Evaluation instruments and evaluation methods

Abstract

The present invention relates to an evaluation instrument and an evaluation method. The evaluation instrument accurately evaluates the ability of cells to produce useful compounds. The evaluation instrument (1) is used to evaluate compounds (3) produced by cells. The evaluation instrument (1) includes a holding space (7) for holding a suspension (5) containing cells. A through hole (11) is formed at the bottom (9) of the holding space (7). The through hole (11) is blocked by a substance (13) that is impermeable to cells and through which the compound (3) can move.

Description

Technical Field

[0001] The present disclosure relates to an evaluation tool and an evaluation method. Background Art

[0002] In order to evaluate the ability of cells to produce useful compounds, for example, a PDMS microchamber is used (see Patent Document 1). In detail, first, a plate-shaped microchamber having a plurality of holes on the surface is used to culture cells in the holes. Then, the microchamber is placed in an inverted state on a culture medium coated with the test bacteria, so that the antibacterial components produced by the cells are transferred to the culture medium. Next, the degree of inhibition of the growth of the test bacteria in the culture medium to which the antibacterial components are transferred is observed, and the ability of the cells to produce antibacterial substances is evaluated.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: Japanese Patent Application Publication No. 2020-202777 Summary of the invention

[0006] Problem that the invention aims to solve

[0007] However, in this method, when the microchamber is inverted and placed on a culture medium coated with test bacteria, cells producing antimicrobial components and the like are also transferred, which may reduce the accuracy of evaluation.

[0008] The present disclosure has been made in view of the above circumstances, and an object of the present disclosure is to improve the accuracy of evaluation. The present disclosure can be implemented as the following aspects.

[0009] Solutions for solving problems

[0010] [1] An evaluation device for evaluating a compound produced by a cell, wherein:

[0011] The evaluation device includes a holding space for holding a suspension containing the cells.

[0012] A through hole is formed at the bottom of the holding space,

[0013] The through-holes are blocked by a substance that is impermeable to the cells but through which the compound can move.

[0014] Effects of the Invention

[0015] According to the evaluation instrument of the present disclosure, the compound produced by the cells is moved to the culture medium by the substance arranged in the through-hole, and since the cells themselves do not move to the culture medium, the accuracy of the evaluation can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a perspective view showing an example of an evaluation tool.

[0017] Figure 2 It is a top view showing an example of an evaluation tool.

[0018] Figure 3 yes Figure 1 A partial enlarged view of .

[0019] Figure 4 It is a cross-sectional view showing an example of an evaluation tool.

[0020] Figure 5 This is a perspective view showing an example of an evaluation tool with a cover.

[0021] Figure 6 This is a schematic diagram showing an evaluation instrument into which a suspension is injected from an inlet.

[0022] Figure 7 This is a schematic diagram showing an evaluation instrument into which air is injected from an inlet.

[0023] Figure 8 This is a schematic diagram (cross-sectional view) showing an example of the evaluation method.

[0024] Fig. 9 This is a schematic diagram (cross-sectional view) showing an example of the evaluation method.

[0025] Fig.10 This is a schematic diagram (cross-sectional view) showing an example of the evaluation method.

[0026] Fig.11 This is a schematic diagram (cross-sectional view) showing an example of the evaluation method.

[0027] Fig.12 It is a perspective view showing an example of an evaluation tool according to another embodiment.

[0028] Description of Reference Numerals

[0029] 1. Evaluation instrument; 3. Compound; 5. Suspension; 7. Holding space; 7A. Holding space; 7B. Holding space; 9. Bottom; 11. Through hole; 13. Substance; 17. Flow path; 19. Inlet; 21. Connecting flow path; 25. Discharge part; 27. Cover; 29. ​​Vent; 31. Inlet; 33. Discharge port; 35. Culture medium; 35A. Cloudy area; 35B. Transparent area; h1. Height; h2. Height; Φ1. Diameter; Φ2. Diameter; Φ3. Diameter. DETAILED DESCRIPTION

[0030] Here, a desirable example of the present disclosure is shown.

[0031] [2] An evaluation instrument, wherein a plurality of the holding spaces are present in the evaluation instrument.

[0032] The evaluation accuracy can be improved by increasing the number n.

[0033] [3] The evaluation tool, wherein the through hole is independently formed in each of the holding spaces.

[0034] The independence of each compound produced in each holding space is guaranteed.

[0035] [4] The evaluation instrument, wherein a plurality of the holding spaces are connected to each other.

[0036] The process of filling the plurality of holding spaces with the suspension can be easily performed.

[0037] [5] The evaluation instrument, wherein an introduction portion for introducing the suspension into the holding space is formed in the evaluation instrument, and the holding space is connected to a flow path extending from the introduction portion.

[0038] The process of filling the plurality of holding spaces with the suspension can be easily performed.

[0039] [6] The evaluation instrument, wherein the flow path extends linearly, and the holding space is connected to both sides of the flow path.

[0040] The plurality of holding spaces can be filled with the suspension approximately equally.

[0041] [7] An evaluation method, wherein an evaluation instrument is placed in a culture medium, and the substance blocking the through-hole is brought into contact with the culture medium,

[0042] The compound produced by the cell is moved by the substance to the culture medium where the compound is evaluated.

[0043] The compound produced by the cells is moved to the culture medium by the substance arranged in the through-hole, but the cells themselves do not move to the culture medium, thereby improving the accuracy of the evaluation method.

[0044] [8] An evaluation method, wherein an evaluation instrument is placed on a culture medium coated with bacteria, so that the substance blocking the through-hole contacts the culture medium,

[0045] The compound produced by the cells is moved to the culture medium through the substance, and the compound is evaluated by observing the states of the turbid area and the transparent area formed in the culture medium.

[0046] The compound produced by the cells is moved to the medium by the substance arranged in the through-hole, but the cells themselves do not move to the medium, thereby improving the accuracy of the evaluation.

[0047] The present disclosure is described in detail below. In addition, in this specification, when "-" is used for a numerical range, unless otherwise stated, the lower limit and the upper limit are included. For example, the description "10-20" includes both "10" as the lower limit and "20" as the upper limit. That is, "10-20" has the same meaning as "10 or more and 20 or less".

[0048] 1.Evaluation equipment 1

[0049] The evaluation instrument 1 of the present disclosure is used for evaluating the compound 3 produced by cells.

[0050] like Figure 1 to Figure 4 As shown, the evaluation instrument 1 includes a holding space 7 for holding a suspension 5 containing cells. A through hole 11 is formed at the bottom 9 of the holding space 7. The through hole 11 is blocked by a substance 13 that is impermeable to cells but allows the compound 3 to move. Figure 1 , 2 , 3, and 5 show the state before the through hole 11 is blocked by the substance 13. Figure 4 , the through hole 11 is shown to be clogged with the substance 13. The through hole 11 is clogged with the substance 13 when the evaluation instrument 1 is used.

[0051] The number of holding spaces 7 is not particularly limited. From the viewpoint of increasing the number n and improving the evaluation accuracy, it is preferable that a plurality of holding spaces 7 exist.

[0052] The shape of the holding space 7 is not particularly limited. From the viewpoint of smoothly filling the suspension 5, it is preferred that the shape of the holding space 7 is substantially cylindrical. When the holding space 7 is substantially cylindrical, the diameter Φ1 and the height h1 are not particularly limited. From the viewpoint of allowing cells to produce a sufficient amount of compound 3 for evaluation, it is preferred that the diameter Φ1 is 0.8 mm to 1.2 mm and the height h1 is 0.3 mm to 0.7 mm.

[0053] like Figure 3 As shown, it is preferred that a through hole 11 is independently formed in 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. Figure 4As shown, it is preferred that the shape of the through hole 11 is a shape whose diameter decreases as it moves downward, such as a truncated cone shape. If it is this shape, it is possible to suppress the substance 13 from escaping from the through hole 11 and falling off. In the case where the through hole 11 is a truncated cone shape, from the viewpoint of suppressing the falling off of the substance 13 and fully ensuring the movement amount of the compound 3 in the substance 13, it is preferred that the diameter Φ2 of the upper end portion is 0.25 mm to 0.35 mm, it is preferred that the diameter Φ3 of the lower end portion is smaller than the diameter Φ2, and it is preferred that the height h2 is 0.3 mm to 0.7 mm.

[0054] In the case where there are multiple holding spaces 7, such as Figure 2 , 3 As shown in the example, it is preferred that a plurality of holding spaces 7 are connected to each other. Figure 2 , 3 In the example, the plurality of holding spaces 7 are connected so that the liquid can flow through the flow path 17 and the connection flow path 21 .

[0055] The evaluation instrument 1 is provided with an introduction portion 19 for introducing the suspension 5 into the holding space 7. The holding space 7 is connected to the flow path 17 extending from the introduction portion 19 via the connection flow path 21. In this structure, by injecting the suspension 5 into the introduction portion 19, the plurality of holding spaces 7 can be easily filled with the suspension 5 (see Figure 6 ).

[0056] Preferably, the flow path 17 extends in a linear shape (for example, a straight line shape), 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. Figure 3 In the embodiment, 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 Fig.12 As shown, the flow path 17 and the connecting flow path 21 may be at different heights from the bottom 9 of the holding space 7. Fig.12 In the embodiment, the bottom 9 of the space 7 is kept lower than the flow path 17 and the connecting flow path 21 .

[0057] The arrangement of the plurality of holding spaces 7 on both sides of the flow path 17 is not particularly limited. Figure 2 As shown, the holding space 7A located on one side of the flow path 17 and the holding space 7B located on the other side of the flow path 17 are arranged alternately with each other along the direction from the introduction part 19 to the discharge part 25. According to this structure, when the suspension 5 is introduced from the introduction part 19, the holding space 7A located on one side of the flow path 17 and the holding space 7B located on the other side of the flow path 17 are filled with the suspension 5 alternately, so that the suspension 5 can flow into each holding space 7 approximately evenly.

[0058] The width W of the flow path 17 is not particularly limited. From the viewpoint of smoothly flowing the suspension 5 into the flow path 17, the width W of the flow path 17 is, for example, 0.1 mm or more and 0.3 mm or less.

[0059] Preferably, the evaluation instrument 1 includes a cover 27. The cover 27 has ventilation holes 29 formed above each holding space 7. The cover 27 has an introduction port 31 formed above the introduction portion 19 and a discharge port 33 formed above the discharge portion 25.

[0060] The material of the evaluation instrument 1 is not particularly limited. As a material, for example, a silicone resin can be preferably used as a main component. Here, the main component refers to a substance with a content (mass %) of 50 mass % or more. As the silicone resin, there is no particular limitation, and preferably, polydimethylsiloxane (PDMS), polymethylphenylsiloxane, polymethylhydrogensiloxane, polymethylmethoxysiloxane, polymethylvinylsiloxane, etc. can be used alone or in combination of two or more. Polydimethylsiloxane (PDMS) is preferred. In addition, the cover 27 can also preferably be made of the same material as the evaluation instrument 1.

[0061] The compound 3 produced by the cell is not particularly limited. The compound 3 is, for example, a useful substance such as an antibacterial substance (antibiotic). The cell that produces the compound 3 is also called a producer cell.

[0062] For substance 13, cells are impermeable and compound 3 can move. For substance 13, agar (agar gel) can be preferably used.

[0063] According to the evaluation instrument 1 of the present disclosure, the compound 3 produced by the cell is moved to the culture medium 35 by the substance 13 disposed in the through hole 11. Since the cell itself does not move to the culture medium 35, the accuracy of the evaluation is improved. That is, according to the evaluation instrument 1 of the present disclosure, the following effects can be expected. According to the evaluation instrument 1, since the production cells do not move to the culture medium 35, the possibility of the production cells and the detection bacteria (test bacteria) mixing in the culture medium 35 is small. Therefore, according to the evaluation instrument 1, the growth disorder of the detection bacteria (test bacteria) in the culture medium 35 can be appropriately evaluated.

[0064] 2. Evaluation Method

[0065] In the evaluation method disclosed herein, the evaluation instrument 1 is placed in a culture medium 35, the substance 13 blocking the through-hole 11 is brought into contact with the culture medium 35, the compound 3 produced by the cell is moved to the culture medium 35 through the substance 13, and the compound 3 is evaluated using the culture medium 35 (see Figure 8 to Figure 11 ).

[0066] As the culture medium 35, for example, an agar medium is preferably used. From the viewpoint of handling, it is preferred that the agar medium is a solid culture medium, and more preferably a plate culture medium. It is preferred that bacteria are coated on the agar medium. The bacteria are also called detection bacteria (tested bacteria), and pathogenic bacteria are preferably used.

[0067] The evaluation method is not particularly limited. For example, Fig.11 As shown, compound 3 was evaluated by observing the states of the turbid region 35A and the transparent region 35B formed on the culture medium 35 .

[0068] In the evaluation method of the present disclosure, the compound 3 produced by the cell is moved to the culture medium 35 by the substance 13 disposed in the through hole 11. Since the cell itself does not move to the culture medium 35, the accuracy of the evaluation is improved. That is, according to the evaluation method of the present disclosure, the following effects can be expected. According to the evaluation method, since the production cells do not move to the culture medium 35, the possibility of mixing the production cells and the detection bacteria (tested bacteria) in the culture medium 35 is small. Therefore, according to the evaluation method, the growth disorder of the detection bacteria (tested bacteria) in the culture medium 35 can be appropriately evaluated.

[0069] Here, a preferred evaluation method is described in further detail.

[0070] The evaluation instrument 1 whose upper surface is closed by the cover 27 is used. Figure 6 , 7 In the embodiment, the cover 27 is omitted for ease of explanation. When the suspension 5 is injected from the inlet 31, as shown in FIG. Figure 6 As shown, the suspension 5 schematically shown in a stippled manner is introduced from the introduction portion 19. The suspension 5 flows smoothly in the linear flow path 17. The holding space 7 close to the introduction portion 19 is filled with the suspension 5 in sequence.

[0071] Next, when air is injected from the inlet 31, Figure 7 As shown, air is injected from the inlet 19. As a result, the air moves roughly straight, and the suspension 5 staying in the linear flow path 17 is smoothly pushed to the discharge part 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 of each other. The cross-sectional view of this state is Figure 8 Then, in this state, when the cells in the suspension 5 are cultured, as Fig. 9 As shown, compound 3 is generated in suspension 5. Fig. 9 The density ratio of the points in the point drawing of the suspension 5 is Figure 8 The density of the dots in the point drawing of the suspension 5 is large, schematically shown in Fig. 9 The suspension 5 produces compound 3.

[0072] In addition, a culture medium 35 coated with bacteria (not shown) is prepared. Fig.10 As shown, the evaluation device 1 is placed on the culture medium 35 so that the lower surface of the substance 13 is in contact with the upper surface of the culture medium 35. Fig.10 As shown by the dotted arrow in FIG. , compound 3 produced by the cells moves (diffuses) to the culture medium 35 through substance 13 .

[0073] Thereafter, the culture medium 35 is kept warm at a temperature suitable for culturing (growing) the test bacteria applied to the culture medium 35, for example, 20°C to 45°C, more preferably 35 to 40°C.

[0074] Then, the culture medium 35 is observed. For example, the states of the turbid region 35A and the transparent region 35B (halo-shaped region) formed in the culture medium 35 are observed.

[0075] If compound 3 is an antibiotic effective against the test bacteria, the area around the medium 35 in contact with the substance 13 becomes a transparent area 35B, and the other areas become turbid areas 35A (opaque areas). In this case, the presence of the transparent area 35B means that the growth of the bacteria (test bacteria) is hindered by the antibiotic. In other words, by observing such transparent areas 35B and turbid areas 35A, it can be confirmed that the bacteria die due to the presence of the antibiotic, and the bacteria reproduce due to the absence of the antibiotic, and as a result, the antibacterial activity of the antibiotic produced by the cells can be evaluated.

[0076] The above examples are for illustrative purposes only and should not be construed as limiting the present disclosure. The present disclosure is illustrated by giving examples of typical implementations, but the language used in the descriptions and illustrations of the present disclosure should be understood as illustrative and exemplary and should not be understood as restrictive language. As described in detail herein, changes can be made within the scope of the appended claims without departing from the scope or spirit of the present invention in this manner. Here, reference is made to specific structures, materials, and embodiments in the detailed description of the present disclosure, but there is no intention to limit the present disclosure to the matters disclosed herein, rather, the present disclosure relates to all of the functionally equivalent structures, methods, and applications within the scope of the appended claims.

[0077] The present disclosure is not limited to the above-described detailed embodiments, and various modifications and changes can be made within the scope of the claims of the present disclosure.

[0078] Industrial Applicability

[0079] The evaluation instrument of the present disclosure can be used for evaluating compounds produced by cells.

Claims

1. An evaluation device for evaluating a compound produced by a cell, wherein: The evaluation device includes a holding space for holding a suspension containing the cells. A through hole is formed at the bottom of the holding space, The through-hole is blocked by a substance that is impermeable to the cell and through which the compound can move, The through hole has a truncated cone shape whose diameter decreases downward, the diameter of the upper end of the through hole is 0.25 mm to 0.35 mm, and the height of the through hole is 0.3 mm to 0.7 mm.

2. The evaluation instrument according to claim 1, wherein: The evaluation tool has a plurality of the holding spaces.

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

4. The evaluation instrument according to claim 2, wherein: A plurality of the holding spaces are connected to each other.

5. The evaluation instrument according to claim 1, wherein: The evaluation instrument is provided with an introduction portion for introducing the suspension into the holding space, and the holding space is connected to a flow path extending from the introduction portion.

6. The evaluation instrument according to claim 5, wherein: The flow path extends linearly, and the holding spaces are connected to both sides of the flow path.

7. An evaluation method, wherein: placing the evaluation instrument according to any one of claims 1 to 6 in a culture medium so that the substance blocking the through-hole comes into contact with the culture medium, The compound produced by the cell is moved by the substance to the culture medium where the compound is evaluated.

8. An evaluation method, wherein: placing the evaluation instrument according to any one of claims 1 to 6 on a culture medium coated with bacteria so that the substance blocking the through-hole comes into contact with the culture medium, The compound produced by the cells is moved to the culture medium through the substance, and the compound is evaluated by observing the states of the turbid area and the transparent area formed in the culture medium.

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