description

TECHNICAL FIELD

[0001] The present application relates to a cell chip or the like. BACKGROUND

[0002] A group of odor substances that characterize a specific disease, mental state, or the like of a human being has been identified, and various odor sensors that use them as targets have become popular because of their high value as diagnostic markers. Since biological olfactory receptors have superior characteristics such as diversity, sensitivity, selectivity, and the like that semiconductor and other odor sensor elements of the past do not have, development of new odor sensors that use olfactory receptors as sensor elements is expected.

[0003] In Patent Literature 1, a cell that expresses an altered olfactory receptor, a lipid bilayer membrane having an altered olfactory receptor, and the like are disclosed as being used as an odor sensor.

[0004] PRIOR ART DOCUMENTS

[0005] PATENT LITERATURE

[0006] Patent Literature 1: International Publication No. 2022 / 024902 SUMMARY

[0007] PROBLEMS TO BE SOLVED BY THE INVENTION

[0008] An odor sensor that has a step of artificially preparing a lipid bilayer membrane having a sensor protein such as an olfactory receptor has not necessarily been sufficiently efficient in terms of production efficiency, and further improvement in production efficiency of odor sensors is required. Therefore, attention is focused on the use of a cell that expresses a sensor protein.

[0009] In the case of using a cell as a chemical sensor such as an odor sensor, from the viewpoint of ease of use, a mode in which the cell is not prepared each time it is used but is prepared in advance and is kept in a container or the like and is used when needed is expected. In the case of the latter mode, it is necessary to keep the cell from drying until the cell is used, and in addition, considering transportation from the manufacturer to the place of use and the like, it is important that the cell be kept stably.

[0010] In addition, from the viewpoint of determination of a disease or the like, it is expected that a plurality of cells that express different olfactory receptors from each other can be used simultaneously. According to this viewpoint, use as a cell chip is expected.

[0011] Therefore, the present disclosure aims to provide a cell chip in which the dry resistance of a cell, the keeping property of a cell, and the detection property of an odor substance or the like are excellent.

[0012] MEANS FOR SOLVING THE PROBLEMS

[0013] During their research, the inventors observed that cell chips comprising partitions containing cells and hydrogel can prevent cell drying and stably maintain the cells. However, it is known that in the presence of hydrogel, the increase in the response activity of sensor proteins after the addition of chemical substances is gradual, making rapid and stable activity measurement impossible. Based on this knowledge, the inventors discovered that cell chips comprising partitions containing cells and soluble gel can solve the aforementioned problems. That is, this disclosure includes the following approach.

[0014] Project 1. Cell chip, which contains partitions containing cells and soluble gel.

[0015] Project 2. The cell chip described in Project 1, wherein the aforementioned gel is a gel that is soluble through temperature control, light irradiation, or the addition of a drug.

[0016] Project 3. The cell chip described in Project 2, wherein the aforementioned gel is a solubilized gel obtained through temperature control.

[0017] Project 4. The cell chip described in Project 3, wherein the sol-gel temperature of the aforementioned gel is below 40°C.

[0018] Project 5. The cell chip described in Project 3, wherein the sol-gel temperature of the aforementioned gel is below 30°C.

[0019] Project 6. The cell chip described in Project 3, wherein the sol-gel transition temperature of the aforementioned gel is 4–25°C.

[0020] Project 7. The cell chip described in Project 1, wherein the aforementioned gel, after sol-gelling, forms a sol with a viscosity of less than 20 mPa·s.

[0021] Project 8. The cell chip described in Project 1, wherein the aforementioned gel has a storage elastic modulus of 0.01–20 kPa at 4°C.

[0022] Item 9. The cell chip described in any one of Items 1 to 8, for the detection of a chemical substance to be tested, the method comprising solubilizing the aforementioned gel and then contacting the aforementioned cells with the aforementioned chemical substance to be tested.

[0023] Item 10. The cell chip described in any one of Items 1 to 8, used for the detection of light from the aforementioned cells.

[0024] Item 11. The cell chip described in any one of Items 1 to 8, wherein the aforementioned cells are insect cells.

[0025] Item 12. The cell chip of any one of Items 1 to 8, wherein the aforementioned cells contain exogenous polynucleotides containing a coding sequence for a sensor protein.

[0026] Project 13. The cell chip described in Project 12, wherein the aforementioned sensor protein is an olfactory receptor protein.

[0027] Item 14. A kit for cell fixation containing soluble gel-forming components.

[0028] Item 15. The kit described in Item 14 is a kit for immobilizing cells on a cell chip.

[0029] Project 16. A method for preparing transport cells, comprising fixing cells with a soluble gel.

[0030] Project 17. A method for detecting light from cells, comprising solubilizing a soluble gel of immobilized cells.

[0031] Item 18. A sol-gelled gel, wherein the gel:

[0032] Solvable by temperature control.

[0033] The sol-gel temperature is below 40℃.

[0034] The sol-gel transition temperature is 4–25℃.

[0035] After sol-gelation, it can form a sol with a viscosity of less than 20 mPa·s, and

[0036] The storage elastic modulus at 4℃ is 0.01–20 kPa.

[0037] Item 19. Sol, which forms the gel described in Item 18.

[0038] Item 20. The kit described in Item 14, which contains the gel described in Item 18 and / or the sol described in Item 19.

[0039] Item 21. The kit described in Item 20 is a kit for immobilizing cells on a cell chip.

[0040] Item 22. The preparation method described in Item 16, wherein the aforementioned gel is the gel described in Item 18.

[0041] Item 23. The method described in Item 17, wherein the aforementioned gel is the gel described in Item 18.

[0042] Invention Effects

[0043] According to this disclosure, a cell chip with excellent cell desiccation resistance, cell retention, and detectability of chemical substances such as odor substances can be provided. Furthermore, according to this disclosure, a kit for immobilizing cells on a cell chip, a method for preparing transport cells, a method for detecting light from cells, a gel suitable for use on the aforementioned cell chip, and a sol for forming the gel can also be provided. Brief description of the attached diagram

[0045] [ Figure 1 The results of the olfactory receptor activity assay for Example 4 are shown. The vertical axis shows fluorescence intensity, and the horizontal axis shows time. Arrows indicate the time points when compound a (the ORA-responsive substance) was added.

[0046] [ Figure 2 The results of the olfactory receptor activity assay in Experiment 5 are shown. The vertical axis shows fluorescence intensity, and the horizontal axis shows time. Arrows indicate the time points when compound a (the ORA-responsive substance) was added.

[0047] [ Figure 3 The results of the olfactory receptor activity assay for Example 6 are shown. The vertical axis shows the average value (average of the background value in the first 20 seconds before compound addition) minus the maximum fluorescence intensity. On the horizontal axis, the percentage represents the gelatin concentration, and "no gel" indicates that a buffer solution was added instead of the gelatin solution.

[0048] [ Figure 4 The results of the olfactory receptor activity assay for Example 7 are shown. The vertical axis shows the average value (average of the background value in the first 20 seconds before compound addition) minus the maximum fluorescence intensity. On the horizontal axis, "no gel" indicates the case where a buffer solution was added instead of gelatin solution, and "other" indicates the presence or absence of a sol after hydrogel liquefaction.

[0049] [ Figure 5 This displays the viscosity measurement results of the sol in Experiment Example 8. The vertical axis shows the measured viscosity value. The horizontal axis shows the fish gelatin concentration of the gelatin solution.

[0050] [ Figure 6 This displays the viscosity measurement results of the sol in Experiment Example 9. The vertical axis shows the measured viscosity value. The horizontal axis shows the fish gelatin concentration of the gelatin solution.

[0051] [ Figure 7 The results of the storage elastic modulus and sol-gel transition temperature measurements for Experiment Example 10 are shown. Detailed Implementation

[0052] In this specification, the expressions “containing” and “comprising” include the concepts of “containing”, “comprising”, “substantially composed of” and “composed of only”.

[0053] In one embodiment, this disclosure relates to cell chips (sometimes referred to in this specification as "cell chips of this disclosure") comprising partitions containing cells and soluble gels. This will be described below.

[0054] There are no particular restrictions on the cells. From the viewpoint of suitability for the detection of chemical substances, animal cells such as insect cells and mammalian cells are preferred. Insect cells are especially preferred because they are easy to manage and do not require CO2 or temperature control.

[0055] As insect cells, for example, Sf cells, MG1 cells, High Five cells are used. TM Cells, such as BmN cells, etc. As Sf cells, for example, Sf9 cells (ATCC CRL1711) and Sf21 cells are used. Insect cells contain coding sequences for insect olfactory receptors within their genomes and can be used as chemical sensory molecules. Among insect cells, cells derived from insects of the family Arctiidae are particularly preferred.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] The cells preferably contain exogenous polynucleotides encoding sequences of sensor proteins. This allows for the expression of any sensor protein, and further enables the increase of expression levels of target sensor proteins and the improvement of detection sensitivity for target chemicals.

[0061] 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.

[0062] In this specification, the polynucleotides, as exemplified below, include not only typical polynucleotides such as endogenous DNA and RNA, but also polynucleotides and artificial polynucleotides that have undergone known chemical modifications. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues (phosphate esters) of each nucleotide can be replaced with chemically modified phosphate residues such as thiophosphate (PS), methylphosphonate, or dithiophosphate. Furthermore, the hydroxyl group at the 2-position of the sugar (ribose) in each ribonucleotide can be replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Further, the base moiety (pyrimidine, purine) can be chemically modified, for example, by introducing a methyl group at the 5-position of the pyrimidine base, introducing a cationic functional group, or replacing the carbonyl group at the 2-position with a thiocarbonyl group. Furthermore, examples of modifications can be made to the phosphate and hydroxyl moieties, such as those by biotin, amino, lower alkylamine, acetyl, etc., but are not limited to these. In addition, it is also possible to fix the conformation of the sugar moieties to the N-type BNA (LNA) by cross-linking the 2' oxygen and 4' carbon of the sugar moieties of the nucleotides.

[0063] 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 particularly preferred as sensor proteins.

[0064] 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).

[0065] 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).

[0066] 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. ,

[0067] 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.

[0068] 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.

[0069] Amino acid mutations, such as substitutions, insertions, additions, or deletions of amino acids, are preferred, with substitutions being particularly preferred, especially conservative substitutions.

[0070] In this specification, "conservative substitution" means replacing an amino acid residue with an amino acid residue having a similar side chain. For example, substitution with each other using amino acid residues with basic side chains, such as lysine, arginine, and histidine, is equivalent to a conservative substitution. Furthermore, substitution with each other using amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine, is also equivalent to a conservative substitution.

[0071] 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.

[0072] In this specification, the “identity” of an amino acid sequence refers to the degree of similarity between two or more comparable amino acid sequences relative to each other. Therefore, the higher the similarity between two amino acid sequences, the higher their sequence identity or similarity. The level of amino acid sequence identity can be determined, for example, using FASTA, a tool for sequence analysis, with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268(1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7(1993)). A program called BLASTX has been developed based on such a BLAST algorithm. The specific methods used in these analyses are well-known and can be found on the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0073] As long as the activity of the sensor protein in responding to chemical substances is not significantly impaired, other amino acid sequences can be added, such as protein tags, fluorescent proteins, luminescent proteins, signal sequences, and other proteins or peptides. Examples of protein tags include biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, and PA tags.

[0074] In this specification, chemical substance 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 olfactory receptors, it refers to the property of an olfactory receptor to recognize a chemical substance, and the olfactory receptor complex formed by the olfactory receptor and its co-receptor to be activated, thereby exhibiting ion channel activity. The chemical substance 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 olfactory receptors, the chemical substance response activity of an 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 light emitted by the cell is measured. The higher the amount of light emitted, the higher the chemical substance response activity of the olfactory receptor is determined. Specifically, it can be measured according to the method described in Patent Document 1.

[0075] 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 polynucleotide comprising a promoter and a coding sequence for the sensor protein configured under the control of that promoter.

[0076] There are no particular restrictions on the type of promoter used; appropriate selection is permissible. For example, various pol II lineage promoters can be used. There are no particular restrictions on pol II lineage promoters; examples include CMV promoters, EF1 promoters, SV40 promoters, MSCV promoters, and promoters of insect-derived genes.

[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 emits fluorescence or light through ions (such as calcium ions) flowing into the cell in response to a sensor protein (particularly, an olfactory receptor protein). 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] To enable drug screening of cells, the exogenous polynucleotide preferably contains the coding sequence of a drug resistance gene. As the drug resistance gene, a resistance gene targeting a drug that can be used in cell-based drug screening can be selected; examples include chloramphenicol resistance genes, tetracycline resistance genes, neomycin resistance genes, erythromycin resistance genes, spectinomycin resistance genes, kanamycin resistance genes, hygromycin resistance genes, and puromycin resistance genes.

[0080] The coding sequences for insect olfactory receptor co-receptors, chromogenic 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.

[0081] 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, and the coding sequence of the drug resistance gene 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).

[0082] 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).

[0083] The gel is solubilizable, and there are no particular limitations in this definition. It is believed that solubilization ensures high diffusivity of the target substance, enabling rapid and stable assays. Furthermore, since it can be easily removed through solubilization, it is possible to replace it with a solution that has even better diffusivity for the target substance. Therefore, in one aspect, this disclosure relates to a kit for immobilizing cells on a cell chip, which contains components that form a solubilizable gel.

[0084] As a gel, examples include gels that can be soluble by temperature control, light irradiation, or the addition of a chemical agent. Among these, gels that can be soluble by temperature control are preferred from the viewpoints of ease of preparing gel components, simplicity of sol-gelation, and reversibility of sol-gelation. Gels that can be soluble by temperature increase are even more preferred.

[0085] From the viewpoint of contacting and more firmly fixing the cells in the cell chip of this disclosure, while suppressing thermal damage to the cells during solidation, the solidation temperature of the gel, which can be solidified by temperature control, is preferably below 40°C, more preferably below 35°C, further preferably below 30°C, and particularly preferably below 28°C. From the viewpoint of being able to stably maintain the gel state at the temperature desired for cell preservation, the lower limit of this temperature is preferably 10°C, 15°C, 20°C, or 25°C.

[0086] In the dynamic viscoelasticity test described in Test Example 10 below, the sol-gelation temperature can be defined as the temperature at which the loss tangent exceeds 1 when the gel is heated at +1℃ / min.

[0087] From the viewpoint of contacting and more firmly fixing the cells in the cell chip of this disclosure, while suppressing thermal damage to the cells during sol preparation and gelation, the sol-gel transition temperature (i.e., the temperature at which the sol transforms into a gel before forming the gel) of the temperature-controlled gel is preferably 35°C or less, more preferably 30°C or less, further preferably 25°C or less, and particularly preferably 20°C or less. There is no particular limitation on the lower limit of this temperature as long as it is above 0°C. From the viewpoint of stable gelation within a temperature range that prevents cell freezing, 4°C or more is preferred, and from the viewpoint of maintaining the gelled state even when the temperature rises during transport, 10°C or more is more preferred.

[0088] The sol-gel transition temperature can be determined according to the method described in Test Example 10 below.

[0089] The solubilizable gel preferably forms a sol with a viscosity of 20 mPa·s or less after solubilization. From the viewpoint of improving the diffusivity of the target substance and facilitating faster and more stable determinations, and further from the viewpoint of improving operability during cell seeding, this viscosity is preferably 15 mPa·s or less, more preferably 10 mPa·s or less, even more preferably 8 mPa·s or less, even more preferably 6 mPa·s or less, and particularly preferably 5 mPa·s or less. There is no particular limitation on the lower limit of this viscosity; for example, 0 mPa·s, 0.1 mPa·s, 0.2 mPa·s, 0.5 mPa·s, or 1 mPa·s.

[0090] Viscosity can be determined according to or based on the methods described in Test Example 8 or Test Example 9. The value determined by either method can be used as the viscosity described above. The viscosity is preferably the value determined according to or based on the method described in Test Example 9 (i.e., the viscosity after standing for 2 days at the sol-gelling temperature). This relatively low viscosity indicates that the viscosity increase over time after sol-gelling is suppressed, resulting in excellent workability.

[0091] There is no particular limitation on the storage elastic modulus of the sol-gelled gel at 4°C, for example, 0.001 to 100 kPa. From the viewpoint of maintaining cells while inhibiting external forces on cells in contact with the gel (i.e., inhibiting cell damage), 0.01 to 20 kPa is preferred, more preferably 0.02 to 10 kPa, further preferably 0.05 to 5 kPa, and even more preferably 0.07 to 2 kPa.

[0092] The storage modulus can be determined according to the method described in Test Example 10 below.

[0093] 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.

[0094] The gel-forming component can be a single component or a combination of two or more components.

[0095] In one aspect of this disclosure, the gelatin content in the soluble gel 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, especially preferably 95% by mass or more (particularly 100% by mass) relative to 100% by mass of the gel-forming component.

[0096] In one aspect of this disclosure, the gelatin content in the soluble gel 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, especially preferably 95% by mass or more (particularly 100% by mass) relative to 100% by mass of the solid portion constituting the gel.

[0097] In the case of a soluble gel containing gelatin, from the viewpoint of easily exhibiting the aforementioned desired properties, the concentration of gelatin in the gel is preferably 0.5 to 10% by mass, more preferably 0.7 to 8% by mass, further preferably 0.8 to 6% by mass, even more preferably 0.9 to 4% by mass, and particularly preferably 1 to 3.5% by mass.

[0098] The solvent for the solubilized gel 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, especially preferably 95% by mass or more (particularly 100% by mass).

[0099] In one embodiment, this disclosure relates to a sol-gellizable gel and a sol forming the gel. More specifically, the gel may be a gellizable by temperature control and satisfying at least one (preferably two, more preferably three, particularly preferably five) of the following requirements:

[0100] (a) The sol-gelation temperature is within a certain range.

[0101] (b) The sol-gel transition temperature is within a certain range.

[0102] (c) After sol-gelation, it can form a sol with a viscosity within a certain range.

[0103] (d) The storage elastic modulus at 4℃ is within a certain range, and

[0104] (e) The viscosity of gelled at 4°C and then re-gelled at 25°C is within a certain range.

[0105] The cell chip disclosed herein comprises cells and soluble gels.

[0106] The form of the partitions is not particularly limited as long as it preserves the cells and gel. From the viewpoints of cell desiccation resistance, cell retention, preparation efficiency, or chemical detection, pore-shaped partitions are preferred.

[0107] There are no particular restrictions on the material used for partitioning, as long as it can preserve the cells and gel. Materials can include, for example, resin and metal.

[0108] Within the partitions, there are no particular restrictions on the configuration of cells and gels, as long as the cell's resistance to desiccation and its retention are guaranteed. For example, in a partition, cells are encapsulated in a gel. There are no particular restrictions on the encapsulation method, as long as at least a portion of the cells are isolated from the external air outside the cell chip by the gel.

[0109] From a detection sensitivity perspective, partitions typically contain multiple cells. The area per cm² of a partition... 2 The cell count (cells) is, for example, 5,000 to 2,000,000 cells / cm³. 2 From the perspectives of detection sensitivity and cell viability, a concentration of 10,000–1,500,000 cells / cm² is preferred. 2 More preferably, 10,000 to 1,000,000 cells / cm³ 2 Further optimization is needed to select cells with a density of 20,000 to 1,000,000 cells / cm³. 2 Further optimization yields 50,000 to 700,000 cells / cm³. 2 Especially preferred is 100,000 to 500,000 cells / cm³. 2 .

[0110] From the perspective of detection sensitivity or preparation efficiency, the bottom area of ​​partition 1 is preferably 0.5–100 mm². 2 More preferably 1 to 30 mm 2 Further optimization is preferred, with a diameter of 1.5–15 mm. 2 Furthermore, in one embodiment of the invention, the upper limit of the area can be 80 mm². 2 60mm 2 Or 40mm 2 .

[0111] From the perspective of detection sensitivity or preparation efficiency, the number of partitions contained in the cell chip is preferably 10 to 2000, more preferably 30 to 1000, and even more preferably 50 to 500.

[0112] The cell chip disclosed herein 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.

[0113] The cell chip disclosed herein can be prepared by gelling the sol in a partition containing a sol and cells that form a sol-gel. The sol can be obtained according to or based on known methods. When the gel-forming component is gelatin, from the viewpoint of easily obtaining a gel / sol with the aforementioned preferred properties, it is preferable to add powdered gelatin to a solvent, stir at 12–30°C (preferably 15–25°C) for 10–60 minutes (preferably 20–40 minutes), then raise the temperature to the gelatin's dissolution temperature (e.g., 30–60°C) and stir until completely dissolved.

[0114] The cell chip disclosed herein can be applied to the detection of chemical substances, particularly odor substances. Chemical substances 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 a sample to a partition of the cell chip of this disclosure, the chemical substances in the sample can reach the cells and come into contact with the cell's sensor proteins. For example, the chemical substances can be detected by detecting ions flowing into the cells (e.g., by detection by proteins that produce color or light from ions).

[0115] As described above, rapid and stable assays of the cell microarray disclosed herein are made possible by sol-gelling the gel. Therefore, the cell microarray of this disclosure can be used in methods for detecting a chemical substance, wherein the method involves contacting the cells with the chemical substance after sol-gelling the gel. After sol-gelling, the chemical substance can be contacted with the cells in the presence of the sol, or the sol can be removed, and other liquids can be added to the compartments as needed before contacting the chemical substance with the cells. In the case of detecting luminescence from cells (by adding luminescence via a substrate (coelin, etc.), the problem of slow and stable assays is more significant. The technology of this invention can be more preferably utilized in this case, where the aforementioned problem can be more effectively eliminated by removing the sol after sol-gelling.

[0116] This disclosed kit for cell fixation includes a solubilizable gel component and reagents and / or apparatus (e.g., cell culture containers) for fixing cells with the gel. The solubilizable gel component included in this kit may be in powder, gel, or sol form. The cell culture container included in this kit has compartments for cell fixation. The bottom area of ​​each compartment is not particularly limited. From the viewpoint of detection sensitivity or preparation efficiency, a size of 0.5–100 mm² is preferred. 2 More preferably 1-30mm 2 Further optimization is preferred, with a diameter of 1.5–15 mm. 2 Furthermore, in one embodiment of the invention, the upper limit of the area can be 80 mm².2 60mm 2 Or 40mm 2 .

[0117] As described above, by including the step of immobilizing cells with a solubilized gel, transport cells that can be transported in a stable state can be prepared. The gel immobilizing the transport cells can also be solubilized and removed after transport and before use. Thus, after stable transport, it becomes possible to utilize the cells in the absence of a gel / sol, and the detection of light from the cells, which is problematic in cases of high gel / sol turbidity, can be performed effectively. Therefore, in one aspect, this disclosure relates to a method for preparing transport cells, which includes immobilizing cells with a solubilized gel. Furthermore, in another aspect, this disclosure relates to a method for detecting light from cells, which includes solubilizing the solubilized gel immobilizing the cells. In the method for detecting light, after solubilization, treatment to induce cell luminescence (e.g., substance addition, light irradiation, etc.) can be performed as needed.

[0118] Example

[0119] The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0120] Test Example 1. Preparation of stable expression cells

[0121] 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.

[0122] Test Example 2. Preparation of gelatin solution

[0123] Powdered fish gelatin (Nitta Gelatin Co., Ltd. Fish Gelatin (Type A)) was added to the PBS solution and stirred at 17°C for 30 minutes. The solution was then heated to 35°C and stirred until completely dissolved to obtain a gelatin solution of the specified concentration. After sterilization through a 0.22 μm filter or autoclave, it was used in the following tests.

[0124] Test Example 3. Preparation of acrylic resin solution

[0125] A MO3N6O / 2AAmLN aqueous solution was prepared by dissolving 2-[[2-(methacryloyloxy)ethyl]dimethylammonium]acetate (monomer: MO3N6O) and N,N'-[oxybis(2,1-ethylenedioxy-3,1-propanediyl)]bisacrylamide (crosslinking agent: 2AAmLN) in an aqueous PBS (Nippon Sui Pharmaceutical #05913). An LPA aqueous solution was prepared by dissolving lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (photopolymerization initiator: LPA) in an aqueous PBS. The two aqueous solutions were mixed and stirred to obtain an acrylic resin solution with a final concentration of 0.8% by mass for MO3N6O, 5.2% by mass for 2AAmLN, and 0.02% by mass for LPA.

[0126] Test Example 4. Assay 1 of olfactory receptor activity

[0127] ORA cells were cultured in 96-well CORNING3903 at a rate of 5 × 10⁶ cells / well. 4 Inoculate with 100 μL per well and incubate at 27°C (without CO2 supply). Use Sf-900III SFM medium. Perform the following steps 24 hours after inoculation.

[0128] (Experimental Group 1) The culture medium was removed from each well of the 96-well plate, and 40 μL of acrylic resin solution was added to each well. UV light irradiation was applied using a device (CCS Corporation 365 nm LED light source) at 110 mW / cm². 2 Irradiation with 365 nm light for 30 seconds formed a hydrogel. 210 μL of culture medium was added to the top of the hydrogel, followed by two washes with the medium.

[0129] (Experimental Group 2) No gel formed and continued culturing.

[0130] 24 hours after the above operation, remove all culture medium. Add 40 μL of 0.1% BSA / 1×Hanks' buffer / 20mM HEPES buffer to each well in experimental group 1 and 80 μL to each well in experimental group 2. Add compound a (the substance that responds to ORA) to each well to a final concentration of 100 μM. Quantify the change in fluorescence intensity before and after addition using a microplate reader (FlexStation3, Molecular Devices).

[0131] The results are shown in Figure 1In the absence of hydrogel (Experimental Group 2), the fluorescence intensity increased sharply after the addition of compound a, allowing for rapid and stable measurement of fluorescence intensity. On the other hand, in the presence of hydrogel (Experimental Group 1), the increase in fluorescence intensity after the addition of compound a was gradual, making rapid and stable measurement of fluorescence intensity impossible.

[0132] Test Example 5. Assay 2 of olfactory receptor activity

[0133] ORA cells were cultured in 96-well CORNING3903 at a rate of 5 × 10⁶ cells / well. 4 Inoculate with 100 μL per well and incubate at 27°C (without CO2 supply). Use Sf-900III SFM medium. Perform the following steps 24 hours after inoculation.

[0134] (Experimental Group A) The culture medium was removed from each well of the 96-well plate, and 40 μL of a gelatin solution (5% fish gelatin sol) at 25°C was added to each well. A gel was formed by standing at 4°C for 3 hours.

[0135] (Experimental Group B) is the same as Experimental Group A.

[0136] (Experimental Group C) No gel formed and continued culturing.

[0137] Following the above procedures, for experimental groups A and B, the gel was dissolved by standing at 27°C for 1 hour. For experimental group A, without removing the sol, 40 μL of 0.1% BSA / 1×Hanks' buffer / 20 mM HEPES buffer was added to each well. For experimental groups B and C, after removing the sol or culture medium, 80 μL of 0.1% BSA / 1×Hanks' buffer / 20 mM HEPES buffer was added to each well. Compound a (the ORA-responsive substance) was added to each well at a final concentration of 100 μM, and the change in fluorescence intensity before and after addition was quantified using a microplate reader (FlexStation3, Molecular Devices).

[0138] The results are shown in Figure 2 In the case of gel sol-gelation (experimental group A), the increase in fluorescence intensity after the addition of compound a was sharp, allowing for rapid and stable measurement of fluorescence intensity. Furthermore, based on a comparison of experimental groups B and C, gel fixation of cells had almost no effect on the speed and stability of chemical substance detection.

[0139] Test Example 6. Assay 3 of olfactory receptor activity

[0140] ORA cells were cultured in 96-well CORNING3903 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.

[0141] Twenty-four hours after inoculation, the culture medium was removed from each well of the 96-well plate, and 40 μL of a gelatin solution (2-8% by mass fish gelatin sol) at 25°C was added to each well. A gel was formed by incubation at 4°C for at least 1 hour. 100 μL of culture medium was added to the top of the gel. A group was also prepared that continued culturing with 100 μL of culture medium without replacing the gelatin solution. After gel formation, the plates were stored at 4°C.

[0142] 72 hours after gel formation, the gel was solubilized by standing at 27°C for 1 hour. After removing the sol, 80 μL of 0.1% BSA / 1×Hanks' buffer / 20 mM HEPES buffer was added to each well. Compound a (the ORA-responsive substance) 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 quantified using a microplate reader (FlexStation3, Molecular Devices).

[0143] The results are shown in Figure 3 Chemical detection is possible for all cases involving the use of gels of various concentrations. Furthermore, chemical detection is possible even when cells are fixed in hydrogels and stored at 4°C.

[0144] Test Example 7. Assay 4 of olfactory receptor activity

[0145] ORA cells were cultured in 96-well CORNING3903 at a rate of 5 × 10⁶ cells / well. 4 Inoculate with 100 μL per well and incubate at 27°C (without CO2 supply). Use Sf-900III SFM medium.

[0146] Twenty-four hours after inoculation, the culture medium was removed from each well of the 96-well plate, and 40 μL of a 25°C gelatin solution (5% by mass fish gelatin sol) was added to each well. A gel was formed by standing at 4°C for 3 hours. Additionally, a group was prepared that continued culturing with 100 μL of culture medium without replacing the gelatin solution.

[0147] After gel formation, the gel was immediately dissolved by standing at 27°C for 1 hour. After removing the sol, 80 μL of 0.1% BSA / 1×Hanks' buffer / 20 mM HEPES buffer was added to each well. Alternatively, after dissolution, 40 μL of 0.1% BSA / 1×Hanks' buffer / 20 mM HEPES buffer was added without removing the sol. 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 change in fluorescence intensity before and after addition was quantified using a microplate reader (FlexStation3, MolecularDevices).

[0148] The results are shown in Figure 4 The detection of chemical substances is possible regardless of whether a sol removal process is performed. However, the detection sensitivity is high when the sol is removed.

[0149] Test Example 8. Assay 1 of viscosity of sol

[0150] Add gelatin solution (2-5% by mass sol of fish gelatin) to the sample tubes for the purpose of eliminating thermal history, and let stand in a column oven at 40°C for 1 hour. Transfer the sample tubes to an incubator at 25°C and let stand for 30 minutes. Store the sample tubes in a refrigerator at 4°C for 1 day to gel. Transfer the sample tubes to an incubator at 25°C and let stand for 1 hour to gel. Measure the viscosity at 25°C using an EMS-1000 viscometer manufactured by Kyoto Electronics Co., Ltd., with a 2mm aluminum probe.

[0151] The results are shown in Figure 5 The gel formed from the gelatin solution (2-5% fish gelatin sol) becomes a sol with relatively low viscosity after solification.

[0152] Test Example 9. Assay 2 of viscosity of sol

[0153] Add gelatin solution (2-5% by mass sol of fish gelatin) to the sample tubes for the purpose of eliminating thermal history, and let stand in a column oven at 40°C for 1 hour. Transfer the sample tubes to an incubator at 25°C and let stand for 30 minutes. Store the sample tubes in a refrigerator at 4°C for 1 day to gel. Transfer the sample tubes to an incubator at 25°C and let stand for 2 days to gel. Measure the viscosity at 25°C using an EMS-1000 viscometer manufactured by Kyoto Electronics Co., Ltd., with a 2mm aluminum probe.

[0154] The results are shown in Figure 6 Based on the results measured 1 hour after sol-gelation. Figure 5 and as measured 2 days after sol-gelation Figure 6 The comparison shows that at higher concentrations, after sol-gelation, the viscosity increases over time.

[0155] Test Example 10. Assay of storage elastic modulus and sol-gel transition temperature of gel

[0156] Anton Paar Japan's MCR302 viscoelasticity measuring apparatus was used with a CP50-1 plate. Measurements were performed with a sample volume of 1 ml, a frequency of 1 Hz, and a deformation of 1%. Viscosity was initially measured at 25°C, followed by temperature dispersion measurement by cooling to 4°C at a rate of -1°C / min. The point where the loss tangent was less than 1 was taken as the sol-gel transition point. Then, the storage modulus was measured 10 minutes after the temperature reached 4°C.

[0157] The results are shown in Figure 7 See Table 1. The storage elastic modulus at 4°C is 5wt%: 1.3kPa, 4wt%: 1.1kPa, 3wt%: 0.3kPa, 2wt%: 0.1kPa, and 1wt%: 0.002kPa.

[0158] [Table 1]

[0159]

Claims

1. Cell chip, which contains partitions containing cells and soluble gel.

2. The cell chip according to claim 1, wherein, The gel is a gel that is soluble through temperature control, light irradiation, or the addition of pharmaceutical agents.

3. The cell chip according to claim 2, wherein, The gel is a solubilized gel produced through temperature control.

4. The cell chip according to claim 3, wherein, The sol-gel temperature of the gel is below 40°C.

5. The cell chip according to claim 3, wherein, The sol-gel temperature of the gel is below 30°C.

6. The cell chip of claim 3, wherein, The sol-gel transition temperature of the gel is 4–25°C.

7. The cell chip of claim 1, wherein, The gel forms a sol with a viscosity of less than 20 mPa·s after solification.

8. The cell chip of claim 1, wherein, The storage elastic modulus of the gel at 4°C is 0.01–20 kPa.

9. The cell chip according to any one of claims 1 to 8, for the detection of a chemical substance to be tested, the method comprising sol-gelling the gel and then contacting the cells with the chemical substance to be tested.

10. The cell chip according to any one of claims 1 to 8, for detecting light from said cells.

11. The cell chip according to any one of claims 1 to 8, wherein, The cells in question are insect cells.

12. The cell chip according to any one of claims 1 to 8, wherein, The cells contain exogenous polynucleotides that encode sensor proteins.

13. The cell chip of claim 12, wherein, The sensor protein is an olfactory receptor protein.

14. A kit for cell fixation containing a soluble gel-forming component.

15. The kit of claim 14, which is a kit for immobilizing cells on a cell chip.

16. A method for preparing transport cells, comprising fixing cells with a soluble gel.

17. A method for detecting light from cells, comprising solubilizing a soluble gel of immobilized cells.

18. A sol-gelled gel, wherein the gel: Solvable by temperature control. The sol-gel temperature is below 40℃. The sol-gel transition temperature is 4–25℃. After sol-gelation, it can form a sol with a viscosity of less than 20 mPa·s, and The storage elastic modulus at 4℃ is 0.01–20 kPa.

19. A sol that forms the gel of claim 18.

20. The kit of claim 14, comprising the gel of claim 18 and / or the sol of claim 19.

21. The kit of claim 20, which is a kit for immobilizing cells on a cell chip.

22. The preparation method according to claim 16, wherein, The gel is the gel according to claim 18.

23. The method of claim 17, wherein, The gel is the gel according to claim 18.

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