WO2020111265A1 - 色素沈着皮膚モデルおよびその製造方法、ならびに皮膚の色素沈着を治療または予防するための因子の評価方法 - Google Patents
色素沈着皮膚モデルおよびその製造方法、ならびに皮膚の色素沈着を治療または予防するための因子の評価方法 Download PDFInfo
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Definitions
- the present invention relates to a pigmented skin model and a method for producing the same.
- the present invention also relates to a method for evaluating factors for treating or preventing skin pigmentation using a pigmented skin model.
- Dermal 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 dryness and harmful substances from invading the living body, and plays an essential role in maintaining life.
- the skin of higher vertebrates is roughly composed of epidermis, dermis, and subcutaneous tissue layers, which are roughly divided from the outermost layer.
- the epidermis is mainly composed of cells called keratinocytes (keratinocytes).
- the keratinocytes divide at the deepest part (basal layer) of the epidermis, and to the upper layer, to the stratum spinosum, granular layer, and stratum corneum. It moves to the surface while differentiating, and eventually becomes dirt and falls off.
- Melanin exists in the basal layer of the epidermis, and melanin is produced by the melanosomes in the melanocyte. The generated melanin is incorporated into the surrounding keratinocytes. The melanin taken in migrates to the stratum corneum with the turnover of keratinocytes and is excreted out of the body in about 40 days.
- Pigmentation of skin spots, freckles, etc. is caused by excessive formation of melanin in melanocytes due to hormonal abnormalities, exposure to ultraviolet rays, local inflammation, etc., and deposition of melanin granules in keratinocytes in the basal layer of the epidermis. Is believed to be the cause.
- a method for treating or preventing pigmentation for example, senile pigment spots due to aging, and a therapeutic agent (whitening agent) have been developed, the expected effect cannot be obtained or a temporary effect is not obtained.
- a therapeutic agent whitening agent
- an object of the present invention is to provide a new pigmented skin model that can be stably cultured for a long period of time and a method for producing the same.
- the condition of the model alone can be changed, and the dermis model and the epidermis model can be freely transferred.
- It is also an object of the present invention to evaluate factors for treating or preventing skin pigmentation using a new pigmented skin model. Is to provide a way to do.
- the present invention includes the following inventions.
- Pigmented skin model [4] The pigmented skin model according to any one of [1] to [3], wherein the light irradiation is UVA light irradiation.
- the second cell group is a second cell group seeded on a second cell culture substrate having a porous membrane. Skin model.
- the first cell group is a first cell group seeded with a hydrogelator.
- the hydrogelator is collagen, gelatin, hyaluronate, hyaluronan, fibrin, alginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen/thrombin, fibrillin, elastin, gum, cellulose, agar.
- a method for producing a pigmented skin model comprising: (1) a step of culturing a first cell group containing fibroblasts damaged by light irradiation, (2) a step of culturing the first cell group obtained in the step (1) on a first cell culture substrate, and (3) a step of culturing the first cell group obtained in the step (2) A step of applying a second cell group containing melanocytes and keratinocytes and culturing, And a manufacturing method.
- the fibroblasts damaged by the irradiation with light are fibroblasts damaged by the irradiation with ultraviolet light in the presence of a photosensitizer.
- Method [12] The method according to any one of [9] to [11], wherein the light irradiation is UVA light irradiation.
- the second cell group is a second cell group seeded on a second cell culture substrate having a porous membrane.
- the first cell culture substrate is a cell culture substrate having a porous membrane.
- the first cell group is a first cell group seeded with a hydrogelator.
- the hydrogelator is collagen, gelatin, hyaluronate, hyaluronan, fibrin, alginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen/thrombin, fibrillin, elastin, gum, cellulose, agar.
- a method for evaluating a factor for treating or preventing skin pigmentation comprising: (1) A step of applying a candidate factor to the pigmented skin model according to any one of [1] to [8] and [18] and culturing, (2) A step of evaluating the therapeutic or preventive effect of the candidate factor, using the degree of pigment production and/or pigmentation in the pigmented skin model as an index. Including the method.
- a method for evaluating a factor for treating or preventing skin pigmentation comprising: (1) culturing a first cell group containing fibroblasts damaged by light irradiation, (2) a step of culturing the first cell group obtained in the step (1) on a first cell culture substrate, (3) A step of applying a second cell group containing melanocytes and keratinocytes onto the first cell group obtained in the step (2) and culturing, (4) transferring the second cell culture group obtained in the step (3) onto a substrate containing a candidate factor, and culturing; (5) A method comprising the step of evaluating the therapeutic or preventive effect of the candidate factor using the degree of pigment production and/or pigment deposition in the second cell group as an index. [21] The method according to [20], wherein the candidate factor is fibroblast.
- the present invention provides a pigmented skin model that can be stably cultured for a long period of time, and by using it, it becomes possible to search for and evaluate factors for treating or preventing skin pigmentation.
- FIG. 1 is a schematic view showing a method for producing a pigmented skin model of one embodiment. Each component mainly represents a cross section.
- FIG. 2 is a schematic diagram showing a method for producing a pigmented skin model of one embodiment. Each component mainly represents a cross section.
- FIG. 3 shows the difference in proliferation of PUVA-treated and untreated fibroblasts.
- A Images of monolayer-cultured PUVA-treated or untreated (control) fibroblasts (culture day 1, 4 and 7).
- FIG. 4 shows the difference in the production level of the senescence-related factor SA- ⁇ -gal in PUVA-treated or untreated (control) fibroblasts.
- FIG. 5 shows the difference in the production level of the melanogenesis promoting factor SCF (Stem Cell Factor) in PUVA-treated or untreated (control) fibroblasts.
- SCF melanogenesis promoting factor
- FIG. 6 shows the difference in the production level of the melanogenesis promoting factor HGF (Hepatocyte growth factor) in PUVA-treated or untreated (control) fibroblasts.
- HGF staining using HGF antibody nuclear staining using DAPI, fusion image (Merge) of HGF staining and nuclear staining (2 sheets each).
- FIG. 7 is a diagram showing pigmentation of a pigmented skin model with or without PUVA treatment.
- A Images of the epidermis model in the pigmented skin model treated with PUVA (day 0 of culture, day 21 of culture).
- (B) Images of the epidermis model in the PUVA untreated pigmented skin model (culture day 0, culture day 21).
- FIG. 8 is an image of the epidermis model after the epidermis model co-cultured with the PUVA-treated dermis model for about 10 days was transferred onto the PUVA-untreated dermis model and cultured for about 10 days.
- FIG. 9 is a pigment of an epidermis model (Replace) obtained by transferring and culturing an epidermis model of a PUVA-treated or untreated (control) pigmented skin model and a PUVA-treated pigmented skin model onto a PUVA-untreated dermis model. Shows deposition.
- A An image of an epidermis model taken under fixed observation conditions in a bright field of a phase contrast microscope.
- FIG. 10 shows an epidermal model (Replace) obtained by transferring and culturing an epidermis model of a PUVA-treated or untreated (control) pigmented skin model and a PUVA-treated pigmented skin model onto a PUVA-untreated dermis model. Shows the melanin granules of.
- A Stain melanin granules in an epidermis model section by the Fontana Masson staining method.
- B The graph which binarized the image of (A) and quantified the ratio of the melanin granule area
- FIG. 11 shows an epidermal model (Replace) in which the epidermis model of the PUVA-treated or untreated (control) pigmented skin model and the PUVA-treated pigmented skin model were transferred onto the PUVA-untreated dermis model and cultured. Shows activated melanocytes of.
- A Image obtained by staining the activated melanocytes of the epidermis model section with the TRP2 (Tyrosine Related Protein 2) antibody and nuclear staining with the hematoxylin staining method.
- B A graph quantifying the proportion of activated melanocytes from the images of (A).
- first the first element is the second element.
- second element may be expressed as the first element, which does not depart from the scope of the present invention.
- FIG. 1 is a schematic diagram illustrating a pigmented skin model 1 and a method for manufacturing the same in one embodiment.
- the pigmented skin model 1 is A first cell group 100 seeded on the first cell culture substrate 11 and containing fibroblasts damaged by light irradiation;
- the pigmented skin model 1 in one embodiment has the following steps: (1) a step of culturing a first cell group 100 containing fibroblasts damaged by light irradiation, (2) A step of culturing the first cell group 100 obtained in the step (1) on the first cell culture substrate 11. (3) A step of applying and culturing a second cell group 200 containing melanocytes and keratinocytes on the first cell group 100 obtained in the step (2), Can be provided.
- the structure including the first cell group 100 or the third cell group 100a in the pigmented skin model 1 imitates the structure of the dermis of the skin, and therefore, the “dermis model” (first dermis model or Second dermis model). Further, in the present specification, the structure including the second cell group 200 in the pigmented skin model 1 imitates the structure of the epidermis of the skin, and thus may be referred to as “epidermis model”.
- the pigmented skin model 1 may have a structure in which the second cell group 200 is directly seeded or laminated on the first cell group 100, and as shown in FIG. 1, the second cell group 200 and the first cell group are included. It may have a structure in which a space in which the medium exists between 100 and 100 is provided. In the latter case, the second cell group 200 is placed on the porous membrane (corresponding to the second porous membrane 210 in FIG. 1) so that the humoral components secreted by the first cell group 100 and the second cell group 200 can pass through each other. It is preferable that the seeds are seeded in.
- step (3) of the method for producing the pigmented skin model 1 in one embodiment the second cell group 200 containing melanocytes and keratinocytes is applied on the first cell group 100 obtained in step (2).
- the step may be a step of directly seeding or stacking the second cell group 200 on the first cell group 100, and the second cell group seeded on the second cell culture substrate 21 having a porous membrane. It may be a step of applying 200 onto the first cell group 100.
- the first cell culture substrate 11 on which the first cell group 100 is seeded a known culture substrate capable of culturing fibroblasts can be used, but cells having a porous membrane It is preferably a culture substrate, and more preferably a cell culture insert. If the first cell culture substrate 11 has the first porous film 110, nutrients and oxygen can be supplied from both above and below the cells seeded on the first porous film 110.
- the second cell culture substrate 21 on which the second cell group 200 is seeded a known culture substrate capable of culturing melanocytes and keratinocytes can be used.
- a cell culture substrate having a membrane is preferable, and a cell culture insert is more preferable. If the second cell culture substrate 21 has the second porous membrane 210, not only can nutrients and oxygen be supplied from both above and below the cells seeded on the second porous membrane 210, The humoral components secreted by the first cell group 100 and the second cell group 200 can pass through each other.
- the dermis model and the epidermis model are separated in the process of maintaining and culturing the pigmented skin model 1 under different conditions. It is possible to culture, and it is also possible to transfer the epidermal model onto a different dermis model and culture. Since the formation of the epidermal layer by the epidermal cell group is affected by the quality of the dermis model, it is possible to control the quality of the epidermal model by replacing the pigmented skin model 1 with a dermis model having any quality. Become.
- the average pore diameter of the first porous membrane 110 and the second porous membrane 210 can be appropriately selected, and for example, the average pore diameter of about 0.01 ⁇ m to about 100 ⁇ m ( For example, 0.01 ⁇ m to 100 ⁇ m, 0.01 ⁇ m to 50 ⁇ m, 0.01 ⁇ m to 10 ⁇ m, 0.1 ⁇ m to 50 ⁇ m, or 0.1 ⁇ m to 10 ⁇ m).
- the density of the pores of the first porous film 110 and the second porous film 210 can be appropriately selected, but for example, 1 ⁇ 10 4 /cm 2.
- the density may be 1 ⁇ 10 4 /cm 2 to 100 ⁇ 10 8 /cm 2 , or 1 ⁇ 10 5 /cm 2 to 100 ⁇ 10 8 /cm 2 .
- the second cell culture substrate 21 when the second cell culture substrate 21 is a cell culture insert, the second cell culture substrate 21 (cell culture insert) smaller than the inner diameter of the first cell culture substrate 11 may be used. As a result, the second cell culture substrate 21 can be used by inserting it into the culture portion of the first cell culture substrate 11.
- the cells used in the present invention may be derived from any animal, but are preferably derived from vertebrates, more preferably mammalian, and most preferably human.
- Fibroblast is one of the cells that make up connective tissue, and is a cell that exists in many organs and tissues. In skin, fibroblasts are mainly contained in dermal tissue. The fibroblasts used in the pigmented skin model 1 of the present invention are preferably dermis-derived fibroblasts.
- Keratinocytes are one of the cells that make up the epidermis. In the epidermal tissue of living organisms, they divide toward the uppermost layers while dividing at the deepest part (basal layer), and to the spinous layer, granular layer, and horny layer. It is a cell that migrates to the surface while differentiating into layers, and eventually becomes dirt and falls off.
- Keanocytes are one of the cells that make up the epidermal tissue, and are cells that are present in the basal layer of the epidermis and form melanin in the living body.
- the fibroblasts, keratinocytes and melanocytes used in the present invention may each be primary cultured cells collected from living tissue, or may be cells that are isolated and/or propagated in advance and are commercially available or distributed. Well, it may be an established cell line, or may be a cell differentiated from a pluripotent stem cell such as an ES cell, an iPS cell, or a Muse cell.
- a pluripotent stem cell such as an ES cell, an iPS cell, or a Muse cell.
- the first cell group 100 may include cells other than fibroblasts, and may include, for example, mast cells, histiocytes, plasma cells, dermal dendritic cells and the like contained in dermal tissue. Good.
- the fibroblasts contained in the first cell group 100 include, for example, a cell number of 1 ⁇ 10 4 to 10 8 cells/cm 2 , preferably 0.1 to 10 ⁇ 10 5 cells/cm 2 .
- the second cell group 200 may include cells other than keratinocytes and melanocytes, and may include Langerhans cells or Merkel cells included in the epidermal composition, for example.
- the second cell group 200 comprises, for example, keratinocyte:melanocyte cells in a ratio of 1:1 to 1000:1, preferably 3:1 to 30:1.
- the second cell group 200 has, for example, 1 ⁇ 10 2 to 10 6 keratinocytes/cm 2 , preferably 1.0 to 10 ⁇ 10 4 cells/cm 2 , and more preferably about 4 to 8 ⁇ 10 4 cells/cm 2 .
- cm 2 and the melanocytes contain 1 to 10 ⁇ 10 3 cells/cm 2 , preferably 4 to 8 ⁇ 10 3 cells/cm 2 .
- fibroblasts damaged by light irradiation are used.
- the fibroblasts damaged by the light irradiation are those damaged by the light irradiation, preferably in the presence of the photosensitizer.
- the photosensitizer used include psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, and gold nanoclusters protected by a tinol group (AUxSRy).
- AUxSRy gold nanoclusters protected by a tinol group
- the light used to damage the fibroblasts may damage the nucleic acids in the cells, such as DNA and RNA, as long as it has a wavelength at which all the cells are not killed.
- Ultraviolet light (about 200 nm to about 400 nm) ), more preferably UVA (about 320 nm to about 400 nm).
- the intensity of light to be irradiated may damage nucleic acids in cells, such as DNA and RNA, but may be such that apoptosis or the like is not induced to kill all cells, such as wavelength, irradiation time, and cell density. It may be adjusted as appropriate.
- irradiation with UVA 0.01J / cm 2 ⁇ 100J / cm 2, preferably 0.1J / cm 2 ⁇ 20J / cm 2, more preferably a 0.5J / cm 2 ⁇ 10J / cm 2 irradiation do it.
- Fibroblasts damaged by light irradiation are aging cells, for example, cell morphology is elongated, proliferation ability is reduced, and melanin production factor (eg, stem cell growth factor (SCF)) production is increased. It shows the features of increased levels (see Figure 3).
- SCF stem cell growth factor
- Cell senescence levels are generally known as markers for cell senescence, such as the expression level of aging-related acid ⁇ -galactosidase (SA- ⁇ gal), constitutive activation of cell cycle check mechanism such as p21/p53 pathway and p16 pathway, and IL. It can be investigated by measuring the expression level of a senescence-associated secretory phenotype (SASP) factor such as -6. By adjusting the light irradiation amount, fibroblasts having a desired senescence level can be obtained.
- SASP senescence-associated secretory phenotype
- the fibroblasts contained in the first cell group 100 constituting the pigmented skin model 1 are fibroblasts that are damaged by light irradiation but survive without being killed. Therefore, the pigmented skin model 1 of the present invention contains substantially homogeneous fibroblasts, and can provide a stable system with little variation in activity.
- the epidermal model 20 constituting the pigmented skin model 1 may be, for example, commercially available TESTSKINTM LSE-melano (TOYOBO), MelanoDermTM (MatTek), or the like. ..
- the pigmented skin model 1 is, for example, a culture medium used for usual keratinocyte culture, such as KG medium, Epilife KG2 (Kurabo), Humedia-KG2 (Kurabo), assay medium (TOYOBO), etc., at about 37° C. It can be performed over 0 to 14 days.
- a culture medium used for usual keratinocyte culture, such as KG medium, Epilife KG2 (Kurabo), Humedia-KG2 (Kurabo), assay medium (TOYOBO), etc.
- KG medium Epilife KG2 (Kurabo), Humedia-KG2 (Kurabo), assay medium (TOYOBO), etc.
- TOYOBO assay medium
- the first cell group 100 is preferably seeded together with a hydrogelator.
- the “hydrogelating agent” refers to a substance added to form a hydrogel.
- the hydrogelating agent used in the present invention include collagen, gelatin, hyaluronate, hyaluronan, fibrin, alginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen/thrombin, fibrillin, elastin, gum.
- step (2) may be performed in the presence of ascorbic acid, an ascorbic acid derivative or salt thereof.
- the presence of ascorbic acid, an ascorbic acid derivative or a salt thereof is preferable because it promotes the proliferation of fibroblasts and the production of collagen, thereby promoting stratification like the structure of the dermis.
- the “ascorbic acid derivative” refers to, for example, ascorbic acid diphosphate, ascorbic acid monophosphate, sodium L-ascorbate, L-ascorbic acid 2-glucoside, and a salt thereof (sodium salt). Salts, magnesium salts, etc.) are also included.
- fibroblasts damaged by light irradiation directly or indirectly act on melanocytes to promote melanin production. Therefore, by measuring the degree of pigment production and/or pigmentation of the pigmented skin model 1, it is possible to evaluate the factors that affect pigmentation.
- the present invention can provide a method for evaluating factors for treating or preventing skin pigmentation using a pigmented skin model.
- the method of the invention comprises (1) A step of applying a candidate factor to the pigmented skin model 1 and culturing, (2) A step of evaluating the therapeutic or preventive effect of the candidate factor, using the degree of pigment production and/or pigmentation in the pigmented skin model 1 as an index. Is included.
- the candidate agent is, for example, a small molecule compound, peptide, nucleic acid, protein, mammalian (eg, mouse, rat, pig, cow, sheep, monkey, human etc.) cell, tissue extract or cell culture. It may be a supernatant, a plant-derived compound or extract (for example, a crude drug extract, a crude drug-derived compound), a microorganism-derived compound or extract or a culture product, and the like.
- the candidate substance is added to the pigmented skin model 1, and after culturing for a desired period of time, the degree of pigment production and/or pigmentation in the pigmented skin model 1 is measured, whereby the therapeutic or preventive effect of pigmentation by the candidate substance is evaluated. Can be evaluated. For example, a pigment added with a candidate substance is compared with the degree of pigment production and/or pigmentation of the pigmented skin model 1 in which the candidate substance is not added or any substance having no therapeutic or preventive effect on pigmentation is added. When the degree of pigment production and/or pigmentation in deposited skin model 1 is high, the candidate substance can be evaluated as having a therapeutic or preventive effect on pigmentation.
- pigment production refers to production of a pigment produced by a pigmented skin model, for example, melanin pigment.
- Melanin pigments are mainly produced by melanocytes.
- the amount of pigment production for example, the amount of melanin can be determined by extracting the melanin pigment from a pigmented skin model, particularly the second cell group (keratinocytes and melanocytes) and measuring the absorbance at 405 nm.
- the amount of pigment production is determined by, for example, the pigmentation skin model, particularly the amount of melanin contained in the second cell group (keratinocytes and melanocytes) or the amount of nucleic acid encoding the same (for example, amount of mRNA), ELISA method, flow cytometer method, western method. It can be measured by using a method such as a blotting method, an immunohistochemistry method, a qPCR method, but is not limited thereto.
- the “pigmentation degree” refers to the lightness of color of a pigmented skin model under visible light, particularly the second cell group (keratinocytes and melanocytes).
- the brightness changes depending on the amount of melanin produced by melanocytes. Therefore, as the amount of melanin increases, the lightness decreases, and the pigmented skin model has a dark color. Conversely, when the amount of melanin decreases, the lightness increases, and the pigmented skin model has a pale color. That is, by comparing the lightness of colors of pigmented skin models, the therapeutic or preventive effect of pigmentation by the added candidate substance can be evaluated.
- the lightness can be quantified by recording a pigmented skin model as an image and using a known image measuring means.
- the method of the present invention comprises (1) a step of culturing a first cell group 100 containing fibroblasts damaged by light irradiation, (2) a step of culturing the first cell group 100 obtained in the step (1) on the first cell culture substrate 11.
- Steps (1) to (3) are common to the steps described in the method for producing a pigmented skin model described above, and therefore description thereof will be omitted here ((A) to (D) in FIG. 2). Further, step (5) is common with step (2) described in the above-mentioned “Method for evaluating factor for treating or preventing skin pigmentation (first aspect)”, and therefore description thereof is omitted here. ..
- the present invention includes a step of applying the second cell group 200 obtained in the step (3) and culturing the obtained pigmented skin model 1a on a substrate containing a candidate factor.
- the candidate factor on the substrate used in the step (3) is, for example, a low molecular compound, a peptide, a nucleic acid, a protein, or a mammal.
- the candidate factor is any cell, eg, a precursor of fibroblasts, including mesenchymal stem cells, a fibroblast (eg, a light cell), as shown in FIGS. 2(E) and (F). Fibroblasts that have not been damaged by irradiation, or fibroblasts that have low damage by irradiation with light).
- the term “fibroblasts that have not been damaged by light irradiation” or “fibroblasts having low damage by light irradiation” are fibroblasts that have not been subjected to the light irradiation step described above, "A fibroblast having a lower senescence level than the "fibroblast damaged by light irradiation”.
- the third cell group 100a as a candidate factor may be seeded on the third cell culture substrate 11a having a porous membrane.
- step (4) may be a step of directly seeding or stacking the second cell group 200 obtained in step (3) on the third cell group 100a as a candidate factor, It may be a step of applying the second cell group 200 seeded on the second cell culture substrate 21 having a porous membrane onto the third cell group 100a as a candidate factor. In the latter case, the second cell culture substrate 21 can be easily transferred onto another dermis model.
- UVE-502S UVE-502S was performed (hereinafter referred to as "PUVA treatment"). Then, after swelling with 1 mL of PBS, the PBS was removed, the medium was replaced with a growth medium, and the cells were cultured for about 2 to 7 days. In the process, cells were partially killed by the effect of PUVA treatment, but thereafter, viable fibroblasts were proliferated. PUVA-untreated fibroblasts from the same donor served as controls for the PUVA-treated fibroblasts (FIGS. 3-6).
- a dermis model was prepared by using PUVA-treated or PUVA-untreated (control) fibroblasts, respectively. Briefly, the above 1. After the procedure of 1., the growth medium was removed and swollen with 1 mL of PBS. Thereafter, PBS was removed, 300 ⁇ L of a cell detachment agent (TrypLE SELECT, manufactured by Thermo Fisher Scientific) was added to each well of a 6-well plate, and the cells were allowed to stand in a 37° C. 5% CO 2 incubator for 5 minutes to perform cell detachment treatment. went. The reaction was stopped by adding a growth medium, and the cell suspension was collected in a 15 ml centrifuge tube.
- a cell detachment agent TrypLE SELECT, manufactured by Thermo Fisher Scientific
- the cell culture insert for preparing a dermis model was placed on a 6-well plate, and 3 mL gel suspension was added into the cell culture insert. After solidifying the gel suspension at 37 °C 5% CO 2 incubator, the growth medium containing ascorbic acid (AA2G) was added 2mL to 6 well plates, incubated for 24 hours at 37 °C 5% CO 2 incubator did.
- A2G ascorbic acid
- the prepared skin model is the same as the above 1-2. Placed on the dermis model of No. 3 (see FIG. 1(D)), placed in a dedicated container (Corning Biocoat, deep well plate, for 6 wells) (Corning, #355467), 9.5 mL three-dimensional A medium for the skin model (medium for exclusive use of the epidermis model (MatTek, #EPI-100-NMM-113) and DMEM mixed at a ratio of 1:1) was added, and the medium was used once every 3 to 4 days. Exchanged.
- FIG. 3 shows fibroblasts after and without treatment with PUVA. Fibroblasts after PUVA treatment were elongated (FIG. 3(A)). When the change in the proliferation number of fibroblasts treated with PUVA and untreated was examined, the proliferation rate of fibroblasts treated with PUVA was remarkably reduced (FIG. 3(B)).
- FIG. 7 is a diagram showing pigmentation of an epidermal model in a pigmented skin model after (A) or not treated (B) with PUVA. It was observed that pigmentation was promoted in the epidermis model in the pigmented skin model treated with PUVA.
- Pigmentation of a pigmented skin model in which PUVA treatment, untreated, and dermis model are replaced Epidermis models of PUVA-treated or untreated (control) pigmented skin model and PUVA-treated pigmented skin model Observation of the appearance of the epidermal model (Replace), which was transferred to the model and cultured, showed that pigmentation was promoted in the epidermis model of the PUVA-treated pigmented skin model, whereas that in the epidermis model of the Replace condition was reduced. The behavior was observed (FIG. 9(A)). Quantification based on the image in FIG. 9(A) showed that the pigmentation skin model with PUVA treatment had the highest proportion of the pigmentation area, whereas the pigmentation skin model under the Replace condition had a pigmentation concentration. It was shown that the area was reduced (FIG. 9(B)).
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Abstract
Description
前記第1細胞群の上に適用された、メラノサイトとケラチノサイトとを含む第2細胞群と、
を含む、色素沈着皮膚モデル。
[2] 前記光照射により損傷が与えられた線維芽細胞が、光増感剤の存在下で、紫外光の照射により損傷が与えられた線維芽細胞である、[1]に記載の色素沈着皮膚モデル。
[3] 前記光増感剤が、ソラレン、NAD、リボフラビン、トリプトファン、葉酸、ポルフィリン、メチレンブルー、チノール基で保護された金ナノクラスター(AUxSRy)からなる群から選択される、[2]に記載の色素沈着皮膚モデル。
[4] 前記光照射が、UVAの光照射である、[1]~[3]のいずれか1項に記載の色素沈着皮膚モデル。
[5] 前記第2細胞群が、多孔膜を有する第2細胞培養基材の上に播種された第2細胞群である、[1]~[4]のいずれか1項に記載の色素沈着皮膚モデル。
[6] 前記第1細胞培養基材が、多孔膜を有する細胞培養基材である、[1]~[5]のいずれか1項に記載の色素沈着皮膚モデル。
[7] 前記第1細胞群が、ハイドロゲル化剤とともに播種された第1細胞群である、[1]~[6]のいずれか1項に記載の色素沈着皮膚モデル。
[8] 前記ハイドロゲル化剤が、コラーゲン、ゼラチン、ヒアルロナート、ヒアルロナン、フィブリン、アルギナート、アガロース、キトサン、キチン、セルロース、ペクチン、デンプン、ラミニン、フィブリノーゲン/トロンビン、フィブリリン、エラスチン、ガム、セルロース、寒天、グルテン、カゼイン、アルブミン、ビトロネクチン、テネイシン、エンタクチン/ニドジェン、糖タンパク質、グリコサミノグリカン、ポリ(アクリル酸)およびその誘導体、ポリ(エチレンオキシド)およびその共重合体、ポリ(ビニルアルコール)、ポリホスファゼン、マトリゲルならびにそれらの組み合わせからなる群から選択される、[7]に記載の色素沈着皮膚モデル。
(1)光照射により損傷が与えられた線維芽細胞を含む第1細胞群を培養する工程、
(2)前記工程(1)で得られる第1細胞群を、第1細胞培養基材の上で、培養する工程、および
(3)前記工程(2)で得られる第1細胞群の上に、メラノサイトとケラチノサイトとを含む第2細胞群を適用し、培養する工程、
を含む、製造方法。
[10] 前記光照射により損傷が与えられた線維芽細胞が、光増感剤の存在下で、紫外光の照射により損傷が与えられた線維芽細胞である、[9]に記載の方法。
[11] 前記光増感剤が、ソラレン、NAD、リボフラビン、トリプトファン、葉酸、ポルフィリン、メチレンブルー、チノール基で保護された金ナノクラスター(AUxSRy)からなる群から選択される、[10]に記載の方法。
[12] 前記光照射が、UVAの光照射である、[9]~[11]のいずれか1項に記載の方法。
[13] 前記第2細胞群が、多孔膜を有する第2細胞培養基材の上に播種された第2細胞群である、[9]~[12]のいずれか1項に記載の方法。
[14] 前記第1細胞培養基材が、多孔膜を有する細胞培養基材である、[9]~[13]のいずれか1項に記載の方法。
[15] 前記第1細胞群が、ハイドロゲル化剤とともに播種された第1細胞群である、[9]~[14]のいずれか1項に記載の方法。
[16] 前記ハイドロゲル化剤が、コラーゲン、ゼラチン、ヒアルロナート、ヒアルロナン、フィブリン、アルギナート、アガロース、キトサン、キチン、セルロース、ペクチン、デンプン、ラミニン、フィブリノーゲン/トロンビン、フィブリリン、エラスチン、ガム、セルロース、寒天、グルテン、カゼイン、アルブミン、ビトロネクチン、テネイシン、エンタクチン/ニドジェン、糖タンパク質、グリコサミノグリカン、ポリ(アクリル酸)およびその誘導体、ポリ(エチレンオキシド)およびその共重合体、ポリ(ビニルアルコール)、ポリホスファゼン、マトリゲルならびにそれらの組み合わせからなる群から選択される、[15]に記載の方法。
[17] 前記工程(2)が、アスコルビン酸、アスコルビン酸誘導体またはそれらの塩の存在下で実施される、[9]~[16]のいずれか1項に記載の方法。
(1)[1]~[8]および[18]のいずれか1項に記載の色素沈着皮膚モデルに、候補因子を適用し、培養する工程、
(2)前記色素沈着皮膚モデルにおける色素産生および/または色素沈着の度合いを指標として、前記候補因子の治療または予防効果を評価する工程、
を含む、方法。
(1)光照射により損傷が与えられた線維芽細胞を含む第1細胞群を培養する工程、
(2)前記工程(1)で得られる第1細胞群を、第1細胞培養基材の上で、培養する工程、
(3)前記工程(2)で得られる第1細胞群の上に、メラノサイトとケラチノサイトとを含む第2細胞群を適用し、培養する工程、
(4)候補因子を含む基材上に、前記工程(3)で得られる前記第2細胞培養群を移し替えて、培養する工程、
(5)前記第2細胞群における色素産生および/または色素沈着の度合いを指標として、前記候補因子の治療または予防効果を評価する工程
を含む、方法。
[21] 前記候補因子が、線維芽細胞である、[20]に記載の方法。
図1は、一実施形態における、色素沈着皮膚モデル1およびそれを製造する方法を説明する概略図である。一実施態様において、色素沈着皮膚モデル1は、
第1細胞培養基材11の上に播種された、光照射により損傷が与えられた線維芽細胞を含む第1細胞群100と;
前記第1細胞群100の上に適用された、メラノサイトとケラチノサイトとを含む第2細胞群200と、を含んでいる(例えば、図1(D)参照)。
(1)光照射により損傷が与えられた線維芽細胞を含む第1細胞群100を培養する工程、
(2)前記工程(1)で得られる第1細胞群100を、第1細胞培養基材11の上で、培養する工程、
(3)前記工程(2)で得られる第1細胞群100の上に、メラノサイトとケラチノサイトとを含む第2細胞群200を適用し、培養する工程、
を含む、方法によって提供することができる。
本発明は、色素沈着皮膚モデルを用いた、皮膚の色素沈着を治療または予防するための因子の評価方法を提供することができる。一実施態様において、本発明の方法は、
(1)色素沈着皮膚モデル1に、候補因子を適用し、培養する工程、
(2)記色素沈着皮膚モデル1における色素産生および/または色素沈着の度合いを指標として、前記候補因子の治療または予防効果を評価する工程、
を含んでいる。
他の実施態様において、本発明の方法は、
(1)光照射により損傷が与えられた線維芽細胞を含む第1細胞群100を培養する工程、
(2)前記工程(1)で得られる第1細胞群100を、第1細胞培養基材11上で、培養する工程、
(3)前記工程(2)で得られる第1細胞群100の上に、メラノサイトとケラチノサイトとを含む第2細胞群200を適用し、培養する工程、
(4)候補因子を含む基材上に、前記工程(3)で得られる前記第2細胞群を適用し、得られる色素沈着皮膚モデル1aを培養する工程、
(5)前記色素沈着皮膚モデル1aにおける色素産生および/または色素沈着の度合いを指標として、前記候補因子の治療または予防効果を評価する工程、
を含んでいる。
1-1.線維芽細胞の培養およびPUVA処理
1×105個の正常ヒト線維芽細胞を増殖用培地(DMEM+10%牛胎児血清)で培養した。100%コンフルエントになる前にソラレン(終濃度25ng/mL)(SigmaAldrich社製)を加えた増殖用培地に置換し、培養した。24時間後に1mLのリン酸緩衝液(PBS)で膨潤させた後、PBSを取り除き、ソラレン(終濃度25ng/mL)を加えた1mLのPBSに置換し、6J/cm2 UVA照射(SAN-EI UVE-502S)を行った(以下、「PUVA処理」という)。その後、1mLのPBSで膨潤させた後、PBSを取り除いて、増殖用培地に置換し、2~7日程度培養した。その過程で、PUVA処理の影響で細胞が一部死滅するが、その後、生存している線維芽細胞が増殖した。PUVA未処理の同一ドナー由来線維芽細胞を上記PUVA処理線維芽細胞に対するコントロールとした(図3~6)。
上記1-1.においてPUVA処理、またはPUVA未処理(コントロール)の線維芽細胞をそれぞれ用いて真皮モデルを作製した。簡単に述べると、上記1.の手順後、増殖用培地を取り除いて1mLのPBSで膨潤させた。その後、PBSを取り除き、細胞剥離剤(TrypLE SELECT、Thermo Fisher Scientific社製)を6穴プレートの1ウェルあたり300μL添加し、37℃ 5%CO2インキュベーター内に5分間静置し、細胞剥離処理を行った。増殖用培地を添加して反応を停止させ、15ml遠沈管に細胞懸濁液を回収した。1000rpmで5分間遠心後、上清を除去し、細胞ペレットを増殖用培地で再懸濁し、5×105個の細胞/mLとなるように調整した。6穴プレートの各ウェルに適用可能な真皮モデルを作製するにあたり、表1の組成からなるゲル懸濁液を氷上にて調製した。
上記真皮モデルとは別に、メラノサイトとケラチノサイトで構成された市販の表皮モデル(MatTek社製)を使用した(図1(C)参照)。
PUVA処理線維芽細胞を用いて作製した真皮モデルと組み合わせて10日間培養した表皮モデルを、PUVA未処理の正常線維芽細胞を用いて作製した真皮モデルと置き換え、さらに10日間培養を行った(図2(E)および(F)参照)。なお、置き換え用真皮モデル(PUVA未処理の線維芽細胞を用いて作製した真皮モデル)は、置き換える前日に作製した。
2-1.PUVA処理後の線維芽細胞
図3は、PUVA処理後及び未処理の線維芽細胞を示している。PUVA処理後の線維芽細胞は、伸長していた(図3(A))。PUVA処理後及び未処理の線維芽細胞の増殖数の変動を調べたところ、PUVA処理後の線維芽細胞は増殖速度が著しく低下していた(図3(B))。
PUVA処理後及び未処理の線維芽細胞それぞれの細胞溶解物中の老化関連因子SA-β-galの酵素活性値について調べたところ、PUVA処理後の線維芽細胞はSA-β-galの酵素活性値が著しく増加していた(図4(A))。また、SA-β-galの染色を行ったところ、PUVA処理後の線維芽細胞は著しく多数のSA-β-gal陽性細胞(青緑色)が観察された(図4(B))。
PUVA処理後及び未処理の線維芽細胞それぞれ単層培養し、PUVA処理1日前、PUVA処理0、1、3、7、14、21日目に培養上清に含まれるSCF量をELISA法で測定し、1細胞あたりの分泌量として算出したところ、PUVA処理後の線維芽細胞のSCFレベルは著しく増加していた(図5(A))。また、SCF抗体を用いてSCF染色、DAPIによる核染色を行い、SCF染色および核染色の融合画像(Merge)を作成した結果、PUVA処理後の線維芽細胞においてSCF陽性細胞が多数観察された(図5(B))。
PUVA処理後及び未処理の線維芽細胞それぞれ単層培養し、HGF抗体を用いてHGF染色、DAPIによる核染色を行い、HGF染色および核染色の融合画像(Merge)を作成した結果、PUVA処理後の線維芽細胞においてHGF陽性細胞が多数観察された(図6(A))。
図7は、PUVA処理後(A)または未処理(B)の色素沈着皮膚モデルにおける、表皮モデルの色素沈着を示す図である。PUVA処理を行った色素沈着皮膚モデルにおける、表皮モデルは、色素沈着が促進されていることが観察された。
PUVA処理線維芽細胞を用いて作製した真皮モデルと組み合わせて10日間培養した表皮モデルを、PUVA未処理の正常線維芽細胞を用いて作製した真皮モデルと置き換え、さらに10日間培養を行った結果、PUVA処理を行った色素沈着皮膚モデルにおける表皮モデルの色素沈着(図7(A))に比べて色素沈着が抑制されていることが観察された(図8)。
PUVA処理または未処理(コントロール)の色素沈着皮膚モデル、およびPUVA処理の色素沈着皮膚モデルの表皮モデルを、PUVA未処理の真皮モデル上に移し替えて培養した表皮モデル(Replace)の外観について観察したところ、PUVA処理の色素沈着皮膚モデルの表皮モデルで色素沈着が促進したのに対し、Replace条件の表皮モデルでは色素沈着が緩和している様子が観察された(図9(A))。図9(A)の画像を元に定量化を行ったところ、PUVA処理の色素沈着皮膚モデルで最も色素濃化領域の割合が高いのに対して、Replace条件の色素沈着皮膚モデルでは色素濃化領域が減少することが示された(図9(B))。
PUVA処理または未処理(コントロール)の色素沈着皮膚モデル、およびPUVA処理の色素沈着皮膚モデルの表皮モデルを、PUVA未処理の真皮モデル上に移し替えて培養した表皮モデル(Replace)で切片を作成し、フォンタナマッソン染色法により表皮モデル切片のメラニン顆粒を染色した(図10(A))。また、図10(A)の染色像を二値化処理し、メラニン顆粒領域の割合を定量化したところ、PUVA処理の色素沈着皮膚モデルで最もメラニン領域の割合が高いのに対して、Replace条件の色素沈着皮膚モデルではメラニン領域が減少することが示された(図10(B))。
PUVA処理または未処理(コントロール)の色素沈着皮膚モデル、およびPUVA処理の色素沈着皮膚モデルの表皮モデルを、PUVA未処理の真皮モデル上に移し替えて培養した表皮モデル(Replace)で切片を作成し、TRP2抗体を用いて表皮モデル切片の活性化メラノサイトを染色し、およびヘマトキシリン染色法で核を染色した(図11(A))。図11(A)の画像から活性化メラノサイトの割合を定量化したところ、PUVA処理の色素沈着皮膚モデルで最も活性化メラノサイト数の割合が高いのに対して、Replace条件の色素沈着皮膚モデルでは活性化メラノサイト数が減少することが示された(図11(B))。
10 第1真皮モデル
100 第1細胞群
11 第1細胞培養基材
110 第1多孔膜
12 第1細胞培養容器
13 第1培地
14 第2培地
10a 第2真皮モデル
100a 第3細胞群
11a 第3細胞培養基材
110a 第3多孔膜
20 表皮モデル
200 第2細胞群
21 第2細胞培養基材
210 第2多孔膜
22 第2細胞培養容器
23 第3培地
24 第4培地
RD 光照射
FB 線維芽細胞
bFB 光照射による損傷を受けた線維芽細胞
KC ケラチノサイト
MC メラノサイト
Claims (21)
- 第1細胞培養基材の上に播種された、光照射により損傷が与えられた線維芽細胞を含む第1細胞群と;
前記第1細胞群の上に適用された、メラノサイトとケラチノサイトとを含む第2細胞群と、
を含む、色素沈着皮膚モデル。 - 前記光照射により損傷が与えられた線維芽細胞が、光増感剤の存在下で、紫外光の照射により損傷が与えられた線維芽細胞である、請求項1に記載の色素沈着皮膚モデル。
- 前記光増感剤が、ソラレン、NAD、リボフラビン、トリプトファン、葉酸、ポルフィリン、メチレンブルー、チノール基で保護された金ナノクラスター(AUxSRy)からなる群から選択される、請求項2に記載の色素沈着皮膚モデル。
- 前記光照射が、UVAの光照射である、請求項1~3のいずれか1項に記載の色素沈着皮膚モデル。
- 前記第2細胞群が、多孔膜を有する第2細胞培養基材の上に播種された第2細胞群である、請求項1~4のいずれか1項に記載の色素沈着皮膚モデル。
- 前記第1細胞培養基材が、多孔膜を有する細胞培養基材である、請求項1~5のいずれか1項に記載の色素沈着皮膚モデル。
- 前記第1細胞群が、ハイドロゲル化剤とともに播種された第1細胞群である、請求項1~6のいずれか1項に記載の色素沈着皮膚モデル。
- 前記ハイドロゲル化剤が、コラーゲン、ゼラチン、ヒアルロナート、ヒアルロナン、フィブリン、アルギナート、アガロース、キトサン、キチン、セルロース、ペクチン、デンプン、ラミニン、フィブリノーゲン/トロンビン、フィブリリン、エラスチン、ガム、セルロース、寒天、グルテン、カゼイン、アルブミン、ビトロネクチン、テネイシン、エンタクチン/ニドジェン、糖タンパク質、グリコサミノグリカン、ポリ(アクリル酸)およびその誘導体、ポリ(エチレンオキシド)およびその共重合体、ポリ(ビニルアルコール)、ポリホスファゼン、マトリゲルならびにそれらの組み合わせからなる群から選択される、請求項7に記載の色素沈着皮膚モデル。
- 色素沈着皮膚モデルの製造方法であって、
(1)光照射により損傷が与えられた線維芽細胞を含む第1細胞群を培養する工程、
(2)前記工程(1)で得られる第1細胞群を、第1細胞培養基材の上で、培養する工程、および
(3)前記工程(2)で得られる第1細胞群の上に、メラノサイトとケラチノサイトとを含む第2細胞群を適用し、培養する工程、
を含む、製造方法。 - 前記光照射により損傷が与えられた線維芽細胞が、光増感剤の存在下で、紫外光の照射により損傷が与えられた線維芽細胞である、請求項9に記載の方法。
- 前記光増感剤が、ソラレン、NAD、リボフラビン、トリプトファン、葉酸、ポルフィリン、メチレンブルー、チノール基で保護された金ナノクラスター(AUxSRy)からなる群から選択される、請求項10に記載の方法。
- 前記光照射が、UVAの光照射である、請求項9~11のいずれか1項に記載の方法。
- 前記第2細胞群が、多孔膜を有する第2細胞培養基材の上に播種された第2細胞群である、請求項9~12のいずれか1項に記載の方法。
- 前記第1細胞培養基材が、多孔膜を有する細胞培養基材である、請求項9~13のいずれか1項に記載の方法。
- 前記第1細胞群が、ハイドロゲル化剤とともに播種された第1細胞群である、請求項9~14のいずれか1項に記載の方法。
- 前記ハイドロゲル化剤が、コラーゲン、ゼラチン、ヒアルロナート、ヒアルロナン、フィブリン、アルギナート、アガロース、キトサン、キチン、セルロース、ペクチン、デンプン、ラミニン、フィブリノーゲン/トロンビン、フィブリリン、エラスチン、ガム、セルロース、寒天、グルテン、カゼイン、アルブミン、ビトロネクチン、テネイシン、エンタクチン/ニドジェン、糖タンパク質、グリコサミノグリカン、ポリ(アクリル酸)およびその誘導体、ポリ(エチレンオキシド)およびその共重合体、ポリ(ビニルアルコール)、ポリホスファゼン、マトリゲルならびにそれらの組み合わせからなる群から選択される、請求項15に記載の方法。
- 前記工程(2)が、アスコルビン酸、アスコルビン酸誘導体またはそれらの塩の存在下で実施される、請求項9~16のいずれか1項に記載の方法。
- 請求項9~17のいずれか1項に記載の方法により得られる、色素沈着皮膚モデル。
- 皮膚の色素沈着を治療または予防するための因子の評価方法であって、
(1)請求項1~8および請求項18のいずれか1項に記載の色素沈着皮膚モデルに、候補因子を適用し、培養する工程、
(2)前記色素沈着皮膚モデルにおける色素産生および/または色素沈着の度合いを指標として、前記候補因子の治療または予防効果を評価する工程、
を含む、方法。 - 皮膚の色素沈着を治療または予防するための因子の評価方法であって、
(1)光照射により損傷が与えられた線維芽細胞を含む第1細胞群を培養する工程、
(2)前記工程(1)で得られる第1細胞群を、第1細胞培養基材の上で、培養する工程、
(3)前記工程(2)で得られる第1細胞群の上に、メラノサイトとケラチノサイトとを含む第2細胞群を適用し、培養する工程、
(4)候補因子を含む基材上に、前記工程(3)で得られる前記第2細胞群を適用し、得られる色素沈着皮膚モデルを培養する工程、
(5)前記色素沈着皮膚モデルにおける色素産生および/または色素沈着の度合いを指標として、前記候補因子の治療または予防効果を評価する工程
を含む、方法。 - 前記候補因子が、線維芽細胞である、請求項20に記載の方法。
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CN201980078315.9A CN113166694A (zh) | 2018-11-30 | 2019-11-29 | 色素沉积皮肤模型及其制造方法、以及用于治疗或预防皮肤的色素沉积的因子的评价方法 |
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