JP5408520B2 - Skin model construct - Google Patents

Description

  The present invention relates to a skin model construct. More specifically, the present invention relates to a skin model construct, a method for producing the skin model construct, and a skin differentiation simulation method.

  It is known that various factors are expressed in animal differentiation and morphogenesis in animals. For example, in mice, epithelial morphogenesis occurs in mesenchymal cells cultured in agglomerated form, whereas epithelial morphogenesis occurs in mesenchymal cells cultured in a monolayer that assumes a flat cell morphology. It is known not to. As a factor involved in the epithelial morphogenesis in the mouse, epimorphin produced by a mesenchymal cell cultured in agglomerated form and not produced by a mesenchymal cell cultured in a monolayer having a flat cell shape is single. (See Patent Document 1). Further, Patent Document 1 discloses human epimorphin as a homolog of mouse epimorphin.

  On the other hand, disease model animals and three-dimensional cultured skin models (see Non-Patent Document 1 and Non-Patent Document 2) are used to elucidate the onset mechanism of skin diseases and develop therapeutic or preventive drugs for the diseases. It has been.

  However, in the case of the disease model animal, there is a drawback that variation among individuals is large and it is difficult to obtain sufficient reproducibility for screening of multiple specimens. Furthermore, in the case of the disease model animal, it may be difficult to establish a model showing a specific disease state in humans, and there is a drawback that there are few types of disease models themselves.

Further, the three-dimensional cultured skin model described in Non-Patent Document 1 and Non-Patent Document 2 is a method in which normal epidermal cells for which a culture method is established are seeded on a support such as a collagen gel layer containing fibroblasts. This is a model in which a structure simulating the skin structure in a normal state is constructed by cultivating and layering. Therefore, the three-dimensional cultured skin model described in Non-Patent Document 1 and Non-Patent Document 2 has less variation between models compared to the disease model animal, and therefore can obtain reproducibility suitable for screening of multiple specimens. . However, the three-dimensional cultured skin model described in Non-Patent Document 1 and Non-Patent Document 2 has a drawback in that it takes time to construct a structure imitating a normal skin structure by layer formation.
Japanese Patent No. 2849517 Bell E et al., “Recipes for reconstituting skin.”, Journal of Biomechanical Engineering, 1991, Vol. 113, pp. 113-119. Schoop VM et al., “Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts.”, Journal of Investigative Journal of Investigative Dermatology, 1991, 112, p.343-353

  This invention is made | formed in view of the said prior art, and it aims at providing the manufacturing method of a skin model construct which can manufacture a skin model construct simply in a short time. Another object of the present invention is to provide a skin model construct that can be easily produced in a short time and that can easily reproduce various skin conditions. It is still another object of the present invention to provide a skin differentiation simulation method that can easily simulate skin differentiation in a short time.

That is, the gist of the present invention is as follows.
(1) A method for producing a skin model construct, comprising culturing cell aggregates of epidermal basal cells in a support body containing a component of an extracellular matrix and serving as a scaffold,
(2) The method for producing a skin model construct according to (1), wherein the epidermal basal cells are gene-introduced epidermal basal cells into which a nucleic acid has been introduced exogenously,
(3) The manufacturing method of the skin model structure as described in said (1) or (2) whose said support body is a collagen gel.
(4) A skin model construct obtained by the production method according to any one of (1) to (3),
(5) a cell cluster comprising epidermal basal cells and differentiated cells of the epidermal basal cells,
A basal layer formed on the outermost part of the cell cluster and containing the epidermal basal cells;
A differentiated cell layer formed inside the basal layer and containing differentiated cells of the epidermal basal cell,
A skin model construct in which a space is formed inside the differentiated cell layer,
(6) The spinous layer, wherein the differentiated cell layer is formed inside the basal layer and contains spinous cells;
A granule layer formed inside the barbed layer and containing granule cells;
The skin model construct according to (5), comprising a keratinized layer formed inside the granule layer, and (7) a cell aggregate of epidermal basal cells containing an extracellular matrix component, and a scaffold A method of simulating the differentiation of skin, characterized by culturing in a support body,
About.

  The method for producing a skin model construct of the present invention has an excellent effect that the skin model construct can be easily produced in a short time. In addition, the skin model construct of the present invention can be easily produced in a short time, and has an excellent effect that various skin states can be easily reproduced. The skin differentiation simulation method of the present invention has an excellent effect that skin differentiation can be simulated easily in a short time.

  The method for producing a skin model construct of the present invention is characterized in that cell aggregates of epidermal basal cells are cultured in a support containing an extracellular matrix component and serving as a scaffold. Therefore, the production method of the present invention has an excellent effect that the skin model construct can be easily produced in a short time.

  Further, in the production method of the present invention, cell aggregates of epidermal basal cells are cultured in the support. Therefore, the production method of the present invention is, for example, a fibroblast as in the conventional method of producing a three-dimensional cultured skin model. Compared to using a support containing cells, it can be performed under more homogenized conditions. Thereby, the manufacturing method of this invention has the outstanding effect that a skin model structure can be manufactured substantially homogeneously with high reproducibility.

  Cell aggregates of epidermal basal cells can be obtained, for example, by rotating and culturing epidermal basal cells on a surface with extremely low adhesion to cells, or stationary culture in a container with extremely low adhesion to cells. . The swivel culture is performed by loading a culture vessel with a rotational motion on a horizontal plane. The adhesion to the cells may be such that it does not adhere to the surface even if the cells are allowed to stand for at least 1 hour after seeding, preferably 10 hours or more after completion of cell mass formation.

  The rotation speed in the rotation culture is preferably 10 rpm or more, more preferably 50 rpm or more from the viewpoint of favorably forming cell aggregates, and preferably 180 rpm or less, more preferably from the viewpoint of maintaining a good cell state. Is 150 rpm or less.

The carbon dioxide (CO ₂ ) concentration in the swirl culture can be set as appropriate. The CO ₂ concentration is preferably 5% by volume.

  The culture temperature in the swirl culture is preferably 36 ° C. to 38 ° C., more preferably 36.5 ° C. to 37.5 ° C. (for example, 37 ° C., etc.) from the viewpoint of favorably growing the cells.

  The medium used for the rotation culture of the epidermal basal cells may be any medium suitable for the growth of epidermal basal cells. The medium is not particularly limited. For example, a mixed medium of Dulbecco's modified medium (DMEM) containing heat-inactivated serum (for example, heat-inactivated FCS) and HamF12 (DMEM / containing heat-inactivated FCS) HamF12 medium), DMEM medium containing heat-inactivated serum, MEM medium containing heat-inactivated serum, and the like. Among these, from the viewpoint of being suitable for cell colony growth, a heat-inactivated FCS-containing DMEM / HamF12 medium is preferable. When the heat-inactivated FCS-containing DMEM / HamF12 medium is used in the three-dimensional culture of the transgenic epidermal basal cells, the heat-inactivated FCS-containing DMEM / HamF12 medium can be obtained, for example, by an antibody against a specific substance. It is possible to perform an operation for inhibiting a substance, which is advantageous in that it is excellent in applicability to elucidation of the onset mechanism of a disease and the development of a therapeutic or preventive drug for the disease.

  From the viewpoint of forming cell aggregates, a culture vessel with low adhesion to cells, preferably a cell non-adhesive culture vessel, is used as the culture vessel in the swirl culture.

  The culture time in the swirl culture can be appropriately set according to the type of epidermal basal cells used.

  The epidermal basal cells may be cells constituting the basal layer of the epidermis, and include keratinocytes and melanocytes. The epidermal basal cells may be keratinocytes alone or melanocytes alone or a mixture of keratinocytes and melanocytes.

  The epidermal basal cells are not particularly limited, and examples thereof include human epidermal basal cells and mouse epidermal basal cells. Of these, human epidermal basal cells having the ability to differentiate normally are preferable from the viewpoint of well reproducing the human skin structure and sufficiently reproducing the human skin state. The human epidermal basal cells are not particularly limited, and examples thereof include HaCaT cells (Breitkreutz D, European Journal of Cell Biology, 1998, Vol. 75, p.273. -286.), Normal human skin epidermal keratinocytes (NHEK) and the like. Among these, the human epidermal basal cells are preferably HaCaT cells from the viewpoint that the cell properties are relatively uniform regardless of the number of passages and the cell differentiation state is normal.

  The epidermal basal cell may be a normal epidermal basal cell or a gene-introduced epidermal basal cell into which a nucleic acid has been introduced exogenously. When the epidermal basal cells are normal epidermal basal cells, the differentiation process from normal epidermal basal cells into stratum corneum cells is reproduced in the process of culturing the cell aggregate in the support in the production method of the present invention. Thus, when the epidermal basal cells are normal epidermal basal cells, according to the production method of the present invention, a skin model construct having a layer similar to normal skin can be obtained. When the epidermal basal cells are gene-introduced epidermal basal cells, according to the production method of the present invention, it is possible to obtain a skin model construct that reflects the behavior according to the gene contained in the introduced nucleic acid.

  When the epidermal basal cell is a normal epidermal basal cell, the normal epidermal basal cell is preferably in an undifferentiated state from the viewpoint of maintaining the proliferative property of the normal epidermal basal cell and obtaining a good skin model construct. And the number of subcultures is low.

  The gene-introduced epidermal basal cell can be obtained by exogenously introducing a nucleic acid into the normal epidermal basal cell. In such gene-introduced epidermal basal cells, proteins derived from the exogenously introduced nucleic acids and the like are expressed.

  The gene-introduced epidermal basal cell exogenously introduces into the normal epidermal basal cell a nucleic acid comprising a transcriptional regulatory element for increasing or decreasing the expression level of a specific endogenous protein possessed by the normal epidermal basal cell. It may be a cell obtained by doing so. In such a transgenic epidermal basal cell, the expression level of a specific endogenous protein in the normal epidermal basal cell is increased or decreased compared to the expression level in a normal state.

  The nucleic acid is not particularly limited, but a nucleic acid encoding a gene for a developmental differentiation regulator or a mutant gene corresponding to the gene, a gene for a skin constituent protein, a nucleic acid encoding a mutant gene corresponding to the gene, or a skin constituent A nucleic acid encoding a gene of a factor involved in the production of a gene or a mutant gene corresponding to the gene, a nucleic acid encoding a gene of a skin disease-related factor or a mutant gene corresponding to the gene, a nucleic acid encoding a gene of a cell death controlling factor , Nucleic acids encoding transcriptional regulatory factors, nucleic acids comprising transcriptional regulatory elements, and the like.

  In the present specification, the developmental differentiation regulator is a molecule involved in differentiation, and refers to a factor that is expressed and functions in the morphogenesis process of an individual. Among the developmental differentiation regulators, developmental differentiation regulators that act on the differentiation of skin tissue are preferable. Moreover, in this specification, a skin constituent protein means the protein which comprises a skin structure. Although it does not specifically limit as said skin constituent protein, For example, collagen, keratin, etc. are mentioned. Furthermore, in the present specification, factors involved in the production of skin constituents include enzymes involved in the synthesis and degradation of skin constituents such as ceramide lipids. In the present specification, the concept of the developmental differentiation control factor and the concept of the skin constituent protein may each include the concept of a skin disease-related factor. The transcriptional regulatory factor refers to a transcriptional regulatory factor involved in the expression control of the developmental differentiation regulator, the skin constituent protein, or the skin disease-related factor. Although it does not specifically limit as said transcriptional regulatory factor, For example, C / EBP etc. are mentioned. Furthermore, the transcriptional regulatory element refers to an element comprising a base sequence involved in the expression control of the developmental differentiation regulator, the skin constituent protein, or the skin disease-related factor.

  Specific examples of the nucleic acid include a nucleic acid encoding an epimorphin gene, a nucleic acid encoding a mutant gene corresponding to a collagen gene, a nucleic acid encoding a mutant gene corresponding to a keratin gene, and a ceramidase gene. And a nucleic acid encoding a mutant gene corresponding to.

  The introduction of the nucleic acid into normal epidermal basal cells is not particularly limited. For example, a method using a viral vector such as a retroviral vector or an adenoviral vector [eg, Molecular Cloning: A Laboratory Manual] For example, see Sambrook et al., Cold Spring Harbor Press, published in 1989). Among the methods using the viral vector, when the expression product of the nucleic acid is stably expressed, a method using a retroviral vector is desirable, and the expression product of the nucleic acid is expressed by freely controlling the expression level. A method using an adenovirus vector is desirable. In the method for producing a skin construct of the present invention, the introduction of the nucleic acid into the normal epidermal basal cell is carried out using a vector obtained by inserting the nucleic acid into the retroviral vector during the rotation culture of the epidermal basal cell. The obtained recombinant retrovirus is added to the culture containing the normal epidermal basal cells and the normal epidermal basal cells are infected with the recombinant retrovirus, or the nucleic acid is inserted into the adenoviral vector. The recombinant adenovirus obtained using the vector obtained above is added to a culture containing the normal epidermal basal cells, and the normal adenovirus is infected with the normal epidermal basal cells. it can.

  The gene-introduced epidermal basal cell can be selected using as an index the presence of the introduced nucleic acid in the obtained cell, the expression of the function of the expression product of the nucleic acid in the cell, and the like.

  The support that contains the extracellular matrix component and serves as a scaffold may be any support that contains the extracellular matrix component that is a basement membrane component and that can grow epidermal basal cells inside. . The support is not particularly limited, and examples thereof include collagen, laminin, an extracellular matrix component substitute, and a synthetic polymer compound containing at least one of these. Although it does not specifically limit as said extracellular matrix component substitute, For example, an EHS sarcoma (Engelbreth-Holm-Swarm sarcoma) extract etc. are mentioned. The synthetic polymer compound is not particularly limited, and examples thereof include methyl cellulose and polyvinyl alcohol. In addition, the support may be a polymer that causes a sol-gel transition by physical stimulation such as temperature and light irradiation as long as it includes a component of the extracellular matrix. Among these, the collagen gel, which is abundantly contained in the dermis, can be prepared easily and inexpensively with high purity and uniformity, and from the viewpoint of favorably promoting cell adhesion and proliferation, preferably the collagen gel, More preferred is a collagen gel comprising type I collagen.

  From the viewpoint of efficiently obtaining a skin model construct from the cell aggregate, the collagen gel is preferably maintained under anchorage-dependent growth conditions by contacting the outermost cell of the cell aggregate with the collagen gel. The gel has a gel strength that allows the cells inside the cell aggregate to be isolated from the collagen gel and maintained under anchorage-independent growth conditions.

  When the support is a collagen gel, the cell aggregate is embedded in the collagen gel. The collagen gel is prepared by adding 100 mM HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid in a 10-fold concentrated medium suitable for culturing epidermal basal cells in an ice water bath, for example. ) Final concentration of buffer solution (pH 7.4): 20 mM, final concentration of sodium bicarbonate: 26 mM, final concentration of sodium hydroxide: 5 mM, 0.5 mass% type I collagen acid solubilized solution of final type I collagen A mixture can be obtained by adding to a concentration of 0.4% by mass, and the obtained mixture can be maintained at 37 ° C. to form a gel. Embedding the cell aggregates in a collagen gel is performed by placing the cell aggregates in the mixture and forming the gel by maintaining the mixture at 37 ° C. during gel formation.

  The medium suitable for culturing the epidermal basal cells used for the production of the collagen gel may be any medium suitable for the growth of epidermal basal cells. The medium is not particularly limited. For example, a mixed medium of Dulbecco's modified medium (DMEM) containing heat-inactivated serum (for example, heat-inactivated FCS) and HamF12 (DMEM / containing heat-inactivated FCS) HamF12 medium), DMEM medium containing heat-inactivated serum, MEM medium containing heat-inactivated serum, and the like. Among these, from the viewpoint of being suitable for cell colony growth, a heat-inactivated FCS-containing DMEM / HamF12 medium is preferable. When the heat-inactivated FCS-containing DMEM / HamF12 medium is used in the three-dimensional culture of the transgenic epidermal basal cells, the heat-inactivated FCS-containing DMEM / HamF12 medium can be obtained, for example, by an antibody against a specific substance. It is possible to perform an operation for inhibiting a substance, which is advantageous in that it is excellent in applicability to elucidation of the onset mechanism of a disease and the development of a therapeutic or preventive drug for the disease. The medium is, for example,

In culturing the cell aggregate in the support, the carbon dioxide (CO ₂ ) concentration can be appropriately set. The CO ₂ concentration is preferably 5% by volume.

  The culture temperature for culturing the cell aggregate in the support is preferably 36 ° C. to 38 ° C., more preferably 36.5 ° C. to 37.5 ° C. (for example, 37 ° C., etc.) from the viewpoint of favorable cell growth. ).

  The cell aggregates in the support are cultured by immersing and maintaining the support in a medium in which epidermal basal cells can grow. The medium used for culturing the cell aggregate in the support is not particularly limited, and examples thereof include a medium obtained by adding about 5 to 20% by mass of fetal calf serum to a serum-free medium for keratinocytes. The serum-free medium for keratinocytes is not particularly limited, and examples thereof include a serum-free Dulbecco's modified medium (DMEM) and HamF12 mixed medium (DMEM / HamF12 medium), α-MEM, DMEM, and the like. The medium may appropriately contain a cell growth promoting factor such as insulin.

  The culture vessel used for culturing the cell aggregate in the support is not particularly limited, and a normal culture vessel coated with agarose, polyhydroxyethyl methacrylate or the like uniformly, an ultra-low adhesion surface Plate (made by Corning, trade name: Ultra Low Attachment Plate) and the like.

  When the epidermal basal cell is a normal epidermal basal cell, the culture time for culturing the cell aggregate in the support is such that there is a space inside the cell cluster formed from the cell aggregate due to the differentiation of the normal epidermal basal cell. It may be a time suitable for forming. The culture time is preferably at least 3 days, more preferably 5 days or more from the viewpoint of forming a space inside the cell cluster formed from cell aggregates, and the viewpoint that differentiation can be followed in a short time Therefore, it is preferably 10 days or less. Thus, the production method of the present invention is, for example, a conventional three-dimensional cultured skin model in which a support such as a collagen gel containing fibroblasts is prepared and normal epidermal cells are three-dimensionally cultured on the support. Compared to the production method, the skin model structure can be obtained in a short time.

  On the other hand, when the epidermal basal cells are gene-introduced epidermal basal cells, the culture time for culturing the cell aggregate in the support can be appropriately set according to the purpose.

  When the epidermal basal cell is a normal epidermal basal cell, when culturing the cell aggregate in the support body, as the culture time elapses, the cell aggregate from the epidermal basal cell and the differentiated cell of the epidermal basal cell A cell cluster containing is formed. In the cell cluster, from the outermost part of the cell cluster toward the inside, the normal layer containing the normal epidermal basal cell and the differentiation containing the differentiated cell of the normal epidermal basal cell, as in normal skin A cell layer, specifically, a spiny layer, a granule layer, and a stratum corneum are formed in order toward the inner direction of the cell aggregate, and a space is formed in the innermost part of the cell aggregate.

  Here, a normal human skin structure is composed of a dermis, a basal layer, a spiny layer, a granular layer, and a stratum corneum in order from the lower layer. In the normal human skin structure, keratinocytes in the basal layer express cytokeratin-5 and cytokeratin-14, and then, in the process of differentiation, in the keratinocytes in the spinous layer of the normal skin structure, cytokeratin -1 and cytokeratin-10 are expressed, and emboplatin, periplakin, and involucrin are expressed. Further, the embossed plakin and periplakin form a heterodimer, the formed heterodimer and the involucrin bind on the cell membrane, and transglutaminase 1 is expressed, in addition to between the heterodimer and the involucrin, A cross-linked structure is formed with other proteins on the cell membrane. In the granular layer of normal skin structure, loricrin is expressed in the upper layer, and the expressed loricrin forms a cross-link with the SPR molecule by the action of transglutaminase-3, thereby generating in the spiny layer. The crosslinked structure is reinforced. In the stratum corneum having a normal skin structure, the cell nuclei and intracellular organelles existing up to the granular layer disappear. In the stratum corneum with normal skin structure, a mature, confined envelope is generated.

  In this way, by culturing cell aggregates of epidermal basal cells in a support body containing an extracellular matrix component and serving as a scaffold, differentiation of skin can be simulated easily in a short time.

  The present invention also includes a method for simulating skin differentiation, which comprises culturing cell aggregates of epidermal basal cells in a support body containing an extracellular matrix component and serving as a scaffold.

  In the skin differentiation simulation method of the present invention, when the epidermal basal cell is a normal epidermal basal cell, the differentiation process of the epidermal basal cell similar to normal skin is reproduced when the cell aggregate is cultured in the support. can do.

  The skin model construct of the present invention is a skin model construct obtained by the production method of the present invention.

The skin model construct of the present invention comprises a cell cluster comprising epidermal basal cells and differentiated cells of the epidermal basal cells,
A basal layer formed on the outermost part of the cell cluster and containing the epidermal basal cells;
A differentiated cell layer formed inside the basal layer and containing differentiated cells of the epidermal basal cell,
It is a construct in which a space is formed inside the differentiated cell layer. Since the skin model construct of the present invention can be obtained by the production method of the present invention, it can be easily produced in a short time.

  Further, since the skin model construct of the present invention comprises a cell cluster including the epidermal basal cells and differentiated cells of the epidermal basal cells, various skin states can be used depending on the types of cells used as the epidermal basal cells. It is possible to easily reproduce the above.

  Examples of the differentiated cells of the epidermal basal cells include spiny cells, granule cells, and corneocytes when the epidermal basal cells are normal epidermal basal cells.

When the epidermal basal cell is a normal epidermal basal cell, the differentiated cell layer is formed inside the basal layer, and includes a spiny layer containing spinous cells,
A granule layer formed inside the barbed layer and containing granule cells;
It consists of a keratinized layer formed inside the granular layer.

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

Example 1
Heat-inactivated FCS was added to DMEM / HamF12 (manufactured by Sigma-Aldrich) to a final concentration of 10% by mass, and a heat-inactivated FCS-containing DMEM / HamF12 medium (hereinafter referred to as “DH10”) was added. Obtained.

HaCaT cells, a normal human epidermal keratinocyte cell line, were cultured in DH10 at 37 ° C. under 5% by volume CO ₂ .

Next, the obtained HaCaT cells were suspended in 350 μl of DH10 containing 1000 U / ml DNase I (manufactured by Sigma-Aldrich). The resulting suspension was cultivated in a 24-well dish (manufactured by Corning, ultra-low adhesion surface) at 100 rpm, rotating at 5 ° C. CO ₂ for 24 hours at 37 ° C., and smooth and round. Cell aggregates were formed.

The formed cell aggregate was embedded in a high-density collagen gel prepared with a 0.5 mass% type IA collagen solution (manufactured by Koken Co., Ltd.). The cell aggregates after embedding were cultured in DH10 containing insulin at a final concentration of 5 μg / ml for 3 days at 37 ° C. under 5 vol% CO ₂ to obtain cell clusters. Moreover, the obtained cell cluster was observed under the phase-contrast microscope. The results are shown in FIG. FIG. 1 is a drawing-substituting photograph showing the observation results of a cell cluster on the third day from the start of culture in Example 1 using a phase contrast microscope. In the figure, the scale bar indicates 50 μm.

  From the results of FIG. 1, it can be seen that the cell cluster on the third day from the start of the culture has a substantially spherical structure in which a plurality of layers are formed and a space is formed in the innermost part.

  As described above, by rotating the epidermal basal cells in advance, cell aggregates are formed and then cultured in a support body that contains extracellular matrix components and serves as a scaffold. It can be seen that a cell cluster having a substantially spherical structure in which a space is formed in the innermost part is obtained.

(Example 2)
(1) Changes in the structure of cell clusters with the passage of the culture period In the same manner as in Example 1, by rotating the epidermal basal cells, smooth and round cell aggregates were formed.

The formed cell aggregate was embedded in a high-density collagen gel prepared with a 0.5 mass% type IA collagen solution (manufactured by Koken Co., Ltd.), and in DH10 containing insulin at a final concentration of 5 μg / ml, While culturing at 37 ° C. under 5% by volume CO ₂ , changes in the structure of cell clusters during the culturing process were observed under an electron microscope. The results are shown in FIG.

  FIG. 2 is a schematic explanatory view schematically showing changes in the structure of cell clusters during the culture process in Example 2. FIG. In the figure, 1 indicates a cell cluster on the first day from the start of culture, 2 indicates a cell cluster on the fourth day from the start of culture, and 3 indicates a cell cluster on the eighth day from the start of culture. In the figure, the scale bar indicates 100 μm.

  In addition, as shown in FIG. 2, the cell cluster had a densely aggregated structure on the first day from the start of the culture, but on the fourth day from the start of the culture, a plurality of layers were formed, and the innermost An almost spherical structure was formed in which a space was formed. After that, on the 8th day from the start of the culture, the space formed inside the cell cluster was larger and the size of the culture itself was larger than the cell cluster on the 4th day from the start of the culture.

  Based on the above, cell aggregates are formed in advance by swirling culture of epidermal basal cells, and then cultured in a support body that contains extracellular matrix components and serves as a scaffold. It can be seen that the structure of the cell cluster resulting from the cell aggregate changes with time.

(2) Expression profiles of various differentiation markers in the cell cluster over the course of the culture period About the cell cluster on the first day from the start of culture, the cell cluster on the fourth day from the start of culture, and the cell cluster on the first day from the start of culture, The expression of differentiation markers at each differentiation stage of skin differentiation was examined.

  The cell clusters on the first day, the fourth day, and the eighth day from the start of the culture were respectively mixed with an SDS sample buffer (composition: 62.5 mM Tris-HCl (pH 6.5), 10% by mass glycerol, 2% by mass SDS, 5 volumes). % 2-mercaptoethanol, 0.0025 mass% bromophenol blue) and boiled for 5 minutes to obtain a whole cell lysate sample.

  Each obtained whole cell lysate sample was loaded on a 4-20 mass% concentration gradient SDS-polyacrylamide gel and then subjected to electrophoresis. Next, all protein bands were transferred to a PVDF membrane (trade name: Immobilon-P, manufactured by Millipore Corporation). The transferred PVDF membrane was incubated with a 5% by weight skim milk TBS solution for 60 minutes at room temperature. The resulting PVDF membrane was then incubated with the primary antibody for 60 minutes at room temperature, and then incubated with the HRP-labeled secondary antibody for 60 minutes at room temperature. As the primary antibody, a polyclonal antibody against cytokeratin-14 (manufactured by Covance), a monoclonal antibody against cytokeratin-5 (manufactured by Covance), a polyclonal antibody against cytokeratin-1 (manufactured by Covans), a monoclonal antibody against involcrine (Santa Cruz) Biotechnology Inc., monoclonal antibody against β-actin (Sigma-Aldrich), procaspace-3 and activated caspase-3, polyclonal antibody against caspase-3 (manufactured by StressGen), For Procaspase-14 and Activated Caspase-14, polyclonal antibodies against Caspase-14 (Imgenex) were used. Further, as the HRP-labeled secondary antibody, an HRP-labeled anti-mouse IgG antibody and an HRP-labeled anti-rabbit IgG antibody were used according to the primary antibody. Cytokeratin-1 is a spiny layer and granule layer, cytokeratin-5 is a basal layer, cytokeratin-14 is a basal layer differentiation marker, involucrin is a spiny layer and granule layer differentiation marker , Β-actin is an internal control, procaspace-3 is a marker for proteases that act in the final stages of apoptosis, and activated caspase-3 is cleaved from procaspace-3 as an executioner Procaspace-14 is a functional marker, and is a stratum corneum differentiation marker expressed in the terminal differentiation of skin. Activated caspase-14 is cleaved from procaspace-14 as an execution factor It is a marker with the function of. The results are shown in FIG.

  FIG. 3 is a drawing-substituting photograph showing the results of immunoblot analysis of various differentiation markers in the cell clusters on day 1, 4 and 8 from the start of culture. In the figure, lane 1, lane 2, and lane 3 correspond to 1, 2, and 3 in FIG. 2, respectively. In the figure, (a) is cytokeratin-14, (b) is cytokeratin-5, (c) is cytokeratin-1, (d) is involucrin, and (e) is β-actin. , (F) shows procaspace-3, (g) shows activated caspase-3, (h) shows procaspace-14, and (i) shows activated caspase-14.

  From the results shown in FIG. 3, compared to the cell cluster on the first day from the start of the culture, the cell cluster on the fourth day from the start of the culture and the cell cluster on the eighth day from the start of the culture in which a space is formed in the innermost part are spiny. It can be seen that expression of involucrin and cytokeratin 1, which are molecular markers of the layer and the granule layer, is increased. Therefore, it is suggested that in the cell cluster, epidermal basal cells are differentiated into spinous layer cells and granule layer cells.

  In addition, from the results shown in FIG. 3, compared with the cell cluster on the 4th day from the start of the culture, the cell cluster on the 8th day from the start of the culture in which the space is formed in the innermost part, the molecular markers of the spinous layer and the granular layer are used. It can be seen that the expression of certain involucrin and cytokeratin 1 is increased. Therefore, it can be seen that differentiation from epidermal basal cells into spinous layer cells and granule layer cells progresses as the culture period elapses.

  Moreover, from the result of FIG. 3, the cytokeratin 5 and the cytokeratin 14 which are the molecular markers of a basal layer are increasing in the cell cluster of the 4th day from the culture start compared with the cell cluster of the 1st day from the culture start. I understand that. Further, from the results of FIG. 3, cytokeratin 5 and cytokeratin 14 which are molecular markers of the basal layer are slightly increased in the cell cluster on the 8th day from the start of the culture as compared with the cell cluster on the 4th day from the start of the culture. I understand that. From these results, with the progress of the culture period, cytokeratin 5 and cytokeratin 14, which are molecular markers of the basal layer, increase in the cell cluster as the space formed in the innermost portion increases. I understand that. From these results, it is suggested that the diameter of the spherical structure of the cell cluster increases with the passage of the culture period, and the number of cells in contact with the collagen gel increases.

  Further, from the results of FIG. 3, changes in the amount of procaspace-3 were observed in the cell cluster on the first day from the start of culture, the cell cluster on the fourth day from the start of culture, and the cell cluster on the eighth day from the start of culture. It can be seen that there is no change in the expression level of activated caspase-3. Therefore, the formation of the space in the innermost part of the cell cluster with the progress of the culture period does not show the generation of activated caspase-3 from procaspase-3, so that in the event caused by apoptosis derived from cytotoxicity Not suggested.

  In addition, from the results shown in FIG. 3, procaspase-14 decreased and activated caspase-14 increased in the cell cluster on the fourth day from the start of the culture as compared to the cell cluster on the first day from the start of the culture. Therefore, it can be seen that activation of caspase-14 occurs. Further, from the results shown in FIG. 3, procaspase-14 decreased and activated caspase-14 increased in the cell cluster on the eighth day from the start of the culture as compared to the cell cluster on the fourth day from the start of the culture. Therefore, it can be seen that activation of caspase-14 occurs. From these results, the activation of caspase-14 is observed with the passage of the culture period in the cell cluster as in the three-dimensional cultured skin model and the stratum corneum of human skin. It is suggested that the formation of the innermost space of the cell cluster with the passage of time is caused by terminal differentiation from epidermal basal cells to stratum corneum cells.

  From the above, cell clusters obtained by swirling culture of epidermal basal cells in advance to form cell aggregates and then culturing in a support body that contains extracellular matrix components and serves as a scaffold As in human skin, it can be seen that it reflects the differentiation state of each layer from the basal layer in contact with collagen to the enucleated stratum corneum. Therefore, it is suggested that the cell cluster is a skin model construct that can be used as a skin model. In addition, since the cell cluster shows a differentiating process similar to the differentiation of epidermal basal cells in the skin as the culture time in the support body elapses, the method for producing the skin model construct includes a process of skin differentiation. Useful as a model.

  The conventional three-dimensional cultured skin model requires complicated operations during production, and requires 20 days for production.

  However, in the skin model construct, the period from the start of swiveling culture of epidermal basal cells to the formation of a layer similar to human skin having a basal layer, a spiny layer, a granule layer, and a stratum corneum is only 4 Days. Therefore, the skin model construct is superior to the conventional three-dimensional cultured skin model in that it is easy to manufacture and requires less time for manufacturing. In plate culture, epidermal basal cells do not form a layer similar to human skin having a basal layer, a spiny layer, a granule layer, and a stratum corneum, and are found in the stratum corneum of human skin. In contrast, the skin model construct forms a layer similar to human skin having a basal layer, a spiny layer, a granule layer, and a stratum corneum. As with the original cultured skin model, activation of caspase-14 is observed. Therefore, the skin model construct is superior in that it is a model closer to the skin than the epidermal basal cells cultured by planar culture.

(Example 3)
The nucleic acid (SEQ ID NO: 4) encoding the IL-2 signal peptide (SEQ ID NO: 3) is converted into epimorphin (SEQ ID NO: 2) in the cDNA (SEQ ID NO: 1) encoding epimorphin, which is a developmental differentiation regulator. Was added to the site corresponding to the N-terminus of the nucleic acid to obtain a nucleic acid encoding IL-2 signal peptide-bound epimorphin. Using the obtained nucleic acid encoding IL-2 signal peptide-bound epimorphin and an adenovirus expression system (Clontech, trade name: Adeno-X Expression System 1), IL-2 signal peptide-bound epimorphin A recombinant adenovirus for expression was constructed.

  HaCaT cells were suspended in 350 μl of DH10 containing 1000 U / ml DNase I (Sigma-Aldrich), and the obtained suspension was suspended at 100 rpm in a 24-well dish (super low adhesion surface, Corning). It was rotated with.

In some wells, the recombinant adenovirus (10 ⁷ pfu) is added, introduced into HaCaT cells, and a transgenic epidermal basal cell (HaCaT-Av) that transiently expresses IL-2 signal peptide-bound epimorphin. -EPM cells).

  In addition, adenovirus without cDNA encoding IL-2 signal peptide-binding epimorphin was added to some wells and introduced into HaCaT cells to obtain HaCaT-Av-control cells.

The obtained HaCaT-Av-EPM cells were cultured in the same manner as in Example 1 to form cell clusters. Thereafter, the formed cell cluster was embedded in a high-density collagen gel prepared with a 0.5 mass% type IA collagen solution (manufactured by Koken Co., Ltd.) in the same manner as in Example 1, and the final concentration was 5 μg. Cell clusters were obtained by culturing for 3 days at 37 ° C. in 5% by volume CO _{2 in} DH10 containing / ml insulin.

  Further, instead of HaCaT-Av-EPM cells, HaCaT-Av-control cells were used to obtain cell clusters in the same manner.

  The cell cluster obtained from the HaCaT-Av-control cell and the cell cluster obtained from the HaCaT-Av-EPM cell were each treated with an affinity purified antibody against involucrin (manufactured by Santa Cruz Biotechnology Inc.) for 60 minutes at room temperature. After incubation, immunocytochemical staining was performed by incubation with AlexaFluor488-labeled secondary antibody (Invitrogen, trade name: AlexaFluor488-labeled anti-mouse IgG antibody) for 60 minutes at room temperature. Cell nuclei were counterstained with propidium iodide. The cell clusters obtained from the HaCaT-Av-control cells and the cell clusters obtained from the HaCaT-Av-EPM cells were observed with a phase contrast microscope and a fluorescence microscope. Thereby, the morphology of the cell cluster when the nucleic acid encoding the developmental differentiation regulator was introduced into the epidermal basal cell and the expression of the marker in the cell cluster were examined. The results are shown in FIG.

  FIG. 4 is a drawing-substituting photograph showing the results of examining the morphology of a cell cluster and the effect of marker expression on the cell cluster when a nucleic acid encoding a developmental differentiation regulator is introduced into epidermal basal cells. In the figure, the scale bars are all 50 μm. FIG. 4 (A) (a) is a drawing-substituting photograph showing the results of phase contrast microscopic observation of a cell cluster obtained from HaCaT-Av-control cells, and (b) is obtained from HaCaT-Av-control cells. 4A is a drawing-substituting photograph showing the result of immunocytochemical staining of the cell cluster, and FIG. 4B is a drawing showing the result of fluorescence microscope observation of the cell cluster obtained from HaCaT-Av-EPM cells. A substitute photo, (b), is a drawing substitute photo showing the results of immunocytochemical staining of a cell cluster obtained from HaCaT-Av-EPM cells. In the figure, 4 is a formed space, 5 is an example of a staining position for involucrin, and 6 is an example of a staining position for propidium iodide.

  From the result of (a) in FIG. 4 (A) and the result of (b) in FIG. 4 (A), a space is formed in the innermost part of the cell cluster obtained from the HaCaT-Av-control cell and is adjacent to the space. It can be seen that there is no cell nucleus in the layer. The results of FIG. 4 and FIG. 3 suggest that the formation of this space is accompanied by enucleation due to terminal differentiation from epidermal basal cells to stratum corneum cells.

  In addition, from the result of (b) of FIG. 4 (A), expression of involucrin, which is a molecular marker of the spinous layer and the granular layer, is observed along the inner side of the formed space without being in contact with the collagen gel. It has been. Therefore, in the cell cluster obtained from the HaCaT-Av-control cells, the basal layer in contact with collagen, the spinal layer and granule layer expressing involucrin, and enucleated were differentiated by the loss of the binding to collagen. It is suggested that a layer similar to human skin having a stratum corneum indicated by a space is formed.

  On the other hand, from the result of (a) in FIG. 4B and the result of (b) in FIG. 4 (B), the cell cluster obtained from the HaCaT-Av-EPM cell does not form a space, and particles of involucrin Has a deposited structure. From the results of (a) and (b) of FIG. 4 (A) and (a) and (b) of FIG. 4 (B), the cell cluster obtained from the HaCaT-Av-EPM cells was introduced exogenously. It is suggested that overexpression of the epimorphin gene inhibits the formation of layers similar to human skin. From these results, production of the skin model construct is carried out in which cell aggregates are formed in advance by swirling culture of epidermal basal cells, and then cultured in a support body containing an extracellular matrix component and serving as a scaffold. In the method, it can be seen that the function of the developmental differentiation regulator can be examined by using, as the epidermal basal cell, a transgenic basal cell into which a nucleic acid encoding a developmental differentiation regulator is introduced. In addition, in the method for producing a skin model construct, it may be possible to easily reproduce a skin disease state by using a gene-introduced epidermal basal cell into which a gene of a disease-related factor is introduced as an epidermal basal cell. It is suggested.

2 is a drawing-substituting photograph showing the observation results of the cell clusters on the eighth day from the start of culture in Example 1 using a phase contrast microscope. In the figure, the scale bar indicates 50 μm. 6 is a schematic explanatory diagram schematically showing changes in the structure of cell clusters during the culture process in Example 2. FIG. In the figure, the scale bar indicates 100 μm. 2 is a drawing-substituting photograph showing the results of immunoblot analysis of various differentiation markers of the cell clusters on the first, fourth, and eighth days from the start of culture in Example 2. FIG. FIG. 6 is a drawing-substituting photograph showing the results of examining the morphology of cell clusters and the effect on marker expression in the cell clusters when a nucleic acid encoding a developmental differentiation regulator is introduced into epidermal basal cells in Example 3. FIG. In the figure, the scale bars are all 50 μm.

  SEQ ID NO: 3 is the amino acid sequence of the IL-2 signal peptide.

  SEQ ID NO: 4 is the base sequence of nucleic acid encoding IL-2 signal peptide.

Claims (7)

  A method for producing a skin model construct, comprising culturing cell aggregates of epidermal basal cells in a support body containing a component of an extracellular matrix and serving as a scaffold.   The method for producing a skin model construct according to claim 1, wherein the epidermal basal cell is a gene-introduced epidermal basal cell into which a nucleic acid has been introduced exogenously.   The method for producing a skin model construct according to claim 1 or 2, wherein the support is a collagen gel.   The skin model structure obtained by the manufacturing method of any one of Claims 1-3. A cell cluster comprising epidermal basal cells and differentiated cells of the epidermal basal cells,
A basal layer formed on the outermost part of the cell cluster and containing the epidermal basal cells;
A differentiated cell layer formed inside the basal layer and containing differentiated cells of the epidermal basal cell,
A skin model construct in which a space is formed inside the differentiated cell layer. The differentiated cell layer is formed inside the basal layer and contains spinous cells;
A granule layer formed inside the barbed layer and containing granule cells;
The skin model construct according to claim 5, comprising a keratinized layer formed inside the granular layer.   A method of simulating skin differentiation, comprising culturing cell aggregates of epidermal basal cells in a support body containing a component of an extracellular matrix and serving as a scaffold.