WO2020204106A1 - Feuille de peau cultivée en trois dimensions, récipient de culture cellulaire à utiliser pour la produire, et son procédé de production - Google Patents

Feuille de peau cultivée en trois dimensions, récipient de culture cellulaire à utiliser pour la produire, et son procédé de production Download PDF

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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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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.

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Abstract

La présente invention concerne un feuillet dermique cultivé en trois dimensions, qui contient au moins des kératinocytes et comporte une pluralité de parties creuses dans au moins une partie d'une base, caractérisé en ce que la largeur moyenne des parties creuses est de 15 à 25 μm et la largeur d'intervalle moyen entre les parties creuses est de 15 à 25 µm. La présente invention concerne également un récipient de culture cellulaire, qui est pourvu d'un film poreux formé au moins dans une partie de la surface de culture et des parties en saillie formées sur le film poreux pour former des parties creuses dans la base d'un feuillet dermique cultivé en trois dimensions, caractérisé en ce que la largeur moyenne des parties en saillie est de 15 à 25 µm et la largeur d'intervalle moyen entre les parties en saillie est de 15 à 25 µm. La présente invention concerne également un procédé permettant de produire un feuillet dermique cultivé en trois dimensions à l'aide du récipient de culture cellulaire.
PCT/JP2020/015109 2019-04-04 2020-04-01 Feuille de peau cultivée en trois dimensions, récipient de culture cellulaire à utiliser pour la produire, et son procédé de production WO2020204106A1 (fr)

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WO2017222065A1 (fr) * 2016-06-24 2017-12-28 株式会社資生堂 Feuille de peau de culture tridimensionnelle, cuve de culture de cellules utilisée pour la production de celle-ci, et procédé de production de feuille de peau de culture tridimensionnelle
JP2018535688A (ja) * 2015-12-04 2018-12-06 プレジデント アンド フェローズ オブ ハーバード カレッジ 組織の機能を模擬するためのオープントップマイクロ流体デバイスおよび方法

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JP2018535688A (ja) * 2015-12-04 2018-12-06 プレジデント アンド フェローズ オブ ハーバード カレッジ 組織の機能を模擬するためのオープントップマイクロ流体デバイスおよび方法
WO2017222065A1 (fr) * 2016-06-24 2017-12-28 株式会社資生堂 Feuille de peau de culture tridimensionnelle, cuve de culture de cellules utilisée pour la production de celle-ci, et procédé de production de feuille de peau de culture tridimensionnelle

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