Method for Visualizing Tight Junctions in a Three-Dimensional Skin Model

Abstract

To provide a method for visualization of a tight junction that enables a tight junction to be observed from the surface of a three-dimensional skin model.SOLUTION: The present invention discloses a method for visualization of a tight junction in a three-dimensional skin model. The method includes a first step for subjecting a keratinocyte to gas-liquid interfacial culture for differentiation induction, and forming a basal layer, a spiny layer, and a granular layer, at which stage the differentiation induction is stopped to make a three-dimensional skin model, and a second step for visualizing a constituent protein of a tight junction in the three-dimensional skin model made in the first step.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for visualizing a tight junction so that the tight junction can be observed from the surface of a three-dimensional skin model. The present invention also relates to a method for evaluating the tight junction-forming ability of a test substance and a method for screening a substance that affects the tight junction-forming ability, using the method for visualizing the tight junction.

Background Art

[0002] The skin is a tissue located in the outermost layer of the living body and is constantly exposed to stresses such as dryness and invasion of foreign substances from the outside. In order to defend the living body from these stresses, the skin has developed a barrier function. Tight junctions, which are one of the intercellular adhesion structures, are localized in the granular layer immediately below the stratum corneum and, together with the stratum corneum, are responsible for the skin barrier function. It has been reported that mice lacking claudin 1, one of the constituent proteins of tight junctions, die after birth due to an increase in transepidermal water loss, and tight junctions are thought to function not only as a barrier function against the outside world but also as a defense against water outflow in the living body. Furthermore, it has also been proposed that a decrease in the constituent proteins of tight junctions is one of the factors causing the onset of atopic dermatitis. Therefore, promoting the formation of tight junctions is considered useful for the health of the skin.

[0003] Conventionally, three-dimensional skin models have been widely used in evaluation tests as a substitute for human skin or experimental animals, which are difficult to obtain. The three-dimensional skin model is a tissue model produced by culturing keratinocytes at the air-liquid interface and inducing differentiation, and a pseudo-epidermal tissue with a basal layer, a spinous layer, a granular layer, and a stratum corneum laminated in order is formed. Conventionally, the formation evaluation of tight junctions has used three-dimensional skin models (see, for example, Patent Document 1). However, since tight junctions are localized in the granular layer below the stratum corneum, in the conventional three-dimensional skin model where the stratum corneum is present on the outermost surface, even if tight junctions are visualized by immunostaining, tight junctions cannot be observed from the surface of the three-dimensional skin model in which the stratum corneum is formed. Therefore, currently, tight junctions are observed from the cross-section of the three-dimensional skin model. Even when observing tight junctions from the cross-section of the three-dimensional skin model in this way, there is a drawback that tight junctions can only be observed as dots or lines in the cross-section, and the degree of development of tight junctions cannot be accurately evaluated.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a method for visualizing tight junctions that enables observation of tight junctions from the surface of a three-dimensional skin model. Another object of the present invention is to provide a method for evaluating the effect of a test substance on the tight junction-forming ability using the method for visualizing tight junctions, and a method for screening substances that affect the tight junction-forming ability.

Means for Solving the Problems

[0006] The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that in a three-dimensional skin model in which keratinocytes are cultured at the air-liquid interface for differentiation induction and the differentiation induction is stopped at the stage where the basal layer, spinous layer, and granular layer are formed, a network structure of tight junctions (network of tight junction strands) can be visualized on the surface (plane of the three-dimensional skin model). Furthermore, by using this method, it has been found that it is possible to evaluate the tight junction-forming ability of a test substance and to screen for substances that affect the tight junction-forming ability. The present invention has been completed by further studies based on such findings.

[0007] That is, the present invention provides an invention in the following aspects. Item 1. A method for visualizing tight junctions in a three-dimensional skin model, comprising: a first step of culturing keratinocytes at the air-liquid interface for differentiation induction, stopping the differentiation induction at the stage where the basal layer, spinous layer, and granular layer are formed, and producing a three-dimensional skin model; and a second step of visualizing the constituent proteins of tight junctions in the three-dimensional skin model produced in the first step, the visualization method. Item 2. The visualization method according to Item 1, wherein the visualization of the constituent proteins of tight junctions in the second step is performed by immunostaining against the constituent proteins. Item 3. A method for evaluating the effect on the tight junction-forming ability of a test sample, comprising: a first A step of culturing keratinocytes at the air-liquid interface for differentiation induction in the presence of a test sample, stopping the differentiation induction at the stage where the basal layer, spinous layer, and granular layer are formed, and producing a three-dimensional skin model; and a second A step of visualizing the constituent proteins of tight junctions in the three-dimensional skin model produced in the first A step, the evaluation method of comparing the degree of development of the visualized network structure of tight junctions between the case where keratinocytes are cultured at the air-liquid interface in the presence of a test sample and the case where keratinocytes are cultured at the air-liquid interface in the absence of a test sample. Item 4. A method for screening a substance that affects the ability to form tight junctions, comprising: A first step 1B of culturing keratinocytes at a gas-liquid interface in the presence of a candidate sample to induce differentiation, stopping the differentiation induction at the stage where the basal layer, spinous layer, and granular layer are formed, and producing a three-dimensional skin model; and A second step 2B of visualizing the constituent proteins of tight junctions in the three-dimensional skin model produced in the first step 1B, The screening method, wherein the degree of development of the visualized network structure of tight junctions is compared between the case where keratinocytes are cultured at a gas-liquid interface in the presence of a candidate sample and the case where keratinocytes are cultured at a gas-liquid interface in the absence of the candidate sample.

Advantages of the Invention

[0008] According to the present invention, the network structure of tight junctions (network of tight junction strands) can be visualized from the surface of the three-dimensional skin model, and the degree of development of tight junctions can be evaluated more accurately. Therefore, it is possible to efficiently evaluate the effect of a test substance on the ability to form tight junctions and screen for substances that affect the ability to form tight junctions.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0010] 1. Method for visualizing tight junctions The method for visualizing tight junctions of the present invention is a method for visualizing tight junctions in a three-dimensional skin model, which comprises a first step of culturing keratinocytes at an air-liquid interface to induce differentiation, stopping the differentiation induction at the stage when the basal layer, spinous layer, and granular layer are formed, and producing a three-dimensional skin model, and a second step of visualizing the constituent proteins of tight junctions in the three-dimensional skin model produced in the first step. Hereinafter, the method for visualizing tight junctions of the present invention will be described in detail.

[0011] [First Step] In the first step, keratinocytes are cultured at an air-liquid interface to induce differentiation, and the differentiation induction is stopped at the stage when the basal layer, spinous layer, and granular layer are formed, thereby producing a three-dimensional skin model. That is, in the first step, a three-dimensional skin model with a granular layer formed on the outermost surface is produced.

[0012] The origin of the keratinocytes used in the first step is not particularly limited, and they may be derived from any of humans, mice, rats, monkeys, etc., but are preferably of human origin from the viewpoint of making a three-dimensional skin model similar to human skin.

[0013] In addition, the keratinocytes used in the first step may be those subjected to genetic modification, drug treatment, etc., as long as they can differentiate into the basal layer, spinous layer, and granular layer.

[0014] In order to culture keratinocytes at an air-liquid interface and induce differentiation in the first step, first, keratinocytes are seeded on a liquid-permeable membrane to proliferate the keratinocytes.

[0015] The liquid-permeable membrane may be a porous membrane that can permeate the culture medium and can serve as a support for keratinocytes. Examples thereof include membranes such as polycarbonate, polyethylene terephthalate, and polystyrene. In addition, the liquid-permeable membrane may be coated with extracellular matrices such as collagen, laminin, and fibronectin, or components that enhance cell adhesion such as poly-L-lysine.

[0016] To grow keratinocytes on a liquid-permeable membrane, it is preferable to use a culture insert having a liquid-permeable membrane on the bottom surface.

[0017] The number of keratinocytes seeded on the liquid-permeable membrane is not particularly limited. For example, 1×10 3 ~1×10 7 cells / cm 2 or so, preferably 1×10 4 ~1×10 6 cells / cm 2 or so, more preferably 1×10 5 ~2×10 5 cells / cm 2 or so can be mentioned.

[0018] The growth of keratinocytes on the liquid-permeable membrane may be carried out by culturing the keratinocytes in a state of being immersed in a growth medium. When using a culture insert, the keratinocytes may be cultured in the culture insert with the growth medium added inside and outside the culture insert.

[0019] The growth medium is not particularly limited as long as keratinocytes can grow. Conventional media used for the growth of keratinocytes may be used, but a serum-free medium with a low calcium concentration is preferred. In addition, the growth medium may contain epinephrine, transferrin, vitamins, amino acids, interleukins, insulin, heparin, heparan sulfate, collagen fibronectin, progesterone, selenite, keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), hepatocyte growth factor (SCF), epidermal growth factor (EGF), tumor necrosis factor (TNF), antibiotics, etc. as growth factors. As the differentiation induction medium, for example, commercially available products such as KGMTM, KGMTM-2, KGM-GoldTM, KGM-CDTM (manufactured by Lonza Co., Ltd.) can be used.

[0020] The proliferation of keratinocytes on the liquid-permeable membrane may be carried out until the keratinocytes become confluent. The culture time is appropriately set according to the type and state of the keratinocytes used. For example, it may be 24 to 168 hours, preferably 48 to 120 hours, and more preferably 72 to 96 hours. Also, the proliferation of keratinocytes on the liquid-permeable membrane may be carried out in a CO 2 incubator at 25 to 40 °C, preferably 36 to 38 °C, and more preferably 37 °C.

[0021] After proliferating keratinocytes on the liquid-permeable membrane, prior to the air-liquid interface culture, the keratinocytes may be cultured in a state of being immersed in a differentiation-inducing medium. Thus, by immersing and culturing keratinocytes in the differentiation-inducing medium before the air-liquid interface culture, the keratinocytes can be efficiently differentiated into the basal layer, spinous layer, and granular layer. In the case of using a culture insert, to culture keratinocytes by immersing them in the differentiation-inducing medium, after proliferating the keratinocytes, the growth medium inside and outside the culture insert may be replaced with the differentiation-inducing medium for culture.

[0022] The differentiation-inducing medium is not particularly limited as long as it can induce the differentiation of confluent keratinocytes into the basal layer, spinous layer, and granular layer, and a conventional medium used for inducing the differentiation of keratinocytes may be used. However, it is preferably a serum-free medium with a calcium concentration of 1 mM or more, preferably about 1.2 to 1.8 mM. As the differentiation-inducing medium, for example, commercially available products such as CnT-Prime 3D Barrier Culture Medium (manufactured by CELLnTEC) can be used.

[0023] Regarding the culture time when culturing in a state of immersing keratinocytes in the differentiation-inducing medium, it is appropriately set according to the type and state of the keratinocytes used. For example, it may be 6 to 72 hours, preferably 12 to 48 hours, and more preferably 18 to 27 hours. Also, the culture is carried out in a CO 2In an incubator, it may be carried out at 25 to 40°C, preferably 36 to 38°C, more preferably 37°C.

[0024] In addition, to induce differentiation by culturing keratinocytes at the air-liquid interface, the side of the liquid-permeable membrane where keratinocytes are present may be exposed to the gas phase (atmosphere), and the side where keratinocytes are not present in the liquid-permeable membrane may be cultured in a state of contacting a differentiation-inducing medium. When using a culture insert, keratinocytes may be cultured in a state where the culture insert does not contain a differentiation-inducing medium and the outside of the culture insert contains a growth medium.

[0025] The air-liquid interface culture of keratinocytes may be carried out while replacing the differentiation-inducing medium with fresh medium once every 1 to 4 days, preferably once every 2 to 3 days. Also, the air-liquid interface culture is carried out in a CO 2 In an incubator, it may be carried out at 25 to 40°C, preferably 36 to 38°C, more preferably 37°C.

[0026] By performing air-liquid interface culture, keratinocytes are induced to differentiate and form the basal layer, spinous layer, and granular layer in this order and become stratified. In the first step, the air-liquid interface culture is stopped at the stage where the basal layer, spinous layer, and granular layer are formed. In the present invention, the "stage where the basal layer, spinous layer, and granular layer are formed" refers to the time point when the granular layer is located on the surface side to such an extent that the constituent proteins of the visualized tight junction can be directly visually recognized from the surface, not only the time point after the formation of the granular layer is completed and before the formation of the stratum corneum is started, but also the time point during the formation of the granular layer as long as the formation of the tight junction is recognized, and further, the time point when a slight stratum corneum that does not adversely affect the visibility of the visualized tight junction is formed on the granular layer. That is, in the first step, the air-liquid interface culture is terminated from the middle of the formation of the granular layer until a slight stratum corneum is formed on the granular layer.

[0027] Regarding the culture time of air-liquid interface culture, it may be set according to the type and state of the keratinocytes used so that it ends at the stage when the basal layer, spinous layer, and granular layer are formed. Usually, when the culture time of air-liquid interface culture exceeds 240 hours, the formation of the stratum corneum starts in the outermost layer, and when the culture time exceeds 270 hours, the visualization of tight junctions tends to become impossible due to sufficient formation of the stratum corneum. Therefore, the culture time of air-liquid interface culture is usually 96 to 270 hours, preferably 170 to 240 hours, and more preferably 190 to 240 hours.

[0028] Thus, by performing the first step, a three-dimensional skin model with a granular layer formed on the outermost surface is produced.

[0029] [Second Step] In the second step, in the three-dimensional skin model produced in the first step, the constituent proteins of tight junctions are visualized.

[0030] Examples of the constituent proteins of tight junctions include OZ-1, occludin, claudin 1, tricellulin, ERM, JAM, etc.

[0031] To visualize the constituent proteins of tight junctions in the three-dimensional skin model, immunostaining may be performed on the three-dimensional skin model using an antibody against the constituent proteins of tight junctions. Immunostaining can be performed by known whole-mount immunostaining.

[0032] Also, when immunostaining the constituent proteins of tight junctions, it is preferable to fix the three-dimensional skin model produced in the first step using methanol or the like.

[0033] Furthermore, in order to efficiently immunostain the constituent proteins of tight junctions, it is preferable to perform surfactant treatment on the three-dimensional skin model after immobilization. Specifically, the surfactant treatment can be carried out by methods such as immersing the three-dimensional skin model after immobilization in a buffer containing a surfactant, or washing the three-dimensional skin model after immobilization with a buffer containing a surfactant. Examples of the type of surfactant used include nonionic surfactants such as polyoxyethylene octyl phenyl ether (Triton X-100). In addition, examples of the content of the surfactant in the buffer containing the surfactant include 0.05 to 1.0% by weight, preferably 0.1 to 0.5% by weight.

[0034] Thus, by visualizing the constituent proteins of the tight junctions of the three-dimensional skin model prepared in the first step, the network structure of tight junctions (the network of tight junction strands) can be observed on the surface of the three-dimensional skin model.

[0035] 2. Method for evaluating the effect on tight junction-forming ability The evaluation method of the present invention is a method for evaluating the effect on the tight junction-forming ability of a test sample, which comprises a first A step of culturing keratinocytes at a gas-liquid interface in the presence of the test sample to induce differentiation, stopping the differentiation induction at the stage where the basal layer, spinous layer, and granular layer are formed, and preparing a three-dimensional skin model, and a second A step of visualizing the constituent proteins of tight junctions in the three-dimensional skin model prepared in the first A step, and comparing the degree of development of the visualized network structure of tight junctions between the case where keratinocytes are cultured at a gas-liquid interface in the presence of the test sample and the case where keratinocytes are cultured at a gas-liquid interface in the absence of the test sample.

[0036] The method for evaluating the tight junction-forming ability of the present invention is a method for evaluating the tight junction-forming ability of a test sample. The tight junction-forming ability refers to the ability to promote the formation of tight junctions or the ability to suppress or inhibit the formation of tight junctions. In the evaluation method of the present invention, it is possible to determine the presence or absence and strength of the action of the test sample on the formation of tight junctions.

[0037] The test sample is a sample to be evaluated for the presence and strength of the tight junction-forming ability, and may be a single compound or a mixture containing two or more components.

[0038] The first A step in the evaluation method of the present invention is the same as the first step in the method for visualizing the tight junctions, except that when preparing a three-dimensional skin model from keratinocytes, air-liquid interface culture is performed in the presence of the test substance.

[0039] In the first A step, to perform air-liquid interface culture of keratinocytes in the presence of the test substance, it is preferable to add the test substance to the differentiation-inducing medium used for air-liquid interface culture, but the test substance may also be directly added to the keratinocytes.

[0040] Also, in the first A step, the test substance and keratinocytes may coexist at any stage during the differentiation induction of keratinocytes. For example, (1) perform air-liquid interface culture from the beginning to the end in the presence of the test substance, (2) start air-liquid interface culture in the absence of the test substance and then perform air-liquid interface culture in the presence of the test substance, (3) start air-liquid interface culture in the absence of the test substance and then perform air-liquid interface culture in the presence of the test substance, etc. It can be implemented by such methods.

[0041] The second A step is the same as the first step in the method for visualizing the tight junctions.

[0042] When the degree of development of the network structure of tight junctions visualized in the second A step is higher when keratinocytes are cultured at the air-liquid interface in the presence of a test sample than when keratinocytes are cultured at the air-liquid interface in the absence of the test sample, the test substance is determined to have an effect of promoting the formation of tight junctions. Also, when the degree of development of the network structure of tight junctions visualized in the second A step is lower when keratinocytes are cultured at the air-liquid interface in the presence of a test sample than when keratinocytes are cultured at the air-liquid interface in the absence of the test sample, the test substance is determined to have an effect of suppressing or inhibiting the formation of tight junctions.

[0043] 3. Method for screening substances that affect tight junction-forming ability The screening method of the present invention is a method for screening a substance that affects the ability to form tight junctions, comprising: a first B step of culturing keratinocytes at the air-liquid interface in the presence of a candidate sample to induce differentiation, stopping the induction of differentiation at the stage where the basal layer, spinous layer, and granular layer are formed, and producing a three-dimensional skin model; and a second B step of visualizing the constituent proteins of tight junctions in the three-dimensional skin model produced in the first B step, and comparing the degree of development of the network structure of the visualized tight junctions when keratinocytes are cultured at the air-liquid interface in the presence of a candidate sample with that when keratinocytes are cultured at the air-liquid interface in the absence of the candidate sample.

[0044] The ability to form tight junctions refers to the ability to promote the formation of tight junctions or the ability to suppress or inhibit the formation of tight junctions. In the screening method of the present invention, a substance that can affect the ability to form tight junctions can be selected from among candidate substances.

[0045] A candidate substance is a sample for which the presence or absence of an effect on the ability to form tight junctions is determined, and it may be a single compound or a mixture containing two or more components.

[0046] In the first step 1B of the screening method of the present invention, when producing a three-dimensional skin model from keratinocytes, it is the same as the first step in the method for visualizing the tight junction, except that air-liquid interface culture is performed in the presence of a candidate substance.

[0047] In the first step 1B, to perform air-liquid interface culture of keratinocytes in the presence of a candidate substance, it is preferable to add the test substance to the differentiation induction medium used for air-liquid interface culture, but the candidate substance may also be directly added to the keratinocytes.

[0048] Also, in the first step 1B, at any stage during the differentiation induction of keratinocytes, the candidate substance and keratinocytes may coexist. For example, (1) perform air-liquid interface culture from the beginning to the end in the presence of the candidate substance, (2) start air-liquid interface culture in the absence of the candidate substance and perform air-liquid interface culture in the presence of the candidate substance from the middle, (3) start air-liquid interface culture in the absence of the candidate substance and perform air-liquid interface culture in the presence of the candidate substance from the middle, etc. It can be implemented by such methods.

[0049] The second step 2B is the same as the first step in the method for visualizing the tight junction.

[0050] When the degree of development of the network structure of the tight junction visualized in the second step 2A is higher when keratinocytes are cultured at the air-liquid interface in the presence of the candidate sample than when keratinocytes are cultured at the air-liquid interface in the absence of the candidate sample, the candidate substance is selected as having an effect of promoting the formation of tight junctions. Also, when the degree of development of the network structure of the tight junction visualized in the second step 2A is lower when keratinocytes are cultured at the air-liquid interface in the presence of the candidate sample than when keratinocytes are cultured at the air-liquid interface in the absence of the candidate sample, the candidate substance is selected as having an effect of suppressing or inhibiting the formation of tight junctions.

Example

[0051] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited thereto.

[0052] Test Example 1: Visualization of tight junctions in a three-dimensional skin model 1. Preparation of a three-dimensional skin model immunostained with tight junction constituent proteins (1) Proliferation of keratinocytes 1 ml of a cell suspension adjusted with a growth medium (KGM-GOLD medium, Lonza) to 1.0×10 5 cells / ml of keratinocytes derived from human neonatal foreskin (Lonza) was seeded into a cell culture insert (Falcon culture insert (353180, for 12 wells, pore size 0.4 μm, culture area 0.9 cm 2 ; Corning). 1.5 ml / well of the growth medium (KGM-GOLD medium, Lonza) was added to the static 12-well plate (outside the culture insert). After seeding, it was cultured in a CO 2 incubator at 37°C for 3 days.

[0053] (2) Culture in a state where keratinocytes are immersed in a differentiation-inducing medium After the culture in (1) above, the media in the culture insert and in the 12-well plate (outside the culture insert) were replaced with a differentiation-inducing medium (CnT-Prime 3D Barrier Culture Medium, CELLnTEC), and cultured in a CO 2 incubator at 37°C for 1 day.

[0054] (3) Induction of keratinocyte differentiation After the culture in (2) above, the medium in the culture insert was removed with an aspirator, and air-liquid interface culture was performed in a CO 2 incubator at 37°C for 10 days. During the air-liquid interface culture, the medium in the 12-well plate (outside the culture insert) was replaced with fresh differentiation-inducing medium once every 2 days. Note that through 10 days of air-liquid interface culture, keratinocytes are induced to differentiate, and the basal layer, spinous layer, and granular layer are formed, but it is in a state before the formation of the stratum corneum.

[0055] (4) Visualization of tight junctions After the gas-liquid interface culture in (3) above, the medium in the 12-well plate (outside the culture insert) was removed with an aspirator, washed twice with PBS(-), and then cold methanol was added to the inside of the cell culture insert and the 12-well plate (outside the culture insert) (100 μl inside the culture insert and 1.5 ml in the 12-well plate), and fixed at -20°C for 10 minutes. After fixation, the cell culture insert was washed three times with PBS(-). After washing, the three-dimensional skin model formed inside the cell culture insert was cut out together with the membrane using a scalpel, and then the membrane was removed from the three-dimensional skin model.

[0056] The excised three-dimensional epidermal model was immersed in PBS containing 0.5% Triton X-100 and treated at room temperature for 1 hour. After treatment, it was washed three times with PBS(-), and then blocked at room temperature for 30 minutes using PBS containing 10% fetal bovine serum (FBS). Next, as primary antibodies, rabbit anti-ZO-1 polyclonal IgG (#61-7300, Invitrogen) and mouse anti-occludin monoclonal IgG (OC-3F10, #33-1500, Invitrogen) were added and reacted overnight at 4°C. After the reaction of the primary antibodies, it was washed with PBS(-), and then as secondary antibodies, Alexa488-labeled donkey anti-rabbit IgG polyclonal antibody (Invitrogen) and Alexa Fluor 568-labeled goat anti-mouse IgG polyclonal antibody (Invitrogen) were used and reacted at room temperature for 1 hour together with DAPI (10236276001, Sigma). Then, it was washed three times with PBS(-) to obtain a three-dimensional skin model immunostained for tight junction proteins (ZO-1 and occludin).

[0057] 2. Observation of the three-dimensional skin model The results of observing the three-dimensional skin model obtained above with a fluorescence microscope are shown in Fig. 1. As a result, it was confirmed that in the surface of the obtained three-dimensional skin model (the plane of the three-dimensional skin model), a network structure of tight junctions (a network of tight junction strands) was clearly observed. That is, from this result, when producing a three-dimensional skin model by inducing the differentiation of keratinocytes, after the formation of the granular layer and before the formation of the stratum corneum, by stopping the induction of keratinocyte differentiation, it became clear that a three-dimensional skin model capable of observing the network structure of tight junctions from the surface can be obtained.

[0058] Test Example 2: Evaluation of the tight junction-forming ability of a test substance using a three-dimensional skin model capable of visualizing tight junctions 1. Preparation of a three-dimensional skin model in the presence of a test substance (1) Proliferation of keratinocytes Keratinocytes were grown in a culture insert under the same conditions as in “(1) Proliferation of keratinocytes” in Test Example 1 above.

[0059] (2) Culture in a state where keratinocytes are immersed in a differentiation-inducing medium The grown keratinocytes were cultured in a state of being immersed in a differentiation-inducing medium under the same conditions as in “(2) Culture in a state where keratinocytes are immersed in a differentiation-inducing medium” in Test Example 1 above.

[0060] (3) Addition of the test substance and induction of keratinocyte differentiation After the culture in (2) above, the medium in the culture insert was removed with an aspirator, and while replacing the medium in a 12-well plate (outside the culture insert) with fresh differentiation-inducing medium (CnT-Prime 3D Barrier Culture Medium, CELLnTEC) once every 2 days, at 37 °C in CO 2Air-liquid interface culture was performed in an incubator for 7 days. After 7 days of air-liquid interface culture, the medium in the 12-well plate (outside the culture insert) was replaced with a differentiation induction medium (CnT-Prime 3D Barrier Culture Medium, CELLnTEC) containing 0.0004 wt% of dipotassium glycyrrhizinate (a test substance known to promote tight junction formation), and further incubated at 37°C in CO 2 Air-liquid interface culture was performed in an incubator for 3 days. Also, as a control, air-liquid interface culture was performed under the same conditions using a differentiation induction medium without dipotassium glycyrrhizinate. By a total of 10 days of air-liquid interface culture, keratinocytes were induced to differentiate and the basal layer, spinous layer, and granular layer were formed, but it was in a state before the formation of the stratum corneum.

[0061] (4) Visualization of tight junctions Under the same conditions as in "4) Visualization of tight junctions" in Test Example 1 above, a three-dimensional skin model in which tight junction constituent proteins were immunostained was obtained.

[0062] 2. Observation of the three-dimensional skin model and evaluation of the tight junction-forming ability of the test substance The results of observing the three-dimensional skin model obtained above with a fluorescence microscope are shown in Figure 2. As a result, in the three-dimensional skin model obtained by performing air-liquid interface culture in the presence of dipotassium glycyrrhizinate, which is known to have an effect of promoting tight junction formation, it was confirmed that the network structure of tight junctions (network of tight junction strands) observed on the surface of the three-dimensional skin model was more developed compared to the control. That is, from these results, it was found that a three-dimensional skin model prepared by stopping the induction of keratinocyte differentiation after the formation of the granular layer and before the formation of the stratum corneum can accurately evaluate the tight junction-forming ability of a test substance.

Claims

1. A method for evaluating the effect on the tight junction-forming ability of a test sample, comprising: a first A step of culturing keratinocytes at a gas-liquid interface in the presence of a test sample to induce differentiation, stopping the differentiation induction at the stage where the basal layer, spinous layer, and granular layer are formed, and producing a three-dimensional skin model; and a second A step of visualizing the constituent proteins of tight junctions in the three-dimensional skin model produced in the first A step, The evaluation method, wherein the degree of development of the visualized tight junction network structure is compared between the case where keratinocytes are cultured at a gas-liquid interface in the presence of a test sample and the case where keratinocytes are cultured at a gas-liquid interface in the absence of a test sample.

2. A method for screening a substance that affects tight junction-forming ability, comprising: a first B step of culturing keratinocytes at a gas-liquid interface in the presence of a candidate sample to induce differentiation, stopping the differentiation induction at the stage where the basal layer, spinous layer, and granular layer are formed, and producing a three-dimensional skin model; and a second B step of visualizing the constituent proteins of tight junctions in the three-dimensional skin model produced in the first B step, The screening method, wherein the degree of development of the visualized tight junction network structure is compared between the case where keratinocytes are cultured at a gas-liquid interface in the presence of a candidate sample and the case where keratinocytes are cultured at a gas-liquid interface in the absence of a candidate sample.

Images