WO2019031500A1 - 不死化汗腺筋上皮細胞 - Google Patents
不死化汗腺筋上皮細胞 Download PDFInfo
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- WO2019031500A1 WO2019031500A1 PCT/JP2018/029594 JP2018029594W WO2019031500A1 WO 2019031500 A1 WO2019031500 A1 WO 2019031500A1 JP 2018029594 W JP2018029594 W JP 2018029594W WO 2019031500 A1 WO2019031500 A1 WO 2019031500A1
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Definitions
- the present invention relates to immortalized sweat gland myoepithelial cells. More particularly, the present invention relates to an immortalized sweat gland myoepithelial cell useful for the development of external preparations such as antiperspirants and deodorants, agents for improving sweat gland function and the like, and a method for producing the same.
- Sweat gland myoepithelial cells are one of the cells that make up the sweat gland.
- the sweat gland myoepithelial cells are involved in the movement of the sweat glands during sweat secretion.
- sweat gland myoepithelial cells are stem cells of sweat glands (see, for example, Non-Patent Document 1). Therefore, it may be considered to use sweat gland myoepithelial cells for evaluation of the function of sweat glands, etc. in order to develop means for improving the dysfunction and hyperfunction of the sweat glands.
- sweat gland myoepithelial cells isolated from sweat glands have the disadvantage of poor availability and handleability due to the small amount present in the sweat glands and the small number of times that can be subcultured. Therefore, it is desirable to immortalize the sweat gland myoepithelial cells to impart long-term cell proliferation ability to the sweat gland myoepithelial cells.
- SV40 simian virus 40
- non-patent document 2 a method of infecting sweat gland cells cultured in a state of being adhered to a culture vessel with a simian virus 40 (hereinafter referred to as "SV40") has been reported (see, for example, non-patent document 2) ).
- the method has the disadvantage that it is difficult to obtain immortalized sweat gland cells having the same function and properties as sweat gland cells in the living body, because the sweat gland cells are cultured under an environment different from the living environment.
- the present inventors have not found documents specifically describing a technique capable of introducing a foreign gene into sweat gland cells in a suspended state.
- NCL-SG3 A human eccrine sweat cell line having transepithelial ion transport ability
- NCL-SG3 human eccrine sweat gland cell line that retains the capacity for transepithelial ion transport
- the present invention has been made in view of the above-mentioned prior art, and has the same function and property as sweat gland myoepithelial cells in vivo, and can immortalize cells having such function and property for a long period of time. It is an object of the present invention to provide a method for producing an transformed sweat gland myoepithelial cell, and an immortalized sweat gland myoepithelial cell capable of producing the immortalized sweat gland myoepithelial cell with high production efficiency.
- the present invention (1) An immortalized sweat gland myoepithelial cell which expresses ⁇ -smooth muscle actin and pancytokeratin and has a sphere-forming ability after at least 5 passages. (2) The immortalized sweat gland myoepithelial cell according to the above (1), which further expresses ATP1a1.
- a method for producing immortalized sweat gland myoepithelial cells (I) introducing an immortalizing gene into the sweat gland myoepithelial cell while culturing the cell structure in which the sweat gland myoepithelial cell is exposed on the surface in a suspended state in a medium to obtain a gene transfer body; And (II) A method for producing immortalized sweat gland myoepithelial cells comprising the step of culturing the gene transfer body obtained in the step (I) suspended in a medium to obtain immortalized sweat gland myoepithelial cells.
- the method further includes the step of removing at least all or part of collagen fibers from the collected skin tissue before performing the step (I) to obtain a sweat gland-containing tissue, wherein the cells in the step (I)
- the method for producing an immortalized sweat gland myoepithelial cell according to the above (3) or (4), wherein the sweat gland-containing tissue is used as a structure, and (6) before performing the step (I), The step of culturing in a suspended state to form a sphere in which the sweat gland myoepithelial cells are exposed on the surface, wherein in the step (I), the sphere is used as the cell structure (3) or (3) It relates to the manufacturing method of immortalized sweat gland myoepithelial cells as described in 4).
- the immortalized sweat gland myoepithelial cell of the present invention has an excellent effect that cells having the same function and nature as the sweat gland myoepithelial cell in vivo can be proliferated for a long period of time. Is played. Moreover, according to the method for producing immortalized sweat gland myoepithelial cells of the present invention, the excellent effect that the immortalized sweat gland myoepithelial cells of the present invention can be produced with high production efficiency is exhibited.
- (A) is a drawing-substituting photograph showing the result of fluorescence microscope observation of the virus-infected sweat gland sphere obtained in Reference Example 1
- (B) shows the result of fluorescence microscope observation of the virus-infected sweat gland cell obtained in Reference Example 2
- Photo substitute for drawing
- (A) is a drawing-substituting photograph showing the results of examining the relationship between the sphere forming ability of the sweat gland cells contained in the virus-infected sweat gland sphere obtained in Example 1 and the passage number in Test Example 2 (1)
- (B ) Is a drawing-substituting photograph showing the results of examining the relationship between the sphere forming ability and passage number of the dissociated sweat gland cells obtained in Comparative Example 1 in Test Example 2 (1).
- (A) is a graph showing the result of examining the relationship between cell type and pancytokeratin expression level
- (B) is a graph showing the result of examining the relationship between cell type and ⁇ -SMA expression level.
- (A) is a drawing-substituting photograph showing the result of examining the relationship between the sphere forming ability of the sweat gland cells contained in the virus-infected tissue obtained in Example 2 and the passage number in Test Example 3
- (B) is the Test Example 10 is a drawing-substituting photograph showing the results of examining the relationship between the sphere forming ability and passage number of the dissociated sweat gland cells obtained in Comparative Example 1 in No. 3.
- the immortalized sweat gland myoepithelial cells of the present invention are characterized in that they express ⁇ -smooth muscle actin and pancytokeratin and have the ability to form spheres after at least 5 passages.
- sweat gland myoepithelial cells isolated from sweat glands refer to sweat gland myoepithelial cells (hereinafter also referred to as “primary sweat gland myoepithelial cells”) separated from the sweat glands and subjected to primary culture.
- ⁇ -smooth muscle actin and pancytokeratin are markers of sweat gland myoepithelial cells in vivo.
- Alpha-smooth muscle actin is involved in the expression of the function of sweat gland myoepithelial cells to cause sweat gland contraction.
- Pancytokeratin also forms the cytoskeleton of sweat gland myoepithelial cells. Since the immortalized sweat gland myoepithelial cells of the present invention express ⁇ -smooth muscle actin and pancytokeratin, they have the same functions and properties as sweat gland myoepithelial cells in vivo.
- the immortalized sweat gland myoepithelial cells of the present invention have the ability to form spheres after at least 5 passages, they have the same function as sweat gland myoepithelial cells in vivo as compared to primary sweat gland myoepithelial cells. And cells with properties can be grown over an extended period of time.
- the immortalized sweat gland myoepithelial cells of the present invention have at least 5, preferably 7 or more, more preferably 9 or more, still more preferably 18 or more, still more preferably 100 or more passages. Even, it has the ability to form a sphere.
- the immortalized sweat gland myoepithelial cells of the present invention maintain their cell proliferation ability for a longer period of time as compared to primary sweat gland myoepithelial cells.
- Examples of the “function similar to the function of the sweat gland myoepithelial cell in the living body” include, for example, contraction movement of the sweat gland at the time of secretion of sweat, but the present invention is not limited to such an illustration only.
- Examples of the “properties similar to the properties of sweat gland myoepithelial cells in vivo” include the ability to differentiate into sweat gland luminal cells etc., self-replication ability, ⁇ -smooth muscle actin expression positive, pan cytokeratin expression positive, sodium / Although the ⁇ subunit of potassium ATPase (ATP1a1) expression is positive and the like, the present invention is not limited to such examples.
- the immortalized sweat gland myoepithelial cells of the present invention further express ATP1a1.
- ATP1a1 is a marker for sweat gland cells because it is not expressed in myoepithelial cells derived from organs other than sweat glands (eg, myoepithelial cells of mammary gland, etc.). Therefore, the immortalized sweat gland myoepithelial cells of the present invention expressing ATP1a1 can be differentiated from myoepithelial cells derived from organs other than sweat glands.
- the immortalized sweat gland myoepithelial cell of the present invention has the same function and property as the sweat gland myoepithelial cell in vivo, it is used, for example, as a method for evaluating the differentiation regulatory action of the sweat gland myoepithelial cell possessed by the test substance. There is expected.
- Such an evaluation method is, for example, (A) culturing the immortalized sweat gland myoepithelial cells of the present invention in a medium suspended state in the absence of a test substance to obtain a cell culture, (B) culturing the immortalized sweat gland myoepithelial cells of the present invention in the presence of a test substance in a suspended state in a medium to obtain a cell culture, and (C) obtaining in the step (A) The expression profile of the differentiation marker in the cell culture (A) and the expression profile of the differentiation marker in the cell culture (B) obtained in the step (B) are examined, and the expression of the differentiation marker in the cell culture (A) Evaluating the differentiation regulatory action of the sweat gland myoepithelial cell possessed by the test substance based on the difference between the profile and the expression profile of the differentiation marker in the cell culture (B).
- the test substance When it is confirmed by the present evaluation method that the test substance has an action to promote differentiation of sweat gland myoepithelial cells, the test substance is, for example, for the improvement of the condition caused by the abnormal enhancement of the function of sweat gland myoepithelial cells. It is expected to be used. In addition, when it is confirmed by the present evaluation method that the test substance has an action of suppressing differentiation of sweat gland myoepithelial cells, the test substance is, for example, for the improvement of a condition caused by dysfunction of sweat gland myoepithelial cells. It is expected to be used.
- the immortalized sweat gland myoepithelial cell of the present invention is, for example, cultured in the medium in which the cell structure in which the sweat gland myoepithelial cell is exposed on the surface is suspended in a culture medium, It can be manufactured by introducing or the like.
- the method for producing immortalized sweat gland myoepithelial cells of the present invention is (I) introducing an immortalizing gene into the sweat gland myoepithelial cell while culturing the cell structure in which the sweat gland myoepithelial cell is exposed on the surface in a suspended state in a medium to obtain a gene transfer body; And (II) a step of culturing the gene transfer body obtained in the step (I) in a suspended state in a medium to obtain immortalized sweat gland myoepithelial cells (hereinafter referred to as “the present invention Also known as
- an operation of introducing an immortalizing gene into the sweat gland myoepithelial cell while culturing the cell structure in which the sweat gland myoepithelial cell is exposed on the surface is suspended in the medium is As they are taken, immortalizing genes can be introduced into sweat gland myoepithelial cells with high transfer efficiency. Therefore, according to the method of the present invention, immortalized sweat gland myoepithelial cells can be produced with high production efficiency.
- the immortalizing gene is introduced into the sweat gland myoepithelial cell to culture a gene transfer body while culturing the cell structure in which the sweat gland myoepithelial cell is exposed on the surface in a suspended state in the medium. obtain.
- the cell structure may contain other sweat gland cells and the like as long as the sweat gland myoepithelial cells are exposed on the surface.
- Other sweat gland cells include, for example, sweat gland luminal cells, sweat gland secretory cells, and the like, but the present invention is not limited to such examples.
- Examples of cell structures include sweat gland-containing tissues in which sweat gland myoepithelial cells are exposed on the surface, spheres in which sweat gland myoepithelial cells are exposed on the surface, and the like, but the present invention is limited to such examples only. It is not something to be done.
- the sweat gland-containing tissue is a tissue of a portion of the skin tissue containing the sweat gland.
- the surface layer of the sweat gland contained in the sweat gland-containing tissue is covered by sweat gland myoepithelial cells.
- sweat gland myoepithelial cells that are more differentiated from the surface to the inside.
- the sweat gland-containing tissue can be separated, for example, by removing at least all or part of collagen fibers from the collected skin tissue.
- the method of the present invention removes at least all or part of collagen fibers from the skin tissue collected prior to step (I) to form a surface of sweat gland myoepithelial cells.
- the method may further include the step of obtaining the exposed sweat gland-containing tissue.
- step (A1) separating tissue pieces including sweat glands from the collected skin tissue; and (a2) removing collagen fibers etc. from the tissue pieces obtained in step (a1) to expose the surface of the sweat gland myoepithelial cells
- the method includes the step of obtaining the sweat gland-containing tissue, the present invention is not limited to such examples.
- the collected skin tissue includes, for example, skin tissue in a living state obtained from surplus skin and the like generated during surgery, and the present invention is not limited to such examples. Absent.
- the collected skin tissue is preferably fresh tissue in terms of better maintaining the function and properties of sweat gland myoepithelial cells in vivo.
- the collected skin tissue is a tissue stored under refrigeration, it is preferably a tissue within 48 hours after excision from the viewpoint of maintaining the function and properties of sweat gland myoepithelial cells in vivo better.
- living skin tissue refers to skin tissue in a state that exhibits biological activity and movement similar to the original biological activity and movement in vivo. Sources of skin tissue include, for example, humans, but the present invention is not limited to such examples.
- a piece of tissue including a sweat gland is separated from the collected skin tissue.
- a staining reagent such as neutral red to visualize the sweat gland.
- the skin tissue is stained, it is preferable to wash away the tissue fragments separated from the skin tissue from the viewpoint of reducing the influence of the staining reagent on the sweat glands and the viewpoint of reducing contamination such as microorganisms.
- step (a2) collagen fibers and the like are removed from the tissue piece obtained in step (a1) to obtain a sweat gland-containing tissue in which the sweat gland myoepithelial cells are exposed on the surface.
- collagen fibers can be removed from the tissue fragments using, for example, enzymes such as dispase and collagenase, physical excision means, and the like.
- Spheres are aggregates of sweat gland cells.
- the spheres have a surface layer of sweat gland myoepithelial cells.
- sweat gland cells that are more differentiated from the surface layer to the inside.
- Spheres can be produced, for example, by culturing sweat gland cells in suspension in a medium.
- the method of the present invention is, prior to step (I), culturing the sweat gland cells suspended in a medium, and exposing the surface of the sweat gland myoepithelial cells to the surface. Can be included.
- a method of manufacturing a sphere for example, (B1) separating a piece of tissue including a sweat gland from the collected skin tissue; (B2) culturing the sweat gland cells obtained in the step (b2) from the tissue piece obtained in the step (b1) in a state in which the sweat gland cells obtained in the step (b2) are suspended
- the method includes a step of forming a sphere in which the myoepithelial cells are exposed on the surface, the present invention is not limited to such an example.
- step (b1) a piece of tissue including a sweat gland is separated from the collected skin tissue.
- the collection of the tissue piece in the step (b1) can be performed by the same method as the collection of the tissue piece in the step (a1) of the method for producing the sweat gland-containing tissue.
- step (b2) the sweat gland cells in the dissociated state are obtained from the tissue piece obtained in step (b1).
- the step (b2) it is possible to obtain sweat gland cells in a dissociated state by dissociating the sweat gland cells from the tissue piece by causing the cell dissociation reagent to act on the tissue piece.
- the cell dissociation reagent include enzymes such as thermolysin, dispase, collagenase, trypsin and the like, but the present invention is not limited to such examples.
- the dissociated sweat gland cells obtained in the step (b2) are cultured in a suspended state in a culture medium for sphere formation, and the spheres in which the sweat gland myoepithelial cells are exposed on the surface are Let it form.
- the medium for sphere formation include, for example, a medium containing an epidermal growth factor, a basic fibroblast growth factor, an artificial basement membrane matrix for cell culture, and a serum-free medium. It is not limited to When the sphere-forming medium is a medium containing an epidermal growth factor, the content of the epidermal growth factor in the sphere-forming medium varies depending on the type of skin tissue source and the like, and therefore can not be determined indiscriminately.
- the content of epidermal growth factor in the culture medium for sphere formation is usually preferably 0.01 ng / mL or more, from the viewpoint of causing cells to proliferate appropriately and differentiate properly.
- the concentration is preferably 1 ng / mL or more, and preferably 1 ⁇ g / mL or less, more preferably 100 ng / mL or less from the viewpoint of suppressing excessive proliferation and differentiation of cells.
- the sphere-forming medium is a medium containing a basic fibroblast growth factor, the content of the basic fibroblast growth factor in the sphere-forming medium varies depending on the type of skin tissue source etc.
- the content of the basic fibroblast growth factor in the culture medium for sphere formation is preferably from the viewpoint of appropriately proliferating cells and suppressing excessive differentiation of cells.
- the concentration is preferably 0.01 ng / mL or more, more preferably 1 ng / mL or more, and preferably 1 ⁇ g / mL or less, more preferably 100 ng / mL or less, from the viewpoint of suppressing excessive proliferation of the cells and appropriately differentiating the cells. is there.
- a serum-free medium for example, trade name of Stem Cell Technologies (Stem Cell Technologies): Complete Mammo Cult Human Medium, trade name of Thermo Fisher Scientific Co., Ltd .: Gibco (registered trademark) Keratinocyte-SFM, etc.
- the present invention is not limited to such examples.
- the culture conditions of the sweat gland cells are different depending on the type of the source of skin tissue and the like and can not be determined generally, it is preferable to appropriately determine according to the type of source of skin tissue and the like.
- the culture conditions of sweat gland cells include, for example, culture temperature, culture time, pH of culture medium, carbon dioxide concentration in culture atmosphere, and the like.
- the culture temperature is preferably 35 ° C. or higher, more preferably 36.5 ° C. or higher, from the viewpoint of maintaining the function and properties of sweat gland myoepithelial cells in vivo better.
- the temperature is 38 ° C. or less, more preferably 37.5 ° C.
- the culture temperature is usually preferably 35 to 38 ° C., more preferably 36.5 to 37.5 ° C., from the viewpoint of maintaining the function and properties of sweat gland myoepithelial cells in vivo better. It is.
- the culture time is different depending on the culture temperature and the like and can not be determined uniquely, so it is preferable to appropriately determine according to the culture temperature and the like.
- the culture time is preferably 60 hours or more, more preferably 144 hours or more from the viewpoint of maintaining the function and properties of sweat gland myoepithelial cells in vivo better, and in the same manner as described above, sweat gland myoepithelial cells in vivo Preferably, it is 672 hours or less, more preferably 168 hours or less from the viewpoint of maintaining the function and properties of Specifically, the culture time is usually preferably 60 to 672 hours, more preferably 144 to 168 hours, from the viewpoint of better maintaining the function and properties of the sweat gland myoepithelial cells in vivo.
- the pH of the culture medium is preferably 6.8 or more, more preferably 7., from the viewpoint of better maintaining the function and properties of sweat gland myoepithelial cells in vivo. From the viewpoint of maintaining the function and properties of the sweat gland myoepithelial cells in vivo better as described above, it is preferably at most 7.6, more preferably at most 7.4. Specifically, from the viewpoint of maintaining the function and properties of sweat gland myoepithelial cells in vivo better, the pH is usually preferably 6.8 to 7.6, more preferably 7.0 to 7.
- the carbon dioxide concentration in the culture atmosphere is preferably 4% by volume or more, more preferably 5% by volume or more, from the viewpoint of maintaining the function and properties of sweat gland myoepithelial cells in vivo better. From the viewpoint of better maintaining the function and properties of sweat gland myoepithelial cells in the body, it is preferably 10% by volume or less, more preferably 7% by volume or less. Specifically, the carbon dioxide concentration is generally preferably 4 to 10% by volume, more preferably 5 to 7% by volume, from the viewpoint of maintaining the function and properties of sweat gland myoepithelial cells in vivo better. is there.
- the “state in which the sweat gland cells are suspended in the medium” is not particularly limited as long as the sweat gland cells are not in contact with the wall surface of the culture vessel used for culturing the sweat gland cells.
- the culture vessel may be a vessel having on its inner surface a substance that inhibits the adhesion of sweat gland cells.
- step (I) introduction of the immortalizing gene into sweat gland myoepithelial cells is carried out while culturing the cell construct in a suspended state in a culture medium.
- the “state in which the cell structure is suspended in the medium” is not particularly limited as long as the cell structure is not in contact with the inner surface of the container used for culturing the cell structure.
- the container used for culturing the cell structure may be a container having a substance on its inner surface that inhibits adhesion of the cell structure.
- the medium for culturing the cell structure in a suspended state is a medium containing a nutrient component that allows the sweat gland myoepithelial cells to survive, gene transfer such as serum
- the medium may be a medium having a low content of the component that inhibits or may be a medium not containing the component.
- the suspension culture medium may be a low serum medium or a serum-free medium supplemented with a nutrient component, or a commercially readily available medium.
- nutritional components include amino acids, vitamins, inorganic salts, saccharides, cell growth promoting factors (eg, epidermal growth factor, basic fibroblast growth factor, hydrocortisone-21-hemisuccinate etc.), etc.
- the content of nutrient components in the suspension culture medium can not be determined indiscriminately because it varies depending on the type of low serum medium or serum-free medium, the type of nutrient component, etc. It is preferable to set appropriately according to the type of nutritional component and the like. These nutritional components may be used alone or in combination of two or more.
- the serum-free medium for example, trade name: Complete Mammo Cult Human Medium manufactured by Stem Cell Technologies, trade name: Thermo Fisher Scientific Co., Ltd .: Gibco (registered trademark) Keratinocyte-SFM, Thermo Fisher Scientific Although trade name manufactured by KK: Opti-MEM (registered trademark) I Reduced Serum Medium, etc.
- the present invention is not limited to such exemplification.
- the low serum medium a medium obtained by adding serum to the aforementioned serum-free medium can be used.
- the serum concentration of the low serum medium is preferably 0.01% by volume or more, more preferably 0.1% by volume or more, from the viewpoint of better maintaining the function and properties of sweat gland myoepithelial cells in vivo.
- it is preferably at most 0.5% by volume, more preferably at most 0.1% by volume, from the viewpoint of better maintaining the function and properties of sweat gland myoepithelial cells in vivo.
- the serum concentration is preferably 0.1 to 0.5% by volume, more preferably 0.01 to 0.1% by volume.
- Examples of methods for introducing immortalizing genes into sweat gland myoepithelial cells include methods using viral vectors, transfection methods and the like, but the present invention is not limited to such examples. Among these methods, a method using a viral vector is preferable because high gene transfer efficiency can be obtained by a simple operation. Therefore, in the step (I), it is preferable to introduce an immortalizing gene into the sweat gland myoepithelial cell via a viral vector.
- viral vectors examples include lentiviral vectors, retroviral vectors and the like, but the present invention is not limited to such examples.
- lentiviral vectors are preferred because they have high gene transfer efficiency to sweat gland myoepithelial cells and can stably transfer immortalizing genes.
- immortalizing genes include human telomerase reverse transcriptase (hTERT) gene, SV40 t (small t) antigen gene, SV40 T (large T) antigen gene, c-myc gene, papilloma virus E6 gene, papilloma virus E7 gene And the like, but the present invention is not limited to such examples.
- These immortalizing genes may be used alone or in combination of two or more.
- the surface of the sweat gland myoepithelial cell is a recombinant virus particle in which the viral vector into which the immortalizing gene has been incorporated is packaged.
- Immortalization genes can be introduced into sweat gland myoepithelial cells by infecting the exposed cell structures.
- a recombinant virus particle is cotransfected into a cell for cotransfection with a recombinant virus vector in which an immortalizing gene has been incorporated into a virus vector and a vector carrying a gene required for virus packaging, It can be prepared by a method of recovering virus particles and the like.
- the recombinant virus particles may be commercially readily available recombinant virus particles.
- the introduction of the immortalizing gene into cell structures can be carried out by contacting recombinant virus particles with cell structures in the presence of a gene transfer agent to infect them.
- Examples of co-transfection cells include, for example, 293T cells, but the present invention is not limited to such examples.
- Examples of the gene transfer adjuvant include polybrene and protamine, but the present invention is not limited to such examples.
- a method of contacting the cell structure with the recombinant virus particle for example, a method of adding the recombinant virus particle to the medium for virus infection containing the cell structure in a suspended state, including the cell structure and the recombinant virus particle
- the method etc. of mixing with the culture medium for virus infection etc. are mentioned, this invention is not limited only to this illustration.
- a medium for virus infection for example, a medium in which the serum-free medium is supplemented with hydrocortisone-21-hemisuccinate, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, glutamic acid, non-essential amino acid and the like, commercial Although the medium etc. which can be obtained easily are mentioned, this invention is not limited only to such an illustration.
- the number of sweat glands contained in the sweat gland-containing tissue per 100 ⁇ L of the mixture of the sweat gland-containing tissue and the recombinant virus particle is a gene
- the number is preferably 1 or more, more preferably 4 or more, from the viewpoint of improving the introduction efficiency to improve the production efficiency, and from the viewpoint of improving the gene introduction efficiency to improve the production efficiency as described above. 20 or less, more preferably 10 or less.
- the number of sweat glands contained in the sweat gland-containing tissue per 100 ⁇ L of the mixture is preferably 1 to 20, more preferably 4 to 10.
- the ratio of the number of infectious virus particles to the number of sweat glands is preferably 1 ⁇ 10 2 to 1 ⁇ 10 10 .
- the number of the spheres per 100 ⁇ L of the mixture of the spheres and the recombinant virus particles improves the gene transfer efficiency to improve the production efficiency From the viewpoint of reducing the number, preferably 1 or more, more preferably 4 or more, and from the viewpoint of improving gene transfer efficiency to improve production efficiency as described above, preferably 20 or less, more preferably 10 or less It is.
- the number of the spheres per 100 ⁇ L of the mixture is preferably 1 to 20, more preferably 4 to 10.
- the ratio of the number of infectious virus particles to the number of spheres is preferably 1 ⁇ 10 2 to 1 ⁇ 10 10 .
- step (II) the gene transfer product obtained in step (I) is cultured in the state of being suspended in a culture medium to obtain immortalized sweat gland myoepithelial cells.
- the medium used in step (II) is the same as the floating culture medium used in step (I).
- the culture conditions for the transgenic body in step (II) are the same as the culture conditions for sweat gland cells in the method for producing spheres.
- the gene transfer body obtained in step (I) includes tissues other than the sweat gland, and so on.
- a step of isolating transformed sweat gland myoepithelial cells can further be performed. Isolation of immortalized sweat gland myoepithelial cells from a gene transfer agent may be carried out, for example, by causing a cell dissociation reagent to act on the gene transfer body, applying a mechanical stimulus to the gene transfer body, a cell dissociation reagent and mechanical force. It can be performed by using in combination with stimulation.
- the cell dissociation reagent is the same as the cell dissociation reagent used in the step (b2) of the method for producing the sphere.
- mechanical stimulation includes stimulation by pipetting and the like, the present invention is not limited to such examples.
- the method of the present invention can further include the step of isolating immortalized sweat gland myoepithelial cells after step (II).
- the method for isolating immortalized sweat gland myoepithelial cells include cell sorting using a sweat gland myoepithelial cell specific marker as an index, but the present invention is not limited to such an example.
- the immortalized sweat gland myoepithelial cells obtained by the method of the present invention are characterized by the characteristics (i) to (iii) of sweat gland myoepithelial cells: (i) ⁇ -smooth muscle actin expression positive (ii) pancytokeratin expression positive and ATP1a1 positive expression, and (iii) It can be identified by examining the ability to form spheres after 5 or more passages.
- the presence or absence of the expression of each of ⁇ -smooth muscle actin, pancytokeratin and ATP1a1 can be confirmed by, for example, fluorescence immunocell staining, real-time RT-PCR, and the like.
- the ability to form spheres can be confirmed by the same method as the method for producing spheres described above.
- the immortalized myoepithelial cell of the present invention it is possible to proliferate cells having the same function and property as in the sweat gland myoepithelial cell in vivo and having the function and the property over a long period of time it can. Further, according to the method for producing immortalized myoepithelial cells of the present invention, the immortalized sweat gland myoepithelial cells of the present invention can be obtained with high production efficiency. Therefore, it is expected that the immortalized myoepithelial cells of the present invention and the method for producing immortalized myoepithelial cells of the present invention can be used for the development of external preparations such as antiperspirants, deodorants and the like, agents for improving sweat gland function, etc. It is expected that the immortalized myoepithelial cells of the present invention and the method for producing immortalized myoepithelial cells of the present invention can be used for the development of external preparations such as antiperspirants, deodorants and the like, agents for improving sweat gland function, etc. It is expected that the immortalized
- Basal medium (Stem Cell Technologies, Inc., trade name: Complete Mammo Cult Human Medium), hydrocortisone-21-hemisuccinate, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, heparin and penicillin / streptomycin mixed Solution (penicillin concentration 10000 units / mL, streptomycin concentration 10000 ⁇ g / mL) at a concentration of 10.5 ⁇ g / mL (hydrocortisone-21-hemisuccinate), 10 ng / mL (recombinant human epidermal growth factor), 10 ng / mL
- the medium (I) was obtained by adding them to a human fibroblast growth factor, 4 ⁇ g / mL (heparin) and 100 ⁇ g / mL (penicillin / streptomycin mixed solution).
- Basal medium (Stem Cell Technologies, Inc., trade name: Complete Mammo Cult Human Medium), hydrocortisone-21-hemisuccinate, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, heparin and penicillin / streptomycin mixed Artificial basement membrane matrix for solution and cell culture (Corning Inc., trade name: Growth Factor Reduced Matrigel Matrix), each concentration 10.5 ⁇ g / mL (hydrocortisone-21-hemisuccinate), 10 ng / ML (recombinant human epidermal growth factor), 10 ng / mL (recombinant human basic fibroblast growth factor), 4 ⁇ g / mL (heparin), 100 ⁇ g / mL (peni
- the medium (II) was obtained by adding it to a mixture of syrin / streptomycin and 2% by volume (artificial basement membrane matrix for cell culture).
- Reference Example 1 (1) Production of Dissected Sweat Gland Cells As skin tissue, immediately after excision from a living body (a 68-year-old human), it was stored refrigerated at 4 ° C., and eyelid skin tissue within 48 hours after excision was used. The sweat glands in the skin tissue were incorporated with neutral red by immersing the skin tissue in PBS containing 10 ⁇ M neutral red. Next, a tissue piece including a sweat gland was separated from the skin tissue using a forceps and scissors under a light microscope. The separated tissue pieces were collected in sterile PBS in 15 mL volumetric tubes. After gently shaking the PBS containing the tissue piece, the tissue piece was washed by centrifuging the PBS at 350 ⁇ g and 4 ° C. for 5 minutes to remove the supernatant.
- the medium (I) in the tube and the tissue fragments after removal of collagen fibers were transferred to a dish of 10 cm in diameter.
- the tissue pieces on the dish were collected using a pipette under a light microscope. Collected tissue pieces were collected in sterile PBS in 15 mL volumetric tubes. After gently shaking the PBS containing the tissue piece, the tissue piece was washed by centrifuging the PBS at 350 ⁇ g and 4 ° C. for 5 minutes to remove the supernatant.
- the washed tissue pieces and 1 mL of the 0.5 mass% trypsin-EDTA solution obtained in Production Example 4 were mixed in a 15 mL volumetric tube.
- the sweat gland cells constituting the sweat glands were dissociated from each other to obtain sweat gland cells in a dissociated state (hereinafter also referred to as "dissociated sweat gland cells").
- the mixture containing the dissociated sweat gland cells in the tube is passed through a cell strainer (mesh size: 40 ⁇ m, manufactured by Corning, trade name: Falcon (registered trademark) 40 ⁇ m cell strainer, blue, sterile, individual packaging). By removing the aggregated cells, a suspension of dissociated sweat gland cells was obtained.
- a cell strainer mesh size: 40 ⁇ m, manufactured by Corning, trade name: Falcon (registered trademark) 40 ⁇ m cell strainer, blue, sterile, individual packaging.
- the obtained mixed solution was placed in a low adhesion plate (manufactured by Corning, trade name: ultra low adhesion plate 24 wells).
- the dissociated sweat gland cells were incubated in a 5% by volume carbon dioxide atmosphere at 37 ° C. while suspended in the medium (II) in the plate.
- the spheres were transferred to a 15 mL volumetric tube.
- the spheres were subjected to centrifugation at 350 ⁇ g and 4 ° C. for 5 minutes to remove liquid components.
- 1 mL of a solution for cell recovery (manufactured by Corning, trade name: Cell Recovery Solution) was mixed with the sphere in the tube to obtain a sphere-containing liquid.
- the tube containing the obtained sphere-containing solution was allowed to stand on ice for 1 to 2 hours.
- Virus infection GFP recombinant virus particle solution (manufactured by Applied Biological Materials, trade name: GFP Control Lentivirus, GFP recombinant virus particle concentration: 1 ⁇ 10 6 U / mL) Concentrated according to polyethylene glycol precipitation method. The resulting concentrate was diluted with the above virus infection culture medium to a GFP recombinant virus particle concentration of 1 ⁇ 10 8 U / mL to obtain a GFP recombinant virus dilution solution.
- the sphere-containing solution obtained in the above (2) "sphere culture” was mixed with 9 mL of PBS. Next, the resulting mixture was subjected to centrifugation at 350 ⁇ g and 4 ° C. for 5 minutes to remove the supernatant. Four to 10 spheres after centrifugation were mixed with 90 ⁇ L of the medium for virus infection obtained in Production Example 3. 10 ⁇ L of the GFP recombinant virus dilution solution was added to the obtained mixed solution to obtain a sphere-virus mixed solution.
- the GFP gene was introduced into the cells constituting the spheres by infecting the sweat gland cells constituting the spheres with the recombinant virus by incubating the sphere-virus mixture in a 5% by volume carbon dioxide atmosphere at 37 ° C. Virus-infected sweat gland spheres were collected 24 hours after the start of virus infection.
- Reference Example 2 The dissociated sweat gland cells obtained in (1) “Production of sweat gland cells in a dissociated state” in Reference Example 1 were mixed with 9 mL of PBS. Next, the resulting mixture was subjected to centrifugation at 350 ⁇ g and 4 ° C. for 5 minutes to remove the supernatant. 5 ⁇ 10 2 to 1 ⁇ 10 5 dissociated sweat gland cells after centrifugation and 90 ⁇ L of the medium for virus infection obtained in Production Example 3 were mixed. To the resulting mixture, 10 ⁇ l of the diluted GFP recombinant virus was added to obtain a sphere-virus mixture.
- the GFP gene was introduced into the dissociated sweat gland cells by incubating the dissociated sweat gland cells with the recombinant virus by incubating the sphere-virus mixture in a 5% by volume carbon dioxide atmosphere at 37 ° C. Virus-infected sweat gland cells were collected 24 hours after the start of viral infection.
- Test Example 1 The fluorescence based on the virus-infected sweat gland sphere obtained in Reference Example 1 and the virus-infected sweat gland cells obtained in Reference Example 2 was observed under a fluorescence microscope. Also, the virus-infected sweat gland sphere obtained in Reference Example 1 and the virus-infected sweat gland cells obtained in Reference Example 2 were observed under a confocal microscope.
- FIG. 1 (A) The results of observing the GFP-based fluorescence in the virus-infected sweat gland sphere obtained in Reference Example 1 under a fluorescence microscope are shown in FIG. 1 (A), the fluorescence-based fluorescence in the virus-infected sweat gland cells obtained in Reference Example 2
- Fig. 1 (B) The results observed under Fig. 1 (B), the virus-infected sweat gland sphere obtained in Reference Example 1 under a confocal microscope are shown in Fig. 1 (C), the virus-infected sweat gland cells obtained in Reference Example 2
- FIG. 1 (D) The result of observation under a confocal microscope is shown in FIG. 1 (D).
- the scale bar indicates 153 ⁇ m.
- arrowheads in FIG. 1 (A) indicate virus-infected sweat gland spheres
- arrowheads in FIG. 1 (B) indicate virus-infected sweat gland cells.
- Example 1 The sphere-containing solution obtained in (2) "sphere culture” of Reference Example 1 was mixed with 9 mL of PBS. Next, the resulting mixture was subjected to centrifugation at 350 ⁇ g and 4 ° C. for 5 minutes to remove the supernatant. Four to 10 spheres after centrifugation were mixed with 100 ⁇ L of the medium for virus infection obtained in Production Example 3.
- hTERT recombinant virus particle solution (trade name of Hightiter Lentivirus containing hTERT, hTERT recombinant virus particle concentration: 1 ⁇ 10 9 U / mL) manufactured by Applied Biological Materials
- SV40 Tt recombinant virus particle solution (trade name of Applied Biological Materials: SV40 large and small T antigens, SV40 Tt recombinant virus particle concentration: 1 ⁇ 10 9 U / mL) 0.5 ⁇ L
- SV40 Tt recombinant virus particle solution (trade name of Applied Biological Materials: SV40 large and small T antigens, SV40 Tt recombinant virus particle concentration: 1 ⁇ 10 9 U / mL)
- the immortalization gene was introduced into the cells constituting the spheres by incubating the sweat gland cells constituting the spheres with the recombinant lentivirus by incubating the sphere-virus mixture in a 5% by volume carbon dioxide atmosphere at 37 ° C. .
- Virus-infected sweat gland spheres were collected 24 hours after the start of virus infection.
- Comparative Example 1 Spheres were obtained by performing the same operation as (1) “production of sweat gland cells in the dissociation state” in Reference Example 1 and “sphere culture” in (2).
- Test example 2 (1) Sphere Passaging Culture Conducting the following (1-1) and (1-2) was defined as "one passaging culture”.
- Example 1 Production of Dissociated Sweat Gland Cells
- the virus-infected sweat gland sphere obtained in Example 1 and 1 mL of the 0.5% by mass trypsin-EDTA solution obtained in Production Example 4 were mixed in a 15 mL volumetric tube.
- the sweat gland cells constituting the sweat glands were dissociated from each other by stirring the sweat glands in the tube for 3 minutes using a pipette to obtain dissociated sweat gland cells.
- the mixture containing the dissociated sweat gland cells in the tube is aggregated through a cell strainer (mesh size: 40 ⁇ m, manufactured by Corning, trade name: Falcon® 40 ⁇ m cell strainer, blue, sterile, individual packaging). By removing the treated cells, a suspension of dissociated sweat gland cells was obtained.
- a cell strainer (mesh size: 40 ⁇ m, manufactured by Corning, trade name: Falcon® 40 ⁇ m cell strainer, blue, sterile, individual packaging).
- the spheres were transferred to a 15 mL volumetric tube.
- the spheres were subjected to centrifugation at 350 ⁇ g and 4 ° C. for 5 minutes to remove liquid components.
- 1 mL of a solution for cell recovery (manufactured by Corning, trade name: Cell Recovery Solution) was mixed with the sphere in the tube to obtain a sphere-containing liquid.
- the tube containing the obtained sphere-containing solution was allowed to stand on ice for 1 to 2 hours.
- FIG. 2 (A) The relationship between sphere forming ability and passage number of sweat gland cells contained in virus-infected sweat gland spheres obtained in Example 1 is shown in FIG. 2 (A).
- Sphere formation of dissociated sweat gland cells obtained in Comparative Example 1 The result of examining the relationship between Noh and passage number is shown in FIG. 2 (B).
- arrowheads indicate spheres.
- Example 1 the sweat gland cells contained in the virus-infected sweat gland sphere obtained in Example 1 form a sphere even when passaged 20 times or more.
- immortalized sweat gland cells can be obtained by infecting sweat gland cells contained in the sphere with a lentivirus having an immortalization gene in a state where the spheres are suspended in the culture medium.
- fluorescence intensity A the intensity of fluorescence based on pancytokeratin in the dissociated sweat gland cells after immunostaining
- fluorescence intensity B the intensity of fluorescence based on ⁇ -SMA
- the pancytokeratin expression level in the cells to be evaluated was calculated.
- the fluorescence intensity B in the dissociated sweat gland cells obtained in Comparative Example 1 was decreased from the fluorescence intensity B in the cells to be evaluated to determine the ⁇ -SMA expression amount in the cells to be evaluated.
- FIG. 3 (A) The results of examining the relationship between the cell type and the amount of pancytokeratin expression are shown in FIG. 3 (A), and the results of examining the relationship between the cell type and the amount of ⁇ -SMA expression are shown in FIG. 3 (B).
- lane 1 represents the pancytokeratin expression level in immortalized sweat gland cells contained in the virus-infected sweat gland sphere obtained in Example 1
- lane 2 represents bread in the dissociated sweat gland cells obtained in Comparative Example 1. It shows cytokeratin expression level.
- lane 1 represents the ⁇ -SMA expression amount in immortalized sweat gland cells contained in the virus-infected sweat gland sphere obtained in Example 1
- lane 2 represents ⁇ in the dissociated sweat gland cells obtained in Comparative Example 1. -Shows SMA expression level.
- the immortalized sweat gland cells contained in the virus-infected sweat gland sphere obtained in Example 1 are pancytokeratin, which is a myoepithelial cell marker. It can be seen that both E. coli and ⁇ -SMA are expressed. From these results, it can be understood that the immortalized sweat gland cells are immortalized sweat gland myoepithelial cells. Therefore, it is understood that immortalized sweat gland myoepithelial cells can be obtained by infecting the sweat gland cells contained in the sphere with the virus in a state where the spheres are suspended in the culture medium.
- DNase / RNase-free purified water manufactured by invitrogen, trade name: UltraPure DNase / RNase-Free Distilled Water
- concentration of the obtained total RNA was 1 ⁇ g / ⁇ L.
- a reverse transcription kit [QIAGEN, trade name: Quatitect Reverse Transcription Kit]
- a PCR kit manufactured by Toyobo Co., Ltd., trade name: THUNDERBIRD SYBR qPCR Mix
- a real-time PCR device manufactured by Applied Biosystems, trade name: ViiA7
- Ct B value The number of cycles (hereinafter referred to as "Ct B value") until the amount of nucleotide synthesis with the template as a template reaches a threshold was measured.
- the thermal profile in real-time RT-PCR method is 40 cycles of 1 cycle of treatment at 95 ° C followed by denaturation at 95 ° C for 5 seconds, annealing at 55 ° C for 10 seconds and elongation at 72 ° C for 20 seconds. It is a reaction.
- Example 1 the expression value of the ATP1a1 gene in immortalized sweat gland myoepithelial cells contained in the virus-infected sweat gland sphere obtained in Example 1 was determined.
- the corrected expression value of the ATP1a1 gene in the immortalized sweat gland myoepithelial cells was determined. Next, based on the corrected expression value, it was evaluated whether the immortalized sweat gland myoepithelial cell expresses the ATP1a1 gene according to the following evaluation criteria. ⁇ Evaluation criteria> "Immortalized sweat gland myoepithelial cells express the ATP1a1 gene” The corrected expression value is a “positive value”. "Immortalized sweat gland myoepithelial cells do not express the ATP1a1 gene” The corrected expression value is “0” or “negative value”.
- ATP1a1 is one of sweat gland myoepithelial cell markers. Therefore, it can be seen that the immortalized sweat gland myoepithelial cells contained in the virus-infected sweat gland sphere obtained in Example 1 express sweat gland myoepithelial cell markers.
- Example 2 (1) Production of sweat gland-containing tissue As skin tissue, immediately after excision from a living body (a 41-year-old human), it was stored refrigerated at 4 ° C., and skin tissue of the eyelid within 48 hours after excision was used. The sweat glands in the skin tissue were incorporated with neutral red by soaking the skin tissue in PBS containing 10 ⁇ M neutral red. Next, a tissue piece including a sweat gland was separated from the skin tissue using a forceps and scissors under a light microscope. The separated tissue pieces were collected in sterile PBS in 15 mL volumetric tubes. After gently shaking the PBS containing the tissue piece, the tissue piece was washed by centrifuging the PBS at 350 ⁇ g and 4 ° C. for 5 minutes to remove the supernatant.
- the medium (I) in the tube and the tissue fragments after removal of collagen fibers were transferred to a dish of 10 cm in diameter.
- the tissue pieces on the dish were collected using a pipette under a light microscope. Collected tissue pieces were collected in sterile PBS in 15 mL volumetric tubes. After gently shaking the PBS containing the tissue piece, the tissue piece was washed by centrifuging the PBS at 350 ⁇ g and 4 ° C. for 5 minutes to remove the supernatant.
- the washed tissue pieces and 10 mL of medium (I) were mixed in a 15 mL volumetric tube.
- To the resulting mixture was added dispase to a concentration of 1 U / mL.
- the obtained mixture was transferred to a dish of 10 cm in diameter, and then the mixture was incubated and incubated in a 5% by volume carbon dioxide atmosphere at 37 ° C. After 8 hours from the start of the incubation, 10 mL of a 2% by mass FBS / PBS solution was added to the mixture to dilute the mixture to stop the enzyme reaction.
- tissue pieces were collected in sterile PBS in 15 mL volumetric tubes. The PBS containing the tissue pieces was gently shaken, and then the PBS was subjected to centrifugation at 350 ⁇ g and 4 ° C. for 5 minutes to remove the supernatant to obtain a sweat gland-containing tissue.
- Virus infection hTERT recombinant virus particle solution and SV40 Tt recombinant virus particle solution in the medium for virus infection hTERT recombinant virus particle concentration is 1 ⁇ 10 8 U / mL and SV40 Tt recombinant virus particle concentration is 1 ⁇ It was diluted to 10 8 U / mL to obtain an immortalized gene-containing recombinant virus dilution.
- the sweat gland-containing tissue obtained in (1) "Production of sweat gland-containing tissue” was mixed with 9 mL of PBS. Next, the resulting mixture was subjected to centrifugation at 350 ⁇ g and 4 ° C. for 5 minutes to remove the supernatant.
- the sweat gland-containing tissue containing 4 to 10 sweat glands after centrifugation was mixed with 100 ⁇ L of the medium for virus infection obtained in Production Example 3.
- To the resulting mixture was added 2 ⁇ L of the immortalized gene-containing recombinant virus dilution to obtain a tissue-virus mixture.
- the tissue-virus mixture was incubated at 37 ° C. in a 5% by volume carbon dioxide atmosphere to infect the sweat gland cells constituting the sweat gland with the recombinant lentivirus.
- the sweat gland cells are infected with the recombinant virus by containing the sweat gland cells in the sweat gland-containing tissue.
- the immortalizing gene was introduced. After 33 hours from the start of viral infection, virus-infected tissue was recovered.
- Test Example 3 In Test Example 2 (1) “Sphere Subculture”, except that the virus-infected tissue obtained in Example 2 was used instead of using the virus-infected sweat gland sphere obtained in Example 1. The same procedure as (1) "sphere passage culture” was carried out, and sphere passage culture was performed.
- FIG. 4 (A) The relationship between the sphere forming ability of the sweat gland cells contained in the virus-infected tissue obtained in Example 2 and the passage number is shown in FIG. 4 (A), and the sphere forming ability of the dissociated sweat gland cells obtained in Comparative Example 1
- FIG. 4 (B) The result of examining the relationship between and the passage number is shown in FIG. 4 (B).
- arrowheads indicate spheres.
- the sweat gland cells contained in the virus-infected tissue obtained in Example 2 can form a sphere even after the seventh passaging, while in Comparative Example 1, they can form spheres. It can be seen that the resulting dissociated sweat gland cells can not form spheres after the fourth passage.
- the sweat gland cells contained in the virus-infected tissue obtained in Example 2 were confirmed to form spheres even when passaged 12 times or more. Further, when the presence or absence of expression of pancytokeratin and ⁇ -SMA in sweat gland cells contained in the virus-infected tissue obtained in Example 2 was examined, it was confirmed that pancytokeratin and ⁇ -SMA were expressed.
- immortalized sweat gland myoepithelial cells can be obtained by infecting the sweat gland myoepithelial cells contained in the sweat gland-containing tissue with the sweat gland-containing tissue suspended in the medium.
- Example 1 Production of dissociated sweat gland cells
- skin tissue of a 20-year-old human eyelid is used instead of skin tissue of a 68-year-old human eyelid (Reference Example 3) Skin tissue of a 71-year-old human eyelid (Reference Example 4), Skin tissue of a 74-year-old human eyelid (Reference Example 5), 51-year-old human abdominal skin tissue (Reference Example 6) or 55
- An operation was performed in the same manner as (1) "Production of sweat gland cells in a dissociated state” in Reference Example 1 except that skin tissue of the abdomen of a year-old human (Reference Example 7) was used to obtain dissociated sweat gland cells.
- (2) Sphere culture in the (2) "sphere culture” of the reference example 1 reference examples 3 to 7 are used instead of using the dissociative sweat gland cells obtained in (1) "production of the sweat gland cells in the dissociation state" of the reference example 1.
- the same procedure as (2) "sphere culture” in Reference Example 1 was carried out except using each of the dissociated sweat gland cells obtained in (1) "production of dissociated sweat gland cells", to obtain a sphere-containing solution. .
- Example 1 the sphere-containing liquid (Example 3) obtained in Reference Example 3 instead of using the sphere-containing liquid obtained in (2) "sphere culture” of Reference Example 1; Sphere-containing solution (Example 4), Sphere-containing solution obtained in Reference Example 5 (Example 5), Sphere-containing solution obtained in Reference Example 6 (Example 6) or Sphere-containing solution obtained in Reference Example 7
- the same procedure as in Example 1 was carried out except using the liquid (Example 7) to obtain a virus-infected sweat gland sphere.
- Comparative Examples 3 to 7 In (1) "Production of sweat gland cells in the disaggregated state" of Reference Example 1, skin tissue of a 20-year-old human eyelid (Comparative Example 3), 71-year-old human instead of using skin tissue of a 68-year-old human eyelid Skin tissue of the eyelids of the present invention (comparative example 4), skin tissue of eyelids of the 74-year-old human (comparative example 5), abdominal skin tissue of the 51-year-old human (comparative example 6) or 55-year-old human abdominal skin tissue An operation similar to (1) “Production of sweat gland cells in a dissociated state” in Comparative Example 1 was performed except that (Comparative Example 7) was used, to obtain dissociated sweat gland cells.
- Test Example 4 In (1) “sphere passage culture” of Test Example 2, a test was conducted except that the virus-infected tissue obtained in Examples 3 to 7 was used instead of using the virus-infected sweat gland sphere obtained in Example 1. The same operation as (1) "sphere passage culture” of Example 2 was performed, and sphere passage culture was performed.
- the spheres used in Examples 1 and 3 to 7 and the sweat gland-containing tissue used in Example 2 both have a structure in which sweat gland myoepithelial cells are exposed on the surface. Therefore, by culturing a cell structure in which sweat gland myoepithelial cells are exposed on the surface in a suspended state in a culture medium, an immortalizing gene is obtained by infecting the cell structure with a virus having the immortalization gene. It can be seen that it can be introduced into myoepithelial cells.
- Test Example 5 (1) Sphere Passaging Culture Conducting the following (1-1) and (1-2) was defined as "one passaging culture”.
- the mixture containing the dissociated sweat gland cells in the tube is aggregated through a cell strainer (mesh size: 40 ⁇ m, manufactured by Corning, trade name: Falcon® 40 ⁇ m cell strainer, blue, sterile, individual packaging). By removing the treated cells, a suspension of dissociated sweat gland cells was obtained.
- a cell strainer (mesh size: 40 ⁇ m, manufactured by Corning, trade name: Falcon® 40 ⁇ m cell strainer, blue, sterile, individual packaging).
- the obtained dissociated sweat gland cells were added to the medium (II) obtained in Production Example 2 so as to be 2.5 ⁇ 10 3 cells / mL, to obtain a mixed solution containing the dissociated sweat gland cells.
- the obtained mixed solution was placed in a low adhesion plate (manufactured by Corning, trade name: ultra low adhesion plate 24 wells).
- the dissociated sweat gland cells were incubated in a 5% by volume carbon dioxide atmosphere at 37 ° C. while suspended in the medium (II) in the plate.
- the spheres were transferred to a 15 mL volumetric tube.
- the spheres were subjected to centrifugation at 350 ⁇ g and 4 ° C. for 5 minutes to remove liquid components.
- 1 mL of a solution for cell recovery (manufactured by Corning, trade name: Cell Recovery Solution) was mixed with the sphere in the tube to obtain a sphere-containing liquid.
- the tube containing the obtained sphere-containing solution was allowed to stand on ice for 1 to 2 hours.
- Test Example 6 (1) Production of dissociated sweat gland cells In (1-1) “Production of dissociated sweat gland cells” in Test Example 2, the sphere obtained in Example 3 instead of using the virus-infected sweat gland sphere obtained in Example 1 ( Except for using the experiment No. 1), the sphere obtained in Example 4 (Experiment No. 2) or the sphere obtained in Example 5 (Experiment No. 3), (1-1) “Dissociation of Test Example 2” The same procedure as in "Production of sweat gland cells” was performed to obtain dissociated sweat gland cells. In the above, the sphere obtained at Example 3 has a sphere of 5 passages, the sphere obtained in Example 4 has a sphere of 8 times, and the sphere obtained in Example 5 has a sphere of 14 times. The sphere of
- Test Example 6 (2) Sphere Formation In Test Example 2 (1-2) “Sphere Culture”, Test Example 6 is used instead of using the dissociative sweat gland cells obtained in (1-1) Test body 2 (Produced dissociated sweat gland cells). The same procedure as (1-2) "sphere culture” in Test Example 2 was performed except that the thawed cells obtained in (2) “freeze storage of dissociated sweat gland cells” were used, and experiment numbers 1 to 3 were used. The sphere-containing solution A was obtained.
- Example 6 is the same as (1-2) "sphere culture” in Test Example 2 except that the dissociative sweat gland cells (Experiment No. 3) obtained in (3-1) "production of dissociated sweat gland cells” in Example 6 are used. The operation was performed to obtain Sphere-containing solution B of Experiment No. 3.
- Spheres contained in the sphere-containing solution A of Experiment No. 1 instead of using the virus-infected sweat gland spheres obtained in Example 1 in (1-1) “Production of dissociated sweat gland cells” in Test Example 2, Experiment No. 2 Except that the spheres contained in the sphere-containing liquid A and the spheres contained in the sphere-containing liquid B of the experiment No. 3 (spheres of the experiment Nos. 1 to 3) were used, The same operation as in "Production of” was performed to obtain subcultured cells. The number of obtained subcultured cells (hereinafter referred to as "cell number D”) was measured.
- immortalization is achieved by introducing an immortalizing gene into the sweat gland myoepithelial cell while culturing the cell structure in which the sweat gland myoepithelial cell is exposed on the surface in a suspended state in the medium. It can be seen that sweat gland myoepithelial cells are obtained.
- immortalized sweat gland myoepithelial cells obtained by performing the above-mentioned operation have the same function and properties as sweat gland myoepithelial cells in vivo, and proliferate cells having the function and nature for a long period of time.
- the method for producing immortalized sweat gland myoepithelial cells and immortalized sweat gland myoepithelial cells of the present invention is expected to be used for the development of external preparations such as antiperspirants and deodorants, agents for improving sweat gland function, etc. It is expected to be used for the development of external preparations such as antiperspirants and deodorants, agents for improving sweat gland function, etc. It is possible to be used for the development of external preparations such as antiperspirants and deodorants, agents for improving sweat gland function, etc. It is expected to be used for the development of external preparations such as antiperspirants and deodorants, agents for improving sweat gland function, etc. It is expected to be used for the development of external preparations such as antiperspirants and deodorants, agents for improving sweat gland function, etc. It is expected to be used for the development of external preparations such as antiperspirants and deodorants, agents for improving sweat gland function, etc. It is expected to be used for the development of external preparations such as antiperspirants and deodorants, agents for
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Abstract
Description
(1)α-平滑筋アクチンおよびパンサイトケラチンを発現し、少なくとも5回の継代後にスフィア形成能を有していることを特徴とする不死化汗腺筋上皮細胞、
(2)ATP1a1をさらに発現している前記(1)に記載の不死化汗腺筋上皮細胞、
(3)不死化汗腺筋上皮細胞を製造する方法であって、
(I)汗腺筋上皮細胞が表面に露出している細胞構造体を培地中に浮遊させた状態で培養しながら、不死化遺伝子を当該汗腺筋上皮細胞に導入して遺伝子導入体を得る工程、および
(II)前記工程(I)で得られた遺伝子導入体を培地中に浮遊させた状態で培養して不死化汗腺筋上皮細胞を得る工程
を含む不死化汗腺筋上皮細胞の製造方法、
(4)前記工程(I)において、ウイルスベクターを介して不死化遺伝子を前記汗腺筋上皮細胞に導入する(3)に記載の不死化汗腺筋上皮細胞の製造方法、
(5)前記工程(I)を行なう前に、採取された皮膚組織から少なくとも膠原繊維の全部または一部を除去して汗腺含有組織を得る工程をさらに含み、前記工程(I)において、前記細胞構造体として前記汗腺含有組織を用いる前記(3)または(4)に記載の不死化汗腺筋上皮細胞の製造方法、ならびに
(6)前記工程(I)を行なう前に、汗腺細胞を培地中に浮遊させた状態で培養し、汗腺筋上皮細胞が表面に露出しているスフィアを形成させる工程をさらに含み、前記工程(I)において、前記細胞構造体として前記スフィアを用いる前記(3)または(4)に記載の不死化汗腺筋上皮細胞の製造方法
に関する。
(A)本発明の不死化汗腺筋上皮細胞を被験物質の非存在下で培地中に浮遊させた状態で培養して細胞培養物を得るステップ、
(B)本発明の不死化汗腺筋上皮細胞を被験物質の存在下で培地中に浮遊させた状態で培養して細胞培養物を得るステップ、および
(C)前記ステップ(A)で得られた細胞培養物(A)における分化マーカーの発現プロファイルと前記ステップ(B)で得られた細胞培養物(B)における分化マーカーの発現プロファイルとを調べ、前記細胞培養物(A)における分化マーカーの発現プロファイルと前記細胞培養物(B)における分化マーカーの発現プロファイルとの違いに基づき、前記被験物質が有する汗腺筋上皮細胞の分化調節作用を評価するステップ
を含む。本評価方法により、被験物質が汗腺筋上皮細胞の分化を促進する作用を有することが確認された場合、当該被験物質は、例えば、汗腺筋上皮細胞の機能の異常亢進に起因する状態の改善に用いられることが期待される。また、本評価方法により、被験物質が汗腺筋上皮細胞の分化を抑制する作用を有することが確認された場合、当該被験物質は、例えば、汗腺筋上皮細胞の機能不全に起因する状態の改善に用いられることが期待される。
(I)汗腺筋上皮細胞が表面に露出している細胞構造体を培地中に浮遊させた状態で培養しながら、不死化遺伝子を当該汗腺筋上皮細胞に導入して遺伝子導入体を得る工程、および
(II)前記工程(I)で得られた遺伝子導入体を培地中に浮遊させた状態で培養して不死化汗腺筋上皮細胞を得る工程
を含むことを特徴とする(以下、「本発明の方法」ともいう)。
(a1)採取された皮膚組織から汗腺を含む組織片を分離する工程、および
(a2)工程(a1)で得られた組織片から膠原繊維などを除去して汗腺筋上皮細胞が表面に露出している汗腺含有組織を得る工程
を含む方法などが挙げられるが、本発明は、かかる例示のみに限定されるものではない。
(b1)採取された皮膚組織から汗腺を含む組織片を分離する工程、
(b2)工程(b1)で得られた組織片から解離状態の汗腺細胞を得る工程、および
(b3)工程(b2)で得られた汗腺細胞を培地中に浮遊させた状態で培養し、汗腺筋上皮細胞が表面に露出しているスフィアを形成させる工程
を含む方法などが挙げられるが、本発明は、かかる例示のみに限定されるものではない。
(ii)パンサイトケラチン発現陽性およびATP1a1発現陽性を示すこと、および
(iii)5回以上の継代後にスフィア形成能を有すること
を調べることによって同定することができる。α-平滑筋アクチン、パンサイトケラチンおよびATP1a1それぞれの発現の有無は、例えば、蛍光免疫細胞染色法、リアルタイムRT-PCR法などによって確認することができる。スフィア形成能は、前述のスフィアの製造方法と同様の方法によって確認することができる。
<略語および用語の説明>
EDTA:エチレンジアミン四酢酸
FBS:ウシ胎児血清
GAPDH:グリセルアルデヒド3リン酸脱水素酵素
GFP:緑色蛍光タンパク質
GFP組換ウイルス:GFP遺伝子を保持する組換レンチウイルス
hTERT組換ウイルス:hTERT遺伝子を保持する組換レンチウイルス
PBS:リン酸緩衝生理食塩水
SV40Tt組換ウイルス:SV40t抗原遺伝子およびSV40T抗原遺伝子を保持する組換レンチウイルス
α-SMA: α-平滑筋アクチン
基礎培地〔ステム・セル・テクノロジーズ社製、商品名:Complete MammoCult HumanMedium〕に、ヒドロコルチゾン-21-ヘミスクシナート、組換ヒト上皮増殖因子、組換ヒト塩基性線維芽細胞増殖因子、ヘパリンおよびペニシリン/ストレプトマイシン混合液〔ペニシリン濃度10000ユニット/mL、ストレプトマイシン濃度10000μg/mL〕をそれぞれの濃度が10.5μg/mL(ヒドロコルチゾン-21-ヘミスクシナート)、10ng/mL(組換ヒト上皮増殖因子)、10ng/mL(組換ヒト塩基性線維芽細胞増殖因子)、4μg/mL(ヘパリン)および100μg/mL(ペニシリン/ストレプトマイシン混合液)となるように添加して培地(I)を得た。
基礎培地〔ステム・セル・テクノロジーズ社製、商品名:Complete MammoCult HumanMedium〕に、ヒドロコルチゾン-21-ヘミスクシナート、組換ヒト上皮増殖因子、組換ヒト塩基性線維芽細胞増殖因子、ヘパリンおよびペニシリン/ストレプトマイシン混合液および細胞培養用人工基底膜マトリックス〔コーニング・インコーポレーティッド(Corning Inc.)製、商品名:Growth Factor Reduced Matrigel Matrix〕をそれぞれの濃度が10.5μg/mL(ヒドロコルチゾン-21-ヘミスクシナート)、10ng/mL(組換ヒト上皮増殖因子)、10ng/mL(組換ヒト塩基性線維芽細胞増殖因子)、4μg/mL(ヘパリン)、100μg/mL(ペニシリン/ストレプトマイシン混合液)および2体積%(細胞培養用人工基底膜マトリックス)となるように添加して培地(II)を得た。
基礎培地〔ステム・セル・テクノロジーズ社製、商品名:Complete MammoCult HumanMedium〕に、ヒドロコルチゾン-21-ヘミスクシナート、組換ヒト上皮増殖因子および組換ヒト塩基性線維芽細胞増殖因子をそれぞれの濃度が10.5μg/mL(ヒドロコルチゾン-21-ヘミスクシナート)、10ng/mL(組換ヒト上皮増殖因子)および10ng/mL(組換ヒト塩基性線維芽細胞増殖因子)となるように添加して培地(III)を得た。ポリブレンをその濃度が10μg/mLとなるように培地(III)に添加してウイルス感染用培地を得た。
トリプシン溶液〔サーモフィッシャーサイエンティフィック株式会社製、商品名:2.5% Trypsin (10×)、no Phenol Red〕、ダルベッコPBS〔サーモフィッシャーサイエンティフィック株式会社製、商品名:DPBS、no calcium、no magnesium〕、およびEDTA溶液〔ニッポンジーン社製、商品名:0.5M EDTA〕を混合することで、0.5質量%トリプシン-EDTA溶液を得た。
粉末状のディスパーゼ〔サーモフィッシャーサイエンティフィック株式会社製、商品名:Dispase II,powder〕をダルベッコPBS〔サーモフィッシャーサイエンティフィック株式会社製、商品名:DPBS,no calcium,no magnesium〕に溶解させることで、5U/mLディスパーゼ液を得た。
(1)解離状態の汗腺細胞の製造
皮膚組織として、生体(68歳のヒト)から切除後すぐに4℃で冷蔵保存され、切除後48時間以内の眼瞼の皮膚組織を用いた。皮膚組織を10μMニュートラルレッド(Neutral Red)含有PBSに浸すことにより、前記皮膚組織中の汗腺にニュートラルレッドを取り込ませた。つぎに、光学顕微鏡下でピンセットとハサミとを用い、前記皮膚組織から汗腺を含む組織片を分離した。分離された組織片を15mL容量チューブ中の無菌PBS内に集めた。前記組織片を含有するPBSを軽く振とうさせた後、当該PBSを350×gおよび4℃で5分間の遠心分離に供して上清を除去することにより、前記組織片を洗浄した。
前記(1)「解離状態の汗腺細胞の製造」で得られた解離汗腺細胞と2質量%FBS/PBS溶液9mLとを混合した。得られた混合液を350×gおよび4℃で5分間の遠心分離に供して当該混合液から上清を除去した。前記チューブ中の解離汗腺細胞に製造例5で得られた5U/mLディスパーゼ液1mLを添加した後、ピペットを用い、前記チューブ中の解離汗腺細胞を撹拌した。つぎに、前記チューブ中の解離汗腺細胞を含有する混合液をセルストレーナー〔メッシュサイズ:40μm、コーニング社製、商品名:Falcon(登録商標)40μmセルストレーナー、ブルー、滅菌、個別包装〕に通して凝集した細胞を除去することにより、解離汗腺細胞の浮遊液を得た。
GFP組換ウイルス粒子溶液〔アプライド・バイオロジカル・マテリアルズ(Applied Biological Materials)社製、商品名:GFP Control Lentivirus、GFP組換ウイルス粒子濃度:1×106U/mL〕をポリエチレングリコール沈殿法にしたがって濃縮した。得られた濃縮物を前記ウイルス感染用培地でGFP組換ウイルス粒子濃度が1×108U/mLになるように希釈することにより、GFP組換ウイルス希釈液を得た。
参考例1の(1)「解離状態の汗腺細胞の製造」で得られた解離汗腺細胞とPBS9mLとを混合した。つぎに、得られた混合液を350×gおよび4℃で5分間の遠心分離に供して上清を除去した。遠心分離後の解離汗腺細胞5×102~1×105個と製造例3で得られたウイルス感染用培地90μLとを混合した。得られた混合液に、前記GFP組換ウイルス希釈液10μLを添加し、スフィア-ウイルス混合液を得た。前記スフィア-ウイルス混合液を37℃の5体積%二酸化炭素雰囲気中でインキュベートして解離汗腺細胞に組換ウイルスを感染させることにより、解離汗腺細胞にGFP遺伝子を導入した。ウイルス感染開始時から24時間経過後、ウイルス感染汗腺細胞を回収した。
参考例1で得られたウイルス感染汗腺スフィアおよび参考例2で得られたウイルス感染汗腺細胞におけるGFPに基づく蛍光を蛍光顕微鏡下に観察した。また、参考例1で得られたウイルス感染汗腺スフィアおよび参考例2で得られたウイルス感染汗腺細胞を共焦点顕微鏡下に観察した。
参考例1の(2)「スフィア培養」で得られたスフィア含有液とPBS9mLとを混合した。つぎに、得られた混合液を350×gおよび4℃で5分間の遠心分離に供して上清を除去した。遠心分離後のスフィア4~10個と製造例3で得られたウイルス感染用培地100μLとを混合した。得られた混合液に、hTERT組換ウイルス粒子溶液〔アプライドバイオロジカルマテリアルズ社製の商品名:High Titer Lentivirus containing hTERT、hTERT組換ウイルス粒子濃度:1×109U/mL〕0.5μLとSV40Tt組換ウイルス粒子溶液〔アプライドバイオロジカルマテリアルズ社製の商品名:High Titer Lentivirus expressing SV40 large and small T antigens、SV40Tt組換ウイルス粒子濃度:1×109U/mL〕0.5μLとを添加し、スフィア-ウイルス混合液を得た。前記スフィア-ウイルス混合液を37℃の5体積%二酸化炭素雰囲気中でインキュベートしてスフィアを構成する汗腺細胞に組換レンチウイルスを感染させることにより、スフィアを構成する細胞に不死化遺伝子を導入した。ウイルス感染開始時から24時間経過後、ウイルス感染汗腺スフィアを回収した。
参考例1の(1)「解離状態の汗腺細胞の製造」および(2)の「スフィア培養」と同様の操作を行なうことにより、スフィアを得た。
(1)スフィア継代培養
以下の(1-1)および(1-2)を行なうことを「1回の継代培養」と定義した。
実施例1で得られたウイルス感染汗腺スフィアと、製造例4で得られた0.5質量%トリプシン-EDTA溶液1mLとを15mL容量チューブ中で混合した。ピペットを用い、前記チューブ中の汗腺を3分間撹拌することにより、汗腺を構成する汗腺細胞を互いに解離させ、解離汗腺細胞を得た。
前記(1-1)「解離汗腺細胞の製造」で得られた解離汗腺細胞と、2質量%FBS/PBS溶液9mLとを混合した。得られた混合液を350×gおよび4℃で5分間の遠心分離に供して上清を除去した。前記チューブ中の解離汗腺細胞に製造例5で得られた5U/mLディスパーゼ液1mLを添加した後、ピペットを用い、前記チューブ中の解離汗腺細胞を撹拌した。つぎに、前記チューブ中の解離汗腺細胞を含有する混合液をセルストレーナー〔メッシュサイズ:40μm、コーニング製、商品名:Falcon(登録商標)40μmセルストレーナー、ブルー、滅菌、個別包装〕に通して凝集した細胞を除去することにより、解離汗腺細胞の浮遊液を得た。
前記(1-1)「解離汗腺細胞の製造」および(1-2)「スフィア培養」からなる一連の操作を繰り返し、前記(1-2)「スフィア培養」におけるスフィアの形成の有無に基づき、スフィア形成能を調べた。
抗パンサイトケラチン抗体、抗パンサイトケラチン抗体に対する蛍光標識二次抗体、抗α-SMA抗体および当該抗α-SMA抗体に対する蛍光標識二次抗体を用いて前記(1-1)「解離汗腺細胞の製造」で得られた解離汗腺細胞および比較例1で得られた解離汗腺細胞の蛍光免疫染色を行なった。つぎに、免疫染色後の解離汗腺細胞におけるパンサイトケラチンに基づく蛍光の強度〔以下、「蛍光強度A」という〕およびα-SMAに基づく蛍光の強度〔以下、「蛍光強度B」という〕を測定した。
前記(1-1)「解離汗腺細胞の製造」で得られた解離汗腺細胞から全RNAを抽出した。得られた全RNAをその濃度が1μg/μLとなるようにDNアーゼ/RNアーゼフリーの精製水〔invitrogen社製、商品名:UltraPure DNase/RNase-Free Distilled Water〕を添加した。逆転写キット〔QIAGEN社製、商品名:Quatitect Reverse Transcription Kit〕を用い、前記全RNAからcDNAを合成し測定試料を得た。
<評価基準>
「不死化汗腺筋上皮細胞がATP1a1遺伝子を発現している」
・・・補正発現値が「正の値」である。
「不死化汗腺筋上皮細胞がATP1a1遺伝子を発現していない」
・・・補正発現値が「0」または「負の値」である。
(1)汗腺含有組織の製造
皮膚組織として、生体(41歳のヒト)から切除後すぐに4℃で冷蔵保存され、切除後48時間以内の眼瞼の皮膚組織を用いた。皮膚組織を10μMニュートラルレッド含有PBSに浸すことにより、前記皮膚組織中の汗腺にニュートラルレッドを取り込ませた。つぎに、光学顕微鏡下でピンセットとハサミとを用い、前記皮膚組織から汗腺を含む組織片を分離した。分離された組織片を15mL容量チューブ中の無菌PBS内に集めた。前記組織片を含有するPBSを軽く振とうさせた後、当該PBSを350×gおよび4℃で5分間の遠心分離に供して上清を除去することにより、前記組織片を洗浄した。
hTERT組換ウイルス粒子溶液とSV40Tt組換ウイルス粒子溶液とを前記ウイルス感染用培地でhTERT組換ウイルス粒子濃度が1×108U/mLおよびSV40Tt組換ウイルス粒子濃度が1×108U/mLとなるように希釈して不死化遺伝子含有組換ウイルス希釈液を得た。
試験例2の(1)「スフィア継代培養」において、実施例1で得られたウイルス感染汗腺スフィアを用いる代わりに実施例2で得られたウイルス感染組織を用いたことを除き、試験例2の(1)「スフィア継代培養」と同様の操作を行ない、スフィア継代培養を行なった。
(1)解離汗腺細胞の製造
参考例1の(1)「解離状態の汗腺細胞の製造」において、68歳のヒトの眼瞼の皮膚組織を用いる代わりに20歳のヒトの眼瞼の皮膚組織(参考例3)、71歳のヒトの眼瞼の皮膚組織(参考例4)、74歳のヒトの眼瞼の皮膚組織(参考例5)、51歳のヒトの腹部の皮膚組織(参考例6)または55歳のヒトの腹部の皮膚組織(参考例7)を用いたことを除き、参考例1の(1)「解離状態の汗腺細胞の製造」と同様の操作を行ない、解離汗腺細胞を得た。
参考例1の(2)「スフィア培養」において、参考例1の(1)「解離状態の汗腺細胞の製造」で得られた解離汗腺細胞を用いる代わりに参考例3~7の(1)「解離汗腺細胞の製造」で得られた各解離汗腺細胞を用いたことを除き、参考例1の(2)「スフィア培養」と同様の操作を行ない、スフィア含有液を得た。
実施例1において、参考例1の(2)「スフィア培養」で得られたスフィア含有液を用いる代わりに参考例3で得られたスフィア含有液(実施例3)、参考例4で得られたスフィア含有液(実施例4)、参考例5で得られたスフィア含有液(実施例5)、参考例6で得られたスフィア含有液(実施例6)または参考例7で得られたスフィア含有液(実施例7)を用いたことを除き、実施例1と同様の操作を行ない、ウイルス感染汗腺スフィアを得た。
参考例1の(1)「解離状態の汗腺細胞の製造」において、68歳のヒトの眼瞼の皮膚組織を用いる代わりに20歳のヒトの眼瞼の皮膚組織(比較例3)、71歳のヒトの眼瞼の皮膚組織(比較例4)、74歳のヒトの眼瞼の皮膚組織(比較例5)、51歳のヒトの腹部の皮膚組織(比較例6)または55歳のヒトの腹部の皮膚組織(比較例7)を用いたことを除き、比較例1の(1)「解離状態の汗腺細胞の製造」と同様の操作を行ない、解離汗腺細胞を得た。
試験例2の(1)「スフィア継代培養」において、実施例1で得られたウイルス感染汗腺スフィアを用いる代わりに実施例3~7で得られたウイルス感染組織を用いたことを除き、試験例2の(1)「スフィア継代培養」と同様の操作を行ない、スフィア継代培養を行なった。
(1)スフィア継代培養
以下の(1-1)および(1-2)を行なうことを「1回の継代培養」と定義した。
試験例2の(1-1)「解離汗腺細胞の製造」において、実施例1で得られたウイルス感染汗腺スフィアを用いる代わりに実施例3~7で得られたウイルス感染汗腺スフィアを用いたことを除き、試験例2の(1-1)「解離汗腺細胞の製造」と同様の操作を行ない、解離汗腺細胞を得た。
前記(1-1)「解離汗腺細胞の製造」で得られた解離汗腺細胞の一部(細胞数Aの解離汗腺細胞)と、2質量%FBS/PBS溶液9mLとを混合した。得られた混合液を350×gおよび4℃で5分間の遠心分離に供して上清を除去した。前記チューブ中の解離汗腺細胞に製造例5で得られた5U/mLディスパーゼ液1mLを添加した後、ピペットを用い、前記チューブ中の解離汗腺細胞を撹拌した。つぎに、前記チューブ中の解離汗腺細胞を含有する混合液をセルストレーナー〔メッシュサイズ:40μm、コーニング製、商品名:Falcon(登録商標)40μmセルストレーナー、ブルー、滅菌、個別包装〕に通して凝集した細胞を除去することにより、解離汗腺細胞の浮遊液を得た。
前記(1-1)「解離汗腺細胞の製造」および(1-2)「スフィア培養」からなる一連の操作をスフィア形成が停止するまで繰り返した。
[各継代間での細胞増殖倍率(KX)]=[細胞数B]/[細胞数A]
(II)
にしたがい、各継代間での細胞増殖倍率(KX)を求めた。各継代間での細胞増殖倍率(KX)を用い、式(III)にしたがい、
[総細胞増殖倍率]=K1×K2・・・×KX (III)
(式中、Xは正の整数を示す)
にしたがい、実施例3~7で得られたスフィアに含まれる汗腺細胞の総細胞増殖倍率を求めた。その結果を表2に示す。
(1)解離汗腺細胞の製造
試験例2の(1-1)「解離汗腺細胞の製造」において、実施例1で得られたウイルス感染汗腺スフィアを用いる代わりに実施例3で得られたスフィア(実験番号1)、実施例4で得られたスフィア(実験番号2)または実施例5で得られたスフィア(実験番号3)を用いたことを除き、試験例2の(1-1)「解離汗腺細胞の製造」と同様の操作を行ない、解離汗腺細胞を得た。なお、前記において、実施例3で得られたスフィアとして継代数5回のスフィア、実施例4で得られたスフィアとして継代数8回のスフィア、実施例5で得られたスフィアとして継代数14回のスフィアを用いた。
試験例6の(1)「解離完成細胞の製造」で得られた解離汗腺細胞を表3に示される濃度となるように凍結保存液〔タカラバイオ(株)製、商品名CELLBANKER(登録商標) 1plus〕1mLに懸濁した。得られた懸濁液を-80℃に保たれたフリーザで凍結させ、凍結細胞を得た。得られた凍結細胞を-80℃に保たれたフリーザまたは液体窒素中で表3に示される期間保存した。その後、凍結物を解凍して実験番号1~3の解凍細胞を得た。得られた解凍細胞の数を測定した。以下において、解凍細胞の数を細胞数Cとして用いた。
試験例2の(1-2)「スフィア培養」において、試験例2の(1-1)「解離汗腺細胞の製造」で得られた解離汗腺細胞を用いる代わりに試験例6の(2)「解離汗腺細胞の凍結保存」で得られた解凍細胞を用いたことを除き、試験例2の(1-2)「スフィア培養」と同様の操作を行ない、実験番号1~3のスフィア含有液Aを得た。
以下の(3-1)および(3-2)を行なうことを「1回の継代培養」と定義した。
試験例2の(1-1)「解離汗腺細胞の製造」において、実施例1で得られたウイルス感染汗腺スフィアを用いる代わりに試験例6の(2)「スフィア形成」で得られたスフィア含有液Aのうち、実験番号3のスフィア含有液Aを用いたことを除き、試験例2の(1-1)「解離汗腺細胞の製造」と同様の操作を行ない、実験番号3の解離汗腺細胞を得た。
試験例2の(1-2)「スフィア培養」において、試験例2の(1-1)「解離汗腺細胞の製造」で得られた解離汗腺細胞を用いる代わりに試験例6の(3-1)「解離汗腺細胞の製造」で得られた解離汗腺細胞(実験番号3)を用いたことを除き、試験例2の(1-2)「スフィア培養」と同様の操作を行ない、実験番号3のスフィア含有液Bを得た。
[細胞増殖倍率]=[細胞数D]/[細胞数C] (IV)
にしたがい、実験番号1~3のスフィアに含まれる不死化汗腺細胞を凍結保存したときの細胞増殖倍率を求めた。細胞増殖能の評価結果を表4に示す。表中、細胞増殖能およびスフィア形成能の評価基準は、以下のとおりである。
<細胞増殖能の評価基準>
+:式(IV)にしたがって算出された細胞増殖率が1より大きい。
-:式(IV)にしたがって算出された細胞増殖率が1以下である。
<スフィア形成能の評価基準>
+:光学顕微鏡下でスフィアの形成が確認される。
-:光学顕微鏡下でスフィアの形成が確認されない。
Claims (6)
- α-平滑筋アクチンおよびパンサイトケラチンを発現し、少なくとも5回の継代後にスフィア形成能を有していることを特徴とする不死化汗腺筋上皮細胞。
- ATP1a1をさらに発現している請求項1に記載の不死化汗腺筋上皮細胞。
- 不死化汗腺筋上皮細胞を製造する方法であって、
(I)汗腺筋上皮細胞が表面に露出している細胞構造体を培地中に浮遊させた状態で培養しながら、不死化遺伝子を当該汗腺筋上皮細胞に導入して遺伝子導入体を得る工程、および
(II)前記工程(I)で得られた遺伝子導入体を培地中に浮遊させた状態で培養して不死化汗腺筋上皮細胞を得る工程
を含む不死化汗腺筋上皮細胞の製造方法。 - 前記工程(I)において、ウイルスベクターを介して不死化遺伝子を前記汗腺筋上皮細胞に導入する請求項3に記載の不死化汗腺筋上皮細胞の製造方法。
- 前記工程(I)を行なう前に、採取された皮膚組織から少なくとも膠原繊維の全部または一部を除去して汗腺筋上皮細胞が表面に露出している汗腺含有組織を得る工程をさらに含み、前記工程(I)において、前記細胞構造体として前記汗腺含有組織を用いる請求項3または4に記載の不死化汗腺筋上皮細胞の製造方法。
- 前記工程(I)を行なう前に、汗腺細胞を培地中に浮遊させた状態で培養し、汗腺筋上皮細胞が表面に露出しているスフィアを形成させる工程をさらに含み、前記工程(I)において、前記細胞構造体として前記スフィアを用いる請求項3または4に記載の不死化汗腺筋上皮細胞の製造方法。
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