WO2018043533A1

WO2018043533A1 - Detection device and cell-containing matter

Info

Publication number: WO2018043533A1
Application number: PCT/JP2017/031079
Authority: WO
Inventors: 昌治竹内; 雄矢森本; 昭太郎吉田; 垠列南; 誠司田畑; 優介平田
Original assignee: 国立大学法人東京大学
Priority date: 2016-09-02
Filing date: 2017-08-30
Publication date: 2018-03-08
Also published as: JPWO2018043533A1; JP6785476B2

Abstract

The purpose of the present invention is to provide a detection device capable of easily detecting intracellular fluorescence. The present invention provides a detection device comprising a gel 11, a plurality of cells 1 that are disposed in the gel 11 and stained with a fluorescent reagent, and a photodetector 2 that detects fluorescence emitted from the fluorescent reagent when the plurality of cells 1 react to a substance.

Description

Detection device and cell-containing object

The present invention relates to a cell technology, and relates to a detection device and a cell-containing object.

When a cell reacts with an extracellular substance, the membrane potential changes, the intracellular ion concentration changes, or a response substance is generated. Therefore, by detecting whether or not a response response of a cell to a substance such as a ligand occurs, it is possible to determine that a substance such as a ligand exists when the response reaction occurs. For this reason, attempts have been made to use cells to detect substances. Further, by detecting a response response of a cell to a substance such as a drug, it is possible to determine that a substance such as a drug acts on the cell when the response reaction occurs. Therefore, attempts have been made to use cells for drug screening (see, for example, Patent Documents 1 to 3).

Japanese Patent No. 5127778 Japanese Patent No. 5603850 Japanese Patent No. 5696953

∙ Cell response to extracellular substances is visualized with fluorescent reagents. However, the intensity of the fluorescence emitted from the fluorescent reagent in the cell is weak, and a highly sensitive detection device is required for fluorescence detection. Therefore, the present invention provides a detection device capable of easily detecting fluorescence in a cell, a cell-containing object, a method for producing a cell-containing object, an array of cell-containing objects, and a method for producing an array of cell-containing objects. One of the purposes.

According to an aspect of the present invention, a gel, a plurality of cells arranged in the gel and stained with a fluorescent reagent, and light detection for detecting fluorescence emitted from the fluorescent reagent when the plurality of cells react with a substance And a detector.

In the above detection device, a plurality of cells may be dispersed in the gel.

In the above detection device, a plurality of cells may form a cell spheroid.

In the above detection device, the gel may have a three-dimensional shape.

In the above detection device, the gel may have a cylindrical shape.

In the above detection device, the gel may contain collagen.

In the above detection device, the substance may be permeable through the gel.

In the above detection apparatus, the gel may be disposed on the photodetector.

The detection device may further include a culture container, and the gel may be disposed in the culture container.

The detection device may include a plurality of gels.

In the above detection device, different types of cells may be held in a plurality of gels.

In the above detection apparatus, different types of cells may be stained with different types of fluorescent reagents.

In the above-described detection device, cells expressing different types of receptors may be retained in a plurality of gels.

In the above detection apparatus, cells expressing different types of receptors may be stained with different types of fluorescent reagents.

In addition, according to an aspect of the present invention, the apparatus includes a cell spheroid composed of a plurality of cells stained with a fluorescent reagent, and a photodetector that detects fluorescence emitted from the fluorescent reagent when the cell spheroid reacts with a substance. A detection device is provided.

The detection device may further include a culture container, and the cell spheroid may be disposed in the culture container.

In the above detection apparatus, the solution may be put in a culture container. The solution may be a culture solution.

In the above detection apparatus, the culture vessel may be disposed on the photodetector.

In the above detection device, the photodetector may include an image sensor. The image sensor may be a solid-state image sensor. The solid-state imaging device may be a CMOS image sensor.

In the above detection apparatus, the substance may be an odor substance, and a plurality of cells may express a receptor for the odor substance.

In the above detection device, the substance may be a drug or a pesticide.

In the above detection apparatus, the fluorescent reagent may be a calcium indicator.

The detection device may further include a perfusion device that perfuses the solution in the culture vessel.

The detection device described above may further include a temperature control device that controls the temperature of the solution.

Moreover, according to the aspect of the present invention, there is provided a cell-containing object comprising a gel and a plurality of cells arranged in the gel and stained with a fluorescent reagent.

In the above cell-containing object, a plurality of cells may be dispersed in the gel.

In the cell-containing object, a plurality of cells may form a cell spheroid.

In the cell-containing object, the gel may have a three-dimensional shape.

In the cell-containing object, the gel may have a cylindrical shape.

In the above-mentioned cell-containing object, the gel may contain collagen.

In the cell-containing object, a plurality of cells may express a receptor.

In the above cell-containing object, the receptor may be a receptor for an odor substance.

In the cell-containing object, the fluorescent reagent may be a calcium indicator.

Moreover, according to the aspect of the present invention, a solution containing a plurality of cells stained with a fluorescent reagent is placed in a mold, the solution is made into a gel, the gel and the mold are separated, and the gel and the gel And obtaining a cell-containing object comprising a plurality of cells stained with a fluorescent reagent, and a method for producing a cell-containing object.

In the method for producing a cell-containing object, a plurality of cells may be dispersed in the gel.

In the above method for producing a cell-containing object, a plurality of cells may form a cell spheroid.

In the above method for producing a cell-containing object, the gel may have a three-dimensional shape.

In the above method for producing a cell-containing object, the gel may have a cylindrical shape.

In the above method for producing a cell-containing object, the gel may contain collagen.

In the method for producing a cell-containing object, a plurality of cells may express a receptor.

In the above method for producing a cell-containing object, the receptor may be an odor substance receptor.

In the above method for producing a cell-containing object, the fluorescent reagent may be a calcium indicator.

According to an aspect of the present invention, there is provided an array of cell-containing objects, comprising a plurality of gels and a plurality of cells stained in a fluorescent reagent arranged in each of the plurality of gels. .

In the above-described array of cell-containing objects, a plurality of cells may be dispersed in the gel.

In the array of cell-containing objects, a plurality of cells may form cell spheroids.

In the above-described array of cell-containing objects, each of the plurality of gels may have a three-dimensional shape.

In the above-described array of cell-containing objects, each of the plurality of gels may have a cylindrical shape.

In the above-described array of cell-containing objects, each of the plurality of gels may contain collagen.

In the cell-containing object array, different types of cells may be held in a plurality of gels.

In the above-described array of cell-containing objects, different types of cells may be stained with different types of fluorescent reagents.

In the array of cell-containing objects described above, cells expressing different types of receptors may be retained in a plurality of gels.

In the array of cell-containing objects, cells expressing different types of receptors may be stained with different types of fluorescent reagents.

In the cell-containing object array, each of different types of receptors may be a receptor for an odor substance.

In the array of cell-containing objects, different types of fluorescent reagents may emit fluorescence in different wavelength bands.

In the cell-containing object array described above, each of the different types of fluorescent reagents may be a calcium indicator.

In addition, according to the aspect of the present invention, the first solution containing the first type of cells stained with the first type of fluorescent reagent is placed in the mold, and the first solution is used using the first photomask. Forming a part of the solution into a first gel, placing a second solution containing cells of the second type stained with the second type of fluorescent reagent into a mold, and a second photomask A method for producing an array of cell-containing objects comprising: forming a second gel from a portion of the second solution.

In the method for manufacturing an array of cell-containing objects, the first type of cells may be dispersed in the first gel.

In the method for producing an array of cell-containing objects, the first type of cells may form a cell spheroid in the first gel.

In the above method for producing an array of cell-containing objects, the second type of cells may be dispersed in the second gel.

In the above method for producing an array of cell-containing objects, the second type of cells may form cell spheroids in the second gel.

In the method for manufacturing an array of cell-containing objects, the first and second gels may have a three-dimensional shape.

In the above method for producing an array of cell-containing objects, the first and second gels may have a cylindrical shape.

In the above-described method for manufacturing an array of cell-containing objects, the first and second gels may contain collagen.

In the above method for producing an array of cell-containing objects, the first and second types of cells may express different types of receptors.

In the above method for producing an array of cell-containing objects, each of the different types of receptors may be a receptor for an odor substance.

In the above method for producing an array of cell-containing objects, the first and second types of fluorescent reagents may emit fluorescence in different wavelength bands.

In the above method for producing an array of cell-containing objects, the first and second types of fluorescent reagents may be calcium indicators.

ADVANTAGE OF THE INVENTION According to this invention, the detection apparatus which can detect the fluorescence in a cell easily, a cell containing object, the manufacturing method of a cell containing object, the array of a cell containing object, and the manufacturing method of the array of a cell containing object can be provided. .

It is a schematic diagram of the detection apparatus which concerns on 1st Embodiment. It is a schematic diagram of the array of the cell containing object which concerns on 1st Embodiment. It is a schematic diagram of the detection apparatus which concerns on 1st Embodiment. It is a schematic diagram of the cell which concerns on 1st Embodiment. It is a schematic diagram which shows the manufacturing process of the array of the cell containing object which concerns on 1st Embodiment. It is a schematic diagram which shows the manufacturing process of the array of the cell containing object which concerns on 1st Embodiment. It is a schematic diagram which shows the manufacturing process of the array of the cell containing object which concerns on 1st Embodiment. It is a schematic diagram which shows the manufacturing process of the array of the cell containing object which concerns on 1st Embodiment. It is a schematic diagram which shows the manufacturing process of the array of the cell containing object which concerns on 1st Embodiment. It is a schematic diagram which shows the manufacturing process of the array of the cell containing object which concerns on 1st Embodiment. It is a schematic diagram which shows the manufacturing process of the array of the cell containing object which concerns on 1st Embodiment. It is a schematic diagram which shows the manufacturing process of the array of the cell containing object which concerns on 1st Embodiment. It is a schematic diagram which shows the manufacturing process of the array of the cell containing object which concerns on 1st Embodiment. It is a bright field image of the column-shaped gel which concerns on the Example of 1st Embodiment. It is a graph which shows distribution of the diameter of the column-shaped gel which concerns on the Example of 1st Embodiment. It is a three-dimensional image of the cylindrical gel which concerns on the Example of 1st Embodiment. It is a graph which shows distribution of the height of the column-shaped gel which concerns on the Example of 1st Embodiment. It is the bright-field image and fluorescence image of the column-shaped gel which concern on the Example of 1st Embodiment. It is a fluorescence image of the column-shaped gel which concerns on the Example of 1st Embodiment. It is a graph which shows the relationship between the height of the column-shaped gel which concerns on the Example of 1st Embodiment, and fluorescence intensity. It is a graph which shows the relationship between the time passage after addition of the odorous substance which concerns on the Example of 1st Embodiment, and fluorescence intensity. It is a graph which shows the density | concentration of the odor substance which concerns on the Example of 1st Embodiment, and the relationship of fluorescence intensity. FIG. 23A is a bright-field image of a gel formed by UV irradiation according to an example of the first embodiment. FIG. 23B is a composite image of a bright field image and a fluorescence image of a gel formed by UV irradiation according to the example of the first embodiment. The cells were stained with Calcein-AM and Ethidium Homodimer. Calcein-AM penetrates the cell membrane of living cells and emits green fluorescence. Ethidium Homodimer permeates the membrane damage part of dead cells and emits red fluorescence. In the original color image of FIG. 23, the majority of cells emitted green fluorescence, confirming that the cells were alive. FIG. 24A is a bright-field image of a gel formed by UV irradiation according to an example of the first embodiment. FIG. 24B is a fluorescence image of a gel formed by UV irradiation according to an example of the first embodiment and including cells stained with different fluorescent reagents. FIG. 24C is a graph showing the position of a gel containing cells stained with different fluorescent reagents and the fluorescence intensity. It is a schematic diagram of the detection apparatus which concerns on 2nd Embodiment. It is a schematic diagram of the detection apparatus which concerns on 2nd Embodiment. It is an image of the spheroid which concerns on the Example of 2nd Embodiment. FIG. 28A is a bright field image of a spheroid according to an example of the second embodiment. FIG. 28B is a fluorescent image of spheroids according to an example of the second embodiment. FIG. 28C is a composite image of a bright field image of a spheroid and a fluorescence image according to an example of the second embodiment. FIG. 28 (d) is a graph showing the fluorescence intensity along the broken line AA ′ in FIG. 28 (b). FIG. 29A is a fluorescence image of a single cell according to an example of the second embodiment. FIG. 29B is a fluorescence image of a spheroid according to an example of the second embodiment. FIG. 29C is a graph showing the intensity of fluorescence emitted from the single cell and spheroid according to the example of the second embodiment. FIG. 30A is a fluorescence image of a spheroid before addition of eugenol using a confocal microscope according to an example of the second embodiment. FIG. 30B is a fluorescence image after addition of eugenol to spheroids using a confocal microscope according to an example of the second embodiment. FIG. 30C is a graph showing the fluorescence intensity before and after the addition of eugenol according to an example of the second embodiment. FIG. 30D is a bright field image of a spheroid using a CMOS image sensor. FIG. 30E is a binary spheroid fluorescence image using a CMOS image sensor.

Embodiments of the present invention will be described below. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

(First embodiment)
As shown in FIG. 1, the detection apparatus according to the first embodiment includes a gel 11, a plurality of cells 1 arranged in the gel 11, and stained with a fluorescent reagent, and the plurality of cells 1 reacted to a substance. And a photodetector 2 that detects fluorescence emitted from the fluorescent reagent.

A plurality of cells 1 may be dispersed in the gel 11. Alternatively, the plurality of cells 1 may form a cell spheroid. A cell spheroid is a cell mass composed of a plurality of cells. In the cell spheroid, cells are adhered to each other. The substance that reacts with the plurality of cells 1 is, for example, a ligand and an odor substance. Each cell of the plurality of cells 1 expresses an odor substance receptor that binds to a specific odor substance. The odorant receptor is also called an olfactory receptor and belongs to the G protein coupled receptor family. The cells are mammalian cells such as HEK293T cells, but are not particularly limited. The cell may be an olfactory receptor cell or a cell in which a specific receptor is expressed by genetic engineering.

The cell incorporates a fluorescent reagent that emits fluorescence when the cell reacts with the substance. The fluorescent reagent taken up by the cells is, for example, a calcium indicator. When an odor substance and a cell odor substance receptor bind to each other, a signal is transmitted within the cell, and the calcium ion concentration increases. Calcium indicators fluoresce as the calcium ion concentration increases. Thus, the fluorescent reagent can visualize a chemical reaction in the cell that occurs when the cell reacts with the substance. Examples of calcium indicators include Fura2-AM, Fluo-3-AM, Fluo-4-AM, and Fluo-8-AM.

Fluorescent reagent is not limited to calcium indicator. The fluorescent reagent may be a membrane potential sensitive fluorescent reagent or a neurotransmitter sensitive fluorescent reagent. The membrane potential sensitive fluorescent reagent emits fluorescence in response to a change in membrane potential that occurs when a cell reacts with a substance. Examples of membrane potential sensitive fluorescent reagents include di-4-ANEPPS and DiBAC4. A neurotransmitter-sensitive fluorescent reagent emits fluorescence in response to a neurotransmitter such as glutamate produced in a cell when the cell reacts with the substance. An example of a neurotransmitter sensitive fluorescent reagent is EOS (glutamate optical sensor).

The gel 11 containing a plurality of cells 1 has an arbitrary three-dimensional shape such as a cylindrical shape. The gel 11 is a hydrogel, for example. As the material of the gel 11, for example, collagen, gelatin, agar, alginic acid, polyethylene glycol diacrylate (PEGDA), or the like can be used. The material of the gel 11 is not particularly limited as long as a substance that reacts with cells can permeate the inside of the gel 11. Moreover, the gel 11 may be permeable to culture components such as nutrient components of the culture solution. The plurality of cells 1 and the gel 11 constitute a cell-containing object.

As shown in FIG. 2, the gel 11 including a plurality of cells 1 is disposed on a transparent substrate 21 that can transmit fluorescence generated in the plurality of cells 1, for example. One gel 11 or a plurality of gels 11 may be arranged on the transparent substrate 21. The plurality of gels 11 may be arranged in an array. The surface of the transparent substrate 21 may be coated with the same material as that of the gel 11. Thereby, the adhesiveness of the gel 11 to the transparent substrate 21 is improved.

It is reported that animals do not recognize one odor in response to one odor substance, but recognize one odor according to a combination pattern of a plurality of odor substances. Therefore, when the detection apparatus includes a plurality of gels 11, different types of cells may be held in the plurality of gels 11. In different types of cells, for example, different types of receptors may be expressed.

In the detection device, for example, a gel that holds a plurality of cells that react with the first odor substance, a gel that holds a plurality of cells that react with the second odor substance, and a plurality of substances that react with the third odor substance. A gel for holding cells may be disposed. You may identify the kind of odorous substance from the position of the gel from which fluorescence was emitted. Moreover, you may identify the kind of odor from the combination of the position of the gel in which fluorescence was emitted. Alternatively, the type of odor substance may be specified from the wavelength band or color of fluorescence by staining different types of cells with different types of fluorescent reagents. Further, the type of odor may be specified from the combination of the fluorescent wavelength band and the color.

As shown in FIG. 1, a frame 22 may be disposed on the transparent substrate 21. In this case, the transparent substrate 21 and the frame 22 constitute a culture vessel. The frame 22 is made of, for example, dimethylpolysiloxane (PDMS), but is not particularly limited. The gel 11 including a plurality of cells 1 is placed in a culture container formed by the transparent substrate 21 and the frame 22. Further, a solution 23 such as a culture solution or a buffer solution may be placed in the culture vessel formed by the transparent substrate 21 and the frame 22. By putting the solution in the frame 22, it is possible to prevent the moisture of the gel 11 from evaporating and supply nutrients to the plurality of cells 1.

The culture vessel formed by the transparent substrate 21 and the frame 22 is placed on the photodetector 2. Therefore, the gel 11 including the plurality of cells 1 is arranged on the photodetector 2. A lens barrel 31 may be disposed between the photodetector 2 and the transparent substrate 21. In the lens barrel 31, a lens 32 that converts the fluorescence generated in the plurality of cells 1 into parallel light, and a lens 33 that converges the fluorescence transmitted through the lens 32 on the photodetector 2 may be disposed.

A band-pass filter 34 that transmits fluorescence generated in a plurality of cells 1 and does not transmit light having a wavelength band different from that of the fluorescence may be disposed on the photodetector 2.

The photodetector 2 is disposed on the circuit board 3, for example. The photodetector 2 may include an image sensor such as a solid-state image sensor. As the solid-state imaging device, a CMOS (Complementary Metal Oxide Semiconductor) image sensor, a CCD (Charge-Coupled Device) image sensor, or the like can be used. By using the small photodetector 2, it is possible to reduce the size of the detection device according to the first embodiment.

A processing device may be connected to the photodetector 2. For example, the processing apparatus may calculate the concentration of the odor substance based on the fluorescence intensity detected by the photodetector 2 using the relationship between the fluorescence intensity acquired in advance and the concentration of the odor substance. .

The detection apparatus according to the first embodiment includes an excitation light source 4 that emits excitation light toward a plurality of cells 1, and a light source 5 that emits light including wavelengths other than excitation light such as white light toward the plurality of cells 1. And may be further provided. The excitation light source 4 emits excitation light having an excitation wavelength corresponding to the fluorescent reagent taken into the cells constituting the plurality of cells 1. When the excitation light source 4 emits excitation light, it is possible to take an image of fluorescence emitted by the plurality of cells 1 with the photodetector 2. Further, when the light source 5 emits white light, the photodetector 2 can capture images such as bright field images and dark field images of the shape of the plurality of cells 1.

As shown in FIG. 3, the gel 11 including a plurality of cells 1 may be arranged on the photodetector 2 without using a lens or the like. The solution 23 filling the periphery of the gel 11 containing a plurality of cells 1 may be perfused with a perfusion device 71 such as a motor pump. Further, the temperature of the solution 23 may be controlled by a temperature control device 72 such as a heater. The culture vessel in which the gel 11 including a plurality of cells 1 is arranged may be covered with a semipermeable membrane 73 that allows gas to permeate and does not allow liquid to permeate. Thereby, it is possible to prevent evaporation of the solution 23 while supplying components necessary for cell culture such as oxygen dioxide and oxygen to the solution 23.

Since the intensity of fluorescence generated in a single cell or in a cell cultured in a single layer is weak, it may be difficult to detect with a conventional detector or large light that is not easy to carry. Sometimes a detector is required. On the other hand, in the detection apparatus according to the first embodiment, a plurality of cells 1 are three-dimensionally arranged in the gel 11, and the plurality of cells 1 are stacked vertically with respect to the photodetector 2. Yes. Therefore, since the fluorescence emitted by the plurality of cells 1 in the gel 11 is superimposed, the fluorescence having a higher intensity than the fluorescence emitted by the cells cultured in the single layer reaches the photodetector 2. Therefore, it is possible to accurately detect the response response of the cell to the substance even with the photodetector 2 with low sensitivity such as a CMOS image sensor. In addition, since it is not always necessary to use a high-sensitivity large photodetector, the detection device according to the first embodiment can be made small enough to be portable.

In the detection apparatus according to the first embodiment, a plurality of cells 1 are held in the gel 11. The gel 11 can be easily bonded to the transparent substrate 21. Therefore, since the gel 11 can be fixed to the transparent substrate 21, even if the solution 23 around the gel 11 is exchanged or a substance that reacts with cells is dropped into the solution 23, the plurality of cells 1 It is possible to suppress the position and orientation from changing with respect to the photodetector 2. Therefore, it is possible to improve reproducibility at the time of fluorescence measurement.

Next, a method for producing a gel 11 containing a plurality of cells 1 will be described. A plasmid incorporating a DNA encoding an odorant receptor is introduced into the cell, and the odorant receptor 13 is expressed in the cell 12, as shown in FIG. Next, the cells are suspended in a gel raw material solution such as a collagen solution to obtain a cell suspension.

As shown in FIG. 5, a mold 51 provided with a plurality of

wells

52a, 52b, 52c. The inner diameter and depth of each of the plurality of

wells

52a, 52b, 52c... Are designed based on the diameter and height of the gel to be produced. The mold 51 is made of, for example, dimethylpolysiloxane (PDMS), but is not particularly limited. The surface of the mold 51 may be coated with bovine serum albumin (BSA) or the like so that the gel can be easily removed. Next, the cell suspension 14 is filled in each of the

wells

52a, 52b, 52c.

As shown in FIGS. 6 and 7, a transparent substrate 21 coated with the same gel material as the gel material of the gel raw material solution is prepared, and the surface of the mold 51 on which the

wells

52a, 52b, 52c. Is adhered to the surface of the transparent substrate 21. Thereafter, the plurality of cells 12 are cultured in the

wells

52a, 52b, 52c. The plurality of cells 12 may be cultured in a dispersed state. Alternatively, the cell spheroid 1 may be formed in a plurality of cells 12. Different types of cells may be cultured in the

wells

52a, 52b, 52c. Furthermore, in order to gel the gel raw material solution into the gel 11, the gel raw material solution is heated. The heating temperature is 37 ° C., for example, and the heating time is 15 minutes, for example.

As shown in FIG. 8, for example, a solution 23 such as a culture solution or a buffer solution is placed around the mold 51. Thereby, it becomes easy to peel off the mold 51 from the transparent substrate 21. Thereafter, as shown in FIG. 9, although not particularly limited, when the mold 51 is pulled up with tweezers 61 or the like, the gel 11 containing a plurality of cells 1 remains on the transparent substrate 21 as shown in FIG.

Alternatively, as shown in FIG. 11, a mold 151 provided with one well 152 is prepared, and the well 152 is filled with a cell suspension 14 containing a gelling agent that gels by light irradiation such as PEGDA. The cell suspension 14 contains the first type of cells stained with the first type of fluorescent reagent. As shown in FIG. 12, the transparent substrate 21 is prepared, and the surface of the mold 151 on the side where the well 152 is provided is brought into close contact with the surface of the transparent substrate 21. Thereafter, the first type of cells 12 are cultured in the well 152 of the mold 151. Optionally, cell spheroids may be formed. Furthermore, light is irradiated from a light source 82 such as a UV irradiation device through the first photomask 81 or the like, and a part of the cell suspension 14 is gelled.

Thereafter, a plurality of gels including a plurality of cells of the first type are placed on the transparent substrate 21 by removing the cell suspension 14 that has not been irradiated with light and has not gelled. Further, the well 152 of the mold 151 is filled with a cell suspension containing a second type of cell different from the first type of cell. The second type of cells are stained with a second type of fluorescent reagent different from the first type of fluorescent reagent. Next, as shown in FIG. 13, a part of the cell suspension is gelled using a second photomask 83 having an opening pattern different from that of the first photomask 81. Thereafter, a plurality of gels including a plurality of cells of the second type are arranged on the transparent substrate 21 by removing the cell suspension that has not been irradiated with light and has not gelled. Thereafter, the same method may be repeated to arrange a plurality of gels each containing different types of cells on the transparent substrate 21.

(Example of the first embodiment)
HEK293T cells in which mOR-EG, an olfactory receptor, was genetically engineered were prepared. The cells were stained with Fluo-8 AM (AAT Bioquest), a calcium indicator. In addition, Cellmatrix Type I-A (Nitta Gelatin Co., Ltd.), 10-fold Hanks buffer, and reconstitution buffer were mixed at a ratio of 8: 1: 1 to prepare a collagen solution containing collagen at a concentration of 2.4 mg / mL. did. Furthermore, a mold made of PDMS provided with an array of wells having a diameter of 200 μm and a depth of 200 μm was prepared. The mold surface was coated with 1% BSA for 30 minutes. Cells were suspended in a collagen solution to a concentration of 10 ⁸ cells / mL to obtain a cell suspension. The obtained cell suspension was put into a mold well, and the surface provided with the mold well was attached to the frame of the glass plate. Cells were cultured for 3 hours in the wells. Thereafter, the collagen solution was heated at 37 ° C. for 30 to 40 minutes in order to gel the collagen solution. After the gel was formed, the culture solution was put in a frame and the mold was peeled off from the surface of the glass plate.

As shown in FIG. 14, it was confirmed with an inverted microscope that columnar gels were arranged in an array on a glass plate. When the distribution of the diameters of a plurality of cylindrical gels formed from wells having the same shape was examined by image analysis, the diameters were almost uniform as shown in FIG. As shown in FIG. 16, the columnar gel is observed with a shape analysis laser microscope (VK-X21, Keyence), and the height distribution of a plurality of columnar gels formed from wells having the same shape is observed. As a result, as shown in FIG. 17, the height was almost uniform.

Next, eugenol was prepared as an odor substance. The chemical formula of eugenol is shown below. Eugenol is generally extracted from cinnamon and used in spices and perfumes. Eugenol was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mmol / L to obtain an odor substance solution.

A detection device having a structure similar to that shown in FIG. 1 was prepared using a CMOS image sensor. A plurality of cylindrical gels having the same diameter and different heights were prepared using molds having different well depths. In order to measure background noise, before dropping the odor substance solution into the culture solution, a plurality of cells were irradiated with excitation light, and a fluorescence image was taken with a CMOS image sensor. Next, after the concentration of the odor substance solution was appropriately diluted and half the amount of the culture solution was sucked so that the liquid level did not change, the same amount of the odor substance solution was dropped into the culture solution. Thereafter, a plurality of cells were irradiated with excitation light, and a fluorescence image was taken with a CMOS image sensor. Further, a difference image between the fluorescence images before and after the odor substance solution was dropped was obtained as a corrected fluorescence image in which background noise was canceled. The obtained bright field image and corrected fluorescence image are shown in FIG. In the bright field image, the outline of the cylindrical gel was observed. In the corrected fluorescence image, the fluorescence emitted by calcein was observed.

Further, as shown in FIG. 19 and FIG. 20, it was confirmed that the fluorescence intensity detected by the CMOS image sensor increases as the height of the columnar gel increases. This is probably because the number of cells contained in the gel increases as the height of the gel increases, so that the intensity of fluorescence generated in the cells is superimposed. Moreover, when the temporal change in fluorescence intensity after dropping the odor substance solution into the culture solution was observed, an increase in fluorescence intensity was confirmed within several tens of seconds as shown in FIG. Therefore, it was shown that the detection device according to the example has good response and can detect an odorous substance in real time.

Furthermore, when the concentration of the odor substance in the odor substance solution was changed, as shown in FIG. 22, it was observed that the higher the odor substance concentration, the stronger the fluorescence intensity. Therefore, it was shown that if the relationship between the concentration of the odor substance and the fluorescence intensity is acquired in advance, the concentration of the odor substance can be calculated from the intensity of the fluorescence observed by the detection apparatus.

In addition, when a cell suspension containing PDMS was prepared and the cell suspension was gelled using a photomask provided with a plurality of circular openings, a cylindrical gel was obtained as shown in FIG. It was. When cells stained with different types of fluorescent reagents were included in adjacent gels, it was confirmed that fluorescence in different wavelength bands was emitted from each gel, as shown in FIG.

(Second Embodiment)
In the first embodiment, an example in which a plurality of cells are held in a gel is shown. On the other hand, when a plurality of cells form a cell spheroid, the cell spheroid does not necessarily have to be retained in the gel. For example, in the detection apparatus according to the second embodiment shown in FIG. 25, the cell spheroid composed of a plurality of cells 1 may be directly placed in the solution 23. Also in FIG. 25, the transparent substrate 21 and the frame 22 constitute a culture container, and the cell spheroid composed of a plurality of cells 1 and the solution 23 such as a culture solution or a buffer solution are the transparent substrate 21 and the frame 22. Is placed in a culture vessel formed. As shown in FIG. 26, in the detection apparatus according to the second embodiment, when a substance 15 such as an odor substance enters the solution 23, a cell spheroid composed of a plurality of cells 1 reacts with the substance 15 and emits fluorescence.

Other components of the detection device according to the second embodiment are the same as those in the first embodiment.

(Example of the second embodiment)
Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich), 10 vol% fetal bovine serum (FBS, Biosera), 100 U / mL penicillin (Kanto Chemical or Wako Pure Chemical), and 100 μg / mL streptomycin (Kanto Chemical or Japanese) Photopure drug) was added and the culture solution was adjusted. In addition, phosphate buffered saline (PBS, Sigma-Aldrich) and eugenol as an odor substance were prepared.

In order to detect eugenol, HEK293T cells were treated with lipofectamine 3000 or 2000 (registered trademark, Invitrogen) with mOR-EG, which is an olfactory receptor, G _α15 , which is a G protein α subunit, and a receptor transporter protein. It was transformed to express a certain RTP1 and a signal amplification molecule, Ric-8. The amount of gene added per mL of medium was 1.5 μg for pME18S-Rho-mOR-EG (when using Lipofectamine 3000) or 1.45 μg (when using Lipofectamine 2000), 1.0 μg for pME18S-G _a15 (Lipofectamine 3000) Used) or 0.87 μg (when using Lipofectamine 2000), pME18S-RTP1 is 0.5 μg (when using Lipofectamine 3000) or 0.58 μg (when using Lipofectamine 2000), pEGFP-N3-Myr-Ric-8A is 0. It was 5 μg (when using Lipofectamine 3000) or 0.29 μg (when using Lipofectamine 2000).

The transformed cells subjected to the adhesion culture were treated with a trypsin-EDTA solution at 37 ° C. for 5 minutes to release the cells from the incubator. Furthermore, the culture solution was added to suspend the cells, followed by centrifugation at 2G for 5 minutes. After centrifugation, the cells were resuspended in the culture solution, and the suspension was dropped into each well of a PDMS incubator in which wells having a diameter of 200 μm and a depth of 200 μm were provided in an array. Thereafter, cell spheroids composed of transformed cells were formed by culturing for 3 hours. A bright field image of the cell spheroid is shown in FIG. Furthermore, cell spheroids were stained with calcium indicator calcein-AM (Thermo Fisher Scientific) or Fluo-8-AM (AAT bioquest).

Using a 3D printer (AGILISTA-3100, Keyence), a detection device of several cm square having the same structure as that shown in FIG. 25 was produced. A CMOS image sensor (WAT-01U2, Watec), a white light source (OSW4XME3C1E, OptoSupply), and a blue excitation light source (OEHB220, OptoSupply) were arranged in the detection device. Cell spheroids collected from the incubator with a pipette were placed in the culture solution in the culture vessel of the detection apparatus. When capturing a bright-field image of spheroid, white light was irradiated to the spheroid through a pinhole. When eugenol was put into the culture solution and a fluorescent image of spheroids was taken, the cell spheroids were irradiated with blue excitation light, and the color in the green wavelength band was extracted.

Fig. 28 (a) shows a bright field image of a cell spheroid, Fig. 28 (b) shows a fluorescence image of the cell spheroid, and Fig. 28 (c) shows a composite image of the bright field image and the fluorescence image. FIG. 28 (d) shows a distribution graph of fluorescence intensity along the broken line AA ′ in FIG. 28 (c). It has been shown that a fluorescence reaction in a cell spheroid can be detected using a CMOS image sensor. Furthermore, as shown in FIG. 29, the intensity of the fluorescence emitted by the cell spheroids was about 4 times stronger than the intensity of the fluorescence emitted by one cell (single cell). Thus, cell spheroids have been shown to improve the sensitivity of the detection device.

Moreover, when it measured using the confocal microscope, as shown to FIG. 30 (a) to (c), compared with before adding eugenol to a culture solution, 2 mmol / L eugenol was added to the culture solution. Later, the intensity of fluorescence emitted by cell spheroids increased more than twice. FIG. 30D shows a bright field image of a cell spheroid imaged by a CMOS image sensor, and FIG. 30E shows an image obtained by binarizing a fluorescent image of the cell spheroid with an image J that is image processing software. From the results of this example, it was shown that a detection apparatus using a CMOS image sensor can be used to detect a reaction between a cell spheroid olfactory receptor and eugenol, which is an odor substance.

(Other embodiments)
Although the present invention has been described by the embodiments as described above, it should not be understood that the description and drawings constituting a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, examples and operational techniques should be apparent to those skilled in the art. For example, in the embodiment, an odor substance is given as an example of a substance with which cells react, but the substance is not limited to this. The substance may be various ligands, ions, or cell membrane permeable substances. The presence of these substances can be detected by detecting the fluorescence emitted due to the intracellular response reaction of these substances.

In addition, the substance with which the cell reacts may be a drug such as a medicine or an agrochemical. It is possible to verify or screen whether or not the drug or pesticide affects the cells by detecting the fluorescence emitted due to the cell response to the drug or pesticide. Thus, it should be understood that the present invention includes various embodiments and the like not described herein.

The detection device according to the embodiment is not particularly limited, but can be used for detection of food contaminants, medical care, environmental measurement, and the like.

DESCRIPTION OF SYMBOLS 1 ... Cell, 2 ... Photodetector, 3 ... Circuit board, 4 ... Excitation light source, 5 ... Light source, 11 ... Gel, 12 ... Cell, 13 ... -Receptor, 14 ... cell suspension, 15 ... substance, 21 ... transparent substrate, 22 ... frame, 23 ... solution, 31 ... lens tube, 32, 33 ... Lens, 34: Band pass filter, 51, 151 ... Mold, 52, 152 ... Well, 61 ... Tweezers, 71 ... Perfusion device, 72 ... Temperature control device, 73 ..Semipermeable membrane, 81, 83 ... Photomask, 82 ... Light source

Claims

Gel and
A plurality of cells stained in a fluorescent reagent disposed in the gel;
A photodetector for detecting fluorescence emitted from the fluorescent reagent when the plurality of cells react with a substance;
A detection device comprising:
The detection device according to claim 1, wherein the plurality of cells are dispersed in a gel.
The detection device according to claim 1, wherein the plurality of cells form a cell spheroid.
The detection device according to any one of claims 1 to 3, wherein the gel contains collagen.
The detection device according to any one of claims 1 to 4, wherein the substance can pass through the gel.
A culture vessel,
The gel is disposed in the culture vessel;
The detection device according to any one of claims 1 to 5.
The detection device according to any one of claims 1 to 6, comprising a plurality of the gels, wherein different types of cells are held in the plurality of gels.
A cell spheroid consisting of a plurality of cells stained with a fluorescent reagent;
A photodetector for detecting fluorescence emitted from the fluorescent reagent when the cell spheroid reacts with a substance;
A detection device comprising:
A culture vessel,
The cell spheroid is disposed in the culture vessel;
The detection device according to claim 8.
The detection device according to any one of claims 1 to 9, wherein the photodetector includes an imaging device.
The detection device according to any one of claims 1 to 10, wherein the substance is an odor substance, and the plurality of cells express receptors of the odor substance.
The detection device according to any one of claims 1 to 10, wherein the substance is a drug or an agrochemical.
The detection device according to any one of claims 1 to 12, wherein the fluorescent reagent is a calcium indicator.
The detection device according to claim 6 or 9, further comprising a temperature control device for controlling the temperature of the solution in the culture vessel.
Gel and
A plurality of cells stained in a fluorescent reagent disposed in the gel;
A cell-containing object comprising: