3D cell culture device and its use

Abstract

To prevent a vapor phase including a test substance from dissolving in a medium inside a well when evaluating the test substance by vapor-phase exposure using a three-dimensional cell.SOLUTION: A culture device (10) includes a gap prevention plate (2) that is positioned between an insert cup (1) and a well plate (3) to fill a gap (3b) created when the insert cup (1) is inserted into the well plate (3).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a three-dimensional cell culture device and its use.

Background Art

[0002] Cultures obtained by growing cultured cells by two-dimensional spreading can be used as research subjects for the growth process and functionality of cells. However, such cultures are far from tissues in vivo and are difficult to use for tissue research. Therefore, a technique for reproducing phenomena in living tissues and evaluating the effects on living tissues using a three-dimensional model obtained by culturing cultured cells three-dimensionally is required in various fields such as drug discovery and regenerative medicine.

[0003] So far, a plurality of techniques related to the culture of three-dimensional models have been reported, and a technique for three-dimensionally culturing cultured cells using a well plate and an insert cup is widely used. In this technique, an insert cup having a porous membrane on the bottom surface is placed in a well of a well plate filled with a culture medium. Then, cultured cells are seeded on the porous membrane of the insert cup and cultured three-dimensionally. For example, in Patent Document 1, a glass ring is fitted into a hole made in a lid for a plate (well plate + insert cup) containing three-dimensional cultured skin, so that only the surface of the three-dimensional cultured skin is directly exposed to the outside air.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, in a three-dimensional cell culture apparatus equipped with well plates and insert cups as described above, the following problems arise when the surface of the three-dimensional cells is exposed to a gas phase containing the test substance.

[0006] In other words, when using commercially available well plates and insert cups, a void is created between the space inside the well and the outside air (gas phase) when the insert cup is inserted into the well plate. Therefore, when gas phase exposure is performed using a 3D cell culture device with such a void, the gas phase containing the test substance dissolves into the culture medium in the well through the void. As a result, the 3D cells in the insert cup come into contact with the test substance not only from the outermost layer of cultured cells, but also from the cultured cells on the culture medium side. Therefore, there is a problem in that it is not possible to accurately evaluate the 3D cells' reaction to the test substance contained in the gas phase when exposing 3D cells to the gas phase.

[0007] One aspect of the present invention aims to realize a three-dimensional cell culture apparatus and its use, which can prevent the gas phase containing the test substance from dissolving into the culture medium in the well when evaluating a test substance in gas phase exposure using three-dimensional cells. [Means for solving the problem]

[0008] As a result of diligent research to solve the above problems, the present inventors have found that by providing a gap-preventing plate between the insert cup and the well plate to fill the gap that occurs when the insert cup is inserted into the well plate, it is possible to prevent the gas phase containing the test substance from dissolving into the culture medium in the well, and thus completed the present invention. Accordingly, one aspect of the present invention is as follows.

[0009] [1] A three-dimensional cell culture apparatus comprising: an insert cup for holding three-dimensional cells; a well plate into which the insert cup is inserted and which has wells for holding culture medium; and a void prevention plate provided between the insert cup and the well plate for filling the voids that occur when the insert cup is inserted into the well plate.

[0010] [2] The three-dimensional cell culture apparatus according to [1], further comprising a spraying device for spraying a test substance onto the three-dimensional cells in the insert cup.

[0011] [3] The three-dimensional cell culture apparatus according to [2], comprising a covering member that covers the insert cup and the gas phase containing the test substance.

[0012] [4] A three-dimensional cell culture apparatus according to any of [1] to [3], wherein the void portion includes at least the gap between the outer circumferential surface of the insert cup and the inner circumferential surface of the well.

[0013] [5] Any three-dimensional cell culture apparatus of [1] to [4], wherein the void portion connects at least the inside of the insert cup and the inside of the well, and includes a connecting portion formed on the side wall of the insert cup.

[0014] [6] A method for evaluating the effects of a test substance on three-dimensional cells, wherein the test substance has the effect on three-dimensional cells, A method for evaluating three-dimensional cells against a test substance, comprising a spraying step of spraying the test substance onto the three-dimensional cells in the insert cup using any three-dimensional cell culture apparatus of [1] to [5]. [Effects of the Invention]

[0015] According to one aspect of the present invention, when evaluating a test substance by gas-phase exposure, it is possible to prevent the gas phase containing the test substance from dissolving into the culture medium in the well. [Brief explanation of the drawing]

[0016] [Figure 1]Fig. 101 shows a configuration example of a three-dimensional cell culture device according to an embodiment of the present invention. Fig. 102 is a sectional view taken along line I-I in Fig. 101, and Fig. 103 is a sectional view taken along line II-II in Fig. 101. [Figure 2] Fig. 201 shows another configuration example of a three-dimensional cell culture device according to an embodiment of the present invention. Fig. 202 is a sectional view taken along line III-III in Fig. 201, Fig. 203 is a sectional view taken along line IV-IV in Fig. 201, and Fig. 204 is a side view showing the configuration of the cup body of the insert cup. [Figure 3] Fig. is a sectional view showing still another configuration example of a three-dimensional cell culture device according to an embodiment of the present invention. [Figure 4] Fig. is a sectional view showing still another configuration example of a three-dimensional cell culture device according to an embodiment of the present invention. [Figure 5] Fig. is a perspective view showing the configuration of a specific example of an air gap prevention plate provided in a three-dimensional cell culture device according to an embodiment of the present invention. [Figure 6] Fig. is a perspective view showing the configuration of another specific example of an air gap prevention plate provided in a three-dimensional cell culture device according to an embodiment of the present invention. [Figure 7] Fig. shows a schematic configuration of a three-dimensional cell culture device used in Example 1.

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] An embodiment of the present invention will be described in detail below. Unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more and B or less". Also, all the documents described in this specification are incorporated herein by reference.

[0018] 〔1. Outline of Embodiment of the Present Invention〕 When the present inventors evaluated a test substance in a gas-phase exposure, they intensively studied a three-dimensional cell culture device that can prevent the gas phase containing the test substance from dissolving in the culture medium in the well. As a result, they found that by providing a void prevention plate that fills the void generated when an insert cup is inserted into a well plate between the insert cup and the well plate, it is possible to prevent the gas phase containing the test substance from dissolving in the culture medium in the well.

[0019] According to an embodiment of the present invention, by installing the void prevention plate on the well plate and further installing an insert cup on the void prevention plate, the culture medium in the well does not communicate with the outside air, and the culture medium and the outside air can be isolated. Therefore, when exposing a gas phase containing a test substance to a three-dimensional cell model such as skin, cornea, nasal mucosa, lung, airway, etc., the influence on gas-phase exposure can be accurately evaluated without considering the test substance dissolved in the culture medium, which is useful in various fields such as the cosmetics field and the medical field.

[0020] Also, according to an embodiment of the present invention, for a gas-phase exposure test of three-dimensional cells, commercially available products may be prepared without using a dedicated well plate and insert cup. Therefore, a gas-phase exposure test of three-dimensional cells can be easily carried out.

[0021] [2. Three-dimensional cell culture device] A three-dimensional cell culture device (hereinafter referred to as "the present culture device") according to an embodiment of the present invention includes an insert cup that holds three-dimensional cells, a well plate having a well into which the insert cup is inserted and that holds a culture solution, and a void prevention plate provided between the insert cup and the well plate that fills the void generated when the insert cup is inserted into the well plate.

[0022] With the above configuration, the gap-preventing plate fills the gaps that occur when the insert cup is inserted into the well plate, so that the culture medium in the wells of the well plate does not come into contact with the outside air, and the culture medium can be isolated from the outside air. As a result, with the above configuration, it is possible to prevent the gas phase containing the test substance from dissolving into the culture medium in the wells. Furthermore, when the outermost surface of a three-dimensional cell is exposed to the gas phase containing the test substance, the effect of gas phase exposure can be accurately evaluated without considering the test substance dissolved in the culture medium.

[0023] Furthermore, until now, the only way to perform gas-phase exposure was to use specialized equipment. Depending on the type of equipment, 3D cells were to be placed directly into the device. Therefore, considering the toxicity to 3D cells, only relatively short-term gas-phase exposure was possible, and it was difficult to expose 3D cells to gas for several hours. In addition, even when attempting to measure extracellular release factors released into the culture medium to evaluate the effects of 3D cell samples, evaluation using culture media was difficult because the aforementioned specialized equipment does not allow for normal culture of 3D cells.

[0024] This culture device allows for prolonged exposure of 3D cells to the gas phase, and also enables simultaneous gas phase exposure of 3D cells while performing normal culture of 3D cells.

[0025] In this specification, "three-dimensional cells" refers to a cell mass or tissue having a three-dimensional structure, where layers of cultured cells are stacked at least two or more times. Furthermore, in addition to cells, an extracellular matrix may also be included. If an extracellular matrix is ​​included, it refers to a structure in which cells are stacked at least two or more times via the extracellular matrix. The extracellular matrix refers to endogenous material that fills the space outside the cell in cells cultured in vivo or in vitro, and performs functions such as skeletal role, scaffolding role, and / or role in holding biofactors. The extracellular matrix is ​​not particularly limited, but examples include collagen, proteoglycans, fibronectin, laminin, hyaluronic acid, tenascin, and elastin.

[0026] In one embodiment of the present invention, the cell aggregates and tissues formed by three-dimensional cells are preferably tissues in which at least a portion is exposed to the outside air within the body. Examples include skin tissue, tracheal epithelial tissue, esophageal tissue, various mucosal tissues (e.g., oral mucosa, nasal mucosa, eye tissue, etc.), and alveolar epithelial tissue.

[0027] The types of cells that make up the three-dimensional cells are not particularly limited, as long as they are cells that make up tissues in which at least a portion is exposed to the outside air, but cells that make up the aforementioned tissues are preferred examples. For example, epidermal cells (e.g., keratinocytes, basal cells), myoepithelial cells, luminal cells, oral mucosa cells, vascular cells, endothelial cells (e.g., vascular endothelial cells), sebaceous cells (e.g., sebaceous gland basal cells, differentiated sebaceous gland cells, mature sebaceous gland cells), epithelial cells, fibroblasts, alveolar epithelial cells, etc. The cells that make up the three-dimensional cells may be obtained from within the body or may be commercially available. They may also be produced by differentiation induction from stem cells (e.g., embryonic stem cells (ES cells), adult stem cells, induced pluripotent stem cells (iPS cells), mesenchymal stem cells), progenitor cells (e.g., neural progenitor cells), etc. The types of cells that make up the three-dimensional cells may be one type or two or more types.

[0028] An embodiment of this culture apparatus will be described below with reference to Figures 1 to 4. It goes without saying that this culture apparatus is not limited to the three-dimensional cell culture apparatus shown in Figures 1 to 4.

[0029] (Example of the configuration of this culture device) Figure 1 shows an example configuration of this culture apparatus. 101 in Figure 1 is a top view seen from above, 102 in Figure 1 is a cross-sectional view along line II at 101 in Figure 1, and 103 in Figure 1 is a cross-sectional view along line II-II at 101 in Figure 1.

[0030] As shown in Figures 101-103, the culture apparatus 10 comprises an insert cup 1, a gap-preventing plate 2, and a well plate 3 having multiple wells 3a. Each well 3a of the well plate 3 is filled with culture medium. The culture apparatus 10 is configured such that one insert cup 1 is inserted into each well 3a of the well plate 3.

[0031] The insert cup 1 is a cup that holds three-dimensional cells. More specifically, the insert cup 1 has a flange 1a, a cup body 1b, and a porous membrane 1c. The flange 1a is flat and extends toward the well plate 3 from two points on the upper end of the cup body 1b. The lower surface of the flange 1a opposite to the cup body 1b contacts the upper surface of the well plate 3. The flange 1a supports the cup body 1b relative to the wells 3a of the well plate 3. The porous membrane 1c is a membrane that has pores that prevent cells from passing through but allow culture media to pass through. It is located at the bottom of the cup body 1b. In this culture apparatus 10, cells constituting three-dimensional cells are seeded and cultured on the porous membrane 1c. In this culture apparatus 10, culture media in the wells 3a can be supplied from the back side of the porous membrane 1c, that is, from the side opposite to the culture surface of the porous membrane 1c. A commercially available insert cup 1 can be used.

[0032] The well plate 3 only needs to have wells 3a into which the cup body 1b of the insert cup 1 can be inserted. The well plate 3 can be of a conventionally known structure, or it may be a commercially available product. The wells 3a of the well plate 3 are filled with a culture medium in an amount sufficient to contact at least the porous membrane 1c of the insert cup 1.

[0033] The gap prevention plate 2 is a plate provided between the insert cup 1 and the well plate 3. Therefore, in a configuration in which the insert cup 1, gap prevention plate 2, and well plate 3 are combined, the gap prevention plate 2 cannot be removed from the configuration without removing either the insert cup 1 or the well plate 3. As shown in Figure 1, 102, the flange portion 1a of the insert cup 1 is placed on the upper surface of the gap prevention plate 2. The gap prevention plate 2 is also placed on the upper surface of the well plate 3. Although not shown in Figures 1, 101 to 103, the gap prevention plate 2 may be provided with a fitting portion that fits with the structure of the lower surface of the flange portion 1a of the insert cup 1.

[0034] Here, when the insert cup 1 is inserted into the well 3a of the well plate 3, a gap is created. In this culture apparatus 10, a gap 3b is created between the outer surface of the cup body 1b of the insert cup 1 and the inner surface of the well 3a. The gap prevention plate 2 has a closure region 2a that fills the gap 3b, that is, closes the gap 3b from above. As shown in 101 of Figure 1, the closure region 2a covers the upper end of the side wall of the well 3a and extends to the insert cup 1. The edge of the closure region 2a on the insert cup 1 side is shaped to match the outer shape of the cup body 1b. In other words, the gap prevention plate 2 has a flat plate-shaped body that closes the gap 3b, and the body has an opening formed in its side wall that matches the outer shape of the cup body 1b.

[0035] According to this culture apparatus 10, the gap prevention plate 2 is configured to fill the gap 3b that occurs when the insert cup 1 is inserted into the well 3a of the well plate 3, thereby isolating the space inside the well 3a from the outside air. Therefore, when the gas phase containing the test substance is exposed to three-dimensional cells, it is possible to prevent the gas phase containing the test substance from dissolving into the culture medium in the well 3a. The gap prevention plate 2 can also be described as an isolation plate that isolates the space inside the well 3a from the outside air, including the space inside the cup body 1b of the insert cup 1, so as not to communicate with it.

[0036] In the evaluation of three-dimensional cells by exposure to a gas phase containing the test substance, the material of the void-preventing plate 2 can be any material that does not react with or absorb the test substance, and can be appropriately set according to the type of test substance. For example, the material of the void-preventing plate 2 can be polystyrene, polypropylene, polyethylene terephthalate, polycarbonate, amorphous polyethylene terephthalate, etc.

[0037] Figure 2 shows another example of the configuration of this culture apparatus. Figure 201 is a top view seen from above, Figure 202 is a cross-sectional view taken along line III-III in Figure 201, Figure 203 is a cross-sectional view taken along line IV-IV in Figure 201, and Figure 204 is a side view showing the configuration of the cup body 1b of the insert cup 1A.

[0038] The culture apparatus 10A shown in Figures 2, 201-203, differs from the configuration shown in Figures 1, 101-103, in that the insert cup 1A and the void prevention plate 2A are configured differently.

[0039] The insert cup 1A has a flange portion 1a, a cup body 1b, a porous membrane 1c, and a recess 1d (communication portion). As shown in 204 of Figure 2, the recess 1d is formed in the side wall of the cup body 1b and is recessed downward from the upper end of the side wall of the cup body 1b.

[0040] Here, in this culture apparatus 10A as well, when the insert cup 1A is inserted into the well 3a of the well plate 3, a void is created. In this culture apparatus 10A, similar to this culture apparatus 10, a gap 3b is created between the outer surface of the cup body 1b of the insert cup 1 and the inner surface of the well 3a. In addition, the recess 1d becomes a communication part that connects the inside of the cup body 1b of the insert cup 1 and the inside of the well 3a. The void prevention plate 2A has a closure region 2a that fills the void 3b and a protrusion 2b that fills the recess 1d. The protrusion 2b is formed protruding from the lower surface of the void prevention plate 2A, and the lower edge of the protrusion 2b is shaped to match the upper shape of the recess 1d. Therefore, when the insert cup 1A is placed on the void prevention plate 2A, the recess 1d formed in the side wall of the cup body 1b fits snugly into the protrusion 2b.

[0041] The recess 1d is provided to improve the operability of the insert cup 1. As shown in Figure 2, the recess 1d is formed on the side wall of the cup body 1b, thereby forming a protrusion 1e. The protrusion 1e functions as a gripping part for grasping the insert cup 1A with tweezers. Note that the recess 1d is not limited to the configuration shown in Figure 2, but is formed on the side wall of the cup body 1b so as to connect the inside of the cup body 1b and the inside of the well 3a when the insert cup 1 is inserted into the well 3a of the well plate 3.

[0042] In this culture apparatus 10A, the gap prevention plate 2A is configured to fill the gap 3b and the recess 1d, which serves as a communication area, that occur when the insert cup 1 is inserted into the well 3a of the well plate 3. Therefore, the gap prevention plate 2A can isolate the outside air from the space inside the well 3a. Consequently, when the gas phase containing the test substance is exposed to the three-dimensional cells, it is possible to prevent the gas phase containing the test substance from dissolving into the culture medium in the well 3a.

[0043] Figure 3 is a cross-sectional view showing yet another configuration example of this culture apparatus. As shown in Figure 3, this culture apparatus 10B differs from the configurations shown in Figures 101-103 in that it includes a spraying device 4 and a covering member 5. The spraying device 4 is a device that sprays the test substance M onto the three-dimensional cells in the insert cup 1. The covering member 5 is a member that covers the insert cup 1 and the gas phase containing the test substance M.

[0044] As shown in Figure 3, the spraying device 4 is attached to the covering member 5. The spraying device 4 sprays the test substance M into the space inside the covering member 5. The spraying device 4 can be any conventionally known device that can spray the test substance M. Examples of spraying devices 4 include nebulizers, aerosols, and dispensers. The spraying device 4 may also be an ultrasonic sprayer. In this case, the diameter of the sprayed particles is not particularly limited, but is preferably 1 to 45 micrometers, and more preferably 1 to 8 micrometers.

[0045] The test substance M can be appropriately set depending on the type of 3D cell being studied. Specific examples of test substance M include fine particles such as PM2.5, propellants (liquids) such as aerosol products, and gases.

[0046] The covering member 5 is a box that covers at least the insert cup 1. The test substance M sprayed by the spraying device 4 remains inside the covering member 5 and is not discharged to the outside of the covering member 5.

[0047] When exposing three-dimensional cells to a test substance M in the gas phase, it is necessary to create a specific space for gas-phase exposure in order to expose the test substance M at a certain density or concentration. Furthermore, the concentration of the test substance M in that space must be considered. In the "Act on Examination and Regulation of Manufacture, etc., of Chemical Substances" (Chemical Substances Control Law) and GHS (Globally Harmonized System of Classification and Labelling of Chemicals), it is common to consider volume when exposing gases or powders. In particular, in the evaluation of three-dimensional cells, since multiple samples are processed simultaneously, the test substance M may concentrate on a specific sample among the multiple samples depending on the distance between the spraying device 4 and the surface of the three-dimensional cells, or the size of the particles sprayed from the spraying device 4. For this reason, depending on the test substance M, it has been difficult to reliably expose multiple samples of three-dimensional cells to the test substance M in the gas phase.

[0048] In this culture apparatus 10B, the coating member 5 maintains a constant concentration of the test substance M exposed to the three-dimensional cells. In this culture apparatus 10B, the space within the coating member 5 becomes a space for exposing the three-dimensional cells to a gas phase containing the test substance M (hereinafter sometimes referred to as the gas phase exposure space).

[0049] The covering member 5 is not particularly limited as long as it is a box that covers at least the insert cups 1. Furthermore, the configuration of the covering member 5 can be appropriately set according to the number and position of the insert cups 1 exposed to the gas phase. For example, the covering member 5 may be a box that covers each individual insert cup 1, or it may be a box that covers the entire well plate 3 and the insert cups 1 arranged on the well plate 3. Alternatively, the covering member 5 may be a box that covers two or more of the insert cups 1 arranged on the well plate 3.

[0050] Furthermore, the covering member 5 is a component that may be provided as appropriate, and a configuration in which the covering member 5 is not provided in the culture apparatus 10B is also included in this culture apparatus. The covering member 5 may be omitted if the three-dimensional cells can be reliably brought into contact with the surface of the three-dimensional cells by exposing them to a gas phase with an extremely high concentration of the test substance M.

[0051] Figure 4 is a cross-sectional view showing yet another configuration example of this culture apparatus. As shown in Figure 4, this culture apparatus 10C differs from the configuration shown in Figure 3 in that it is equipped with a base 6. The base 6 is a stand on which the apparatus body, which consists of an insert cup 1, a gap prevention plate 2, and a well plate 3, is placed. The covering member 5 is also placed on the base 6. In this culture apparatus 10C, the space formed by the covering member 5 and the base 6 becomes the gas phase exposure space.

[0052] A CO2 generating agent may be stored in the base 6. This supplies CO2 to the gas-phase exposure space. The CO2 generating agent adjusts the CO2 concentration in the gas-phase exposure space to, for example, 1.0 to 10.0%, preferably 3.0 to 8.0%, and more preferably 4.0 to 5.0%. This allows for, for example, long-term (e.g., overnight) gas-phase exposure while culturing three-dimensional cells.

[0053] (Specific examples of gap prevention plates) As described above, in this culture apparatus, the void-preventing plate is designed to fill the voids that occur when the insert cup is inserted into the well plate. Therefore, the shape of the void-preventing plate conforms to the shape of the insert cup and the well plate.

[0054] Here, among the various commercially available well plates, (1) the shape of the outer periphery and (2) the structure and shape around each well differ from one product to another. Similarly, among the various commercially available insert cups, the outer periphery shape also differs from one product to another. For this reason, it is preferable that the thickness or shape of the void prevention plate be set in a way that is a greatest common denominator to accommodate the shapes of various commercially available well plates and insert cups. In other words, it is preferable that the thickness or shape of the void prevention plate be set to accommodate a shape common to various commercially available well plates and insert cups.

[0055] Figure 5 is a perspective view showing the configuration of one specific example of a gap prevention plate. The gap prevention plate 2B shown in Figure 5 is a specific example of the gap prevention plate 2 shown in Figure 1. The gap prevention plate 2B corresponds to 4 x 3 wells. A closure region 2a is provided for each of these wells. In addition, a notch 2c is formed in the gap prevention plate 2B. The notch 2c functions as a fitting portion that fits with the structure of the lower surface of the flange portion of the insert cup.

[0056] Figure 6 is a perspective view showing the configuration of another specific example of a void prevention plate. The void prevention plate 2C shown in Figure 6 is a specific example of the void prevention plate 2A shown in Figure 2. Similar to the void prevention plate 2B, the void prevention plate 2C corresponds to 4 x 3 wells. The closure region 2a and the protrusion 2b are provided for each of these wells. Also, similar to the void prevention plate 2B, the void prevention plate 2C has a notch 2c.

[0057] [3. Evaluation Method for Test Substances in 3D Cells] An embodiment of the present invention provides a method for evaluating the effects of a test substance on three-dimensional cells (hereinafter sometimes referred to as "this evaluation method"), which is a method for evaluating the effects of a test substance on three-dimensional cells. This evaluation method includes a spraying step of spraying the test substance onto the three-dimensional cells in the insert cup using the three-dimensional cell culture apparatus described above.

[0058] With the above configuration, the test substance does not dissolve into the culture medium, allowing for accurate evaluation of the effect of the test substance on the outermost layer of cells in a three-dimensional cell structure.

[0059] The culture apparatus used in this evaluation method is not particularly limited, but examples include the culture apparatuses 10, 10A, 10B, and 10C shown in Figures 1 to 4.

[0060] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Examples]

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0062] (Example 1) Figure 7 shows a schematic configuration of the 3D cell culture apparatus 10D used in Example 1. As shown in Figure 7, in the 3D cell culture apparatus 10D, the apparatus body, which consists of an insert cup 1, a gap prevention plate 2, and a 24-well plate, is placed on a base 6. The covering member 5 is configured to cover both the apparatus body and the base 6 from above. A CO2 generator (Anelopack®) is placed below the base 6. A nebulizer head is attached to the covering member 5 as a spraying device 4.

[0063] First, a 3D skin model (LabCyte EPI-MODEL: manufactured by Japan Tissue Engineering Co., Ltd.) was cultured in insert cup 1. Then, insert cup 1 containing the cultured 3D skin model was placed in a 24-well plate along with void-preventing plate 2, and set in 3D cell culture device 10D. A cytotoxicity test was then performed using this 3D cell culture device 10D.

[0064] First, 10 ml of 40% ethanol solution was placed in a nebulizer head (nebulizer 4), and the entire volume was exposed to the 3D skin model over 1 hour at a constant spray rate. Subsequently, as a post-culture, the 3D skin model was cultured for 24 hours in a 37°C incubator. The cytotoxicity of the 3D skin model after 24 hours of culture was measured based on cell viability using a cytotoxicity assay kit (Cell Counting Kit-8: manufactured by Dojin Chemical Laboratories).

[0065] (Comparative Example 1) The cytotoxicity test was performed in the same manner as in Example 1, except that the insert cup 1 containing the cultured 3D skin model was placed in a 24-well plate without the use of the void-preventing plate 2.

[0066] (result) In Example 1, the cell viability of the 3D skin model was 103% compared to the cell viability of an untreated 3D skin model that was not exposed to the gas phase. On the other hand, in Comparative Example 1, the cell viability of the 3D skin model was 79% compared to the cell viability of an untreated 3D skin model. From the results of Comparative Example 1, cytotoxicity was observed when the ethanol solution was mixed into the culture medium.

[0067] From the cell viability of the 3D skin model in Example 1, it was found that spray exposure to a 40% ethanol solution did not cause cytotoxicity to the 3D skin model when exposed only from the stratum corneum. On the other hand, in Comparative Example 1, cytotoxicity occurred to the 3D skin model when the ethanol solution was mixed into the culture medium and exposed from the basal layer side. As a result, it is considered that the cytotoxicity that should have been measured was not properly evaluated in Comparative Example 1. On the other hand, in Example 1, it is considered that cytotoxicity can be properly evaluated when spray exposure is performed on the 3D skin model by placing the void-preventing plate 2. [Industrial applicability]

[0068] This invention can be widely used in the fields of cosmetics, medicine, and other areas. [Explanation of symbols]

[0069] 1. 1A Insert Cup 1d Recess (void, connecting part) 2, 2A, 2B, 2C Gap prevention plate 2b Convex part 3-well plate 3a well 3b Gap (void) 4 Spraying device 5 Covering member 10, 10A, 10B, 10C Main culture device (3-dimensional cell culture device)

Claims

1. An insert cup that holds a three-dimensional cell, The insert cup is inserted into a well plate having wells for holding culture medium, The system includes a gap-preventing plate provided between the insert cup and the well plate, which fills the gap created when the insert cup is inserted into the well plate, The void portion includes at least the gap between the outer circumferential surface of the insert cup and the inner circumferential surface of the well. The gap-preventing plate fills the gap between the outer surface of the insert cup and the inner surface of the well. 3D cell culture device.

2. The three-dimensional cell culture apparatus according to claim 1, further comprising a spraying device for spraying a test substance onto the three-dimensional cells in the insert cup.

3. The three-dimensional cell culture apparatus according to claim 2, further comprising a covering member for covering the insert cup and the gas phase containing the test substance.

4. The three-dimensional cell culture apparatus according to any one of claims 1 to 3, wherein the void portion connects at least the inside of the insert cup and the inside of the well, and includes a communication portion formed on the side wall of the insert cup.

5. A method for evaluating the effects of a test substance on three-dimensional cells, A method for evaluating three-dimensional cells against a test substance, comprising a spraying step of spraying the test substance onto the three-dimensional cells in the insert cup using a three-dimensional cell culture apparatus according to any one of claims 1 to 4.

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