JP2020202754A - Method for manufacturing three-dimensional cultured skin, and three-dimensional cultured skin obtained by the method - Google Patents

Abstract

To provide a method for stably manufacturing a three-dimensional cultured skin having a structure similar to a natural skin.SOLUTION: A method for manufacturing three-dimensional cultured skin includes: (1) a process of inoculating fibroblast on a porous membrane of a cell culture vessel having the porous membrane to form a fibroblast layer, and charging culture medium inside and outside the cell culture vessel to culture the medium; (2) a process of pouring solution containing the fibroblast and hydrogelling agent onto the fibroblast layer to form a hydrogel layer containing the fibroblast, and charging culture medium inside and outside the cell culture vessel to culture the medium; (3) a process of inoculating keratinocyte on the hydrogel layer to form a keratinocyte layer, and charging culture medium inside and outside the cell culture vessel to culture the medium; and (4) a process of removing the culture medium on the keratinocyte layer, bringing the culture medium into contact with an outside surface of the porous membrane of the cell culture vessel while exposing the keratinocyte layer to a vapor phase, and culturing the medium.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for producing a three-dimensional cultured skin and the three-dimensional cultured skin obtained thereby.

The skin is an organ that covers the body surface that separates the environment inside and outside the body. The skin acts as a physical barrier, protects against dryness and the invasion of harmful substances into the body, and plays an essential role in sustaining life.

Natural skin is roughly divided into two layers, the epidermis and the dermis, and a thin and delicate membrane called the epidermis basement membrane exists between the epidermis and the dermis. The epidermis basement membrane is a very thin structure of about 0.1 μm, and exists in a sheet shape at the junction between the epidermis and the dermis. The epidermal basement membrane is composed of keratinocytes hemidesmosomes, anchoring filaments, anchoring fibers, etc., in addition to the basic structures lamina densa and laminar densa, and the basic structure is particularly type IV. It is composed of collagen, various laminins, and proteoglycans. Laminin 5 is the main component of the anchoring filament that binds the epidermal basal cells to the basic structure, especially the laminadensa, and the basic structure and the collagen fibers of the dermis are connected by anchoring fibers containing VII type collagen as the main component. ing. Anchoring filaments and anchoring fibers can also bind to each other, forming a complex called an anchoring complex. Due to such a structure, the skin existing in the outermost layer of the living body has the strength to withstand the mechanical stress from the outside world.

There is an increasing demand for artificial skin to be used as a substitute for natural skin (ie, natural skin) that has been damaged for some reason. In addition, there is an increasing demand for artificial skin as an experimental material for testing the action of medicines and cosmetics on the skin and drugs. For all applications, the development of artificial skin that mimics the structure of natural skin as much as possible is strongly desired.

Methods for producing various artificial skins (three-dimensional cultured skins) that imitate the skin structure have been developed, and some have already been put into practical use. For example, a method of culturing normal human epidermal keratinocytes on a type I collagen gel containing human fibroblasts to form an epidermal layer is known, but the artificial skin obtained by this method is collagen that mimics the dermis. It is known that the basement membrane is not sufficiently formed between the gel and the epidermis layer that mimics the epidermis. Therefore, when such artificial skin is used, it is possible to promote the reformation of the skin basal membrane structure by administering a matrix metalloprotease inhibitor or both a matrix metalloprotease inhibitor and a matrix protein production enhancer. Yes (Patent Document 1). It is also known that substances that inhibit serine protease and substances that increase the production of type IV, VII collagen or laminin 5, which are the main constituents of the epidermal basement membrane component, promote basement membrane formation by the matrix metalloproteinase inhibitor. (Patent Document 2). It is also known that the formation of higher-order structures of the epidermal basement membrane and the dermis is promoted by using a medium to which a matrix metalloproteinase inhibitor and a heparanase inhibitor are added (Patent Document 3). However, the artificial skin produced by these methods has a problem that the gel shrinks and the quality obtained varies widely.

Further, as another method, there is also known a method in which an extracellular matrix is formed in pre-sown fibroblasts and keratinocytes are seeded on the extracellular matrix to prepare artificial skin (Non-Patent Document 1). However, this method has problems such as it takes a long time to prepare and the cell density of the dermis of the obtained artificial skin is too high. Further, the artificial skin produced by this method also has a large variation in quality and cannot be said to be sufficient.

It is known that the barrier function of the human epidermis equivalent is improved by culturing the human epidermis equivalent under low humidity conditions (Non-Patent Document 2), but the method is applied to the human epidermis equivalent and is naturally occurring. No artificial skin has been obtained that mimics the skin structure of.

Japanese Unexamined Patent Publication No. 2001-269398 Japanese Unexamined Patent Publication No. 2004-75661 Japanese Patent No. 5744409

El Ghalbzouri A. , Et al. , Replacement of animal-developed collagen matrix by human fibroblast-derivated demomal matrix for human skin equivalent products. Biomaterials. 2009 Jan; 30 (1): 71-78 Sun R. , Et al. , Lowered humidity products human epidermal equivalents with enhanced barrier properties. Tissue Eng Part C Methods. 2015 Jan; 21 (1): 15-22

An object of the present invention is to provide a method for stably producing three-dimensional cultured skin having a structure similar to that of natural skin.

In order to solve the above problems, the present inventors have conducted research and development by examining from various angles. As a result, after seeding and culturing fibroblasts, a hydrogel layer containing fibroblasts is formed, and keratinocytes are further seeded to form a keratinocyte layer, thereby forming a three-dimensional structure similar to natural skin. It has been found that cultured skin can be stably produced. That is, the present invention includes the following inventions.

[1] A method for producing three-dimensional cultured skin.
(1) A step of seeding fibroblasts on the porous membrane of a cell culture vessel having a porous membrane to form a fibroblast layer, filling the inside and outside of the cell culture vessel with a medium, and culturing. ;
(2) A solution containing fibroblasts and a hydrogelling agent is poured onto the fibroblast layer to form a hydrogel layer containing fibroblasts, and a medium is formed inside and outside the cell culture vessel. The process of filling and culturing;
(3) A step of seeding keratinocytes on the hydrogel layer to form a keratinite layer, filling the inside and outside of the cell culture vessel with a medium, and culturing the cells; and (4) on the keratinocytes layer. A step of removing the medium and bringing the medium into contact with the outer surface of the porous membrane of the cell culture vessel while exposing the keratinocyte layer to the gas phase for culturing.
Including methods.
[2] The method according to [1], wherein in the step (4), the medium contains a matrix metalloproteinase inhibitor.
[3] The method according to [1] or [2], wherein the medium contains a heparanase inhibitor in the step (4).
[4] The method according to any one of [1] to [3], wherein the gas phase has a relative humidity of 45% to 75% in the step (4).
[5] The method according to [4], wherein the cell culture container has means for preventing water vapor derived from a medium filled on the outside of the cell culture container from being mixed into the inside of the cell culture container.
[6] The method according to any one of [1] to [5], wherein the fibroblast is a dermal fibroblast.
[7] The hydrogelling agent is collagen, gelatin, hyaluronate, hyaluronan, fibrillin, arginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen / thrombin, fibrillin, elastin, gum, cellulose, agar. , Gluten, Casein, Albumin, Vitronectin, Tenesin, Entactin / Nidogen, Glycoprotein, Glycosaminoglycan, Poly (acrylic acid) and its derivatives, Poly (ethylene oxide) and its copolymers, Poly (Vinyl alcohol), Polyphosphazene The method according to any one of [1] to [6], which is selected from the group consisting of Matrigel and combinations thereof.

[8] A three-dimensional cultured skin obtained by the method according to any one of [1] to [7].

According to the present invention, it is possible to stably provide three-dimensional cultured skin having a structure closer to that of natural skin than the three-dimensional cultured skin obtained by a conventional method.

FIG. 1 is a schematic view showing a method for producing a three-dimensional cultured skin of the present invention in one embodiment. FIG. 2 shows a three-dimensional cultured skin obtained by a conventional method (comparative example). (A) Shrinked three-dimensional cultured skin in a comparative example taken from above. (B) HE-stained image of three-dimensional cultured skin in a comparative example. FIG. 3 shows the three-dimensional cultured skin in one embodiment obtained by the method of the present invention. (A) Three-dimensional cultured skin obtained by the method of the present invention, taken from above. (B) HE-stained image of three-dimensional cultured skin (14th day of culture) obtained by the method of the present invention. (C) HE-stained image of three-dimensional cultured skin (21st day of culture) obtained by the method of the present invention. (D) Overall image of HE staining of three-dimensional cultured skin (14th day of culture) obtained by the method of the present invention. FIG. 4 shows a fluorescence-stained image of three-dimensional cultured skin obtained by the method of the present invention and the conventional method. Upper: Three-dimensional cultured skin obtained by the present invention, Lower: Three-dimensional cultured skin obtained by a conventional method. Left column: Anti-Type VII collagen antibody (green), anti-Ki67 antibody (red), Hoechst33342 (nucleus) (blue). Middle row: anti-TGase-1 antibody (green), anti-Involurin antibody (red), Hoechst33342 (nucleus) (blue). Right column: anti-Laminin332 antibody (green), anti-Corneodesmosin antibody (red), Hoechst33342 (nucleus) (blue). FIG. 5 is a cross-sectional view of a culture vessel used in the method for producing a three-dimensional cultured skin of the present invention in one embodiment. FIG. 6 shows an HE-stained image of three-dimensional cultured skin cultured at 100% relative humidity (RH) or 50% RH. (A) 100% RH, cultured for 12 days. (B) Incubate at 100% RH for 14 days. (C) Incubate at 50% RH for 5 days. (D) Incubate at 50% RH for 7 days. FIG. 7 shows a fluorescence-stained image of three-dimensional cultured skin cultured at 100% RH or 50% RH. (A, C, E, G) 3D cultured skin cultured at 100% RH, (B, D, F, H) 3D cultured skin cultured at 50% RH. (A, B) Green: anti-Filaggrin antibody, red: anti-BH antibody, blue: Hoechst33342 (nucleus). (C, D) Green: anti-TGase-1 antibody, red: anti-Involurin antibody, blue: Hoechst33342 (nucleus). (E, F) Red: anti-Corneodesmosin antibody, blue: Hoechst33342 (nucleus). (G, H) Red: anti-Ki67 antibody, blue: Hoechst33342 (nucleus). FIG. 8 is an electron micrograph of three-dimensional cultured skin cultured at 100% RH or 50% RH, and human skin. SC: stratum granulosum, SG-SP: stratum granulosum-stratum spinosum, SB-derm: basal layer-dermis. FIG. 9 shows a fluorescence-stained image of three-dimensional cultured skin cultured at 100% RH or 50% RH. (A, C, E) 3D cultured skin cultured at 100% RH, (B, D, F) 3D cultured skin cultured at 50% RH. (A, B) Green: anti-Laminin332 antibody, blue: Hoechst33342 (nucleus). (C, D) Green: Anti-Type VII collagen antibody, Blue: Hoechst 33342 (nucleus). (E, F) Green: anti-Fibrillin-1 antibody, blue: Hoechst33342 (nucleus). FIG. 10 is an electron micrograph of three-dimensional cultured skin cultured at 100% RH or 50% RH, and near the basement membrane of human skin. Arrowhead: Hemidesmosome, LD: Laminadensa, CF: Collagen fiber. FIG. 11 shows an HE-stained image of three-dimensional cultured skin cultured at 100% RH or 70% RH. (A) 100% RH, cultured for 14 days. (B) 70% RH, 14 days culture. FIG. 12 shows an HE-stained image of three-dimensional cultured skin cultured at 100% RH or 40% RH. (A) 100% RH, cultured for 14 days. (B) 40% RH, culture for 14 days.

Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings, but the technical scope of the present invention is not limited to the following embodiments. As used herein, a value with "about" means that the value also includes values in the range ± 20%, more preferably 10%.

<Manufacturing method of 3D cultured skin>
In one embodiment, the present invention is a method for producing three-dimensional cultured skin.
(1) A step of seeding fibroblasts on the porous membrane of a cell culture vessel having a porous membrane to form a fibroblast layer, filling the inside and outside of the cell culture vessel with a medium, and culturing. ;
(2) A solution containing fibroblasts and a hydrogelling agent is poured onto the fibroblast layer to form a hydrogel layer containing fibroblasts, and a medium is formed inside and outside the cell culture vessel. The process of filling and culturing;
(3) A step of seeding keratinocytes on the hydrogel layer to form a keratinite layer, filling the inside and outside of the cell culture vessel with a medium, and culturing the cells; and (4) on the keratinocytes layer. A step of removing the medium and bringing the medium into contact with the outer surface of the porous membrane of the cell culture vessel while exposing the keratinocyte layer to the gas phase for culturing.
Provide a method including.

The three-dimensional cultured skin obtained by a conventional method for producing three-dimensional cultured skin (for example, a method of culturing normal human keratinocytes on a collagen gel containing human fibroblasts to form an epidermal layer) can be obtained during culturing. The collagen gel contracted and became smaller, and for example, the amount of water evaporation (TEWL), which is an index of the barrier function, could not be measured (see, for example, FIG. 2 (A)). In addition, the degree of contraction of the obtained three-dimensional cultured skin was different between the samples, and the quality, for example, the thickness of the obtained three-dimensional cultured skin varied widely (see, for example, FIG. 2B).

However, according to the method of the present invention, it is possible to produce a three-dimensional cultured skin in which the shrinkage of the obtained three-dimensional cultured skin is suppressed, the thickness is substantially uniform, and the quality is less variable. In addition, it becomes possible to stably provide three-dimensional cultured skin having a structure similar to that of natural skin.

FIG. 1 shows a method for producing a three-dimensional cultured skin of the present invention in one embodiment. Fibroblasts are seeded on the porous membrane of a cell culture vessel having a porous membrane (for example, a cell culture insert) to form a fibroblast layer, and the inside and outside of the cell culture vessel are filled with a medium. , Culturing (step (1)). The cell culture insert used in one embodiment of the present invention refers to a cell culture vessel provided with a membrane having porous pores through which cells cannot permeate but a medium or the like can permeate. The medium or the like can also be supplied from the opposite side of the culture surface of the porous membrane, that is, the back side of the attachment surface of the adherent cells. As the cell culture container used in the present invention, for example, a cell culture insert, a commercially available one may be used.

As the medium used in the present invention, any medium conventionally used for producing three-dimensional cultured skin can be used, for example, Dalveco Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum; 10%. DMEM-Ham's F12 (3: 1) containing fetal bovine serum, transferase 5 μg / ml, insulin 5 μg / ml, tri-iodothyronine 2 nM, choleratoxin 0.1 nM, hydrocortisone 0.4 μg / ml; keratinocyte growth medium ( A medium in which KGM) and DMEM containing 10% fetal bovine serum are mixed 1: 1 can be used, but the medium is not limited thereto.

The number of fibroblasts seeded in step (1) is, for example, 0.01 × 10 ^{6 to} 10.0 × 10 ⁶ cells / cm ² , preferably 0.05 × 10 ^{6 to} 5.0 × 10. Seed in an amount of ⁶ pcs / cm ² , more preferably 0.1 × 10 ^{6 to} 1.0 × 10 ⁶ pcs / cm ² . The cells seeded in step (1) include fibroblasts and other cells contained in the dermis, such as mast cells, Meissner bodies, tissue spheres, nerve cells, dendritic cells, vascular endothelial cells and plasma cells. It may contain one or more cells selected from the group consisting of.

The culture period of step (1) is preferably a period in which fibroblasts become confluent or subconfluent and produce extracellular matrix, for example, 1 to 14 days, preferably 3 to 10 days, more preferably. Is 5 to 9 days, for example about 7 days. As a result, the proliferated fibroblasts can cover the culture surface and produce an extracellular matrix.

After the step (1), a solution containing fibroblasts and a hydrogelling agent is poured onto the fibroblast layer to form a hydrogel layer containing fibroblasts, and the inside of the cell culture vessel. And the outside is filled with a medium and cultured (step (2)). Hydrogelling agents that can be used in the present invention include, for example, collagen, gelatin, hyaluronate, hyaluronan, fibrillin, arginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen / thrombin, fibrillin, elastin, etc. Gum, Cellulose, Agar, Gluten, Casein, Albumin, Vitronectin, Tenesin, Entactin / Nidgen, Glycoprotein, Glycosaminoglycan, Poly (acrylic acid) and its derivatives, Poly (ethylene oxide) and its copolymers, Poly (Vinyl) It can be selected from the group consisting of alcohol), polyphosphazene, matrigel and combinations thereof, preferably collagen.

The solution containing the fibroblasts and the hydrogelling agent in step (2) is, for example, 0.01 × 10 ^{6 to} 10.0 × 10 ⁶ pieces / mL, preferably 0.05 × 10 ^{6 to} 5.0. It contains fibroblasts having a density of × 10 ⁶ cells / mL, more preferably 0.05 × 10 ^{6 to} 1.0 × 10 ⁶ cells / mL. The solution is poured onto the fibroblast layer obtained in step (1) to form a hydrogel layer. The thickness of the hydrogel layer formed in the step (2) may be adjusted to, for example, 1 mm to 10 mm in consideration of vertical shrinkage in the subsequent culturing stage. The cells seeded in step (2) include fibroblasts and other cells contained in the dermis, such as mast cells, Meissner bodies, tissue spheres, nerve cells, dendritic cells, vascular endothelial cells and plasma cells. It may contain one or more cells selected from the group consisting of. The fibroblasts used in steps (1) and (2) are preferably dermal fibroblasts.

The culture period of step (2) is, for example, 1 to 7 days, preferably 2 to 5 days, more preferably 2 to 4 days, for example, about 3 days.

After the step (2), keratinocytes (epidermal keratinocytes) are seeded on the hydrogel layer to form a keratinocyte layer, and the inside and outside of the cell culture vessel are filled with a medium and cultured (step). (3)). The number of keratinocytes seeded in step (3) is, for example, 0.01 × 10 ^{6 to} 10.0 × 10 ⁶ cells / cm ² , preferably 0.05 × 10 ^{6 to} 5.0 × 10 ⁶ cells. Seed in an amount of / cm ² , more preferably 0.1 × 10 ^{6 to} 1.0 × 10 ⁶ pieces / cm ² . In addition to keratinocytes, the cells seeded in step (3) may include other cells contained in the epidermis, for example, one or more cells selected from the group consisting of melanocytes, Langerhans cells, and Merkel cells.

Step (3) is preferably cultured for a period in which keratinocytes become confluent or subconfluent, for example, 1 to 7 days, preferably 2 to 5 days, more preferably 2 to 4 days, for example, about 3 days. Is.

The cell used in the present invention may be a primary cell, a cell obtained by subculturing the primary cell and proliferating, and from pluripotent stem cells such as ES cells, iPS cells, or Muse cells. A cell obtained by inducing differentiation may be used, or a cell that has been established may be used. The cells used may be of any animal origin, but are preferably of vertebrate origin, more preferably of mammalian origin, and most preferably of human origin.

After the step (3), the medium on the keratinocytes layer is removed, and the medium is brought into contact with the outer surface of the porous membrane of the cell culture vessel while exposing the keratinocytes layer to the gas phase, and the cells are cultured (step). (4)). This promotes the three-dimensionalization and keratinization of keratinocytes, resulting in thicker three-dimensional cultured skin. The step (4) may be carried out by culturing for a period in which the three-dimensionalization and keratinization of keratinocytes are promoted, for example, 1 to 28 days, preferably 3 to 21 days, more preferably 7 to 21 days, for example. It may be 14 days.

In one embodiment, the medium used in step (4) may contain a matrix metalloproteinase inhibitor (MMP inhibitor). The medium contains a matrix metalloproteinase inhibitor at a concentration sufficient to promote regeneration and repair of the basement membrane and dermis of the skin, typically 0.000000001 to 10% by weight based on the medium, preferably. It is contained in an amount of 0.000001 to 10% by weight.

The matrix metalloproteinase inhibitor used in the present invention is not particularly limited as long as it is a substance having an inhibitory activity against the matrix metalloproteinase. Examples of the matrix metalloprotease include gelatinase, collagenase, stromelysin, matrylicin and the like. Therefore, the matrix metalloproteinase inhibitor can be selected as a substance that inhibits, for example, gelatinase, collagenase, stromelysin, matrylicin and the like.

Specific examples of the matrix metalloproteinase inhibitor include CGS27023A substance (N-hydroxy-2-[[(4-methoxyphenyl) sulfonyl] 3-picoryl) amino] -3-methylbutaneamide hydrochloride) (J. Med. Chem. 1997, Vol. 40, p. 2525-2532), MMP-Inhibitor (p-NH2-Bz-Gly-Pro-D-Leu-Ala-NHOH) (FN-437) (BBRC, 1994, Vol. 199) , P.1442-1446) and the like. Preferably, the matrix metalloproteinase inhibitor is a CGS27023A substance.

In one embodiment, the medium used in step (4) may contain a heparanase inhibitor. The medium contains a heparanase inhibitor at a concentration sufficient to promote regeneration and repair of the basement membrane and dermis of the skin, typically 0.000000001 to 10% by weight based on the medium, preferably 0. It is contained in 0.0001 to 10% by weight.

The heparanase inhibitor used in the present invention may be any substance having an inhibitory activity against heparanase, and is not particularly limited. Heparanase is an enzyme that exists in various cells and specifically decomposes the heparan sulfate chain of various heparan sulfate proteoglycans. In the skin, epidermal keratinocytes constituting the epidermis, fibroblasts of the dermis, vascular endothelial cells and the like are produced.

Specific examples of the heparanase inhibitor include 1- [4- (1H-benzimidazol-2-yl) -phenyl] -3- [4- (1H-benzimidazol-2-yl) -phenyl] -urea (" (Also referred to as "BITBIPU") and SF-4 (also referred to as "Hepalastatin hydrochloride").

In one embodiment, step (4) may be performed, for example, in a gas phase with a relative humidity of 45% to 75% (45% RH to 75% RH). By performing step (4) in a gas phase of 45% RH to 75% RH, the thickness of the stratum corneum is further increased to that of human skin as compared with the conventional three-dimensional cultured skin cultured at 100% RH. A dense, three-dimensional cultured skin that is close to the thickness is obtained (see, for example, the arrow in FIG. 8). Further, by carrying out the step (4) in the gas phase of 45% RH to 75% RH, not only the structure of the epidermis layer but also the basement membrane formed between the epidermis layer and the hydrogel layer becomes a dense structure. , Laminadensa is also thickened, expression of laminin 332 and type VII collagen is also increased, basement membrane reconstruction is promoted (see FIGS. 9 to 10), and three-dimensional cultured skin closer to the natural skin structure than before is obtained. Be done.

The temperature of the step (4) may be typically a temperature at which cell culture is performed, and is, for example, 20 ° C to 42 ° C, preferably 30 ° C to 39 ° C, for example, about 37 ° C.

The relative humidity of the gas phase can be adjusted, for example, by using an incubator that can control the relative humidity of the gas phase. For example, step (4) may be carried out by placing the cell culture vessel in an incubator controlled in a gas phase of 45% RH to 75% RH. In this case, it is preferable to use a cell culture container having a means for preventing the water vapor derived from the medium filled on the outside of the cell culture container from being mixed inside the cell culture container. As an example of the cell culture vessel having such a means, for example, a cell culture vessel having the structure shown in FIG. 5 can be used. Briefly, the lid of the cell culture vessel is provided with a hole having a diameter substantially equal to or larger than the inner diameter of the cell culture insert, and the hole is in contact with the cell culture insert and is outside the cell culture vessel. A glass ring or the like is provided to prevent the water vapor derived from the medium filled in the cell culture vessel from being mixed into the inside of the cell culture container. This makes it possible to strictly control the gas phase on the stratum corneum side of the three-dimensional cultured skin.

The three-dimensional cultured skin obtained by the method of the present invention as described above can be used as one of alternative methods for animal experiments, for example, as a skin model. For example, it can be used as a method for evaluating the reactivity of skin to chemical substances (for example, cosmetics, industrial products, household products, drugs, external preparations for skin, etc.). In addition, the three-dimensional cultured skin obtained by the method of the present invention can obtain a tissue having an enhanced barrier function as compared with the conventional three-dimensional cultured skin, and is therefore used as a useful skin model in basic research of dermatology. It is possible. Furthermore, since the three-dimensional cultured skin obtained in the present invention has a structure closer to that of human skin as compared with the conventional three-dimensional cultured skin, it has a high barrier function from the outside and heals burns, wounds, etc. It is also useful as a three-dimensional cultured skin.

<Method of evaluating a target substance that improves and / or restores the barrier function of the skin using three-dimensional cultured skin>
In one embodiment, it is possible to provide a method for evaluating a target substance that improves and / or restores the barrier function of the skin by adding the target substance to the three-dimensional cultured skin of the present invention.

For example, after adding the target substance to the three-dimensional cultured skin of the present invention, the change in the barrier function of the skin can be evaluated by measuring the amount of water evaporation that evaporates from the surface of the three-dimensional cultured skin. In addition, after adding the target substance to the three-dimensional cultured skin of the present invention, the expression of known markers related to the barrier function of the skin (for example, Filaggrin, Lolicrin, ZO-1, Claudin-1, etc.) is examined for the skin. Changes in barrier function can be evaluated.

In this embodiment, the target substances for improving and / or restoring the barrier function of the skin include, for example, low molecular weight compounds, peptides, proteins, mammals (for example, mice, rats, pigs, cows, sheep, monkeys, humans and the like). ) Tissue extract or cell culture supernatant, plant-derived compound or extract (for example, crude drug extract, crude drug-derived compound), and microorganism-derived compound or extract or culture product.

Hereinafter, the present invention will be described in more detail based on examples, but these are not intended to limit the present invention in any way.

<Example 1>
1-1. Three-dimensional culture method for manufacturing a cell culture insert of the skin (12mm, an average pore diameter of the porous membrane: 0.4 .mu.m) in seeded human dermal fibroblasts (0.2 × 10 ⁶ cells), 200 [mu] M ascorbic acid 2-phosphate The medium was changed once every two days using magnesium (APM) and 10% FBS-DMEM, and the cells were cultured for one week. A 0.5% type I collagen-10% FBS-DMEM solution containing human dermal fibroblasts was dispensed thereto to prepare a collagen gel on the human dermal fibroblasts, which was cultured for 3 days.

Furthermore, epidermal keratinocytes dispersed in Humania-KG2 (Kurabou) medium were seeded on a collagen gel so as to be 5 × 10 ⁵ cells / well, and Humandia-KG2 and 10% FBS-DMEM were placed outside the insert. Medium mixed at 1: 1 and added with 200 μM APM was added to the same liquid level as the inside and cultured for 3 days.

Then, the medium in the insert or the glass ring is removed, and 200 μM in the medium for skin model (10% FBS-DMEM and Human-KG2 EGF (-) are mixed 1: 1 to prepare Ca 1.8 mM) on the outside. APM, 10 μM N-hydroxy-2-[[(4-methoxyphenyl) sulfonyl] 3-picoryl) amino] -3-methylbutaneamide hydrochloride (CGS27023A (MMP inhibitor)), 10 μM BIPBIPU (heparanase inhibitor) The mixture was added up to the height of the bottom surface of the insert, and gas-liquid boundary culture was performed with the inside of the insert exposed to air. The medium was changed once every 2 to 3 days and the cells were cultured for 2 weeks.

1-2. Results In the three-dimensional cultured skin obtained by the conventional method (a method of culturing keratinocytes on a collagen gel containing fibroblasts to form an epidermal layer), the collagen gel contracts during the culture and peels off from the culture surface. However, the thickness and the structure of the epidermis and dermis were uneven (Fig. 2). On the other hand, the three-dimensional cultured skin obtained by the method of the present invention has suppressed shrinkage even on the 14th and 21st days of culture, and the thickness and the structure of the epidermis and dermis are almost uniform throughout the three-dimensional cultured skin. There was (Fig. 3). In addition, the deposition of basement membrane components such as type VII collagen and laminin 332 is better than that of the three-dimensional cultured skin obtained by the conventional method, and maintenance of proliferative (Ki67-positive) cells in the epidermis and expression of differentiation markers such as transglutaminase Was also good. (Fig. 4).

<Example 2>
2-1. Low-humidity culture method The three-dimensional cultured skin prepared in Example 1 was subjected to gas-liquid boundary culture for about one week, and then placed in an incubator controlled to an atmosphere of 37 ° C. and 50% RH or 37 ° C. and 100% RH. Culture was performed. Humidity in the incubator was controlled using a humidity control device (KITZ Microfilter, AHCU-2). The plate containing the three-dimensional cultured skin had a hole in the lid and a glass ring was fitted so that only the surface of the three-dimensional cultured skin was in direct contact with the outside air (see FIG. 5).

2-2. Results The 3D cultured skin cultured at 50% RH has a thinner keratinocyte layer and dermis layer (collagen gel layer) than the 3D cultured skin cultured at 100% RH, and the morphology of epidermal cells is overall. It had a flatter shape (Fig. 6).

In addition, epidermal differentiation markers (filaggrin, bleomycin hydrolytic enzyme (BH), transglutaminase-1 (TGase-1), involucrin, Corneodesmosin), and Ki67, which is a cell proliferation marker, As a result of examining the expression by immunostaining, the epidermal differentiation markers were densely stained in the flat granular layer in the three-dimensional cultured skin cultured at 50% RH as compared with the three-dimensional cultured skin cultured at 100% RH. It was (Fig. 7). The expression of Ki67, which is a cell proliferation marker, was slightly reduced in the three-dimensional cultured skin cultured at 50% RH, but the expression was maintained (Fig. 7).

When the morphology of the epidermal layers of both was further examined using an electron microscope, the stratum corneum was thicker and swollen than normal human skin in the three-dimensional cultured skin cultured at 100% RH, and the cell gap was widened. In contrast to the morphology, the three-dimensional cultured skin cultured at 50% RH lost its characteristics, and it became clear that it had an epidermal structure closer to that of natural skin (see the arrow in FIG. 8). ).

In addition, the extracellular matrix of the basement membrane and dermis was examined. It was clarified that laminin 332 and type VII collagen were accumulated and strongly expressed in the vicinity of the basement membrane in the three-dimensional cultured skin cultured at 50% RH (Fig. 9). In addition, when the morphology near the basement membrane was examined using an electron microscope, the laminadenser was thicker and the extracellular matrix was denser in the three-dimensional cultured skin cultured at 50% RH. It was revealed that it has a structure closer to that of natural skin (Fig. 10).

<Example 3>
3-1. Low-humidity culture method When the three-dimensional cultured skin prepared in Example 1 was subjected to gas-liquid boundary culture for about one week, the atmosphere was changed to 37 ° C.40% RH, 37 ° C./70% RH, or 37 ° C./100% RH. Culturing was performed in a controlled incubator. Humidity in the incubator was controlled using a humidity control device (KITZ Microfilter, AHCU-2). The plate containing the three-dimensional cultured skin had a hole in the lid and a glass ring was fitted so that only the surface of the three-dimensional cultured skin was in direct contact with the outside air (see FIG. 5).

3-2. Results Three-dimensional cultured skin cultured at 70% RH has a thinner keratinocyte layer and dermis layer (collagen gel layer) and a flatter overall epidermal cell morphology than three-dimensional cultured skin cultured at 100% RH. It had a similar shape and showed the same characteristics as the above-mentioned three-dimensional cultured skin cultured at 50% RH (Fig. 11). On the other hand, the three-dimensional cultured skin cultured at 40% RH had almost no epidermal cell layer and dermis layer, and did not maintain its shape as a cultured skin (FIG. 12).

From the above results, it is clear that culturing in a low humidity environment affects the reconstruction of the epidermis layer, dermis layer and basement membrane, and provides three-dimensional cultured skin with a structure similar to that of natural skin. became.

Claims (6)

A method for producing three-dimensional cultured skin.
(1) A step of seeding fibroblasts on the porous membrane of a cell culture vessel having a porous membrane to form a fibroblast layer, filling the inside and outside of the cell culture vessel with a medium, and culturing. ;
(2) A solution containing fibroblasts and a hydrogelling agent is poured onto the fibroblast layer to form a hydrogel layer containing fibroblasts, and a medium is formed inside and outside the cell culture vessel. The process of filling and culturing;
(3) A step of seeding keratinocytes on the hydrogel layer to form a keratinite layer, filling the inside and outside of the cell culture vessel with a medium, and culturing the cells; and (4) on the keratinocytes layer. A step of removing the medium and bringing the medium into contact with the outer surface of the porous membrane of the cell culture vessel while exposing the keratinocyte layer to the gas phase for culturing.
Including methods. The method of claim 1, wherein in step (4), the medium comprises a matrix metalloproteinase inhibitor. The method according to claim 1 or 2, wherein in the step (4), the medium contains a heparanase inhibitor. The method according to any one of claims 1 to 3, wherein the fibroblast is a dermal fibroblast. The hydrogelling agents include collagen, gelatin, hyalronate, hyaluronan, fibrin, arginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen / thrombin, fibrillin, elastin, gum, cellulose, agar, gluten, Casein, albumin, bitronectin, tenesin, entactin / nidogen, glycoprotein, glycosaminoglycan, poly (acrylic acid) and its derivatives, poly (ethylene oxide) and its copolymers, poly (vinyl alcohol), polyphosphazene, matrigel and The method according to any one of claims 1 to 4, which is selected from the group consisting of a combination thereof. A three-dimensional cultured skin obtained by the method according to any one of claims 1 to 5.