WO2020204106A1 - Three-dimensionally cultured skin sheet, cell culture container to be used for producing same, and method for producing same - Google Patents

Three-dimensionally cultured skin sheet, cell culture container to be used for producing same, and method for producing same Download PDF

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Publication number
WO2020204106A1
WO2020204106A1 PCT/JP2020/015109 JP2020015109W WO2020204106A1 WO 2020204106 A1 WO2020204106 A1 WO 2020204106A1 JP 2020015109 W JP2020015109 W JP 2020015109W WO 2020204106 A1 WO2020204106 A1 WO 2020204106A1
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skin sheet
cultured skin
cell culture
average
dimensional cultured
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PCT/JP2020/015109
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French (fr)
Japanese (ja)
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傳田　光洋
淳一 熊本
雅晴 長山
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株式会社 資生堂
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a three-dimensional cultured skin sheet.
  • the present invention also relates to a cell culture container 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 body surface that separates the environment inside and outside the body.
  • the skin acts as a physical barrier, protects against dryness and the invasion of harmful substances into the body, and plays an essential role in sustaining life.
  • the skin of higher vertebrates is roughly divided from the outermost layer to the epidermis, dermis, and subcutaneous tissue.
  • the interface between the epidermis and the dermis has undulations, and it is known that the undulations flatten with aging (see, for example, Non-Patent Document 1).
  • the epidermis is mainly composed of cells called keratinocytes, and the keratinocytes divide in the deepest part (basal layer) of the epidermis, and the surface is differentiated into the stratum spinosum, the stratum granulosum, and the stratum granulosum toward the upper layer. It moves to, and eventually becomes dirt and falls off. This cycle takes approximately 4 weeks for humans.
  • Patent Document 1 Attempts have been made to construct various cultured skin models that imitate the epidermis structure and its function in vitro (for example, Patent Document 1 and Non-Patent Document 2).
  • Patent Document 2 Attempts have been made to construct various cultured skin models that imitate the epidermis structure and its function in vitro (for example, Patent Document 1 and Non-Patent Document 2).
  • a thickened three-dimensional cultured skin model can be obtained by culturing epidermal cells in a porous membrane having a convex portion on the culture surface (see Patent Document 2).
  • Patent Document 3 the surface shape of the culture surface affects the differentiation of epidermal stem cells.
  • the cultured skin model is used in safety tests of cosmetics and drugs applied to the skin, basic research, etc., and is attracting attention as a model to replace animals.
  • the present inventors have conducted research and development by examining from various angles in order to solve the above problems. As a result, by culturing the epidermal cells in a porous membrane with an optimized design of the convex portion on the culture surface, a three-dimensional cultured skin sheet having a thickened or enhanced barrier function as compared with the conventional culture method was obtained. I found that it was possible. That is, the present invention includes the following inventions.
  • the cell culture vessel is characterized in that the width of the convex portions is on average 15 ⁇ m to 25 ⁇ m, and the interval width of the convex portions is on average 15 ⁇ m to 25 ⁇ m.
  • the cell culture vessel according to [10] wherein the height of the convex portion is 5 ⁇ m to 40 ⁇ m on average.
  • [15] A method for producing a three-dimensional cultured skin sheet. (1) A step of seeding cells containing 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 a medium in contact with the outside of the porous membrane of the cell culture container. Including methods. [16] Furthermore (3) A step of removing the medium on the porous membrane of the cell culture container and culturing the cells while exposing them to air. The method according to [15]. [17] The method according to [15] or [16], wherein the keratinocytes contain keratinocytes having 2 or more passages after being isolated and cultured from a living tissue.
  • a three-dimensional cultured skin sheet having a thickened and / or barrier function is obtained as compared with the conventional three-dimensional cultured skin sheet.
  • a three-dimensional cultured skin sheet having an enhanced thickening and / or barrier function can be provided stably and inexpensively as compared with the conventional three-dimensional cultured skin sheet.
  • the culture method of the present invention it becomes possible to provide a three-dimensional cultured skin sheet having a thickened and / or barrier function enhanced more stably and inexpensively than the conventional three-dimensional cultured skin sheet.
  • the conceptual diagram which shows one Embodiment of this invention is shown.
  • A A cross-sectional view of the cell culture vessel of the present invention in one embodiment is shown.
  • B is an enlarged view of a part of (a).
  • It is a conceptual diagram which shows the structure of the 3D culture skin sheet of this invention.
  • It is a conceptual diagram which shows the cell culture container of this invention in one Embodiment.
  • It is a conceptual diagram which shows the cell culture container of this invention in one Embodiment.
  • the HE-stained image of the three-dimensional culture skin sheet cultured on the porous membrane having a convex part is shown.
  • Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width ⁇ spacing and an average pore diameter of 0.4 ⁇ m: (A) control (no convex portion), (B) convex width 20 ⁇ m ⁇ spacing 100 ⁇ m, ( C) Convex width 25 ⁇ m ⁇ interval 25 ⁇ m, (D) convex width 50 ⁇ m ⁇ interval 20 ⁇ m, (E) convex width 20 ⁇ m ⁇ interval 20 ⁇ m, (F) convex width 30 ⁇ m ⁇ interval 30 ⁇ m, (G) convex width 50 ⁇ m ⁇ interval 50 ⁇ m.
  • SC stratum granulosum
  • SG stratum granulosum
  • SS stratum spinosum
  • SB stratum basale.
  • the HE-stained image and the immuno-stained image of the three-dimensional cultured skin sheet cultured on the porous membrane having the convex portion are shown.
  • Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width ⁇ spacing and an average pore diameter of 0.4 ⁇ m: (A) control (no convex portion), (B) convex width 20 ⁇ m ⁇ spacing 20 ⁇ m. The height of the convex portions is all 30 ⁇ m.
  • Scale bar 50 ⁇ m.
  • the HE-stained image of the three-dimensional culture skin sheet cultured on the porous membrane having a convex part is shown. Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width ⁇ spacing and an average pore diameter of 1.0 ⁇ m: (A) control (no convex portion), (B) convex width 20 ⁇ m ⁇ spacing 20 ⁇ m, ( C) Convex width 30 ⁇ m ⁇ interval 30 ⁇ m, (D) convex width 15 ⁇ m ⁇ interval 15 ⁇ m, (E) convex width 25 ⁇ m ⁇ interval 25 ⁇ m, (F) convex width 50 ⁇ m ⁇ interval 50 ⁇ m.
  • the HE-stained image (lower magnification than FIG. 8) of the three-dimensional culture skin sheet cultured on the porous membrane having a convex portion is shown.
  • Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width ⁇ spacing and an average pore diameter of 1.0 ⁇ m: (A) control (no convex portion), (B) convex width 15 ⁇ m ⁇ spacing 15 ⁇ m, ( C) Convex width 20 ⁇ m ⁇ interval 20 ⁇ m, (D) convex width 30 ⁇ m ⁇ interval 30 ⁇ m, (E) convex width 50 ⁇ m ⁇ interval 20 ⁇ m. The height of the convex portions is all 30 ⁇ m. An immunostaining image of a three-dimensional cultured skin sheet cultured on a porous membrane having a convex portion is shown.
  • Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width ⁇ spacing and an average pore diameter of 1.0 ⁇ m: (A) control (no convex portion), (B) convex width 20 ⁇ m ⁇ spacing 20 ⁇ m, ( C) Convex width 25 ⁇ m ⁇ interval 25 ⁇ m. Left figure: anti-Filaggrin antibody (red) and DAPI (blue), right figure: anti-Filaggrin antibody (red) and DAPI (blue). The height of the convex portions is all 30 ⁇ m.
  • the result of evaluating the barrier function (the amount of water evaporation) of the three-dimensional culture skin sheet cultured on the porous membrane having a convex part is shown.
  • the amount of water evaporation of a three-dimensional cultivated skin sheet obtained by culturing keratinocytes having a number of passages of 4 on a porous membrane having a convex portion and an average pore diameter of 1.0 ⁇ m was examined.
  • the amount of transepidermal water loss (mg / cm 2 / hour) is shown for (A) 0 to 2 hours, (B) 2 to 4 hours, and (C) 4 to 6 hours.
  • the present invention is a three-dimensional cultured skin sheet containing at least keratinocytes and having a plurality of recesses in at least a part of a basal portion.
  • a three-dimensional cultured skin sheet characterized in that the width of the recesses is an average of 15 ⁇ m to 25 ⁇ m and the spacing width of the recesses is an average of 15 ⁇ m to 25 ⁇ m.
  • FIG. 1 is a cross-sectional view showing the cell culture container 1 used in the present embodiment and the culture surface cross-sectional enlarged portion 6 of a part thereof.
  • the cell culture vessel 1 contains a cell culture insert 2 for seeding cells and a bottom well 4 for filling the outside of the cell culture insert 2 with a medium.
  • the cell culture insert 2 includes a porous membrane 3. It is possible to supply nutrients, oxygen, and the like to the cells on the porous membrane 3 from the medium 5 that fills the outside of the cell culture insert 2 through the porous membrane 3.
  • the surface of the porous film 3 is provided with a convex portion 7 for forming irregularities on the three-dimensional cultured skin sheet 10 of the present invention.
  • the epidermal cells are layered, and at least a part of the base portion 11 (see FIG. 2, thick line) of the three-dimensional cultured skin sheet 10 is uneven. A three-dimensional cultured skin sheet 10 having the same is obtained.
  • the "base” of the three-dimensional cultured skin sheet 10 refers to a surface of the epidermal cells formed in a sheet shape in contact with the surface of the cell culture vessel when the epidermal cells are seeded in the cell culture vessel 1, for example. , The surface of the porous membrane 3 shown in FIG. 1 and / or the surface in contact with the convex portion 7 (see FIG. 2, 11, thick line).
  • the “basement membrane” is a structure corresponding to the basement membrane formed between the epidermis and the dermis in the skin tissue of the living body, but does not necessarily have the same membrane structure as the basement membrane of the living body. You don't have to have it.
  • the “basement portion” in the present invention includes a "epidermal basement membrane-like structure” having an uneven shape at least in part, and the epidermal basement membrane-like structure contains epidermal cells.
  • the epidermal basement membrane-like structure may include a basement membrane formed by epidermal cells in the skin tissue of a living body.
  • FIG. 2 is a cross-sectional view showing a three-dimensional cultured skin sheet 10 excluding the porous film 3 and the convex portion 7 from FIG. 1 (b).
  • the three-dimensional cultured skin sheet 10 contains a stratum corneum 9 having no cell nucleus and epidermal cells 8 other than the stratum corneum 9.
  • the convex portion 7 provided on the porous membrane 3 described above forms an epidermal cell non-existent region V in the three-dimensional cultured skin sheet 10.
  • the "unevenness" possessed by the base portion 11 of the three-dimensional cultured skin sheet 10 is formed in at least a part of the surface of the three-dimensional cultured skin sheet of the present invention in contact with the culture surface of the cell culture vessel.
  • the undulations are larger than the undulations obtained accidentally when the epidermal cells are cultured on the flat surface of the culture equipment.
  • the concave portion 110 of the three-dimensional cultured skin sheet 10 refers to a portion of the base portion 11 that is depressed toward the air exposure (upper layer) direction of the cell culture container, and is a convex portion on the porous membrane 3 of FIG. It is a part formed by 7 (see FIG. 2).
  • the convex portion 111 of the three-dimensional cultured skin sheet means a portion of the base portion 11 other than the concave portion 110.
  • the distance between the most depressed portion of any concave portion 110 and the tip end portion of the adjacent convex portion 111 in the vertical direction with respect to the culture surface can be referred to as the height H'of the concave portion 110.
  • the shape and height H'of the recess 110 formed in the three-dimensional cultured skin sheet 10 depends on the shape and height H of the convex portion 7 provided on the porous film 3.
  • the spacing W'of the recesses 110 formed in the three-dimensional cultured skin sheet 10 depends on the spacing width W of the protrusions 7 on the porous film 3.
  • the width Y'of the concave portion 110 formed on the three-dimensional cultured skin sheet 10 depends on the width Y of the convex portion 7 formed by the convex portion 7 on the porous film 3.
  • the height H', spacing width W', and / or width Y'of the recesses 110 of the three-dimensional cultured skin sheet are measured, for example, by preparing a HE-stained tissue section and using a commercially available optical microscope. It may be stained by an immunohistochemical method and observed and measured using a fluorescence microscope, a confocal microscope or a two-photon laser microscope, or it may be observed and measured using an electron microscope, and in particular. Not limited.
  • the height H'of the recess 110 of the three-dimensional cultured skin sheet can be measured from an image acquired by a device equipped with a camera in a normal microscope, a fluorescence microscope, or the like.
  • Olympus Corporation System Industry Microscope combined with epi-illumination transmission
  • BX51 can be used.
  • the camera for example, the Olympus Corporation microscope digital camera "DP71" can be used.
  • the acquired image can be imported into computer analysis software and analyzed by a standard image analysis method.
  • three-dimensional means that the cells are vertical, unlike the state of a substantially one-layer cell layer obtained by culturing adherent cells in a cell culture dish or the like, that is, a two-dimensional cell layer. It refers to a mode in which layers are stacked in a direction.
  • the three-dimensional cultured skin sheet of the present invention refers to a three-dimensional structure in which the epidermal basement membrane-like structure has irregularities and epidermal cells are layered to have a thickness.
  • the "three-dimensional cultured skin sheet” is distinguished from the skin tissue that is naturally present in the living body, and is obtained by decomposing the adhesive protein between cells into pieces.
  • a cell group containing cells derived from skin tissue and / or cells constituting the skin obtained by inducing differentiation from pluripotent stem cells is seeded in a cell culture vessel or the like, cultured in the cell culture vessel, and re-cultured.
  • the cells constituting the three-dimensional cultured skin sheet include epidermal cells, and can also be referred to as a three-dimensional cultured epidermal sheet.
  • the three-dimensional cultured skin sheet or the three-dimensional cultured epidermis sheet refers to cells other than epidermal cells, for example, cells other than epidermal cells (melanin cells, Langerhans cells, Merkel cells, etc.) and / or dermis.
  • the cells contained in the tissue may be contained.
  • the three-dimensional cultured skin sheet of the present invention may further include a porous film 3 having a convex portion 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 mammals, and most preferably humans.
  • the "epidermis cell” refers to a cell containing all cells constituting the epidermis at different differentiation stages.
  • Epidermal cells are mainly composed of cells also called keratinocytes or keratinocytes.
  • epidermal tissue is formed by layering epidermal cells having different differentiation stages.
  • the deepest part of the epidermis is called the basal layer or the basal cell layer (hereinafter referred to as "basal layer”), and it is considered that columnar cells form one layer and contain stem cells.
  • the basal layer is also the interface with the dermis, and the stratum spinosum exists above it.
  • the papillary layer constituting the dermis exists just below the basal layer.
  • the stratum spinosum is also called the stratum spinosum, and is composed of about 2 to 10 layers in living tissue. This layer is called the stratum spinosum because it appears to be connected to each other by spines. Only cells in the basal layer have proliferative capacity, and epidermal cells move to the outer layer while changing to a flat shape as the differentiation stage progresses.
  • a granule layer (granule cell layer) having keratohyalin granules and lamellar granules is formed.
  • the granular layer is composed of about 2 to 3 layers in a living tissue.
  • stratum granulosum the cell nucleus disappears and the stratum granulosum (stratum cell layer) is formed.
  • the epidermis is roughly divided into the above-mentioned four types of layers, and the epidermis tissue contains melanocytes, Langerhans cells, Merkel cells, etc. in addition to epidermal cells, and each of them means that ultraviolet rays reach the dermis. It prevents, plays an important role in the immune function of the skin, and is involved in perception.
  • the three-dimensional cultured skin sheet may contain cells other than epidermal cells, and can be appropriately changed depending on the intended use.
  • the "barrier function" of the skin generally refers to a function of preventing the loss of water and biological components in the body and a function of preventing foreign substances (microorganisms, viruses, dust, etc.) from entering the living body from outside the living body. ..
  • the barrier function of the three-dimensional cultured skin sheet of the present invention can be evaluated by examining the transepidermal water evaporation amount (Transepidermal water loss: TEWL). It is shown that the smaller the transepidermal water evaporation amount from the three-dimensional cultured skin sheet, that is, the water permeation amount, the higher the barrier function.
  • a general method used by those skilled in the art can be used (for example, Kumamoto J., Tsusumi 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 see).
  • the average height H'of the recesses 110 of the three-dimensional cultured skin sheet is, for example, in the range of 1 ⁇ m to 50 ⁇ m, preferably 5 ⁇ m to 40 ⁇ m.
  • the height H'of the recess 110 is within the above range, a three-dimensional cultured skin sheet with enhanced thickening and / or barrier function can be obtained.
  • the average of the spacing width W'of the recesses 110 of the three-dimensional cultured skin sheet is in the range of 15 ⁇ m to 25 ⁇ m, more preferably 17.5 ⁇ m to 22.5 ⁇ m, and most preferably 20 ⁇ m.
  • the interval width W'of the recesses 110 is within the above range, a three-dimensional cultured skin sheet having an enhanced thickening and / or barrier function can be obtained.
  • the average width Y'of the recesses 110 of the three-dimensional cultured skin sheet is in the range of 15 ⁇ m to 25 ⁇ m, more preferably 17.5 ⁇ m to 22.5 ⁇ m, and most preferably 20 ⁇ m.
  • the interval width W'of the recesses 110 is within the above range, a three-dimensional cultured skin sheet having an enhanced thickening and / or barrier function can be obtained.
  • the three-dimensional cultured skin sheet of the present invention has a recess 110 in the basal portion 11 at least in a part thereof, the epidermal cell layer on the basal portion 11 (for example, as compared with the one having no recess 110).
  • a three-dimensional cultured skin sheet in which the spinous layer, the stratum granulosum and / or the stratum corneum) is thickened is obtained.
  • the three-dimensional cultured skin sheet obtained by the present invention is physically formed on the base 11 by a convex portion composed of a non-biological substance, not by a convex portion composed of a biological substance such as a cell or a cell adhesion protein. It exerts an excellent effect of thickening only by forming the recess 110.
  • the convex portion is a convex portion composed of a non-biological substance
  • the convex portion is not decomposed even during the culture period and when used thereafter, and the base portion of the three-dimensional cultured skin sheet is stably obtained.
  • the shape of the recess can be maintained, and the effect of the present invention can be maintained.
  • the average height, shape and spacing width of the recesses 110 of the three-dimensional cultured skin sheet of the present invention can be controlled by the height, shape and spacing width of the protrusions provided on the surface of the porous membrane of the cell culture vessel. is there.
  • the three-dimensional cultured skin sheet obtained by the present invention has a smaller amount of transepidermal water loss and a higher barrier function than the conventional three-dimensional cultured skin sheet.
  • the barrier function is enhanced.
  • the base portion 11 of the three-dimensional cultured skin sheet is preferably in close contact with the culture surface (the culture surface including the porous film and the convex portion).
  • the barrier function is further improved. More preferably, the base portion 11 of the three-dimensional cultured skin sheet is in close contact with the entire surface of the culture surface.
  • the convex portion provided on the surface of the porous membrane may be composed of a biological substance or a non-biological substance, and may be composed of a combination of the biological substance and the non-biological substance. It may be a thing.
  • non-biomaterial refers to a biomaterial, that is, a biopolymer (nucleic acid, protein, polysaccharide and its components (nucleotides, nucleosides, amino acids, sugars)) constituting the living body, vitamins and the like. Refers to substances excluding.
  • the non-biomaterial used to form the convex portion of the present invention is preferably a biocompatible substance that does not affect cell culture.
  • the convex portion provided on the surface of the porous membrane is further coated with biological substances such as collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (D-form, L-form), thrombospondin and the like. It may be the one that has been done.
  • the cell used in the present invention may be a primary representative skin cell, an epidermal cell obtained by subculturing and proliferating the primary representative skin cell, and is pluripotent such as ES cell, iPS cell, or Muse cell.
  • Epidermal cells obtained by inducing differentiation from sex stem cells may be used, or may be established epidermal cells.
  • it is a primary cultured epidermal cell collected from a living tissue and seeded, or a subcultured representative skin cell obtained by further subculturing the primary cultured cell.
  • Epidermal cells obtained from living tissues are likely to maintain the same properties as living organisms, and are used for conducting tests to investigate the efficacy and side effects of drugs and for conducting basic research. It is convenient at the time.
  • the primary cultured epidermal cells are epidermal cells collected from a living tissue and then cultured and collected only once in a cell culture vessel, and the epidermis has "0 number of passages (or 1st generation)". Also called a cell. Epidermal cells that have become confluent or subconfluent and have 0 passages can be further amplified and cultured by subconfluence.
  • the epidermal cells obtained by one passage operation are referred to as epidermal cells having "passage number 1 (or 2nd generation)". Corresponding to the number of passage operations, it can be expressed as "number of passages 2, 3, 4 ... n (n (integer) is the number of passages) (n + 1 generation)".
  • primary cultured cells for example, frozen NHEK (NB) manufactured by Kurabo Industries Ltd., catalog number: KK-4009
  • primary cultured cells having a number of passages of 0 to about 2.
  • the number of passages may be referred to the number of passages or the number of generations described in the attached documents and the like.
  • a step of freezing the cells may be included between each passage operation.
  • the primary cultured cells are cells directly isolated from living tissues, and the number of passages is small, so that the number of cells that can be supplied is limited. Not only is the size and amount of tissue that can be harvested limited due to ethical issues, but there are also variations in the properties of cells obtained by donors. Although some primary cultured cells are commercially available, they are inevitably expensive in terms of supply. Therefore, when it is necessary to construct a large amount of three-dimensional cultured skin sheet, it is necessary to obtain a large amount of expensive cells, which poses a problem in terms of cost. In order to produce the cells at low cost, it is possible to subculture and proliferate the primary cultured cells for use, but as described above, the obtained cells do not necessarily exhibit the same properties as the primary cultured cells. Is not always.
  • the first representative skin cells and kits for making the cells three-dimensional are commercially available, and it is possible to purchase and prepare them (for example, keratinocyte three-dimensional).
  • Culture starter kit, CELLnTEC When epidermal cells with two or more passages are used, the property of spontaneously becoming three-dimensional is weakened, and a thick three-dimensional cultured skin model (also referred to as a three-dimensional cultured skin sheet) is created. There are problems such as not being able to obtain it.
  • the thickening that has conventionally been obtained only with primary cultured cells for example, the number of passages is 0 or 1.
  • the three-dimensional cultured skin sheet is constructed even when epidermal cells having a number of passages of 2 or more are used after isolation and culture from a living tissue.
  • primary cultured cells for example, the number of passages are 0 or 1
  • a thickened three-dimensional cultured skin sheet according to the present invention can be obtained.
  • the number of passages of epidermal cells used may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more, and the spinous layer of the 3D cultured skin sheet.
  • Granular layer, and / or stratum granulosum is not limited as long as it thickens.
  • the animal species of the primary epidermal cells used is not particularly limited, but is preferably derived from humans.
  • the primary epidermal cells used may be of any of fetal, neonatal, minor, adult-derived, etc., but preferably fetal, neonatal, or minor-derived cells. Is.
  • cells derived from human minors for example, it is preferable to use cells under 20 years old, 1-19 years old, 1-10 years old, 1-5 years old. Even when cells derived from human adults are used, it is preferable to use young cells, for example, cells aged 20 to 29, 30 to 39, and 40 to 49.
  • the average thickness of the three-dimensional cultured skin sheet is 25 ⁇ m or more, 30 ⁇ m or more, 35 ⁇ m or more, 40 ⁇ m or more, 45 ⁇ m or more, 50 ⁇ m or more, 55 ⁇ m or more, 60 ⁇ m or more, 65 ⁇ m or more, 70 ⁇ m or more.
  • the preferred average thickness is 50 ⁇ m or greater.
  • the upper limit of the average thickness of the three-dimensional cultured skin sheet is not particularly limited because it changes depending on the animal species of the epidermal cells, the number of passages, the age, and the like.
  • the present invention A porous membrane provided on at least a part of the culture surface, It is provided with a convex portion formed on the porous membrane and for forming a concave portion at the base portion of the three-dimensional cultured skin sheet.
  • a cell culture container for producing a three-dimensional cultured skin sheet characterized in that the width of the convex portions is 15 ⁇ m to 25 ⁇ m on average and the interval width of the convex portions is 15 ⁇ m to 25 ⁇ m on average. provide.
  • FIG. 3 shows an embodiment of the cell culture vessel 1 of the present invention.
  • a convex portion 71 for forming a concave portion 110 in the three-dimensional cultured skin sheet of the present invention is directly formed on the porous film 3.
  • the convex portion 71 has a cylindrical structure.
  • the convex portion 71a can be arranged so as to be located at a square grid point.
  • the convex portion 71b is arranged so as to be located at a grid point of an equilateral triangle.
  • the convex spacing width W1b it is possible to control the convex spacing width W'of the above-mentioned three-dimensional cultured skin sheet of the present invention.
  • 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.
  • FIG. 4 shows an embodiment of the cell culture vessel 1 of the present invention.
  • a convex portion 72 for forming irregularities on the three-dimensional cultured skin sheet of the present invention is arranged on the porous film 3.
  • the convex portion 72 has a cubic shape, which is a kind of prismatic shape.
  • the convex portion 72a can be arranged so as to be located at a square grid point.
  • the convex spacing width W2a By adjusting the convex spacing width W2a, it is possible to control the convex spacing width W'of the above-mentioned three-dimensional cultured skin sheet of the present invention.
  • the center of gravity of the convex portion 72b can be arranged so as to be located at the lattice point of an equilateral triangle.
  • the convex spacing width W2b By adjusting the convex spacing width W2b, it is possible to control the convex spacing width W'of the above-mentioned three-dimensional cultured skin sheet of the present invention.
  • the convex portion width Y2 it is possible to control the concave portion width Y'of the three-dimensional cultured skin sheet of the present invention.
  • FIG. 5 is a cross-sectional view showing an embodiment of a convex portion (73, 74, 75) provided in the cell culture vessel 1 of the present invention.
  • FIG. 5A shows a convex portion 73 having a pyramid shape (triangular pyramid, quadrangular pyramid, or cone).
  • FIG. 5B shows a convex portion 74 having a frustum shape.
  • FIG. 5 (c) shows a convex portion 75 having a bell shape.
  • the shape of the convex portion (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) is not limited to the above, but for example, a substantially hemispherical shape, a substantially rectangular parallelepiped shape, or the like. Is also included.
  • Plasma treatment or the like may be performed on the surface of the convex portion (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) by a known method so that cells can easily adhere to the surface.
  • the average of the spacing widths (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 ⁇ m to 25 ⁇ m, more preferably 17.5 ⁇ m to 22.5 ⁇ m, and most preferably 20 ⁇ m.
  • the spacing width (W, W1, W2, W3, W4, W5) of the protrusions is within the above range, a three-dimensional cultured skin sheet with enhanced thickening and / or barrier function can be obtained.
  • the average height of the protrusions is, for example, in the range of 1 ⁇ m to 50 ⁇ m, preferably 5 ⁇ m to 40 ⁇ m.
  • the height of the protrusions (H, H1, H2, H3, H4, H5) is in the above range, a three-dimensional cultured skin sheet with enhanced thickening and / or barrier function can be obtained.
  • the average of the convex widths (Y, Y1, Y2, Y3, Y4, Y5) is in the range of 15 ⁇ m to 25 ⁇ m, more preferably 17.5 ⁇ m to 22.5 ⁇ m, most preferably. It is preferably 20 ⁇ m.
  • the convex width (Y, Y1, Y2, Y3, Y4, Y5) is in the above range, a three-dimensional cultured skin sheet having an enhanced thickening and / or barrier function can be obtained.
  • the protrusions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) are preferably formed directly on the porous film 3, for example, 3D printer technology or semiconductor process technology (3D printer technology or semiconductor process technology).
  • 3D printer technology or semiconductor process technology 3D printer technology or semiconductor process technology.
  • it can be formed by a method used in a lithography process (photolithography, maskless photolithography, etc.) and dry etching (for example, a method combining reactive ion etching) or wet etching).
  • the material of the convex portion (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be made of a biological substance, may be made of a non-biological substance, or may be a living body. It may be composed of a combination of a substance and a non-biological substance.
  • polyester or polyethylene terephthalate (PET) polycarbonate, polystyrene, zirconia, glass-insoluble collagen, silicone rubber, resin (for example, epoxy resin, acrylic resin, etc.) can be mentioned, and if cell culture is possible, it is formed of other materials. May be done.
  • the convex portions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) are directly formed on the porous film 3. Since the protrusions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) are formed directly on the porous membrane 3, epidermal cells do not sneak into the lower part of the protrusions. , Thickening and / or the effect of improving the barrier function is remarkably 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 usually used for the cell culture vessel.
  • a material usually used for the cell culture vessel For example, glass, polystyrene, polypropylene, polycarbonate and the like.
  • the shape of the cell culture vessel used in the present invention is not particularly limited, and examples thereof include a dish type, a multi-well plate type, and a flask type. Since the cell culture vessel of the present invention has a porous membrane as a part of the cell culture vessel, it is preferably a cell culture insert type.
  • the cell culture insert used in the present invention refers to a cell culture container having a membrane having porous pores through which cells cannot permeate but a culture solution or the like can permeate.
  • the culture solution or the like 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 a commercially available one may be used, or a cell culture insert having a membrane having a convex portion directly on the culture surface of the porous membrane may be used.
  • the average pore size of the porous membrane is 0.1 ⁇ m to 5.0 ⁇ m, preferably 0.2 ⁇ m to 3.0 ⁇ m, and more preferably 0.3 to 1.5 ⁇ m.
  • the porosity of the porous membrane is, for example, 1 ⁇ 10 5 to 1 ⁇ 10 9 pieces / cm 2 , preferably 5 ⁇ 10 5 to 5 ⁇ 10 8 pieces / cm 2 , and more preferably 1 ⁇ 10 6 to 5. ⁇ 10 8 pieces / cm 2 .
  • the average thickness of the porous membrane is, for example, 1 to 50 ⁇ m, preferably 3 to 25 ⁇ m, and more preferably 5 to 20 ⁇ m.
  • polyester or polyethylene terephthalate (PET), polycarbonate, polystyrene or the like may be used as in the case of the porous membrane used for conventional cell culture.
  • the culture surface may be coated so that the cells can easily adhere to and proliferate in the cell culture container having the convex portion on the porous membrane 3.
  • collagen for example, collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (D-form, L-form), thrombospondin, fibrin and the like can be mentioned.
  • the present invention (1) A step of seeding cells containing keratinocytes suspended in a medium on the porous membrane of the above cell culture vessel. (2) A step of culturing a medium in contact with the outside of the porous membrane of the cell culture container. Provided is a method for producing a three-dimensional cultured skin sheet including.
  • the present invention further comprises (3) removing the medium on the porous membrane of the cell culture vessel and culturing the cells while exposing them to air. It may be a method including.
  • the number of cells containing keratinocytes used in the method of the present invention may be according to a known method.
  • the cells are 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 ⁇ . Seed at an amount of 10 6 to 1.0 ⁇ 10 6 pieces / cm 2 .
  • the medium (also referred to as a culture medium) used in the present invention is a medium usually used for culturing epidermal cells, such as KG medium, EpilieKG2 (Kurabou), Human-KG2 (Kurabou), assay medium (TOYOBO), and the like.
  • CnT-Prime Epithelial culture medium (CELLnTEC) and the like can be used, and the operation can be performed at about 37 ° C. for 0 to 14 days.
  • DMEM medium GEBCO
  • CnT-Prime, 3D barrier medium CELLnTEC
  • CELLnTEC 3D barrier medium
  • epidermal cells in order to culture epidermal cells and promote keratinization by making them three-dimensional (multilayered), they are seeded in a cell culture vessel 1 containing the above-mentioned cell culture insert 2 and proliferated and cultured. Just do it.
  • epidermal cells are suspended in a medium and seeded on a cell culture insert 2.
  • the medium is also added to the bottom well 4 so that the medium is brought into contact with the outside of the porous membrane 3 of the cell culture insert, and the cell culture insert 2 is immersed and cultured. As a result, the medium is supplied from both the top and bottom of the epidermal cells and cultured.
  • the epidermal cells on the cell culture insert 2 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. Then, in order to further promote the three-dimensionalization (layering) of the cells, it is preferable to replace the medium inside and outside the cell culture insert with, for example, CnT-Prime, 3D barrier medium (CELLnTEC). This further promotes the three-dimensionalization of epidermal cells. After exchanging the medium with CnT-Prime and 3D barrier medium (CELLnTEC) and culturing, 1 to 36 hours, preferably 6 to 24 hours, more preferably 12 to 18 hours, of the cell culture insert.
  • CnT-Prime CELLnTEC
  • the culture temperature of each culture step in the present invention may be close to the body temperature of the origin animal, and specifically, in the case of human cells, it is preferably about 33 to 38 ° C.
  • the three-dimensional cultured skin sheet obtained by the present invention as described above can be used as one of alternative methods for animal experiments, for example, as a skin model.
  • it can be used as a method for evaluating the reactivity of skin to chemical substances (for example, cosmetics, industrial products, household products, drugs, external preparations for skin, etc.).
  • chemical substances for example, cosmetics, industrial products, household products, drugs, external preparations for skin, etc.
  • the three-dimensional cultured skin sheet of the present invention can obtain a tissue having a thicker and / or enhanced barrier function as compared with the conventional three-dimensional cultured skin sheet, the skin is also useful in basic research of dermatology. It can be used as a model.
  • the three-dimensional cultured skin sheet obtained in the present invention is thicker than the conventional three-dimensional cultured skin sheet, it has a high barrier function from the outside and heals burns, wounds, etc. It is also useful as a three-dimensional cultured skin sheet.
  • 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.
  • the three-dimensional cultured skin sheet produced in the cell culture container is separated from the porous membrane with the convex portion in contact with the base portion thereof. Therefore, the base portion of the three-dimensional cultured skin sheet can be moved while maintaining the uneven structure.
  • the convex portion is a biological substance containing a biological substance such as collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (D-form, L-form), thrombospondin and the like. Is preferable, and insoluble collagen is more preferable.
  • ⁇ Method of evaluating target substances that improve and / or restore the barrier function of the skin using a three-dimensional cultured skin sheet> it is possible to provide a method for evaluating a target substance that improves and / or restores the barrier function of the skin by adding the target substance to the three-dimensional cultured skin sheet of the present invention.
  • changes in the barrier function of the skin can be evaluated by measuring the amount of water evaporation that evaporates from the surface of the three-dimensional cultured skin sheet after adding the target substance to the three-dimensional cultured skin sheet of the present invention (Kumamoto). See J., et al., Arch. Dermatol. Res. 308: 49-54, 2016).
  • the skin is examined by examining the expression of known markers (for example, Filaggrin, Lolicrin, ZO-1, Claudin-1, etc.) related to the barrier function of the skin. Changes in barrier function can be evaluated.
  • the target substances for improving and / or restoring the barrier function of the skin include, for example, low molecular weight compounds, peptides, proteins, mammals (for example, mice, rats, pigs, cows, sheep, monkeys, humans and the like). ) Tissue extract or cell culture supernatant, plant-derived compound or extract (for example, crude drug extract, crude drug-derived compound), and microorganism-derived compound or extract or culture product.
  • keratinocytes neonatal-derived keratinocytes (hereinafter, “keratinocytes”. Kurabo Industries, Ltd., product name: frozen NHEK (NB), catalog number: KK-4009) were used. Subculture was performed according to the instructions provided by the sales company.
  • Example 1 1. Materials and experimental methods 1-1. Preparation of Cell Culture Insert with Convex (Porosity Size: 0.4 ⁇ m) (1) Only the porous membrane is peeled off from 12-Well Millicell Hanging Cell Culture inserts PET (Porosity size: 0.4 ⁇ m, Millipore) to make it porous. The film was heated to 80 ° C. and a photosensitive resin sheet (thickness of 30 ⁇ m or 50 ⁇ m) was pressed by a roller to laminate. (2) A photomask provided with circular holes having an intended width of the convex portion and a width of the interval between the convex portions is covered with a porous film laminated with a photosensitive resin sheet, and UV light is emitted through the photomask.
  • CELLstart CTS (gibco) was diluted 50-fold with DPBS (gibco), and 86 ⁇ L was added dropwise to one cell culture insert. The condition was maintained at 37 ° C. for 2 hours.
  • CELLstart CTS was removed, and 220,000 to 250,000 neonatal-derived keratinocytes with 4 passages were dispersed in 500 ⁇ L of CnT-Prime, Epithelial culture medium (CELLnTEC), dropped into a cell culture insert, and the same medium (CnT) -Prime, Epithelium culture medium) was added dropwise to the outside of a 1 mL cell culture insert, and the cells were cultured in a CO 2 incubator for 72 hours at 37 ° C.
  • CnT-Prime and Epithelium culture medium inside and outside the cell culture insert were removed, replaced with CnT-Prime, 3D barrier medium (CELLnTEC), and cultured in a 37 ° C. CO 2 incubator for 16 hours.
  • a three-dimensional film made of a porous film having no convex portion and a porous film having convex portions designed with a convex portion height of 30 ⁇ m, a convex portion width of 20 ⁇ m, and a convex portion spacing of 20 ⁇ m.
  • Immunohistochemical staining was performed on the cultured skin sheet (Fig. 7). Results of staining with anti-Filaggrin antibody (marker antibody for granule cells), anti-Loricrin antibody (marker antibody for granule layer), anti-ZO-1 antibody and anti-Claudin-1 antibody (marker antibody for tight junction) In particular, ZO-1 and Claudin-1 were more clearly expressed in a wider range with a unique structure.
  • Example 2 A three-dimensional cultured skin sheet was prepared and observed by the same procedure as in Example 1 except that 12-Well Millicell Hanging Cell Culture inserts PET (1.0 ⁇ m, Millipore) was used.
  • Convex portions with varying height (30 ⁇ m), width (15 ⁇ m to 50 ⁇ m) and spacing (15 ⁇ m to 30 ⁇ m) were formed on a porous membrane having pores with an average of 1.0 ⁇ m, and keratinocytes were cultured on the convex portions. 8 and 9).
  • a porous film having convex portions designed with a convex portion height of 30 ⁇ m, a convex portion width of 20 to 25 ⁇ m, and a convex portion interval of 20 to 25 ⁇ m formed the stratum corneum of the three-dimensional cultured skin sheet. The effect of thickening was the highest.
  • a porous membrane having no convex portion a porous membrane having convex portions designed with a height: 30 ⁇ m, a width: 20 ⁇ m, and an interval: 20 ⁇ m, and a height: 30 ⁇ m, a width: 20 ⁇ m, an interval: 20 ⁇ m.
  • Immunohistochemical staining was performed on a three-dimensional cultured skin sheet prepared of a porous membrane having a convex portion designed in (FIG. 10).
  • anti-filaggrin antibody marker antibody for granule cells
  • anti-Loricrin antibody marker antibody for granule layer
  • Example 3 Evaluation of Barrier Function A three-dimensional cultured skin sheet was prepared by the same procedure as in Example 1 except that 12-Well Millicell Hanging Cell Cultureinserts PET (1.0 ⁇ m, Millipore) was used.
  • the cell culture insert on which the three-dimensional cultured skin sheet was constructed was fitted into a silicone rubber container containing the culture solution at the bottom so that water evaporation occurred only from the surface of the epidermis model. Then, the weight of the container in which the insert was fitted was measured every two hours, and the reduced weight was taken as the amount of water evaporation and converted into a value per unit area.
  • the barrier function of a commercially available three-dimensional skin model (Episkin (registered trademark), Nicoderm Research) was also evaluated.
  • the obtained results were statistically analyzed (ANOVA ANOVA and then Scheffe's multiple test analysis), and compared with the three-dimensional cultured skin sheet obtained by culturing only with a membrane, the results of the multiple test, Those with p ⁇ 0.05 were judged to have a significant difference.

Abstract

The present invention provides a three-dimensionally cultured skin sheet, which contains at least keratinocyte and has a plurality of recessed portions in at least a part of a base, characterized in that the average width of the recessed portions is 15-25 μm and the average interval width between the recessed portions is 15-25 μm. The present invention also provides a cell culture container, which is provided with a porous film formed at least in a part of the culture surface and protruded portions formed on the porous film for forming recessed portions in the base of a three-dimensionally cultured skin sheet, characterized in that the average width of the protruded portions is 15-25 μm and the average interval width between the protruded portions is 15-25 μm. The present invention also provides a method for producing a three-dimensionally cultured skin sheet using the cell culture container.

Description

三次元培養皮膚シート、その製造に使用するための細胞培養容器及びその製造方法Three-dimensional cultured skin sheet, cell culture container for use in its production, and its production method
　本発明は、三次元培養皮膚シートに関する。また、本発明は、三次元培養皮膚シートの製造に使用するための細胞培養容器に関する。また、本発明は、三次元培養皮膚シートの製造方法に関する。 The present invention relates to a three-dimensional cultured skin sheet. The present invention also relates to a cell culture container 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.
　皮膚は生体内と生体外の環境を分ける体表を覆う器官である。皮膚は、物理的なバリアとして働き、乾燥や有害物質が生体内へ侵入することから守り、生命の維持に不可欠な役割を果たしている。 The skin is an organ that covers the body surface that separates the environment inside and outside the body. The skin acts as a physical barrier, protects against dryness and the invasion of harmful substances into the body, and plays an essential role in sustaining life.
　高等脊椎動物の皮膚は、最外層から大別すると表皮、真皮、皮下組織の層から形成されている。表皮と真皮の境界面には起伏を有しており、この起伏は、老化に伴い平坦化することが知られている（例えば、非特許文献１参照。）。表皮は、主にケラチノサイトと呼ばれる細胞から構成されており、表皮の最深部（基底層）でケラチノサイトが分裂しながら、上層に向かって有棘層、顆粒層、そして角層へと分化しながら表面へと移動し、やがて垢となって脱落する。このサイクルはヒトの場合概ね４週間で行われる。 The skin of higher vertebrates is roughly divided from the outermost layer to the epidermis, dermis, and subcutaneous tissue. The interface between the epidermis and the dermis has undulations, and it is known that the undulations flatten with aging (see, for example, Non-Patent Document 1). The epidermis is mainly composed of cells called keratinocytes, and the keratinocytes divide in the deepest part (basal layer) of the epidermis, and the surface is differentiated into the stratum spinosum, the stratum granulosum, and the stratum granulosum toward the upper layer. It moves to, and eventually becomes dirt and falls off. This cycle takes approximately 4 weeks for humans.
　表皮構造、及びその機能を生体外で模倣した様々な培養皮膚モデルの構築が試みられている（例えば、特許文献１及び非特許文献２）。近年、培養表面上に凸部を備えた多孔性膜で表皮細胞を培養することによる、肥厚化した三次元培養皮膚モデルが得られることが報告されている（特許文献２参照）。また、培養表面の表面形状が、表皮幹細胞の分化に影響を与えることが報告されている（非特許文献３）。 Attempts have been made to construct various cultured skin models that imitate the epidermis structure and its function 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 in a porous membrane having a convex portion on the culture surface (see Patent Document 2). In addition, it has been reported that the surface shape of the culture surface affects the differentiation of epidermal stem cells (Non-Patent Document 3).
　培養皮膚モデルは、化粧品や皮膚へ塗布する薬剤の安全性試験や、基礎研究等で用いられており、動物を代替するモデルとして注目を集めている。 The cultured skin model is used in safety tests of cosmetics and drugs applied to the skin, basic research, etc., and is attracting attention as a model to replace animals.
特開２０１２－２３５９２１号公報Japanese Unexamined Patent Publication No. 2012-235921 国際公開第２０１７／２２２０６５号International Publication No. 2017/22065
　継代回数が少ない初代培養細胞を用いた場合に限らず、皮膚を構成する任意の細胞を用いたとしても、生体と同等又はそれに近い厚さを有し、バリア機能を備えた三次元培養皮膚を、安価、安定的かつ簡便に得る技術の提供を目的とする。 Not only when primary cultured cells with a small number of passages are used, but also when any cells constituting the skin are used, three-dimensional cultured skin having a thickness equal to or close to that of a living body and having a barrier function. It is an object of the present invention to provide a technique for obtaining cheap, stable and easy.
　本発明者らは、上記課題を解決するために、種々の角度から検討を加えて研究開発を行ってきた。その結果、培養表面上の凸部の設計を最適化した多孔性膜で表皮細胞を培養することにより、従来の培養方法と比較して肥厚化又はバリア機能が亢進した三次元培養皮膚シートが得られることを見出した。すなわち、本発明は以下の発明を包含する。 The present inventors have conducted research and development by examining from various angles in order to solve the above problems. As a result, by culturing the epidermal cells in a porous membrane with an optimized design of the convex portion on the culture surface, a three-dimensional cultured skin sheet having a thickened or enhanced barrier function as compared with the conventional culture method was obtained. I found that it was possible. That is, the present invention includes the following inventions.
　［１］　少なくともケラチノサイトを含み、かつ、基底部の少なくとも一部に複数の凹部を有する三次元培養皮膚シートであって、
　前記凹部の幅が平均１５μｍ～２５μｍであり、かつ、前記凹部の間隔幅が平均１５μｍ～２５μｍであることを特徴とする、三次元培養皮膚シート。
　［２］　前記凹部の高さが、平均５μｍ～４０μｍである、［１］に記載の三次元培養皮膚シート。
　［３］　前記凹部の高さが、平均２５μｍ～３５μｍである、［１］に記載の三次元培養皮膚シート。
　［４］　前記三次元培養皮膚シートの平均の厚さが、５０μｍ以上である、［１］～［３］のいずれか１項に記載の三次元培養皮膚シート。
　［５］　前記基底部に密着させた、複数の凸部を有する多孔性膜をさらに備え、
　ここで前記凸部の幅が平均１５μｍ～２５μｍであり、かつ、前記凸部の間隔幅が平均１５μｍ～２５μｍであり、
　前記凸部が、前記基底部の前記凹部と密着していることを特徴とする、［１］～［４］のいずれか１項に記載の三次元培養皮膚シート。
　［６］　前記凸部の高さが、平均５μｍ～４０μｍである、［５］に記載の三次元培養皮膚シート。
　［７］　前記凸部の高さが、平均２５μｍ～３５μｍである、［５］に記載の三次元培養皮膚シート。
　［８］　前記多孔性膜が、平均０．２μｍ～３μｍの平均孔径を有する、［５］～［７］のいずれか１項に記載の三次元培養皮膚シート。
　［９］　前記凸部が、樹脂からなる、［５］～［８］のいずれか１項に記載の三次元培養皮膚シート。 [1] A three-dimensional cultured skin sheet containing at least keratinocytes and having a plurality of recesses in at least a part of the base.
A three-dimensional cultured skin sheet, wherein the width of the recesses is an average of 15 μm to 25 μm, and the spacing width of 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 5 μm to 40 μm on average.
[3] The three-dimensional cultured skin sheet according to [1], wherein the height of the recesses is 25 μm to 35 μm on average.
[4] The three-dimensional cultured skin sheet according to any one of [1] to [3], wherein the average thickness of the three-dimensional cultured skin sheet is 50 μm or more.
[5] Further provided with a porous membrane having a plurality of convex portions, which is in close contact with the base portion,
Here, the width of the convex portion is 15 μm to 25 μm on average, and the interval width of the convex portion is 15 μm to 25 μm on average.
The three-dimensional cultured skin sheet according to any one of [1] to [4], wherein the convex portion is in close contact with the concave portion of the base portion.
[6] The three-dimensional cultured skin sheet according to [5], wherein the height of the convex portion is 5 μm to 40 μm on average.
[7] The three-dimensional cultured skin sheet according to [5], wherein the height of the convex portion is 25 μm to 35 μm on average.
[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 on average.
[9] The three-dimensional cultured skin sheet according to any one of [5] to [8], wherein the convex portion is made of a resin.
　［１０］　［１］～［９］のいずれか１項に記載の三次元培養皮膚シートを製造するための細胞培養容器であって、
　培養表面の少なくとも一部に設けられた多孔性膜と、
　前記多孔性膜の上に形成され、三次元培養皮膚シートの基底部に凹部を形成するための凸部と、
を備え、
　ここで前記凸部の幅が平均１５μｍ～２５μｍであり、かつ、前記凸部の間隔幅が平均１５μｍ～２５μｍであることを特徴とする、細胞培養容器。
　［１１］　前記凸部の高さが、平均５μｍ～４０μｍである［１０］に記載の細胞培養容器。
　［１２］　前記凸部の高さが、平均２５μｍ～３５μｍである、［１０］に記載の細胞培養容器。
　［１３］　前記多孔性膜が、平均０．２μｍ～３μｍの平均孔径を有する、［１０］～［１２］のいずれか１項に記載の細胞培養容器。
　［１４］　前記凸部が、樹脂からなる、［１０］～［１３］のいずれか１項に記載の細胞培養容器。 [10] A cell culture container for producing the three-dimensional cultured skin sheet according to any one of [1] to [9].
A porous membrane provided on at least a part of the culture surface,
A convex portion formed on the porous membrane and for forming a concave portion in the base portion of the three-dimensional cultured skin sheet,
With
Here, the cell culture vessel is characterized in that the width of the convex portions is on average 15 μm to 25 μm, and the interval width of the convex portions is on average 15 μm to 25 μm.
[11] The cell culture vessel according to [10], wherein the height of the convex portion is 5 μm to 40 μm on average.
[12] The cell culture vessel according to [10], wherein the height of the convex portion 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 on average.
[14] The cell culture container according to any one of [10] to [13], wherein the convex portion is made of resin.
　［１５］　三次元培養皮膚シートの製造方法であって、
　（１）［１０］～［１４］のいずれか１項に記載の細胞培養容器の多孔膜の上に、培地に懸濁したケラチノサイトを含む細胞を播種する工程、
　（２）前記細胞培養容器の前記多孔性膜の外側に培地を接触させて培養する工程、
を含む、方法。
　［１６］　さらに、
　（３）前記細胞培養容器の前記多孔膜の上の培地を除去して、前記細胞を空気に暴露させながら培養する工程、
を含む、［１５］に記載の方法。
　［１７］　前記ケラチノサイトが、生体組織から単離培養後、継代回数２以上のケラチノサイトを含む、［１５］または［１６］に記載の方法。 [15] A method for producing a three-dimensional cultured skin sheet.
(1) A step of seeding cells containing 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 a medium in contact with the outside of the porous membrane of the cell culture container.
Including methods.
[16] Furthermore
(3) A step of removing the medium on the porous membrane of the cell culture container and culturing the cells while exposing them to air.
The method according to [15].
[17] The method according to [15] or [16], wherein the keratinocytes contain keratinocytes having 2 or more passages after being isolated and cultured from a living tissue.
　本発明によれば、従来の三次元培養皮膚シートよりも、肥厚化及び／又はバリア機能が亢進した三次元培養皮膚シートが得られる。これにより、化粧品や皮膚へ塗布する薬剤の安全性試験、基礎研究に有用な三次元皮膚モデルが提供可能となる。また、本発明の細胞培養容器を用いれば、従来の三次元培養皮膚シートよりも、肥厚化及び／又はバリア機能が亢進した三次元培養皮膚シートが安定的かつ安価に提供可能となる。さらにまた、本発明の培養方法を用いれば、従来の三次元培養皮膚シートよりも、肥厚化及び／又はバリア機能が亢進した三次元培養皮膚シートが安定的かつ安価に提供可能となる。 According to the present invention, a three-dimensional cultured skin sheet having a thickened and / or barrier function is obtained as compared with the conventional three-dimensional cultured skin sheet. This makes it possible to provide a three-dimensional skin model useful for safety tests and basic research of cosmetics and drugs applied to the skin. Further, by using the cell culture container of the present invention, a three-dimensional cultured skin sheet having an enhanced thickening and / or barrier function can be provided stably and inexpensively as compared with the conventional three-dimensional cultured skin sheet. Furthermore, by using the culture method of the present invention, it becomes possible to provide a three-dimensional cultured skin sheet having a thickened and / or barrier function enhanced more stably and inexpensively than the conventional three-dimensional cultured skin sheet.
本発明の一実施態様を示す概念図を示す。（ａ）一実施態様における本発明の細胞培養容器の断面図を示す。（ｂ）（ａ）の一部を拡大した図である。The conceptual diagram which shows one Embodiment of this invention is shown. (A) A cross-sectional view of the cell culture vessel of the present invention in one embodiment is shown. (B) is an enlarged view of a part of (a). 本発明の三次元培養皮膚シートの構造を示す概念図である。It is a conceptual diagram which shows the structure of the 3D culture skin sheet of this invention. 一実施態様における本発明の細胞培養容器を示す概念図である。It is a conceptual diagram which shows the cell culture container of this invention in one Embodiment. 一実施態様における本発明の細胞培養容器を示す概念図である。It is a conceptual diagram which shows the cell culture container of this invention in one Embodiment. 一実施態様における本発明の細胞培養容器を示す概念図である。It is a conceptual diagram which shows the cell culture container of this invention in one Embodiment. 凸部を有する多孔性膜上で培養した三次元培皮膚シートのＨＥ染色像を示す。以下の凸幅×間隔を有する平均孔径０．４μｍの多孔性膜上で継代回数４のケラチノサイトを培養した：（Ａ）コントロール（凸部無し）、（Ｂ）凸幅２０μｍ×間隔１００μｍ、（Ｃ）凸幅２５μｍ×間隔２５μｍ、（Ｄ）凸幅５０μｍ×間隔２０μｍ、（Ｅ）凸幅２０μｍ×間隔２０μｍ、（Ｆ）凸幅３０μｍ×間隔３０μｍ、（Ｇ）凸幅５０μｍ×間隔５０μｍ。なお、凸部の高さは、（Ａ）～（Ｆ）：３０μｍ、（Ｇ）：５０μｍである。スケールバー＝５０μｍ。ＳＣ：角層、ＳＧ：顆粒層、ＳＳ：有棘層、ＳＢ：基底層。The HE-stained image of the three-dimensional culture skin sheet cultured on the porous membrane having a convex part is shown. Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width × spacing and an average pore diameter of 0.4 μm: (A) control (no convex portion), (B) convex width 20 μm × spacing 100 μm, ( C) Convex width 25 μm × interval 25 μm, (D) convex width 50 μm × interval 20 μm, (E) convex width 20 μm × interval 20 μm, (F) convex width 30 μm × interval 30 μm, (G) convex width 50 μm × interval 50 μm. The heights of the convex portions are (A) to (F): 30 μm and (G): 50 μm. Scale bar = 50 μm. SC: stratum granulosum, SG: stratum granulosum, SS: stratum spinosum, SB: stratum basale. 凸部を有する多孔性膜上で培養した三次元培皮膚シートのＨＥ染色像及び免疫染色像を示す。以下の凸幅×間隔を有する平均孔径０．４μｍの多孔性膜上で継代回数４のケラチノサイトを培養した：（Ａ）コントロール（凸部無し）、（Ｂ）凸幅２０μｍ×間隔２０μｍ。なお、凸部の高さは、全て３０μｍである。１段目：ＨＥ染色、２段目：抗Ｆｉｌａｇｇｒｉｎ抗体（赤）及びＤＡＰＩ（青）、３段目：抗Ｌｏｒｉｃｒｉｎ抗体（赤）及びＤＡＰＩ（青）、４段目：抗ＺＯ－１抗体（赤）、抗Ｃｌａｕｄｉｎ－１抗体（緑）及びＤＡＰＩ（青）。スケールバー＝５０μｍ。The HE-stained image and the immuno-stained image of the three-dimensional cultured skin sheet cultured on the porous membrane having the convex portion are shown. Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width × spacing and an average pore diameter of 0.4 μm: (A) control (no convex portion), (B) convex width 20 μm × spacing 20 μm. The height of the convex portions is all 30 μm. 1st stage: HE stain, 2nd stage: anti-Filaggrin antibody (red) and DAPI (blue), 3rd stage: anti-Lolicrin antibody (red) and DAPI (blue), 4th stage: anti-ZO-1 antibody (red) ), Anti-Claudin-1 antibody (green) and DAPI (blue). Scale bar = 50 μm. 凸部を有する多孔性膜上で培養した三次元培皮膚シートのＨＥ染色像を示す。以下の凸幅×間隔を有する平均孔径１．０μｍの多孔性膜上で継代回数４のケラチノサイトを培養した：（Ａ）コントロール（凸部無し）、（Ｂ）凸幅２０μｍ×間隔２０μｍ、（Ｃ）凸幅３０μｍ×間隔３０μｍ、（Ｄ）凸幅１５μｍ×間隔１５μｍ、（Ｅ）凸幅２５μｍ×間隔２５μｍ、（Ｆ）凸幅５０μｍ×間隔５０μｍ。なお、凸部の高さは、全て３０μｍである。スケールバー＝５０μｍ。The HE-stained image of the three-dimensional culture skin sheet cultured on the porous membrane having a convex part is shown. Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width × spacing and an average pore diameter of 1.0 μm: (A) control (no convex portion), (B) convex width 20 μm × spacing 20 μm, ( C) Convex width 30 μm × interval 30 μm, (D) convex width 15 μm × interval 15 μm, (E) convex width 25 μm × interval 25 μm, (F) convex width 50 μm × interval 50 μm. The height of the convex portions is all 30 μm. Scale bar = 50 μm. 凸部を有する多孔性膜上で培養した三次元培皮膚シートのＨＥ染色像（図８よりも低倍率）を示す。以下の凸幅×間隔を有する平均孔径１．０μｍの多孔性膜上で継代回数４のケラチノサイトを培養した：（Ａ）コントロール（凸部無し）、（Ｂ）凸幅１５μｍ×間隔１５μｍ、（Ｃ）凸幅２０μｍ×間隔２０μｍ、（Ｄ）凸幅３０μｍ×間隔３０μｍ、（Ｅ）凸幅５０μｍ×間隔２０μｍ。なお、凸部の高さは、全て３０μｍである。The HE-stained image (lower magnification than FIG. 8) of the three-dimensional culture skin sheet cultured on the porous membrane having a convex portion is shown. Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width × spacing and an average pore diameter of 1.0 μm: (A) control (no convex portion), (B) convex width 15 μm × spacing 15 μm, ( C) Convex width 20 μm × interval 20 μm, (D) convex width 30 μm × interval 30 μm, (E) convex width 50 μm × interval 20 μm. The height of the convex portions is all 30 μm. 凸部を有する多孔性膜上で培養した三次元培皮膚シートの免疫染色像を示す。以下の凸幅×間隔を有する平均孔径１．０μｍの多孔性膜上で継代回数４のケラチノサイトを培養した：（Ａ）コントロール（凸部無し）、（Ｂ）凸幅２０μｍ×間隔２０μｍ、（Ｃ）凸幅２５μｍ×間隔２５μｍ。左図：抗Ｆｉｌａｇｇｒｉｎ抗体（赤）及びＤＡＰＩ（青）、右図：抗Ｌｏｒｉｃｒｉｎ抗体（赤）及びＤＡＰＩ（青）。なお、凸部の高さは、全て３０μｍである。An immunostaining image of a three-dimensional cultured skin sheet cultured on a porous membrane having a convex portion is shown. Keratinocytes having a number of passages of 4 were cultured on a porous membrane having the following convex width × spacing and an average pore diameter of 1.0 μm: (A) control (no convex portion), (B) convex width 20 μm × spacing 20 μm, ( C) Convex width 25 μm × interval 25 μm. Left figure: anti-Filaggrin antibody (red) and DAPI (blue), right figure: anti-Filaggrin antibody (red) and DAPI (blue). The height of the convex portions is all 30 μm. 凸部を有する多孔性膜上で培養した三次元培皮膚シートのバリア機能（水分蒸散量）を評価した結果を示す。凸部を有する平均孔径１．０μｍの多孔性膜上で継代回数４のケラチノサイトを培養して得た三次元培皮膚シートの水分蒸散量を調べた。（Ａ）０～２時間まで、（Ｂ）２～４時間まで、（Ｃ）４～６時間までの経皮水分蒸散量（ｍｇ／ｃｍ２／時間）を示している。Ｃｏｎｔｒｏｌ：凸部無し、１５－２０：凸幅１５μｍ×間隔２０μｍ、２０－２０：凸幅２０μｍ×間隔２０μｍ、２５－２５：凸幅２５μｍ×間隔２５μｍ、３０－３０：凸幅３０μｍ×間隔３０μｍ、５０－５０：凸幅５０μｍ×間隔５０μｍ、ＥＰＳ：ＥｐｉＳｋｉｎ（登録商標）。なお、凸部の高さは、全て３０μｍである。The result of evaluating the barrier function (the amount of water evaporation) of the three-dimensional culture skin sheet cultured on the porous membrane having a convex part is shown. The amount of water evaporation of a three-dimensional cultivated skin sheet obtained by culturing keratinocytes having a number of passages of 4 on a porous membrane having a convex portion and an average pore diameter of 1.0 μm was examined. The amount of transepidermal water loss (mg / cm 2 / hour) is shown for (A) 0 to 2 hours, (B) 2 to 4 hours, and (C) 4 to 6 hours. Trade: No convex part, 15-20: Convex width 15 μm x interval 20 μm, 20-20: Convex width 20 μm × interval 20 μm, 25-25: Convex width 25 μm × interval 25 μm, 30-30: Convex width 30 μm × interval 30 μm, 50-50: Convex width 50 μm x spacing 50 μm, EPS: EpiSkin®. The height of the convex portions is all 30 μm.
　以下、本発明を実施するための形態について図面を参照しつつ詳細に説明するが、本発明の技術的範囲は下記の形態のみに限定されることはない。なお、以下の「三次元培養皮膚シート」、「三次元培養皮膚シートを製造するための細胞培養容器」及び「三次元培養皮膚シートの製造方法」のそれぞれで説明されている事項は、「三次元培養皮膚シート」、「三次元培養皮膚シートを製造するための細胞培養容器」及び「三次元培養皮膚シートの製造方法」のいずれにおいても適用され得る。 Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings, but the technical scope of the present invention is not limited to the following embodiments. The matters explained in the following "3D cultured skin sheet", "cell culture container for producing 3D cultured skin sheet", and "method for producing 3D cultured skin sheet" are described in "3D". It can be applied to any of "former cultured skin sheet", "cell culture container for producing 3D cultured skin sheet", and "method for producing 3D cultured skin sheet".
＜三次元培養皮膚シート＞
　一実施態様において、本発明は、少なくともケラチノサイトを含み、かつ、基底部の少なくとも一部に複数の凹部を有する三次元培養皮膚シートであって、
　前記凹部の幅が平均１５μｍ～２５μｍであり、かつ、前記凹部の間隔幅が平均１５μｍ～２５μｍであることを特徴とする、三次元培養皮膚シートを提供する。 <Three-dimensional cultured skin sheet>
In one embodiment, the present invention is a three-dimensional cultured skin sheet containing at least keratinocytes and having a plurality of recesses in at least a part of a basal portion.
Provided is 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 spacing width of the recesses is an average of 15 μm to 25 μm.
　図１は、本実施形態で使用する細胞培養容器１、及び、その一部の培養表面断面拡大部６を示す断面図である。細胞培養容器１は、細胞を播種するセルカルチャーインサート２と、セルカルチャーインサート２の外部に培地を満たすためのボトムウェル４とを含む。セルカルチャーインサート２は多孔性膜３を備えている。多孔性膜３上の細胞へ、多孔性膜３を通してセルカルチャーインサート２の外部を満たす培地５から、栄養分及び酸素等を供給することが可能である。本発明の実施形態において、多孔性膜３の表面には、本発明の三次元培養皮膚シート１０に凹凸を形成するための凸部７を備えている。本発明の細胞培養容器１に表皮細胞を播種して培養することで、表皮細胞が重層化され、三次元培養皮膚シート１０の基底部１１（図２参照、太線）の少なくとも一部に凹凸を有する、三次元培養皮膚シート１０が得られる。 FIG. 1 is a cross-sectional view showing the cell culture container 1 used in the present embodiment and the culture surface cross-sectional enlarged portion 6 of a part thereof. The cell culture vessel 1 contains a cell culture insert 2 for seeding cells and a bottom well 4 for filling the outside of the cell culture insert 2 with a medium. The cell culture insert 2 includes a porous membrane 3. It is possible to supply nutrients, oxygen, and the like to the cells on the porous membrane 3 from the medium 5 that fills the outside of the cell culture insert 2 through the porous membrane 3. In the embodiment of the present invention, the surface of the porous film 3 is provided with a convex portion 7 for forming irregularities on 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 at least a part of the base portion 11 (see FIG. 2, thick line) of the three-dimensional cultured skin sheet 10 is uneven. A three-dimensional cultured skin sheet 10 having the same is obtained.
　本発明において、三次元培養皮膚シート１０の「基底部」とは、表皮細胞を細胞培養容器１に播種した際、シート状に形成される表皮細胞の細胞培養容器表面に接する面をいい、例えば、図１に示す多孔性膜３の表面、及び／又は、凸部７に接する面をいう（図２参照、１１、太線）。また、本発明において、「基底部」とは、生体の皮膚組織における、表皮と真皮の間に形成された基底膜に相当する構造体であるが、必ずしも生体の基底膜と同様の膜構造を有する必要はない。すなわち、本発明における「基底部」は、少なくとも一部に凹凸形状を有する「表皮基底膜様構造体」を含み、該表皮基底膜様構造体は、表皮細胞を含んでいる。また、該表皮基底膜様構造体は、生体の皮膚組織において表皮細胞が形成する基底膜を含んでもよい。 In the present invention, the "base" of the three-dimensional cultured skin sheet 10 refers to a surface of the epidermal cells formed in a sheet shape in contact with the surface of the cell culture vessel when the epidermal cells are seeded in the cell culture vessel 1, for example. , The surface of the porous membrane 3 shown in FIG. 1 and / or the surface in contact with the convex portion 7 (see FIG. 2, 11, thick line). Further, in the present invention, the "basement membrane" is a structure corresponding to the basement membrane formed between the epidermis and the dermis in the skin tissue of the living body, but does not necessarily have the same membrane structure as the basement membrane of the living body. You don't have to have it. That is, the "basement portion" in the present invention includes a "epidermal basement membrane-like structure" having an uneven shape at least in part, and the epidermal basement membrane-like structure contains 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.
　図２は、図１（ｂ）から多孔性膜３及び凸部７を除いた三次元培養皮膚シート１０を示す断面図である。三次元培養皮膚シート１０は、細胞核を有しない角層９、及び、角層９以外の表皮細胞８を含んでいる。上述の多孔性膜３上に備えた凸部７によって、三次元培養皮膚シート１０は、表皮細胞非存在領域Ｖが形成される。 FIG. 2 is a cross-sectional view showing a three-dimensional cultured skin sheet 10 excluding the porous film 3 and the convex portion 7 from FIG. 1 (b). The three-dimensional cultured skin sheet 10 contains a stratum corneum 9 having no cell nucleus and epidermal cells 8 other than the stratum corneum 9. The convex portion 7 provided on the porous membrane 3 described above forms an epidermal cell non-existent region V in the three-dimensional cultured skin sheet 10.
　本発明において、三次元培養皮膚シート１０の基底部１１が有する「凹凸」とは、本発明の三次元培養皮膚シートにおいて、細胞培養容器の培養面に接する面の少なくとも一部に構成されるものであって、表皮細胞を平坦な培養器材表面で培養したときに偶発的に得られる起伏よりも大きな起伏をいう。本発明において、三次元培養皮膚シート１０の凹部１１０とは、細胞培養容器の空気暴露（上層）方向に向かって陥没した基底部１１の部分をいい、図１の多孔性膜３上の凸部７によって形成される部分である（図２参照）。一方、本発明において、三次元培養皮膚シートの凸部１１１とは、基底部１１の凹部１１０以外の部分をいう。任意の凹部１１０の最陥没部と、その隣接する凸部１１１の先端部の、培養面に対する垂直方向の距離を凹部１１０の高さＨ’ということができる。三次元培養皮膚シート１０に形成される凹部１１０の形状及び高さＨ’は、多孔性膜３上に備えられた凸部７の形状及び高さＨに依存する。また、三次元培養皮膚シート１０に形成される凹部１１０の間隔Ｗ’は、多孔性膜３上の凸部７の間隔幅Ｗに依存する。また、三次元培養皮膚シート１０に形成される凹部１１０の幅Ｙ’は、多孔性膜３上の凸部７により形成される凸部７の幅Ｙに依存する。 In the present invention, the "unevenness" possessed by the base portion 11 of the three-dimensional cultured skin sheet 10 is formed in at least a part of the surface of the three-dimensional cultured skin sheet of the present invention in contact with the culture surface of the cell culture vessel. The undulations are larger than the undulations obtained accidentally when the epidermal cells are cultured on the flat surface of the culture equipment. In the present invention, the concave portion 110 of the three-dimensional cultured skin sheet 10 refers to a portion of the base portion 11 that is depressed toward the air exposure (upper layer) direction of the cell culture container, and is a convex portion on the porous membrane 3 of FIG. It is a part formed by 7 (see FIG. 2). On the other hand, in the present invention, the convex portion 111 of the three-dimensional cultured skin sheet means a portion of the base portion 11 other than the concave portion 110. The distance between the most depressed portion of any concave portion 110 and the tip end portion of the adjacent convex portion 111 in the vertical direction with respect to the culture surface can be referred to as the height H'of the concave portion 110. The shape and height H'of the recess 110 formed in the three-dimensional cultured skin sheet 10 depends on the shape and height H of the convex portion 7 provided on the porous film 3. Further, the spacing W'of the recesses 110 formed in the three-dimensional cultured skin sheet 10 depends on the spacing width W of the protrusions 7 on the porous film 3. Further, the width Y'of the concave portion 110 formed on the three-dimensional cultured skin sheet 10 depends on the width Y of the convex portion 7 formed by the convex portion 7 on the porous film 3.
　三次元培養皮膚シートの凹部１１０の高さＨ’、間隔幅Ｗ’、及び／又は、幅Ｙ’は、例えば、ＨＥ染色を行った組織切片を作製し、市販の光学顕微鏡を用いて測定してもよく、免疫組織化学法によって染色し、蛍光顕微鏡、共焦点顕微鏡若しくは二光子レーザー顕微鏡を用いて観察して測定してもよく、電子顕微鏡を用いて観察して測定してもよく、特に限定されない。三次元培養皮膚シートの凹部１１０の高さＨ’は、通常の顕微鏡、蛍光顕微鏡等にカメラを装備した装置により取得される画像から測定することができる。顕微鏡としては、例えば、オリンパス株式会社　システム工業顕微鏡（落射透過兼用）ＢＸ５１を用いることができる。カメラとしては、例えば、オリンパス株式会社顕微鏡用デジタルカメラ「ＤＰ７１」を用いることができる。取得した画像は、画像解析の定法によりコンピュータ解析ソフトに取り込み、解析をすることができる。 The height H', spacing width W', and / or width Y'of the recesses 110 of the three-dimensional cultured skin sheet are measured, for example, by preparing a HE-stained tissue section and using a commercially available optical microscope. It may be stained by an immunohistochemical method and observed and measured using a fluorescence microscope, a confocal microscope or a two-photon laser microscope, or it may be observed and measured using an electron microscope, and in particular. Not limited. The height H'of the recess 110 of the three-dimensional cultured skin sheet can be measured from an image acquired by a device equipped with a camera in a normal microscope, a fluorescence microscope, or the like. As the microscope, for example, Olympus Corporation System Industry Microscope (combined with epi-illumination transmission) BX51 can be used. As the camera, for example, the Olympus Corporation microscope digital camera "DP71" can be used. The acquired image can be imported into computer analysis software and analyzed by a standard image analysis method.
　本明細書において、「三次元」とは、従来の付着細胞を細胞培養皿等での培養で得られるほぼ１層の細胞層、つまり、二次元の細胞層の状態とは異なり、細胞が垂直方向へと積み重なり、重層化した態様をいう。例えば、本発明の三次元培養皮膚シートは、表皮基底膜様構造に凹凸を有し、表皮細胞が重層化されて厚みをもった三次元化した構造体をいう。 In the present specification, "three-dimensional" means that the cells are vertical, unlike the state of a substantially one-layer cell layer obtained by culturing adherent cells in a cell culture dish or the like, that is, a two-dimensional cell layer. It refers to a mode in which layers are stacked in a direction. For example, the three-dimensional cultured skin sheet of the present invention refers to a three-dimensional structure in which the epidermal basement membrane-like structure has irregularities and epidermal cells are layered to have a thickness.
　本明細書において、「三次元培養皮膚シート」とは、生体に生来的に備わっている皮膚組織とは区別されるものであって、細胞間の接着タンパク質を分解してバラバラにして得られた皮膚組織由来の細胞、及び／又は、多能性幹細胞から分化誘導して得られた皮膚を構成する細胞を含む細胞群を、細胞培養容器等に播種し、細胞培養容器上で培養して再構築したシート状の三次元皮膚様組織をいう。本発明において、三次元培養皮膚シートを構成する細胞は表皮細胞を含んでおり、三次元培養表皮シートということもできる。本発明において、三次元培養皮膚シート又は三次元培養表皮シートは、表皮細胞以外の細胞、例えば、表皮を構成する表皮細胞以外の細胞（メラニン細胞、ランゲルハンス細胞、メルケル細胞等）及び／又は、真皮組織に含まれる細胞（線維芽細胞、神経細胞、肥満細胞、形質細胞、血管内皮細胞、組織球、ｍｅｉｓｓｎｅｒ小体、等）が含まれていてもよい。また、本発明の三次元培養皮膚シートは、さらに基底部１１と接触した凸部を有する多孔性膜３を備えるものであってもよい。 In the present specification, the "three-dimensional cultured skin sheet" is distinguished from the skin tissue that is naturally present in the living body, and is obtained by decomposing the adhesive protein between cells into pieces. A cell group containing cells derived from skin tissue and / or cells constituting the skin obtained by inducing differentiation from pluripotent stem cells is seeded in a cell culture vessel or the like, cultured in the cell culture vessel, and re-cultured. The constructed sheet-like three-dimensional skin-like tissue. In the present invention, the cells constituting the three-dimensional cultured skin sheet include epidermal cells, and can also be referred to as a three-dimensional cultured epidermal sheet. In the present invention, the three-dimensional cultured skin sheet or the three-dimensional cultured epidermis sheet refers to cells other than epidermal cells, for example, cells other than epidermal cells (melanin cells, Langerhans cells, Merkel cells, etc.) and / or dermis. The cells contained in the tissue (fibroblasts, nerve cells, obesity cells, plasma cells, vascular endothelial cells, tissue spheres, meshner bodies, etc.) may be contained. Further, the three-dimensional cultured skin sheet of the present invention may further include a porous film 3 having a convex portion in contact with the base portion 11.
　本発明の三次元培養皮膚シートを構成する細胞は、いずれの動物由来であってもよいが、脊椎動物由来が好ましく、哺乳動物由来がより好ましく、ヒト由来であることが最も好ましい。 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 mammals, and most preferably humans.
　本発明において、「表皮細胞」とは、表皮を構成する分化段階の異なる全ての細胞を含む細胞をいう。表皮細胞は、主に、角化細胞又はケラチノサイトとも呼ばれる細胞から構成されている。生体において、表皮組織は、分化段階の異なる表皮細胞が層状に重なって形成されている。表皮の最深部は基底層又は基底細胞層（以下、「基底層」という。）と呼ばれ、円柱状の細胞が一層を形成し、幹細胞を含むと考えられている。基底層は真皮との境界面でもあり、その上層には有棘層が存在する。また、生体においては、基底層の直下に真皮を構成する乳頭層が存在する。 In the present invention, the "epidermis cell" refers to a cell containing all cells constituting the epidermis at different differentiation stages. Epidermal cells are mainly composed of cells also called keratinocytes or keratinocytes. In a living body, epidermal tissue is formed by layering epidermal cells having different differentiation stages. The deepest part of the epidermis is called the basal layer or the basal cell layer (hereinafter referred to as "basal layer"), and it is considered that columnar cells form one layer and contain stem cells. The basal layer is also the interface with the dermis, and the stratum spinosum exists above it. Moreover, in the living body, the papillary layer constituting the dermis exists just below the basal layer.
　有棘層とは、有棘細胞層とも呼ばれ、生体組織においては２～１０層程度から構成されている。この層は、互いに棘で繋がっているように見えるために有棘層と呼ばれる。基底層の細胞のみが増殖能を有しており、表皮細胞は分化段階が進むにつれて扁平な形状に変化しながら外層へと移動する。 The stratum spinosum is also called the stratum spinosum, and is composed of about 2 to 10 layers in living tissue. This layer is called the stratum spinosum because it appears to be connected to each other by spines. Only cells in the basal layer have proliferative capacity, and epidermal cells move to the outer layer while changing to a flat shape as the differentiation stage progresses.
　有棘層より分化段階が進むと、ケラトヒアリン顆粒、ラメラ顆粒を有する顆粒層（顆粒細胞層）を形成する。顆粒層は、生体組織においては２～３層程度で構成されている。顆粒層よりさらに分化段階が進むと、細胞核が消失し、角層（角層細胞層）を形成する。表皮は大別して上述の４種類の層を構成するが、表皮組織は、表皮細胞の他にも、メラニン細胞、ランゲルハンス細胞、メルケル細胞等が含まれており、それぞれ紫外線が真皮へ到達することを防いだり、皮膚の免疫機能に重要な役割を果たしたり、知覚に関与したりする。本発明においては、三次元培養皮膚シートは、表皮細胞以外の細胞が含まれても良く、使用する用途に応じて適宜変更することが可能である。 When the differentiation stage progresses from the spinous layer, a granule layer (granule cell layer) having keratohyalin granules and lamellar granules is formed. The granular layer is composed of about 2 to 3 layers in a living tissue. When the differentiation stage progresses further than the stratum granulosum, the cell nucleus disappears and the stratum granulosum (stratum cell layer) is formed. The epidermis is roughly divided into the above-mentioned four types of layers, and the epidermis tissue contains melanocytes, Langerhans cells, Merkel cells, etc. in addition to epidermal cells, and each of them means that ultraviolet rays reach the dermis. It prevents, plays an important role in the immune function of the skin, and is involved in perception. In the present invention, the three-dimensional cultured skin sheet may contain cells other than epidermal cells, and can be appropriately changed depending on the intended use.
　皮膚が備える「バリア機能」とは、一般に、体内の水分や生体成分の損失を防止する機能や、生体外部から生体内部へ異物（微生物、ウイルス、ホコリ等）が入ることを防止する機能をいう。本明細書において、本発明の三次元培養皮膚シートが有するバリア機能は、経表皮水分蒸散量（Ｔｒａｎｓｅｐｉｄｅｒｍａｌ　ｗａｔｅｒ　ｌｏｓｓ：ＴＥＷＬ）を調べることにより評価することができる。三次元培養皮膚シートからの経表皮水分蒸散量、すなわち、水分透過量が少ないほど、バリア機能が高いことを示す。本発明において、経表皮水分蒸散量を調べる方法は、当業者に用いられる一般的な方法を用いることができる（例えば、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を参照のこと）。 The "barrier function" of the skin generally refers to a function of preventing the loss of water and biological components in the body and a function of preventing foreign substances (microorganisms, viruses, dust, etc.) from entering the living body from outside the living body. .. In the present specification, the barrier function of the three-dimensional cultured skin sheet of the present invention can be evaluated by examining the transepidermal water evaporation amount (Transepidermal water loss: TEWL). It is shown that the smaller the transepidermal water evaporation amount from the three-dimensional cultured skin sheet, that is, the water permeation amount, the higher the barrier function. In the present invention, as a method for examining the transepidermal water loss amount, a general method used by those skilled in the art can be used (for example, Kumamoto J., Tsusumi 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 see).
　本発明において、三次元培養皮膚シートの凹部１１０の高さＨ’の平均は、例えば、１μｍ～５０μｍ、好ましくは５μｍ～４０μｍの範囲である。凹部１１０の高さＨ’が上記の範囲であれば、肥厚化及び／又はバリア機能が亢進した三次元培養皮膚シートが得られる。 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. When the height H'of the recess 110 is within the above range, a three-dimensional cultured skin sheet with enhanced thickening and / or barrier function can be obtained.
　本発明において、三次元培養皮膚シートの凹部１１０の間隔幅Ｗ’の平均は、１５μｍ～２５μｍの範囲であり、より好ましくは、１７．５μｍ～２２．５μｍ、最も好ましくは２０μｍである。凹部１１０の間隔幅Ｗ’が上記の範囲であれば、肥厚化及び／又はバリア機能が亢進した三次元培養皮膚シートが得られる。 In the present invention, the average of the spacing width W'of the recesses 110 of 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. When the interval width W'of the recesses 110 is within the above range, a three-dimensional cultured skin sheet having an enhanced thickening and / or barrier function can be obtained.
　本発明において、三次元培養皮膚シートの凹部１１０の幅Ｙ’の平均は、１５μｍ～２５μｍの範囲であり、より好ましくは、１７．５μｍ～２２．５μｍ、最も好ましくは２０μｍである。凹部１１０の間隔幅Ｗ’が上記の範囲であれば、肥厚化及び／又はバリア機能が亢進した三次元培養皮膚シートが得られる。 In the present invention, the average width Y'of the recesses 110 of 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. When the interval width W'of the recesses 110 is within the above range, a three-dimensional cultured skin sheet having an enhanced thickening and / or barrier function can be obtained.
　本発明の三次元培養皮膚シートは、その少なくとも一部に、基底部１１に凹部１１０を有することより、凹部１１０を有さないものと比較して、基底部１１上の表皮細胞層（例えば、有棘層、顆粒層及び／又は角質層）が、肥厚化した三次元培養皮膚シートが得られる。特に、本発明によって得られる三次元培養皮膚シートは、細胞や細胞接着タンパク質などの生体物質から構成される凸部によらず、非生体物質から構成される凸部によって物理的に基底部１１に凹部１１０を形成させるだけで肥厚化するという優れた効果を発揮する。また、凸部が、非生体物質から構成される凸部である場合、培養期間中及びその後使用する場合においても、凸部が分解されることなく、安定的に三次元培養皮膚シートの基底部に凹部の形状を維持することができ、本発明の効果を持続させることができる。本発明の三次元培養皮膚シートの凹部１１０の平均の高さ、形状及び間隔幅は、細胞培養容器の多孔性膜表面に設けられた凸部の高さ、形状及び間隔幅等により制御可能である。また、本発明により得られる三次元培養皮膚シートは、従来の三次元培養皮膚シートと比較して経皮水分蒸散量が少なく、バリア機能が高い。三次元培養皮膚シートの基底部１１の少なくとも一部に凹部１１０を有することにより、バリア機能が亢進する。三次元培養皮膚シートの基底部１１は、培養表面（多孔性膜と凸部とを含む培養面）と密着していることが好ましい。三次元培養皮膚シートの基底部１１が培養表面に密着していると、バリア機能がより向上する。より好ましくは、三次元培養皮膚シートの基底部１１が培養表面の全面に密着している状態である。 Since the three-dimensional cultured skin sheet of the present invention has a recess 110 in the basal portion 11 at least in a part thereof, the epidermal cell layer on the basal portion 11 (for example, as compared with the one having no recess 110). A three-dimensional cultured skin sheet in which the spinous layer, the stratum granulosum and / or the stratum corneum) is thickened is obtained. In particular, the three-dimensional cultured skin sheet obtained by the present invention is physically formed on the base 11 by a convex portion composed of a non-biological substance, not by a convex portion composed of a biological substance such as a cell or a cell adhesion protein. It exerts an excellent effect of thickening only by forming the recess 110. In addition, when the convex portion is a convex portion composed of a non-biological substance, the convex portion is not decomposed even during the culture period and when used thereafter, and the base portion of the three-dimensional cultured skin sheet is stably obtained. The shape of the recess can be maintained, and the effect of the present invention can be maintained. The average height, shape and spacing width of the recesses 110 of the three-dimensional cultured skin sheet of the present invention can be controlled by the height, shape and spacing width of the protrusions provided on the surface of the porous membrane of the cell culture vessel. is there. Further, the three-dimensional cultured skin sheet obtained by the present invention has a smaller amount of transepidermal water loss and a higher barrier function than the conventional three-dimensional cultured skin sheet. By having the recess 110 in at least a part of the base portion 11 of the three-dimensional cultured skin sheet, the barrier function is enhanced. The base portion 11 of the three-dimensional cultured skin sheet is preferably in close contact with the culture surface (the culture surface including the porous film and the convex portion). When the base portion 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 portion 11 of the three-dimensional cultured skin sheet is in close contact with the entire surface of the culture surface.
　本発明において、多孔性膜表面に設けられた凸部は、生体物質により構成されたものでもよく、非生体物質により構成されたものでもよく、生体物質と非生体物質とを組み合わせて構成されたものであってもよい。本明細書において、「非生体物質」とは、生体物質、すなわち、生体を構成する生体高分子（核酸、タンパク質、多糖及びこれらの構成要素（ヌクレオチド、ヌクレシド、アミノ酸、糖））や、ビタミンなどを除く物質をいう。本発明の凸部を構成するために用いられる非生体物質は、細胞の培養に影響を与えない生体適合性物質であることが好ましい。本発明において、多孔性膜表面に設けられた凸部は、さらに、表面に生体物質、例えば、コラーゲン、フィブロネクチン、ラミニン、ゼラチン、ビトロネクチン、ポリリジン（Ｄ体、Ｌ体）、トロンボスポンジン等がコートされたものであってもよい。 In the present invention, the convex portion provided on the surface of the porous membrane may be composed of a biological substance or a non-biological substance, and may be composed of a combination of the biological substance and the non-biological substance. It may be a thing. In the present specification, the term "non-biomaterial" refers to a biomaterial, that is, a biopolymer (nucleic acid, protein, polysaccharide and its components (nucleotides, nucleosides, amino acids, sugars)) constituting the living body, vitamins and the like. Refers to substances excluding. The non-biomaterial used to form the convex portion of the present invention is preferably a biocompatible substance that does not affect cell culture. In the present invention, the convex portion provided on the surface of the porous membrane is further coated with biological substances such as collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (D-form, L-form), thrombospondin and the like. It may be the one that has been done.
　従来の三次元培養皮膚シートは、細胞外マトリックスを構成するタンパク質を含むゲルを、凹凸を有するモールドに流し込んで形成した凹凸面に、表皮細胞を播種することで作製していた（例えば、米国特許出願公開第２０１１／０１７１１８０号明細書）。しかし、当該方法では、培養面全体がタンパク質を含むゲルによって形成されているため、これらが培養中に分解されてしまい、その結果、細胞が接着できず、三次元培養皮膚シートが収縮するという問題があった。一方、本発明では、該凸部が、伸縮しない多孔性膜表面上に設けられており、培養しても培養面は分解されないため、収縮していない三次元培養皮膚シートを得ることができる。 Conventional three-dimensional cultured skin sheets have been prepared by inoculating epidermal cells on an uneven surface formed by pouring a gel containing proteins constituting an extracellular matrix into a mold having irregularities (for example, US patent). Application Publication No. 2011/0171180). However, in this method, since the entire culture surface is formed by a gel containing proteins, these are decomposed during the culture, and as a result, cells cannot adhere and the three-dimensional cultured skin sheet contracts. was there. On the other hand, in the present invention, the convex portion is provided on the surface of the porous membrane that does not expand and contract, and the cultured surface is not decomposed even when cultured, so that a non-shrinkable three-dimensional cultured skin sheet can be obtained.
　本発明において用いられる細胞は、初代表皮細胞であってもよく、初代表皮細胞を継代操作して増殖させた表皮細胞であってもよく、ＥＳ細胞、ｉＰＳ細胞、又はＭｕｓｅ細胞等の多能性幹細胞から分化誘導して得られる表皮細胞を用いてもよく、株化された表皮細胞であってもよい。好ましくは、生体組織から採取して播種した初代培養表皮細胞、又は該初代培養細胞をさらに継代処理して得られた継代表皮細胞である。生体組織から採取して得られた表皮細胞であれば、生体と同様の性質を維持している可能性が高く、薬剤の効能、副作用を調べる試験を行ったり、基礎研究を行う上で使用する際に都合が良い。 The cell used in the present invention may be a primary representative skin cell, an epidermal cell obtained by subculturing and proliferating the primary representative skin cell, and is pluripotent such as ES cell, iPS cell, or Muse cell. Epidermal cells obtained by inducing differentiation from sex stem cells may be used, or may be established epidermal cells. Preferably, it is a primary cultured epidermal cell collected from a living tissue and seeded, or a subcultured representative skin cell obtained by further subculturing the primary cultured cell. Epidermal cells obtained from living tissues are likely to maintain the same properties as living organisms, and are used for conducting tests to investigate the efficacy and side effects of drugs and for conducting basic research. It is convenient at the time.
　本明細書において、初代培養表皮細胞とは、生体組織から採取した後に細胞培養容器で一度だけ培養されて回収された表皮細胞であり、「継代回数０（又は、第１世代）」の表皮細胞ともいう。コンフルエント又はサブコンフルエントとなった継代回数０の表皮細胞を、継代操作により、さらに増幅培養することが可能である。１度の継代操作により得られた表皮細胞は、「継代回数１（又は、第２世代）」の表皮細胞という。継代操作数に対応して「継代回数２、３、４・・・ｎ（ｎ（整数）は継代回数）（第ｎ＋１世代）」と表現することができる。市販の初代培養細胞（例えば、クラボウ社の凍結ＮＨＥＫ（ＮＢ）、カタログ番号：ＫＫ－４００９）は、継代回数０～継代回数２程度のものを初代培養細胞として提供されている。市販の初代培養細胞を本発明に用いる場合、継代回数は、添付の書類等に記載されている継代回数又は世代数を参考すればよい。各継代操作の間に、細胞を凍結処理する工程が含まれていてもよい。 In the present specification, the primary cultured epidermal cells are epidermal cells collected from a living tissue and then cultured and collected only once in a cell culture vessel, and the epidermis has "0 number of passages (or 1st generation)". Also called a cell. Epidermal cells that have become confluent or subconfluent and have 0 passages can be further amplified and cultured by subconfluence. The epidermal cells obtained by one passage operation are referred to as epidermal cells having "passage number 1 (or 2nd generation)". Corresponding to the number of passage operations, it can be expressed as "number of passages 2, 3, 4 ... n (n (integer) is the number of passages) (n + 1 generation)". Commercially available primary cultured cells (for example, frozen NHEK (NB) manufactured by Kurabo Industries Ltd., catalog number: KK-4009) are provided as primary cultured cells having a number of passages of 0 to about 2. When a commercially available primary cultured cell is used in the present invention, the number of passages may be referred to the number of passages or the number of generations described in the attached documents and the like. A step of freezing the cells may be included between each passage operation.
　通常、株化されていない体細胞の多くは、継代操作を繰り返すことによって性質が変化したり、構成される細胞の割合が変化したりするため、継代回数が少ない細胞集団とは異なる細胞集団へと変化する。これは継代を繰り返して細胞分裂を繰り返す度にテロメアが短くなったり、コンフルエントになること及び／又はトリプシン処理等によって、生物学的なストレス及び／又は物理的なストレスが加わったことによって細胞の老化が引き起こされることが原因と考えられている。そのため、通常は、培養細胞を使用した組織モデルを作製する場合は、継代回数が少ない細胞が用いられる。 Usually, many unstrained somatic cells are different from the cell population with a small number of passages because their properties change and the proportion of cells composed changes by repeating the passage operation. Transform into a group. This is due to the shortening of telomeres, confluence, and / or the addition of biological and / or physical stress due to trypsin treatment, etc., with each successive passage and repeated cell division. It is believed that the cause is aging. Therefore, usually, when a tissue model using cultured cells is prepared, cells with a small number of passages are used.
　しかしながら、初代培養細胞は、直接生体組織から単離された細胞であり、継代回数が少ないために、供給可能な細胞数に限界がある。倫理的な問題から採取できる組織の大きさや量が限られているだけでなく、ドナーによっても得られる細胞の性質にばらつきも存在する。初代培養細胞は市販されているものもあるが、供給量の点から、必然的に高価格となる。そのため、大量の三次元培養皮膚シートを構築する必要がある場合は、高価な細胞を大量に入手する必要があり、コスト面で問題があった。安価に製造するためには、初代培養細胞を継代して増殖させて使用することも可能であるが、上述のように、得られた細胞は、必ずしも初代培養細胞と同様の性質を示すとは限らない。表皮細胞の三次元培養皮膚モデルを作製する場合、初代表皮細胞やその細胞を三次元化するキットが市販されており、それを購入して作製することが可能であるが（例えば、ケラチノサイト三次元培養スターターキット、ＣＥＬＬｎＴＥＣ社）、２以上の継代回数の表皮細胞を用いた場合、自発的に三次元化する性質が弱まり、厚い三次元培養皮膚モデル（三次元培養皮膚シートともいう。）が得られない等の問題がある。 However, the primary cultured cells are cells directly isolated from living tissues, and the number of passages is small, so that the number of cells that can be supplied is limited. Not only is the size and amount of tissue that can be harvested limited due to ethical issues, but there are also variations in the properties of cells obtained by donors. Although some primary cultured cells are commercially available, they are inevitably expensive in terms of supply. Therefore, when it is necessary to construct a large amount of three-dimensional cultured skin sheet, it is necessary to obtain a large amount of expensive cells, which poses a problem in terms of cost. In order to produce the cells at low cost, it is possible to subculture and proliferate the primary cultured cells for use, but as described above, the obtained cells do not necessarily exhibit the same properties as the primary cultured cells. Is not always. When creating a three-dimensional cultured skin model of epidermal cells, the first representative skin cells and kits for making the cells three-dimensional are commercially available, and it is possible to purchase and prepare them (for example, keratinocyte three-dimensional). Culture starter kit, CELLnTEC) When epidermal cells with two or more passages are used, the property of spontaneously becoming three-dimensional is weakened, and a thick three-dimensional cultured skin model (also referred to as a three-dimensional cultured skin sheet) is created. There are problems such as not being able to obtain it.
　本発明の実施形態において、三次元培養皮膚シートの基底部１１に凹部１１０を有したものであれば、従来、初代培養細胞（例えば、継代回数０又は１）でしか得られなかった肥厚化した三次元培養皮膚シートが、生体組織から単離培養後、継代回数２以上の表皮細胞を用いた場合であっても構築される。初代培養細胞（例えば、継代回数０又は１）を用いた場合も、本発明によって肥厚化した三次元培養皮膚シートが得られる。使用される表皮細胞の継代回数は、例えば、１、２、３、４、５、６、７、８、９、１０、又は１０以上であってよく、三次元培養皮膚シートの有棘層、顆粒層、及び／又は角層が肥厚化するものであれば限定されない。本発明の実施形態において、使用される初代表皮細胞の動物種は、特に限定されないが、好ましくはヒト由来である。また、本発明の実施形態において、使用される初代表皮細胞は、胎児、新生児、未成年、又は成人由来等いずれのものであってもよいが、好ましくは胎児、新生児、又は未成年由来の細胞である。ヒトの未成年由来の細胞を用いる場合、例えば、２０歳未満、１～１９歳、１～１０歳、１～５歳の細胞を用いるのが好ましい。ヒトの成人由来の細胞を用いる場合であっても、若年、例えば、２０歳～２９歳、３０歳～３９歳、４０歳～４９歳の細胞を用いることが好ましい。 In the embodiment of the present invention, if the base portion 11 of the three-dimensional cultured skin sheet has a recess 110, the thickening that has conventionally been obtained only with primary cultured cells (for example, the number of passages is 0 or 1). The three-dimensional cultured skin sheet is constructed even when epidermal cells having a number of passages of 2 or more are used after isolation and culture from a living tissue. When primary cultured cells (for example, the number of passages are 0 or 1) are also used, a thickened three-dimensional cultured skin sheet according to the present invention can be obtained. The number of passages of epidermal cells used may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more, and the spinous layer of the 3D cultured skin sheet. , Granular layer, and / or stratum granulosum is not limited as long as it thickens. In the embodiment of the present invention, the animal species of the primary epidermal cells used is not particularly limited, but is preferably derived from humans. Further, in the embodiment of the present invention, the primary epidermal cells used may be of any of fetal, neonatal, minor, adult-derived, etc., but preferably fetal, neonatal, or minor-derived cells. Is. When using cells derived from human minors, for example, it is preferable to use cells under 20 years old, 1-19 years old, 1-10 years old, 1-5 years old. Even when cells derived from human adults are used, it is preferable to use young cells, for example, cells aged 20 to 29, 30 to 39, and 40 to 49.
　本発明の一実施形態において、三次元培養皮膚シートの平均の厚さは、２５μｍ以上、３０μｍ以上、３５μｍ以上、４０μｍ以上、４５μｍ以上、５０μｍ以上、５５μｍ以上、６０μｍ以上、６５μｍ以上、７０μｍ以上、７５μｍ以上、８０μｍ以上、８５μｍ以上、９０μｍ以上、９５μｍ以上、１００μｍ以上、１０５μｍ以上、１１０μｍ以上、１１５μｍ以上、１２０μｍ以上、１２５μ以上、１５０μｍ以上、１７５μｍ以上、２００μｍ以上、２２５μｍ以上、２５０μｍ以上、２７５μｍ以上、３００μｍ以上であってもよい。好ましい平均の厚さは５０μｍ以上である。三次元培養皮膚シートの平均の厚さの上限値は、表皮細胞の動物種、継代回数、年齢等により変化するため、特に限定されない。 In one embodiment of the present invention, the average thickness of the three-dimensional cultured skin sheet is 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 , 300 μm or more. The preferred average thickness is 50 μm or greater. The upper limit of the average thickness of the three-dimensional cultured skin sheet is not particularly limited because it changes depending on the animal species of the epidermal cells, the number of passages, the age, and the like.
　＜三次元培養皮膚シートを製造するための細胞培養容器＞
　一実施態様において、本発明は、
　培養表面の少なくとも一部に設けられた多孔性膜と、
　前記多孔性膜の上に形成され、三次元培養皮膚シートの基底部に凹部を形成するための凸部と、を備え、
　ここで前記凸部の幅が平均１５μｍ～２５μｍであり、かつ、前記凸部の間隔幅が平均１５μｍ～２５μｍであることを特徴とする、三次元培養皮膚シートを製造するための細胞培養容器を提供する。 <Cell culture container for manufacturing 3D cultured skin sheet>
In one embodiment, the present invention
A porous membrane provided on at least a part of the culture surface,
It is provided with a convex portion formed on the porous membrane and for forming a concave portion at the base portion of the three-dimensional cultured skin sheet.
Here, a cell culture container for producing a three-dimensional cultured skin sheet, characterized in that the width of the convex portions is 15 μm to 25 μm on average and the interval width of the convex portions is 15 μm to 25 μm on average. provide.
　図３は、本発明の細胞培養容器１の一実施態様を示す。多孔性膜３上には、本発明の三次元培養皮膚シートに凹部１１０を形成するための凸部７１が直接形成されている。凸部７１は、円柱構造を有している。凸部７の高さＨ１を調節することによって、本発明の三次元培養皮膚シートの凹部１１０の高さＨ’を制御することが可能である。例えば、図３（ｂ）に示すように、凸部７１ａは正方形の格子点に位置するように配置可能である。凸部間隔幅Ｗ１ａを調整することによって、前述の本発明の三次元培養皮膚シートの凸部間隔幅Ｗ’を制御することが可能である。例えば、図３（ｃ）に示すとおり、凸部７１ｂは、正三角形の格子点に位置するように配置されている。凸部間隔幅Ｗ１ｂを調整することによって、前述の本発明の三次元培養皮膚シートの凸部間隔幅Ｗ’を制御することが可能である。また、凸部幅Ｙ１を調整することによって、本発明の三次元培養皮膚シートの凹部幅Ｙ’を制御することが可能である。 FIG. 3 shows an embodiment of the cell culture vessel 1 of the present invention. A convex portion 71 for forming a concave portion 110 in the three-dimensional cultured skin sheet of the present invention is directly formed on the porous film 3. 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. 3B, the convex portion 71a can be arranged so as to be located at a square grid point. By adjusting the convex spacing width W1a, it is possible to control the convex spacing width W'of the above-mentioned three-dimensional cultured skin sheet of the present invention. For example, as shown in FIG. 3C, the convex portion 71b is arranged so as to be located at a grid point of an equilateral triangle. By adjusting the convex spacing width W1b, it is possible to control the convex spacing width W'of the above-mentioned three-dimensional cultured skin sheet of the present invention. Further, 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.
　図４は、本発明の細胞培養容器１の一実施態様を示す。多孔性膜３上には、本発明の三次元培養皮膚シートに凹凸を形成するための凸部７２が配置されている。凸部７２は、角柱形状の一種である立方体形状を有している。凸部の高さＨ２を調節することによって、本発明の三次元培養皮膚シートの凹凸の高さＨ’を制御することが可能である。例えば、図４（ｂ）に示すように、凸部７２ａは正方形の格子点に位置するように配置可能である。凸部間隔幅Ｗ２ａを調整することによって、前述の本発明の三次元培養皮膚シートの凸部間隔幅Ｗ’を制御することが可能である。例えば、図４（ｃ）に示すとおり、凸部７２ｂの重心が、正三角形の格子点に位置するように配置可能である。凸部間隔幅Ｗ２ｂを調整することによって、前述の本発明の三次元培養皮膚シートの凸部間隔幅Ｗ’を制御することが可能である。また、凸部幅Ｙ２を調整することによって、本発明の三次元培養皮膚シートの凹部幅Ｙ’を制御することが可能である。 FIG. 4 shows an embodiment of the cell culture vessel 1 of the present invention. On the porous film 3, a convex portion 72 for forming irregularities on the three-dimensional cultured skin sheet of the present invention is arranged. The convex portion 72 has a cubic shape, which is a kind 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 FIG. 4B, the convex portion 72a can be arranged so as to be located at a square grid point. By adjusting the convex spacing width W2a, it is possible to control the convex spacing width W'of the above-mentioned three-dimensional cultured skin sheet of the present invention. For example, as shown in FIG. 4C, the center of gravity of the convex portion 72b can be arranged so as to be located at the lattice point of an equilateral triangle. By adjusting the convex spacing width W2b, it is possible to control the convex spacing width W'of the above-mentioned three-dimensional cultured skin sheet of the present invention. Further, by adjusting the convex portion width Y2, it is possible to control the concave portion width Y'of the three-dimensional cultured skin sheet of the present invention.
　図５は、本発明の細胞培養容器１に設けられる凸部（７３、７４、７５）の実施形態を示す断面図である。例えば、図５（ａ）は、錐体形状（三角錐、四角錐、若しくは円錐）を有する凸部７３を示す。例えば、図５（ｂ）は、錐台形状を有する凸部７４を示す。例えば、図５（ｃ）は、釣鐘型を有する凸部７５を示す。それぞれの凸部の高さ（Ｈ３、Ｈ４、Ｈ５）を調整することにより、本発明の三次元培養皮膚シートの凹凸の高さＨ’を制御することが可能である。凸部間隔幅（Ｗ３、Ｗ４、Ｗ５）を調整することによって、本発明の三次元培養皮膚シートの凹部間隔幅Ｗ’を制御することが可能である。また、凸部幅（Ｙ３、Ｙ４、Ｙ５）を調整することによって、本発明の三次元培養皮膚シートの凹部幅Ｙ’を制御することが可能である。それぞれの凸部（７３、７４、７５）の平面上の配置は、図３及び図４と同様に配置することが可能であるが、これに限定されない。 FIG. 5 is a cross-sectional view showing an embodiment of a convex portion (73, 74, 75) provided in the cell culture vessel 1 of the present invention. For example, FIG. 5A shows a convex portion 73 having a pyramid shape (triangular pyramid, quadrangular pyramid, or cone). For example, FIG. 5B 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 height of each convex portion (H3, H4, H5), 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 spacing widths (W3, W4, W5), it is possible to control the concave spacing width W'of the three-dimensional cultured skin sheet of the present invention. Further, by adjusting the convex portion widths (Y3, Y4, Y5), it is possible to control the concave portion width Y'of the three-dimensional cultured skin sheet of the present invention. The arrangement of the respective convex portions (73, 74, 75) on the plane can be arranged in the same manner as in FIGS. 3 and 4, but is not limited thereto.
　本発明の実施態様において、凸部（７、７１、７１ａ、７１ｂ、７２、７２ａ、７２ｂ、７３、７４、７５）の形状については上記に限定されないが、例えば、略半球形、略直方体形等も含まれる。 In the embodiment of the present invention, the shape of the convex portion (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) is not limited to the above, but for example, a substantially hemispherical shape, a substantially rectangular parallelepiped shape, or the like. Is also included.
　凸部（７、７１、７１ａ、７１ｂ、７２、７２ａ、７２ｂ、７３、７４、７５）の表面に、細胞が接着しやすいように公知の方法によってプラズマ処理等が行われていても良い。 Plasma treatment or the like may be performed on the surface of the convex portion (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) by a known method so that cells can easily adhere to the surface.
　本発明の細胞培養容器１において、凸部（７、７１、７１ａ、７１ｂ、７２、７２ａ、７２ｂ、７３、７４、７５）の間隔幅（Ｗ、Ｗ１、Ｗ２、Ｗ３、Ｗ４、Ｗ５）の平均は、１５μｍ～２５μｍの範囲であり、より好ましくは、１７．５μｍ～２２．５μｍ、最も好ましくは２０μｍである。凸部の間隔幅（Ｗ、Ｗ１、Ｗ２、Ｗ３、Ｗ４、Ｗ５）が上記の範囲であれば、肥厚化及び／又はバリア機能が亢進した三次元培養皮膚シートが得られる。 In the cell culture vessel 1 of the present invention, the average of the spacing widths (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. When the spacing width (W, W1, W2, W3, W4, W5) of the protrusions is within the above range, a three-dimensional cultured skin sheet with enhanced thickening and / or barrier function can be obtained.
　本発明の細胞培養容器１において、凸部の高さ（Ｈ、Ｈ１、Ｈ２、Ｈ３、Ｈ４、Ｈ５）の平均は、例えば、１μｍ～５０μｍ、好ましくは５μｍ～４０μｍの範囲である。凸部の高さ（Ｈ、Ｈ１、Ｈ２、Ｈ３、Ｈ４、Ｈ５）が上記の範囲であれば、肥厚化及び／又はバリア機能が亢進した三次元培養皮膚シートが得られる。 In the cell culture vessel 1 of the present invention, the average height of the protrusions (H, H1, H2, H3, H4, H5) is, for example, in the range of 1 μm to 50 μm, preferably 5 μm to 40 μm. When the height of the protrusions (H, H1, H2, H3, H4, H5) is in the above range, a three-dimensional cultured skin sheet with enhanced thickening and / or barrier function can be obtained.
　本発明の細胞培養容器１において、凸部幅（Ｙ、Ｙ１、Ｙ２、Ｙ３、Ｙ４、Ｙ５）の平均は、１５μｍ～２５μｍの範囲であり、より好ましくは、１７．５μｍ～２２．５μｍ、最も好ましくは２０μｍである。凸部幅（Ｙ、Ｙ１、Ｙ２、Ｙ３、Ｙ４、Ｙ５）が上記の範囲であれば、肥厚化及び／又はバリア機能が亢進した三次元培養皮膚シートが得られる。 In the cell culture vessel 1 of the present invention, the average of the convex widths (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, most preferably. It is preferably 20 μm. When the convex width (Y, Y1, Y2, Y3, Y4, Y5) is in the above range, a three-dimensional cultured skin sheet having an enhanced thickening and / or barrier function can be obtained.
　凸部（７、７１、７１ａ、７１ｂ、７２、７２ａ、７２ｂ、７３、７４、７５）は、多孔質膜３上に直接形成されていることが好ましく、例えば、３Ｄプリンタ技術又は半導体プロセス技術（例えば、リソグラフィープロセス（フォトリソグラフィ、マスクレスフォトリソグラフィなど）及びドライエッチング（例えば、反応性イオンエッチング）又はウエットエッチングを組み合わせた方法等）等で使用される方法で形成することができる。凸部（７、７１、７１ａ、７１ｂ、７２、７２ａ、７２ｂ、７３、７４、７５）の材質については、生体物質により構成されたものでもよく、非生体物質により構成されたものでもよく、生体物質と非生体物質とを組み合わせて構成されたものであってもよい。例えば、ポリエステル若しくはポリエチレンテレフタレート（ＰＥＴ）、ポリカーボネート、ポリスチレン、ジルコニア、ガラス不溶性コラーゲン、シリコンゴム、樹脂（例えば、エポキシ樹脂、アクリル樹脂等）が挙げられ、細胞培養が可能であれば、その他材質で形成されてもよい。凸部（７、７１、７１ａ、７１ｂ、７２、７２ａ、７２ｂ、７３、７４、７５）は、多孔性膜３の上に直接形成されていることが好ましい。凸部（７、７１、７１ａ、７１ｂ、７２、７２ａ、７２ｂ、７３、７４、７５）が、多孔性膜３の上に直接形成されていることによって、表皮細胞が凸部の下部に潜り込まず、肥厚化及び／又はバリア機能の向上効果が著しく発揮される。 The protrusions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) are preferably formed directly on the porous film 3, for example, 3D printer technology or semiconductor process technology (3D printer technology or semiconductor process technology). For example, it can be formed by a method used in a lithography process (photolithography, maskless photolithography, etc.) and dry etching (for example, a method combining reactive ion etching) or wet etching). The material of the convex portion (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) may be made of a biological substance, may be made of a non-biological substance, or may be a living body. It may be composed of a combination of a substance and a non-biological substance. For example, polyester or polyethylene terephthalate (PET), polycarbonate, polystyrene, zirconia, glass-insoluble collagen, silicone rubber, resin (for example, epoxy resin, acrylic resin, etc.) can be mentioned, and if cell culture is possible, it is formed of other materials. May be done. It is preferable that the convex portions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) are directly formed on the porous film 3. Since the protrusions (7, 71, 71a, 71b, 72, 72a, 72b, 73, 74, 75) are formed directly on the porous membrane 3, epidermal cells do not sneak into the lower part of the protrusions. , Thickening and / or the effect of improving the barrier function is remarkably exhibited.
　本発明の実施形態において使用される細胞培養容器１の材質は、通常細胞培養容器に用いられる材質であれば特に限定されない。例えば、ガラス、ポリスチレン、ポリプロピレン、ポリカーボネート等である。本発明に用いられる細胞培養容器の形状は特に限定されないが、ディッシュ型、マルチウエルプレート型、フラスコ型等が挙げられる。本発明の細胞培養容器は、その細胞培養容器の一部に多孔性膜を有するため、セルカルチャーインサート型であることが好ましい。本発明に用いられるセルカルチャーインサートとは、細胞は透過できないが、培養液等は透過できる多孔性の孔を有する膜を備えた細胞培養容器をいう。多孔性膜の培養表面の反対側、つまり、付着細胞の付着面の裏側からも培養液等を供給することが可能である。本発明に用いられるセルカルチャーインサートは、市販のものを使用してもよく、多孔性膜の培養表面上に直接凸部を設けた膜を有するセルカルチャーインサートであってもよい。多孔性膜の平均孔径は、０．１μｍ～５．０μｍ、好ましくは０．２μｍ～３．０μｍ、さらに好ましくは０．３～１．５μｍである。この範囲の細孔を有する多孔性膜を用いることにより、肥厚化及び／又はバリア機能が向上した三次元培養皮膚シートが得られる。多孔性膜の細孔密度は、例えば、１×１０５～１×１０９個／ｃｍ２、好ましくは５×１０５～５×１０８個／ｃｍ２、より好ましくは１×１０６～５×１０８個／ｃｍ２である。多孔性膜の平均の厚さは、例えば１～５０μｍ、好ましくは３～２５μｍ、より好ましくは５～２０μｍである。多孔性膜の素材は、従来の細胞培養に用いられる多孔性膜と同様、例えば、ポリエステル若しくはポリエチレンテレフタレート（ＰＥＴ）、又は、ポリカーボネート、ポリスチレン等を用いれば良い。 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 usually used for the cell culture vessel. For example, glass, polystyrene, polypropylene, polycarbonate and the like. The shape of the cell culture vessel used in the present invention is not particularly limited, and examples thereof include a dish type, a multi-well plate type, and a flask type. Since the cell culture vessel of the present invention has a porous membrane as a part of the cell culture vessel, it is preferably a cell culture insert type. The cell culture insert used in the present invention refers to a cell culture container having a membrane having porous pores through which cells cannot permeate but a culture solution or the like can permeate. It is possible to supply the culture solution or the like 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. As the cell culture insert used in the present invention, a commercially available one may be used, or a cell culture insert having a membrane having a convex portion directly on the culture surface of the porous membrane may be used. 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 having an improved thickening and / or barrier function can be obtained. The porosity of the porous membrane is, for example, 1 × 10 5 to 1 × 10 9 pieces / cm 2 , preferably 5 × 10 5 to 5 × 10 8 pieces / cm 2 , and more preferably 1 × 10 6 to 5. × 10 8 pieces / cm 2 . The average thickness of the porous membrane is, for example, 1 to 50 μm, preferably 3 to 25 μm, and more preferably 5 to 20 μm. As the material of the porous membrane, for example, polyester or polyethylene terephthalate (PET), polycarbonate, polystyrene or the like may be used as in the case of the porous membrane used for conventional cell culture.
　本発明の実施形態において、多孔性膜３上に凸部を有する細胞培養容器に細胞が付着して増殖しやすいように、培養表面をコーティングしてもよい。例えば、コラーゲン、フィブロネクチン、ラミニン、ゼラチン、ビトロネクチン、ポリリジン（Ｄ体、Ｌ体）、トロンボスポンジン、フィブリン等が挙げられる。 In the embodiment of the present invention, the culture surface may be coated so that the cells can easily adhere to and proliferate in the cell culture container having the convex portion on the porous membrane 3. For example, collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (D-form, L-form), thrombospondin, fibrin and the like can be mentioned.
　＜三次元培養皮膚シートの製造方法＞
　一実施態様において、本発明は、
　（１）上記の細胞培養容器の多孔膜の上に、培地に懸濁したケラチノサイトを含む細胞を播種する工程、
　（２）前記細胞培養容器の前記多孔性膜の外側に培地を接触させて培養する工程、
を含む、三次元培養皮膚シートの製造方法を提供する。 <Manufacturing method of 3D cultured skin sheet>
In one embodiment, the present invention
(1) A step of seeding cells containing keratinocytes suspended in a medium on the porous membrane of the above cell culture vessel.
(2) A step of culturing a medium in contact with the outside of the porous membrane of the cell culture container.
Provided is a method for producing a three-dimensional cultured skin sheet including.
　他の実施態様において、本発明は、上記工程に加え、さらに
　（３）前記細胞培養容器の前記多孔膜の上の培地を除去して、前記細胞を空気に暴露させながら培養する工程、
を含む、方法であってもよい。 In another embodiment, in addition to the above steps, the present invention further comprises (3) removing the medium on the porous membrane of the cell culture vessel and culturing the cells while exposing them to air.
It may be a method including.
　本発明の方法において用いられるケラチノサイトを含む細胞の数は、公知の方法に従えばよい。例えば、細胞を、０．０１×１０６～１０．０×１０６個／ｃｍ２、好ましくは０．０５×１０６～５．０×１０６個／ｃｍ２、より好ましくは０．１×１０６～１．０×１０６個／ｃｍ２の量で播種する。本発明において用いられる培地（培養液ともいう）は、表皮細胞の培養に通常使用される培地、例えばＫＧ培地、ＥｐｉｌｉｆｅＫＧ２（クラボウ社）、Ｈｕｍｅｄｉａ－ＫＧ２（クラボウ社）、アッセイ培地（ＴＯＹＯＢＯ社）、ＣｎＴ－Ｐｒｉｍｅ，Ｅｐｉｔｈｅｌｉａｌ　ｃｕｌｔｕｒｅ　ｍｅｄｉｕｍ（ＣＥＬＬｎＴＥＣ社）などを用いることができる、約３７℃で０～１４日間かけて行うことができる。培地としては、その他にＤＭＥＭ培地（ＧＩＢＣＯ社）又は２－０－ａ－Ｄ－グルコピラノシル－Ｌ－アスコルビン酸含有ＫＧＭとＤＭＥＭを１：１混合した培地などが使用できる。表皮細胞の三次元化（重層化）には、ＣｎＴ－Ｐｒｉｍｅ、３Ｄ　ｂａｒｒｉｅｒ　ｍｅｄｉｕｍ（ＣＥＬＬｎＴＥＣ社）を用いても良い。その他、培地を使用することが可能である。 The number of cells containing keratinocytes used in the method of the present invention may be according to a known method. For example, the cells are 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 ×. Seed at an amount of 10 6 to 1.0 × 10 6 pieces / cm 2 . The medium (also referred to as a culture medium) used in the present invention is a medium usually used for culturing epidermal cells, such as KG medium, EpilieKG2 (Kurabou), Human-KG2 (Kurabou), assay medium (TOYOBO), and the like. CnT-Prime, Epithelial culture medium (CELLnTEC) and the like can be used, and the operation can 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 1: 1 can be used. CnT-Prime, 3D barrier medium (CELLnTEC) may be used for three-dimensionalization (multi-walling) of epidermal cells. In addition, it is possible to use a medium.
　本発明の実施形態において、表皮細胞を培養し、三次元化（重層化）して角質化を促進するために、上述のセルカルチャーインサート２を含む細胞培養容器１に播種し、増殖培養させればよい。具体的には、表皮細胞を培地に懸濁し、セルカルチャーインサート２上に播種する。セルカルチャーインサートの多孔性膜３の外側に培地を接触させるように、ボトムウェル４にも培地を添加して、セルカルチャーインサート２を浸漬させて培養する。これにより、表皮細胞の上部及び底部の両方から培地が供給されて培養される。 In the embodiment of the present invention, in order to culture epidermal cells and promote keratinization by making them three-dimensional (multilayered), they are seeded in a cell culture vessel 1 containing the above-mentioned cell culture insert 2 and proliferated and cultured. Just do it. Specifically, epidermal cells are suspended in a medium and seeded on a cell culture insert 2. The medium is also added to the bottom well 4 so that the medium is brought into contact with the outside of the porous membrane 3 of the cell culture insert, and the cell culture insert 2 is immersed and cultured. As a result, the medium is supplied from both the top and bottom of the epidermal cells and cultured.
　セルカルチャーインサート２上の表皮細胞が、コンフルエント又はサブコンフルエントになるまで数日間程度（１～６日間程度、好ましくは２～４日間程度）培養させることが好ましい。その後、細胞を三次元化（重層化）をさらに促進するために、セルカルチャーインサート内外の培地を、例えば、ＣｎＴ－Ｐｒｉｍｅ、３Ｄ　ｂａｒｒｉｅｒ　ｍｅｄｉｕｍ（ＣＥＬＬｎＴＥＣ社）へ交換することが好ましい。これにより、さらに表皮細胞の三次元化が促進される。ＣｎＴ－Ｐｒｉｍｅ、３Ｄ　ｂａｒｒｉｅｒ　ｍｅｄｉｕｍ（ＣＥＬＬｎＴＥＣ社）へと培地を交換して培養後、１～３６時間後、好ましくは６時間～２４時間後、さらに好ましくは１２時間～１８時間後、セルカルチャーインサートの内部の培地のみを除去して表皮細胞の上面を気相に暴露して培養することが好ましい。これによって、表皮細胞の三次元化及び角質化が促進され、より厚い三次元培養皮膚シートが得られる。 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. Then, in order to further promote the three-dimensionalization (layering) of the cells, it is preferable to replace the medium inside and outside the cell culture insert with, for example, CnT-Prime, 3D barrier medium (CELLnTEC). This further promotes the three-dimensionalization of epidermal cells. After exchanging the medium with CnT-Prime and 3D barrier medium (CELLnTEC) and culturing, 1 to 36 hours, preferably 6 to 24 hours, more preferably 12 to 18 hours, of the cell culture insert. It is preferable to remove only the internal medium and expose the upper surface of the epidermal cells to the gas phase for culturing. This promotes the three-dimensionalization and keratinization of epidermal cells, resulting in a thicker three-dimensional cultured skin sheet.
　なお、本発明における各培養工程の培養温度は由来動物の体温付近であればよく、具体的にはヒト細胞の場合は、３３～３８℃程度とするのが好適である。 The culture temperature of each culture step in the present invention may be close to the body temperature of the origin animal, and specifically, in the case of human cells, it is preferably about 33 to 38 ° C.
　上記のようにして本発明によって得られる三次元培養皮膚シートは、動物実験代替法の一つ、例えば皮膚モデルとして用いることができる。例えば、皮膚の化学物質（例えば、化粧料、工業製品、家庭用品、薬剤、皮膚外用剤等）に対する反応性を評価する方法に用いることができる。また、本発明の三次元培養皮膚シートは、従来の三次元培養皮膚シートと比較してより肥厚化及び／又はバリア機能が亢進した組織が得られるため、皮膚科学の基礎研究においても有用な皮膚モデルとして使用することが可能である。さらにまた、本発明で得られた三次元培養皮膚シートは、従来の三次元培養皮膚シートと比較して肥厚化していることから、外部からのバリア機能も高く、熱傷、創傷等を治癒するための三次元培養皮膚シートとしても有用である。 The three-dimensional cultured skin sheet obtained by the present invention as described above can be used as one of alternative methods for animal experiments, for example, as a skin model. For example, it can be used as a method for evaluating the reactivity of skin to chemical substances (for example, cosmetics, industrial products, household products, drugs, external preparations for skin, etc.). Further, since the three-dimensional cultured skin sheet of the present invention can obtain a tissue having a thicker and / or enhanced barrier function as compared with the conventional three-dimensional cultured skin sheet, the skin is also useful in basic research of dermatology. It can be used as a model. Furthermore, since the three-dimensional cultured skin sheet obtained in the present invention is thicker than the conventional three-dimensional cultured skin sheet, it has a high barrier function from the outside and heals burns, wounds, etc. It is also useful as a three-dimensional cultured skin sheet.
　一実施態様において、本発明に用いられる細胞培養容器は、該凸部と該多孔性膜とが培養終了後に分離可能な細胞培養容器であっても良い。この場合、該細胞培養容器で製造した三次元培養皮膚シートは、その基底部に該凸部を接触させたまま該多孔性膜から分離される。そのため、該三次元培養皮膚シートの基底部は、凹凸構造を維持したまま移動させることが可能となる。本発明に用いられる細胞培養容器は、該凸部が、生体物質、例えば、コラーゲン、フィブロネクチン、ラミニン、ゼラチン、ビトロネクチン、ポリリジン（Ｄ体、Ｌ体）、トロンボスポンジン等を含む生体物質であることが好ましく、特に、不溶性コラーゲンであることがより好ましい。 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 container is separated from the porous membrane with the convex portion in contact with the base portion thereof. Therefore, the base portion 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 a biological substance containing a biological substance such as collagen, fibronectin, laminin, gelatin, vitronectin, polylysine (D-form, L-form), thrombospondin and the like. Is preferable, and insoluble collagen is more preferable.
　＜三次元培養皮膚シートを用いた皮膚のバリア機能を改善及び／又は回復させる対象物質を評価する方法＞
　一実施態様において、本発明の三次元培養皮膚シートに、対象物質を添加することによって、皮膚のバリア機能を改善及び／又は回復させる対象物質を評価する方法を提供することができる。 <Method of evaluating target substances that improve and / or restore the barrier function of the skin using a three-dimensional cultured skin sheet>
In one embodiment, it is possible to provide a method for evaluating a target substance that improves and / or restores the barrier function of the skin by adding the target substance to the three-dimensional cultured skin sheet of the present invention.
　例えば、本発明の三次元培養皮膚シートに対象物質を添加後、三次元培養皮膚シートの表面から蒸発する水分蒸散量を測定することによって、皮膚のバリア機能の変化を評価することができる（Ｋｕｍａｍｏｔｏ　Ｊ．，ｅｔ　ａｌ．，Ａｒｃｈ．Ｄｅｒｍａｔｏｌ．Ｒｅｓ．３０８：４９－５４，２０１６を参照）。また、本発明の三次元培養皮膚シートに対象物質を添加後、皮膚のバリア機能に関連する公知のマーカー（例えば、Ｆｉｌａｇｇｒｉｎ、Ｌｏｒｉｃｒｉｎ、ＺＯ－１、Ｃｌａｕｄｉｎ－１など）の発現を調べることによって皮膚のバリア機能の変化を評価することができる。 For example, changes in the barrier function of the skin can be evaluated by measuring the amount of water evaporation that evaporates from the surface of the three-dimensional cultured skin sheet after adding the target substance to the three-dimensional cultured skin sheet of the present invention (Kumamoto). See J., et al., Arch. Dermatol. Res. 308: 49-54, 2016). In addition, after adding the target substance to the three-dimensional cultured skin sheet of the present invention, the skin is examined by examining the expression of known markers (for example, Filaggrin, Lolicrin, ZO-1, Claudin-1, etc.) related to the barrier function of the skin. Changes in barrier function can be evaluated.
　本実施態様において、皮膚のバリア機能を改善及び／又は回復させる対象物質としては、例えば、低分子化合物、ペプチド、タンパク質、哺乳動物（例えば、マウス、ラット、ブタ、ウシ、ヒツジ、サル、ヒトなど）の組織抽出物又は細胞培養上清、植物由来の化合物又は抽出物（例えば、生薬エキス、生薬由来の化合物）、及び微生物由来の化合物もしくは抽出物又は培養産物などであってもよい。 In this embodiment, the target substances for improving and / or restoring the barrier function of the skin include, for example, low molecular weight compounds, peptides, proteins, mammals (for example, mice, rats, pigs, cows, sheep, monkeys, humans and the like). ) Tissue extract or cell culture supernatant, plant-derived compound or extract (for example, crude drug extract, crude drug-derived compound), and microorganism-derived compound or extract or culture product.
　以下に、本発明を実施例に基づいて更に詳しく説明するが、これらは本発明を何ら限定するものではない。 Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.
＜細胞の準備＞
　本発明に用いられる表皮細胞は、新生児由来ケラチノサイト（以下、「ケラチノサイト」。クラボウ社、製品名：凍結ＮＨＥＫ（ＮＢ）、カタログ番号：ＫＫ－４００９）を使用した。継代培養は、販売会社から提供されたインストラクションに従い行った。 <Cell preparation>
As the epidermal cells used in the present invention, neonatal-derived keratinocytes (hereinafter, “keratinocytes”. Kurabo Industries, Ltd., product name: frozen NHEK (NB), catalog number: KK-4009) were used. Subculture was performed according to the instructions provided by the sales company.
　＜実施例１＞
　１．材料及び実験方法
　１－１．凸部を有するセルカルチャーインサート（細孔サイズ：０．４μｍ）の作製
　（１）１２－Ｗｅｌｌ　Ｍｉｌｌｉｃｅｌｌ　Ｈａｎｇｉｎｇ　Ｃｅｌｌ　Ｃｕｌｔｕｒｅ　ｉｎｓｅｒｔｓ　ＰＥＴ（細孔サイズ：０．４μｍ、Ｍｉｌｌｉｐｏｒｅ）から多孔性膜のみ剥がし、多孔性膜を８０℃に加熱して光感光性樹脂シート（３０μｍ又は５０μｍの厚さ）をローラーで押し当ててラミネートした。
　（２）意図する凸部の幅と凸部の間隔幅の円形の孔が設けられているフォトマスクを、光感光性樹脂シートをラミネートした多孔性膜に被せ、フォトマスクを介してＵＶ光を照射した。これにより、ＵＶ光の通過／遮光部のパターンが光感光性樹脂シートに転写された。ＵＶ光が照射された光感受性樹脂のみが硬化した。
　（３）（２）で得られた多孔性膜を有機溶剤に浸漬して現像した。これにより、ＵＶ光照射部のみがパターンとして残り、遮光部は現像によって除去された。
　（４）（３）で得られた多孔性膜をエタノールに浸漬して、洗浄した。その後、凸部が形成された多孔性膜をセルカルチャーインサートの枠に再び取り付けた。 <Example 1>
1. 1. Materials and experimental methods 1-1. Preparation of Cell Culture Insert with Convex (Porosity Size: 0.4 μm) (1) Only the porous membrane is peeled off from 12-Well Millicell Hanging Cell Culture inserts PET (Porosity size: 0.4 μm, Millipore) to make it porous. The film was heated to 80 ° C. and a photosensitive resin sheet (thickness of 30 μm or 50 μm) was pressed by a roller to laminate.
(2) A photomask provided with circular holes having an intended width of the convex portion and a width of the interval between the convex portions is covered with a porous film laminated with a photosensitive resin sheet, and UV light is emitted through the photomask. Irradiated. As a result, the pattern of the UV light passing / light-shielding portion was transferred to the photosensitive resin sheet. Only the photosensitive resin irradiated with UV light was cured.
(3) The porous membrane obtained in (2) was immersed in an organic solvent for development. As a result, only the UV light irradiation part remained as a pattern, and the light-shielding part was removed by development.
(4) The porous membrane obtained in (3) was immersed in ethanol and washed. Then, the porous membrane on which the protrusion was formed was reattached to the frame of the cell culture insert.
　１－２．凸部を有するセルカルチャーインサートを用いた三次元培養皮膚シートの作製及び観察
　（１）ＣＥＬＬｓｔａｒｔ　ＣＴＳ　（ｇｉｂｃｏ社）をＤＰＢＳ（ｇｉｂｃｏ社）で５０倍希釈し、１つのセルカルチャーインサートに８６μＬ滴下し、２時間３７℃の条件に維持した。
　（２）ＣＥＬＬｓｔａｒｔ　ＣＴＳを除去し、継代回数４の新生児由来ケラチノサイト２２～２５万個をＣｎＴ－Ｐｒｉｍｅ，Ｅｐｉｔｈｅｌｉａｌ　ｃｕｌｔｕｒｅ　ｍｅｄｉｕｍ（ＣＥＬＬｎＴＥＣ社）　５００μＬに分散し、セルカルチャーインサート内に滴下、同じ培地（ＣｎＴ－Ｐｒｉｍｅ，Ｅｐｉｔｈｅｌｉａｌ　ｃｕｌｔｕｒｅ　ｍｅｄｉｕｍ）を１ｍＬセルカルチャーインサート外部に滴下し、７２時間３７℃、ＣＯ２インキュベーターで培養した。
　（３）セルカルチャーインサート内外のＣｎＴ－Ｐｒｉｍｅ、Ｅｐｉｔｈｅｌｉａｌ　ｃｕｌｔｕｒｅ　ｍｅｄｉｕｍを除去し、ＣｎＴ－Ｐｒｉｍｅ、３Ｄ　ｂａｒｒｉｅｒ　ｍｅｄｉｕｍ（ＣＥＬＬｎＴＥＣ社）で置き換え、１６時間３７℃ＣＯ２インキュベーターで培養した。
　（４）セルカルチャーインサート内外の培地を除去し、セルカルチャーインサート内は空気に曝露し、セルカルチャーインサート外に５００μＬ　ＣｎＴ－Ｐｒｉｍｅ、３Ｄ　ｂａｒｒｉｅｒ　ｍｅｄｉｕｍを入れた。
　（５）毎日セルカルチャーインサート外の５００μＬ　ＣｎＴ－Ｐｒｉｍｅ、３Ｄ　ｂａｒｒｉｅｒ　ｍｅｄｉｕｍを交換しながら３７℃ＣＯ２インキュベーターで７日間培養した。
　（６）セルカルチャーインサート内膜ごと三次元培養皮膚シートを切り取り、４％パラフォルムアルデヒドＰＢＳ溶液で固定し、組織切片標本を作製して、公知の方法従ってヘマトキシリン・エオジン（ＨＥ）染色又は免疫組織染色（抗Ｆｉｌａｇｇｒｉｎ抗体（ｓｃ－３０２２９，Ｓａｎｔａ　Ｃｒｕｚ，Ｄａｌｌａｓ，ＵＳＡ）、抗Ｌｏｒｉｃｒｉｎ抗体（ＰＲＢ－１４５Ｐ，Ｂｉｏｌｅｇｅｎｄ，Ｓａｎ　Ｄｉｅｇｏ，ＵＳＡ）、抗ＺＯ－１抗体（＃３３－９１００，Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ，ＵＳＡ）、抗Ｃｌａｕｄｉｎ－１抗体（＃５１－９０００，Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ，ＵＳＡ）又はＤＡＰＩ（Ｒ３７６０６，Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ，ＵＳＡ）を行い、顕微鏡にて観察した。 1-2. Preparation and Observation of 3D Cultured Skin Sheet Using Cell Culture Insert with Convex (1) CELLstart CTS (gibco) was diluted 50-fold with DPBS (gibco), and 86 μL was added dropwise to one cell culture insert. The condition was maintained at 37 ° C. for 2 hours.
(2) CELLstart CTS was removed, and 220,000 to 250,000 neonatal-derived keratinocytes with 4 passages were dispersed in 500 μL of CnT-Prime, Epithelial culture medium (CELLnTEC), dropped into a cell culture insert, and the same medium (CnT) -Prime, Epithelium culture medium) was added dropwise to the outside of a 1 mL cell culture insert, and the cells were cultured in a CO 2 incubator for 72 hours at 37 ° C.
(3) CnT-Prime and Epithelium culture medium inside and outside the cell culture insert were removed, replaced with CnT-Prime, 3D barrier medium (CELLnTEC), and 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 CnT-Prime and 3D barrier medium were placed outside the cell culture insert.
(5) The cells were cultured in a 37 ° C. CO 2 incubator for 7 days while exchanging 500 μL CnT-Prime and 3D barrier medium outside the cell culture insert every day.
(6) Cell culture insert A three-dimensional cultured skin sheet together with the intima is cut out and fixed with a 4% paraformaldehyde PBS solution to prepare a tissue section sample, and hematoxylin / eosin (HE) staining or immunohistochemistry is performed according to a known method. Staining (anti-Filaggrin antibody (sc-30229, Santa Cruz, Dollars, USA), anti-Lolicrin antibody (PRB-145P, Invitrogen, San Diego, USA), anti-ZO-1 antibody (# 33-9100, Invitrogen, Carlsbad) ), Anti-Claudin-1 antibody (# 51-9000, Invitrogen, Carlsbad, USA) or DAPI (R37606, Invitrogen, Carlsbad, USA) was performed and observed under a microscope.
　２．結果
　平均０．４μｍの細孔を有する多孔性膜上に高さ（３０μｍ又は５０μｍ）、幅（２０μｍ～５０μｍ）及び間隔（１５μｍ～５０μｍ）を変えた凸部を形成し、その上でケラチノサイトを培養した（図６）。その結果、凸部の高さ：３０μｍ、凸部の幅：２０μｍ、凸部の間隔：２０μｍで設計された凸部を有する多孔性膜が、三次元培養皮膚シートを肥厚化させる効果が最も高かった。 2. Results Convex portions of varying height (30 μm or 50 μm), width (20 μm to 50 μm) and spacing (15 μm to 50 μm) were formed on a porous membrane with average pores of 0.4 μm, on which keratinocytes were formed. It was cultured (Fig. 6). As a result, the porous membrane having convex parts designed with the height of the convex parts: 30 μm, the width of the convex parts: 20 μm, and the distance between the convex parts: 20 μm is most effective in thickening the three-dimensional cultured skin sheet. It was.
　また、凸部を有さない多孔性膜と、凸部の高さ：３０μｍ、凸部の幅：２０μｍ、凸部の間隔：２０μｍで設計された凸部を有する多孔性膜で作製した三次元培養皮膚シートについて、免疫組織染色を行った（図７）。抗フィラグリン（Ｆｉｌａｇｇｒｉｎ）抗体（顆粒細胞のマーカー抗体）、抗ロリクリン（Ｌｏｒｉｃｒｉｎ）抗体（顆粒層のマーカー抗体）、抗ＺＯ－１抗体及び抗Ｃｌａｕｄｉｎ－１抗体（タイトジャンクションのマーカー抗体）で染色した結果、より広い範囲に特にＺＯ－１、Ｃｌａｕｄｉｎ－１が特有の構造をもって、より明確に発現していた。 In addition, a three-dimensional film made of a porous film having no convex portion and a porous film having 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. Immunohistochemical staining was performed on the cultured skin sheet (Fig. 7). Results of staining with anti-Filaggrin antibody (marker antibody for granule cells), anti-Loricrin antibody (marker antibody for granule layer), anti-ZO-1 antibody and anti-Claudin-1 antibody (marker antibody for tight junction) In particular, ZO-1 and Claudin-1 were more clearly expressed in a wider range with a unique structure.
　＜実施例２＞
　１２－Ｗｅｌｌ　Ｍｉｌｌｉｃｅｌｌ　Ｈａｎｇｉｎｇ　Ｃｅｌｌ　Ｃｕｌｔｕｒｅ　ｉｎｓｅｒｔｓ　ＰＥＴ（１．０μｍ、Ｍｉｌｌｉｐｏｒｅ）を用いること以外、実施例１と同じ手順により三次元培養皮膚シートを作製し、観察した。 <Example 2>
A three-dimensional cultured skin sheet was prepared and observed by the same procedure as in Example 1 except that 12-Well Millicell Hanging Cell Culture inserts PET (1.0 μm, Millipore) was used.
　平均１．０μｍの細孔を有する多孔性膜上に高さ（３０μｍ）、幅（１５μｍ～５０μｍ）及び間隔（１５μｍ～３０μｍ）を変えた凸部を形成し、その上でケラチノサイトを培養した（図８及び９）。その結果、凸部の高さ：３０μｍ、凸部の幅：２０～２５μｍ、凸部の間隔：２０～２５μｍで設計された凸部を有する多孔性膜が、三次元培養皮膚シートの角層を肥厚化させる効果が最も高かった。 Convex portions with varying height (30 μm), width (15 μm to 50 μm) and spacing (15 μm to 30 μm) were formed on a porous membrane having pores with an average of 1.0 μm, and keratinocytes were cultured on the convex portions. 8 and 9). As a result, a porous film having convex portions designed with a convex portion height of 30 μm, a convex portion width of 20 to 25 μm, and a convex portion interval of 20 to 25 μm formed the stratum corneum of the three-dimensional cultured skin sheet. The effect of thickening was the highest.
　また、凸部を有さない多孔性膜、高さ：３０μｍ、幅：２０μｍ、間隔：２０μｍで設計された凸部を有する多孔性膜、及び、高さ：３０μｍ、幅：２０μｍ、間隔：２０μｍで設計された凸部を有する多孔性膜で作製した三次元培養皮膚シートについて、免疫組織染色を行った（図１０）。抗フィラグリン（Ｆｉｌａｇｇｒｉｎ）抗体（顆粒細胞のマーカー抗体）、抗ロリクリン（Ｌｏｒｉｃｒｉｎ）抗体（顆粒層のマーカー抗体）で染色した結果、それぞれの発現は認められたが、Ｆｉｌａｇｇｒｉｎ及びＬｏｒｉｃｒｉｎの発現の範囲は狭かった。 Further, a porous membrane having no convex portion, a porous membrane having convex portions designed with a height: 30 μm, a width: 20 μm, and an interval: 20 μm, and a height: 30 μm, a width: 20 μm, an interval: 20 μm. Immunohistochemical staining was performed on a three-dimensional cultured skin sheet prepared of a porous membrane having a convex portion designed in (FIG. 10). As a result of staining with anti-filaggrin antibody (marker antibody for granule cells) and anti-Loricrin antibody (marker antibody for granule layer), their respective expressions were observed, but the range of expression of Filaggrin and Loriclin was narrow. It was.
　＜実施例３＞
　バリア機能の評価
　１２－Ｗｅｌｌ　Ｍｉｌｌｉｃｅｌｌ　Ｈａｎｇｉｎｇ　Ｃｅｌｌ　Ｃｕｌｔｕｒｅ　ｉｎｓｅｒｔｓ　ＰＥＴ（１．０μｍ、Ｍｉｌｌｉｐｏｒｅ）を用いること以外、実施例１と同じ手順により三次元培養皮膚シートを作製した。 <Example 3>
Evaluation of Barrier Function A three-dimensional cultured skin sheet was prepared by the same procedure as in Example 1 except that 12-Well Millicell Hanging Cell Cultureinserts PET (1.0 μm, Millipore) was used.
　三次元培養皮膚シートを構築したセルカルチャーインサートを、底部に培養液を入れたシリコンゴム容器にはめこみ、水分蒸散が表皮モデル表面からのみ起きるようにした。その後、２時間おきにインサートをはめこんだ容器の重量を測定し、減少した重量を水分蒸散量とし、単位面積当たりの数値に換算した。この手法はＫｕｍａｍｏｔｏの論文（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を参照のこと）に記載された方法と同じ原理である。 The cell culture insert on which the three-dimensional cultured skin sheet was constructed was fitted into a silicone rubber container containing the culture solution at the bottom so that water evaporation occurred only from the surface of the epidermis model. Then, the weight of the container in which the insert was fitted was measured every two hours, and the reduced weight was taken as the amount of water evaporation and converted into a value per unit area. This approach Kumamoto of 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 It is the same principle as the method described in ex vivo. Arch. Dermatol. Res. 308: 49-54, 2016).
　比較例として、市販の三次元皮膚モデル（Ｅｐｉｓｋｉｎ（登録商標）、ニコダームリサーチ）のバリア機能も評価した。 As a comparative example, the barrier function of a commercially available three-dimensional skin model (Episkin (registered trademark), Nicoderm Research) was also evaluated.
　得られた結果は、統計学的に解析を行い、（ＡＮＯＶＡ分散解析後Ｓｃｈｅｆｆｅによる多重検定解析）、膜のみで培養して得られた三次元培養皮膚シートと比較して、多重検定の結果、ｐ＜０．０５であるものを有意差があるものと判断した。 The obtained results were statistically analyzed (ANOVA ANOVA and then Scheffe's multiple test analysis), and compared with the three-dimensional cultured skin sheet obtained by culturing only with a membrane, the results of the multiple test, Those with p <0.05 were judged to have a significant difference.
　その結果、凸部の高さ：３０μｍ、凸部の幅：２０μｍ、凸部の間隔：２０μｍで設計された凸部を有する多孔性膜（平均細孔サイズ：１．０μｍ）を用いて作製した三次元培養皮膚シートが、最も経皮水分蒸散量が少なく、高角層バリア機能を有していることが明らかとなった(図１１）。 As a result, it was produced using a porous film having convex portions (average pore size: 1.0 μm) designed with a convex portion height: 30 μm, a convex portion width: 20 μm, and a convex portion interval: 20 μm. It was clarified that the three-dimensional cultured skin sheet has the smallest amount of transdermal water evaporation and has a high stratum corneum barrier function (Fig. 11).
　１　　細胞培養容器
　２　　セルカルチャーインサート
　３　　多孔性膜
　４　　ボトムウェル
　５　　培地
　６　　培養表面断面拡大部
　７、７１、７１ａ、７１ｂ、７２、７２ａ、７２ｂ、７３～７５　　凸部
　８　　表皮細胞
　９　　角層
　１０　　三次元培養皮膚シート
　１１　　基底部（太線）
　１１０　　三次元培養皮膚シートの凹部
　１１１　　三次元培養皮膚シートの凸部
　Ｈ～Ｈ５　　凸部の高さ
　Ｈ’　　三次元培養皮膚シートの凹部の高さ
　Ｖ　　表皮細胞非存在領域
　Ｗ、Ｗ１、Ｗ１ａ、Ｗ１ｂ、Ｗ２、Ｗ２ａ、Ｗ２ｂ、Ｗ３～Ｗ５　　凸部間隔幅
　Ｗ’　　三次元培養皮膚シート凸部間隔幅
　Ｙ、Ｙ１～Ｙ５　　凸部幅
　Ｙ’　　三次元培養皮膚シート凹部の幅 1 Cell culture container 2 Cell culture insert 3 Porous membrane 4 Bottom well 5 Medium 6 Culture surface cross-section enlargement part 7, 71, 71a, 71b, 72, 72a, 72b, 73-75 Convex part 8 Epidermal cell 9 Corneal layer 10 Tertiary Original cultured skin sheet 11 Base (thick line)
110 Concave part of 3D cultured skin sheet 111 Convex part of 3D cultured skin sheet H to H5 Height of convex part H'Height of concave part of 3D cultured skin sheet V Area where epidermal cells are absent W, W1, W1a, W1b , W2, W2a, W2b, W3 to W5 Convex spacing width W'Three-dimensional cultured skin sheet Convex spacing width Y, Y1 to Y5 Convex width Y'Three-dimensional cultured skin sheet concave width

Claims (17)

  1. 　少なくともケラチノサイトを含み、かつ、基底部の少なくとも一部に複数の凹部を有する三次元培養皮膚シートであって、
    　前記凹部の幅が平均１５μｍ～２５μｍであり、かつ、前記凹部の間隔幅が平均１５μｍ～２５μｍであることを特徴とする、三次元培養皮膚シート。     A three-dimensional cultured skin sheet containing at least keratinocytes and having a plurality of recesses in at least a part of the base.
    A three-dimensional cultured skin sheet, wherein the width of the recesses is an average of 15 μm to 25 μm, and the spacing width of the recesses is an average of 15 μm to 25 μm.
  2. 　前記凹部の高さが、平均５μｍ～４０μｍである、請求項１に記載の三次元培養皮膚シート。 The three-dimensional cultured skin sheet according to claim 1, wherein the height of the recesses is 5 μm to 40 μm on average.
  3. 　前記凹部の高さが、平均２５μｍ～３５μｍである、請求項１に記載の三次元培養皮膚シート。 The three-dimensional cultured skin sheet according to claim 1, wherein the height of the recesses is 25 μm to 35 μm on average.
  4. 　前記三次元培養皮膚シートの平均の厚さが、５０μｍ以上である、請求項１～３のいずれか１項に記載の三次元培養皮膚シート。 The three-dimensional cultured skin sheet according to any one of claims 1 to 3, wherein the average thickness of the three-dimensional cultured skin sheet is 50 μm or more.
  5. 　前記基底部に密着させた、複数の凸部を有する多孔性膜をさらに備え、
    　ここで前記凸部の幅が平均１５μｍ～２５μｍであり、かつ、前記凸部の間隔幅が平均１５μｍ～２５μｍであり、
    　前記凸部が、前記基底部の前記凹部と密着していることを特徴とする、請求項１～４のいずれか１項に記載の三次元培養皮膚シート。     Further provided with a porous membrane having a plurality of convex portions, which is in close contact with the base portion,
    Here, the width of the convex portion is 15 μm to 25 μm on average, and the interval width of the convex portion is 15 μm to 25 μm on average.
    The three-dimensional cultured skin sheet according to any one of claims 1 to 4, wherein the convex portion is in close contact with the concave portion of the base portion.
  6. 　前記凸部の高さが、平均５μｍ～４０μｍである、請求項５に記載の三次元培養皮膚シート。 The three-dimensional cultured skin sheet according to claim 5, wherein the height of the convex portion is 5 μm to 40 μm on average.
  7. 　前記凸部の高さが、平均２５μｍ～３５μｍである、請求項５に記載の三次元培養皮膚シート。 The three-dimensional cultured skin sheet according to claim 5, wherein the height of the convex portion is 25 μm to 35 μm on average.
  8. 　前記多孔性膜が、平均０．２μｍ～３μｍの平均孔径を有する、請求項５～７のいずれか１項に記載の三次元培養皮膚シート。 The three-dimensional cultured skin sheet according to any one of claims 5 to 7, wherein the porous membrane has an average pore size of 0.2 μm to 3 μm on average.
  9. 　前記凸部が、樹脂からなる、請求項５～８のいずれか１項に記載の三次元培養皮膚シート。 The three-dimensional cultured skin sheet according to any one of claims 5 to 8, wherein the convex portion is made of resin.
  10. 　請求項１～９のいずれか１項に記載の三次元培養皮膚シートを製造するための細胞培養容器であって、
    　培養表面の少なくとも一部に設けられた多孔性膜と、
    　前記多孔性膜の上に形成され、三次元培養皮膚シートの基底部に凹部を形成するための凸部と、
    を備え、
    　ここで前記凸部の幅が平均１５μｍ～２５μｍであり、かつ、前記凸部の間隔幅が平均１５μｍ～２５μｍであることを特徴とする、細胞培養容器。     A cell culture container for producing the three-dimensional cultured skin sheet according to any one of claims 1 to 9.
    A porous membrane provided on at least a part of the culture surface,
    A convex portion formed on the porous membrane and for forming a concave portion in the base portion of the three-dimensional cultured skin sheet,
    With
    Here, the cell culture vessel is characterized in that the width of the convex portions is on average 15 μm to 25 μm, and the interval width of the convex portions is on average 15 μm to 25 μm.
  11. 　前記凸部の高さが、平均５μｍ～４０μｍである、請求項１０に記載の細胞培養容器。 The cell culture vessel according to claim 10, wherein the height of the convex portion is 5 μm to 40 μm on average.
  12. 　前記凸部の高さが、平均２５μｍ～３５μｍである、請求項１０に記載の細胞培養容器。 The cell culture vessel according to claim 10, wherein the height of the convex portion is 25 μm to 35 μm on average.
  13. 　前記多孔性膜が、平均０．２μｍ～３μｍの平均孔径を有する、請求項１０～１２のいずれか１項に記載の細胞培養容器。 The cell culture vessel according to any one of claims 10 to 12, wherein the porous membrane has an average pore size of 0.2 μm to 3 μm on average.
  14. 　前記凸部が、樹脂からなる、請求項１０～１３のいずれか１項に記載の細胞培養容器。 The cell culture container according to any one of claims 10 to 13, wherein the convex portion is made of resin.
  15. 　三次元培養皮膚シートの製造方法であって、
    　（１）請求項１０～１４のいずれか１項に記載の細胞培養容器の多孔膜の上に、培地に懸濁したケラチノサイトを含む細胞を播種する工程、
    　（２）前記細胞培養容器の前記多孔性膜の外側に培地を接触させて培養する工程、
    を含む、方法。     A method for manufacturing a three-dimensional cultured skin sheet.
    (1) A step of seeding cells containing keratinocytes suspended in a medium on the porous membrane of the cell culture vessel according to any one of claims 10 to 14.
    (2) A step of culturing a medium in contact with the outside of the porous membrane of the cell culture container.
    Including methods.
  16. 　さらに、
    　（３）前記細胞培養容器の前記多孔膜の上の培地を除去して、前記細胞を空気に暴露させながら培養する工程、
    を含む、請求項１５に記載の方法。     further,
    (3) A step of removing the medium on the porous membrane of the cell culture container and culturing the cells while exposing them to air.
    15. The method of claim 15.
  17. 　前記ケラチノサイトが、生体組織から単離培養後、継代回数２以上のケラチノサイトを含む、請求項１５または１６に記載の方法。 The method according to claim 15 or 16, wherein the keratinocytes contain keratinocytes having 2 or more passages after being isolated and cultured from a living tissue.
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