Laminate

Abstract

Provided is a detection technique excellent in cell drying resistance, cell retention, and detection of a chemical substance such as an odorant. More specifically, provided are: a laminate containing

Description

Title of Invention: LAMINATE Technical Field

[0001] The present invention relates to a layered construct containing cells and the like. Background Art

[0002] Groups of odorous substances associated with specific human diseases and mental states have been identified. Owing to their high value as test markers, there has been growing interest in developing various odor sensors that target these substances. Biological odorant receptors exhibit excellent characteristics, including diversity, sensitivity, and selectivity, which are not found in conventional odor sensor elements such as semiconductors; thus, these odorant receptors offer a promising foundation for the development of novel odor sensors using them as sensor elements.

[0003] PTL 1 discloses the use of cells expressing modified odorant receptors or lipid bilayer membranes incorporating modified odorant receptors as odor sensors. Citation List Patent Literature

[0004] PTL 1: WO2022 / 024902A Summary of Invention Technical Problem

[0005] Odor sensors that rely on the process of artificially preparing lipid bilayer membranes equipped with sensor proteins (e.g., odorant receptors) are often inefficient to produce, and there has been demand for further improvement in the production efficiency of odor sensors. The inventors then focused on using cells that express sensor proteins.

[0006] From the perspective of convenience in use, the cells used as a chemical sensor such as an odor sensor are desirably those prepared in advance and retained in containers, which can be used when needed, rather than cells that are cultured and prepared each time for use. In the former case, it is necessary to keep the cells from drying out until they are used, and it is also important that the cells are stably maintained, considering factors such as transportation from the manufacturer to the place of use.

[0007] Accordingly, an object of the present disclosure is to provide detection technology excellent in cell desiccation resistance, cell retention, and detection of chemical substances, such as odorants . Solution to Problem

[0008] In the course of conducting research, the present inventors focused on the fact that a cell chip containing compartments each containing cells and a hydrogel can prevent cells from drying out and stably retain the cells. From the perspective of mass production and production efficiency, it is desirable to use a hydrogel in which cells are dispersed. However, hydrogels containing dispersed cells were found to have low detectability of chemical substances. Based on this finding, the inventors discovered that a layered construct comprising a cell layer and a gel layer placed on the cell layer could achieve the object and solve the problem. Specifically, the present disclosure encompasses the following embodiments.

[0009] Item 1. A layered construct comprising a cell layer and a gel layer placed on the cell layer.

[0010] Item 2. The layered construct according to Item 1, wherein the cell layer and the gel layer are each a monolayer.

[0011] Item 3. The layered construct according to Item 1, wherein the cell layer has a cell concentration of 10000 to 1000000 cells / cm2.

[0012] Item 4. The layered construct according to Item 1, wherein a gel forming the gel layer has a storage elastic modulus of 0.001 to 100 kPa.

[0013] Item 5. The layered construct according to Item 1, which is placed within a compartment.

[0014] Item 6. The layered construct according to Item 5, wherein the cell layer is in contact with the bottom surface of the compartment.

[0015] Item 7. The layered construct according to Item 1, wherein a cell contained in the cell layer is an insect cell.

[0016] Item 8. The layered construct according to Item 1, wherein a cell contained in the cell layer comprises a polynucleotide containing a coding sequence for a sensor protein.

[0017] Item 9. The layered construct according to Item 8, wherein the sensor protein is an odorant receptor protein.

[0018] Item 10. The layered construct according to Item 1, for use in measurement of sensor protein activity.

[0019] Item 11. The layered construct according to Item 10, for use in the measurement by adding a substance that alters the sensor protein activity from the gel-layer side.

[0020] Item 12. The layered construct according to Item 10, for use in the measurement wherein the sensor protein activity is converted into light.

[0021] Item 13. The layered construct according to Item 12, wherein the light is detected on the cell-layer side.

[0022] Item 14. A cell chip comprising a compartment that contains the layered construct of any one of Items 1 to 13.

[0023] Item 15. The cell chip according to Item 14, wherein the upper part of the compartment is sealed.

[0024] Item 16. The cell chip according to Item 14 or 15, which is packaged.

[0025] Item 17. A method for producing the layered construct of any one of Items 1 to 13, comprising a cell layer formation step comprising placing a cell suspension, and a gel layer formation step comprising forming a gel layer on a cell layer.

[0026] Item 18. suspension The method according to Item 17, wherein the cell is a sol capable of forming a gel.

[0027] Item 19. The method according to Item 17, comprising the cell layer formation step comprising placing a suspension of a cell in a sol capable of forming a gel on a substrate, and allowing the cell to settle on the substrate, and the gel layer formation step comprising gelling the sol.

[0028] Item 20. The method according to Item 19, wherein the substrate comprises a compartment with a polypeptide-coated surface.

[0029] Item 21. A layered construct obtained according to the method of Item 20, the layered construct being placed on the substrate.

[0030] Item 22. The method according to Item 17, comprising the cell layer formation step comprising placing a suspension of a cell in a liquid that does not form a gel on a substrate and allowing the cell to settle on the substrate, and the gel layer formation step comprising stacking a sol capable of forming a gel on a cell layer and then gelling the sol.

[0031] Item 23. The method according to Item 22, wherein the substrate comprises a compartment with a surface having an oxygen-to-carbon molar ratio (O / C) of 0.150 or more.

[0032] Item 24. A layered construct obtained according to the method of Item 23, the layered construct being placed on the substrate. Advantageous Effects of Invention

[0033] The present disclosure provides detection technology excellent in cell desiccation resistance, cell retention, and detection of chemical substances, such as odorants. Specifically, the present disclosure provides a layered construct containing cells and a cell chip containing the layered construct. Brief Description of Drawings

[0034] Fig. 1 shows the results of measuring the activity of an odorant receptor in Test Example 6. The vertical axis indicates the value determined by subtracting the average background value (the average of a period of 20 seconds before the compound was added) from the maximum fluorescence intensity. On the horizontal axis, "Gel A," "Gel B," and "Gel C" indicate the cases in which acrylic resin solutions A, B, and C were used, respectively. Fig. 2 shows the results of measuring the activity of an odorant receptor in Test Example 7. The vertical axis indicates the value determined by subtracting the average background value (the average of a period of 20 seconds before the compound was added) from the maximum fluorescence intensity. On the horizontal axis, "Gel A", "Gel B," and "Gel C" indicate the cases in which acrylic resin solutions A, B, and C were used, respectively. Fig. 3 shows images of cells observed immediately after a cell-seeded plate was left to stand at 27°C overnight in Test Example 9-1. The gelatin concentration in the medium is shown on the left of the photographic images. Fig. 4 shows images of cells observed after the medium was replaced with an assay buffer in Test Example 9-1. The gelatin concentration in the medium is shown on the left of the photographic images . Fig. 5 shows the results of measuring the activity of an odorant receptor in Test Example 9-1. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the gelatin concentration in the medium. The legend indicates the concentration of compound a. Fig. 6 shows images of cells observed immediately after a cell-seeded plate was left to stand at 27°C overnight in Test Example 9-2. The gelatin concentration in the medium is shown on the left of the photographic images. Fig. 7 shows images of cells observed after the medium was replaced with an assay buffer in Test Example 9-2. The gelatin concentration in the medium is shown on the left of the photographic images . Fig. 8 shows the results of measuring the activity of an odorant receptor in Test Example 9-2. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the gelatin concentration in the medium. The legend indicates the concentration of compound a. Fig. 9 shows images of cells observed immediately after a cell-seeded plate was left to stand at 27°C overnight in Test Example 9-3. The gelatin concentration in the medium is shown on the left of the photographic images. Fig. 10 shows images of cells observed after the medium was replaced with an assay buffer in Test Example 9-3. The gelatin concentration in the medium is shown on the left of the photographic images. Fig. 11 shows the results of measuring the activity of an odorant receptor in Test Example 9-3. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the gelatin concentration in the medium. The legend indicates the concentration of compound a. Fig. 12 shows images of cells observed immediately after a cell-seeded plate was left to stand at 27°C overnight in Test Example 9-4. The gelatin concentration in the medium is shown on the left of the photographic images. Fig. 13 shows images of cells observed after the medium was replaced with an assay buffer in Test Example 9-4. The gelatin concentration in the medium is shown on the left of the photographic images. Fig. 14 shows the results of measuring the activity of an odorant receptor in Test Example 9-4. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the gelatin concentration in the medium. The legend indicates the concentration of compound a. Fig. 15 shows images of cells observed in Test Example 10-2. The plate types at the top of the images and cell counts 1 and 2 on the left of the images correspond to those in Table 1. Fig. 16 shows the results of measuring the activity of an odorant receptor in Test Example 10-2. The vertical axis indicates fluorescence intensity. The horizontal axis indicates the plate type and cell count. The plate types correspond to those in Table 1. The legend indicates the concentration of compound a. Description of Embodiments

[0035] In the present specification, the terms "comprising," "containing," and "including" include the concepts of comprising, containing, consisting essentially of, and consisting of.

[0036] In an embodiment, the present disclosure relates to a layered construct comprising a cell layer and a gel layer placed on the cell layer ("the layered construct of the present disclosure" in the present specification). The following provides an explanation of the layered construct.

[0037] The cell layer is not particularly limited as long as it is a layer containing cells.

[0038] The cell chip is not particularly limited as long as it includes one or more compartments each containing cells.

[0039] The cell is not particularly limited. From the perspective of chemical detection suitability, the cell is preferably animal cells such as insect cells or mammalian cells, with insect cells being particularly preferred due to the ease of management, such as not requiring CO2 or temperature control.

[0040] The insect cells for use include, for example, Sf cells, MG1 cells, High Five™ cells, and BmN cells. Sf cells for use include, for example, Sf9 cells (ATCC CRL1711) and Sf21 cells. Of the insect cells, those derived from insects of the family Arctiidae are particularly preferred.

[0041] The cells derived from insects of the family Arctiidae are not particularly limited as long as they are primary cultured cells or established cell lines of biological constituent cells derived from insects of the family Arctiidae.

[0042] Examples of the family Arctiidae include subfamilies such as Arctiinae, Lithosiinae, and Syntominae, among which the subfamily Arctiinae is preferred. The subfamily Arctiinae is, for example, preferably the genus Spilosoma, Spilarctia, or Rhagonis, with the genus Spilosoma being particularly preferable. While there is no particular limitation on the genus Spilosoma, Spilosoma imparilis is particularly preferable.

[0043] The cells derived from insects of the family Arctiidae can be obtained from known biological banks or collected and cultured from living insects of the family Arctiidae according to or with reference to known methods; if necessary, they can be established as cell lines.

[0044] Examples of cells derived from Spilosoma imparilis include FFPRI-Splm-2AM-SF cells (MAFF No.: 275052) and FFPRI- Splm-2AM-IPL411 cells (MAFF No.: 275053) from the Genebank Project, National Agriculture and Food Research Organization.

[0045] The cell preferably contains an exogenous polynucleotide containing a coding sequence for a sensor protein. This enables the expression of any sensor protein and enables increased expression levels of a target sensor protein, thereby enhancing the detection sensitivity for a target chemical substance.

[0046] The "exogenous polynucleotide" is not particularly limited as long as it refers to a polynucleotide containing a base sequence that is not derived from the genomic DNA (in particular, chromosomal genomic DNA) of an insect cell.

[0047] In the present specification, the polynucleotide includes not only typical polynucleotides such as DNA and RNA inherent in organisms, but also polynucleotides with known chemical modifications, artificial polynucleotides, and like polynucleotides, as listed below. To prevent degradation due to hydrolases such as nucleases, the phosphate residue of each nucleotide can be substituted with a chemically modified phosphate residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. The hydroxyl group at position 2 of the ribose of each ribonucleotide may also be substituted with -OR (R indicates, for example, CH3 (2'-O-Me) , CH2CH2OCH3 (2 '-O-MOE) , CH2CH2NHC (NH)NH2, CH3CONHCH3, or CH2CH2CN) . Additionally, the nucleobase moiety (pyrimidine, purine) may be chemically modified, by, for example, introduction of a methyl group or a cationic functional group into position 5 of the pyrimidine base, or substitution of the carbonyl group at position 2 with thiocarbonyl. Additionally, the polynucleotide of the present invention also includes, but is not limited to, those formed by modifying the phosphate moiety or the hydroxyl moiety, for example, with biotin, an amino group, a lower alkyl amine group, or an acetyl group. The polynucleotide for use can also be, for example, BNA (LNA), which is prepared by crosslinking the 2' oxygen and the 4' carbon in the ribose moiety of a nucleotide to fix the ribose moiety in N-conformation.

[0048] The sensor protein may be selected from proteins capable of detecting the presence of a chemical substance, such as a receptor protein that uses a chemical substance as a ligand. The sensor protein is particularly preferably an odorant receptor protein.

[0049] The insect odorant receptor protein is a membrane protein with a seven-transmembrane structure and functions as an odor sensor in the organisms. The odorant receptor protein is composed of the following components that are sequentially linked from the amino terminus ("N-terminus" below) to the carboxyl terminus ("C-terminus" below): N-terminal region (NT), first transmembrane domain (TM1), first extracellular loop (ECI), 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 the present disclosure, each region is determined by structural prediction using TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membrane spanning proteins segments, Biol. Chern. Hoppe-Seyler, 374 (1993), p. 166, https: / 1embnet.vital-it. ch / softwareZTMPRED_form.html) (with default conditions).

[0050] The insect of origin for the insect odorant receptor protein is preferably an insect in the order Diptera, such as the family Culicidae and the family Drosophilidae; an insect in the order Lepidoptera, such as the family Bombycidae; an insect in the order Hymenoptera, such as the family Apidae; an insect in the order Orthoptera, such as the family Acrididae; and an insect in the order Hemiptera, such as the family Cimicidae, and more preferably an insect in the order Diptera, such as the family Culicidae and the family Drosophilidae; an insect in the order Orthoptera, such as the family Acrididae; and an insect in the order Hemiptera, such as the family Cimicidae. Examples of insects in the family Culicidae include Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. Examples of insects in the family Drosophilidae include Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila virilis. Examples of insects in the family Bombycidae include Bornbyx mori, Bornbyx mandarina, and Trilocha varians. Examples of insects in the family Apidae include Apis mellifera, Apis florea, Apis dorsata, and Bombus terrestris. Examples of insects in the family Acrididae include Locusta migratoria. Examples of insects in the family Cimicidae include Cimex lectularius.

[0051] Specifically, examples of wild-type insect odorant receptor proteins include the following: AaORl, AaOR2, AaOR4, AaOR5, AaOR6, AaOR8, AaOR9, AaORl0a, AaORl5, AaOR22, AaOR24, AaOR25, AaOR2 6, AaOR27, AaOR28, AaOR30, AaOR34, AaOR36, AaOR38, AaOR41a, AaOR41b, AaOR42, AaOR43, AaOR44, AaOR47, AaOR49, AaOR50, AaOR52, AaOR54, AaOR58, AaOR59, AaOR60, AaOR61, AaOR64, AaOR65, AaOR66, AaOR67a, AaOR69a, AaOR70, AaOR71, AaOR72a, AaOR73, AaOR74, AaOR75, AaOR77, AaOR78, AaOR79, AaOR81, AaOR83b, AaOR84, AaOR85, AaOR86, AaOR87, AaOR91, AaOR95, AaOR97, AaOR96, AaOR99, AaORlOO, AaOR102, AaOR103, AaOR104a, AaOR105, AaOR107, AaOR108, AaOR109, AaORllO, AaOR112, AaOR114, AaORllS, AaOR117, AaOR118, AaOR122, AaOR125, AaOR128, AgORl, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR8, AgOR9, AgORlO, AgORlla, AgOR12a, AgOR12b, AgOR13, AgORl 4, AgORl 5, AgORl 6a, AgORl 7, AgORl 8, AgOR20, AgOR21, AgOR23, AgOR25, AgOR2 6, AgOR27, AgOR28, AgOR30, AgOR34, AgOR36, AgOR37, AgOR38, AgOR39a, AgOR40, AgOR42, AgOR44, AgOR45, AgOR4 6, AgOR47, AgOR49, AgOR50, AgOR54, AgOR56a, AgOR57, AgOR60, AgOR61, AgOR62, AgOR63, AgOR64, AgOR65, AgOR69, AgOR70, AgOR71, AgOR72, AgOR74, AgOR75, AgOR76a, AmORl, AmOR3, AmOR9, AmORlO, AmOR13, AmOR41, AmOR51, AmOR52, AmOR55, AmOR71, AmOR73, AmOR78, AmOR85, AmOR89, AmOR90, AmOR114, AmOR115, AmOR118, AmOR120, AmOR121, AmOR161, BmORl, BmOR2, BmOR3, BmOR4, BmOR5, BmOR8, BmOR9, BmORlO, BmOR13, BmORl7, BmORl8, BmOR23, BmOR24, BmOR25, BmOR35, BmOR36, BmOR42, BmOR45, BmOR49, BmOR51, BmOR52, BmOR55, BmOR56, BmOR61, DmORla, DmOR9a, DmOR19a, DmOR22a, DmOR22b, DmOR22c, DmOR24a, DmOR30a, DmOR33a, DmOR33b, DmOR33c, DmOR35a, DmOR42b, DmOR43a, DmOR45a, DmOR45b, DmOR47a, DmOR49b, DmOR59b, DmOR65b, DmOR65c, DmOR67b, DmOR67c, DmOR69a, DmOR71a, DmOR74a, DmOR82a, DmOR83a, DmOR83c, DmOR85a, DmOR85c, DmOR85e, DmOR85f, DmOR88a, DmOR92a, DmOR94a, DmOR94b, and DmOR98b.

[0052] In the present specification, OR denotes "odorant receptor." Dm indicates derivation from Drosophila melanogaster, Bm indicates derivation from Bombyx mori, Ag indicates derivation from Anopheles gambiae, and Aa indicates derivation from Aedes aegypti. The amino acid sequences of various odorant receptor proteins, including these, and their coding sequences are either known or can be readily identified through sequence identity searches based on known sequences.

[0053] The sensor protein may contain one or more amino acid mutations in its wild-type amino acid sequence, provided that the chemical response activity is not significantly decreased. The phrase "not significantly decreased" means, for example, that the chemical response activity of a sensor protein containing amino acid mutations is, for example, at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, and yet more preferably at least 90% of the chemical response activity of the wild-type sensor protein, taken as 100%.

[0054] The amino acid mutation is, for example, substitution, insertion, addition, or deletion of an amino acid, preferably substitution, and particularly preferably conservative substitution.

[0055] In the present specification, "conservative substitution" refers to the substitution of an amino acid residue with another amino acid residue having a similar side chain. For example, the substitution between amino acid residues having a basic side chain such as lysine, arginine, or histidine is considered to be a conservative substitution. The following substitutions between other amino acid residues are also considered to be a conservative substitution: the substitution between amino acid residues having an acidic side chain such as aspartic acid and glutamic acid; the substitution between amino acid residues having an uncharged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine; the substitution between amino acid residues having a nonpolar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; the substitution between amino acid residues having a |3-branched side chain such as threonine, valine, or isoleucine; and the substitution between amino acid residues having an aromatic side chain such as tyrosine, phenylalanine, tryptophan, or histidine.

[0056] The sensor protein may contain its wild-type amino acid sequence or an amino acid sequence having, for example, at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, and particularly preferably at least 99% identity to the wildtype amino acid sequence.

[0057] In the present specification, the "identity" of amino acid sequences refers to the degree to which two or more contrastable amino acid sequences match each other. Thus, the higher the degree of match between two amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, by using FASTA, which is a tool for sequence analysis, with default parameters. Alternatively, the level of amino acid sequence identity can be determined by using the BLAST algorithm by Karlin and Altschul (Karlin S., Altschul S. F. Methods for assessing the statistical significance of molecular sequence features by using general scorings schemes, Proc. Natl. Acad. Sci. USA. 87: 2264-2268 (1990); Karlin S., Altschul S. F. Applications and statistics for multiple high-scoring segments in molecular sequences, Proc Natl Acad Sci USA. 90: 5873-7 (1993)) . A program called "BLASTX," based on this BLAST algorithm, has been developed. The specific techniques of these analysis methods are known and can be found on the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0058] The sensor protein may contain other amino acid sequences, such as protein tags, fluorescent proteins, luminescent proteins, signal sequences, or other proteins or peptides, attached thereto, provided that the chemical response activity is not significantly impaired. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, and PA tag.

[0059] In the present specification, "chemical response activity" refers to the property of a sensor protein to recognize a chemical substance and initiate signal transduction activity (e.g., ion channel activity) either alone or in conjunction with other proteins. Tin odorant receptor functioning as a sensor protein may be a G protein-coupled receptor or an ion channel receptor. In the case of insect odorant receptors, "chemical response activity" refers to the property of an odorant receptor to recognize a chemical substance and form an odorant receptor complex, together with an olfactory receptor co-receptor, thereby exhibiting ion channel activity upon activation. The chemical response activity of a sensor protein can be measured by using the signal transduction activity of the sensor protein in contact with a chemical substance as an indicator (e.g., by quantifying and evaluating the amount of signal molecules). In the case of insect odorant receptors, their chemical response activity can be measured using the ion channel activity of an odorant receptor complex formed of an odorant receptor in contact with a chemical substance and an olfactory receptor co-receptor as an indicator. For example, a chemical substance is brought into contact with cells that express a protein that becomes fluorescent or luminescent in response to the influx of ions (e.g., calcium ions) into the cells when (a) an odorant receptor, (b) an olfactory receptor co-receptor, and (c) an odorant receptor complex respond, and then the amount of fluorescence or luminescence of the cells is measured. The greater the measured amount of fluorescence or luminescence, the higher the response activity of the odorant receptor to the chemical substance is determined to be. Specifically, the chemical response activity can be measured according to the method described in PTL 1.

[0060] The coding sequence for the sensor protein is not particularly limited as long as it is a base sequence encoding the sensor protein. In an embodiment, the exogenous polynucleotide contains an expression cassette for a sensor protein. The expression cassette is not particularly limited as long as it is a polynucleotide capable of expressing the sensor protein within a cell. A typical example of expression cassettes for a sensor protein is a polynucleotide containing a promoter and the coding sequence for the sensor protein placed under control of the promoter.

[0061] The promoter is not particularly limited and can be selected as appropriate. The promoter for use can be selected from various types of pol II promoters, for example. Examples of pol II promoters include, but are not particularly limited to, CMV promoter, EFl promoter, SV40 promoter, MSCV promoter, and promoters derived from insect genes.

[0062] When the sensor protein is an insect odorant receptor, it is preferred that the exogenous polynucleotide contain a coding sequence for an insect olfactory receptor co-receptor. The insect olfactory receptor co-receptor is a membrane protein with a seven-transmembrane structure as with the odorant receptor and functions by forming a hetero-complex with an odorant receptor. The odorant receptor complex, which is a hetero-complex composed of an odorant receptor and an olfactory receptor co-receptor, has ion channel activity that is activated by odorous substances. When activated, the odorant receptor complex allows the influx of cations such as sodium ions (Na+) and calcium ions (Ca2+) into cells.

[0063] It is preferred that the exogenous polynucleotide include a coding sequence for a protein that becomes fluorescent or luminescent in response to the influx of ions (e.g., calcium ions) into the cell when the sensor protein (in particular, the odorant receptor protein) responds. Examples of such proteins include aequorin, Yellow Cameleon (YC) , and GCaMP. Alternatively, the cell preferably contains an ion-dependent fluorescent dye, such as calcium ion-dependent fluorescent dyes (e.g., Fura-2, Fluo-3, and Fluo-4).

[0064] The exogenous polynucleotide preferably contains a coding sequence for a drug-resistant gene to allow for drug screening of cells. For the drug-resistant gene, a gene resistant to a drug that can be used for drug screening of cells may be selected. Examples of drug-resistant genes include chloramphenicol resistance gene, tetracycline resistance gene, neomycin resistance gene, erythromycin resistance gene, spectinomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene, and puromycin resistance gene.

[0065] It is preferred that the exogenous polynucleotide contains coding sequences such as a coding sequence for an insect olfactory receptor co-receptor, a coding sequence for a fluorescent or luminescent protein, and a coding sequence for a drug-resistant gene in the form of an expression cassette. The structure of the expression cassette is similar to that of the expression cassette for the sensor protein. The promoter of the expression cassette can be shared among multiple coding sequences.

[0066] The exogenous polynucleotide is preferably integrated into the genomic DNA (particularly preferably chromosomal genomic DNA). This enables stable expression of the sensor protein, making it suitable for chemical detection. In this case, the polynucleotide can be a single continuous region within the genomic DNA or a combination of two or more continuous regions (e.g., a form in which the sensor protein-coding sequence is included in continuous region A, and the coding sequence for a drug-resistant gene is included in continuous region B, which is separate from continuous region A, or a form in which the sensor protein-coding sequence is included in both continuous region A and continuous region B).

[0067] In another embodiment, the polynucleotide may be in a state in which it is not integrated into the genomic DNA. In this case, the exogenous polynucleotide can be in the form of a vector, for example. In this case, the polynucleotide can be a single polynucleotide molecule, or two or more polynucleotide molecules (e.g., a form in which the sensor protein-coding sequence is included in polynucleotide molecule A, while the coding sequence for a drug-resistant gene is included in polynucleotide molecule B, which is a separate molecule from polynucleotide molecule A, or a form in which the sensor proteincoding sequence is included in both polynucleotide molecule A and polynucleotide molecule B).

[0068] The cell concentration of the cell layer is not particularly limited as long as it is a concentration that allows for the detection of a chemical substance. The cell concentration of the cell layer (cell count per square centimeter of the cell layer) is, for example, 5000 to 2000000 cells / cm2. The cell concentration of the cell layer is preferably 10000 to 1500000 cells / cm2, more preferably 10000 to 1000000 cells / cm2, even more preferably 20000 to 1000000 cells / cm2, still more preferably 50000 to 700000 cells / cm2, and particularly preferably 100000 to 500000 cells / cm2, from the viewpoint of chemical detection, cell viability, and other factors.

[0069] The area of the cell layer is not particularly limited. From the viewpoint of detection sensitivity or production efficiency, the area of the cell layer is preferably 0.5 to 100 mm2, more preferably 1 to 30 mm2, and even more preferably 1.5 to 15 mm2 . In an embodiment of the present invention, the upper limit of the area can be set to 80 mm2, 60 mm2, or 40 mm2.

[0070] From the viewpoint of chemical detection, it is desirable for the cell layer to be thinner. The thickness of the cell layer is preferably 5 to 500 pm, more preferably 5 to 300 pm, even more preferably 5 to 200 pm, still more preferably 5 to 150 pm, particularly preferably 8 to 100 pm, and yet more preferably 10 to 70 pm.

[0071] The cell layer may include components other than cells. Other components are not particularly limited as long as they do not have a significant adverse effect on cell survival; examples include solvents, culture medium components, and gel-forming components. In an embodiment of the present disclosure, the cell layer may be a gel containing cells. Regarding the gel, for example, the definition of the gel layer described below can be applied.

[0072] The cell layer may be a monolayer or a multilayer composed of two or more cell layers with different configurations. From the viewpoint of production efficiency, the cell layer is a monolayer.

[0073] The gel layer is not particularly limited as long as it is placed on one side of the cell layer and is a layer in a gel form. It is preferred that no other layer including a gel layer be placed on the other side of the cell layer on which the gel layer is not placed.

[0074] The component that forms a gel (gel-forming component: a component cross-linked to form a network) is not particularly limited; examples include gelatin, polysaccharides, such as agar, carrageenan, starch, and xanthan gum, and water-soluble synthetic polymers, such as PVA, PEG, and acrylic polymers.

[0075] The component that forms a gel can be a single type or a combination of two or more types.

[0076] From the perspective of cell desiccation resistance, cell retention, production efficiency, or chemical detection, the content of the gel-forming component is preferably 50 mass! or more, more preferably 70 mass% or more, still more preferably 80 mass! or more, even more preferably 85 mass% or more, particularly preferably 90 mass! or more, and especially preferably 95 mass! or more (in particular, 100 mass!) based on 100 mass% of the solid content that constitutes the gel layer.

[0077] When the gel layer contains gelatin, the concentration of gelatin in the gel layer is preferably 0.5 to 10 mass%, more preferably 0.7 to 8 mass%, even more preferably 0.8 to 6 mass%, still more preferably 0.9 to 4 mass%, and particularly preferably 1 to 3.5 mass%.

[0078] The solvent for the gel is not particularly limited as long as it does not adversely affect cell viability to a significant degree. It is preferred that the solvent contains water. The water content in the solvent is preferably 50 mass! or more, more preferably 70 mass! or more, still more preferably 80 mass% or more, even more preferably 85 mass! or more, particularly preferably 90 mass% or more, and especially preferably 95 mass! or more (in particular, 100 mass!) .

[0079] The storage elastic modulus of the gel forming the gel layer is not particularly limited, and is, for example, 0.001 to 100 kPa. From the viewpoint of suppressing external forces on cells in contact with the gel (i.e., suppressing damage to cells) while retaining the cells, the storage elastic modulus of the gel forming the gel layer is preferably 0.01 to 20 kPa, more preferably 0.02 to 10 kPa, even more preferably 0.05 to 5 kPa, and yet more preferably 0.07 to 2 kPa.

[0080] The storage elastic modulus can be the storage elastic modulus at 4°C. From the viewpoint of cell preservation, it is preferred that the layered construct of the present disclosure be transported and stored at relatively low temperatures, and therefore the storage elastic modulus at relatively low temperatures is more important.

[0081] The area of the gel layer is not particularly limited. From the viewpoint of detection sensitivity or production efficiency, the area of the gel layer is preferably 0.5 to 100 mm2, more preferably 1 to 30 mm2, and even more preferably 1.5 to 15 mm2. In an embodiment of the present invention, the upper limit of the area can be set to 80 mm2, 60 mm2, or 40 mm2. The area of the gel layer is typically equivalent to the area of the cell layer, and is, for example, 70 to 150, 80 to 120, or 90 to 110 relative to the area of the cell layer taken as 100.

[0082] The thickness of the gel layer is preferably 0.1 to 30 mm, more preferably 0.2 to 20 mm, even more preferably 0.3 to 15 mm, and still more preferably 0.5 to 12 mm, from the viewpoints of chemical detection, cell desiccation resistance, cell retention, and other factors. Furthermore, the thickness can be further reduced while the advantages are maintained, from the perspective of production efficiency and other factors. The upper limit of the thickness is preferably 10 mm, more preferably 7 mm, even more preferably 5 mm, still more preferably 3 mm, and particularly preferably 1 mm.

[0083] The gel layer can be a monolayer or a multilayer composed of two or more gel layers with different configurations. From the viewpoint of production efficiency, the gel layer is a monolayer.

[0084] The method for producing the layered construct of the present disclosure is not particularly limited. The layered construct of the present disclosure can be produced, for example, according to a method comprising a cell layer formation step comprising placing a cell suspension, and a gel layer formation step comprising forming a gel layer on the cell layer.

[0085] In an embodiment of the present disclosure, a sol capable of forming a gel can be used as a cell suspension. In this case, a cell suspension is placed on a suitable substrate (the bottom surface of wells for cells) and then left to stand for a predetermined period of time in a sol state to allow the cells to settle onto the substrate, followed by gelling the sol to obtain the layered construct of the present disclosure.

[0086] In an embodiment of the present disclosure, a liquid not forming a gel (e.g., culture medium) can be used as a cell suspension. In this case, a cell suspension is placed on a suitable substrate (the bottom surface of swells for cells) and then left to stand for a predetermined period of time to allow the cells to settle on the substrate, followed by removing the supernatant and subsequently forming a gel on the cell layer (e.g., by stacking a sol capable of forming a gel and then gelling it), thereby obtaining the layered construct of the present disclosure.

[0087] From the viewpoint of cell desiccation resistance, cell retention, and other factors, it is preferred that the layered construct of the present disclosure be placed within a compartment (e.g., in a well for cells). From the viewpoint of cell desiccation resistance, cell retention, chemical detection, and other factors, it is preferable for the cell layer to be in contact with the bottom surface of the compartment, and it is more preferable for the cell layer to be in contact with both the bottom surface and side surfaces of the compartment. When the cell layer is not in contact with the side surfaces of the compartment or when the compartment does not have sides, it is preferable for the sides of the cell layer to be covered with the gel layer.

[0088] The layered construct of the present disclosure can be used for measuring sensor protein activity, enabling the detection of chemical substances (in particular odorants). The chemical substances can be, for example, those in a sample such as body fluids (e.g., urine, blood, and saliva), air (e.g., indoor air and air inside packaging), and water (e.g., river water, seawater, tap water, clean water, and sewage). In this case, for example, the layered construct can be used in measuring sensor protein activity by adding a substance that alters sensor protein activity from the gel-layer side. In this measurement, the chemical substance permeates through the gel layer to reach the cell layer and come into contact with the sensor protein in the cells. In this measurement, for example, the chemical substance can be detected by detecting ions flowing into a cell (e.g., by detecting a protein that becomes colored or luminescent in response to ions). The layered construct of the present invention is suitable for use in measurement in which sensor protein activity is converted into light. In this case, from the viewpoint of chemical detection of the layered construct of the present invention, it is particularly preferable to detect light on the cell-layer side.

[0089] It is preferred that the layered construct of the present disclosure be included in a cell chip. In an embodiment, the present disclosure relates to a cell chip comprising a compartment including the layered construct of the present disclosure.

[0090] The configuration of the compartment is not particularly limited as long as it is capable of retaining cells and a gel. From the perspectives of cell desiccation resistance, cell retention, production efficiency, or chemical detection, the compartment is preferably in the form of wells.

[0091] The material of the compartment is not particularly limited as long as the material can retain cells and a gel. The material can be, for example, resin or metal.

[0092] From the perspective of detection sensitivity, the compartment typically contains multiple cells. The number of cells per unit area (cm2) in a compartment is the same as that in the cell layer.

[0093] From the perspective of detection sensitivity or production efficiency, the bottom surface area of a single compartment is preferably 0.5 to 100 mm2, more preferably 1 to 30 mm2, and even more preferably 1.5 to 15 mm2. In an embodiment of the present invention, the upper limit of the area can be set to 80 mm2, 60 mm2, or 40 mm2.

[0094] From the perspective of detection sensitivity or production efficiency, the number of compartments included in the cell chip is preferably 10 to 2000, more preferably 30 to 1000, and even more preferably 50 to 500.

[0095] It is preferred that the cell chip of the present disclosure include two or more (more preferably three or more, even more preferably four or more, still more preferably five or more, ten or more, 15 or more, or 20 or more) types of cells that differ from each other in the type of sensor protein.

[0096] The upper part of the compartments of the cell chip may be sealed. The term "sealed" refers to a state in which the compartments are covered by a film or similar material that has the function of preventing contamination between compartments and that has gas permeability. Examples of film materials include rayon, polyolefin, and polyester.

[0097] The entire cell chip may be packaged. The container for packaging can be a deformable container, a container with a fixed shape, etc., although there is no particular limitation. The container for packaging is preferably a deformable container (e.g., a bag). A surface of the container may be in tight contact with the upper part of the compartment, thereby sealing the upper part of the compartment. The material of the container is not particularly limited as long as it is a commercially available common material, such as polyethylene.

[0098] In an embodiment, the present disclosure relates to a method for producing a layered construct (in particular, the layered construct of the present disclosure), comprising a cell layer formation step comprising placing a suspension of a cell in a sol capable of forming a gel on a substrate, and allowing the cell to settle on the substrate, and a gel layer formation step comprising gelling the sol (one-step method). [ 0099] In the one-step method, from the viewpoint of detection of chemical substances, such as odorants, it is particularly preferred that the substrate contain a compartment with a polypeptide-coated surface. The layered construct placed on the substrate and obtained according to the one-step method is also an embodiment of the present disclosure.

[0100] The polypeptide is not particularly limited as long as it is a chain structure composed of multiple amino acids linked by peptide bonds .

[0101] The amino acids that constitute the polypeptide are not particularly limited, and include not only a-amino acids but also p-amino acids, y-amino acids, 5-amino acids, etc. Examples of a- amino acids include amino acids with a basic side chain, such as lysine, arginine, or histidine; amino acids with an acidic side chain, such as aspartic acid or glutamic acid; amino acids with an uncharged polar side chain, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine; amino acids with a nonpolar side chain, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; amino acids with a p-branched side chain, such as threonine, valine, or isoleucine; and amino acids with an aromatic side chain, such as tyrosine, phenylalanine, tryptophan, or histidine.

[0102] The number of amino acids (the number of amino acid residues) that constitute the polypeptide is, for example, 3 to 5000. From the viewpoint of detection of chemical substances, the number of amino acid residues is preferably 10 to 3000, more preferably 15 to 2000, and even more preferably 20 to 1500. In particular, in a preferable embodiment of the present disclosure, the number of amino acid residues is preferably 10 or more, more preferably 100 or more, and even more preferably 500 or more.

[0103] The polypeptide may be in a single-strand form or in a form of a complex of two or more strands.

[0104] The polypeptide may be chemically modified as long as it has the effect of improving chemical detection. Whether the polypeptide has an effect of improving chemical detection can be determined by measuring the activity of an odorant receptor as described in Test Example 2 below, using a cell substrate containing compartments having a surface coated with a test substance, and comparing the result with that of a cell substrate not coated with the test substance.

[0105] The C-terminus of the polypeptide may be a carboxyl group (-COOH) , a carboxylate (-COCF) , an amide (-CONH2) , or an ester (-COOR).

[0106] The R in the ester may be, for example, a Ci-e alkyl group, such as methyl, ethyl, n-propyl, isopropyl, or n-butyl; a C3-8 cycloalkyl group, such as cyclopentyl or cyclohexyl; a C6u2 aryl group, such as phenyl or a-naphthyl; a phenyl-Ci^2 alkyl group, such as benzyl or phenethyl; a C7-14 aralkyl group, such as an a-naphthyl-Ci-2 alkyl group, such as a-naphthylmethyl; or a pivaloyloxymethyl group.

[0107] The carboxyl groups (or carboxylates) other than the C-terminus of the polypeptide may be amidated or esterified. In this case, the esters, such as the C-terminal esters described above, can be used.

[0108] Specific examples of the polypeptide include, for instance, polypeptides having cell adhesion properties and their partial peptides. Examples of such polypeptides include extracellular matrix proteins, their partial peptides, and polypeptides that adhere to cells through electrostatic interactions with cells.

[0109] Examples of extracellular matrix proteins include collagen, elastin, fibronectin, laminin, fibrinogen, fibrin, tenascin, ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs), and proteoglycans. As long as the partial peptides of the extracellular matrix proteins can adhere to cells through binding with cell receptors, their site and length are not particularly limited. The region responsible for binding with cell receptors is known or can be readily estimated based on known information. These extracellular matrix proteins may have mutations (e.g., substitutions, deletions, and insertions, preferably conservative substitutions) in their wild-type amino acid sequences as long as they can adhere to cells through binding to cell receptors. Specifically, such extracellular matrix proteins may include mutant amino acid sequences with, for example, at least 90%, at least 95%, or at least 99% identity to the wild-type amino acid sequences.

[0110] Examples of polypeptides that adhere to cells through electrostatic interaction with the cells include, specifically, positively charged polypeptides (the percentage of positively charged amino acid residues (e.g., lysine, arginine, and histidine) of the total number of amino acid residues taken as 100% is, for example, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100%).

[0111] From the viewpoint of enabling cells to be stably arranged on the substrate to detect chemical substances, the polypeptide is preferably an extracellular matrix protein. Of the extracellular matrix proteins, collagen is particularly preferred.

[0112] The polypeptide may be a single type or a combination of two or more types.

[0113] The amount of the polypeptide coating the surface of the substrate is not particularly limited and can be, for example, 0.1 pg / cm2 or more. From the viewpoint of chemical detection, the amount of the polypeptide coating the surface of the substrate is preferably 0.5 pg / cm2 or more, more preferably 1 pg / cm2 or more, even more preferably 2 pg / cm2 or more, and still more preferably 3 pg / cm2 or more. The upper limit for the amount of coating is not particularly limited, and can be, for example, 200 pg / cm2, 100 pg / cm2, 50 pg / cm2, 30 pg / cm2, 20 pg / cm2, or 10 pg / cm2.

[0114] The method for coating the target surface with the polypeptide can be performed according to or with reference to a known method.

[0115] A compartment containing a greater amount of a polypeptide-coated surface enables more effective manifestation of the intended effects of the present disclosure. From this perspective, the area of the polypeptide-coated surface relative to the area of the compartment taken as 100% (the area of the region in which cells are placed (e.g., the region in which a cell layer is formed or cells are adhered)) is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, still more preferably 95% or more, yet more preferably 99% or more, and particularly preferably 100%.

[0116] In the one-step method, the method for placing cells on the bottom surface of the compartment to form a cell layer is not particularly limited in the method for allowing cells in a cell suspension to settle. The cells may be allowed to freely fall by settling, or may be moved to the bottom surface by centrifugal force, for example, by centrifugation.

[0117] For performing the solution replacement or addition described below, it is preferable to allow the cells to adhere.

[0118] For cells that adhere to the bottom surface of the compartment, there is no particular limitation on the adhesion state of the cells with respect to the bottom surface.

[0119] When cells placed on the bottom surface are adhered to the bottom by letting them stand at a cell culture temperature, the time period for letting them stand is preferably 1 to 48 hours, more preferably 4 to 36 hours, even more preferably 12 to 28 hours, and particularly preferably 16 to 24 hours.

[0120] In an embodiment, the present disclosure relates to a method for producing a layered construct (in particular, the layered construct of the present disclosure), comprising a cell layer formation step comprising placing a suspension of cells in a liquid that does not form a gel on a substrate and allowing the cells to settle on the substrate, and a gel layer formation step comprising stacking a sol capable of forming a gel on the cell layer and then gelling the sol (two-step method).

[0121] In the two-step method, from the viewpoint of detection of chemical substances, such as odorants, it is particularly preferred that the substrate includes a compartment with a surface having a molar ratio of oxygen atoms to carbon atoms (O / C) of 0.150 or more. The layered construct placed on the substrate, obtained according to the two-step method, is also an embodiment of the present disclosure.

[0122] From the viewpoint of chemical detection, the molar ratio (O / C) of the surface is preferably 0.160 or more, more preferably 0.170 or more, and even more preferably 0.180 or more. The molar ratio (O / C) within the above range is particularly preferably 0.195 or more, and especially preferably 0.205 or more, from the viewpoint of detection of chemical substances (in particular, the detection of chemical substances at lower concentrations). While there is no specific limitation on the upper limit of the molar ratio (O / C) , the upper limit is preferably 0.300, more preferably 0.270, even more preferably 0.250, still more preferably 0.240, yet more preferably 0.230, and particularly preferably 0.220.

[0123] Without wishing to be bound by limited interpretation, the molar ratio (O / C) represents, in one aspect, the amount of oxidized carbon groups (C-O, O-C=O, C-O, C=O), and it appears that the amount of these groups or functional groups containing these groups above a certain level affects cell adhesion and the manner of cell adhesion, thereby contributing to improved cellular response in the detection of chemical substances.

[0124] From the perspective of chemical detection, it is preferred that the surface with the molar ratio (O / C) described above has a molar ratio of nitrogen atoms to carbon atoms (N / C) of 0.020 or more. The molar ratio (N / C) of the surface with the molar ratio (O / C) described above is more preferably 0.030 or more, even more preferably 0.035 or more, and still more preferably 0.040 or more. While there is no specific limitation on the upper limit of the molar ratio (N / C), the upper limit is preferably 0.100, more preferably 0.080, even more preferably 0.070, still more preferably 0.060, and particularly preferably 0.050.

[0125] From the viewpoint of chemical detection, the surface with the molar ratio (O / C) described above has a ratio of preferably 0.15 or more, more preferably 0.16 or more, still more preferably 0.17 or more, even more preferably 0.18 or more, yet more preferably 0.19 or more, particularly more preferably 0.20 or more, still particularly more preferably 0.21 or more, even particularly more preferably 0.22 or more, and especially preferably 0.23 or more, the ratio being a ratio of the sum of the state ratios of (2) to (4) in the Cis spectrum to the sum of (1) to (4) in the Cis spectrum wherein the state ratios of (2) to (4) in the Cis spectrum are the ratios to the sum of the following groups taken as 100: the groups measured in the Cis spectrum ((1) C-C, C-H; (2) C-O, C-N; (3) O-C=O; (4) CO3) , the groups measured in the Ols spectrum (C-O, C=O), and the groups measured in the Nls spectrum ((1) C-N, C=N; (2) quaternary ammonium salt; (3) NOX) . The sum of the state ratios is not particularly limited, and is preferably 0.4 or less, more preferably 0.35 or less, and even more preferably 0.3 or less.

[0126] The molar ratio (O / C), molar ratio (N / C), and state ratios of the surface are values as measured according to the method described in Test Example 4 below or a method equivalent to that method.

[0127] In an embodiment of the present disclosure, the water contact angle of the surface with the above molar ratio (O / C) is preferably 60° or less. The water contact angle is more preferably 55° or less, even more preferably 50° or less, and still more preferably 45° or less. The lower limit of the water contact angle is preferably 30°, more preferably 35°, and particularly preferably 40°.

[0128] The water contact angle of the surface is a value as measured according to the method described in Test Example 5 below or a method equivalent to that method.

[0129] The material that constitutes the surface with the above molar ratio (O / C) is not particularly limited as long as it is a material the molar ratio (O / C) of which can be adjusted to fall within a predetermined range. Examples of such materials include resins (e.g., polystyrene, polyethylene, polypropylene, poly(acrylic acid esters), poly(methacrylic acid esters), polyacrylamide, polyacrylonitrile, polyethylene terephthalate, poly(L-lactic acid), poly(glycolic acid), poly (s-caprolactone), poly(ethylene glycol), and copolymers thereof), metals (gold, silver, copper, iron, zinc, aluminum, nickel, and their alloys or oxides), glass, silica, silicon, and composite materials of these. From the viewpoint of ease of adjusting the molar ratio (O / C) to fall within a predetermined range, the material is preferably a resin.

[0130] The surface with the molar ratio (O / C) described above can be obtained, for example, by performing a treatment to increase the molar ratio (O / C) on a resin surface. While there are no particular limitations on such a treatment, examples include plasma treatment, corona treatment, and UV-ozone treatment. Alternatively, a surface with the molar ratio (O / C) described above can also be obtained by using a resin obtained through a reaction that introduces functional groups containing oxygen atoms and / or by adding a resin containing a relatively large number of functional groups containing oxygen atoms as the material.

[0131] A compartment containing a greater amount of the surface with the molar ratio (O / C) described above enables more effective manifestation of the intended effects of the present disclosure. From this perspective, the area of the surface with the above molar ratio (O / C) relative to the area of the compartment taken as 100% (the area of the region in which cells are placed (e.g., adhered)) is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, still more preferably 95% or more, particularly preferably 99% or more, and especially preferably 100%.

[0132] In the two-step method, the method for placing cells on the bottom surface of the compartment to form a cell layer is not particularly limited in the method for allowing cells in a cell suspension to settle. The cells may be allowed to freely fall by settling, or may be moved to the bottom by centrifugal force, for example, by centrifugation.

[0133] It is particularly preferred that the cells placed on the bottom surface of the compartment be adhered to the bottom surface of the compartment.

[0134] When cells placed on the bottom surface are adhered to the bottom by letting them stand at a cell culture temperature, the time period for letting them stand is preferably 1 to 48 hours, more preferably 4 to 36 hours, even more preferably 12 to 28 hours, and particularly preferably 16 to 24 hours.

[0135] In an embodiment of the present disclosure, the layered construct placed on a substrate, obtained according to the one-step method or two-step method, can be used for solution replacement or addition with cells placed on the surface of the present disclosure. In this case, for example, after solvent replacement or addition, the layered construct can be used for measuring the activity of a sensor protein. Solutions (e.g., culture mediums) or gels suitable for retaining cells may contain substances that adversely affect chemical detection or may have physical properties that could adversely affect chemical detection. Thus, to mitigate the risk of such adverse effects, it is preferable to perform solution replacement or addition before activity measurement. Gels are appropriately converted into their liquid form (e.g., by raising the temperature) before performing solution replacement or addition. This allows for the replacement of solutions such as culture mediums with solutions that can control the risks mentioned above (e.g., buffer solutions); alternatively, dilution can also mitigate the risks. Examples

[0136] The present invention will be described in detail below with reference to Examples. However, the present invention is not limited to these Examples.

[0137] In the following test examples, Sf-900 III SEM medium was used for culture medium.

[0138] Test Example 1: Preparation of Stably Expressing Cell Cells derived from Spilosoma imparilis (Splm cells) were transfected with a transposon vector containing the coding sequence for an odorant receptor protein (ORA) , the coding sequence for an olfactory receptor co-receptor, the coding sequence for a calcium sensor fluorescence protein, and the coding sequence for a puromycin resistance gene that were all placed under control of a promoter sequence and that were positioned between the 5' ITR (inverted terminal repeat) and 3' ITR. Selection was performed with puromycin, thereby preparing Splm cells stably expressing odorant receptors in which the foreign DNA containing the above coding sequences and promoter sequence was integrated into the chromosomal genomic DNA ("ORA cells" below). The odorant receptor is an insect-derived odorant receptor and is a receptor for compound a. The ORA cells emit fluorescence in response to compound a.

[0139] Test Example 2: Preparation of Acrylic Resin Solution A Acrylamide (monomer) and N-[tris(3-acrylamide propoxymethyl)methyl]acrylamide (crosslinking agent: 4AAmST) were dissolved in an aqueous PBS (Nissui Pharmaceutical Co. Ltd., #05913) solution to prepare an aqueous acrylamide / 4AAmST solution. Lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (photoinitiator: LPA) was dissolved in an aqueous PBS solution to prepare an aqueous LPA solution. The two aqueous solutions were mixed and stirred so as to achieve a final acrylamide concentration of 0.06 mmol / g, a final 4AAmST concentration of 0.14 mmol / g, and a final LPA concentration of 0.0006 mmol / g, thereby obtaining acrylic resin solution A.

[0140] Test Example 3: Preparation of Acrylic Resin Solution B Acrylic resin solution B was obtained in the same manner as in Test Example 2, except that N,N-bis(2-acrylamide methyl)acrylamide (3AAmST) was used instead of 4AAmST as the crosslinking agent.

[0141] Test Example 4: Preparation of Acrylic Resin Solution C Acrylic resin solution C was obtained in the same manner as in Test Example 2, except that N,N’-[oxybis(2,1-ethanediyloxy-3,1-propanediyl)]bisacrylamide (2AAmLN) was used instead of 4AAmST as the crosslinking agent.

[0142] Test Example 5: Preparation of Gelatin Solution Powdered fish gelatin was added to ultrapure water and stirred at 17°C for 30 minutes, followed by heating the mixture to 40°C with stirring until complete dissolution, thereby obtaining a gelatin solution with a fish gelatin concentration of 20%. After being autoclaved for sterilization, the gelatin solution was used in the following tests.

[0143] Test Example 6: Measurement of Odorant Receptor Activity 1 ORA cells were suspended in culture medium, and the obtained cell suspension was centrifuged (150G for 5 min), followed by removing the culture medium (supernatant). Then, one of acrylic resin solutions A to C was added to obtain a cell-suspended acrylic resin solution of 5 * 104 cells / 40 pL. The cell-suspended acrylic resin solution was seeded into a 96-well plate (Corning, catalog number: 3903) at 5 x 104 cells / 40 pL / well and irradiated with UV (110 mW / cm for 30 seconds) using a UV irradiation device (light source, 365 nm, LED manufactured by CCS Inc.) to turn the cell-suspended acrylic resin solution into a gel. Thereby, a gelled cell layer (thickness: about 1.25 mm) was formed in the wells as a monolayer, which is composed of cells dispersed in the gel (gel-containing group). 100 pL of culture medium was added on top of the gelled cell layer. Additionally, a group was also prepared for which ORA cells were seeded in the same manner as described above using culture medium instead of the acrylic resin solution (gel-free group).

[0144] After 24 hours from seeding, the culture medium was removed, and an assay buffer (0.1% BSA / lx Hanks' buffer / 20 mM HEPES buffer) was added in an amount of 40 pL to the gelcontaining groups and 80 pL to the gel-free group. Compound a (a substance to which ORA cells respond) or buffer free of compound a was added to each well so as to achieve a final concentration of 0, 10, or 100 pM, and the changes in fluorescence intensity before and after addition were measured using a microplate reader (FlexStation 3, Molecular Devices). With the microplate reader, the wells were irradiated with excitation light from their bottom to detect fluorescence on the bottom side of the wells.

[0145] Fig. 1 shows the results. In the gelled cell layers, which were a monolayer in which cells were dispersed within the gel (Gel A to Gel C), no increase in fluorescence intensity due to addition of compound a was observed.

[0146] Test Example 7: Measurement of Odorant Receptor Activity 2 ORA cells were suspended in culture medium, seeded at 5 x 104 cells / 100 pL / well in a 96-well plate (Corning, catalog number: 3903), and then left to stand in a 27°C incubator (without CO2 supply).

[0147] After 48 hours from seeding, only the culture medium of the upper layer was removed without removing the cells that had settled on the bottom surface (cell layer) , and 40 pL of acrylic resin solution A, B, or C was gently added on top of the cell layer so as not to cause the cells to come up. The cell-suspended acrylic resin solution was then irradiated with UV (110 mW / cm2 for 30 seconds) using a UV irradiation device (light source, 365 nm, LED manufactured by CCS Inc.) to turn the cell-suspended acrylic resin solution into a gel. Thereby, a layered construct composed of a cell layer (thickness: about 10 to 50 pm), which is a monolayer, and a gel layer (free of cells) (thickness: about 1.25 mm) placed on top of the cell layer was formed in the wells (gel-containing group). 100 pL of culture medium was added on top of the gel layer. Additionally, a group was also prepared for which culture was continued in the medium without performing the above operation (gel-free group).

[0148] After 24 hours from the operation above, the culture medium was removed, and 0.1% BSA / lx Hanks’ buffer / 20 mM HEPES buffer was added in an amount of 40 pL to the gel-containing groups and in an amount of 80 pL to the gel-free group. The changes in fluorescence intensity before and after addition of compound a were measured in the same manner as in Test Example 6.

[0149] Fig. 2 shows the results. When the layered construct composed of a cell layer (monolayer) and a gel layer (free of cells) placed on top of the cell layer (Gel A to Gel C) was used, an increase in fluorescence intensity due to the addition of compound a was observed in a manner similar to the case without gel.

[0150] Test Example 8: Production of Layered Construct An ORA cell suspension and a 20% gelatin solution were mixed to achieve a final gelatin concentration of 3.3%, seeded into a 96-well plate (Corning 3903) at 5 x 104 cells / 120 pL / well, and then left to stand in a 27°C incubator (without CO2 supply) .

[0151] After 24 hours from seeding, the cells were confirmed to have settled on the bottom and formed a cell layer. Thereafter, the plate was then left to stand at 4°C to form a gelatin gel. Thereby, a layered construct composed of a cell layer (thickness: about 10 to 50 pm), which is a monolayer, and a gel layer (free of cells) (thickness: about 1.25 mm) placed on top of the cell layer was formed in the wells.

[0152] Test Example 9: Study of Substrates Suitable for Production of Layered Construct According to 1-Step Method Test Example 9-1: Measurement of Odorant Receptor Activity with Tissue Culture (TC) ORA cells were detached from a culture flask and mixed with medium (Sf-900 III, Gibco) containing 0 to 2% fish gelatin to prepare a cell suspension. The cell density of the cell suspension was counted using a cell counter. The cell density was adjusted so as to be in a range of 5 x 105 to 1 * 106 cells / mL. Subsequently, the cells were seeded into a TC-treated 384-well plate (Greiner, catalog number: 781098) using a dispenser (ASSIST PLUS, Integra Biosciences), with an equal number of cells per well (40 pL / well). Table 1 shows the seeded cell density. After seeding, the plate was left to stand at 27°C overnight to allow the cells to adhere to the plate. The adherence of the cells was observed under a microscope. Fig. 3 shows the observed images. Subsequently, the plate was stored at 4°C.

[0153] After the plate was stored at 4°C for 3 days and then left at 27°C for 2 hours, the medium was replaced with an assay buffer (40 pL / well) using a dispenser. Subsequently, the condition of the cells was observed under a microscope. Fig. 4 shows the observed images.

[0154] A solution of compound a (a substance to which ORA cells respond) was added to each well to achieve final concentrations of 0, 0.1, 1, or 10 pM, or a solution identical to the solution of compound a except for not containing compound a was added to each well, and the changes in fluorescence intensity before and after addition were measured using a microplate reader (FlexStation 3, Molecular Devices). Fig. 5 shows the results.

[0155] When solution replacement is performed with cells adhered, mechanical stress is applied to the cells. As a result, cells detach especially in wells containing gelatin (Fig. 4). Although cell detachment was not noticeable in wells free of gelatin (Fig. 4), it is assumed to occur at a certain frequency. In wells containing gelatin, the detection of the chemical substance decreased (0.1 pM of compound a could not be detected (Fig. 5)).

[0156] Test Example 9-2: Measurement of Odorant Receptor Activity with Amine Coating The results of Test Example 9-1 suggested that improving cell adhesion could limit reductions in chemical detection in gelatin-containing wells. Thus, a test was conducted in the same manner as in Test Example 9-1 using a 384-well plate with a coating to improve cell adhesion (amine coat, Falcon, catalog number: 356719) . Fig. 6 shows images of the cells observed immediately after the cell-seeded plate was left overnight at 27°C. Fig. 7 shows images of the cells observed after the medium was replaced with an assay buffer. Fig. 8 shows the results of measuring the odorant receptor activity.

[0157] Although amine coating improved cell adhesion (comparison between Fig. 4 and Fig. 6), it did not lead to detection of 0.1 pM of compound a (Fig. 8), thus not resulting in increased chemical detection.

[0158] Test Example 9-3: Measurement of Odorant Receptor Activity with Poly-D-Lysine Coating Based on the results of Test Example 9-2, a different coating was tried. A test was conducted in the same manner as in Test Example 9-1 using a 384-well plate coated with Poly-D-Lysine (Falcon, catalog number: 356660). Fig. 9 shows images of the cells observed immediately after the cell-seeded plate was left overnight at 27°C. Fig. 10 shows images of the cells observed after the medium was replaced with an assay buffer. Fig. 11 shows the results of measuring the odorant receptor activity.

[0159] Poly-D-Lysine coating improved cell adhesion to a degree comparable to that of amine coating (comparison between Fig. 6 and Fig. 10). However, despite this, Poly-D-Lysine coating unexpectedly enabled the detection of 0.1 pM of compound a (Fig. 11) , indicating enhancement in chemical detection.

[0160] Test Example 9-4: Measurement of Odorant Receptor Activity with Collagen Coating Based on the results of Test Example 9-3, it was assumed that polypeptide coating would improve chemical detection. Thus, a test was conducted in the same manner as in Test Example 9-1 using a 384-well plate coated with collagen, which is a polypeptide (Falcon, catalog number: 356702) . Fig. 12 shows images of the cells observed immediately after the cell-seeded plate was left overnight at 27°C. Fig. 13 shows images of the cells observed after the medium was replaced with an assay buffer. Fig. 14 shows the results of measuring the odorant receptor activity.

[0161] As seen with the Poly-D-Lysine coating, the collagen coating also enabled the detection of 0.1 pM of compound a (Fig. 14), indicating enhancement in chemical detection. In particular, collagen coating appeared to result in less cell detachment (Fig. 13) and enable more stable detection.

[0162] Test Example 10: Study of Substrates Suitable for Production of Layered Construct According to Two-Step Method Test Example 10-1: Cell Adhesion to Plate ORA cells were detached from a culture flask and mixed with medium (Sf-900 III, Gibco) to prepare a cell suspension. The cell density of the cell suspension was measured using a cell counter. The cell density was adjusted so as to be in a range of 5 x 105 to 1 x io6 cells. Subsequently, the cells were seeded into three types of TC (tissue culture)-treated 384-well plates (resin-made, plates A to C) using a dispenser (ASSIST PLUS, Integra Biosciences), with an equal number of cells per well (40 pL / well). Table 1 shows the seeded cell count. After seeding, the plates were left to stand at 27°C overnight to allow for cell adhesion. Subsequently, the plates were stored at 4°C.

[0163] Table 1 Plate A Plate B Plate C Cell Count 1 (per well) 22500 22500 22500 Cell Count 2 (per well) 30000 30000 30000

[0164] Test Example 10-2: Measurement of Odorant Receptor Activity 1 The plates stored at 4°C for 3 days in Test Example 10-1 were retrieved and left to stand at 27°C for 2 hours. After the cells were returned to room temperature, the medium was replaced with an assay buffer (Hanks’ buffer containing 0.1% BSA and 20 niM HEPES) using a dispenser (40 pL / well). Thereafter, the adhesion of the cells was observed under a microscope. Fig. 15 shows the observed images.

[0165] A solution of compound a (a substance to which ORA cells respond) was added to each well to achieve a final concentration of 0, 0.1, 1, or 10 pM, or a solution identical to the solution of compound a except for not containing compound a was added to each well, and the changes in fluorescence intensity before and after the addition were measured using a microplate reader (FlexStation 3, Molecular Devices).

[0166] Fig. 16 shows the results. Of plates A to C, using plates A and B (in particular, plate A) resulted in higher fluorescence intensities as compared to using plate C.

[0167] Test Example 10-3: Measurement of Physical Properties of Plate Surface 1 The chemical composition and electronic state of the elements present on the cell-adhered surfaces of the three types of plates (plates A to C with no cells seeded) were non-destructively measured using an X-ray photoelectron spectrometer (Thermo Fisher Scientific, K-Alpha+) . The details of the measurement method are shown below. First, a utility knife was inserted from the back side of the plate, and the bottom of the plate was cut out along the shape of the well. The cut-out piece was then set in a vacuum container to secure a vacuum condition. Subsequently, the measurement surface was inspected using a camera equipped inside the X-ray photoelectron spectrometer to ensure that contaminants such as cutting debris were not analyzed. The measurement was performed using an AlKa X-ray source with the X-ray spot diameter set to 300 pm and irradiation onto the measurement surface. To obtain more accurate measurement results, a neutralizer gun was used to remove static electricity accumulated on the sample surface during measurement. A survey scanning measurement (survey scan spectrum) was performed to identify the types of elements present in the sample. Additionally, a narrow scanning measurement (narrow scan spectrum) was also performed to analyze the chemical bonding states of the elements detected in the survey scanning.

[0168] Table 2 shows the results. As a result of survey scanning measurement, peaks of Cis, Ols, and Nls were confirmed in all plates. Through narrow scanning measurement, curve fitting analysis of the Cis and Ols spectra detected peaks attributable to C-O, O-C=O, and CO3, suggesting the presence of oxygencontaining functional groups. In regards to Nls, the presence of C-N and C=N was suggested in all plates. The proportions of different chemical states for each element were shown as state ratios (with the total amount of all elements set to 100). To calculate the state ratio, the peak area of each element state obtained from narrow scanning spectra was used to determine the proportion of each state. The results indicated that plates A and B (in particular, plate A) had a higher molar ratio of oxygen atoms to carbon atoms (O / C) as compared to plate C.

[0169] Table 2 Photoelectron Presumed Attribution Plate A Plate B Plate C State Ratio State Ratio State Ratio Cis (i) C-C, C-H 59.3 64.2 76.2 (ii) C-O, C-N 12.7 10.8 7.2 (iii) O-C=O 6.1 5.2 2.1 (iv) co3 1.6 1.7 2.5 Ols (i) c-o, c=o - - - (ii) 16.7 15.4 11.2 Nls (i) C-N, C=N - - - (ii) 3.6 2.7 0.6 (iii) Quaternary Ammonium Salt - - 0.2 (iv) NOx - - - O / C (Ratio of Total 0 to Total C) 0.210 0.188 0.127 N / C (Ratio of Total 0 to Total N) 0.045 0.033 0.009 Sum of ( (ii ((i) to (iv relative to to (iv) of Cis) / sum of of Cis) (C=O / C+N / C shifted total C) 0.256 0.216 0.134

[0170] 5 Test Example 10-4: Measurement of Physical Properties of Plate Surface 2 The cell-adhered surface of the plate (with no cells seeded) was punched out using a biopsy trephine and placed horizontally on a contact angle meter (DMo-702 manufactured by 10 Kyowa Interface Science Co., Ltd.), followed by adding 0.2 pL of ultrapure water dropwise onto the surface. An image was captured, and the contact angle was calculated. Because the calculated values were expected to include error of about 5°, the measurement was performed using plates of the same type (n = 3 to 15 4), and the average was calculated. Table 3 shows the results.

[0171] Table 3 Contact Angle (°) Plate A Plate B Plate C 1 38.4 53.5 64.7 2 40.8 53.5 56.1 3 41.6 52.1 66.3 4 52.6 Average 43.4 53.0 62.4 S.D. 6.3 0.8 5.5

[0172] Test Example 10-5: Measurement of Odorant Receptor Activity 2 A mixture of cells and culture medium was seeded onto a 5 TC-treated plate, and the plate was left to stand at 27°C overnight to allow for cell adhesion. Subsequently, the culture medium was aspirated using a dispenser, and 1 to 2% fish gelatin was added, followed by storing the plate at 4°C. A cell-adhered plate was prepared in the same manner as in Test Example 10-1, 10 except for the removal of the medium and the addition of fish gelatin. Using the cell-adhered plate, the odorant receptor activity was measured in the same manner as in Test Example 10-2. The results indicated the same trend as that observed as in Test Example 10-2. 15

Claims

1. A layered construct comprising a cell layer and a gel layer placed on the cell layer.cell layer

2. The layered and the gel construct layer are according to claim each a monolayer. 1, wherein the cell layer cells / cm2.

3. The layered has a cell construct according to concentration of 10000 claim 1, wherein to 1000000 the

4. The layered construct according to claim 1, wherein agel forming the gel layer has a storage elastic modulus of 0.001 to 100 kPa.

5. The layered construct according to claim 1, which is placed within a compartment.

6. The layered construct according to claim 5, wherein the cell layer is in contact with the bottom surface of the compartment.

7. The layered construct according to claim 1, wherein a cell contained in the cell layer is an insect cell.

8. The layered construct according to claim 1, wherein a cell contained in the cell layer comprises a polynucleotide containing a coding sequence for a sensor protein.

9. The layered construct according to claim 8, wherein the sensor protein is an odorant receptor protein.

10. The layered construct according to claim 1, for use in measurement of sensor protein activity.

11. The layered construct according to claim 10, for use inthe measurement by adding a substance that alters the sensorprotein activity from the gel-layer side.

12. The layered construct according to claim 10, for use in the measurement wherein the sensor protein activity is converted into light.

13. The layered construct according to claim 12, wherein the light is detected on the cell-layer side.

14. A cell chip comprising a compartment that contains the layered construct of any one of claims 1 to 13.

15. The cell chip according to claim 14, wherein the upper part of the compartment is sealed.

16. The cell chip according to claim 14 or 15, which is packaged.

17. A method for producing the layered construct of any one of claims 1 to 13, comprisinga cell layer formation step comprising placing a cell suspension, anda gel layer formation step comprising forming a gel layer on a cell layer.

18. The method according to claim 17, wherein the cell suspension is a sol capable of forming a gel.

19. The method according to claim 17, comprising the cell layer formation step comprising placing a suspension of a cell in a sol capable of forming a gel on a substrate, and allowing the cell to settle on the substrate, and the gel layer formation step comprising gelling the sol.

20. The method according to claim 19, wherein the substrate comprises a compartment with a polypeptide-coated surface.

21. A layered construct obtained according to the method of claim 20, the layered construct being placed on the substrate.

22. The method according to claim 17, comprisingthe cell layer formation step comprising placing a suspension of a cell in a liquid that does not form a gel on a substrate and allowing the cell to settle on the substrate, andthe gel layer formation step comprising stacking a5 sol capable of forming a gel on a cell layer and then gelling the sol.

23. The method according to claim 22, wherein the substrate 10 comprises a compartment with a surface having an oxygen-to-carbon molar ratio (O / C) of 0.150 or more.

24. A layered construct obtained according to the method of15 claim 23, the layered construct being placed on the substrate.INTERNATIONAL SEARCH REPORT International application No. PCT / JP2024 / 017602A. CLASSIFICATION OF SUBJECT MATTER C12M 1 / 00(2006.01)1; C12M / / 34(2006.01 )i: C12NIW(2006.01)i; C12N5 / 07(2OlO.Ol)i; C12N 5 / 10(2006.01)1; C12N15 / 12(2006.01)i FI: C12M1 / 00 C; C12N5 / 10; C12M1 / 34; C12N5 / 07; C12N1 / 00 B; C12N15 / 12 According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) C12M1 / 00; C12M1 / 34; C12N1 / 00; C12N5 / 07; C12N5 / 10; C12N15 / 12 Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Published examined utility model applications of Japan 1922-1996 Published unexamined utility model applications of Japan 1971-2024 Registered utility model specifications of Japan 1996-2024 Published registered utility model applications of Japan 1994-2024 Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) JSTP1 us / JMEDPlus / JST7580 (JDreamlII) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X Y X JP 2021-145645 A (RICOH COMPANY, LTD.) 27 September 2021 (2021-09-27) claims, fig. 1, paragraphs [0016], [0021], [0029], [0034], examples JP 2021-078377 A (RICOH COMPANY, LTD.) 27 May 2021 (2021-05-27) claims, examples, fig. 1, 2, paragraphs [0018], [0049], [0050] 1-6, 8, 10-24 7-24 1-6, 14-24 X JP 2016-052271 A (TERUMO KABUSHIKI KAISHA) 14 April 2016 (2016-04-14) claims, example 1 1-6, 14-24 X WO 2014 / 192909 Al (IHEART JAPAN CORPORATION) 04 December 2014 (2014-12-04) claims, examples, fig. 1, 2 1-6, 14-24 Y WO 2022 / 024902 Al (SUMITOMO CHEMICAL COMPANY, LIMITED) 03 February 2022 (2022-02-03) claims, paragraphs [0002]-[0006], [0036], [0089]-[0103] 7-24 | | Further documents are listed in the continuation of Box C. | Z | See patent family annex. * Special categories of cited documents: “T” later document published after the international filing date or priority “A” document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand the to be of particular relevance principle or theory underlying the invention “D” document cited by the applicant in die international application “X” document of particular relevance; the claimed invention cannot be “E" earlier application orpatent but published on or after the international considered novel or cannot be considered to involve an inventive step filing date when the document is taken alone •SL” document which may throw doubts on priority claim(s) or which is “Y” document of particular relevance; the claimed invention cannot be cited to establish the publication date of another citation or other considered to involve an inventive step when the document is special reason (as specified) combined with one or more other such documents, such combination “O” document referring to an oral disclosure, use, exhibition or other being obvious to a person skilled in the art means document member of the same patent family “P” document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search 04 July 2024 Date of mailing of the international search report 23 July 2024 Name and mailing address of the ISA / JP Japan Patent Office (ISA / JP) 3-4-3 Kasumigaseki, Chiyoda-ku, Tokyo 100-8915 Japan Authorized officer Telephone No.INTERNATIONAL SEARCH REPORT Information on patent family membersInternational application No.PCT / J1’2024 / 017602Patent document cited in search report Publication date (day / month / year) Patent family member)s) Publication date (day / month / year) JP 2021-145645 A 27 September 2021 (Family: none) JP 2021-078377 A 27 May 2021 US 2021 / 0147795 Al claims, examples, fig. 1, 2, paragraphs [0044], [0073], [0074] EP 3828542 Al JP 2016-052271 A 14 April 2016 US 2016 / 0058908 Al claims, example 1 WO 2014 / 192909 Al 04 December 2014 US 2016 / 0121025 Al claims, examples, fig. 1, 2 EP 3006559 Al WO 2022 / 024902 Al 03 February 2022 US 2023 / 0331793 Al claims, paragraphs [0002], [0003], [0008], [0067], [0120]-[0134] EP 4190900 Al

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