JP6817610B2 - Three-dimensional cultured epidermis model and its manufacturing method, and how to use the three-dimensional cultured epidermis model - Google Patents

Description

The present invention relates to a three-dimensional cultured epidermis model capable of observing cells in each layer constituting the epidermis from multiple directions in real time, a method for producing the same, and a method for using the three-dimensional cultured epidermis model.

The skin is roughly divided into a three-layer structure of epidermis, dermis, and subcutaneous tissue. The epidermis, which exists in the outermost layer of the skin, is mainly composed of keratinocytes (epidermis keratinocytes), and also includes melanocytes that produce melanin pigment, Langerhans cells that are antigen-presenting cells, and Merkel cells related to tactile sensation. .. Keratinocytes divide in the basal layer, which is the lowest layer of the epidermis, move to the upper layer as they mature, keratinize, and eventually peel off (keratinization). Therefore, the epidermis is composed of a plurality of layers (basal layer, spinous layer, granular layer, stratum corneum) composed of epidermal keratinocytes at different maturation stages. Stem cells of keratinocytes are present in the basal layer, proliferate and differentiate as needed, constantly supplying new cells to the epidermis, and as a result, the skin is constantly regenerating. However, when abnormalities occur in the proliferation and differentiation of keratinocytes, they cause various diseases. For example, psoriasis is a chronic inflammatory disease characterized by overgrowth and poor differentiation of keratinocytes.

In recent years, from the viewpoint of animal protection, an alternative method for animal experiments, in which an evaluation test for the safety and effectiveness of pharmaceuticals and cosmetic raw materials is conducted without conducting animal experiments, has attracted much attention. Among them, various three-dimensional cultured skins have been developed for evaluation of the efficacy and toxicity of drugs on the skin or application to dermatological research.

However, in order to observe and evaluate the condition of the skin, the conventional three-dimensional cultured skin requires a very complicated series of operations such as fixation with paraformaldehyde, embedding with paraffin, section preparation, and dyeing. (Patent Documents 1 and 2). Moreover, from the stained image of the tissue section, only limited information could be obtained from the surface observed from one direction. Furthermore, with the conventional method, the prepared three-dimensional cultured skin could not be observed alive, and information on living cells and tissues could not be acquired in real time.

JP-A-2009-294200 Japanese Unexamined Patent Publication No. 2010-193822

Therefore, in view of the above-mentioned circumstances, it is an object of the present invention to provide a three-dimensional cultured epidermis model capable of observing epidermal cells from multiple directions in real time while alive and more accurately evaluating the state of the epidermis. To do.

As a result of diligent research to solve the above problems, the present inventors prepared keratinocytes dyed with a fluorescent dye in advance or introduced a fluorescent protein, and cultured the keratinocytes three-dimensionally. In the epidermis, we have found that cells or organelles in each layer are three-dimensionally visualized with a fluorescent substance, and their morphology and distribution can be observed in real time from multiple directions while living, and the present invention has been completed.

That is, the present invention includes the following inventions.
[1] A three-dimensional cultured epidermis model in which cells or organelles in each layer constituting the epidermis are three-dimensionally visualized with a fluorescent substance.
[2] The three-dimensional cultured epidermis model according to [1], wherein the fluorescent substance is a fluorescent dye or a fluorescent protein.
[3] The three-dimensional cultured epidermis model according to [1] or [2], wherein the cells are keratinocytes or melanocytes.
[4] A method for producing a three-dimensional cultured epidermis model, which comprises the following steps.
(1) Step of staining keratinocytes with a fluorescent dye or introducing a fluorescent protein expression vector into keratinocytes
(2) Step of three-dimensional culture of keratinocytes obtained in step (1)
[5] The method for producing a three-dimensional cultured epidermis model according to [4], wherein the three-dimensional culture of keratinocytes is carried out together with melanocytes into which a fluorescent protein expression vector has been introduced.
[6] Further comprising the step of stimulating the keratinocytes at at least one step selected from before the start of the 3D culture, during the 3D culture, and after the end of the 3D culture [4] or [4] or [ 5] The method for producing a three-dimensional cultured epidermis model.
[7] The test substance is brought into contact with the three-dimensional cultured epidermis model according to any one of [1] to [3], the epidermal cells or organelles of the model are observed by fluorescence imaging, and the test is performed based on the observation results. A method of assessing the efficacy or safety of a substance.
[8] The test substance is brought into contact with the three-dimensional cultured epidermis model according to any one of [1] to [3], the epidermis cells or organelles of the model are observed by fluorescence imaging, and the epidermis function is based on the observation results. How to screen for improving substances.
[9] A skin evaluation kit comprising the three-dimensional cultured epidermis model according to any one of [1] to [3].

According to the present invention, there is provided a three-dimensional cultured epidermis model in which cells or organelles in each layer constituting the epidermis are three-dimensionally visualized with a fluorescent substance, and a method for producing the same. Unlike the conventional three-dimensional cultured epidermis model, the three-dimensional cultured epidermis model of the present invention does not require complicated operations such as fixation, embedding, section preparation, and staining when observing and evaluating the skin condition. Living cells (living cells) can be observed from multiple directions, and cell information (number, morphology, localization, migration, etc.) can be obtained from multiple directions. Further, in the three-dimensional cultured epidermis model of the present invention, since it is possible to observe cells alive, it is also possible to observe cell morphological changes and movements in real time by time-lapse imaging. In addition, by stimulating the three-dimensional cultured epidermis model with a drug or the like, it is possible to obtain an epidermis model that reproduces a skin disease such as a wound or keratosis. Therefore, the three-dimensional cultured epidermis model of the present invention can be used for developing cosmetics and drugs for promoting skin turnover and suppressing melanin production, studying the mechanism of skin turnover and whitening action, psoriasis and contact dermatitis, etc. It is useful for elucidating the pathogenic mechanism of intractable skin diseases.

FIG. 1A shows a confocal laser scanning microscope image of keratinocytes stained with cell tracker orange (CTO), a schematic diagram of three-dimensional culture, and a confocal laser scanning microscope image of a three-dimensional cultured epidermis prepared using fluorescently stained keratinocytes. Shows a stereoscopic image (R: cells stained red). FIG. 1B shows a confocal laser scanning microscope image in the depth direction (horizontal direction) of the prepared three-dimensional cultured epidermis (values in the image indicate the distance from the stratum corneum). FIG. 2A shows a confocal laser scanning microscope image of keratinocytes (G-HEK) into which a green fluorescent protein GFP expression vector (pTagGFP2-C) has been introduced, and a three-dimensional cultured epidermis (G-) prepared by culturing the keratinocytes in three dimensions. A confocal laser scanning microscope image of a vertical cross section of a HEK-derived three-dimensional cultured epidermis (G: cells glowing green) is shown. FIG. 2B shows a confocal laser scanning microscope image of a cross section of the G-HEK-derived three-dimensional cultured epidermis in the depth direction (horizontal direction) (numerical values in the image indicate the distance from the stratum corneum). FIG. 3A shows a confocal laser scanning microscope image of mixed cells of G-HEK and keratinocytes (R-HEK) into which a red fluorescent protein RFP expression vector (pTagRFP-C) was introduced, and three-dimensional culture of the mixed cells. A confocal laser scanning microscope image of a vertical cross section of the three-dimensional cultured epidermis is shown (cells surrounded by dotted lines: R-HEK, unmarked cells: G-HEK). FIG. 3B shows an image obtained by sterically observing the morphology of R-HEK in a three-dimensional cultured epidermis prepared by three-dimensionally culturing the mixed cells with a confocal laser scanning microscope. FIG. 4A shows a three-dimensional cultured epidermis (NG-HEK-derived three-dimensional cultured epidermis) prepared by three-dimensionally culturing keratinocytes (NG-HEK) into which a GFP expression vector (pAcGFP1-Nuc Vector) to which a nuclear localization signal has been added has been introduced. ) Is shown as a stereoscopic image by a confocal laser scanning microscope. FIG. 4B shows a confocal laser scanning microscope image of a cross section of the NG-HEK-derived three-dimensional cultured epidermis in the depth direction (horizontal direction) (numerical values in the image indicate the distance from the stratum corneum). FIG. 5 shows a three-dimensional cell prepared by three-dimensionally culturing a mixed cell of keratinocytes (R-NG-HEK) into which pTagRFP-C and pAcGFP1-Nuc Vector were simultaneously introduced and normal HEK (without gene transfer). A confocal laser scanning microscope image of a vertical cross section of the cultured epidermis is shown (NC: nucleated cells, ENC: enucleated cells, Y1: yellow glowing nucleus, Y2: entire yellow glowing cytoplasm). FIG. 6 shows the confocal cross-section of the cross section in the depth direction (horizontal direction) by a confocal laser scanning microscope after treating the three-dimensional cultured epidermis prepared by three-dimensionally culturing NG-HEK with SDS, which is a skin stimulant. A laser microscope image is shown (the numbers in the image indicate the distance from the stratum corneum, and the arrowheads indicate the cells that have undergone apoptosis). FIG. 7 shows stereoscopic images of a three-dimensionally cultured epidermis prepared by three-dimensionally culturing mixed cells of G-HEK and melanocytes (R-HEM) into which an RFP expression vector has been introduced by a confocal laser scanning microscope from various angles. Shows the observed image (cells surrounded by dotted lines: R-HEM, unmarked cells: G-HEK). FIG. 8 shows a fluorescence microscope image of a tissue section of a three-dimensionally cultured epidermis prepared by three-dimensionally culturing ordinary keratinocytes and stained with an anti-tyrosinase antibody and DAPI (cells surrounded by a dotted line: melanocytes).

1. 1. Three-dimensional cultured epidermis model The three-dimensional cultured epidermis model of the present invention is characterized in that cells (epidermis cells) or organelles in each layer constituting the epidermis are three-dimensionally visualized with a fluorescent substance.

Fluorescent substances include fluorescent dyes and fluorescent proteins. The fluorescent dye is not particularly limited as long as it can observe the morphology and localization of cells, is not toxic to living cells, and is suitable for staining cells, but for example, CellTracker; Each series of registered trademarks) (Orange CMRA, Green CMFDA, Blue CMAC, Violet BMQC, Red CMTPX, etc.), Alexa Fluor (registered trademark) series, Cy3, Cy5, Hoechst 33342, Hoechst 33258, etc. Be done. Examples of the fluorescent protein include green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyanide fluorescent protein (CFP) and the like. As these fluorescent substances, one kind may be used, and two or more kinds may be used.

The layers that make up the epidermis are the basal layer (1 layer), the spinous layer (5 to 10 layers), the stratum granulosum (2 to 3 layers), and the stratum corneum (about 10 layers) in order from the deepest to the skin surface. Refers to all layers included.

Epidermal cells include epidermal keratinocytes (keratinocytes), pigment cells (melanocytes), Langerhans cells, Merkel cells, hair matrix cells, hair papilla cells and the like. In addition, organelles (organelles) include nuclei, endoplasmic reticulum, ribosomal granules, cytoskeleton (actins, microtubules, intermediate filaments), Golgi apparatus, endosomes, mitochondria and the like.

2. 2. Method for producing a three-dimensional cultured epidermis model The method for producing a three-dimensional cultured epidermis model of the present invention includes (1) staining keratinocytes with a fluorescent dye or introducing a fluorescent protein expression vector into keratinocytes, and (2) step (1). Including the step of three-dimensionally culturing the keratinocytes obtained in 1.

First, in step (1), keratinocytes are stained with a fluorescent dye or a fluorescent protein expression vector is introduced into the keratinocytes. The keratinocytes may be immortalized keratinocytes or normal keratinocytes, but immortalized keratinocytes are preferred. The immortalization method is not limited as long as it immortalizes cultured cells of epithelial cells such as keratinocytes and does not induce cell death. For example, telomerase reverse transcriptase (TERT) and viral genes (SV40T, HPV) E6-E7, EBV, etc.) can be mentioned. The origin of the cells is not particularly limited as long as it is a mammal, and examples thereof include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses, but humans are preferable. ..

The types of fluorescent dyes and fluorescent proteins are as described above. For the introduction of fluorescent protein into keratinocytes, a vector into which a gene encoding the fluorescent protein is inserted (fluorescent protein expression vector) is prepared, and this is introduced into keratinocytes using an electroporation method, a transfection method, a microinjection method, or the like. It can be done by doing. When observing a specific organelle or intracellular structure in epidermal cells, for example, a gene encoding a fluorescent protein is bound to a DNA encoding various organelle translocation signal peptides or a gene encoding various organelle constituent proteins. The gene may be inserted into an appropriate vector and introduced into keratinocytes by the above method. In addition, genes encoding fluorescent proteins are related to various genes related to skin function and skin diseases (for example, genes related to the onset of atopic dermatitis and skin barrier dysfunction, genes causing pigmentation disorders, and inflammation. (Genes, etc.) When a gene bound downstream of a specific promoter sequence is introduced into keratinocytes, it is possible to monitor the expression pattern of the various genes in three-dimensional cultured skin in real time.

Next, in step (2), three-dimensional culture is carried out using keratinocytes stained with a fluorescent dye or keratinocytes into which a fluorescent protein expression vector has been introduced. The three-dimensional culture can be carried out according to the following method for producing three-dimensional cultured skin, which is generally used in the art, in which keratinocytes are layered by air exposure to reconstruct the skin.

Preparation of three-dimensional cultured skin consists of a cell proliferation culture step and a differentiation induction step. In the growth culture step, keratinocytes stained with a fluorescent dye or keratinocytes introduced with a fluorescent protein expression vector (hereinafter, simply referred to as keratinocytes) are grown and cultured in a culture vessel such as a culture insert until they become confluent on the bottom surface. Specifically, keratinocytes are dispersed in a cell growth medium, the cell dispersion is seeded in a culture insert having a liquid permeable membrane on the bottom surface, and the outside of the culture insert is also filled with the same cell growth medium to prepare the liquid. The keratinocytes on the permeable membrane are cultured in a state of being immersed in a cell growth medium. The liquid permeable membrane keeps the inside and outside of the culture insert in communication so that the medium can permeate. Here, the number of keratinocytes added to the cell culture insert is not particularly limited, but is usually preferably 15 × 10 ^{4 to} 120 × 10 ⁴ cells / cm ² , preferably 30 × 10 ^{4 to} 90 × 10 ⁴ cells / cm ^2. More preferred.

The liquid-permeable membrane of the culture insert is not particularly limited as long as the seeded keratinocytes are adhered or fixed, and the keratinocytes can grow on it and can serve as a support, but for example, polycarbonate, polyethylene terephthalate, polystyrene, etc. Membrane is mentioned. Further, 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.

Further, in addition to using a culture insert as a support, a collagen gel, a collagen sponge, or a cell-free dermis from which epithelium and fibroblasts have been removed may be used. When these are used as supports, fibroblasts may be incorporated as appropriate. Further, it is preferable to install a glass ring on the surface of these supports and add a cell suspension of keratinocytes to the glass ring.

The growth culture is carried out, for example, for 1 to 6 days, preferably 2 to 4 days. Further, during this period, the medium may be changed as appropriate. Whether or not the keratinocytes grown in the culture insert are in a confluent state can be confirmed by a cell staining reagent such as CnT-ST-100 stain kit (manufactured by CELLn TEC). It can also be determined that keratinocytes have become confluent when the liquid level of the culture solution in 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 medium for cell proliferation to the medium for cell differentiation, and keratinocytes are immersed and cultured in the medium for about 6 to 48 hours, and then the inside of the culture insert. And all external medium was removed with an aspirator, medium for cell differentiation was added to the outside of the insert, keratinocytes inside the culture insert were exposed to air (air), cultured for 5 to 12 days, and stratified epidermal cells. Induce differentiation into.

The medium for cell proliferation is not particularly limited as long as it is a basal medium suitable for keratinocyte growth and subculture, but is preferably a serum-free and low calcium concentration basal medium, for example. Commercially available Keratinocyte-SFM (Thermo Fisher Scientific), MCDB153 medium (Sigma), Humedia-KG2 (Kurabou), serum-free medium for normal human epidermal keratinocytes (DS Pharma Biomedical), etc. A medium may be used. The medium may contain basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), Stem Cell Factor (SCF) and the like as growth factors. In addition, epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferase, heparin, heparan sulfate, collagen, bovine serum are required to increase the growth rate. Albumin (BSA), L-glutamine, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement may be contained. In addition, antibiotics may be added if necessary. The calcium concentration of the cell growth medium is preferably about 0.03 to 0.15 mM.

The above-mentioned medium for cell differentiation is not particularly limited as long as it is a basal medium suitable for inducing differentiation of keratinocytes, but if a commercially available medium such as CnT-Prime 3D Barrier Culture Medium (manufactured by CELLn TEC) is used. Good. The calcium concentration of the cell differentiation medium is preferably about 1.2 to 1.5 mM.

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

The steps of culturing keratinocytes stained with the above fluorescent dye or keratinocytes introduced with fluorescent protein to prepare stratified epidermal cells are Millicelle cell culture insert (manufactured by Millipore) and keratinocytes three-dimensional culture starter kit (manufactured by Funakoshi). ) And the like, a commercially available culture epidermis preparation kit may be used, and the culture medium and culture insert packed in the kit can be used according to the instructions attached to the kit.

In addition, as another embodiment, by performing the above-mentioned three-dimensional culture by mixing two types of keratinocytes into which two different fluorescent proteins (for example, GFP and RFP) have been introduced, it is easy to observe the morphology of each cell. A three-dimensional cultured epidermis model can be prepared. When three-dimensional culture is performed using two types of keratinocytes, for example, the ratio of the number of cells of RFP-introduced keratinocytes to the number of cells of GFP-introduced keratinocytes varies depending on the purpose of use of the three-dimensional cultured epidermis, for example, about 0.001. It is preferably ~ 50%, more preferably 0.01-10%.

As yet another aspect, a three-dimensional cultured epidermis model containing melanocytes can be prepared by performing the above-mentioned three-dimensional culture by mixing keratinocytes into which a fluorescent protein has been introduced and melanocytes into which a fluorescent protein has been introduced. In this case, by using a different type of fluorescent protein to be introduced into melanocytes from the fluorescent protein to be introduced into keratinocytes, it becomes easy to observe the localization of melanocytes in the epidermal tissue. The origin of melanocytes is not particularly limited as long as it is the above-mentioned mammal, but human origin is preferable. The ratio of the number of melanocytes to the number of cells of keratinocytes when keratinocytes and melanocytes are cultured together varies depending on the purpose of use of the three-dimensional cultured epidermis, but is preferably about 0.1 to 30%, for example. 5 to 20% is more preferable.

In addition, by stimulating the keratinocytes at at least one stage selected before the start of the three-dimensional culture of keratinocytes, during the three-dimensional culture, and after the end of the three-dimensional culture, wounds and keratoses are abnormal. It is also possible to use an epidermis model that reproduces or imitates a skin disease such as. Stimulation includes UV irradiation, drying, heat, and stimulation with chemicals (surfactants, organic solvents, etc.).

The above-mentioned three-dimensional cultured epidermis model of the present invention may be made into a kit, and the kit describes, for example, a medium or container suitable for culturing epidermal cells, positive or negative standard samples, and instructions on how to use the kit. Calligraphy etc. can be included.

3. 3. How to use the 3D cultured skin model The 3D cultured skin model of the present invention can be used as an alternative method for animal experiments to evaluate the effectiveness and safety of cosmetics, external preparations for skin, and chemical substances (detergents, dyes for clothes, etc.). Can be used. For example, the evaluation of efficacy includes skin barrier function, retention / regulation function of water or oil, wrinkle prevention / improvement function, presence / absence of water permeability, etc., and evaluation of safety includes erythema, redness, inflammation. , Pigmentation, swelling, presence or absence of rash, etc. In addition, the three-dimensional cultured epidermis model of the present invention can also be used for screening for substances that improve epidermis function. Improvement of epidermis function includes improvement of epidermis barrier function (water retention function, prevention function of invasion of ultraviolet rays, chemical substances, bacteria, etc. from the outside), normalization function of skin turnover, normalization function of melanin metabolism, etc. Can be mentioned.

In the present invention, the efficacy and safety of cosmetics and pharmaceuticals are evaluated and screened by contacting the test substance with the three-dimensional cultured epidermis model of the present invention and observing changes in the epidermal cells or organelles of the model by fluorescence imaging. It can be done, and changes in epidermal cells or organelles are indicators of evaluation. At this time, if 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 compared with the observation result, the evaluation becomes more accurate. Changes in epidermal cells or organelles include changes in their number, morphology, distribution, localization, migration, disappearance, etc. For example, if the cell death or proliferation inhibition of epidermal cells is used as an index, the test substance can be evaluated as a substance that stimulates the epidermis, and if the disappearance of the nucleus (enucleation) is used as an index, the test substance can be used. It can be screened as a candidate substance for a skin turnover promoter, and if the decrease in the number of melanocytes is used as an index, the test substance can be screened as a candidate substance for a whitening agent. The test substance is brought into contact with the three-dimensional cultured epidermis model formed in the culture insert from the surface side of the stratum corneum and / or from the surface side of the basal layer. Specifically, it can be carried out by a method of administering or applying the test substance from above to the epidermis model in the culture insert, or a method of adding the test substance to the culture solution outside the culture insert.

The test substance is mainly intended for components 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, peptide mimetics, etc.) And non-naturally occurring molecules (eg, low molecular weight organic compounds made using combinatorial chemistry techniques and the like); and mixtures thereof. In addition, as the test substance, a single test substance may be tested independently, or a mixture (including a library) of several candidate test substances may be tested. Examples of the library containing a plurality of test substances include a synthetic compound library and a peptide library.

Observation of cells by fluorescence imaging can be performed using a microscope capable of accurately reproducing and visualizing a three-dimensional epidermal tissue structure, and it is preferable to use a confocal laser scanning microscope. The confocal laser scanning microscope includes an optical microscope main body, a confocal scanning module, and a detector, and is not particularly limited as long as it is a commercially available fluorescence imaging device for observing living cells. For example, the confocal laser scanning microscope LSM510 ( ZEISS) and the like. The observation may be performed by directly fluorescing the cultured epidermis model with the naked eye, or by taking a photograph to obtain an image and performing the observation based on the image. When observing based on the image obtained by photography, the image may be acquired by taking a picture only at a certain time, but the image is taken continuously over time (time-lapse photography) at a certain time interval. By acquiring, dynamic visualization of changes in cells can be performed. Further, the fluorescence can be observed by appropriately changing the measurement conditions and time depending on the type of cell or organelle for visualization, the type of the test substance, and the type of the fluorescent substance.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto.

(Example 1) Preparation of three-dimensional cultured epidermis using keratinocytes stained with a fluorescent indicator and observation with a confocal laser scanning microscope First, normal with a cell immortalization reagent (Lenti-hTERT Virus Cell Immortalization Reagent, manufactured by Applied Biological Materials). Human epidermal keratinocytes (HEK, manufactured by Kurabou) were immortalized according to the manufacturer's protocol. All HEKs described in the following examples are this immortalized HEK. HEK was cultured using Keratinocyte-SFM (manufactured by Thermo Fisher Scientific). HEK was stained by adding Cell Tracker Orange (CTO, manufactured by Thermo Fisher Scientific), which is a fluorescent indicator that specifically stains the cytoplasm, to the medium and culturing for 1 hour to prepare a HEK that glows in the cytoplasm.

Next, a three-dimensional cultured epidermis was prepared by a conventional method using this stained HEK. Specifically, 200,000 of the above HEKs were sown on a millicell culture insert (for a 24-well plate), cultured in Keratinocyte-SFM for 3 days (medium volume: 400 μL inside the insert, 1000 μL outside the insert), and then media inside and outside the insert. Was replaced with CnT-Prime 3D barrier medium (manufactured by CELLnTEC), which is a differentiation medium (medium volume is 400 μL inside the insert and 1000 μL outside the insert). 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, and air exposure was performed for 10 days to prepare a three-dimensional cultured epidermis (Fig. 1A). When the prepared three-dimensional cultured epidermis was observed using a confocal laser scanning microscope LSM510, the morphology of the epidermal cells in each layer could be observed three-dimensionally from both the vertical and horizontal directions while alive (Fig.). 1A, 1B). On the other hand, even if you try to stain the epidermis tissue with CTO after preparing a three-dimensional cultured epidermis using normal HEK that has not been stained with CTO, the epidermis tissue once formed has a strong barrier function, so CTO. Did not enter the tissue, it only stained the surface of the tissue.

(Example 2) Preparation of three-dimensional cultured epidermis using keratinocytes introduced with a fluorescent protein expression vector and observation with a confocal laser scanning microscope
Using a Gene Pulser Xcell (manufactured by BIORAD), a GFP expression vector (pTagGFP2-C, manufactured by Evrogen) was introduced into an immortalized HEK by an electroporation method. A stable expression strain was established by drug selection, and HEK (G-HEK) in which the entire cell glowed green was established. Next, using the cells, a three-dimensional cultured epidermis was prepared in the same manner as in Example 1 (FIG. 2A). When the prepared three-dimensional cultured epidermis was observed using LSM510, the morphology of the epidermal cells in each layer could be observed three-dimensionally from both the vertical and horizontal directions while alive (FIGS. 2A and 2B). .. The cell image could be observed more clearly because the fluorescence did not decrease due to the culture as compared with the fluorescence indicator of Example 1.

In addition, an RFP expression vector (pTagRFP-C, manufactured by Evrogen) was introduced into HEK to establish HEK (R-HEK) in which the entire cell glows red, and the cells were converted to G-HEK (200,000). The cells were mixed at a ratio of 0.01% (20 cells) to prepare a three-dimensional cultured epidermis in the same manner as in Example 1 (Fig. 3A). When we focused on R-HEK that glows red using LSM510 and observed its morphology, we were able to observe the morphological changes of each cell in more detail (FIGS. 3A and 3B). Furthermore, it was possible to observe cell morphological changes and movements in real time by time-lapse imaging.

(Example 3) Preparation of three-dimensional cultured epidermis using keratinocytes in which organelles specifically shine, and observation with a confocal laser scanning microscope
A GFP expression vector (pAcGFP1-Nuc Vector, manufactured by Clontech) to which a nuclear localization signal was added was introduced into HEK, drug selection was performed, and HEK (NG-HEK) in which the nucleus shines specifically was established.

Next, a three-dimensional cultured epidermis was prepared using NG-HEK in the same manner as in Example 1, and observed using LSM510. As a result, the morphology of the epidermal cells in each layer could be observed in detail alive (FIGS. 4A and 4B). It was also confirmed that when NG-HEK differentiated and moved to the outermost stratum corneum, the nuclei disappeared due to enucleation (Fig. 4B: distance from the stratum corneum around 10.5 μm).

In order to observe this phenomenon in more detail, the above-mentioned pTagRFP-C and pAcGFP1-Nuc Vector were simultaneously introduced into HEK to establish HEK in which the whole cell glows red and the nucleus glows green (R-NG-HEK). R-NG-HEK is mixed at a ratio of 0.01% (20 cells) to normal HEK (200,000 cells) without gene transfer to prepare a three-dimensional cultured epidermis in the same manner as in Example 1. did. As a result, cells that have not undergone enucleation are observed to have a red cytoplasm and a yellow nucleus (the result of a mixture of red and green looks yellow), whereas cells that have undergone enucleation have an entire cytoplasm observed to be yellow. It was found that (Fig. 5). This is thought to be because GFP with a nuclear localization signal stays in the cytoplasm because enucleated cells do not have a nucleus. Therefore, by using the three-dimensional cultured epidermis of the present invention, the state of enucleation of epidermal cells can be analyzed in great detail while alive. In order to know the positions of the stratum corneum and the basal layer of the epidermal tissue, the prepared epidermal tissue was stained with the fluorescent indicator wheat germ agglutinin, Alexa Fluor ^TM 488 conjugate (WGA) (manufactured by Thermo Fisher Scientific). (Green). As shown in Example 1, since the fluorescent indicator does not enter the tissue due to the strong barrier of the epidermal tissue and stains the surface of the tissue, the positions of the basal layer and the stratum corneum can be discriminated.

In addition, using vectors for visualizing actin and mitochondria (pAcGFP1-Actin Vector, pDsRed2-Mito Vector, manufactured by Clontech), HEKs in which those organelles shine were prepared, and similar experiments were performed. We were able to observe the morphological changes of the organelles.

As shown above, according to the three-dimensional cultured epidermis prepared using keratinocytes in which organelles specifically shine, it was possible to determine the state of the epidermis alive and very clearly. Furthermore, it was possible to observe the morphological changes and movements of cell organelles in real time by time-lapse imaging.

(Example 4) Evaluation of a test substance using a three-dimensional cultured epidermis prepared using keratinocytes in which the nucleus shines specifically Three-dimensionally using NG-HEK in which the nucleus shines specifically in the same manner as in Example 3. A cultured epidermis was prepared, and it was examined whether the effect of the test substance on the epidermis could be evaluated using the three-dimensional cultured epidermis. Specifically, SDS (sodium dodecyl sulfate, manufactured by Wako Pure Chemical Industries, Ltd.), a typical substance that causes a skin irritation reaction from the stratum corneum side, was added to the three-dimensional cultured epidermis on the 10th day of air exposure at a concentration of 0.1%. Allowed to stand for minutes. Then, the epidermis was washed twice with PBS, cultured again in CnT-Prime 3D barrier medium, and observed with LSM510 24 hours later. As a result, in the SDS-treated epidermis, nuclear condensation was confirmed, and the appearance of apoptotic epidermal cells was confirmed in detail (Fig. 6). In addition, the proportion of cells with condensed nuclei tended to increase depending on the concentration of SDS (Table 1).

From the above results, it was clarified that the evaluation of whether or not it is a stimulant can be performed in great detail and easily by using a three-dimensional cultured epidermis prepared using NG-HEK in which the nucleus shines specifically. .. In addition, since this evaluation method allows observation of the three-dimensional cultured epidermis alive, it is possible to observe the damage received by SDS and the recovery process from the damage over time using the same sample. On the other hand, in the conventional method, observation of the three-dimensional cultured epidermis requires fixation treatment, embedding treatment, section preparation treatment, etc., and in order to perform observation over time, a sample is prepared for each number of days. And it was not possible to observe the same sample over time.

(Example 5) Preparation of melanocyte-containing three-dimensional cultured epidermis and observation with a confocal laser scanning microscope
Using Gene Pulser Xcell, pTagRFP-C was introduced into human melanocytes (HEM; manufactured by Kurabou Co., Ltd.) by the electroporation method to establish HEM (R-HEM) in which the entire cell glows red. A three-dimensional cultured epidermis was prepared in the same manner as in Example 1 by mixing at a ratio of 10% (20,000 cells) to G-HEK (200,000 cells). As a result of observing R-HEM (melanocytes) that glow red using LSM510, the number, morphology, and localization of melanocytes could be observed alive (Fig. 7). When the effects of melanocyte growth factors (SCF, endothelin, WNT) were confirmed using this melanocyte-containing three-dimensional cultured epidermis model, it was quantitatively found that the number of melanocytes per unit area was increased by these factors. I was able to analyze it.

For comparison, the preparation of the three-dimensional cultured epidermis by the conventional method and the observation using it were performed as follows. Normal human melanocyte HEM was mixed at a ratio of 10% (20,000 pieces) to normal keratinocyte HEK (200,000 pieces) stained with a fluorescent dye or not having a fluorescent protein introduced into keratinocytes, and the same as in Example 1. To prepare a three-dimensional cultured epidermis. The prepared cultured epidermis was fixed with 4% paraformaldehyde for 30 minutes. The tissue was then placed in a Tissue Tech Cliomold plastic embedding dish (Sakura Finetech), poured with O.C.T. compound (Sakura Finetech) and frozen at -80 ° C (15 minutes). Next, sections were prepared by cryostat microtome and dried with a dryer. Tissue sections were washed with PBS and then reacted with an antibody against the melanocyte marker protein tyrosinase (anti-Tyrosinase antibody, manufactured by Santa Cruz) for 1 hour (37 ° C), and then an anti-IgG antibody labeled with Alexa Fluor 594 (Life Technologies). It was reacted with (manufactured by OLYMPUS) for 30 minutes and observed with a fluorescence microscope (manufactured by OLYMPUS) (Fig. 8). In addition, HEK and HEM nuclei were stained with DAPI (manufactured by dojindo). As a result, the localization of melanocytes was confirmed, but only information from one direction could be obtained, and only information on fixed and dead tissues could be obtained.

Table 2 shows a comparison between the conventional method for observing the three-dimensional cultured epidermis and the method for observing the three-dimensional cultured epidermis of the present invention.

As shown in Table 2, the three-dimensional cultured epidermis of the present invention has many excellent advantages as compared with the conventional three-dimensional cultured epidermis.

In the three-dimensional cultured epidermis model of the present invention, epidermal cells can be observed alive from multiple directions in real time, and the state of the epidermis can be evaluated more accurately. Therefore, according to the three-dimensional cultured epidermis model of the present invention, it is possible to carry out an evaluation test of the safety and effectiveness of pharmaceuticals and cosmetic raw materials without conducting animal experiments, and the epidermis model is used in the fields of manufacturing cosmetics and pharmaceuticals. It can be used in dermatology research fields.

Claims (7)

Three-dimensional, in which keratinocytes whose nuclei are stained with a fluorescent dye or keratinocytes expressing a fluorescent protein in the nuclei are layered, and the nuclei of keratinocytes in each layer constituting the epidermis are three-dimensionally visualized with a fluorescent substance. Cultured epidermis model. A method for producing a three-dimensional cultured epidermis model, which comprises the following steps.
(1) A step of staining the nucleus of keratinocytes with a fluorescent dye or introducing an expression vector into which a gene encoding a fluorescent protein is expressively inserted in the nucleus of keratinocytes (2) Obtained in step (1). Step of three-dimensional culture of keratinocytes The three-dimensional structure according to claim 2 , further comprising a step of stimulating keratinocytes at at least one step selected from before the start of the three-dimensional culture, during the three-dimensional culture, and after the end of the three-dimensional culture. A method for producing a cultured epidermis model. The three-dimensional culture skin model according to claim 1 is contacted with the test substance, the keratinocytes nuclei of the model was observed by fluorescence imaging, to evaluate the efficacy or safety of test substances on the basis of the observations Method. How the three-dimensional culture skin model according to claim 1 is contacted with the test substance, the keratinocytes nuclei of the model was observed by fluorescence imaging, screening improver epidermal function based on the observation results. In the three-dimensional cultured epidermis model according to claim 1, when the fluorescent substance whose nucleus is visualized is observed in the entire cytoplasm of keratinocytes, it is determined that the keratinocytes are differentiated and / or enucleated. A method for determining differentiation and / or enucleation of keratinocytes. A skin evaluation kit including the three-dimensional cultured epidermis model according to claim 1 .

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