JP7506661B2 - Three-dimensional cultured skin sheet, cell culture vessel used for the production thereof and method for producing the same - Google Patents

Description

The present invention relates to a three-dimensional cultured skin sheet. The present invention also relates to a cell culture vessel for use in the production of a three-dimensional cultured skin sheet. The present invention also relates to a method for producing a three-dimensional cultured skin sheet.

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

The skin of higher vertebrates is roughly divided into the epidermis, dermis, and subcutaneous tissue layers, starting from the outermost layer. The boundary between the epidermis and dermis has undulations, and it is known that these undulations flatten with age (see, for example, Non-Patent Document 1). The epidermis is mainly composed of cells called keratinocytes, which divide in the deepest part of the epidermis (basal layer) and differentiate into the spinous layer, granular layer, and stratum corneum as they move upwards toward the surface, eventually becoming dandruff and falling off. In humans, this cycle takes approximately four weeks.

Attempts have been made to construct various cultured skin models that mimic the epidermal structure and its functions in vitro (for example, Patent Document 1 and Non-Patent Document 2). In recent years, it has been reported that a thickened three-dimensional cultured skin model can be obtained by culturing epidermal cells on a porous membrane with convexities on the culture surface (see Patent Document 2). It has also been reported that the surface shape of the culture surface affects the differentiation of epidermal stem cells (Non-Patent Document 3).

Cultured skin models are used in safety testing of cosmetics and drugs applied to the skin, as well as in basic research, and are attracting attention as an alternative to animals.

JP 2012-235921 A International Publication No. 2017/222065

Lavker R. M. Structural alterations in exposed and unexposed aged skin. J Invest Dermatol. 1979 Jul;73(1):59-66. Bell E. , et al. , The reconstitution of living skin. J Invest Dermatol. 1983 Jul;81(1 Suppl):2s-10s. Zijl S. , et al. , Acta Biomater. 2019 Jan 15;84:133-145

The objective of the present invention is to provide a technology that can inexpensively, stably and simply obtain three-dimensional cultured skin with a thickness equivalent to or close to that of a living body and a barrier function, not only when primary cultured cells with a small number of passages are used, but also when any cell that constitutes skin is used.

The present inventors have conducted research and development by examining the above-mentioned problems from various angles. As a result, they have found that by culturing epidermal cells on a porous membrane with an optimized design of the convex portions on the culture surface, a three-dimensional cultured skin sheet with enhanced thickening or barrier function can be obtained compared to conventional culture methods. That is, the present invention encompasses the following inventions.

[1] A three-dimensional cultured skin sheet comprising at least keratinocytes and having a plurality of recesses in at least a portion of a base,
A three-dimensional cultured skin sheet, characterized in that the width of the recesses is an average of 15 μm to 25 μm, and the interval between the recesses is an average of 15 μm to 25 μm.
[2] The three-dimensional cultured skin sheet according to [1], wherein the height of the recesses is an average of 5 μm to 40 μm.
[3] The three-dimensional cultured skin sheet according to [1], wherein the height of the recesses is an average of 25 μm to 35 μm.
[4] The three-dimensional cultured skin sheet according to any one of [1] to [3], wherein the three-dimensional cultured skin sheet has an average thickness of 50 μm or more.
[5] Further comprising a porous membrane having a plurality of protrusions attached to the base,
wherein the width of the protrusions is 15 μm to 25 μm on average, and the interval between the protrusions is 15 μm to 25 μm on average,
The three-dimensional cultured skin sheet according to any one of [1] to [4], characterized in that the convex portion is in close contact with the concave portion of the base.
[6] The three-dimensional cultured skin sheet according to [5], wherein the height of the convex portions is an average of 5 μm to 40 μm.
[7] The three-dimensional cultured skin sheet according to [5], wherein the height of the convex portions is an average of 25 μm to 35 μm.
[8] The three-dimensional cultured skin sheet according to any one of [5] to [7], wherein the porous membrane has an average pore size of 0.2 μm to 3 μm.
[9] The three-dimensional cultured skin sheet according to any one of [5] to [8], wherein the convex portions are made of resin.

[10] A cell culture vessel for producing the three-dimensional cultured skin sheet according to any one of [1] to [9],
A porous membrane provided on at least a portion of the culture surface;
A protrusion formed on the porous membrane for forming a recess in the base of the three-dimensional cultured skin sheet;
Equipped with
The cell culture vessel is characterized in that the width of the convex portions is 15 μm to 25 μm on average, and the spacing between the convex portions is 15 μm to 25 μm on average.
[11] The cell culture vessel according to [10], wherein the height of the convex portions is 5 μm to 40 μm on average.
[12] The cell culture vessel according to [10], wherein the height of the convex portions is 25 μm to 35 μm on average.
[13] The cell culture vessel according to any one of [10] to [12], wherein the porous membrane has an average pore size of 0.2 μm to 3 μm.
[14] The cell culture vessel according to any one of [10] to [13], wherein the convex portion is made of a resin.

[15] A method for producing a three-dimensional cultured skin sheet, comprising:
(1) seeding cells, including keratinocytes, suspended in a medium on the porous membrane of the cell culture vessel according to any one of [10] to [14];
(2) a step of culturing the cells by contacting a medium with the outside of the porous membrane of the cell culture vessel;
A method comprising:
[16] Furthermore,
(3) removing the medium on the porous membrane of the cell culture vessel and culturing the cells while exposing them to air;
The method according to [15], comprising:
[17] The method according to [15] or [16], wherein the keratinocytes include keratinocytes that have been passaged twice or more after isolation and culture from a biological tissue.

According to the present invention, a three-dimensional cultured skin sheet with enhanced thickening and/or barrier function compared to conventional three-dimensional cultured skin sheets can be obtained. This makes it possible to provide a three-dimensional skin model that is useful for safety testing of cosmetics and drugs to be applied to the skin, and for basic research. Furthermore, by using the cell culture vessel of the present invention, a three-dimensional cultured skin sheet with enhanced thickening and/or barrier function compared to conventional three-dimensional cultured skin sheets can be provided stably and inexpensively. Furthermore, by using the culture method of the present invention, a three-dimensional cultured skin sheet with enhanced thickening and/or barrier function compared to conventional three-dimensional cultured skin sheets can be provided stably and inexpensively.

1 shows a schematic diagram illustrating an embodiment of the present invention, (a) shows a cross-sectional view of a cell culture vessel according to an embodiment of the present invention, and (b) shows an enlarged view of a part of (a). FIG. 1 is a conceptual diagram showing the structure of the three-dimensional cultured skin sheet of the present invention. FIG. 1 is a conceptual diagram showing a cell culture vessel of the present invention in one embodiment. FIG. 1 is a conceptual diagram showing a cell culture vessel of the present invention in one embodiment. FIG. 1 is a conceptual diagram showing a cell culture vessel of the present invention in one embodiment. This shows an HE staining image of a three-dimensional cultured skin sheet cultured on a porous membrane with convex parts. Keratinocytes of passage number 4 were cultured on a porous membrane with an average pore size of 0.4 μm and the following convex width x interval: (A) control (no convex parts), (B) convex width 20 μm x interval 100 μm, (C) convex width 25 μm x interval 25 μm, (D) convex width 50 μm x interval 20 μm, (E) convex width 20 μm x interval 20 μm, (F) convex width 30 μm x interval 30 μm, (G) convex width 50 μm x interval 50 μm. The height of the convex parts is (A) to (F): 30 μm, (G): 50 μm. Scale bar = 50 μm. SC: stratum corneum, SG: stratum granulosum, SS: stratum spinosum, SB: stratum basale. HE staining and immunostaining images of three-dimensional cultured skin sheets cultured on a porous membrane with convexities are shown. Keratinocytes at passage number 4 were cultured on a porous membrane with an average pore size of 0.4 μm and the following convex width x interval: (A) control (no convexities), (B) convex width 20 μm x interval 20 μm. The height of all convexities is 30 μm. First row: HE staining, second row: anti-Filaggrin antibody (red) and DAPI (blue), third row: anti-Loricrin antibody (red) and DAPI (blue), fourth row: anti-ZO-1 antibody (red), anti-Claudin-1 antibody (green) and DAPI (blue). Scale bar = 50 μm. This shows an HE staining image of a three-dimensional cultured skin sheet cultured on a porous membrane with convex parts. Keratinocytes of passage number 4 were cultured on a porous membrane with an average pore size of 1.0 μm and the following convex width x interval: (A) control (no convex parts), (B) convex width 20 μm x interval 20 μm, (C) convex width 30 μm x interval 30 μm, (D) convex width 15 μm x interval 15 μm, (E) convex width 25 μm x interval 25 μm, (F) convex width 50 μm x interval 50 μm. The height of the convex parts is all 30 μm. Scale bar = 50 μm. This shows an HE stained image (at a lower magnification than in FIG. 8) of a three-dimensional cultured skin sheet cultured on a porous membrane with convex portions. Keratinocytes at passage number 4 were cultured on a porous membrane with an average pore size of 1.0 μm and the following convex width x spacing: (A) control (no convex portions), (B) convex width 15 μm x spacing 15 μm, (C) convex width 20 μm x spacing 20 μm, (D) convex width 30 μm x spacing 30 μm, (E) convex width 50 μm x spacing 20 μm. The height of the convex portions is all 30 μm. This shows an immunostained image of a three-dimensional cultured skin sheet cultured on a porous membrane with convex parts. Keratinocytes at passage number 4 were cultured on a porous membrane with an average pore size of 1.0 μm and the following convex width x interval: (A) control (no convex parts), (B) convex width 20 μm x interval 20 μm, (C) convex width 25 μm x interval 25 μm. Left: anti-Filaggrin antibody (red) and DAPI (blue), right: anti-Loricrin antibody (red) and DAPI (blue). The height of the convex parts is 30 μm in all cases. 1 shows the results of evaluating the barrier function (amount of water evaporation) of a three-dimensional cultured skin sheet cultured on a porous membrane having convex portions. The amount of water evaporation of a three-dimensional cultured skin sheet obtained by culturing keratinocytes at passage number 4 on a porous membrane having convex portions and an average pore size of 1.0 μm was examined. (A) Transepidermal water evaporation (mg/cm ² /hour) from 0 to 2 hours, (B) from 2 to 4 hours, and (C) from 4 to 6 hours are shown. Control: no convex portions, 15-20: convex width 15 μm x interval 20 μm, 20-20: convex width 20 μm x interval 20 μm, 25-25: convex width 25 μm x interval 25 μm, 30-30: convex width 30 μm x interval 30 μm, 50-50: convex width 50 μm x interval 50 μm, EPS: EpiSkin (registered trademark). The height of all the projections is 30 μm.

Below, the form 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 only the form described below. Note that the matters described in each of the following "Three-dimensional cultured skin sheet", "Cell culture vessel for producing three-dimensional cultured skin sheet" and "Method for producing three-dimensional cultured skin sheet" can be applied to any of the "Three-dimensional cultured skin sheet", "Cell culture vessel for producing three-dimensional cultured skin sheet" and "Method for producing three-dimensional cultured skin sheet".

<Three-dimensional cultured skin sheet>
In one embodiment, the present invention provides a three-dimensional cultured skin sheet comprising at least keratinocytes and having a plurality of recesses in at least a portion of the base,
The three-dimensional cultured skin sheet is characterized in that the recesses have an average width of 15 μm to 25 μm and the interval between the recesses is an average width of 15 μm to 25 μm.

1 is a cross-sectional view showing a cell culture vessel 1 used in this embodiment and a part of the enlarged culture surface cross-section 6. The cell culture vessel 1 includes a cell culture insert 2 in which cells are seeded, and a bottom well 4 for filling the outside of the cell culture insert 2 with a culture medium. The cell culture insert 2 is provided with a porous membrane 3. Nutrients, oxygen, etc. can be supplied to the cells on the porous membrane 3 from the culture medium 5 filling the outside of the cell culture insert 2 through the porous membrane 3. In an embodiment of the present invention, the surface of the porous membrane 3 is provided with a convex portion 7 for forming an unevenness in the three-dimensional cultured skin sheet 10 of the present invention. By seeding and culturing epidermal cells in the cell culture vessel 1 of the present invention, the epidermal cells are layered, and a three-dimensional cultured skin sheet 10 having unevenness in at least a part of the base portion 11 (see FIG. 2, thick line) of the three-dimensional cultured skin sheet 10 is obtained.

In the present invention, the "base" of the three-dimensional cultured skin sheet 10 refers to the surface of the epidermal cells formed in a sheet shape when the epidermal cells are seeded in the cell culture vessel 1, which contacts the surface of the cell culture vessel, for example, the surface of the porous membrane 3 shown in FIG. 1 and/or the surface contacting the convex portion 7 (see FIG. 2, 11, thick line). In addition, in the present invention, the "base" is a structure equivalent to the basement membrane formed between the epidermis and dermis in the skin tissue of a living body, but does not necessarily have to have the same membrane structure as the basement membrane of a living body. That is, the "base" in the present invention includes an "epidermal basement membrane-like structure" having at least a partially uneven shape, and the epidermal basement membrane-like structure includes epidermal cells. In addition, the epidermal basement membrane-like structure may include a basement membrane formed by epidermal cells in the skin tissue of a living body.

Figure 2 is a cross-sectional view showing the three-dimensional cultured skin sheet 10 from Figure 1 (b) excluding the porous membrane 3 and the convex portions 7. The three-dimensional cultured skin sheet 10 contains a stratum corneum 9 that does not have cell nuclei, and epidermal cells 8 other than the stratum corneum 9. The convex portions 7 provided on the above-mentioned porous membrane 3 form epidermal cell-free regions V in the three-dimensional cultured skin sheet 10.

In the present invention, the "unevenness" of the base 11 of the three-dimensional cultured skin sheet 10 refers to the unevenness that is formed on at least a part of the surface of the three-dimensional cultured skin sheet of the present invention that contacts the culture surface of the cell culture vessel, and refers to unevenness that is larger than the unevenness that is accidentally obtained when epidermal cells are cultured on a flat culture vessel surface. In the present invention, the recess 110 of the three-dimensional cultured skin sheet 10 refers to the part of the base 11 that is recessed toward the air exposure (upper layer) direction of the cell culture vessel, and is the part formed by the protrusion 7 on the porous membrane 3 in FIG. 1 (see FIG. 2). On the other hand, in the present invention, the protrusion 111 of the three-dimensional cultured skin sheet refers to the part other than the recess 110 of the base 11. The vertical distance between the most depressed part of any recess 110 and the tip of the adjacent protrusion 111 with respect to the culture surface can be called the height H' of the recess 110. The shape and height H' of the recesses 110 formed in the three-dimensional cultured skin sheet 10 depend on the shape and height H of the protrusions 7 provided on the porous membrane 3. In addition, the interval W' of the recesses 110 formed in the three-dimensional cultured skin sheet 10 depends on the interval width W of the protrusions 7 on the porous membrane 3. In addition, the width Y' of the recesses 110 formed in the three-dimensional cultured skin sheet 10 depends on the width Y of the protrusions 7 formed by the protrusions 7 on the porous membrane 3.

The height H', spacing width W', and/or width Y' of the recesses 110 of the three-dimensional cultured skin sheet may be measured, for example, by preparing a tissue slice stained with HE and using a commercially available optical microscope, or by staining the tissue slice by immunohistochemistry and observing and measuring the same using a fluorescent microscope, a confocal microscope, or a two-photon laser microscope, or by observing and measuring the same using an electron microscope, and are not particularly limited. The height H' of the recesses 110 of the three-dimensional cultured skin sheet can be measured from an image obtained by a device equipped with a camera such as a normal microscope or a fluorescent microscope. As a microscope, for example, Olympus Corporation's System Industrial Microscope (for both reflected and transmitted light) BX51 can be used. As a camera, for example, Olympus Corporation's microscope digital camera "DP71" can be used. The obtained image can be imported into computer analysis software according to a standard method of image analysis and analyzed.

As used herein, "three-dimensional" refers to a state in which cells are stacked vertically and stratified, unlike the nearly single cell layer obtained by conventional culture of adherent cells on cell culture dishes, i.e., a two-dimensional cell layer. For example, the three-dimensional cultured skin sheet of the present invention refers to a three-dimensional structure having unevenness in the epidermal basement membrane-like structure and having epidermal cells stratified to give it thickness.

In this specification, the term "three-dimensional cultured skin sheet" is distinct from skin tissue inherent to a living body, and refers to a sheet-like three-dimensional skin-like tissue obtained by seeding a group of cells, including cells derived from skin tissue obtained by decomposing and breaking apart adhesive proteins between cells, and/or cells constituting skin obtained by inducing differentiation from pluripotent stem cells, in a cell culture vessel or the like, and culturing and reconstructing the cells on the cell culture vessel. In the present invention, the cells constituting the three-dimensional cultured skin sheet include epidermal cells, and can also be called a three-dimensional cultured epidermal sheet. In the present invention, the three-dimensional cultured skin sheet or three-dimensional cultured epidermal sheet may contain cells other than epidermal cells, for example, cells other than epidermal cells constituting the epidermis (melanocytes, Langerhans cells, Merkel cells, etc.) and/or cells contained in dermal tissue (fibroblasts, nerve cells, mast cells, plasma cells, vascular endothelial cells, histiocytes, Meissner corpuscles, etc.). Furthermore, the three-dimensional cultured skin sheet of the present invention may further comprise a porous membrane 3 having convex portions in contact with the base portion 11 .

The cells constituting the three-dimensional cultured skin sheet of the present invention may be derived from any animal, but are preferably derived from vertebrates, more preferably from mammals, and most preferably from humans.

In the present invention, "epidermal cells" refers to cells including all cells at different differentiation stages that constitute the epidermis. Epidermal cells are mainly composed of cells also called keratinocytes or keratinocytes. In living organisms, epidermal tissue is formed by layers of epidermal cells at different differentiation stages. The deepest part of the epidermis is called the basal layer or basal cell layer (hereinafter referred to as the "basal layer"), which forms a single layer of columnar cells and is thought to contain stem cells. The basal layer is also the boundary surface with the dermis, and the spinous layer is located above it. In living organisms, the papillary layer that constitutes the dermis is located directly below the basal layer.

The stratum spinosum, also known as the spinous cell layer, is made up of 2-10 layers in biological tissue. This layer is called the stratum spinosum because it appears to be connected to each other by spines. Only the cells in the basal layer have the ability to proliferate, and as the differentiation stage progresses, epidermal cells move to the outer layer while changing into a flattened shape.

When differentiation progresses beyond the spinous layer, a granular layer (granular cell layer) containing keratohyalin granules and lamellar granules is formed. In living tissue, the granular layer is composed of about 2 to 3 layers. When differentiation progresses further beyond the granular layer, the cell nuclei disappear and the stratum corneum (stratum corneum cell layer) is formed. The epidermis is roughly divided into the above-mentioned four layers, but in addition to epidermal cells, epidermal tissue also contains melanocytes, Langerhans cells, Merkel cells, etc., which prevent ultraviolet rays from reaching the dermis, play an important role in the immune function of the skin, and are involved in perception. In the present invention, the three-dimensional cultured skin sheet may contain cells other than epidermal cells, and can be appropriately modified depending on the purpose of use.

The "barrier function" of the skin generally refers to the function of preventing the loss of moisture and biological components in the body, and the function of preventing foreign substances (microorganisms, viruses, dust, etc.) from entering the body from outside. In this specification, the barrier function of the three-dimensional cultured skin sheet of the present invention can be evaluated by examining the transepidermal water loss (TEWL). The lower the transepidermal water loss from the three-dimensional cultured skin sheet, i.e., the lower the amount of water permeation, the higher the barrier function. In the present invention, the method for examining the amount of transepidermal water loss can be a general method used by those skilled in the art (for example, Kumamoto J., Tsutsumi M., Goto M., Nagayama M., Denda M. Japanese Cedar (Cryptomeria japonica) pollen allergen induces elevation of intracellular calcium in human keratinocytes and impairs epidermal barrier function of human skin ex). (see J. Vivo. Arch. Dermatol. Res. 308:49-54, 2016).

In the present invention, the average height H' of the recesses 110 of the three-dimensional cultured skin sheet is, for example, in the range of 1 μm to 50 μm, preferably 5 μm to 40 μm. If the height H' of the recesses 110 is in the above range, a three-dimensional cultured skin sheet with enhanced thickening and/or barrier function can be obtained.

In the present invention, the average spacing width W' of the recesses 110 in the three-dimensional cultured skin sheet is in the range of 15 μm to 25 μm, more preferably 17.5 μm to 22.5 μm, and most preferably 20 μm. If the spacing width W' of the recesses 110 is in the above range, a three-dimensional cultured skin sheet with enhanced thickening and/or barrier function can be obtained.

In the present invention, the average width Y' of the recesses 110 in the three-dimensional cultured skin sheet is in the range of 15 μm to 25 μm, more preferably 17.5 μm to 22.5 μm, and most preferably 20 μm. If the spacing width W' of the recesses 110 is in the above range, a three-dimensional cultured skin sheet with enhanced thickening and/or barrier function can be obtained.

The three-dimensional cultured skin sheet of the present invention has a recess 110 in at least a part of the base 11, and thus a three-dimensional cultured skin sheet is obtained in which the epidermal cell layer (e.g., spinous layer, granular layer and/or stratum corneum) on the base 11 is thickened compared to one not having the recess 110. In particular, the three-dimensional cultured skin sheet obtained by the present invention has an excellent effect of being thickened only by physically forming a recess 110 in the base 11 by a protrusion made of a non-biological material, not by a protrusion made of a biological material such as a cell or cell adhesion protein. In addition, when the protrusion is made of a non-biological material, the protrusion is not decomposed during the culture period and when used thereafter, and the shape of the recess can be stably maintained in the base of the three-dimensional cultured skin sheet, and the effect of the present invention can be sustained. The average height, shape and spacing of the recess 110 of the three-dimensional cultured skin sheet of the present invention can be controlled by the height, shape and spacing of the protrusions provided on the porous membrane surface of the cell culture vessel. In addition, the three-dimensional cultured skin sheet obtained by the present invention has a lower transepidermal water loss and a higher barrier function than conventional three-dimensional cultured skin sheets. The barrier function is enhanced by having a recess 110 in at least a part of the base 11 of the three-dimensional cultured skin sheet. The base 11 of the three-dimensional cultured skin sheet is preferably in close contact with the culture surface (culture surface including a porous membrane and a convex portion). When the base 11 of the three-dimensional cultured skin sheet is in close contact with the culture surface, the barrier function is further improved. More preferably, the base 11 of the three-dimensional cultured skin sheet is in close contact with the entire surface of the culture surface.

In the present invention, the convex portion provided on the porous membrane surface may be composed of a biological material, may be composed of a non-biological material, or may be composed of a combination of a biological material and a non-biological material. In this specification, "non-biological material" refers to a biological material, that is, a material other than biopolymers that constitute living organisms (nucleic acids, proteins, polysaccharides and their components (nucleotides, nucleosides, amino acids, sugars)) and vitamins. The non-biological material used to form the convex portion of the present invention is preferably a biocompatible material that does not affect cell culture. In the present invention, the convex portion provided on the porous membrane surface may further be coated with a biological material, such as collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (D-form, L-form), thrombospondin, etc.

Conventional three-dimensional cultured skin sheets were produced by pouring a gel containing proteins that constitute the extracellular matrix into a mold with projections and recesses to form an uneven surface, and then seeding epidermal cells on the uneven surface (see, for example, U.S. Patent Application Publication No. 2011/0171180). However, in this method, the entire culture surface is formed of a gel containing proteins, which are decomposed during culture, resulting in the problem that cells cannot adhere and the three-dimensional cultured skin sheet shrinks. On the other hand, in the present invention, the projections are provided on a non-stretching porous membrane surface, and the culture surface does not decompose even when cultured, so that a non-shrinking three-dimensional cultured skin sheet can be obtained.

The cells used in the present invention may be primary epidermal cells, epidermal cells proliferated by subculturing primary epidermal cells, epidermal cells obtained by inducing differentiation from pluripotent stem cells such as ES cells, iPS cells, or Muse cells, or established epidermal cell lines. Preferably, primary cultured epidermal cells are collected from biological tissue and seeded, or passaged epidermal cells are obtained by further subculturing the primary cultured cells. Epidermal cells collected from biological tissue are likely to maintain the same properties as those in the living body, and are convenient for use in tests to investigate the efficacy and side effects of drugs and in basic research.

In this specification, primary cultured epidermal cells are epidermal cells that have been harvested from biological tissue and then cultured once in a cell culture vessel, and are also referred to as "passage number 0 (or first generation)" epidermal cells. Confluent or subconfluent epidermal cells with passage number 0 can be further expanded and cultured by passage operations. Epidermal cells obtained by one passage operation are referred to as "passage number 1 (or second generation)" epidermal cells. Corresponding to the number of passage operations, they can be expressed as "passage number 2, 3, 4, ... n (n (integer) is the number of passages) (n+1 generation)". Commercially available primary cultured cells (for example, Kurabo's frozen NHEK (NB), catalog number: KK-4009) are provided as primary cultured cells with a passage number of about 0 to 2. When commercially available primary cultured cells are used in the present invention, the number of passages may be determined by referring to the number of passages or the number of generations described in the attached documents. A step of freezing the cells may be included between each passaging procedure.

Usually, the properties of many unestablished somatic cells change as a result of repeated passaging operations, or the ratio of the cells that make up the cells changes, resulting in a cell population that differs from that of cells with fewer passages. This is thought to be due to the fact that repeated passaging and cell division shortens telomeres, or that cells age due to the addition of biological and/or physical stress caused by confluency and/or trypsin treatment, etc. Therefore, when creating tissue models using cultured cells, cells with fewer passages are usually used.

However, primary cultured cells are cells directly isolated from living tissues, and because they have been subcultured only a few times, there is a limit to the number of cells that can be supplied. Not only are the size and amount of tissue that can be collected limited due to ethical issues, but the properties of the cells obtained also vary depending on the donor. Some primary cultured cells are commercially available, but they are inevitably expensive due to the supply volume. Therefore, if it is necessary to construct a large number of three-dimensional cultured skin sheets, it is necessary to obtain a large amount of expensive cells, which creates a problem in terms of cost. In order to produce them cheaply, it is possible to subculture and grow primary cultured cells for use, but as mentioned above, the obtained cells do not necessarily exhibit the same properties as primary cultured cells. When preparing a three-dimensional cultured skin model of epidermal cells, primary epidermal cells and kits for making those cells three-dimensional are commercially available, and it is possible to purchase these and prepare the model (for example, Keratinocyte Three-Dimensional Culture Starter Kit, CELLnTEC). However, when epidermal cells that have been passaged two or more times are used, the ability to spontaneously become three-dimensional is weakened, and there are problems such as the inability to obtain a thick three-dimensional cultured skin model (also called a three-dimensional cultured skin sheet).

In an embodiment of the present invention, if the three-dimensional cultured skin sheet has a recess 110 in the base 11, a thickened three-dimensional cultured skin sheet that was previously only obtained using primary cultured cells (e.g., 0 or 1 passage) can be constructed even when epidermal cells with 2 or more passages are used after isolation and culture from a living tissue. A thickened three-dimensional cultured skin sheet can be obtained by the present invention even when primary cultured cells (e.g., 0 or 1 passage) are used. The passage number of the epidermal cells used may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more, and is not limited as long as the spinous layer, granular layer, and/or stratum corneum of the three-dimensional cultured skin sheet are thickened. In an embodiment of the present invention, the animal species of the primary epidermal cells used is not particularly limited, but is preferably human-derived. In addition, in an embodiment of the present invention, the primary epidermal cells used may be any of those derived from a fetus, a newborn, a minor, or an adult, but are preferably cells derived from a fetus, a newborn, or a minor. When using cells derived from a human minor, it is preferable to use cells from, for example, a person younger than 20 years old, 1 to 19 years old, 1 to 10 years old, or 1 to 5 years old. Even when using cells derived from a human adult, it is preferable to use cells from a young person, for example, 20 to 29 years old, 30 to 39 years old, or 40 to 49 years old.

In one embodiment of the present invention, the average thickness of the three-dimensional cultured skin sheet may be 25 μm or more, 30 μm or more, 35 μm or more, 40 μm or more, 45 μm or more, 50 μm or more, 55 μm or more, 60 μm or more, 65 μm or more, 70 μm or more, 75 μm or more, 80 μm or more, 85 μm or more, 90 μm or more, 95 μm or more, 100 μm or more, 105 μm or more, 110 μm or more, 115 μm or more, 120 μm or more, 125 μm or more, 150 μm or more, 175 μm or more, 200 μm or more, 225 μm or more, 250 μm or more, 275 μm or more, or 300 μm or more. The preferred average thickness is 50 μm or more. The upper limit of the average thickness of the three-dimensional cultured skin sheet is not particularly limited, as it varies depending on the animal species, number of passages, age, etc. of the epidermal cells.

<Cell culture vessel for producing three-dimensional cultured skin sheets>
In one embodiment, the present invention provides
A porous membrane provided on at least a portion of the culture surface;
A convex portion is formed on the porous membrane to form a concave portion at the base of the three-dimensional cultured skin sheet;
Here, there is provided a cell culture vessel for producing a three-dimensional cultured skin sheet, characterized in that the width of the convex portions is an average of 15 μm to 25 μm, and the spacing between the convex portions is an average of 15 μm to 25 μm.

3 shows one embodiment of the cell culture vessel 1 of the present invention. On the porous membrane 3, a convex portion 71 for forming a concave portion 110 in the three-dimensional cultured skin sheet of the present invention is directly formed. The convex portion 71 has a cylindrical structure. By adjusting the height H1 of the convex portion 7, it is possible to control the height H' of the concave portion 110 of the three-dimensional cultured skin sheet of the present invention. For example, as shown in FIG. 3(b), the convex portion 71a can be arranged so as to be located at a square lattice point. By adjusting the convex portion interval width W1a, it is possible to control the convex portion interval width W' of the three-dimensional cultured skin sheet of the present invention described above. For example, as shown in FIG. 3(c), the convex portion 71b is arranged so as to be located at a regular triangular lattice point. By adjusting the convex portion interval width W1b, it is possible to control the convex portion interval width W' of the three-dimensional cultured skin sheet of the present invention described above. In addition, by adjusting the convex portion width Y1, it is possible to control the concave portion width Y' of the three-dimensional cultured skin sheet of the present invention.

Figure 4 shows one embodiment of the cell culture vessel 1 of the present invention. On the porous membrane 3, a convex portion 72 for forming unevenness in the three-dimensional cultured skin sheet of the present invention is arranged. The convex portion 72 has a cubic shape, which is a type of prismatic shape. By adjusting the height H2 of the convex portion, it is possible to control the height H' of the unevenness of the three-dimensional cultured skin sheet of the present invention. For example, as shown in Figure 4 (b), the convex portion 72a can be arranged so as to be located at a square lattice point. By adjusting the convex portion interval W2a, it is possible to control the convex portion interval W' of the three-dimensional cultured skin sheet of the present invention described above. For example, as shown in Figure 4 (c), the center of gravity of the convex portion 72b can be arranged so as to be located at a lattice point of an equilateral triangle. By adjusting the convex portion interval W2b, it is possible to control the convex portion interval W' of the three-dimensional cultured skin sheet of the present invention described above. In addition, by adjusting the convex portion width Y2, it is possible to control the recess width Y' of the three-dimensional cultured skin sheet of the present invention.

5 is a cross-sectional view showing an embodiment of the convex portions (73, 74, 75) provided in the cell culture vessel 1 of the present invention. For example, FIG. 5(a) shows a convex portion 73 having a pyramidal shape (triangular pyramid, square pyramid, or cone). For example, FIG. 5(b) shows a convex portion 74 having a frustum shape. For example, FIG. 5(c) shows a convex portion 75 having a bell shape. By adjusting the heights (H3, H4, H5) of the respective convex portions, it is possible to control the height H' of the unevenness of the three-dimensional cultured skin sheet of the present invention. By adjusting the convex portion interval width (W3, W4, W5), it is possible to control the recess interval width W' of the three-dimensional cultured skin sheet of the present invention. In addition, by adjusting the convex portion width (Y3, Y4, Y5), it is possible to control the recess width Y' of the three-dimensional cultured skin sheet of the present invention. The arrangement of the respective convex portions (73, 74, 75) on a plane can be arranged in the same manner as in FIG. 3 and FIG. 4, but is not limited thereto.

In an embodiment of the present invention, the shape of the convex portions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) is not limited to the above, but may also include, for example, an approximately hemispherical shape, an approximately rectangular parallelepiped shape, etc.

The surfaces of the convex portions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be subjected to plasma treatment or the like by a known method to facilitate cell adhesion.

In the cell culture vessel 1 of the present invention, the average spacing width (W, W1, W2, W3, W4, W5) of the convex portions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) is in the range of 15 μm to 25 μm, more preferably 17.5 μm to 22.5 μm, and most preferably 20 μm. If the spacing width (W, W1, W2, W3, W4, W5) of the convex portions is within the above range, a three-dimensional cultured skin sheet with enhanced thickening and/or barrier function can be obtained.

In the cell culture vessel 1 of the present invention, the average height of the convex portions (H, H1, H2, H3, H4, H5) is, for example, in the range of 1 μm to 50 μm, preferably 5 μm to 40 μm. If the height of the convex portions (H, H1, H2, H3, H4, H5) is within the above range, a three-dimensional cultured skin sheet with enhanced thickening and/or barrier function can be obtained.

In the cell culture vessel 1 of the present invention, the average convex width (Y, Y1, Y2, Y3, Y4, Y5) is in the range of 15 μm to 25 μm, more preferably 17.5 μm to 22.5 μm, and most preferably 20 μm. If the convex width (Y, Y1, Y2, Y3, Y4, Y5) is in the above range, a three-dimensional cultured skin sheet with enhanced thickening and/or barrier function can be obtained.

The convex portions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) are preferably formed directly on the porous membrane 3, and can be formed, for example, by a method used in 3D printer technology or semiconductor process technology (for example, a method combining a lithography process (photolithography, maskless photolithography, etc.) and dry etching (for example, reactive ion etching) or wet etching, etc.). The material of the convex portions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be composed of a biological material, a non-biological material, or a combination of a biological material and a non-biological material. For example, polyester or polyethylene terephthalate (PET), polycarbonate, polystyrene, zirconia, glass insoluble collagen, silicone rubber, resin (for example, epoxy resin, acrylic resin, etc.), and other materials may be used as long as cell culture is possible. The convex portions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) are preferably formed directly on the porous membrane 3. By forming the convex portions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) directly on the porous membrane 3, epidermal cells do not creep under the convex portions, and the effect of improving the thickening and/or barrier function is significantly exhibited.

The material of the cell culture vessel 1 used in the embodiment of the present invention is not particularly limited as long as it is a material that is usually used for cell culture vessels. For example, glass, polystyrene, polypropylene, polycarbonate, etc. The shape of the cell culture vessel used in the present invention is not particularly limited, but examples include dish type, multi-well plate type, flask type, etc. The cell culture vessel of the present invention is preferably a cell culture insert type because it has a porous membrane in a part of the cell culture vessel. The cell culture insert used in the present invention refers to a cell culture vessel equipped with a membrane having porous holes that cannot be permeated by cells but can be permeated by culture fluid, etc. It is possible to supply culture fluid, etc. from the opposite side of the culture surface of the porous membrane, that is, the back side of the adhesion surface of the adherent cells. The cell culture insert used in the present invention may be a commercially available one, or may be a cell culture insert having a membrane with a convex portion directly provided on the culture surface of the porous membrane. The average pore size of the porous membrane is 0.1 μm to 5.0 μm, preferably 0.2 μm to 3.0 μm, and more preferably 0.3 to 1.5 μm. By using a porous membrane having pores in this range, a three-dimensional cultured skin sheet with improved thickening and/or barrier function can be obtained. The pore density of the porous membrane is, for example, 1×10 ⁵ to 1×10 ⁹ pores/cm ² , preferably 5×10 ⁵ to 5×10 ⁸ pores/cm ² , more preferably 1×10 ⁶ to 5×10 ⁸ pores/cm ² . The average thickness of the porous membrane is, for example, 1 to 50 μm, preferably 3 to 25 μm, more preferably 5 to 20 μm. The material of the porous membrane may be the same as that of the porous membrane used in conventional cell culture, for example, polyester or polyethylene terephthalate (PET), or polycarbonate, polystyrene, etc.

In an embodiment of the present invention, the culture surface may be coated so that cells can easily adhere to and grow on the cell culture vessel having protrusions on the porous membrane 3. Examples of such coatings include collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (D- and L-forms), thrombospondin, and fibrin.

<Method of manufacturing three-dimensional cultured skin sheet>
In one embodiment, the present invention provides
(1) seeding cells, including keratinocytes, suspended in a medium on the porous membrane of the cell culture vessel;
(2) a step of culturing the cells by contacting a medium with the outside of the porous membrane of the cell culture vessel;
The present invention provides a method for producing a three-dimensional cultured skin sheet, comprising:

In another embodiment, the present invention further comprises, in addition to the above steps, (3) removing the medium on the porous membrane of the cell culture vessel and culturing the cells while exposing them to air;
The method may include:

The number of cells including keratinocytes used in the method of the present invention may be determined according to a known method. For example, the cells are seeded at an amount of 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 , more preferably 0.1×10 ⁶ to 1.0×10 ⁶ cells/cm ² . The medium (also called culture solution) used in the present invention may be a medium commonly used for culturing epidermal cells, such as KG medium, EpilifeKG2 (Kurabo Industries, Ltd.), Humedia-KG2 (Kurabo Industries, Ltd.), Assay medium (TOYOBO Corporation), CnT-Prime, Epithelial culture medium (CELLnTEC Corporation), etc., and the culture may be performed at about 37° C. for 0 to 14 days. As the medium, DMEM medium (GIBCO) or a medium in which 2-0-a-D-glucopyranosyl-L-ascorbic acid-containing KGM and DMEM are mixed at a ratio of 1:1 can be used. For three-dimensionalization (stratification) of epidermal cells, CnT-Prime, 3D barrier medium (CELLnTEC) can be used. Other media can be used.

In an embodiment of the present invention, epidermal cells are cultured, three-dimensionalized (stratified) and cultured to promote keratinization by seeding them in a cell culture vessel 1 including the cell culture insert 2 described above. Specifically, epidermal cells are suspended in culture medium and seeded on the cell culture insert 2. Culture medium is also added to the bottom well 4 so that the culture medium is in contact with the outside of the porous membrane 3 of the cell culture insert, and the cell culture insert 2 is immersed and cultured. This allows the epidermal cells to be supplied with culture medium from both the top and bottom, and cultured.

It is preferable to culture the epidermal cells on the cell culture insert 2 for several days (about 1 to 6 days, preferably about 2 to 4 days) until they become confluent or subconfluent. Thereafter, in order to further promote the three-dimensionalization (stratification) of the cells, it is preferable to replace the culture medium inside and outside the cell culture insert with, for example, CnT-Prime, 3D barrier medium (CELLnTEC). This further promotes the three-dimensionalization of the epidermal cells. After the culture medium is replaced with CnT-Prime, 3D barrier medium (CELLnTEC), and the cells are cultured, it is preferable to remove only the culture medium inside the cell culture insert and expose the upper surface of the epidermal cells to the gas phase after 1 to 36 hours, preferably 6 to 24 hours, and more preferably 12 to 18 hours, and then culture the cells. This promotes the three-dimensionalization and keratinization of the epidermal cells, and a thicker three-dimensional cultured skin sheet is obtained.

In addition, the culture temperature for each culture step in the present invention may be close to the body temperature of the originating animal, and specifically, in the case of human cells, a temperature of approximately 33 to 38°C is preferable.

The three-dimensional cultured skin sheet obtained by the present invention as described above can be used as an alternative to animal experiments, for example, as a skin model. For example, it can be used in a method for evaluating the reactivity of skin to chemical substances (e.g., cosmetics, industrial products, household products, medicines, skin topicals, etc.). In addition, the three-dimensional cultured skin sheet of the present invention can be used as a skin model useful in basic research in dermatology, since it can obtain tissue with greater thickening and/or enhanced barrier function compared to conventional three-dimensional cultured skin sheets. Furthermore, the three-dimensional cultured skin sheet obtained by the present invention is thicker than conventional three-dimensional cultured skin sheets, and therefore has a high barrier function against the outside, and is also useful as a three-dimensional cultured skin sheet for healing burns, wounds, etc.

In one embodiment, the cell culture vessel used in the present invention may be a cell culture vessel in which the convex portion and the porous membrane can be separated after the culture is completed. In this case, the three-dimensional cultured skin sheet produced in the cell culture vessel is separated from the porous membrane while the convex portion is in contact with the base. Therefore, the base of the three-dimensional cultured skin sheet can be moved while maintaining the uneven structure. In the cell culture vessel used in the present invention, the convex portion is preferably a biological material including a biological material such as collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (D-form, L-form), thrombospondin, etc., and more preferably insoluble collagen.

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

For example, after adding a target substance to the three-dimensional cultured skin sheet of the present invention, the amount of water evaporation from the surface of the three-dimensional cultured skin sheet can be measured to evaluate changes in the skin barrier function (see Kumamoto J., et al., Arch. Dermatol. Res. 308: 49-54, 2016). In addition, after adding a target substance to the three-dimensional cultured skin sheet of the present invention, changes in the skin barrier function can be evaluated by examining the expression of known markers related to the skin barrier function (e.g., Filaggrin, Loricrin, ZO-1, Claudin-1, etc.).

In this embodiment, the target substance for improving and/or restoring the skin barrier function may be, for example, a low molecular weight compound, a peptide, a protein, a tissue extract or cell culture supernatant of a mammal (e.g., mouse, rat, pig, cow, sheep, monkey, human, etc.), a compound or extract derived from a plant (e.g., a herbal extract, a compound derived from a herbal drug), and a compound or extract derived from a microorganism, or a culture product.

The present invention will be explained in more detail below with reference to examples, which are not intended to limit the present invention in any way.

<Cell preparation>
The epidermal cells used in the present invention were neonatal keratinocytes (hereinafter referred to as "keratinocytes"; Kurabo Industries, Ltd., product name: Frozen NHEK (NB), catalog number: KK-4009). Subculture was performed according to the instructions provided by the distributor.

Example 1
1. Materials and Experimental Methods 1-1. Preparation of Cell Culture Inserts with Convex Parts (Pore Size: 0.4 μm) (1) 12-Well Millicell Hanging Cell Culture Inserts Only the porous membrane was peeled off from PET (pore size: 0.4 μm, Millipore), and the porous membrane was heated to 80° C. and laminated with a photosensitive resin sheet (thickness of 30 μm or 50 μm) by pressing it with a roller.
(2) A photomask with circular holes of the intended width and spacing between the convex portions was placed on the porous film laminated with the photosensitive resin sheet, and UV light was irradiated through the photomask. This transferred the pattern of UV light passing/shielding areas to the photosensitive resin sheet. Only the photosensitive resin irradiated with UV light was cured.
(3) The porous film obtained in (2) was developed by immersing it in an organic solvent, so that only the UV-irradiated areas remained as a pattern, and the light-shielded areas were removed by development.
(4) The porous membrane obtained in (3) was immersed in ethanol and washed. After that, the porous membrane with the convex portions formed thereon was attached again to the frame of the cell culture insert.

1-2. Preparation and observation of three-dimensional cultured skin sheet using cell culture insert with convex portions (1) CELLstart CTS (Gibco) was diluted 50-fold with DPBS (Gibco), and 86 μL was dropped onto one cell culture insert and maintained at 37° C. for 2 hours.
(2) CELLstart CTS was removed, and 220,000 to 250,000 neonatal-derived keratinocytes at passage 4 were dispersed in 500 μL of CnT-Prime, Epithelial culture medium (CELLnTEC), which was then dropped into a cell culture insert. 1 mL of the same medium (CnT-Prime, Epithelial culture medium) was then dropped onto the outside of the cell culture insert, and the cells were cultured at 37° C. for 72 hours in a _CO2 incubator.
(3) CnT-Prime and epithelial culture medium inside and outside the cell culture insert were removed and replaced with CnT-Prime and 3D barrier medium (CELLnTEC), and the cells were cultured in a 37° C. CO ₂ incubator for 16 hours.
(4) The medium inside and outside the cell culture insert was removed, the inside of the cell culture insert was exposed to air, and 500 μL of CnT-Prime and 3D barrier medium were placed outside the cell culture insert.
(5) The cells were cultured for 7 days in a 37° C. CO _incubator while replacing 500 μL of CnT-Prime and 3D barrier medium outside the cell culture insert every day.
(6) The three-dimensional cultured skin sheet was cut out together with the inner membrane of the cell culture insert, fixed in 4% paraformaldehyde in PBS, and tissue slice specimens were prepared. The tissue slice specimens were stained with hematoxylin and eosin (HE) or immunohistochemically stained with anti-Filaggrin antibody (sc-30229, Santa Cruz, Dallas, USA) or anti-Loricrin antibody (PRB-145P, Biolegend, San Diego, CA) according to known methods. Diego, USA), anti-ZO-1 antibody (#33-9100, Invitrogen, Carlsbad, USA), anti-Claudin-1 antibody (#51-9000, Invitrogen, Carlsbad, USA) or DAPI (R37606, Invitrogen, Carlsbad, USA) were used and observed under a microscope.

2. Results Convex portions of varying heights (30 μm or 50 μm), widths (20 μm to 50 μm), and intervals (15 μm to 50 μm) were formed on a porous membrane with pores of an average size of 0.4 μm, and keratinocytes were cultured on the convex portions ( FIG. 6 ). As a result, the porous membrane with convex portions designed with a convex portion height of 30 μm, a convex portion width of 20 μm, and a convex portion interval of 20 μm had the greatest effect of thickening the three-dimensional cultured skin sheet.

We also performed immunohistochemical staining on three-dimensional cultured skin sheets made with a porous membrane without convexities and a porous membrane with convexities designed with a convexity height of 30 μm, a convexity width of 20 μm, and a convexity interval of 20 μm (Figure 7). Staining with anti-filaggrin antibody (a marker antibody for granular cells), anti-loricrin antibody (a marker antibody for the granular layer), anti-ZO-1 antibody, and anti-Claudin-1 antibody (a marker antibody for tight junctions) revealed that ZO-1 and Claudin-1 in particular were more clearly expressed with a unique structure over a wider range.

Example 2
A three-dimensional cultured skin sheet was prepared and observed in the same manner as in Example 1, except that 12-well Millicell Hanging Cell Culture inserts PET (1.0 μm, Millipore) were used.

Keratinocytes were cultured on a porous membrane with pores of an average size of 1.0 μm, on which convexities of different heights (30 μm), widths (15 μm to 50 μm) and intervals (15 μm to 30 μm) were formed (Figures 8 and 9). As a result, a porous membrane with convexities designed with a convexity height of 30 μm, a convexity width of 20 to 25 μm and a convexity interval of 20 to 25 μm had the greatest effect of thickening the stratum corneum of the three-dimensional cultured skin sheet.

In addition, immunohistochemical staining was performed on three-dimensional cultured skin sheets made of a porous membrane without convexities, a porous membrane with convexities designed with a height of 30 μm, a width of 20 μm, and a spacing of 20 μm, and a porous membrane with convexities designed with a height of 30 μm, a width of 20 μm, and a spacing of 20 μm (Figure 10). Staining with anti-filaggrin antibody (a marker antibody for granular cells) and anti-loricrin antibody (a marker antibody for the granular layer) revealed that each antibody was expressed, but the ranges of expression of filaggrin and loricrin were narrow.

Example 3
Evaluation of Barrier Function A three-dimensional cultured skin sheet was prepared in the same manner as in Example 1, except that 12-well Millicell Hanging Cell Culture inserts PET (1.0 μm, Millipore) were used.

The cell culture insert with the three-dimensional cultured skin sheet constructed was placed in a silicone rubber container with culture medium at the bottom, so that water evaporation occurred only from the surface of the epidermis model. After that, the weight of the container with the insert was measured every two hours, and the weight loss was regarded as the amount of water evaporation, which was converted into a value per unit area. This technique is based on the same principle as the method described in Kumamoto's paper (Kumamoto J., Tsutsumi M., Goto M., Nagayama M., Denda M. Japanese Cedar (Cryptomeria japonica) pollen allergen induces elevation of intracellular calcium in human keratinocytes and impairs epidermal barrier function of human skin ex vivo. Arch. Dermatol. Res. 308: 49-54, 2016).

As a comparative example, the barrier function of a commercially available three-dimensional skin model (Episkin (registered trademark), NicoDerm Research) was also evaluated.

The results obtained were statistically analyzed (ANOVA analysis of variance followed by Scheffe's multiple test analysis), and results of the multiple test with p<0.05 were determined to indicate a significant difference when compared to three-dimensional cultured skin sheets obtained by culturing only the membrane.

As a result, it was revealed that the three-dimensional cultured skin sheet prepared using a porous membrane (average pore size: 1.0 μm) with convex portions designed with a convex portion height of 30 μm, a convex portion width of 20 μm, and a convex portion spacing of 20 μm had the least amount of transepidermal water loss and possessed a high stratum corneum barrier function (Figure 11).

1 Cell culture vessel 2 Cell culture insert 3 Porous membrane 4 Bottom well 5 Culture medium 6 Enlarged cross-sectional area of culture surface 7, 71, 71a, 71b, 72, 72a, 72b, 73 to 75 Convex portion 8 Epidermal cell 9 Stratum corneum 10 Three-dimensional cultured skin sheet 11 Basal portion (thick line)
110: Concave portion of three-dimensional cultured skin sheet 111: Convex portion of three-dimensional cultured skin sheet H-H5: Height of convex portion H': Height of concave portion of three-dimensional cultured skin sheet V: Region where no epidermal cells are present W, W1, W1a, W1b, W2, W2a, W2b, W3-W5: Spacing width between convex portions W': Spacing width between convex portions of three-dimensional cultured skin sheet Y, Y1-Y5: Width of convex portion Y': Width of concave portion of three-dimensional cultured skin sheet

Claims (11)

A three-dimensional cultured skin sheet comprising at least keratinocytes and having a plurality of recesses in at least a portion of a base,
A three-dimensional cultured skin sheet , wherein the width of the recess is 15 μm to 25 μm on average, and the interval between the recesses is 15 μm to 25 μm on average ,
Further comprising a porous membrane having a plurality of protrusions attached to the base,
wherein the width of the convex portions is 15 μm to 25 μm on average, the spacing between the convex portions is 15 μm to 25 μm on average, and the height of the convex portions is 25 μm to 35 μm on average;
A three-dimensional cultured skin sheet, characterized in that the convex portion is in close contact with the concave portion of the base . The three-dimensional cultured skin sheet of claim 1, wherein the height of the recesses is an average of 25 μm to 35 μm. The three-dimensional cultured skin sheet according to any one of claims 1 to 2 , wherein the three-dimensional cultured skin sheet has an average thickness of 50 µm or more. The three-dimensional cultured skin sheet according to any one of claims 1 to 3 , wherein the porous membrane has an average pore size of 0.2 μm to 3 μm. The three-dimensional cultured skin sheet according to any one of claims 1 to 4 , wherein the convex portions are made of resin. A cell culture vessel for producing the three-dimensional cultured skin sheet according to any one of claims 1 to 5 ,
A porous membrane provided on at least a portion of the culture surface;
A protrusion formed on the porous membrane for forming a recess in the base of the three-dimensional cultured skin sheet;
Equipped with
The cell culture vessel is characterized in that the width of the convex portions is 15 μm to 25 μm on average, the spacing between the convex portions is 15 μm to 25 μm on average, and the height of the convex portions is 25 μm to 35 μm on average . The cell culture vessel according to claim 6 , wherein the porous membrane has an average pore size of 0.2 μm to 3 μm. The cell culture vessel according to claim 6 , wherein the protrusion is made of a resin. A method for producing a three-dimensional cultured skin sheet, comprising:
(1) A step of seeding cells including keratinocytes suspended in a medium on the porous membrane of the cell culture vessel according to any one of claims 6 to 8 ;
(2) a step of culturing the cells by contacting a medium with the outside of the porous membrane of the cell culture vessel;
A method comprising: moreover,
(3) removing the medium on the porous membrane of the cell culture vessel and culturing the cells while exposing them to air;
10. The method of claim 9 , comprising: The method according to claim 9 or 10 , wherein the keratinocytes include keratinocytes that have been passaged twice or more after being isolated and cultured from a biological tissue.

Images