WO2024150682A1

WO2024150682A1 - Cell chip

Info

Publication number: WO2024150682A1
Application number: PCT/JP2023/046919
Authority: WO
Inventors: 康彦高橋; 時郎石川; 尊弘王
Original assignee: 住友化学株式会社
Priority date: 2023-01-11
Filing date: 2023-12-27
Publication date: 2024-07-18
Also published as: JP2024098966A

Abstract

The present invention provides a cell chip that has excellent cell drying resistance, cell retention properties, and production efficiency, and that can also be used for detecting a chemical substance such as an odor substance.　Provided is a cell chip comprising a section that contains a cell and a hydrogel which contains a (meth)acrylic resin.

Description

Cell Chip

This disclosure relates to a cell chip suitable for detecting chemical substances.

　Groups of odorants that characterize certain human diseases and mental states have been identified, and because of their usefulness as diagnostic markers, there has been active development of various odor sensors targeting these. Since the olfactory receptors of living organisms have superior characteristics in terms of diversity, sensitivity, selectivity, etc. that are not found in conventional odor sensor elements such as semiconductors, there are high expectations for the development of new odor sensors that use olfactory receptors as sensor elements.

Patent Document 1 discloses the use of cells expressing modified olfactory receptors and lipid bilayer membranes containing modified olfactory receptors as odor sensors.

International Publication No. 2022/024902

Artificially preparing a lipid bilayer membrane containing olfactory receptors is not desirable from the standpoint of manufacturing efficiency. Therefore, we focused on using cells that express olfactory receptors.

When using cells as odor sensors, from the standpoint of ease of use, it is preferable to prepare cells in advance in a container or the like and use them when necessary, rather than culturing and preparing the cells each time they are used. In the latter case, it is necessary not to dry the cells until they are to be used, and considering the transportation from the manufacturer to the place of use, it is important that the cells are kept stably, and it is also important to be able to manufacture more uniform cells more efficiently.

In addition, from the perspective of using the cells to identify diseases, etc., it is desirable to be able to simultaneously use multiple types of cells that express different olfactory receptors. From this perspective, it is desirable to use them as cell chips.

The objective of the present disclosure is to provide a cell chip that has excellent resistance to drying of cells, cell retention, and production efficiency, and can also be used to detect chemical substances such as odor substances.

In the course of their research, the inventors noticed that a cell chip including a compartment containing cells and hydrogel could prevent cells from drying out and hold the cells stably. However, when forming a hydrogel to hold cells, problems such as low manufacturing efficiency depending on the type of hydrogel, damage to cells during hydrogel formation, and inability to detect chemical substances due to low permeability of the hydrogel to chemical substances can arise.

The inventors have conducted intensive research in consideration of the above problems, and have surprisingly found that the above problems can be solved by a cell chip that includes a compartment containing cells and a hydrogel containing a (meth)acrylic resin. In other words, the present disclosure encompasses the following aspects:

Item 1. A cell chip including a compartment containing cells and a hydrogel containing a (meth)acrylic resin.

Item 2. The cell chip according to Item 1, wherein the cells are encapsulated in the hydrogel.

Item 3. The cell chip according to Item 1, wherein the cells are cells that contain a polynucleotide that includes a coding sequence for an olfactory receptor protein.

Item 4. The cell chip according to Item 3, wherein the olfactory receptor protein is an insect olfactory receptor.

Item 5. The cell chip according to Item 1, wherein the cells are insect cells.

Item 6. The cell chip according to Item 1, in which the hydrogel is formed by subjecting a compartment containing a solution of (meth)acrylic monomers and/or oligomers thereof and cells to a polymerization treatment.

Item 7. The cell chip according to Item 6, wherein the polymerization treatment is UV irradiation.

Item 8. The cell chip according to Item 6, wherein the concentration of the photopolymerization initiator in the solution of the (meth)acrylic monomer and/or its oligomer is 0.1% by mass or less.

Item 9. The cell chip according to Item 6, wherein the total concentration of the polyfunctional (meth)acrylic monomer and the oligomer in the solution of the (meth)acrylic monomer and/or its oligomer is 0.1 to 25% by mass.

Item 10. The cell chip according to Item 1, wherein the number of compartments is 10 to 2000.

Item 11. The cell chip according to any one of items 1 to 10, which is used for detecting chemical substances.

Item 11A. A method for detecting a chemical substance, comprising contacting a chemical substance with an olfactory receptor on a cell contained in the cell chip according to any one of items 1 to 10.

Item 11B. Use of the cell chip described in any one of items 1 to 10 as a chemical substance detection chip.

Item 11C. Use of the cell chip described in any one of items 1 to 10 for the manufacture of a chemical detection chip.

Item 12. A method for producing a cell chip according to any one of items 1 to 10, comprising polymerizing a compartment containing cells and a (meth)acrylic resin monomer and/or an oligomer thereof to form a hydrogel.

Item 13. The method according to Item 12, wherein the polymerization treatment is UV irradiation.

Item 14. The method according to Item 13, wherein the light source for the UV irradiation is an LED.

The present disclosure provides a cell chip that has excellent cell drying resistance, cell retention, and manufacturing efficiency, and can also be used to detect chemical substances such as odorants, as well as a manufacturing method thereof.

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

In one aspect, the present disclosure relates to a cell chip (sometimes referred to herein as the "cell chip of the present disclosure") that includes a compartment that contains cells and a hydrogel that contains a (meth)acrylic resin. This is described below.

The cells are not particularly limited. From the viewpoint of suitability for detecting chemical substances, animal cells such as insect cells and mammalian cells are preferred, with insect cells being particularly preferred. Mammalian cells include human-derived HeLa cells, HEK293 cells, HepG2 cells, KBM-7 cells, Chinese hamster-derived CHO cells, mouse-derived NIH3T3 cells, Min6 cells, and the like. Insect cells include, for example, SpIm cells derived from Spilosoma imparilis, the Sf cell line derived from Spodoptera frugiperda, the HighFive cell line derived from Trichoplusia ni, AeAe cells derived from Aedes aegypti, S2 cells derived from Drosophila melanogaster, olfactory nerve cells, and the like. Among these, Splm cells are particularly preferred.

When the cell chip of the present disclosure is used to detect chemical substances, it is preferable to use cells that contain a polynucleotide that includes a coding sequence for an olfactory receptor protein.

Olfactory receptor proteins are membrane proteins with seven transmembrane structures that act as odor sensors in living organisms.

From the viewpoint of suitability for detecting chemical substances, the olfactory receptor protein is particularly preferably an insect olfactory receptor protein. Insects from which the insect olfactory receptor protein is derived are preferably dipteran insects such as Culicidae and Drosophilidae; lepidopteran insects such as Bombycidae; Hymenopteran insects such as Apidae; Orthopteran insects such as Acrididae; Hemiptera insects such as Cimexidae, and more preferably dipteran insects such as Culicidae and Drosophilidae; Orthopteran insects such as Acrididae; Hemiptera insects such as Cimexidae. Examples of insects from the Culicidae family include Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. Examples of insects in the Drosophilidae family include the fruit fly (Drosophila melanogaster), the light brown fruit fly (Drosophila pseudoobscura), and the black fruit fly (Drosophila virillis). Examples of insects in the Bombycidae family include the silk moth (Bombyx mori), the mulberry silk moth (Bombyx mandarina), and the fig cassock (Trilocha varians). Examples of insects in the Apidae family include the European honeybee (Apis mellifera), the swarm honeybee (Apis florea), the giant honeybee (Apis dorsata), and the European bumblebee (Bombus terrestris). Examples of insects in the Acrididae family include the migratory locust (Locusta migratoria), and examples of insects in the Cimex family include the bedbug (Cimex lectularius).

Specific examples of wild-type insect olfactory receptor proteins include AaOR1, AaOR2, AaOR4, AaOR5, AaOR6, AaOR7, AaOR8, AaOR9, AaOR10a, AaOR15, AaOR22, AaOR24, AaOR25, AaOR26, AaOR27, AaOR28, AaOR30, AaOR34, AaOR36, AaOR38, AaOR41a, AaOR41b, AaOR42, AaOR43, AaOR 44, AaOR47, AaOR49, AaOR50, AaOR52, AaOR54, AaOR58, AaOR59, AaOR60, AaOR61, AaOR64, AaOR65, AaOR66, AaOR67a, AaOR69a, AaOR70, AaOR71, AaOR72a, AaOR73, AaOR74, A aOR75, AaOR77, AaOR78, AaOR79, AaOR81, AaOR83b, AaOR84, AaOR85, AaOR 86, AaOR87, AaOR91, AaOR95, AaOR97, AaOR96, AaOR99, AaOR100, AaOR102, AaOR103, AaOR104a, AaOR105, AaOR107, AaOR108, AaOR109, AaOR110, AaOR112, AaOR114, AaOR1 16, AaOR117, AaOR118, AaOR122, AaOR125, AaOR128, AgOR1, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR7, AgOR8, AgOR9, AgOR10, AgOR11a, AgOR12a, AgOR12b, AgOR13, AgOR14, AgOR15, AgOR16a, AgOR17, AgOR18, AgOR20, AgOR21, AgOR23, AgOR25, AgOR26 ,AgOR27,AgOR28,AgOR30,AgOR34,AgOR36,AgOR37,AgOR38,AgOR39 a, 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,AmO R13, AmOR41, AmOR51, AmOR52, AmOR55, AmOR71, AmOR73, AmOR78, AmOR85, AmOR89, AmOR90, AmOR114, AmOR115, AmOR118, AmOR120, AmOR121, AmOR161, BmOR1, BmOR2, BmOR3, BmOR4, BmOR5, BmOR8, BmOR 9, BmOR10, BmOR13, BmOR17, BmOR18, BmOR23, B mOR24, BmOR25, BmOR35, BmOR36, BmOR42, BmOR45, BmOR49, BmOR51, BmOR52, BmOR55, BmOR56, BmOR61, DmOR1a, DmOR9a, DmOR19a, DmOR22a, DmOR22b, DmOR22c, DmOR24a, DmOR3 0a, DmOR33a, DmOR33b, DmOR33c, DmOR35a, DmOR42b, DmOR43a, DmOR45a , DmOR45b, DmOR47a, DmOR49b, DmOR59b, DmOR65b, DmOR65c, DmOR67b, DmOR67c, DmOR69a, DmOR71a, DmOR74a, DmOR82a, DmOR83a, DmOR83b, DmOR83c, DmOR85a, DmOR85c, DmOR85e, DmOR85f, DmOR88a, DmOR92a, DmOR94a, DmOR94b, DmOR98b, etc. In these names, Dm indicates that it is derived from Drosophila melanogaster, Bm indicates that it is derived from Bombyx mori, Ag indicates that it is derived from Anopheles gambiae, and Aa indicates that it is derived from Aedes aegypti. The amino acid sequences and coding sequences of various olfactory receptor proteins, including these, are publicly known or can be easily identified by sequence identity searches based on the publicly known sequences.

　Insect olfactory receptor proteins can contain amino acid mutations so long as the chemical response activity is not significantly reduced. "Not significantly reduced" means, for example, that the chemical response activity of an olfactory receptor protein containing amino acid mutations is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more, compared to 100% of the chemical response activity of a wild-type olfactory receptor protein.

In this specification, chemical substance response activity refers to the property that an olfactory receptor recognizes a chemical substance, and the olfactory receptor complex formed by the olfactory receptor and the olfactory receptor co-receptor is activated and exhibits ion channel activity. The chemical substance response activity of an olfactory receptor can be measured using as an index the ion channel activity of the olfactory receptor complex formed by the olfactory receptor and the olfactory receptor co-receptor that have come into contact with a chemical substance. For example, a chemical substance is contacted with a cell expressing a protein that changes color or emits light in response to ions (such as calcium ions) that flow into the cell when (a) the olfactory receptor, (b) the olfactory receptor co-receptor, and (c) the olfactory receptor complex responds, and the amount of luminescence of the cell is measured. The greater the amount of luminescence measured, the higher the chemical substance response activity of the olfactory receptor is determined to be. Specifically, it can be measured according to the method described in Patent Document 1.

The coding sequence of the olfactory receptor protein is not particularly limited, so long as it is a polynucleotide consisting of a base sequence that encodes the olfactory receptor protein of the present disclosure. In one embodiment, the polynucleotide includes an expression cassette for the olfactory receptor protein. The expression cassette is not particularly limited, so long as it is a polynucleotide that is capable of expressing the olfactory receptor protein in a cell. A typical example of an expression cassette for an olfactory receptor protein is a polynucleotide that includes a promoter and the coding sequence for the olfactory receptor protein of the present disclosure that is placed under the control of the promoter.

As for the more specific configuration of the cells, for example, the configuration described in Patent Document 1 can be adopted.

The hydrogel is not particularly limited as long as it contains a (meth)acrylic resin. From the viewpoint of cell drying resistance, cell retention, production efficiency, or chemical substance detectability, the content of the (meth)acrylic resin is preferably 50% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, even more preferably 85% by mass or more, particularly preferably 90% by mass or more, and especially preferably 95% by mass or more (among which, 100% by mass) based on 100% by mass of the solid content constituting the hydrogel. The solid content includes the (meth)acrylic resin, cells, and other solid content. The term "(meth)acrylic" means methacryl and/or acrylic. Details of the (meth)acrylic resin will be described later.

The compartment of the cell chip disclosed herein contains cells and a hydrogel containing a (meth)acrylic resin.

The shape of the compartment is not particularly limited as long as it is capable of retaining cells and hydrogel. From the viewpoints of cell desiccation resistance, cell retention, production efficiency, and chemical substance detectability, it is preferable that the compartment be in the form of a well.

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

The arrangement of the cells and the hydrogel in the compartment is not particularly limited, so long as the cells' resistance to drying and cell retention are ensured. For example, in the compartment, the cells are encapsulated in the hydrogel. The manner of encapsulation is not particularly limited, so long as at least a portion of the cells are isolated from the outside air outside the cell chip by the hydrogel. A specific example of the manner of encapsulation is one in which the cells are dispersed in the hydrogel.

From the viewpoint of detection sensitivity, each compartment usually contains a plurality of cells. The number of cells (cells) per volume ( ^cm3 ) of the hydrogel in the compartment is preferably 1× ¹⁰⁴ to 1× ¹⁰⁹ cells/ ^cm3 , more preferably 1× ¹⁰⁵ to 1× ¹⁰⁸ cells/ ^cm3 , and even more preferably 1× ¹⁰⁶ to 2× ¹⁰⁷ cells/ ^cm3 .

The bottom area of one compartment is preferably 0.5 to 100 mm ² , more preferably 1 to 30 mm ² , and even more preferably 1.5 to 10 mm ² , from the viewpoint of detection sensitivity or production efficiency.

The number of compartments contained in a cell chip is preferably 10 to 2000, more preferably 30 to 1000, and even more preferably 50 to 500, from the viewpoint of detection sensitivity or manufacturing efficiency.

The compartment containing cells and a hydrogel containing a (meth)acrylic resin can be obtained by polymerizing the compartment containing cells and a monomer of the (meth)acrylic resin ((meth)acrylic monomer) and/or its oligomer to form a hydrogel. The oligomer is not particularly limited, but can be, for example, a polymer formed by polymerization of about 2 to 20 monomers. From the viewpoint of production efficiency, it is preferable to use a monomer rather than an oligomer.

From the viewpoint of production efficiency, the compartment containing cells and (meth)acrylic monomers and/or oligomers thereof is preferably a compartment containing a solution of (meth)acrylic monomers and/or oligomers thereof and cells, and is preferably a compartment containing a suspension of cells suspended in a solution of (meth)acrylic monomers and/or oligomers thereof.

From the viewpoint of cell desiccation resistance, cell retention, production efficiency, or chemical substance detectability, the total content of the (meth)acrylic monomer and its oligomer is preferably 50% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, even more preferably 85% by mass or more, particularly preferably 90% by mass or more, and especially preferably 95% by mass or more (among which, 100% by mass is the total of the monomer and its oligomer).

The (meth)acrylic monomer is a monomer containing an acrylic group and/or a methacrylic group. There are no particular limitations on the (meth)acrylic monomer, but from the viewpoint of hydrogel formability, it is preferable that the (meth)acrylic monomer contains a hydrophilic structure (e.g., a hydroxyl group, a carboxyl group, an amino group, an amine, a phosphate group, a sulfo group, an amide bond, a polyethylene glycol structure, etc.).

The (meth)acrylic monomer is usually a monomer containing multiple acrylic and/or methacrylic groups (e.g. 2-5, 2-4, or 2-3 groups) (multifunctional monomer). Examples of multifunctional monomers include polyethylene glycol diacrylate (Mn500) (PEGDA500), polyethylene glycol diacrylate (Mn1000) (PEGDA1000), N-[tris(3-acrylamidopropoxymethyl)methyl]acrylamide (4AAmST), N,N-bis(2-acrylamidomethyl)acrylamide (3AAmST), N,N'-[oxybis(2,1-ethanediyloxy)-3,1-propanediyl]bisacrylamide (2AAmLN), and N,N'-1,2-ethanediylbis{N-[2-(acryloylamino)ethyl]acrylamide} (4AAmLN). The polyfunctional monomers can be used alone or in combination of two or more.

From the viewpoints of monomer reaction rate, pre-exposure fluorescence intensity, sensitivity, etc., preferred polyfunctional monomers are those having 2 to 3 (more preferably 2) acrylic and/or methacrylic groups, a polyalkylene glycol structure (e.g., a polyethylene glycol structure), and a molecular weight of 100 to 2000 (preferably 200 to 1500, more preferably 300 to 1200). Examples of such polyfunctional monomers include 2AAmLN, PEGDA500, and PEGDA1000.

The concentration of the polyfunctional monomer in the solution of the (meth)acrylic monomer and/or its oligomer is preferably 0.1 to 25% by mass from the viewpoints of cell drying resistance, cell retention, production efficiency, and chemical substance detectability. The concentration is more preferably 0.2 to 15% by mass, even more preferably 0.5 to 10% by mass, and even more preferably 1 to 8% by mass. In addition, from the viewpoints of monomer reaction rate, pre-exposure fluorescence intensity, sensitivity, etc., the concentration is more preferably 1 to 20% by mass, even more preferably 2 to 15% by mass, even more preferably 4 to 12% by mass, and particularly preferably 5 to 10% by mass.

It is also preferred that the (meth)acrylic monomer further contains a monomer having one acrylic and/or methacrylic group (monofunctional monomer). Examples of monofunctional monomers include (meth)acrylic acid, 2-hydroxyethyl (meth)acrylate, 3-hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 2-(2-hydroxyethoxy)ethyl (meth)acrylate, 2- or 3-chloro-2-hydroxypropyl (meth)acrylate, 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), acrylamide, polyethylene glycol monomethyl ether acrylate (PEG acrylate), 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1-sulfonate (MAm4N9S), 2-[[2-(methacryloyloxy)ethyl]dimethylammonio]a acetate (MO3N6O), 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate (MO3N7O), 3-[(3-acrylamidopropyl)dimethylammonio]propanoic acid (AAm4N8O), 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate (MO3N7S), 4-[[2-(methacryloyloxy)ethyl]dimethylammonio]butane-1-sulfonate (MO3N8S), 3-[[2-(acryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate (AO3N7S), 3-[(3-methacrylamidopropyl)dimethylammonio]propane-1-sulfonate (MAm4N8S), etc. Monofunctional monomers can be used alone or in combination of two or more.

The total concentration of the monofunctional monomer and its oligomer in the solution of the (meth)acrylic monomer and/or its oligomer is preferably 0.1 to 20% by mass from the viewpoint of cell drying resistance, cell retention, production efficiency, or chemical substance detectability. The concentration is more preferably 0.1 to 10% by mass, even more preferably 0.2 to 5% by mass, and even more preferably 0.3 to 5% by mass.

From the viewpoint of production efficiency, the solution of the (meth)acrylic monomer and/or its oligomer preferably contains a polymerization initiator. Examples of the polymerization initiator include photopolymerization initiators and thermal polymerization initiators, but from the viewpoint of production efficiency or chemical substance detectability, photopolymerization initiators are preferred. Examples of the photopolymerization initiator include benzoin, isopropyl benzoin ether, isobutyl benzoin ether, benzophenone, Michler's ketone, chlorothioxanthone, dodecyl thioxanthone, dimethyl thioxanthone, diethyl thioxanthone, acetophenone diethyl ketal, benzyl dimethyl ketal, 1-hydroxycyclohexyl phenyl ketone, 2-hydroxy-2-methyl-1-phenylpropan-1-one, etc., among which benzyl dimethyl ketal, 1-hydroxycyclohexyl phenyl ketone, 2-hydroxy-2-methyl-1-phenylpropan-1-one, etc. are preferably used. In addition, as cationic photopolymerization initiators, initiators such as onium salts, tri(substituted)phenylsulfoniums, diazosulfones, and iodoniums are preferably used. As anionic photopolymerization initiators, organometallic initiators such as alkyllithiums are preferably used. The photopolymerization initiators can be used alone or in combination of two or more.

The concentration of the photopolymerization initiator in the solution of the (meth)acrylic resin monomer and/or its oligomer is preferably 0.1% by mass or less from the viewpoint of cell drying resistance, cell retention, production efficiency, and chemical substance detectability. The concentration is more preferably 0.005 to 0.1% by mass, and even more preferably 0.01 to 0.05% by mass.

The solvent for the solution of the (meth)acrylic resin monomer and/or its oligomer is preferably water. The solution preferably contains a buffer. From the viewpoint of chemical substance detectability, a preferred example of the buffer is a phosphate buffer.

The polymerization treatment is not particularly limited as long as radical polymerization between monomers of the (meth)acrylic resin and/or their oligomers proceeds, but examples include light irradiation and heat treatment. From the viewpoint of production efficiency or chemical substance detectability, the polymerization treatment is preferably light irradiation, and particularly preferably light (preferably ultraviolet light) irradiation.

From the viewpoint of manufacturing efficiency or chemical substance detectability, the wavelength of the irradiated light is preferably 280 to 400 nm, more preferably 320 to 385 nm, and even more preferably 350 to 380 nm. From the same viewpoint, it is also preferable that the light source is an LED (light-emitting diode).

From the viewpoint of production efficiency or chemical substance detectability, the irradiation amount is preferably 50 to 200 mW/cm ² , and more preferably 80 to 150 mW/cm ² .

From the viewpoint of manufacturing efficiency or chemical substance detectability, the irradiation time is preferably 5 to 60 seconds, and more preferably 15 to 45 seconds.

The water content of the hydrogel without cells is preferably 75 to 99.9% by mass, more preferably 80 to 98% by mass, from the viewpoints of cell drying resistance, cell retention, production efficiency, and chemical substance detectability.

The cell chip of the present disclosure can be used to detect chemical substances (especially odor substances). The chemical substances can be, for example, chemical substances in bodily fluids (e.g., urine). In this case, for example, by adding bodily fluid to a compartment of the cell chip of the present disclosure, the chemical substances in the bodily fluid can diffuse through the hydrogel, reach the cells, and come into contact with the olfactory receptors on the cells. This allows the chemical substances to be detected by detecting ions flowing into the cells (for example, by detecting proteins that color or emit light in response to ions).

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

Example 1. Production and evaluation of cell chip 1
<1-1. Preparation of pre-reaction gel solution>
4-[(3-Methacrylamidopropyl)dimethylammonio]butane-1-sulfonate (monomer: MAm4N9S) and N,N'-1,2-Ethanediylbis{N-[2-(acryloylamino)ethyl]acrylamide} (crosslinker: 4AAmLN) were dissolved in PBS (Nissui Pharmaceutical Co., Ltd. #05913) aqueous solution to prepare MAm4N9S/4AAmLN aqueous solution. Lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (photopolymerization initiator: LPA) was dissolved in PBS aqueous solution to prepare LPA aqueous solution. The above two aqueous solutions were mixed and stirred so that the final concentration of MAm4N9S was 2.1% by mass, the final concentration of 4AAmLN was 5.6% by mass, and the final concentration of LPA was 0.02% by mass, to obtain a pre-reaction gel solution.

<1-2. Preparation of gel solution before cell suspension reaction>
Insect cells (SpIm strain derived from Spilosoma imparilis) (genetically modified organisms expressing olfactory receptor proteins, olfactory receptor co-receptor proteins and calcium-dependent fluorescent proteins: hereafter referred to as sensor cells, or genetically modified organisms expressing fluorescent proteins (GFP): hereafter referred to as GFP cells) attached to the culture dish were detached from the surface of the dish by physical impact and suspended in a small amount of medium to obtain a cell suspension. The cell suspension was centrifuged at 1000 rpm for 3 minutes to separate the supernatant medium and precipitated cells, and the supernatant was then removed. The precipitated cells were resuspended in the pre-reaction gel solution to obtain a cell suspension pre-reaction gel solution with a concentration of 5,000,000 cells/ml.

<1-3. Preparation of samples for measuring reaction rate, storage and loss modulus, and haze>
1 mL of the pre-reaction gel solution was added to each well of a 12-well plate. Using a UV light irradiation device (CCS Co., Ltd., 365 nm LED light source), the wells were irradiated with light of 365 nm wavelength at 110 mW/ ^cm2 for 30 seconds to produce hydrogels.

<1-4. Preparation of samples for measuring pre-exposure fluorescence intensity and sensitivity>
5 ul of cell suspension pre-reaction gel solution was filled into a 2 mm diameter hole in a 2 mm thick acrylic plate. The cell suspension pre-reaction gel solution was filled into the well without creating any large air bubbles. Using a UV light irradiation device (CCS Co., Ltd., 365 nm LED light source), the well was irradiated with 365 nm wavelength light at 110 mJ/ ^cm2 for 30 seconds to produce a cell-encapsulated hydrogel.

<1-5. Measurement of monomer reaction rate and water content>
The hydrogel was prepared, washed with ion-exchanged water, and then vacuum-dried (60°C, reduced pressure) for 8 hours or more. The monomer reaction rate (= 100 x weight of hydrogel after drying/prepared amount) and water content (= 100 x (weight of hydrogel before drying-weight of hydrogel after drying)/weight of hydrogel before drying) were calculated from the dried mass.

<1-6. Measurement of turbidity (Haze)>
The hydrogel was placed in a Konica Minolta petri dish (CM-A128) and ion-exchanged water was added to the extent that the hydrogel was submerged. The air around the hydrogel was removed, and measurements were performed at 25°C using a haze meter (HZ-V3, Suga Test Instruments Co., Ltd.).

<1-7. Measurement of storage modulus and loss modulus>
The measurements were performed using an elasticity measuring device (MCR302, Kitahama Seisakusho Co., Ltd.). The measurement conditions were as follows: fixture; PP12/P2, plate; SS/P2, measurement temperature; 25°C. Specifically, the hydrogel was placed on the plate, the fixture was lowered, and measurements were performed using strain dispersion measurement (frequency 1 Hz) and frequency dispersion measurement (strain: 0.04%) while applying a pressure of 0.03 N. The storage modulus and loss modulus were measured at a frequency of 0.736 Hz and a strain of 0.04%.

<1-8. Sensitivity measurement>
The fluorescence intensity data (pre-exposure fluorescence intensity and post-exposure fluorescence intensity) of all cell-encapsulated hydrogels containing sensor cells were calculated as relative values based on the fluorescence intensity of the cell-encapsulated hydrogel containing GFP cells. The fluorescence intensity was measured using a lumino image analyzer (LAS4000, Fujifilm Corporation).

The cell-encapsulated hydrogel containing the sensor cells was immersed in Hank's Balanced Salt Solution (1x Hank's Balanced Salt Solution (gibco #14025-092), 20 mM HEPES (gibco #15630-080), 0.1% Bovine Serum Albumin (Sigma #7888)) for 10 minutes, after which the fluorescence intensity was measured and used as the pre-exposure fluorescence intensity. On the other hand, the hydrogel containing the sensor cells was immersed in the above-mentioned Hank's solution containing an odorant (the odorant to which the above-mentioned olfactory receptor protein responds: Safranal) at a concentration of 400 nM for 3 minutes, after which the fluorescence intensity was measured and used as the post-exposure fluorescence intensity. The value obtained by dividing the post-exposure fluorescence intensity by the pre-exposure fluorescence intensity was used as the sensitivity.

<1-9. Measurement results>
The measurement results are as follows. The sensitivity was much higher than 1, indicating that odor substances could be detected.
Monomer reaction rate: 99% or more Water content: 91.8%
Sensitivity:2.1.

Example 2. Production and evaluation of cell chip 2
The test was conducted in the same manner as in Example 1, except that the type of monomer, the concentration of monomer, the type of crosslinking agent, the concentration of crosslinking agent, and the type of photopolymerization initiator were changed. As a result, the sensitivity was found to be significantly higher than 1, and it was found that odor substances could be detected.

Example 3. Production and evaluation of cell chip 3
Tests were conducted in the same manner as in Example 1 using a crosslinking agent containing one or more members selected from the group consisting of N,N'-[oxybis(2,1-ethanediyloxy)-3,1-propanediyl]bisacrylamide (2AAmLN), polyethylene glycol diacrylate (Mn500) (PEGDA500), and polyethylene glycol diacrylate (Mn1000) (PEGDA1000).

　Monomers include 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (MPC), acrylamide, polyethylene glycol monomethyl ether acrylate (PEG acrylate), 4-[(3-methacrylamidopropyl)dimethylammonio]butane-1-sulfonate (MAm4N9S), 2-[[2-(methacryloyloxy)ethyl]dimethylammonio]acetate (MO3N6O), 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate (MO3N7O), 3-[[3-acrylamidopropyl]dimethylammonio]propionate (MO3N7O), The following compounds were used alone or in combination: 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propanoic acid (AAm4N8O), 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate (MO3N7S), 4-[[2-(methacryloyloxy)ethyl]dimethylammonio]butane-1-sulfonate (MO3N8S), 3-[[2-(acryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate (AO3N7S), 3-[(3-methacrylamidopropyl)dimethylammonio]propane-1-sulfonate (MAm4N8S).

The crosslinker concentration in the gel solution before reaction, monomer reaction rate, pre-exposure fluorescence intensity, and sensitivity are shown in Table 1. In the table, blank spaces indicate a concentration of 0.0 wt%. Note that the type and/or composition of monomers differs between samples. A pre-exposure fluorescence intensity above a certain level indicates that there are relatively few factors that reduce the fluorescence intensity (such as gel turbidity), and therefore data errors due to such factors can be further reduced, further improving the reliability of the data.

Claims

A cell chip comprising a compartment containing cells and a hydrogel including a (meth)acrylic resin.
The cell chip of claim 1, wherein the cells are encapsulated in the hydrogel.
The cell chip according to claim 1, wherein the cells are cells that contain a polynucleotide that includes a coding sequence for an olfactory receptor protein.
The cell chip of claim 3, wherein the olfactory receptor protein is an insect olfactory receptor.
The cell chip of claim 1, wherein the cells are insect cells.
The cell chip of claim 1, wherein the hydrogel is formed by polymerizing a solution of (meth)acrylic monomers and/or oligomers thereof in a compartment containing cells.
The cell chip of claim 6, wherein the polymerization treatment is UV irradiation.
The cell chip according to claim 6, wherein the photopolymerization initiator concentration in the solution of the (meth)acrylic monomer and/or its oligomer is 0.1% by mass or less.
The cell chip according to claim 6, wherein the total concentration of the polyfunctional (meth)acrylic monomer and the oligomer in the solution of the (meth)acrylic monomer and/or its oligomer is 0.1 to 25% by mass.
The cell chip of claim 1, wherein the number of compartments is 10 to 2000.
The cell chip according to any one of claims 1 to 10, which is used for detecting chemical substances.
A method for producing a cell chip according to any one of claims 1 to 10, comprising polymerizing a compartment containing cells and a (meth)acrylic resin monomer and/or an oligomer thereof to form a hydrogel.
The method of claim 12, wherein the polymerization treatment is UV irradiation.
The method of claim 13, wherein the source of UV radiation is an LED.