JP7412096B2 - Method for producing skin-like tissue and skin-like tissue obtained thereby - Google Patents

Description

The present invention relates to a method for producing skin-like tissue and the skin-like tissue obtained thereby.

The skin is an organ that covers the body surface and separates the internal and external environments. The skin acts as a physical barrier, protecting the body from dryness and harmful substances entering the body, and plays an essential role in sustaining life.

Natural skin is roughly divided into two layers: the epidermis and the dermis, and between the epidermis and the dermis there is a thin and delicate membrane called the epidermal basement membrane. The epidermal basement membrane is a very thin structure of approximately 0.1 μm, and exists in the form of a sheet at the junction between the epidermis and the dermis. The epidermal basement membrane is composed of basic structures such as lamina densa and lamina densa, as well as keratinocyte hemidesmosomes, anchoring filaments, and anchoring fibers. It is composed of collagen, various laminins, proteoglycans, etc. The main component of the anchoring filaments that connect the epidermal basal cells and basic structures, especially the laminadensa, is laminin-5, and the basic structures and collagen fibers in the dermis are connected by anchoring fibers whose main component is type VII collagen. ing. Anchoring filaments and anchoring fibers can also bind to each other, forming a complex called an anchoring complex. Due to this structure, the skin, which is the outermost layer of the living body, has the strength to withstand mechanical stress from the outside world.

There is an increasing demand for artificial skin to be used as a substitute when natural skin (ie, natural skin) is damaged for some reason. Additionally, there is a growing demand for artificial skin as an experimental material for testing the effects of pharmaceuticals and cosmetics on the skin, as well as for testing drugs. For both applications, there is a strong desire to develop artificial skin that mimics the structure of natural skin as much as possible.

Various methods for producing artificial skin (three-dimensional cultured skin) that imitate skin structure have been developed, and some have already been put into practical use. For example, a method is known in which normal human epidermal keratinocytes are cultured on type I collagen gel containing human fibroblasts to form an epidermal layer. It is known that a basement membrane is not sufficiently formed between the gel and the epidermal layer that mimics the epidermis. Therefore, when using such artificial skin, it is possible to promote the re-formation of the skin basement membrane structure by administering a matrix metalloprotease inhibitor, or both a matrix metalloprotease inhibitor and a matrix protein production enhancer. It is possible (Patent Document 1). It is also known that substances that inhibit serine protease and substances that increase the production of type IV and type VII collagen or laminin 5, which are the main constituents of epidermal basement membrane components, promote basement membrane formation caused by matrix protease inhibitors. (Patent Document 2). It is also known that the formation of higher-order structures in the epidermal basement membrane and dermis can be promoted by using a medium containing a matrix metalloprotease inhibitor and a heparanase inhibitor (Patent Document 3). However, the artificial skin produced by these methods has wider gaps in the stratum corneum and lower density than natural skin, and cannot be said to imitate the structure of natural skin.

Another method is known in which artificial skin is produced by forming an extracellular matrix using pre-seeded fibroblasts and then seeding keratinocytes thereon (Non-Patent Document 1). However, this method has problems such as it takes a long time to produce and the cell density of the dermis of the obtained artificial skin is too high. Furthermore, the quality of the artificial skin produced by this method varied greatly, and it could not be said to be satisfactory.

It is known that the barrier function of human epidermal equivalents is improved by culturing them under low humidity conditions (Non-Patent Document 2); Artificial skin that mimics the skin structure of humans has not been obtained.

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

El Ghalbzouri A. , et al. , Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent product cts. Biomaterials. 2009 Jan; 30 (1): 71-78 Sun R. , et al. , Lowered humidity produces 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 producing skin-like tissue and the skin-like tissue obtained thereby.

In order to solve the above problems, the present inventors have conducted research and development by considering from various angles. As a result, the surface of the stratum corneum side of the skin-like tissue containing the epidermal layer or epidermal layer equivalent and the dermis layer or dermal layer equivalent placed on the porous membrane of the cell culture container having a porous membrane is heated to relative humidity. Skin-like tissue having a structure similar to natural skin is grown by contacting the outer surface of the porous membrane of the cell culture container with a medium and performing gas phase culture while being exposed to a gas phase of 45% to 75%. It has been found that it can be produced stably. That is, the present invention includes the following inventions.

[1] A method for producing skin-like tissue, comprising:
The surface of the stratum corneum side of the skin-like tissue including the epidermis layer and the dermis layer placed on the porous membrane of a cell culture container having a porous membrane is exposed to a gas phase with a relative humidity of 45% to 75%. while bringing a medium into contact with the outer surface of the porous membrane of the cell culture container to perform gas phase culture;
including methods.
[2] The method according to [1], wherein the cell culture container has a means for preventing water vapor derived from a medium filled on the outside of the cell culture container from entering the inside of the cell culture container.
[3] The method according to [1] or [2], wherein the skin-like tissue is isolated skin tissue or three-dimensionally cultured skin.
[4] The skin-like tissue is
(1) A step of seeding fibroblasts on the porous membrane of a cell culture container having a porous membrane to form a fibroblast layer, filling the inside and outside of the cell culture container 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 placed inside and outside the cell culture container. and (3) seeding keratinocytes on the hydrogel layer to form a keratinocyte layer, filling the inside and outside of the cell culture container with a medium, and culturing.
The method according to [1] or [2], wherein the three-dimensional cultured skin is produced by a step comprising:
[5] The hydrogelling agent is collagen, gelatin, hyaluronate, hyaluronan, fibrin, alginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen/thrombin, fibrillin, elastin, gum, cellulose, agar. , gluten, casein, albumin, vitronectin, tenascin, entactin/nidogen, glycoprotein, glycosaminoglycan, poly(acrylic acid) and its derivatives, poly(ethylene oxide) and its copolymers, poly(vinyl alcohol), polyphosphazene , Matrigel, and combinations thereof.
[6] The method according to any one of [1] to [5], wherein in the gas phase culturing step, the medium contains a matrix metalloprotease inhibitor.
[7] The method according to any one of [1] to [6], wherein in the gas phase culturing step, the medium contains a heparanase inhibitor.

[8] A skin-like tissue obtained by the method according to any one of [1] to [7].

According to the present invention, it is possible to stably provide skin-like tissue that has a structure closer to natural skin than skin-like tissue obtained by conventional methods.

FIG. 1 is a cross-sectional view of a culture vessel used in the method for producing skin-like tissue of the present invention in one embodiment. FIG. 2 is a schematic diagram showing a method for producing skin-like tissue (three-dimensional cultured skin) of the present invention in one embodiment. FIG. 3 shows HE staining images of three-dimensional cultured skin cultured at 100% relative humidity (RH) or 50% RH. (A) Cultured at 100% RH for 12 days. (B) Cultured for 14 days at 100% RH. (C) Cultured at 50% RH for 5 days. (D) Cultured at 50% RH for 7 days. FIG. 4 shows fluorescent staining images of three-dimensional cultured skin cultured at 100% RH or 50% RH. (A, C, E, G) Three-dimensional cultured skin cultured at 100% RH, (B, D, F, H) Three-dimensional cultured skin cultured at 50% RH. (A, B) Green: anti-Filagrin antibody, red: anti-BH antibody, blue: Hoechst 33342 (nucleus). (C, D) Green: anti-TGase-1 antibody, red: anti-Involurin antibody, blue: Hoechst 33342 (nucleus). (E, F) Red: anti-Corneodesmosin antibody, blue: Hoechst 33342 (nuclei). (G,H) Red: anti-Ki67 antibody, blue: Hoechst33342 (nucleus). FIG. 5 is an electron micrograph of three-dimensional cultured skin and human skin cultured at 100% RH or 50% RH. SC: stratum corneum, SG to SP: stratum granulosum to stratum spinosum, SB to derm: stratum basale to dermis. FIG. 6 shows fluorescent staining images of three-dimensional cultured skin cultured at 100% RH or 50% RH. (A, C, E) Three-dimensional cultured skin cultured at 100% RH, (B, D, F) Three-dimensional 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 (nuclei). FIG. 7 is an electron micrograph of three-dimensional cultured skin cultured at 100% RH or 50% RH, and the vicinity of the basement membrane of human skin. Arrowhead: hemidesmosome, LD: laminadensa, CF: collagen fiber. FIG. 8 shows HE staining images of three-dimensional cultured skin cultured at 100% RH or 70% RH. (A) Cultured at 100% RH for 14 days. (B) Cultured at 70% RH for 14 days. FIG. 9 shows HE staining images of three-dimensional cultured skin cultured at 100% RH or 40% RH. (A) Cultured at 100% RH for 14 days. (B) Cultured at 40% RH 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 only to the following embodiments. As used herein, a value with "about" is meant to include a range of ±20% of that value, more preferably 10%.

<Method for manufacturing skin-like tissue>
In one embodiment, the present invention provides a method for producing skin-like tissue, comprising:
The surface of the stratum corneum side of the skin-like tissue containing the epidermal layer and the dermal layer placed on the porous membrane of the cell culture container having the porous membrane is heated to a relative humidity of 45% to 75% (for example, a relative humidity of about 50%). %) of the cell culture container while exposing the porous membrane to a gas phase, the method includes the step of bringing a medium into contact with the outer surface of the porous membrane of the cell culture container to carry out gas phase culture.

Conventional skin-like tissue cultured in an environment of 100% relative humidity has wider gaps in the stratum corneum and lower density than natural skin, and cannot be called artificial skin that imitates the structure of natural skin. (See, for example, the electron micrograph of the 100% RH column in Figure 7).

However, with the method of the present invention, the thickness of the stratum corneum is closer to that of natural skin and the density of the skin is higher than that of skin-like tissue produced under a conventional 100% RH atmosphere. A similar tissue is obtained (see, for example, the arrow in FIG. 7). In addition, the skin-like tissue produced in an atmosphere of 45% RH to 75% RH not only has a structure of the epidermal layer but also a dense basement membrane and a thick laminadensa. Furthermore, the expression of laminin 332, type VII collagen, etc. is also increased, and a skin-like tissue with a structure similar to that of natural skin in which basement membrane reorganization is promoted is obtained (see FIGS. 6 and 7).

As used herein, "skin-like tissue" refers to a structure that imitates natural skin structure, including an epidermal layer or a dermal layer, such as skin tissue isolated from a living body or regenerated in vitro. This is a constructed three-dimensional cultured skin. As used herein, the term "epidermal layer" refers to a layer mainly containing keratinocytes. Furthermore, in this specification, the term "dermal layer" refers to a layer mainly containing fibroblasts. The dermal layer may be a layer containing collagen produced from fibroblasts, or it may be a layer containing an externally added hydrogel, such as a collagen gel. Moreover, in this specification, "basement membrane" refers to a membranous structure formed between the epidermis layer and the dermis layer.

In the method of the present invention, the temperature for culturing may be any temperature typically used for cell culture, for example 20°C to 42°C, preferably 30°C to 39°C, for example about 37°C.

In one embodiment, the gas phase culture may be carried out for a period during which the reconstitution of the basement membrane is promoted, for example, from 1 day to 28 days, preferably from 3 days to 21 days, more preferably from 7 days to 21 days. For example, it may be 14 days.

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, humidity can be controlled by placing the cell culture container in an incubator controlled to have a gas phase of 45% RH to 75% RH. In this case, it is preferable to use a cell culture container having means for preventing water vapor derived from the medium filled on the outside of the cell culture container from entering the inside of the cell culture container. As an example of a cell culture container having such means, a cell culture container having the structure shown in FIG. 1 can be used, for example. Briefly, the lid of the cell culture container is provided with a hole that is approximately the same diameter as or larger than the inner diameter of the cell culture insert, and in contact with the cell culture insert, the outside of the cell culture container is A glass ring or the like is provided to prevent water vapor derived from the culture medium filled into the cell culture container from entering the inside of the cell culture container. This makes it possible to strictly control the gas phase on the stratum corneum side of skin-like tissue (for example, three-dimensionally cultured skin).

In one embodiment, the skin-like tissue used in the present invention is
(1) A step of seeding fibroblasts on the porous membrane of a cell culture container having a porous membrane to form a fibroblast layer, filling the inside and outside of the cell culture container 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 placed inside and outside the cell culture container. and (3) seeding keratinocytes on the hydrogel layer to form a keratinocyte layer, filling the inside and outside of the cell culture container with a medium, and culturing.
The three-dimensional cultured skin may be produced by a process including (see FIG. 2). Seed fibroblasts on the porous membrane of a cell culture container (e.g., cell culture insert) having a porous membrane to form a fibroblast layer, and fill the inside and outside of the cell culture container with a medium. , and culture (step (1)). The cell culture insert used in one embodiment of the present invention refers to a cell culture container equipped with a membrane having porous pores that cannot be penetrated by cells but can be penetrated by medium and the like. The culture medium and the like can also be supplied from the opposite side of the culture surface of the porous membrane, that is, from the back side of the surface to which adherent cells are attached. Commercially available cell culture containers, such as cell culture inserts, may be used in the present invention.

The medium used in the present invention can be any medium conventionally used for producing skin-like tissue, such as Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum; DMEM-Ham's F12 (3:1) containing fetal bovine serum, transferrin 5 μg/ml, insulin 5 μg/ml, tri-iodothyronine 2 nM, cholera toxin 0.1 nM, hydrocortisone 0.4 μg/ml; keratinocyte growth medium (KGM ) and DMEM containing 10% fetal bovine serum 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 The seeds are sown at an amount of ⁶ seeds/cm ² , more preferably 0.1×10 ⁶ to 1.0×10 ⁶ seeds/cm ² . In addition to fibroblasts, the cells seeded in step (1) include other cells contained in the dermis, such as mast cells, Meissner bodies, histiocytes, 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 in step (1) is preferably cultured for a period during which the 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. This allows the proliferated fibroblasts to cover the culture surface and also to produce extracellular matrix.

After 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 container is Then, 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, fibrin, alginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen/thrombin, fibrillin, elastin, Gums, cellulose, agar, gluten, casein, albumin, vitronectin, tenascin, entactin/nidogen, glycoproteins, glycosaminoglycans, poly(acrylic acid) and its derivatives, poly(ethylene oxide) and its copolymers, poly(vinyl (alcohol), polyphosphazene, matrigel and combinations thereof, preferably collagen.

The solution containing fibroblasts and a hydrogelling agent in step (2) is, for example, 0.01×10 ⁶ to 10.0×10 ⁶ cells/mL, preferably 0.05×10 ⁶ to 5.0 cells/mL. It contains fibroblasts at 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 step (2) may be adjusted to, for example, 1 mm to 10 mm, taking into account vertical shrinkage in the subsequent culture stage. In addition to fibroblasts, the cells seeded in step (2) include other cells contained in the dermis, such as mast cells, Meissner bodies, histiocytes, 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 in 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 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 container is 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/cm 2 . The seeds are sown at an amount of 0.1×10 ⁶ to 1.0×10 6 seeds/cm ² , more preferably 0.1×10 6 to 1.0×10 ⁶ seeds/cm ² . In addition to keratinocytes, the cells seeded in step (3) may include other cells contained in the epidermis, such as one or more cells selected from the group consisting of melanocytes, Langerhans cells, and Merkel cells.

In step (3), keratinocytes are preferably cultured for a period of time until they 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. It is.

The cells used in the present invention may be primary cells, or may be cells obtained by subculturing and proliferating primary cells, and may be derived from pluripotent stem cells such as ES cells, iPS cells, or Muse cells. Cells obtained by inducing differentiation may be used, or established cell lines may be used. Furthermore, the cells used may be derived from any animal, but are preferably derived from vertebrates, more preferably from mammals, and most preferably from humans.

In one embodiment, the medium used in the step of vapor phase culturing may contain a matrix metalloprotease inhibitor (MMP inhibitor). The medium contains a matrix metalloprotease inhibitor at a concentration sufficient to promote regeneration and repair of the skin basement membrane and dermis, typically 0.0000001 to 10% by weight, preferably 0.0000001 to 10% by weight based on the medium. It is contained in an amount of 0.000001 to 10% by weight.

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

Specific examples of matrix metalloprotease inhibitors include substance CGS27023A (N-hydroxy-2-[[(4-methoxyphenyl)sulfonyl]3-picolyl)amino]-3-methylbutanamide 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). Preferably, the matrix metalloprotease inhibitor is substance CGS27023A.

In one embodiment, the medium used in the gas phase culturing step may contain a heparanase inhibitor. The medium contains a heparanase inhibitor at a concentration sufficient to promote regeneration and repair of the skin basement membrane and dermis, typically 0.0000001 to 10% by weight, preferably 0.0000001 to 10% by weight of the medium. It is contained in an amount of 000001 to 10% by weight.

The heparanase inhibitor used in the present invention is not particularly limited as long as it has inhibitory activity against heparanase. Heparanase is an enzyme that exists in various cells and specifically degrades the heparan sulfate chains of various heparan sulfate proteoglycans. In the skin, epidermal keratinocytes that make up the epidermis, fibroblasts in the dermis, vascular endothelial cells, etc. are produced.

Specific examples of heparanase inhibitors include 1-[4-(1H-benzimidazol-2-yl)-phenyl]-3-[4-(1H-benzimidazol-2-yl)-phenyl]-urea ( (also referred to as "BIPBIPU") and SF-4 (also referred to as "Heparastatin hydrochloride").

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

<Method of evaluating target substances that improve and/or restore skin barrier function using skin-like tissue>
In one embodiment, a method for evaluating a target substance that improves and/or restores skin barrier function can be provided by adding the target substance to the skin-like tissue of the present invention.

For example, after adding a target substance to the skin-like tissue of the present invention, changes in the barrier function of the skin can be evaluated by measuring the amount of water evaporated from the surface of the skin-like tissue. Furthermore, after adding the target substance to the skin-like tissue of the present invention, the expression of known markers related to the skin barrier function (for example, Filaggrin, Loricrin, ZO-1, Claudin-1, etc.) can be investigated to determine the skin barrier function. Changes in function can be assessed.

In this embodiment, target substances that improve and/or restore skin barrier function include, for example, low molecular weight compounds, peptides, proteins, mammals (e.g., mice, rats, pigs, cows, sheep, monkeys, humans, etc.). ), tissue extracts or cell culture supernatants, plant-derived compounds or extracts (e.g., crude drug extracts, crude drug-derived compounds), and microorganism-derived compounds or extracts or culture products.

EXAMPLES The present invention will be explained in more detail below based on examples, but these are not intended to limit the invention in any way.

<Example 1>
1. Materials and experimental methods 1-1. Method for producing three-dimensional cultured skin Human dermal fibroblasts (0.2 x 10 ⁶ cells) were seeded in a cell culture insert (φ12 mm, average pore diameter of porous membrane: 0.4 μm), and 200 μM ascorbic acid-2-phosphate was added. The cells were cultured for one week using magnesium (APM) and 10% FBS-DMEM with medium exchange once every two days. A 0.5% type I collagen-10% FBS-DMEM solution containing human dermal fibroblasts was dispensed onto it to prepare a collagen gel on top of the human dermal fibroblasts, and cultured for 3 days.

Furthermore, epidermal keratinocytes dispersed in Humedia-KG2 (Kurabo Industries) medium were seeded onto the collagen gel at 5 x ¹⁰ cells/well, and Humedia-KG2 and 10% FBS-DMEM were added to the outside of the insert. A medium mixed at 1:1 and supplemented with 200 μM APM was added to the same liquid level as the inside, and cultured for 3 days.

Then, remove the medium inside the insert or glass ring, and add 200 μM of Ca to the outside of the skin model medium (mix 1:1 of 10% FBS-DMEM and Humedia-KG2 EGF (-) to make Ca 1.8 mM). APM, 10 μM N-hydroxy-2-[[(4-methoxyphenyl)sulfonyl]3-picolyl)amino]-3-methylbutanamide hydrochloride (CGS27023A (MMP inhibitor)), 10 μM BIPBIPU (heparanase inhibitor). The mixture was added to the bottom of the insert, and air-liquid boundary culture was performed with the inside of the insert exposed to air. Culture was continued for 2 weeks with medium exchange once every 2 to 3 days.

1-2. Low Humidity Cultivation Method After the three-dimensional cultured skin prepared in 1-1 has been subjected to air-liquid boundary culture for about one week, it is placed in an incubator controlled to an atmosphere of 37°C and 50% RH, or 37°C and 100% RH. Culture was performed. The humidity in the incubator was controlled using a humidity controller (Kitz Microfilter, AHCU-2). A hole was made in the lid of the plate containing the three-dimensionally cultured skin and a cloning ring was fitted so that only the surface of the three-dimensionally cultured skin was exposed to the outside air (see Figure 1).

2. Results In the three-dimensional cultured skin cultured at 50% RH, the keratinocyte layer and dermal layer (collagen gel layer) are thinner than the three-dimensional cultured skin cultured at 100% RH, and the overall morphology of the epidermal cells is It had a generally flatter shape (Figure 3).

In addition, epidermal differentiation markers (Filagrin, bleomycin hydrolase (BH), transglutaminase-1 (TGase-1), Involucrin, Corneodesmosin) and cell proliferation marker Ki67 As a result of investigating the expression by immunostaining, it was found that epidermal differentiation markers were densely stained in the flat granular layer in the three-dimensional cultured skin cultured at 50% RH compared to the three-dimensional cultured skin cultured at 100% RH. (Figure 4). Although the expression of Ki67, a cell proliferation marker, was slightly decreased in the three-dimensional cultured skin cultured at 50% RH, the expression was maintained (FIG. 4).

Using an electron microscope, we further examined the morphology of the epidermal layers of both types, and found that in the three-dimensional cultured skin cultured at 100% RH, the stratum corneum was thicker and swollen than in normal human skin, and the cell gap was widened, which was characteristic of the three-dimensionally cultured skin. In contrast, the three-dimensional cultured skin cultured at 50% RH lost this characteristic, and it became clear that it had an epidermal structure more similar to natural skin (see the arrow in Figure 5). ).

Furthermore, the extracellular matrix of the basement membrane and dermis was investigated. It was revealed that laminin 332 and type VII collagen were more strongly expressed and accumulated near the basement membrane in three-dimensional cultured skin cultured at 50% RH (Figure 6). Furthermore, when we examined the morphology of the area around the basement membrane using an electron microscope, we found that the laminadensa was thicker and the extracellular matrix was denser in the three-dimensional cultured skin cultured at 50% RH. It was revealed that the skin had a structure closer to that of natural skin (Figure 7).

<Example 2>
1. Materials and experimental methods 1-1. Method for producing three-dimensional cultured skin It was produced using the same materials and production method as in Example 1.

1-2. Low Humidity Cultivation Method After the three-dimensional cultured skin prepared in 1-1 was air-liquid boundary cultured for about one week, it was placed in an atmosphere of 37°C/40% RH, 37°C/70% RH, or 37°C/100% RH. The cells were placed in a controlled incubator and cultured. The humidity in the incubator was controlled using a humidity controller (Kitz Microfilter, AHCU-2). A hole was made in the lid of the plate containing the three-dimensionally cultured skin and a cloning ring was fitted so that only the surface of the three-dimensionally cultured skin was exposed to the outside air (see Figure 1).

2. Results Compared to the three-dimensional cultured skin cultured at 100% RH, the three-dimensional cultured skin cultured at 70% RH has a thinner keratinocyte layer and dermal layer (collagen gel layer), and the overall morphology of the epidermal cells is flatter. It had a similar shape to the three-dimensional cultured skin cultured at 50% RH described above (Figure 8). On the other hand, the three-dimensional cultured skin cultured at 40% RH had almost no epidermal cell layer and dermal layer, and did not maintain the shape of cultured skin (FIG. 9).

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

Claims (5)

A method for producing three-dimensional cultured skin, comprising:
The surface of the stratum corneum side of the three-dimensionally cultured skin, including the epidermal layer and the dermal layer, placed on the porous membrane of a cell culture container having a porous membrane, is exposed to a gas phase with a relative humidity of 45% to 75%. while bringing a medium into contact with the outer surface of the porous membrane of the cell culture container to perform gas phase culture;
including ;
The three-dimensional cultured skin is
(1) A step of seeding fibroblasts on the porous membrane of a cell culture container having a porous membrane to form a fibroblast layer, filling the inside and outside of the cell culture container 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 placed inside and outside the cell culture container. a step of filling and culturing; and
(3) seeding keratinocytes on the hydrogel layer to form a keratinocyte layer, filling the inside and outside of the cell culture container with a medium, and culturing;
A three-dimensional cultured skin produced by a process comprising : 2. The method according to claim 1, 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 entering the inside of the cell culture container. The hydrogelling agent is collagen, gelatin, hyaluronate, hyaluronan, fibrin, alginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen/thrombin, fibrillin, elastin, gum, cellulose, agar, gluten, casein, albumin, vitronectin, tenascin, entactin/nidogen, glycoproteins, glycosaminoglycans, poly(acrylic acid) and its derivatives, poly(ethylene oxide) and its copolymers, poly(vinyl alcohol), polyphosphazene, matrigel, and 3. The method according to claim 1 or 2 , wherein the method is selected from the group consisting of combinations thereof. The method according to any one of claims 1 to 3 , wherein in the gas phase culturing step, the medium contains a matrix metalloprotease inhibitor. The method according to any one of claims 1 to 4 , wherein in the gas phase culturing step, the medium contains a heparanase inhibitor.