Test method, gel-like composition, and test device

Abstract

Provided is a test method using a test device provided with a cylindrical cup (2) that has openings in the vertical direction, a porous membrane (3) that is provided inside or at the lower end of the cup and that separates spaces on the upper end side and the lower end side of the cup, a skin cell sheet (4) placed on the upper surface of the porous membrane, and a culture solution (M) provided in contact with the lower surface of the porous membrane. The test method involves placing a gel-like composition (5) containing a test substance and a gelling agent on the upper surface of the skin cell sheet and causing the test substance to permeate into the skin cell sheet from the gel-like composition. In the test method, outflow of the test substance from the gel-like composition to the inner wall side of the cup is prevented by the gel characteristics of the gel-like composition so that the test substance does not come into contact with the inner wall of the cup.

Description

Test method, gel composition, and test device

[0001] The present invention relates to a test method, a gel composition, and a test device for a cell sheet forming a skin tissue model. This application claims priority from Japanese Patent Application No. 2024-184213 filed in Japan on October 18, 2024, and the contents thereof are incorporated herein by reference.

[0002] Conventionally, in order to test the effects of drugs and cosmetics on the skin, the use of a cultured skin model has been proposed. Manufacturers of cultured skin models are attempting to improve the culture vessels for skin cells in order to make the structure of the cultured skin model closer to that of human skin tissue. For example, Patent Document 1 discloses a cell culture vessel capable of forming a cultured skin sheet with an increased three-dimensional thickness by forming convex portions on the surface of a porous membrane to which a cultured skin cell sheet adheres during culture and providing irregularities on the base portion of the skin cell sheet. Patent Document 2 also discloses an evaluation method in which a liquid containing a test substance is dropped onto a cultured skin sheet produced by the same method, exposed for a predetermined time, and the effects are examined.

[0003] International Publication No. 2017 / 222065 Japanese Unexamined Patent Application Publication No. 2021-141877

[0004] As a result of the intensive studies by the present inventors, it has been found that when a test substance is dropped onto a cultured skin sheet as disclosed in Patent Document 2, the test substance flows along the surface of the cultured skin sheet and flows out from the edge of the cultured skin sheet to the lower part of the culture vessel, resulting in a problem that appropriate evaluation cannot be performed.

[0005] The present invention has been made in view of the above circumstances, and provides a test method in which a test substance applied to a skin cell sheet is suppressed from flowing out from the edge along its surface.

[0006] [1] A test apparatus comprising a cylindrical cup opening vertically, a porous membrane provided inside or at the lower end of the cup and separating the space between the upper and lower ends of the cup, a skin cell sheet placed on the upper surface of the porous membrane, and a culture medium placed in contact with the lower surface of the porous membrane, wherein a gel-like composition containing the test substance and a gelling agent is placed on the upper surface of the skin cell sheet, and the gel properties of the gel-like composition prevent the test substance from flowing out of the gel-like composition to the inner wall of the cup, or prevent the gel-like composition itself from flowing and contacting the inner wall of the cup, so that the test substance does not come into contact with the inner wall of the cup. [2] The test method according to [1], wherein the test substance in the culture medium that has permeated the skin cell sheet in the cup is measured qualitatively or quantitatively. [3] The test method according to [1] or [2], wherein the storage modulus G' of the gel-like composition at 37°C is 50 or more. [4] The test method according to [3], wherein tanδ is 0.20 or less. [5] The test method according to any one of [1] to [4], wherein the gelling agent comprises a crosspolymer obtained by crosslinking a copolymer containing polyacrylic acid or a salt thereof, alginic acid or a salt thereof, bentonite, carboxyvinyl polymer, or alkyl acrylate as repeating units with an allyl ether of a disaccharide or an allyl ether of a sugar alcohol. [6] The test method according to any one of [1] to [4], wherein the gelling agent comprises a carboxyvinyl polymer. [7] The test method according to any one of [1] to [4], wherein the gelling agent comprises a sodium alginate polymer. [8] The test method according to any one of [1] to [4], wherein the gelling agent comprises smectite. [9] The test method according to any one of [1] to [4], wherein the gelling agent comprises an (acrylates / alkyl acrylate (C10-30)) crosspolymer.

[10] A gel-like composition for use on the surface of a skin cell sheet, comprising a test substance for investigating the effect on the skin cell sheet, water, and a gelling agent, wherein the gelling agent comprises 0.20 to 2.00% (w / w) of a carboxyvinyl polymer relative to the total mass of the gel-like composition.

[11] The gel-like composition according to

[10] , further comprising a glycerin fatty acid ester.

[12] A test apparatus for investigating the effect of a test substance on a skin cell sheet, comprising: a container containing a culture medium; a cylindrical cup whose lower end is immersed in the culture medium; a porous membrane provided inside or at the lower end of the cup, separating the space between the upper and lower ends of the cup; the skin cell sheet placed on the upper surface of the porous membrane; and a gel-like composition containing the test substance and a gelling agent placed on the upper surface of the skin cell sheet, wherein the space at the upper end of the cup is filled with an arbitrary gas, and the culture medium is introduced into the space at the lower end of the cup so as to be in contact with the porous membrane.

[0007] According to the test method of the present invention, the permeability of the test substance to the skin cell sheet can be appropriately evaluated by suppressing the outflow of the test substance from the edges along its surface. As a result, the effect on the skin cell sheet by the test substance that has permeated and penetrated through the skin cell sheet can be appropriately evaluated.

[0008] This is a cross-sectional view of an example embodiment of the test apparatus of the present invention.

[0009] Hereinafter, an example of an embodiment according to the present invention will be described with reference to the drawings. For the sake of explanation, the drawings may show enlarged versions of characteristic parts, and the dimensional ratios of each component may not be the same as in reality.

[0010] ≪Test Method≫ The first aspect of the present invention is a test method in which a gel-like composition containing a test substance and a gelling agent is placed on the upper surface of a skin cell sheet, and the test substance is permeated from the gel-like composition into the skin cell sheet. The test method of this aspect can be used, for example, to investigate the permeability of the test substance to the skin cell sheet. The test method of this aspect can also be used, for example, to investigate the biological effects of the test substance on the cell sheet. Examples of biological effects include any changes that occur within cells. Specific examples include the amount of change in melanin pigment synthesized in melanocytes and the amount of change in melanin pigment deposition.

[0011] As an example of a preferred embodiment of this design, a method can be used that utilizes a test apparatus comprising a skin cell sheet placed on the upper surface of a porous membrane and a culture medium placed in contact with the lower surface of the porous membrane. Using this test apparatus, the test substance that has permeated the skin cell sheet diffuses into the culture medium, allowing for qualitative or quantitative measurement of the test substance contained in the culture medium.

[0012] As an example of a measurement method, if the test substance exhibits color or emits fluorescence in the culture medium, the presence or absence of a change in the color of the culture medium, or the degree of the color change, can be optically observed to measure whether or not the test substance is transmitted and the amount transmitted.

[0013] As another example of a measurement method, the presence or absence and amount of the test substance that has permeated into the culture medium can be measured using known measuring instruments such as liquid chromatography, gas chromatography, mass spectrometry, and scintillation counters. The selection of measuring instruments is made appropriately according to the properties of the test substance to be detected.

[0014] The cultured skin sheet can be any sheet containing cells (e.g., keratinocytes) that make up the skin of a living organism such as a human or animal. In recent years, skin cell sheets, also called skin tissue models, have been developed that mimic skin tissue in living organisms from various perspectives (e.g., those having a multilayer structure composed of multiple cell types, those with thickness, etc.), and such skin tissue models may be used as the skin cell sheet in this embodiment.

[0015] The porous membrane is not particularly limited as long as it can support the skin cell sheet and allows the culture medium to permeate or diffuse through it, and various known porous membranes used in the manufacture and sale of skin cell sheets can be applied.

[0016] The porous membrane is preferably supported by, for example, a cylindrical cup (which may be called a well). Specific examples include a cylindrical cup with an opening in the vertical direction and a porous membrane provided inside or at the lower end of the cup, separating the space between the upper and lower ends of the cup. The culture medium is in contact with the porous membrane, permeates and diffuses through the porous membrane, comes into contact with the lower surface of the skin cell sheet placed on the upper surface of the porous membrane, and supplies nutrients from the culture medium to the skin cell sheet. Since a gel-like composition is placed on the upper surface of the skin cell sheet, it is preferable that the culture medium is in contact with the lower surface of the skin cell sheet while the upper surface of the skin cell sheet is in contact with the gas phase. The composition of the gas phase is suitable for the survival of the skin cell sheet, with appropriate temperature, humidity, and CO2 levels. ２ It is preferable that the culture medium has a known composition that takes into account the concentration and other factors. Similarly, it is preferable that the culture medium has a known composition that takes into account the temperature, nutrients, osmotic pressure, pH, and other factors suitable for the survival of the skin cell sheet.

[0017] In the test method of this embodiment, the gel properties of the gel-like composition prevent the test substance from flowing out of the gel-like composition and flowing downward from the edge along the surface of the skin cell sheet. Specifically, the gel properties of the gel-like composition prevent the test substance from flowing out of the gel-like composition toward the inner wall of the cup so that the test substance does not come into contact with the inner wall of the cup. Furthermore, the gel properties of the gel-like composition prevent the gel-like composition itself from flowing and flowing downward from the edge along the surface of the skin cell sheet. Specifically, it prevents the gel-like composition from flowing toward the inner wall of the cup.

[0018] Here, "gel properties" refer to the physical or chemical properties resulting from the gel state, which makes it less likely to flow on the surface of an object (e.g., a film surface, a skin cell sheet surface, etc.) compared to a general liquid (e.g., water or alcohol). An example of gel properties is the viscoelasticity of the gel-like composition.

[0019] The gel-like composition comprises the test substance and a gelling agent. From the viewpoint of enhancing the penetration of the test substance into the skin cell sheet, it is preferable that the gel-like composition further comprises a liquid medium such as an aqueous solvent (e.g., water) or an oily solvent (e.g., alcohol).

[0020] The test substance is the substance whose effect on the skin cell sheet is to be evaluated, and is a substance different from the gelling agent and the liquid medium. The test substance may be a naturally derived component or a non-natural synthetic compound. The test substance may be a biologically derived component or a non-biologically derived component. The test substance may be a single substance or a mixture of multiple substances. Preferably, the test substance can be dispersed together with the gelling agent in the gel-like composition. The molecular weight of the test substance is not particularly limited, and may be a low molecular weight compound with a molecular weight of 1000 or less, or a high molecular weight compound with a molecular weight of 1000 or more. The test substance may be anionic, nonionic, or cationic. The test substance may be, for example, a known substance whose desirable or undesirable effects on human or animal skin are unknown, expected, or have already been confirmed.

[0021] Specific test substances include, for example, skin medications or candidate compounds thereof.

[0022] The gelling agent can be any substance capable of imparting the gel properties to the gel-like composition, such as polymers and clays. From the viewpoint of easily dispersing the test substance and imparting excellent gel properties, preferred gelling agents include, for example, [a] poly(meth)acrylic acid or a salt thereof, [b] alginic acid or a salt thereof, [c] bentonite, [d] carboxyvinyl polymer, and [e] a crosspolymer formed by crosslinking a copolymer containing alkyl acrylate as a repeating unit.

[0023] [a] Polyacrylic acid refers to a polymer in which acrylic acid is repeated as a unit. The carboxyl groups of these polymers may form salts. Examples of countercations constituting the salts include alkali metal ions such as sodium and potassium, and ammonium ions. If it is necessary to eliminate the effect of countercations on the skin cell sheet, it is preferable to use an acid rather than a salt. The weight-average molecular weight Mw of the polymer in [a] above can be appropriately selected, for example, in the range of 5,000 to 500,000.

[0024] [b] Alginic acid is a polysaccharide found in brown algae. Generally, it is described as a linear polymer in which blocks of β-D-mannuronic acid bonds and blocks of α-L-guluronic acid, which is its C-5 epimer, are linked by glycosidic bonds. Examples of countercations that make up the salt include alkali metal ions such as sodium and potassium, and ammonium ions. From the viewpoint of increasing hydrophilicity, it is preferable that alginic acid forms a salt.

[0025] [c] Bentonite is generally known as a clay that absorbs water and swells, and it is thought that the presence of layered phyllosilicates (e.g., aluminum salts) is the reason for its water absorption. Bentonite is preferably a clay whose main component is montmorillonite (smectite) of the smectite group.

[0026] [d] Carboxyvinyl polymer, sometimes called carbomer, is a crosspolymer having carboxyl groups, with polyacrylic acid as the main chain and these main chains being crosslinked together. The above crosslinking can be carried out using, for example, pentaerythristyl allyl ether (also known as allylpentaerythritol), sucrose allyl ether, or propylene allyl ether (also known as allylpropyl ether), but is not limited to these. The carboxyl groups of the carbomer may form salts as in [a] above. The weight-average molecular weight Mw of the carbomer in [d] above can be appropriately selected, for example, in the range of 5,000 to 500,000.

[0027] In this specification and in the claims, "crosspolymer" means a polymer formed by crosslinking two or more polymers with respect to each other. The crosslinking points in a crosspolymer are preferably located at locations other than the ends of the polymers before crosslinking. Typically, crosslinking points are formed by a reaction with a crosslinking agent.

[0028] [e] A crosspolymer formed by crosslinking copolymers containing alkyl acrylate as repeating units is a polymer formed by crosslinking copolymers, which are polymers of alkyl acrylate and other monomers. The number of carbon atoms in the alkyl group constituting the alkyl acrylate is preferably 5 to 50, more preferably 8 to 40, and even more preferably 10 to 30. When the number of carbon atoms of the alkyl group is within the above range, the lipophilicity is increased, and the compatibility and dispersibility with lipid-soluble test substances can be further improved. Examples of monomers other than alkyl acrylate include acrylic acid, methacrylic acid, or simple esters thereof. As for simple esters, those in which an alkyl group with 1 to 3 carbon atoms is substituted for a hydrogen atom of the carboxyl group are preferred. The above crosslinking can be carried out using, for example, pentaerythristyl allyl ether (also known as allylpentaerythritol), sucrose allyl ether, or propylene allyl ether (also known as allylpropyl ether), but is not limited to these. An example of a commercially available crosspolymer corresponding to [e] is (acrylates / alkyl acrylate (C10-30)) crosspolymer. This crosspolymer has alkyl acrylate as its constituent unit, which has an alkyl group with 10 to 30 carbon atoms.

[0029] The preferred viscoelasticity of the gel-like composition (37°C) used in the test method of this embodiment is expressed within the following ranges of storage modulus G' and tanδ. Specifically, it is preferable that the storage modulus G' is 50 or higher. Furthermore, it is more preferable that tanδ is 0.20 or lower. Also, it is preferable that the storage modulus G' is 70 or higher, more preferably 90 or higher, even more preferably 110 or higher, and particularly preferable 130 or higher. The upper limit of the storage modulus G' is not particularly limited, but 500 can be given as one guideline. Also, it is preferable that tanδ is 0.17 or lower, more preferably 0.13 or lower, even more preferably 0.10 or lower, and particularly preferable 0.07 or lower. The lower limit of tanδ is not particularly limited, but 0.01 can be given as one guideline. Examples of preferred ranges for the storage modulus G' include 50 to 500, 70 to 500, 90 to 500, 110 to 500, or 130 to 500. Preferred ranges for tanδ include, for example, 0.01 to 0.20, 0.01 to 0.17, 0.01 to 0.13, 0.01 to 0.10, or 0.01 to 0.07. When tanδ is above the lower limit of the above range, the effects of the present invention are further enhanced. When tanδ is below the upper limit of the above range, it becomes easier to place the gel-like composition on the skin cell sheet, and the gel-like composition can be sufficiently adhered to the skin cell sheet.

[0030] The viscoelasticity of the gel-like composition can be measured by the following method: Using a rheometer, set the temperature to 37°C and the strain to 1%, perform frequency dispersion measurements in the range of 0.1 to 1 rad / s, and calculate the storage modulus G' and tanδ.

[0031] ≪Gel-like Composition≫ The second aspect of the present invention is a gel-like composition used for application on the surface of a skin cell sheet. The gel-like composition of this aspect can be used as the gel-like composition used in the first aspect. Regarding the gel-like composition, any explanation that overlaps with the explanation of the gel-like composition in the first aspect will be omitted.

[0032] As an example of a preferred embodiment of this design, a gel-like composition comprising a test substance for investigating the effect on the skin cell sheet, water, and a gelling agent is provided.

[0033] The description of the test substance included in this embodiment is the same as the description of the test substance in the first embodiment. The effect of the test substance in this embodiment on the skin cell sheet may be the permeability of the skin cell sheet by the test substance, any effect exerted when the test substance is absorbed by the cells constituting the skin cell sheet (for example, biological effects such as changes occurring within cells), or any effect exerted when the test substance remains between the cells constituting the skin cell sheet.

[0034] The content of the test substance relative to the total mass of the gel-like composition in this embodiment can be appropriately set according to the type of test substance, and preferably falls within the range of viscoelasticity described above. As a guideline, for example, 0.01 to 100 mg / mL relative to the total amount of the gel-like composition can be given. If it is within the above preferred range, it becomes easy to obtain a gel-like composition with viscoelasticity within the preferred range described in the first embodiment. The test substance contained in the gel-like composition in this embodiment may be one type or two or more types.

[0035] The gel-like composition of this embodiment may contain a skin permeability enhancer that enhances the permeability of the test substance to the cells constituting the skin cell sheet or the permeability of the skin cell sheet. Examples of such skin permeability enhancers include glyceryl monocaprate (abbreviated as C10G) and glyceryl monocaprylate (abbreviated as C8G), which are glycerin fatty acid esters. In this case, the test substance is something other than a glycerin fatty acid ester.

[0036] The content of the skin permeability enhancer relative to the total mass of the gel-like composition in this embodiment is preferably, for example, 0.001 to 1% (w / w), more preferably 0.005 to 0.5% (w / w), and even more preferably 0.01 to 0.1% (w / w). If it is above the lower limit of the above range, the effect of the skin permeability enhancer is obtained even more. If it is below the upper limit of the above range, it becomes easy to obtain a gel-like composition with a viscoelastic modulus within the preferred range described in the first embodiment.

[0037] <<Test Apparatus>> The third aspect of the present invention is a test apparatus for investigating the effect of a test substance on a skin cell sheet. The test apparatus of this aspect can be used with the test method of the first aspect. A preferred embodiment of this aspect will be described below with reference to the drawings, but descriptions of each component constituting the test apparatus that overlap with the descriptions of the first or second aspect described above will be omitted.

[0038] As an example of a preferred embodiment of this design, the test apparatus 10 shown in Figure 1 is provided. The test apparatus 10 comprises a container 1 containing a culture medium M, a cylindrical cup 2 whose lower end is immersed in the culture medium M, a porous membrane 3 provided inside or at the lower end of the cup 2 and separating the space between the upper and lower ends of the cup 2, a skin cell sheet 4 placed on the upper surface of the porous membrane 3, and a gel-like composition 5 containing the test substance and a gelling agent placed on the upper surface of the skin cell sheet 4.

[0039] In the illustrated example, one cup 2 is provided in one container 1, but two or more cups 2 may be provided in one container 1. The two or more cups 2 may be in an independent form or in a connected form. Furthermore, there may be multiple containers 1 each containing a cup 2, which are held together by another substrate (not shown).

[0040] In the illustrated example, the shape of cup 2 is a cylinder with an opening in the vertical direction, but it is not limited to this; it may be a cylindrical shape that tapers downwards, or another cylindrical shape with side walls that have an opening in the vertical direction. The diameter of the opening of cup 2 is not particularly limited, for example, 1 to 10 cm. The reason why cup 2 is called a cup even though its standalone shape is cylindrical is that when the porous membrane 3 is provided, the porous membrane 3 forms the bottom surface of cup 2.

[0041] In the illustrated example, the porous membrane 3 is located inside the cup 2, at a height below the middle, dividing the internal space of the cup 2 into two, separating the upper space s1 and the lower space s2. The porous membrane 3 may be directly attached to the inner wall of the cup 2, or it may be attached to the inner wall via a flange portion (not shown) that protrudes from the inner wall of the cup 2 toward the central axis of the cylindrical cup 2.

[0042] In the illustrated example, the upper space s1 in the cup 2 is filled with an arbitrary gas. From the viewpoint of maintaining the good state of the skin cell sheet 4, it is preferable that a gas phase with adjusted temperature, humidity, CO ２ concentration, etc. is introduced. From the above viewpoint, a lid body (not shown) that covers the opening above the container 1 may be provided.

[0043] In the lower space s2 of the internal space of the cup 2, the culture solution M is introduced so as to contact the porous membrane 3. A notch, or a leg serving as an alternative to the notch, or a through-hole may be provided on the inner wall below the porous membrane 3 that constitutes the cup 2. In this form, the culture solution M in the lower space s2 of the cup 2 and the culture solution M outside the cup 2 pass through the above notch or through-hole and mix with each other (indicated by double arrows in the figure).

[0044] Although not shown, the porous membrane 3 may be installed at the lower end of the cylindrical cup 2. In this case, the porous membrane 3 preferably covers the lower opening of the cup 2. The porous membrane 3 installed at the lower end of the cup 2 separates the upper space (i.e., the internal space of the cup 2) of the cup 2 from the lower space (i.e., the space below the cup 2) of the cup 2. When installing the cup 2 in the container 1, a pedestal-like scaffold for supporting a part of the edge at the lower end of the cup 2 may be installed so that the porous membrane 3 does not contact the bottom surface of the container 1. If the porous membrane 3 is thus made to float from the bottom surface of the container 1, the culture solution M injected into the container 1 can be made to contact the porous membrane 3.

[0045] As the porous membrane 3, the same one as the porous membrane in the first aspect described above can be applied. As the skin cell sheet 4, the same one as the skin cell sheet in the first aspect described above can be applied. As the gel composition 5, the same one as the gel composition in the first aspect or the second aspect described above can be applied.

[0046] <Test Example 1> Using the test apparatus 10 illustrated in FIG. 1, a gel composition 5 containing uranine was prepared, applied to the center of the skin cell sheet 4, and then the spreading state of the gel composition after 24 hours was evaluated. This will be described in detail below. As the test apparatus 10, a commercially available product (manufactured by MatTek Corporation, model number: EPI-2000) equipped with a skin cell sheet 4 composed of human normal epidermal keratinocytes that mimics human epidermis was used. Sterilized ion-exchanged water was added with the gelling agent described in Table 1 (final concentration 0.13% by mass or 0.50% by mass) and uranine (final concentration 100 μg / mL), and the uniformly mixed product was used as the gel composition 5. The test apparatus 10 was cultured under conditions of 37 °C and 5% CO ２ , high humidity, 25 μL of the gel composition 5 was applied to the center of the skin cell sheet 4, and the spreading state of the gel composition 5 immediately after application, after 24 hours, and after 48 hours was visually confirmed. The evaluation results obtained in the following three steps are shown in the table below.

[0047] A: The distance from the edge of the applied gel composition 5 to the inner wall of the cup 2 is sufficiently far. B: The distance from the edge of the applied gel composition 5 to the inner wall of the cup 2 is short, but there is no contact. C: The edge of the applied gel composition 5 touches the inner wall of the cup 2.

[0048] The viscoelasticity of each prepared gel composition 5 was measured. As the apparatus, a rheometer MCR302 (manufactured by Anton Paar) was used, and measurement was performed under the conditions of 1% strain, a frequency range of 0.1 to 10 rad / s, and 37 °C by the frequency dispersion measurement method. The measurement results are shown in the table below. As indices of viscoelasticity, G' and tan δ were calculated as the average values of the measured values at 0.1 to 1 rad / s.

[0049]

[0050] In the table, CP-U10 is Carbomer Carbopol Ultrez 10 manufactured by Lubrizol. In the table, CP-U20 is Acrylates / Alkyl Acrylate (C10-30) Crosspolymer Carbopol Ultrez 20 manufactured by Lubrizol.

[0051] After 48 hours had elapsed since application, the amount of uranine contained in the culture medium M in the internal space s2 of cup 2 was calculated using fluorescence chromatography. The results are shown in the table below.

[0052]

[0053] In the above test results, Test Examples 1-3 and 1-5 showed excellent results. Specifically, in Test Examples 1-3 and 1-5, the outflow of uranine, the test substance applied to the skin cell sheet, from the edges along its surface was suppressed. From these results, it is clear that the present invention allows for appropriate evaluation of the permeability of the test substance to the skin cell sheet. Here, appropriate evaluation means being able to distinguish whether the test substance truly permeated the skin cell sheet or merely flowed down from the edges of the skin cell sheet.

[0054] <Test Example 2> Sterilized deionized water was mixed with the gelling agent listed in Table 3 (final concentration 0.25-2.0% by mass) and Malva sylvestris extract ferment (hereinafter, MSEF) (final concentration 13.2 mg / mL), and the mixture was used as gel composition 5. Except for using the above gel composition 5, the procedure was the same as in Test Example 1, with 25 μL of gel composition 5 applied to the center of the skin cell sheet 4. The spread of gel composition 5 was visually confirmed immediately after application, and after 24 hours and 48 hours, and evaluated according to the same criteria as above. The results are shown in Table 3.

[0055]

[0056] In the table, Algi-Na refers to sodium alginate 500-600 manufactured by Tokyo Chemical Industries Co., Ltd. In the table, Smectite refers to Sumecton-SWF, a type of chemically synthesized bentonite manufactured by Kunimine Industries Co., Ltd.

[0057] The above Malva sylvestris lactic acid fermentation solution was prepared by the following method: Malva sylvestris extract was prepared by adding 20 times the volume (by mass ratio) of deionized water to commercially available dried powder of Malva sylvestris L. flowers and heating in an autoclave. The extract for pre-culture was heated at 121°C for 15 minutes, and the extract for main culture was heated at 98°C for 100 minutes. 1 / 200 volume (by mass ratio) of Lactiplantibacillus plantarum culture solution was added to the pre-culture extract, and pre-culture was carried out at 30°C for 24 hours. Then, 1 / 200 volume (by mass ratio) of the pre-culture solution was added to the main culture extract, and main culture was carried out at 30°C for 72 hours. The culture solution was centrifuged at 4°C and 15100 × g for 60 minutes, and the recovered supernatant was obtained as a fermented liquid of Malva sylvestris lactic acid bacteria.

[0058] The results of Test Examples 1 and 2 above support the finding that excellent results can be obtained for the viscoelasticity of the gel-like composition 5 when its storage modulus G' is 50 or higher and its tanδ is 0.2 or lower.

[0059] 1...Container, 2...Cup, 3...Porous membrane, 4...Skin cell sheet, 5...Gel-like composition, M...Culture medium

Claims

1. A test apparatus comprising: a cylindrical cup with an opening in the vertical direction; a porous membrane provided inside or at the lower end of the cup, separating the space between the upper and lower ends of the cup; a skin cell sheet placed on the upper surface of the porous membrane; and a culture medium placed in contact with the lower surface of the porous membrane; wherein a gel-like composition containing the test substance and a gelling agent is placed on the upper surface of the skin cell sheet; and the gel properties of the gel-like composition prevent the test substance from flowing out of the gel-like composition to the inner wall of the cup, or prevent the gel-like composition itself from flowing and coming into contact with the inner wall of the cup, so that the test substance does not come into contact with the inner wall of the cup.

2. The test method according to claim 1, wherein the test substance in the culture medium that has permeated through the skin cell sheet in the cup is measured qualitatively or quantitatively.

3. The test method according to claim 1 or 2, wherein the storage modulus G' of the gel-like composition at 37°C is 50 or more.

4. The test method according to claim 3, further comprising the condition that tanδ is 0.20 or less.

5. The test method according to claim 1 or 2, wherein the gelling agent comprises a crosspolymer in which a copolymer containing polyacrylic acid or a salt thereof, alginic acid or a salt thereof, bentonite, carboxyvinyl polymer, or alkyl acrylate as repeating units is crosslinked with an allyl ether of a disaccharide or an allyl ether of a sugar alcohol.

6. The test method according to claim 1 or 2, wherein the gelling agent comprises a carboxyvinyl polymer.

7. The test method according to claim 1 or 2, wherein the gelling agent comprises a sodium alginate polymer.

8. The test method according to claim 1 or 2, wherein the gelling agent comprises smectite.

9. The test method according to claim 1 or 2, wherein the gelling agent comprises (acrylates / alkyl acrylate (C10-30)) crosspolymer.

10. A gel-like composition for use on the surface of a skin cell sheet, comprising a test substance for investigating its effect on the skin cell sheet, water, and a gelling agent, wherein the gelling agent contains 0.20 to 2.00% (w / w) of a carboxyvinyl polymer relative to the total mass of the gel-like composition.

11. The gel-like composition according to claim 10, further comprising a glycerin fatty acid ester.

12. A test apparatus for investigating the effect of a test substance on a skin cell sheet, comprising: a container containing a culture medium; a cylindrical cup whose lower end is immersed in the culture medium; a porous membrane provided inside or at the lower end of the cup, separating the space between the upper and lower ends of the cup; the skin cell sheet placed on the upper surface of the porous membrane; and a gel-like composition containing the test substance and a gelling agent placed on the upper surface of the skin cell sheet, wherein the space at the upper end of the cup is filled with an arbitrary gas, and the culture medium is introduced into the space at the lower end of the cup so as to be in contact with the porous membrane.

Images