JP7246066B2 - Three-dimensional cultured epidermis model and manufacturing method thereof - Google Patents

Description

The present invention relates to a three-dimensional cultured epidermis model that reproduces specific skin conditions such as rough skin and wrinkles for each level, a method for producing the same, and a method for using the same.

In recent years, the technology to artificially construct functional three-dimensional tissue by culturing cells in three dimensions has been used not only in regenerative medicine such as organ replacement and tissue regeneration, but also in evaluating the safety and efficacy of cosmetics and pharmaceuticals. Since it can also be used in tests, research and development are being actively carried out. For example, cultured epidermis, cultured dermis, and composite cultured skin combining these have been developed as models imitating human skin tissue. These skin models are used in the medical field for wound closure in burns and skin ulcers, and in the development of cosmetics and pharmaceuticals, they are used as tools for predicting and evaluating the efficacy and irritation of drugs on the skin. ing.

Today, various types of cultured epidermis (skin) models are commercially available, and by using these, it is possible to conduct skin irritation tests and percutaneous absorption tests in vitro without using experimental animals. is possible. Injured skin models that reproduce skin damage conditions such as rough skin and inflammation have also been developed for the purpose of evaluating the efficacy and screening of cosmetics and pharmaceuticals. is used. For example, a three-dimensional skin model comprising an epidermis-like structure (Patent Document 1), keratinocytes and fibroblasts that has been damaged by immersion in a physiological saline solution containing SDS (sodium dodecyl sulfate) under certain conditions A parakeratosis and/or epidermal hyperplasia three-dimensional skin model (Patent Document 2) produced by irradiating UVB under the environment, and a horny sheet obtained by isolating the epidermal tissue maintained under culture and drying it under low humidity. A rough skin epidermis model (Patent Document 3) consisting of a skin disease accompanied by inflammation induction and hyperproliferation, which was produced by inducing inflammatory cytokines by allowing S100A and S100A9 of the calcium-binding protein S100 protein family to act on cultured keratinocytes. An in vitro model (Patent Document 4) and the like have been proposed.

However, it is difficult to control the intensity of UV irradiation and the concentration of chemical agents in injured skin models prepared by the above method, and the variation between lots is large, so the accuracy of evaluation tests using them is not necessarily high. It was not something that could be said. In addition, there are large differences in skin quality among individuals, such as those who are prone to rashes, those who are prone to dryness, and those who are prone to pigmentation. It changes with age, season and environment. For example, even low-irritant cosmetics may cause troubles depending on individual skin quality and conditions. Therefore, in order to accurately confirm the actions and effects of medicines and cosmetics, and to provide medicines and cosmetics suitable for individual skin conditions, it is necessary to accurately reproduce specific skin conditions. The development and operation of epidermal models are awaited.

JP-A-2008-26092 JP 2009-240271 A Japanese Patent Application Laid-Open No. 2010-172200 WO2008/096868

Therefore, in view of the above-described circumstances, an object of the present invention is to provide a three-dimensional cultured epidermis model that accurately reproduces specific skin conditions such as rough skin and wrinkles for each level.

As a result of intensive research to solve the above problems, the present inventors have found a three-dimensional cultured epidermis made from epidermal keratinocytes modified using genome editing technology that can arbitrarily edit genes, which has been attracting attention in recent years. By creating , various skin conditions can be reproduced according to the type of target gene and genome editing. By arbitrarily changing the condition of the skin can be accurately reproduced for each level, and have completed the present invention.

That is, the present invention includes the following inventions.
(1) A three-dimensional cultured epidermal model containing genome-edited epidermal keratinocytes whose target genes have been modified by genome editing and normal epidermal keratinocytes.
(2) The ratio of the number of cells occupied by the genome-edited epidermal keratinocytes to the total cell number of the genome-edited epidermal keratinocytes and the normal epidermal keratinocytes is 5% or more. The three-dimensional cultured epidermis model according to (1).
(3) The three-dimensional cultured epidermis model according to (1) or (2), wherein the target gene is a gene associated with skin barrier function.
(4) A method for producing a three-dimensional cultured epidermis model including the following steps.
(a) A step of modifying the target gene of epidermal keratinocytes by genome editing to produce genome-edited epidermal keratinocytes
(b) step of mixing the genome-edited epidermal keratinocytes prepared in step (a) and normal epidermal keratinocytes in an arbitrary ratio
(c) the step of three-dimensionally culturing the cell mixture obtained in step (b); (5) the production method according to (4), wherein the epidermal keratinocytes are immortalized cells;
(6) The production method according to (4) or (5), wherein the target gene is a gene associated with skin barrier function.
(7) The production method according to any one of (4) to (6), wherein the genome editing is performed by CRISPR-Cas9.
(8) The production method according to any one of (4) to (7), wherein the modification of the target gene is deletion, insertion or substitution.
(9) The test substance is brought into contact with the three-dimensional cultured epidermis model according to any one of (1) to (3), and changes in epidermal keratinocytes of the model are measured. A method for evaluating efficacy or safety.
(10) Improvement of epidermal function, characterized by bringing a test substance into contact with the three-dimensional cultured epidermis model according to any one of (1) to (3), and measuring changes in epidermal keratinocytes in the model. Methods of screening substances.

According to the present invention, there is provided a three-dimensional cultured epidermis model that reproduces specific skin conditions such as rough skin and wrinkles for each level and that does not vary between lots. Therefore, the three-dimensional cultured epidermis model of the present invention can provide reproducible and reliable evaluation results in skin irritation tests and evaluation tests of substances that promote skin turnover.

FIG. 1 shows stained images of cultured epidermis models with arbitrary levels of rough skin prepared by changing the ratio of genome-edited cells to all cells.

1. 3. Three-dimensional cultured epidermis model and method for producing same The three-dimensional cultured epidermis model of the present invention is characterized by comprising genome-edited epidermal keratinocytes whose target genes have been modified by genome editing and normal epidermal keratinocytes. In the present invention, the term "genome-edited epidermal keratinocytes" refers to epidermal keratinocytes in which target genes on the genome of epidermal keratinocytes have been genetically modified by genome editing. (including deletion and partial deletion), insertion (including insertion of a foreign gene), substitution, and the like. Moreover, in the present invention, the term “normal epidermal keratinocytes” refers to epidermal keratinocytes that have not undergone genome editing as described above.

Genome editing introduces a double strand break (DSB) into any DNA base sequence using an artificial enzyme that can cleave a specific site in the genomic DNA, and the repair mechanism (homologous group) of the cleaved DNA Recombination (homologous recombination (HR) or non-homologous end jointing (NHEJ) technology to modify the target gene in the process. The three-dimensional cultured epidermis model of the present invention is targeted by such genome editing It contains gene-edited epidermal keratinocytes in an arbitrary ratio to the total cells that make up the cultured epidermis, and reproduces various skin conditions corresponding to the type of target gene and the type of modification. By changing the ratio, the skin condition level can be arbitrarily controlled. It is characterized by having a stratified structure of epidermal keratinocytes near the epidermis.

In the three-dimensional cultured epidermal model of the present invention, the ratio of the number of genome-edited epidermal keratinocytes to the total number of cells, which is the sum of the number of genome-edited epidermal keratinocytes and the number of normal epidermal keratinocytes, is It may be 5% or more, and in the range of less than 100%, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, It can be 65%, 70%, 75%, 80%, 85%, 90%, 95%, and the like.

In the present invention, target genes to be modified by genome editing are not particularly limited as long as they are expressed in epidermal keratinocytes (keratinocytes) and are associated with specific skin conditions. In addition, the above target genes include other cells (melanocytes, etc.) in the epidermis other than epidermal keratinocytes (keratinocytes), and basement membrane cells that affect the functions of the epidermis and the entire skin (elasticity, water retention, etc.). Also included are genes expressed in cells within the skin and dermis that are associated with specific skin conditions. In the present invention, "skin condition" refers to barrier function, wrinkles, sagging, inflammation, pigmentation (spots and freckles), dryness, sebum content, open pores, roughness of texture, and the like. Target genes include, for example, the following genes, and one or more of these may be used. Genome editing makes it possible to modify multiple genes simultaneously.

<Genes related to skin barrier function>
Filaggrin (FLG): NM_002016.1
Involucrin (IVL): NM_005547.3
Loricrin (LOR): NM_000427.2
Transglutaminase-2 (TGM2): NM_001323316.1
tight junction protein-1 (TJP1): NM_001301025.2
Claudin 1 (CLDN1): NM_021101.4
Occludin (OCLN): NM_001205254.2
Aquaporin 1 (AQP1): NM_001329872.1
Aquaporin 3 (AQP3): NM_001318144.1
3-ketodihydrosphingosine reductase (KDSR): NM_002035.4
Ceramide synthase-1 (CERS1): NM_001290265.1
Ceramide synthase-3 (CERS3): NM_001290341.2
Ceramide synthase-6 (CERS6): NM_001256126.1
Sphingosine 1-phosphate phosphatase (SGPP2): NM_001320833.1
Alkaline ceramidase 1 (ACER1): NM_133492.2
Caspase 14 (CASP14): NM_012114.3
small proline-rich proteins 1A (SPRR1A): NM_001199828.1

<Genes related to wrinkles and sagging>
Type I collagen α1 (COL1A1): NM_000088.3
Type III collagen α1 (COL3A1): NM_000090.3
Hyaluronic acid synthase-1 (HAS1): NM_001297436.1
Elastin (ELN): NM_000501.3
Matrix metalloproteinase-1 (MMP1): NM_001145938.1
Hyaluronidase 3 (HYAL3): NM_001200029.1
Galectin 9: NM_001330163.1

<Genes associated with aging and oxidative stress>
Sirtuin (SIRT): NM_001142498.1
kelch-like ECH associated protein 1 (KEAP1): NM_012289.3
Lamin A (LMNA): NM_001257374.2
ATM Serine/Threonine Protein Kinase (ATM): NM_000051.3
ATR Serine/Threonine Protein Kinase (ATR): NM_001184.4
WRN: NM_000553.5
Telomerase reverse transcriptase (TERT): NM_001193376.1
Catalase (CAT): NM_001752.3
Laminin α3 (LAMA3): NM_000227.4
Laminin β3 (LAMB3): NM_000228.2
Laminin γ2 (LAMC2): NM_005562.2
Type IV collagen α1 (COL4A1): NM_001303110.1
Type V collagen α1 (COL5A1): NM_000093.4
Integrin β1 (ITGB1): NM_002211.3
tumor protein p53 (TP53): NM_000546.5
Cyclin dependent kinase inhibitory protein 1A (CDKN1A): NM_000389.4
Cyclin-dependent kinase inhibition 2A (CDKN2A): NM_000077.4)
Superoxide dismutase-2 (SOD2): NM_000636.4
Glutathione peroxidase 1 (GPX1): NM_000581.4

<Genes related to age spots and freckles>
Dopachrome tautomerase (DCT): NM_001129889.2
Ras-related protein Rab-27A (RAB27A): NM_004580.4
Wnt family member 1 (WNT1): NM_005430.3
Microphthalmia-associated transcription factor (MITF): NM_000248.3
Tyrosinase-related protein-1 (TYRP1): NM_000550.2
Tyrosinase (TYR): NM_000372.4

<Genes related to enlarged pores and oily skin>
Steroid-5α-reductase-1 (SRD5A1): NM_001047.4
Peroxisome proliferator-activated receptor gamma (PPARG): NM_001330615.1
fatty acid binding protein 2 (FABP2): NM_000134.3

The method for producing a three-dimensional cultured epidermis model of the present invention includes (a) a step of modifying a target gene of epidermal keratinocytes by genome editing to produce genome-edited epidermal keratinocytes, and (b) produced in step (a). and (c) three-dimensionally culturing the cell mixture of step (b). Hereinafter, the method for producing the three-dimensional cultured epidermis model of the present invention will be described in order of steps.

Step (a):
In step (a), target genes of epidermal keratinocytes are modified by genome editing. The epidermal keratinocytes used in the present invention are preferably immortalized epidermal cells that do not lose their proliferative ability and differentiation ability even after repeated passages and do not undergo cell senescence. Immortalized epidermal keratinocytes can be established by subculturing epidermal keratinocytes isolated from skin tissue of humans or the like, into which an immortalization gene has been introduced, in an appropriate medium. Here, the term "immortalizing gene" refers to a gene that immortalizes cells and acquires the ability to proliferate infinitely, even if it is a gene that immortalizes cultured epithelial cells such as epidermal keratinocytes and does not induce cell death. is not particularly limited. An immortalizing gene is an exogenous gene and means an immortalizing gene newly introduced from outside the cell. Furthermore, the immortalizing gene may be a non-human-derived immortalizing gene, or an immortalizing gene modified to be expressible in target cells. Examples of immortalizing genes used in the present invention include telomerase reverse transcriptase (TERT) gene, genes regulating telomerase expression or activity (e.g., Myc gene, Ras gene, etc.), viral genes (SV40T, HPV E6-E7 , EBV, etc.), preferably the telomerase reverse transcriptase (TERT) gene, more preferably the human telomerase reverse transcriptase (hTERT) gene. The origin of the epidermal keratinocytes used in the present invention is not particularly limited as long as it is a mammal, and examples include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses. but preferably human.

In addition to the immortalizing gene, it is preferable to further introduce a gene encoding a cell cycle transition promoting factor. As a cell cycle transition promoting factor (positive regulator of the cell cycle),
Cyclin-Dependent Kinase (CDK) involved in cell cycle progression from G1 to S phase and its binding partner Cyclin (CCN). Cyclin dependent kinase (CDK) and cyclin (CCN) can be either one or both. Cyclin-dependent kinases (CDKs) used in the present invention include CDK1, CDK2, CDK3, CDK4, CDK6 and CDK7, among which CDK4 and CDK6 are preferred, and CDK4 is more preferred. Cyclins (CCNs) may be those that can bind to and activate the above CDKs, such as D-type cyclins (CCND1, CCND2, CCND3). Nucleotide sequence information for CDK genes and/or CCN genes used in the present invention is available from the NCBI database. In addition, the CDK gene and/or the CCN gene are preferably derived from mammals, more preferably from humans.

The method for introducing the immortalizing gene or the gene encoding the cell cycle transition-enhancing factor into epidermal keratinocytes is not limited as long as it is a method generally used for gene introduction. For example, a virus vector is used. Methods include the lipofection method, calcium phosphate coprecipitation method, electroporation method, and the like, but the method using a viral vector is preferred. Viral vectors include lentiviral vectors, retroviral vectors, adeno-associated virus (AAV) vectors, adenoviral vectors and the like.

In the present invention, modification of the target gene of epidermal keratinocytes is performed by genome editing of the target locus. Genome editing is a technology to modify genes by utilizing DNA double-strand breaks and repair errors, and uses a nuclease that can break DNA double-strands and a DNA recognition component that binds or complexes with the nuclease. do. Examples of such genome editing techniques include CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas9, TALEN (transcription activator-like effector nuclease), ZFN (zinc-finger nuclease), etc. However, the design of the target sequence CRISPR-Cas9 is preferably used from the viewpoint of the ease and efficiency of the method. Genome editing kits based on these technologies are commercially available and can be purchased from, for example, Thermo Fisher Scientific, ORIGENE, Clontech, and the like.

In CRISPR-Cas9, a guide RNA (gRNA) having a base sequence complementary to the target base sequence adjacent to the PAM sequence (NGG) on the genome and a Cas9 nuclease are used to induce double-strand cleavage in the target DNA sequence. (DSB). Guide RNA (gRNA) sequences were generated using the online CRISPR guide RNA design tool (https://apps.thermofisher.com/apps/crispr/index.html#/search), Thermo's Invitrogen® GeneArt® ) can be designed using a known guide RNA design tool such as CRISPR Search and Design Tool.

After introduction of a double-strand break, repair of the non-homologous end joining (NHEJ) pathway induces disruption of gene function (knockout) by inducing frameshifts and stop codons based on mutations such as base deletions and insertions. be able to. In addition, in repairing the homologous recombination (HR) pathway, it is possible to introduce another gene (knock-in) or replace SNPs at the cut site by co-introducing a donor vector.

In the present invention, in order to reproduce a specific skin condition, genome editing is carried out to delete, inactivate, or enhance the expression of the target gene. For example, a cultured epidermis model that reproduces a rough skin condition can be produced by performing genome editing that disrupts (knocks out) all or part of the function of the filaggrin gene, which is a gene responsible for skin barrier function. In addition, by performing genome editing that increases the expression of the matrix metalloproteinase-1 (MMP1) gene, which is a wrinkle-forming gene (for example, editing that increases the expression level of the transcriptional activator or strong promoter of the gene), the wrinkle state can be improved. It is possible to create a cultured epidermis model that reproduces the

Step (b):
In step (b), the genome-edited epidermal keratinocytes prepared in step (a) and normal cells are mixed at an arbitrary ratio. The mixing ratio of genome-edited epidermal keratinocytes and normal cells is not particularly limited. can range from 99:1 to 1:99.

Step (c):
Next, in step (c), three-dimensional culture is performed using a cell mixture of genome-edited epidermal keratinocytes and normal epidermal keratinocytes (hereinafter referred to as "cell mixture"). Three-dimensional culture can be carried out according to the following three-dimensional cultured skin preparation method generally used in the art, in which epidermal keratinocytes (keratinocytes) are stratified by exposure to air to reconstruct the skin. .

The production of three-dimensional cultured skin consists of a cell proliferation culture step and a differentiation induction step. In the growth culture step, the cell mixture is grown and cultured in a culture vessel such as a culture insert until the bottom becomes confluent. Specifically, the cell mixture is dispersed in a cell growth medium, the cell dispersion is seeded in a culture insert having a liquid-permeable membrane on the bottom, and the outside of the culture insert is also filled with the same cell growth medium, The cell mixture on the liquid-permeable membrane is cultured while immersed in a cell growth medium. The liquid-permeable membrane maintains medium-permeable communication between the interior and exterior of the culture insert. Here, the number of cells in the cell mixture added to the cell culture insert is not particularly limited, but is usually preferably 15×10 ⁴ to 120×10 ⁴ cells/cm ² , and preferably 30×10 ⁴ to 90×10 ⁴ cells/cm 2 . ² is more preferred.

The liquid-permeable membrane of the culture insert is not particularly limited as long as the cell mixture can be adhered or fixed, the cell mixture can grow on it, and it can serve as a support. Examples include polycarbonate, polyethylene terephthalate, polystyrene, and the like. membranes. In addition, the membrane may be coated with an extracellular matrix such as collagen, laminin or fibronectin, or a substance that assists cell adhesion such as poly-L-lysine.

Furthermore, in addition to using a culture insert as a support, collagen gel, collagen sponge, or acellular dermis from which epithelium and fibroblasts have been removed may be used. When these are used as supports, fibroblasts may be incorporated as appropriate. These supports are also preferably provided with a glass ring on the surface of the support into which the suspension of the cell mixture is added.

Proliferation culture is performed, for example, for 1 to 6 days, preferably 2 to 4 days. In addition, during this period, the medium may be exchanged as appropriate. Whether or not the mixture of cells grown in the culture insert is confluent can be confirmed using a cell staining reagent such as CnT-ST-100 stain kit (manufactured by CELLn TEC). It can also be determined that the cell mixture has become confluent when the liquid level of the culture solution inside the culture insert is higher than the liquid level of the culture solution outside the culture insert.

Next, in the differentiation induction step, the medium inside and outside the culture insert is changed from the cell growth medium to the cell differentiation medium, and the cell mixture is immersed and cultured in the medium for about 6 to 48 hours. All media inside and outside were removed with an aspirator, cell differentiation medium was added to the outside of the insert, and the cell mixture inside the culture insert was exposed to air (atmosphere), cultured for 5 to 12 days, and layered. Induce differentiation into epidermal keratinocytes.

The cell growth medium is not particularly limited as long as it is a basal medium suitable for epidermal keratinocyte proliferation and subculture, but is preferably a serum-free, low-calcium concentration basal medium. , For example, commercially available Keratinocyte-SFM (manufactured by Thermo Fisher Scientific), MCDB153 medium (manufactured by Sigma), Humedia-KG2 (manufactured by Kurabo), serum-free medium for normal human keratinocytes (manufactured by DS Pharma Biomedical), etc. A culture medium may be used. The above medium may contain growth factors such as keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), stem cell factor (SCF), and the like. Epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, bovine serum, as needed to increase proliferation rate. Albumin (BSA), L-glutamine, fibronectin, progesterone, selenite, B27-supplements, N2-supplements, ITS-supplements may be included. Moreover, you may add an antibiotic as needed. The calcium concentration of the cell growth medium is preferably about 0.03-0.15 mM.

The cell differentiation medium is not particularly limited as long as it is a basal medium suitable for inducing the differentiation of epidermal keratinocytes. should be used. Also, the calcium concentration of the cell differentiation medium is preferably about 1.2 to 3.0 mM.

The culture temperature for proliferation and induction of differentiation varies depending on the origin of the cells, but is preferably 30-40°C, more preferably 36-38°C, for human-derived cells. Also, the CO ₂ gas concentration is, for example, preferably about 1 to 10%, more preferably about 2 to 5%.

In the step of culturing the above cell mixture to prepare a stratified epidermal tissue, commercially available cultured epidermis preparation kits such as Millicell cell culture insert (manufactured by Millipore) and keratinocyte three-dimensional culture starter kit (manufactured by Funakoshi) are used. The culture medium and culture insert packed in the kit can be used and the instructions attached to the kit can be followed.

The three-dimensional cultured epidermis model of the present invention may be made into a kit, and the kit includes, for example, a medium and container suitable for culturing epidermal keratinocytes, positive and negative standard samples, and a method for using the kit. can include written instructions, etc.

2. How to use the three-dimensional cultured epidermis model The three-dimensional cultured epidermis model of the present invention can be used as an alternative to animal experiments to evaluate the efficacy and safety of cosmetics, topical skin preparations, and chemical substances (detergents, dyes for clothes, etc.). can be used. For example, evaluation of efficacy includes skin barrier function, moisture or oil retention/regulation function, wrinkle prevention/improvement function, presence or absence of moisture permeability, etc. Evaluation of safety includes erythema, redness, inflammation. , presence or absence of pigmentation, swelling, and rash. In addition, the three-dimensional cultured epidermis model of the present invention can also be used for screening substances that improve epidermal function. Epidermal function improvement includes improvement of epidermal barrier function (moisture retention function, function to prevent invasion of ultraviolet rays, chemical substances, bacteria, etc. from the outside, etc.), normalization function of skin turnover, normalization function of melanin metabolism, etc. mentioned.

The three-dimensional cultured epidermis model of the present invention is a model that accurately reproduces specific skin conditions for each level by mixing ratios of genome-edited epidermal keratinocytes and normal epidermal keratinocytes. Therefore, by using the three-dimensional cultured epidermis model of the present invention, skin irritation substances that cause rough skin can be identified for each level of skin condition (for example, severe rough skin, moderate rough skin, and mild rough skin). The three-dimensional cultured epidermis model of the present invention provides a novel evaluation system that has never existed before.

In the present invention, evaluation and screening of the safety and efficacy of cosmetics and pharmaceuticals are carried out by contacting the three-dimensional cultured epidermis model of the present invention with a test substance and measuring changes in epidermal keratinocytes of the model. changes in epidermal keratinocytes can be used as an index for evaluation. At this time, the three-dimensional cultured epidermis model of the present invention, which is not brought into contact with the test substance, is used as a control, and the evaluation results are compared with the measurement results for more accurate evaluation. Changes in epidermal keratinocytes include changes in their number, morphology, distribution, localization, migration, disappearance, etc., and specific genes in epidermal keratinocytes (e.g., Involucrin gene, Filaggrin gene, It includes changes in the expression level of epidermal keratinocyte-specific marker genes such as the Loricrin gene. For example, if cell death or growth inhibition of epidermal keratinocytes is used as an index, the test substance can be evaluated as a substance that irritates the epidermis, promoting the proliferation of epidermal keratinocytes and increasing the thickness of the stratum corneum. can be used as an index, the test substance can be screened as a candidate substance for a skin turnover accelerator. The test substance is brought into contact with the three-dimensional cultured epidermis model formed in the culture insert from the stratum corneum surface side and/or from the basal layer surface side. Specifically, it can be carried out by administering or applying the test substance to the epidermis model in the culture insert from above, or by adding the test substance to the culture medium outside the culture insert.

Measurement of changes in epidermal keratinocytes is not particularly limited. It can be performed by known methods such as cell growth/survival activity test, live cell measurement method such as trypan blue dye exclusion test method, and cytotoxicity test such as LDH assay.

The test substance is mainly intended for ingredients that can be used in cosmetics and / or pharmaceuticals, for example, a mixture containing multiple compounds such as animal / plant tissue extracts or microbial cultures, and preparations purified from them; Naturally occurring molecules (eg, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, steroids, glycoproteins, proteoglycans, etc.); or synthetic analogs or derivatives of naturally occurring molecules (eg, peptidomimetics, etc.) and non-naturally occurring molecules (eg, low-molecular-weight organic compounds prepared using combinatorial chemistry techniques and the like); and mixtures thereof. As the test substance, a single test substance may be independently tested, or a mixture of several candidate test substances (including a library, etc.) may be tested. Libraries containing multiple test substances include synthetic compound libraries and peptide libraries.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these.

(Example 1) Preparation of three-dimensional cultured epidermis using genome-edited epidermal keratinocytes
(1) Immortalization and Genome Editing of Epidermal Keratinocytes Normal human epidermal keratinocytes (NHEK, manufactured by Kurabo Industries) were transfected with the telomerase reverse transcriptase (TERT) gene (GenBank number: Nucleotide NM_198253.2, nucleotide Sequence: SEQ ID NO: 1, amino acid sequence of coding region: SEQ ID NO: 2), cyclin-dependent kinase 4 (CDK4) gene (GenBank number: Nucleotide NM_000075.3, nucleotide sequence: SEQ ID NO: 3, amino acid sequence of coding region: SEQ ID NO: 4), and cyclin D1 (CCND1) gene (GenBank number: Nucleotide NM_053056.2, base sequence: SEQ ID NO: 5, amino acid sequence of coding region: SEQ ID NO: 6) were introduced to prepare immortalized epidermal keratinocytes. Each gene was incorporated into a separate lentiviral vector (using Lentiviral High Titer Packaging Mix with pLVSIN series) and introduced into epidermal keratinocytes by the vector.

Next, a monoclonal cell line of immortalized epidermal keratinocytes was obtained using the limiting dilution method. Thermo Fisher Scientific's genome editing design software (online CRISPR guided RNA design tool; https://apps.thermofisher.com/apps/crispr/index.html#/search) and A deletion mutation of the filaggrin gene was induced using kit reagents (Gene Art Precision gRNA Synthesis Kit, A29377, Gene Art Platinum Cas9 Nuclease, B25640, Lipofectamine CRISPERMAX Cas9 Reagent, CMAX00003, Gene Art Genomic Cleavage Detection Kit, A24372). Monoclonal cell lines are obtained from single cells after genome editing, sequence analysis is performed to confirm the presence or absence of mutations, and cell lines in which deletion mutations are not induced are normal epidermal keratinocytes (hereinafter referred to as normal cells). , the cells in which the deletion mutation was induced were defined as genome-edited epidermal keratinocytes (hereinafter referred to as genome-edited cells).

The base sequences used for preparing the guide RNA (gRNA) used for genome editing, and the base sequences used for mutation confirmation PCR and sequence analysis are shown below.
(sequence for gRNA production)
Forward: TAATACGACTCACTATAGTCAACCATATCTGGGT (SEQ ID NO: 7)
Reverse: TTCTAGCTCTAAAACGATGACCCAGATATGGTTG (SEQ ID NO: 8)
(mutation confirmation sequence)
Forward primer: AGTCTTGTTTTTCTCTTT (SEQ ID NO: 9)
Reverse primer: TTCTTTGTTCACATATAAC (SEQ ID NO: 10)

(2) Preparation of three-dimensional cultured epidermis
A three-dimensional cultured epidermis was produced from the genome-edited cells prepared in (1). The three-dimensional cultured epidermis was prepared using a keratinocyte three-dimensional culture starter kit (manufactured by Funakoshi Co., Ltd.) according to the attached protocol. Specifically, 200,000 cells of each cell line were seeded in a milli-cell cell culture insert (for 24-well plate), cultured in Keratinocyte-SFM for 3 days (medium volume: 400 μL inside the insert, 1000 μL outside the insert). The medium was removed and replaced with a differentiation medium, CnT-Prime 3D barrier medium (manufactured by CELLnTEC) (medium volume: 400 µL inside the insert, 1000 µL outside the insert). On the next day, the medium inside and outside the insert was removed, 700 μL of CnT-Prime 3D barrier medium was added only to the outside of the insert, air exposure was performed for 10 days, and a three-dimensional cultured epidermis was produced using genome-edited cells. Similarly, three-dimensional cultured epidermis was prepared by changing the number of genome-edited cells by 5% with respect to the total number of seeded cells (total of genome-edited cells and normal cells).

(Example 2) Evaluation of three-dimensional cultured epidermis prepared using genome-edited epidermal keratinocytes (observation of tissue morphology)
The quality of the structure of the three-dimensional cultured epidermis prepared in Example 1 was evaluated by observing the morphology of the tissue. Specifically, the cultured epidermis after preparation was fixed with 4% PFA/PBS, and then embedded in paraffin using a vacuum rotary to prepare a paraffin block. After preparation, a tissue section was prepared using a microtome, deparaffinization was performed, and the morphology of the tissue was observed by hematoxylin-eosin staining (Sakura Fine Tech). The staining results are shown in FIG.

As shown in FIG. 1, as the percentage of genome-edited cells (mutant cells) increases, the barrier function is more disrupted, the epidermis layer is thin and disordered, and the rough skin condition is worsened to create an epidermis model. was done. In other words, by using genome-edited cells and changing the ratio of genome-edited cells and normal cells, the rough skin model, which had large variations between lots depending on the time and concentration of SLS exposure, was transformed into epidermis with different levels of rough skin. It was shown that the model can be made.

(Example 3) Skin Safety Test Using Three-Dimensional Cultured Epidermis Containing Genome-Edited Epidermal Keratinocytes Using the three-dimensional cultured epidermis prepared in Example 1, a skin safety (skin irritation) test was conducted. Sodium dodecyl sulfate (SDS), which is a skin irritant, was applied to the surface of the cultured epidermis prepared 9 days after exposure to air at 0.025% (w/v) for weak stimulation, 0.1% (w/v) for moderate stimulation, and 0.4% for strong stimulation. % (w/v) concentration, the cultured epidermis was collected after 15 minutes, and the transepidermal water loss of the collected epidermis model was measured using an evaporator. In addition, MTT assay was used to detect tissue cell viability after measuring transepidermal water loss. MTT assay was performed using a commercially available MTT assay kit (manufactured by Cosmo Bio) according to the attached protocol.

Table 1 shows the measurement results of transepidermal water loss, and Table 2 shows the results of the MTT assay.

As shown in Table 1, as the ratio of genome-edited cells to the total cells increased, the amount of water loss in the cultured epidermis produced increased, indicating that the skin barrier function was impaired. It was found that exposure to SDS, a skin irritant, further increases the amount of water loss and impairs the barrier function. In addition, even with a weak stimulus that does not show reactivity in cultured epidermis prepared using normal cells (the ratio of genome-edited cells to the total cells is 0%), when the ratio of genome-edited cells increases, reactivity increases. began to show In addition, it was shown that even when the same level of stimulation is given, it is possible to arbitrarily adjust the strength of reactivity by changing the ratio of genome-edited cells to all cells.

Similarly, as shown in Table 2, the reactivity of the cultured epidermis with arbitrarily adjusted levels of rough skin to skin stimulation (strong, medium, and weak stimulation) was accurately reflected in the cell viability. I found out.

From these results, the cultured epidermis containing the genome-edited cells of the present invention is stable without lot-to-lot variations, unlike rough skin models prepared by SLS, etc., and accurately reproduces the state of the epidermis. Therefore, skin irritation tests and product evaluation tests can be performed with high accuracy.

According to the three-dimensional cultured epidermis model of the present invention, it is possible to conduct evaluation tests for the safety and efficacy of cosmetics and pharmaceutical raw materials without conducting animal experiments. Therefore, the present invention can be used in the field of manufacturing cosmetics and pharmaceuticals, the field of dermatological research, and the like.

Claims (8)

A three-dimensional cultured epidermal model comprising genome-edited epidermal keratinocytes whose target genes have been modified by genome editing and normal epidermal keratinocytes, wherein the target genes are filaggrin (FLG), involucrin (IVL), loricrin (LOR), transglutaminase-2, tight junction protein-1 (TJP1), claudin 1 (CLDN1), occludin (OCLN), aquaporin 1 (AQP1), aquaporin 3 (AQP3), 3-ketodihydrosphingosine reductase ( KDSR), ceramide synthase-1 (CERS1), ceramide synthase-3 (CERS3), ceramide synthase-6 (CERS6), sphingosine 1-phosphate phosphatase (SGPP2), alkaline ceramidase 1 (ACER1), caspase 14 ( A three-dimensional cultured epidermis model containing one or more skin barrier function-related genes selected from CASP14) and small proline-rich proteins 1A (SPRR1A). The ratio of the number of cells occupied by the genome-edited epidermal keratinocytes to the total cell number of the genome-edited epidermal keratinocytes and the number of normal epidermal keratinocytes is 5% or more. The three-dimensional cultured epidermis model according to item 1. A method for producing a three-dimensional cultured epidermis model including the following steps.
(a) A step of modifying a target gene of epidermal keratinocytes by genome editing to produce genome-edited epidermal keratinocytes, wherein the target genes are filaggrin (FLG), involucrin (IVL), loricrin (LOR) , transglutaminase-2, tight junction protein-1 (TJP1), claudin 1 (CLDN1), occludin (OCLN), aquaporin 1 (AQP1), aquaporin 3 (AQP3), 3-ketodihydrosphingosine reductase (KDSR), ceramide synthase-1 (CERS1), ceramide synthase-3 (CERS3), ceramide synthase-6 (CERS6), sphingosine 1-phosphate phosphatase (SGPP2), alkaline ceramidase 1 (ACER1), caspase 14 (CASP14), and One or more genes associated with skin barrier function selected from small proline-rich proteins 1A (SPRR1A)
(b) step of mixing the genome-edited epidermal keratinocytes prepared in step (a) and normal epidermal keratinocytes in an arbitrary ratio
(c) three-dimensional culturing of the cell mixture obtained in step (b) The production method according to claim 3 , wherein the epidermal keratinocytes are immortalized cells. The production method according to claim 3 or 4 , wherein the genome editing is performed by CRISPR-Cas9. The production method according to any one of claims 3 to 5 , wherein the modification of the target gene is deletion, insertion or substitution. A test substance is brought into contact with the three-dimensional cultured epidermis model according to claim 1 or 2 , and changes in epidermal keratinocytes of the model are measured to evaluate the efficacy or safety of the test substance. Method. 3. A method for screening skin barrier function-improving substances, comprising contacting a test substance with the three-dimensional cultured epidermis model according to claim 1 or 2 , and measuring changes in epidermal keratinocytes of the model.

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