Evaluation equipment and evaluation method

Abstract

To accurately evaluate the production ability of useful compounds, which is provided in a cell.SOLUTION: An evaluation instrument 1 is used to evaluate a compound 3 produced by cells. The evaluation instrument 1 is provided with a holding space 7 that holds a suspension 5 containing the cells. A through-hole 11 is formed in a bottom 9 of the holding space 7. The through-hole 11 is blocked with a substance 13 impermeable to the cells and capable of transferring the compound 3.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] The present disclosure relates to an assessment instrument and an assessment method. [Background technology]

[0002] To evaluate the production capacity of useful compounds possessed by cells, for example, a microchamber made of PDMS is used (see Patent Document 1). In detail, first, cells are cultured in a plate-shaped microchamber having multiple wells on its surface. Then, the microchamber is placed upside down on a medium to which a test bacterium has been applied, and antibacterial components and the like produced by the cells are transferred to the medium. Next, the degree of inhibition of the growth of the test bacterium in the medium to which the antibacterial components and the like have been transferred is observed, and the antibacterial substance production capacity of the cells is evaluated. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2020-202777 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, with this method, when the microchamber is turned upside down and placed on the culture medium on which the test bacteria has been applied, the cells that produce antibacterial components, etc. are also transferred along with the microchamber, which poses the risk of low evaluation accuracy. The present disclosure has been made in view of the above-mentioned circumstances, and aims to improve the accuracy of evaluation. The present disclosure can be realized in the following forms. [Means for solving the problem]

[0005] [1] An instrument for evaluating a compound produced by a cell, comprising: A holding space for holding a suspension containing the cells is provided, A through hole is formed in the bottom of the holding space, The through-holes are blocked with a substance that does not permeate the cells and that allows the compound to migrate therethrough. Effect of the Invention

[0006] According to the evaluation device of the present disclosure, the substance placed in the through-holes allows compounds produced by the cells to migrate to the medium, but the cells themselves do not migrate to the medium, thereby improving the accuracy of the evaluation. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 is a perspective view showing an example of an evaluation tool. [Diagram 2] FIG. 2 is a plan view showing an example of an evaluation tool. [Diagram 3] FIG. 3 is a partially enlarged view of FIG. [Figure 4] FIG. 4 is a cross-sectional view showing an example of an evaluation device. [Diagram 5] FIG. 5 is a perspective view showing an example of an evaluation tool with a lid. [Figure 6] FIG. 6 is a schematic diagram showing the evaluation device into which the suspension was injected from the inlet. [Figure 7] FIG. 7 is a schematic diagram showing the evaluation device into which air is injected from the inlet. [Figure 8] FIG. 8 is a schematic diagram (cross-sectional view) showing an example of an evaluation method. [Figure 9] FIG. 9 is a schematic diagram (cross-sectional view) showing an example of an evaluation method. [Figure 10] FIG. 10 is a schematic diagram (cross-sectional view) showing an example of an evaluation method. [Figure 11] FIG. 11 is a schematic diagram (cross-sectional view) showing an example of an evaluation method. [Figure 12] FIG. 13 is a perspective view showing an example of an evaluation instrument according to another embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Here, a preferred example of the present disclosure is given. [2] An evaluation device in which there are multiple holding spaces. The evaluation accuracy can be improved by increasing the number n. [3] An evaluation device, wherein the through holes are formed independently for each of the holding spaces. The independence of each compound produced in each holding space is guaranteed. [4] An evaluation device, wherein the multiple holding spaces are connected to each other. It is easy to fill multiple holding spaces with suspension. [5] An evaluation device, wherein an inlet portion for introducing the suspension into the holding space is formed, and the holding space is connected to a flow path extending from the inlet portion. It is easy to fill multiple holding spaces with suspension. [6] An evaluation device, wherein the flow path extends linearly and the retention space is connected to both sides of the flow path. The suspension can be filled into the multiple holding spaces approximately evenly. [7] Place an evaluation device on the culture medium and bring the substance blocking the through-hole into contact with the culture medium; The evaluation method comprises transferring the compound produced by the cell to the medium via the substance, and evaluating the compound in the medium. The substance placed in the through-holes allows the compounds produced by the cells to migrate to the medium, but the cells themselves do not migrate to the medium, improving the accuracy of the evaluation method. [8] Place an evaluation device on the culture medium to which the bacteria has been applied, and bring the substance blocking the through-hole into contact with the culture medium; The compound produced by the cells is transferred to the medium via the substance, and the compound is evaluated by observing the state of the cloudy and transparent regions formed in the medium. The substance placed in the through-holes allows compounds produced by the cells to migrate to the medium, but the cells themselves do not migrate to the medium, improving the accuracy of the evaluation.

[0009] The present disclosure will be described in detail below. In this specification, a description using "~" for a numerical range includes the lower limit and the upper limit unless otherwise specified. For example, the description "10 to 20" includes both the lower limit "10" and the upper limit "20". In other words, "10 to 20" has the same meaning as "10 or more and 20 or less".

[0010] 1. Evaluation equipment 1 The evaluation device 1 of the present disclosure is used to evaluate a compound 3 produced by a cell. As shown in Figs. 1 to 4, the evaluation device 1 is provided with a holding space 7 that holds a suspension 5 containing cells. A through-hole 11 is formed in a bottom 9 of the holding space 7. The through-hole 11 is blocked with a substance 13 that does not permeate cells and can move compounds 3. Figs. 1, 2, 3, and 5 show the through-hole 11 in a state before being blocked with the substance 13, and Fig. 4 shows the through-hole 11 in a state blocked with the substance 13. When the evaluation device 1 is used, the through-hole 11 is in a state where it is blocked with the substance 13.

[0011] There is no particular limitation on the number of holding spaces 7. From the viewpoint of increasing the number n and improving the evaluation accuracy, it is preferable that a plurality of holding spaces 7 exist. The shape of the retention space 7 is not particularly limited. From the viewpoint of smoothly filling the suspension 5, the shape of the retention space 7 is preferably approximately cylindrical. When the retention space 7 is approximately cylindrical, the diameter φ1 and the height h1 are not particularly limited. From the viewpoint of allowing the cells to produce a sufficient amount of compound 3 for evaluation, the diameter φ1 is preferably 0.8 mm to 1.2 mm, and the height h1 is preferably 0.3 mm to 0.7 mm. As shown in FIG. 3, the through-hole 11 is preferably formed independently for each holding space 7. This is to ensure the independence of each compound 3 produced in each holding space. The shape of the through-hole 11 is not particularly limited. As shown in FIG. 4, the shape of the through-hole 11 is preferably a shape in which the diameter becomes smaller toward the bottom, for example, a truncated cone shape. With this shape, the substance 13 is prevented from slipping out of the through-hole 11 and falling off. The shape of the through-hole 11 is not particularly limited. When the through-hole 11 is a truncated cone shape, from the viewpoint of preventing the substance 13 from falling off and sufficiently securing the amount of movement of the compound 3 in the substance 13, the diameter φ2 of the upper end is preferably 0.25 mm to 0.35 mm, the diameter φ3 of the lower end is preferably smaller than the diameter φ2, and the height h2 is preferably 0.3 mm to 0.7 mm.

[0012] When a plurality of holding spaces 7 are present, the plurality of holding spaces 7 are preferably connected to each other as illustrated in Figures 2 and 3. In the example of Figures 2 and 3, the plurality of holding spaces 7 are connected to each other via a flow path 17 and a connecting flow path 21 so that the liquid can flow therethrough.

[0013] The evaluation device 1 is formed with an introduction portion 19 for introducing the suspension 5 into the holding space 7. The holding space 7 is connected to a flow path 17 extending from the introduction portion 19 via a connecting flow path 21. In this configuration, the suspension 5 can be easily filled into the multiple holding spaces 7 by injecting the suspension 5 into the introduction portion 19 (see FIG. 6). It is preferable that the flow path 17 extends linearly (for example, straight), and the holding space 7 is connected to both sides of the flow path 17. The flow path 17 and the holding space 7 are connected by a connecting flow path 21. In FIG. 3, the flow path 17 and the connecting flow path 21 are at the same height as the bottom 9 of the holding space 7, but as shown in FIG. 12, the flow path 17 and the connecting flow path 21 may be at a different height from the bottom 9 of the holding space 7. In FIG. 12, the bottom 9 of the holding space 7 is lower than the flow path 17 and the connecting flow path 21. The arrangement of the multiple holding spaces 7 on both sides of the flow path 17 is not particularly limited. As shown in Fig. 2, holding spaces 7A located on one side of the flow path 17 and holding spaces 7B located on the other side of the flow path 17 are alternately arranged along the direction from the introduction portion 19 to the discharge portion 25. With this structure, when the suspension 5 is introduced from the introduction portion 19, the holding spaces 7A located on one side of the flow path 17 and the holding spaces 7B located on the other side of the flow path 17 are alternately filled with the suspension 5, so that the suspension 5 can be poured into each holding space 7 approximately evenly. There is no particular limitation on the width W of the flow channel 17. From the viewpoint of allowing the suspension 5 to flow smoothly into the flow channel 17, the width W of the flow channel 17 is set to, for example, 0.1 mm or more and 0.3 mm or less.

[0014] The evaluation device 1 preferably includes a lid 27. The lid 27 has air holes 29 formed above each holding space 7. The lid 27 has an inlet 31 formed above the introduction section 19 and an outlet 33 formed above the outlet section 25.

[0015] The material of the evaluation tool 1 is not particularly limited. For example, a silicone resin is preferably used as the main component of the material. Here, the main component refers to a substance with a content (mass%) of 50 mass% or more. The silicone resin is not particularly limited, but polydimethylsiloxane (PDMS), polymethylphenylsiloxane, polymethylhydrogensiloxane, polymethylmethoxysiloxane, polymethylvinylsiloxane, etc. are preferable. These silicone resins may be used alone or in combination of two or more. Polydimethylsiloxane (PDMS) is preferable. The lid 27 can also be preferably made of the same material as the evaluation tool 1.

[0016] The compound 3 produced by the cells is not particularly limited. The compound 3 is, for example, a useful substance such as an antibacterial substance (antibiotic). The cells that produce the compound 3 are also called producer cells.

[0017] The substance 13 does not permeate cells and is capable of transporting the compound 3. As the substance 13, agar (agar gel) can be suitably used.

[0018] According to the evaluation device 1 of the present disclosure, the compound 3 produced by the cells moves to the medium 35 by the substance 13 arranged in the through-hole 11, but the cells themselves do not move to the medium 35, improving the accuracy of the evaluation. That is, according to the evaluation device 1 of the present disclosure, the following effects can be expected. According to the evaluation device 1, the producing cells do not move to the medium 35, so there is little risk of the producing cells and the detection bacteria (test bacteria) mixing in the medium 35. Therefore, according to the evaluation device 1, it is possible to properly evaluate the growth disorder, etc. of the detection bacteria (test bacteria) in the medium 35.

[0019] 2. Evaluation method The evaluation method of the present disclosure involves placing an evaluation device 1 on culture medium 35, bringing substance 13 blocking through-hole 11 into contact with culture medium 35, transferring compound 3 produced by the cells to culture medium 35 via substance 13, and evaluating compound 3 in culture medium 35 (see Figures 8 to 11). As the medium 35, for example, an agar medium is preferably used. From the viewpoint of ease of handling, the agar medium is preferably a solid medium, and a plate medium is more preferable. It is preferable that bacteria are applied to the agar medium. This bacteria is also called detection bacteria (subject bacteria), and pathogenic bacteria or the like are preferably used. The evaluation method is not particularly limited. For example, as shown in FIG. 11, the compound 3 is evaluated by observing the state of a cloudy region 35A and a transparent region 35B formed on a medium 35. In the evaluation method of the present disclosure, the compound 3 produced by the cells moves to the medium 35 by the substance 13 arranged in the through-hole 11, but the cells themselves do not move to the medium 35, improving the accuracy of the evaluation. That is, the evaluation method of the present disclosure can be expected to have the following effects. According to this evaluation method, the producing cells do not move to the medium 35, so there is little risk of the producing cells and the detection bacteria (test bacteria) mixing in the medium 35. Therefore, according to this evaluation method, it is possible to properly evaluate the growth disorder, etc. of the detection bacteria (test bacteria) in the medium 35.

[0020] A suitable evaluation method will now be described in more detail. An evaluation tool 1 is used with its upper surface covered with a lid 27. The lid 27 is omitted in Figs. 6 and 7 for ease of explanation. When the suspension 5 is injected from the inlet 31, the suspension 5, which is shown in a schematic dotted manner in Fig. 6, is introduced from the inlet 19. The suspension 5 flows smoothly through the linear flow path 17. The holding space 7 closest to the inlet 19 is filled with the suspension 5 in order. Next, when air is injected from the inlet 31, as shown in FIG. 7, air is injected from the inlet 19. Then, the air moves in a substantially straight line, and the suspension 5 that has been stopped in the linear flow path 17 is smoothly pushed out to the outlet 25, and the suspension 5 filled in each holding space 7 becomes unconnected. That is, the suspension 5 filled in each holding space 7 becomes independent from each other. A cross-sectional view of this state is shown in FIG. 8. Then, when cells in the suspension 5 are cultured in this state, compound 3 is generated in the suspension 5 as shown in FIG. 9. The density of the dots in the dotted pattern of the suspension 5 in FIG. 9 is made higher than the density of the dots in the dotted pattern of the suspension 5 in FIG. 8, which shows the generation of compound 3 in the suspension 5 in FIG. 9. Separately, a medium 35 coated with bacteria (not shown) is prepared. As shown in Fig. 10, the evaluation device 1 is placed on the medium 35, and the lower surface of the substance 13 is brought into contact with the upper surface of the medium 35. Compound 3 produced by the cells moves (diffuses) into the medium 35 via the substance 13, as shown by the dashed arrow in Fig. 10.

[0021] Thereafter, the medium 35 is kept at a temperature suitable for the cultivation (growth) of the detection bacteria applied to the medium 35, for example, 20°C to 45°C, more preferably 35°C to 40°C. Then, the medium 35 is observed. For example, the states of the opaque region 35A and the transparent region 35B (halo) formed in the medium 35 are observed. If compound 3 is an antibiotic effective against the detected bacteria, the area around the region in contact with substance 13 in medium 35 will be a transparent region 35B, and the other regions will be a cloudy white region 35A (opaque region). In this case, the presence of transparent region 35B means that the growth of the bacteria (detected bacteria) is inhibited by the antibiotic. In other words, the observation of such transparent region 35B and cloudy white region 35A confirms that the presence of the antibiotic causes the bacteria to die, and that the absence of the antibiotic causes the bacteria to grow, ultimately making it possible to evaluate the antibacterial activity of the antibiotic produced by the cells.

[0022] The above examples are merely illustrative and should not be construed as limiting the present disclosure. Although the present disclosure has been described with examples of typical embodiments, the words used in describing and illustrating the present disclosure are understood to be descriptive and exemplary, rather than limiting. As detailed herein, changes may be made within the scope of the appended claims without departing from the scope or nature of the present disclosure in its form. Although specific structures, materials, and examples have been referred to in the detailed description of the present disclosure, it is not intended that the present disclosure be limited to the disclosures herein, but rather that the present disclosure extends to all functionally equivalent structures, methods, and uses within the scope of the appended claims.

[0023] The present disclosure is not limited to the above-described embodiments, and various modifications and variations are possible within the scope of the claims of the present disclosure. [Industrial Applicability]

[0024] The evaluation apparatus of the present disclosure can be used to evaluate compounds produced by cells. [Explanation of symbols]

[0025] 1 …Evaluation equipment 3...Compound 5. Suspension 7...Holding space 7A…Holding space 7B…Holding space 9…bottom 11 ...Through hole 13...Substance 17 ... Flow path 19 ... Introduction 21 ... Connecting channel 25...Discharge section 27 …lid 29...Ventilation holes 31 ... Entrance 33…Exhaust port 35 ... Culture medium 35A…Cloudy area 35B…Transparent area h1 ...height h2 …height φ1...Diameter φ2 …Diameter φ3 …Diameter

Claims

1. An apparatus for evaluating a compound produced by a cell, comprising: A holding space for holding a suspension containing the cells is provided, A through hole is formed in the bottom of the holding space, the through-holes are blocked with a substance that does not permeate the cells and that allows the compound to move through; the evaluation device is placed on a culture medium to which a bacterium has been applied, and the substance blocking the through-holes is brought into contact with the culture medium; The compound produced by the cells is transferred to the medium via the substance, and the compound is evaluated by observing the state of the cloudy and transparent regions formed in the medium.

2. The evaluation method according to claim 1 , wherein the holding space is a plurality of spaces.

3. The evaluation method according to claim 2 , wherein the through hole is formed independently for each of the holding spaces.

4. The evaluation method according to claim 2 or 3, wherein the plurality of holding spaces are connected to each other.

5. The evaluation method according to claim 1 , wherein an inlet for introducing the suspension is formed into the holding space, and the holding space is connected to a flow path extending from the inlet.

6. The evaluation method according to claim 5 , wherein the flow path extends linearly, and the holding space is connected to both sides of the flow path.

7. 1. An apparatus for evaluating a compound produced by a cell, comprising: A holding space for holding a suspension containing the cells is provided, A through hole is formed in the bottom of the holding space, The through-hole is filled with agar, There are a plurality of the holding spaces, The through holes are formed independently for each of the holding spaces, The agars filling the through-holes are kept independent and do not come into contact with each other, in this evaluation device.

8. An evaluation device as described in claim 7, wherein the through hole has a shape in which its diameter becomes smaller as it moves away from the bottom of the holding space.

9. 9. The evaluation device according to claim 7, wherein an introduction portion for introducing the suspension into the holding space is formed, and the holding space is connected to a flow path extending from the introduction portion.

10. The evaluation device according to claim 9 , wherein the flow path extends linearly, and the holding space is connected to both sides of the flow path.

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