JP6785476B2 - Detector and cell-containing object - Google Patents

Description

The present invention relates to 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, and a responsive substance is produced. Therefore, by detecting whether or not a cell response reaction to a substance such as a ligand occurs, it is possible to determine that the substance such as a ligand is present when the response reaction occurs. Therefore, attempts have been made to use cells to detect substances. Further, by detecting the response reaction of cells to a substance such as a drug, it is possible to determine that the substance such as a drug acts on the cells 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. 5127783 Japanese Patent No. 5603850 Japanese Patent No. 5696953

The response reaction of the cell to the extracellular substance is visualized by a fluorescent reagent or the like. However, the intensity of fluorescence emitted by the fluorescence reagent inside the cell is weak, and a highly sensitive detection device is required to detect the fluorescence. Therefore, the present invention provides a detection device capable of easily detecting intracellular fluorescence, 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. It is 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 photodetection for detecting fluorescence emitted from the fluorescent reagent when the plurality of cells react with a substance. A detection device comprising a device is provided.

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 cell spheroids.

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 detector, the gel may contain collagen.

In the above detector, the substance may be permeable in the gel.

In the above detector, the gel may be placed on the photodetector.

The above detector may further include a culture vessel and the gel may be placed in the culture vessel.

The above-mentioned detection device may include a plurality of gels.

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

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

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

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

Further, according to the aspect of the present invention, a cell spheroid composed of a plurality of cells stained with a fluorescent reagent and a photodetector for detecting the fluorescence emitted from the fluorescent reagent when the cell spheroid reacts with a substance are provided. , A detector is provided.

The above-mentioned detection device may further include a culture vessel, and cell spheroids may be placed in the culture vessel.

In the above detection device, the solution may be placed in the culture vessel. The solution may be a culture solution.

In the above detector, the culture vessel may be placed 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 image sensor may be a CMOS image sensor.

In the above detection device, the substance may be an odorant, and a plurality of cells may express receptors for the odorant.

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

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

The above-mentioned detection device may further include a perfusion device for perfusing the solution in the culture vessel.

The above-mentioned detection device may further include a temperature control device for controlling the temperature of the solution.

Also, according to aspects of the invention, a cell-containing object comprising a gel and a plurality of cells stained with a fluorescent reagent placed in the gel is provided.

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

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

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

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

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

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

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

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

Further, 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 are contained. Provided is a method for producing a cell-containing object, comprising obtaining a cell-containing object comprising a plurality of cells stained with a fluorescent reagent arranged in.

In the above 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 cell spheroids.

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

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

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

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

Also, according to aspects of the invention, there is provided an array of cell-containing objects comprising a plurality of gels and a plurality of cells stained with a fluorescent reagent placed within each of the plurality of gels. ..

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

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

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

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

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

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

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

In the array of cell-containing objects described above, each of the different types of receptors may be a receptor for an odorant.

In the array of cell-containing objects described above, different types of fluorescent reagents may fluoresce in different wavelength bands.

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

Further, according to the aspect of the present invention, a first solution containing a first type of cell stained with a first type of fluorescent reagent is placed in a mold, and a first photomask is used. A part of the solution of the above is made into a first gel, a second solution containing a second type of cells stained with a second type of fluorescent reagent is placed in a mold, and a second photomask is used. Provided is a method of making an array of cell-containing objects, comprising making a portion of the second solution into a second gel using.

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

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

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

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

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

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

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

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

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

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

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

According to the present invention, it is possible to provide a detection device capable of easily detecting intracellular fluorescence, 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. ..

It is a schematic diagram of the detection device 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 device 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 columnar gel which concerns on Example of 1st Embodiment. It is a graph which shows the diameter distribution of the columnar gel which concerns on Example of 1st Embodiment. 6 is a three-dimensional image of a columnar gel according to an embodiment of the first embodiment. It is a graph which shows the height distribution of the columnar gel which concerns on Example of 1st Embodiment. It is a bright field image and a fluorescence image of a columnar gel which concerns on Example of 1st Embodiment. 6 is a fluorescence image of a columnar gel according to an embodiment of the first embodiment. It is a graph which shows the relationship between the height of the columnar gel which concerns on Example of 1st Embodiment, and the fluorescence intensity. It is a graph which shows the relationship between the time passage after the addition of the odor substance which concerns on Example of 1st Embodiment, and the fluorescence intensity. It is a graph which shows the relationship between the concentration of the odor substance and the fluorescence intensity which concerns on Example of 1st Embodiment. FIG. 23A is a bright-field image of the gel formed by UV irradiation according to the embodiment of the first embodiment. FIG. 23 (b) is a composite image of a bright-field image and a fluorescence image of the gel formed by UV irradiation according to the embodiment of the first embodiment. Cells were stained with Calcein-AM and Ethidium Bromidemer. Casein-AM permeates the cell membrane of living cells and emits green fluorescence. Ethidium Bromide penetrates the membrane-damaged portion of dead cells and emits red fluorescence. In the original color image of FIG. 23, the majority of cells fluoresce green, confirming that the cells are alive. FIG. 24A is a bright field image of the gel formed by UV irradiation according to the embodiment of the first embodiment. FIG. 24B is a fluorescence image of a gel formed by UV irradiation according to the embodiment of the first embodiment and containing cells stained with different fluorescent reagents. FIG. 24 (c) is a graph showing the positions of gels containing cells stained with different fluorescent reagents and the fluorescence intensity. It is a schematic diagram of the detection device which concerns on 2nd Embodiment. It is a schematic diagram of the detection device which concerns on 2nd Embodiment. It is an image of a spheroid according to an embodiment of the second embodiment. FIG. 28A is a bright-field image of the spheroid according to the embodiment of the second embodiment. FIG. 28B is a fluorescence image of the spheroid according to the embodiment of the second embodiment. FIG. 28 (c) is a composite image of the bright-field image and the fluorescence image of the spheroid according to the embodiment of the second embodiment. FIG. 28 (d) is a graph showing the fluorescence intensity at the dashed line AA'in FIG. 28 (b). FIG. 29 (a) is a single-cell fluorescence image according to the embodiment of the second embodiment. FIG. 29B is a fluorescence image of the spheroid according to the embodiment of the second embodiment. FIG. 29 (c) is a graph showing the intensity of fluorescence emitted by the single cell and the spheroid according to the embodiment of the second embodiment. FIG. 30A is a fluorescence image of spheroids before addition of eugenol using a confocal microscope according to the embodiment of the second embodiment. FIG. 30B is a fluorescence image of spheroids after addition of eugenol using a confocal microscope according to the embodiment of the second embodiment. FIG. 30 (c) is a graph showing the fluorescence intensity before and after the addition of eugenol according to the embodiment of the second embodiment. FIG. 30D is a bright-field image of the spheroid using a CMOS image sensor. FIG. 30 (e) is a fluorescence image of a binary spheroid using a CMOS image sensor.

Embodiments of the present invention will be described below. In the description of the drawings below, the same or similar parts are represented by the same or similar reference numerals. However, the drawings are schematic. Therefore, the specific dimensions and the like should be determined in light of the following explanations. In addition, it goes without saying that the drawings include parts having different dimensional relationships and ratios from each other.

(First Embodiment)
In the detection device according to the first embodiment, as shown in FIG. 1, the gel 11, the plurality of cells 1 arranged in the gel 11 and stained with the fluorescent reagent, and the plurality of cells 1 reacted with the substance. A photodetector 2 for detecting fluorescence sometimes emitted from a fluorescent reagent is provided.

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

The cell incorporates a fluorescent reagent that fluoresces when the cell reacts with the substance. The fluorescent reagent taken up by the cell is, for example, a calcium indicator. When the odorant and the odorant receptor of the cell bind, a signal is transmitted inside the cell and the calcium ion concentration increases. Calcium indicators fluoresce as the calcium ion concentration increases. In this way, the fluorescent reagent can visualize the intracellular chemical reaction that occurs when a cell reacts with a substance. Examples of calcium indicators include Flua2-AM, Fluo-3-AM, Fluo-4-AM, and Fluo-8-AM.

Fluorescent reagents are not limited to calcium indicators. The fluorescent reagent may be a membrane potential sensitive fluorescent reagent or a neurotransmitter sensitive fluorescent reagent. The voltage-sensitive fluorescent reagent fluoresces in response to changes in the membrane potential that occur when cells react with a substance. Examples of voltage-sensitive fluorescent reagents include di-4-ANEPPS and DiBAC4. Neurotransmitter-sensitive fluorescent reagents fluoresce in response to neurotransmitters such as glutamic acid that are produced intracellularly when cells react with the substance. Examples of neurotransmitter-sensitive fluorescent reagents include EOS (glutamic 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, for example, a hydrogel. As the material of the gel 11, for example, collagen, gelatin, agar, alginic acid, polyethylene glycol diacrylate (PEGDA) and the like can be used. The material of the gel 11 is not particularly limited as long as the substance that reacts with the cells can permeate the inside of the gel 11. Further, the gel 11 may be permeable to culture components such as nutritional 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 containing the plurality of cells 1 is arranged on, for example, a transparent substrate 21 capable of transmitting the fluorescence generated by the plurality of cells 1. One gel 11 may be arranged on the transparent substrate 21, or a plurality of gels 11 may be arranged. 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. As a result, the adhesiveness of the gel 11 to the transparent substrate 21 is improved.

It has been reported that animals do not recognize one odor in response to one odorant, but recognize one odor according to the pattern of combination of multiple odorants. Therefore, when the detection device includes a plurality of gels 11, different types of cells may be retained 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 holding a plurality of cells reacting with a first odorant, a gel holding a plurality of cells reacting with a second odorant, and a plurality of gels holding a plurality of cells reacting with a third odorant. A gel that holds the cells and a gel that holds the cells may be arranged. The type of odorant may be specified from the position of the fluorescent gel. In addition, the type of odor may be specified from the combination of the positions of the fluorescent gels. Alternatively, the type of odorant may be specified from the wavelength band and 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 wavelength band of fluorescence and the combination of colors.

As shown in FIG. 1, the frame 22 may be arranged on the transparent substrate 21. In this case, the transparent substrate 21 and the frame 22 form a culture container. The frame 22 is made of, for example, dimethylpolysiloxane (PDMS), but is not particularly limited. The gel 11 containing the plurality of cells 1 is placed in the culture vessel 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 container formed by the transparent substrate 21 and the frame 22. By putting the solution in the frame 22, it is possible to prevent the evaporation of water in the gel 11 and to supply nutrients to a plurality of cells 1.

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

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

The photodetector 2 is arranged, for example, on the circuit board 3. The photodetector 2 may include an image sensor such as a solid-state image sensor. As the solid-state image sensor, 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, the detection device according to the first embodiment can be made small.

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

The detection device 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 white light or other wavelengths other than the excitation 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 incorporated into the cells constituting the plurality of cells 1. When the excitation light source 4 emits excitation light, the photodetector 2 can capture an image of fluorescence emitted by a plurality of cells 1. Further, when the light source 5 emits white light, the photodetector 2 can capture an image such as a bright-field image or a dark-field image in the shape of a plurality of cells 1.

As shown in FIG. 3, the gel 11 containing the plurality of cells 1 may be arranged on the photodetector 2 without using a lens or the like. The solution 23 that fills the periphery of the gel 11 containing the plurality of cells 1 may be perfused by 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 containing the plurality of cells 1 is arranged may be covered with a semipermeable membrane 73 that is permeable to gas and impermeable to liquid. Thereby, it is possible to prevent the evaporation of the solution 23 while supplying the 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 a cell cultured in a single layer is weak, it may be difficult to detect with a photodetector by the conventional technique, or a large-sized light that is not easy to carry. Sometimes a detector is needed. On the other hand, in the detection device according to the first embodiment, a plurality of cells 1 are arranged three-dimensionally in the gel 11, and the plurality of cells 1 are laminated in the direction perpendicular to the photodetector 2. There is. 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 reaction of cells to a substance even with a photodetector 2 having low sensitivity such as a CMOS image sensor. Further, since it is not always necessary to use a large-sized photodetector with high sensitivity, the detection device according to the first embodiment can be made small enough to be portable.

Further, in the detection device according to the first embodiment, a plurality of cells 1 are held in the gel 11. The gel 11 can be easily adhered 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 can be fixed. It is possible to suppress changes in the position and orientation with respect to the photodetector 2. Therefore, it is possible to improve the 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 ... Is prepared. 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 wells 52a, 52b, 52c ... Of the mold 51 are filled with the cell suspension 14.

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 the side where the wells 52a, 52b, 52c ... Are provided. Is brought into close contact with the surface of the transparent substrate 21. Then, a plurality of cells 12 are cultured in the wells 52a, 52b, 52c ... Of the mold 51. 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. In addition, cells of different types may be cultured in the wells 52a, 52b, 52c ... Further, the gel raw material solution is heated in order to gel the gel raw material solution into gel 11. The heating temperature is, for example, 37 ° C., and the heating time is, for example, 15 minutes.

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. This makes it easier to peel off the mold 51 from the transparent substrate 21. After that, as shown in FIG. 9, 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, a 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. Then, the cells 12 of the first type are cultured in the well 152 of the mold 151. Optionally, cell spheroids may be formed. Further, light is irradiated from a light source 82 such as a UV irradiation device via the first photomask 81 or the like to gel a part of the cell suspension 14.

Then, by removing the cell suspension 14 that has not been gelled without being irradiated with light, a plurality of gels containing the plurality of cells of the first type are arranged on the transparent substrate 21. Also, 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 cell is 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 a different opening pattern from the first photomask 81. Then, by removing the cell suspension that has not been gelled without being irradiated with light, a plurality of gels containing the plurality of cells of the second type are arranged on the transparent substrate 21. After that, the same method may be repeated to place a plurality of gels containing different types of cells on the transparent substrate 21.

(Example of the first embodiment)
HEK293T cells expressing the olfactory receptor mOR-EG by genetic engineering were prepared. Cells were stained with the calcium indicator Fluo-8 AM (AAT Bioquest). In addition, Cellmatic Type I-A (Nitta Gelatin), 10 times Hanks buffer, and a buffer solution for reconstruction 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. Further, 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 surface of the mold was coated with 1% BSA for 30 minutes. So that the cells to a concentration of 10 ⁸ cells / mL, were suspended in a collagen solution to obtain a cell suspension. The obtained cell suspension was placed in the wells of the mold, and the surface provided with the wells of the mold was attached into the frame of the glass plate. Cells were cultured in wells for 3 hours. Then, in order to gel the collagen solution, the collagen solution was heated at 37 ° C. for 30 to 40 minutes. After the gel was formed, the culture solution was placed in the frame and the mold was peeled off from the surface of the glass plate.

As shown in FIG. 14, it was confirmed by an inverted microscope that the columnar gels were arranged in an array on the glass plate. When the diameter distribution of a plurality of columnar gels formed from wells having the same shape was examined by image analysis, the diameters were almost uniform as shown in FIG. Further, as shown in FIG. 16, a columnar gel was 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 was observed. Upon examination, as shown in FIG. 17, the height was almost uniform.

Next, eugenol was prepared as an odorant. The chemical formula of eugenol is shown below. Eugenol is commonly 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 odorant solution.

A detection device having a structure similar to that shown in FIG. 1 was manufactured using a CMOS image sensor. Using molds having different well depths, a plurality of columnar gels having the same diameter and different heights were prepared. In order to measure the background noise, a plurality of cells were irradiated with excitation light and a fluorescence image was taken with a CMOS image sensor before dropping the odorant solution into the culture solution. Next, the concentration of the odorant solution was appropriately diluted, half the amount of the culture solution was sucked up so that the height of the liquid level did not change, and then the same amount of the odorant solution was added dropwise to the culture solution. Then, a plurality of cells were irradiated with excitation light, and a fluorescence image was taken with a CMOS image sensor. Further, an image of the difference between the fluorescence images before and after the odorant solution was dropped was obtained as a corrected fluorescence image in which the background noise was cancelled. The obtained bright field image and the corrected fluorescence image are shown in FIG. In the bright-field image, the contour of the columnar gel was observed. Moreover, in the corrected fluorescence image, the fluorescence emitted by calcein was observed.

Further, as shown in FIGS. 19 and 20, it was confirmed that the higher the height of the columnar gel, the stronger the fluorescence intensity detected by the CMOS image sensor. It is considered that this is 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 time change of the fluorescence intensity after dropping the odorant substance solution into the culture solution was observed, as shown in FIG. 21, an increase in the fluorescence intensity was confirmed within several tens of seconds. Therefore, it was shown that the detection device according to the embodiment has good responsiveness and can detect odorous substances in real time.

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

Further, 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 columnar gel was obtained as shown in FIG. 23. Was done. When cells stained with different types of fluorescent reagents were included in the adjacent gels, it was confirmed that each gel emitted fluorescence in a different wavelength band, as shown in FIG. 24.

(Second Embodiment)
In the first embodiment, an example in which a plurality of cells are retained 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 device according to the second embodiment shown in FIG. 25, the cell spheroid composed of a plurality of cells 1 may be directly put into 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 by. As shown in FIG. 26, in the detection device according to the second embodiment, when a substance 15 such as an odorant 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 of the first embodiment.

(Example of the second embodiment)
10% by volume of fetal bovine serum (FBS, Biosera), 100 U / mL penicillin (Kanto Chemical or Wako Pure Chemical), and 100 μg / mL streptomycin (Kanto Chemical or Japanese) in Dalveco's modified Eagle's medium (DMEM, Sigma Aldrich). Photopure drug) was added to prepare the culture solution. In addition, phosphate buffered saline (PBS, Sigma-Aldrich) and eugenol, which is an odorant, were prepared.

To detect eugenol, HEK293T cells were used with lipofectamine 3000 or 2000 (registered trademark, Invitrogen) on the olfactory receptor mOR-EG, the G protein α subunit G _α15 , and the receptor transporter protein. It was transformed to express a certain RTP1 and a signal amplification molecule, Ric-8. The amount of genes added per medium 1mL is, pME18S-Rho-mOR-EG is 1.5 [mu] g (Lipofectamine 3000 is used) or 1.45Myug (Lipofectamine 2000 is used), _{pME18S-G a15} is 1.0 [mu] g (Lipofectamine 3000 When used) or 0.87 μg (when using Lipofectamine 2000), 0.5 μg of pME18S-RTP1 (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 adherently cultured transformed cells were treated with trypsin-EDTA solution at 37 ° C. for 5 minutes to release the cells from the incubator. In addition, culture medium was added to suspend the cells and centrifuged at 2G for 5 minutes. After centrifugation, the cells were suspended again in the culture medium, and the suspension was added dropwise to 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. Then, the cells were cultured for 3 hours to form cell spheroids composed of transformed cells. A brightfield image of the cell spheroid is shown in FIG. In addition, cell spheroids were stained with the 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 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. The cell spheroids collected from the incubator with a pipette were placed in the culture medium in the culture vessel of the detector. When capturing a bright-field image of the spheroid, the spheroid was irradiated with white light through a pinhole. When eugenol was added to the culture medium and a fluorescent image of the spheroid was imaged, the cell spheroid was irradiated with blue excitation light to extract a color in the green wavelength band.

FIG. 28 (a) shows a bright-field image of the cell spheroid, FIG. 28 (b) shows a fluorescent image of the cell spheroid, and FIG. 28 (c) shows a composite image of the bright-field image and the fluorescent image. Further, FIG. 28 (d) shows a distribution graph of fluorescence intensity in the dashed line AA'in FIG. 28 (c). It has been shown that fluorescence reactions within cell spheroids can be detected using CMOS image sensors. Furthermore, as shown in FIG. 29, the intensity of fluorescence emitted by the cell spheroid was about four times stronger than the intensity of fluorescence emitted by one cell (single cell). Therefore, it was shown that cellular spheroids improve the sensitivity of the detector.

Further, when measured using a confocal microscope, as shown in FIGS. 30 (a) to 30 (c), 2 mmol / L eugenol was added to the culture solution as compared with before the addition of eugenol to the culture solution. Later, the intensity of fluorescence emitted by the cell spheroids increased more than twice. FIG. 30 (d) shows a bright-field image of the cell spheroid captured by the CMOS image sensor, and FIG. 30 (e) shows an image obtained by binarizing the fluorescent image of the cell spheroid with the image processing software ImageJ. From the results of this example, it was shown that a detection device using a CMOS image sensor can be used to detect the reaction between the olfactory receptor of cell spheroids and the odorant eugenol.

(Other embodiments)
Although the present invention has been described according to embodiments as described above, the descriptions and drawings that form part of this disclosure should not be understood to limit the invention. This disclosure should reveal to those skilled in the art various alternative embodiments, examples and operational techniques. For example, in the embodiment, an odorant substance has been mentioned as an example of a substance with which cells react, but the substance is not limited thereto. The substance may be various ligands, ions, or cell membrane penetrating substances. It is possible to detect the presence of these substances by detecting the fluorescence emitted due to the intracellular response reaction of these substances.

Further, the substance with which the cells react may be a drug such as a drug or a pesticide. By detecting the fluorescence emitted by the response reaction of cells to drugs and pesticides, it is possible to verify and screen whether the drugs and pesticides affect cells. As described above, it should be understood that the present invention includes various embodiments not described here.

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

1 ... cell, 2 ... light detector, 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 barrel, 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 ・ ・ ・ Photo mask, 82 ・ ・ ・ Light source

Claims (12)

With gel
A plurality of cells stained with a fluorescent reagent placed in the gel and
A photodetector that detects the fluorescence emitted from the fluorescent reagent when the plurality of cells react with a substance, and
With
The plurality of cells are stacked in the direction perpendicular to the photodetector,
The plurality of cells express the receptor for the substance,
When the substance binds to the receptor, the fluorescence is emitted.
Detection device. Wherein the plurality of cells, are dispersed in the gel, the detection device according to claim 1. 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 is permeable in the gel. With more culture vessels
The gel is placed 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, further comprising a plurality of the gels, wherein different types of cells are retained in the plurality of gels. The detection device according to any one of claims 1 to 7 , wherein the photodetector includes an image sensor. The detection device according to any one of claims 1 to 8 , wherein the substance is an odorant, and the plurality of cells express a receptor for the odorant. The detection device according to any one of claims 1 to 8 , wherein the substance is a drug or a pesticide. The detection device according to any one of claims 1 to 10 , wherein the fluorescent reagent is a calcium indicator. The detection device according to claim 6, further comprising a temperature control device for controlling the temperature of the solution in the culture vessel.