JP7246629B2 - Method for culturing mesenchymal cells, method for producing activated mesenchymal cells, method for producing hair follicle primordium, method for activating mesenchymal cells, and method for activating epithelial cells - Google Patents

Description

The present invention relates to a method for culturing mesenchymal cells, a method for producing activated mesenchymal cells, a method for producing hair follicle primordia, a method for activating mesenchymal cells, and a method for activating epithelial cells.

Techniques for forming hair follicle primordium in vitro and transplanting the hair follicle primordium into the living body to regenerate hair are being developed.

Patent Document 1 discloses a process of seeding mesenchymal cells and epithelial cells on a micro-intaglio plate consisting of regularly arranged micro-concavities and culturing them while supplying oxygen to form a hair follicle primordium. A method for producing aggregates of regenerated follicle primordia is described, comprising:

Patent Document 2 discloses a dermal papilla culture method characterized by culturing and subculturing dermal papilla cells in the presence of basic fibroblast growth factor (bFGF), and sphering the dermal papilla cells. Are listed.

Patent Document 3 discloses a culture of dermal papilla cells maintaining hair-inducing ability, characterized by culturing dermal papilla cells in the presence of FGF-based materials, BMP-2/BMP-4-based materials, and WNT-based materials. method is described.

WO2017/073625 JP 2007-274949 A WO2010/021245

However, a culture method for effectively improving the hair growth-related properties of dermal papilla cells has not yet been established.

The present invention has been made in view of the above problems, and includes a method for culturing mesenchymal cells that improves hair growth-related properties, a method for producing activated mesenchymal cells, a method for producing hair follicle primordia, One object of the present invention is to provide a method for activating leaf cells and a method for activating epithelial cells.

A method for culturing mesenchymal cells according to an embodiment of the present invention for solving the above problems comprises culturing mesenchymal cells adhered to the surface of an electrode while applying an electric current through the electrode. include. INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for culturing mesenchymal cells that improves hair growth-related properties of mesenchymal cells.

In the method, the current may be a pulse current. In the method, the current may be applied via the electrode as the first electrode and a second electrode arranged at a position facing the first electrode.

A method for producing activated mesenchymal cells according to an embodiment of the present invention for solving the above problems is to culture mesenchymal cells adhered to the surface of an electrode while applying an electric current through the electrode. thereby obtaining said mesenchymal cells that are activated compared to mesenchymal cells cultured in the same manner except that no current is applied. INDUSTRIAL APPLICABILITY According to the present invention, a method for producing activated mesenchymal cells with improved hair growth-related properties is provided.

In the method, the current may be a pulse current. In the method, the current may be applied via the electrode as the first electrode and a second electrode arranged at a position facing the first electrode. In the method, the activated mesenchymal cells have an increased expression level of at least one hair growth-related gene compared to mesenchymal cells cultured in the same manner except that no current is applied. It may be a leaf cell.

A method for producing a hair follicle primordium according to one embodiment of the present invention for solving the above-mentioned problems is to co-culture mesenchymal cells produced by any of the above methods and epithelial cells to produce hair follicles. Including forming a follicle. INDUSTRIAL APPLICABILITY According to the present invention, a method for producing a hair follicle primordium having effectively improved hair growth-related properties is provided.

In the method, the hair follicle primordium may be formed by co-cultivating the mesenchymal cells and the epithelial cells on a cell non-adhesive surface. In the method, the hair follicle primordium formed by the co-culture is the same method except that the mesenchymal cells are produced by the same method as the mesenchymal cells except that no current is applied. The activated epithelial cells may be contained in comparison with the epithelial cells contained in the hair follicle primordium formed in . In this case, the activated epithelial cells are mesenchymal cells produced by the same method as the mesenchymal cells, except that the expression level of at least one hair growth-related gene is not applied to the mesenchymal cells. It may be an epithelial cell that is more increased than that of the epithelial cells contained in the hair follicle primordium formed by the same method except that the hair follicle was formed.

In the method, the hair follicle primordium formed by the co-culture is the same method except that the mesenchymal cells are produced by the same method as the mesenchymal cells except that no current is applied. The activated mesenchymal cells may be contained in comparison with the mesenchymal cells contained in the hair follicle primordium formed in . In this case, the activated mesenchymal cells were formed by the same method except that the mesenchymal cells were produced by the same method as the mesenchymal cells except that no current was applied. The mesenchymal cells may have an increased expression level of at least one hair growth-related gene compared to the mesenchymal cells contained in the hair follicle primordium.

A method for activating mesenchymal cells according to an embodiment of the present invention for solving the above problems is to culture mesenchymal cells adhered to the surface of an electrode while applying an electric current through the electrode. activates the mesenchymal cells. INDUSTRIAL APPLICABILITY According to the present invention, a method for activating mesenchymal cells that effectively improves hair growth-related properties of mesenchymal cells is provided.

A method for activating epithelial cells according to one embodiment of the present invention for solving the above problems is characterized in that, in a hair follicle primordium formed by co-culturing mesenchymal cells and epithelial cells, the mesenchymal The epithelial cells contained in the hair follicle primordium are activated by using the mesenchymal cells produced by any of the above methods as the cells. INDUSTRIAL APPLICABILITY According to the present invention, a method for activating epithelial cells is provided that effectively improves the hair growth-related properties of epithelial cells.

According to the present invention, there are provided a method for culturing mesenchymal cells, a method for producing activated mesenchymal cells, a method for producing hair follicle primordia, a method for activating mesenchymal cells, and an epithelium for improving hair growth-related properties. Lineage cell activation methods are provided.

1 is an explanatory view schematically showing a cross section of an example of a culture apparatus used in this embodiment; FIG. FIG. 10 is an explanatory diagram showing the results of evaluating gene expression levels in dermal papilla cells when the timing of starting to apply a current is changed in Example 2 according to the present embodiment. FIG. 10 is an explanatory diagram showing the results of evaluating the gene expression level of dermal papilla cells when the applied current value is changed in Example 2 according to the present embodiment. FIG. 4 is an explanatory diagram showing the results of evaluating the gene expression level of dermal papilla cells when the current application time was changed in Example 2 according to the present embodiment. FIG. 4 is an explanatory diagram showing the results of evaluating gene expression levels of dermal papilla cells and epithelial cells contained in hair follicle primordium in Example 3 according to the present embodiment. FIG. 3 is an explanatory diagram showing a photograph of hair growth from a hair follicle primordium formed using dermal papilla cells cultured by applying an electric current and transplanted to a mouse in Example 3 according to the present embodiment. be. Explanatory drawing showing a photograph of hair growth from a hair follicle primordium formed using dermal papilla cells cultured without applying current and transplanted to a mouse in Example 3 according to the present embodiment. is. FIG. 10 is an explanatory diagram showing the results of evaluating the number of hair growth from hair follicle primordia transplanted to mice in Example 3 according to the present embodiment.

A method (hereinafter referred to as "this method") according to one embodiment of the present invention will be described below. Note that the present invention is not limited to this embodiment.

This method includes a method for culturing mesenchymal cells, which includes culturing mesenchymal cells adhering to the surface of an electrode while applying an electric current through the electrode. That is, the inventors of the present invention have extensively studied technical means for improving the hair growth-related properties of mesenchymal cells. The present inventors have independently found that leaf cells are activated and their hair growth-related properties are effectively improved, leading to the completion of the present invention.

Therefore, the present method activates the mesenchymal cells by culturing the mesenchymal cells adhered to the surface of the electrode while applying an electric current through the electrode. including conversion methods.

In addition, the present method involves culturing mesenchymal cells adhering to the surface of an electrode while applying an electric current through the electrode, thereby culturing the mesenchymal cells by the same method except that no electric current is applied. It includes a method for producing activated mesenchymal cells, comprising obtaining said mesenchymal cells that have been activated compared to.

Furthermore, this method includes a method for producing a hair follicle primordium, which comprises forming a hair follicle primordium by co-culturing the mesenchymal cells produced by the above method and epithelial cells. .

In addition, the inventors of the present invention unexpectedly found that the above-mentioned activated mesenchymal cells and epithelial cells were co-cultured to form hair follicle primordia. uniquely found that epithelial cells that are

Therefore, the present method uses the mesenchymal cells produced by the present method as the mesenchymal cells in the hair follicle primordium formed by co-culturing the mesenchymal cells and the epithelial cells. It includes a method for activating epithelial cells, which activates the epithelial cells contained in the hair follicle primordium.

The mesenchymal cells used in this method should have hair growth-related properties (e.g., expression of hair growth-related genes and/or formation of hair follicle primordium by co-culture with epithelial cells). It is not particularly limited. The mesenchymal cells may be cells derived from adult hair follicle tissue (e.g., dermal papilla and/or hair bulb root sheath), and skin tissue (fetal, juvenile, or adult skin tissue). ) derived from stem cells (e.g., induced pluripotent (iPS) stem cells, embryonic stem (ES) cells, or embryonic germ (EG) cells) in vitro. cells may be used. Mesenchymal cells may be primary cells collected from a living body, or pre-cultured cells (eg, subcultured cells and/or established cells). Mesenchymal cells are identified as cells expressing, for example, Versican and ALP (alkaline phosphatase). Specifically, as the mesenchymal cells, dermal papilla cells and/or hair bulb hair root sheath cells are preferably used. Dermal papilla cells and hair bulb root sheath cells express Versican and ALP.

The mesenchymal cells are preferably derived from humans, but may be derived from non-human animals (animals other than humans). Non-human animals are not particularly limited, but are preferably non-human vertebrates (vertebrates other than humans). Non-human vertebrates are not particularly limited, but preferably non-human mammals. Non-human mammals include, but are not limited to, primates (e.g., monkeys), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), carnivores (e.g., dogs, cats), or ungulates. (eg, porcine, bovine, equine, goat, ovine).

The culture medium used for culturing the mesenchymal cells is not particularly limited as long as it has a composition suitable for maintaining the survival and hair growth-related characteristics of the mesenchymal cells. A medium for cell culture is preferably used.

The surface of the electrode used in the present method (hereinafter referred to as "culture electrode") to which mesenchymal cells adhere (hereinafter referred to as "culture surface") can adhere and culture mesenchymal cells. , and a conductive surface to which an electric current can be applied. Preferably, the culture electrode comprises a metal membrane. In this case, the metal constituting the metal film is not particularly limited as long as it imparts conductivity to the culture electrode. For example, it is selected from the group consisting of gold, platinum, silver, copper, titanium, chromium and nickel. , or alloys thereof are preferably used.

The culture electrode may include a conductive polymer membrane. That is, for example, the culture electrode may include a metal film and a conductive polymer film formed on the metal film. The conductive polymer constituting the conductive polymer film is not particularly limited, but for example, polypyrrole-based polymer, polythiophene-based polymer, polyaniline-based polymer, polyacetylene-based polymer, and polyparaphenylene-based polymer. One or more selected from the group consisting of is preferably used.

The culture electrode may include a cell adhesive membrane. That is, for example, the culture electrode may include a metal film and a cell adhesive substance film formed on the metal film. Further, for example, the culture electrode may include a metal film, a conductive polymer film formed on the metal film, and a cell adhesive substance film formed on the conductive polymer film. The cell adhesive substance is not particularly limited as long as it is a substance that promotes the adhesion of mesenchymal cells to the electrode surface. Amino acid sequences (eg, arginine, glycine, aspartic acid (so-called RGD) sequences, etc.) and specific sugar chain sequences exhibiting cell adhesiveness are preferably used.

Even when the culture electrode does not contain a cell adhesive substance film, for example, the cell adhesive substance contained in the culture solution in contact with the culture surface of the culture electrode is adsorbed to the culture surface, and the mesenchyme Lineage cells can adhere to the culture surface. That is, the culture surface is not particularly limited as long as it is a conductive surface that exhibits cell adhesiveness when in contact with the culture medium.

Adhesion of mesenchymal cells to the culture surface is performed by seeding the mesenchymal cells on the culture surface and retaining the mesenchymal cells on the culture surface. Specifically, a culture solution containing mesenchymal cells is dropped onto the culture surface, and the mesenchymal cells that have settled on the culture surface in the culture solution are treated under conditions suitable for culturing the mesenchymal cells. The mesenchymal cells are allowed to adhere to the culture surface by holding for a predetermined period of time (for example, a temperature suitable for culturing mesenchymal cells, a gas phase composition in contact with the culture solution, and a composition of the culture solution). The mesenchymal cells before adhering to the culture surface are spherical, but the mesenchymal cells adhered to the culture surface are flatter than before adhesion.

Cultivation of the mesenchymal cells in this method is performed while applying an electric current through the culture electrodes to which the mesenchymal cells are adhered. Conditions for applying a current are not particularly limited as long as the effects of the present invention can be obtained. Conditions that increase the amount of mesenchymal cells more than those of mesenchymal cells cultured in the same manner except that they are not used are preferably used.

The timing of starting the application of current is not particularly limited as long as the effects of the present invention can be obtained. It is preferably after 10 hours or more, more preferably after 15 hours or more, and particularly preferably after 20 hours or more.

In addition, the timing to start applying the current is, for example, preferably 170 hours after seeding the mesenchymal cells on the electrode surface, more preferably 120 hours before, and 80 hours before. is more preferable, and it is particularly preferable that it is before 50 hours have elapsed. The timing to start applying the current may be specified by arbitrarily combining any of the above lower limits and any of the above upper limits.

The total time of current application (when the current is applied in multiple times during the culture period, the total time of the multiple current application times) is not particularly limited as long as the effects of the present invention can be obtained. However, for example, it is preferably 5 minutes or longer, more preferably 10 minutes or longer, even more preferably 15 minutes or longer, and particularly preferably 20 minutes or longer.

In addition, the total time for applying the current is, for example, preferably 120 hours or less, more preferably 80 hours or less, even more preferably 50 hours or less, and particularly preferably 20 hours or less. preferable. The total time for which the current is applied may be specified by any combination of any of the above lower limits and any of the above upper limits.

The time to apply the current in one electrical stimulation (when the current is applied in multiple times during the culture period, the time to continuously apply the current in one of the multiple current applications) is Although it is not particularly limited as long as the effect of the present invention is obtained, for example, it is preferably 5 minutes or more, more preferably 10 minutes or more, even more preferably 15 minutes or more, and 20 minutes. Minutes or more are particularly preferred.

In addition, the time for which the current is applied in one electrical stimulation is, for example, preferably 120 hours or less, more preferably 80 hours or less, even more preferably 50 hours or less, and 20 hours or less. is particularly preferred. The duration of current application in one electrical stimulation may be specified by arbitrarily combining any of the above lower limits and any of the above upper limits.

The current value applied per unit area (cm ² ) of the culture surface (electrode surface) is not particularly limited as long as the effect of the present invention can be obtained, but is preferably 10 μA/cm ² or more, for example. , more preferably 20 μA/cm ² or more, even more preferably 30 μA/cm ² or more, and particularly preferably 40 μA/cm ² or more.

Further, the applied current value is, for example, preferably 1000 μA/cm ² or less, more preferably 700 μA/cm ² or less, even more preferably 400 μA/cm ² or less, and 200 μA/cm ^{2 or less} . The following are particularly preferred. The current value to be applied may be specified by arbitrarily combining any of the above lower limits and any of the above upper limits.

The applied current is preferably a pulse current. The pulse current is not particularly limited as long as it is a current applied in a pulsed manner. For example, one pulse is composed of the following (1), (2) and (3), ), or may consist of (1) and (3): (1) first applying a positive or negative current of a first value for a first time; and (2) then applying a second value of negative or positive current for a second time period; (3) applying no current for a subsequent third time period; Note that the descriptions of "positive current or negative current" in (1) and "negative current or positive current" in (2) mean that when a positive current is applied in (1), a negative current is applied in (2). When a negative current is applied in (1), it means that a positive current is applied in (2).

Although the first and second values of the pulse current described above may be different, they are preferably the same. Moreover, although the first time and the second time may be different, they are preferably the same. The third time can be different or the same as the first time and/or the second time.

The frequency of the pulse current is not particularly limited as long as the effects of the present invention can be obtained. For example, it is preferably 2 Hz or more, more preferably 3 Hz or more, and even more preferably 4 Hz or more. It is preferably 5 Hz or more, and particularly preferably 5 Hz or more. Note that 1 Hz means that the pulse current is applied once per second.

Further, the frequency of the pulse current is, for example, preferably 100 Hz or less, more preferably 50 Hz or less, even more preferably 25 Hz or less, and particularly preferably 10 Hz or less. The frequency of the pulse current may be specified by arbitrarily combining any of the above lower limits and any of the above upper limits.

The current application via the culture electrode is performed using, for example, the culture electrode as the first electrode and the second electrode. In this case, for example, the first electrode (culture electrode) is used as a working electrode and the second electrode is used as a counter electrode. A reference electrode may also be used. The positional relationship between the first electrode and the second electrode is not particularly limited as long as the first electrode and the second electrode are in contact with the culture solution containing mesenchymal cells.

In this method, for example, a current may be applied via a culture electrode as a first electrode and a second electrode arranged at a position facing the first electrode. That is, in this case, between the first electrode and the second electrode, mesenchymal cells adhered to the culture surface of the first electrode, and the mesenchymal cells in contact with the first electrode and the second electrode A culture medium containing mesenchymal cells is placed.

FIG. 1 schematically shows an example of a culture apparatus for culturing mesenchymal cells while applying an electric current. In the example shown in FIG. 1, the culture apparatus 1 includes a first electrode 10, which is a culture electrode used as a working electrode, and a counter electrode, which is arranged at a position facing the culture surface 11 of the first electrode 10. and a second electrode 20 which is The first electrode 10 is composed of a metal film such as a gold thin film formed on a substrate 12 composed of a glass plate or the like. The second electrode 20 is made of metal such as platinum.

The culture device 1 includes multiple (four) culture wells 30 . Each culture well 30 has a partition wall 31 for retaining the culture medium M on the first electrode 10 . In each culture well 30 , the mesenchymal cells C are cultured in the culture medium M while adhering to the culture surface 11 . The first electrode 10 and the second electrode 20 are each connected to a power source 40 . That is, the first electrode 10 and the second electrode 20 are connected to the power source 40 by the first wiring 41 and the second wiring 42, respectively. In the example shown in FIG. 1, since the second electrode 20 of each of the four culture wells 30 is connected to the power supply 40 by the wiring 42, current can be applied to the four culture wells 30 under different conditions. .

Then, by applying a predetermined potential between the first electrode 10 and the second electrode 20 in each culture well 30 by the power supply 40, through the first electrode 10 and the second electrode 20 A current can be applied. In this method, such a culture device may be used to culture mesenchymal cells adhering to the culture surface of the culture electrode while applying an electric current.

The time for culturing the mesenchymal cells on the culture surface is not particularly limited as long as the effects of the present invention can be obtained. For example, it is preferably 5 hours or more, more preferably 20 hours or more. It is preferably 50 hours or longer, and particularly preferably 70 hours or longer.

In addition, the time for culturing the mesenchymal cells on the culture surface is, for example, preferably 400 hours or less, more preferably 300 hours or less, even more preferably 200 hours or less, and 100 hours. The following are particularly preferred. The time for culturing the mesenchymal cells on the culture surface may be specified by arbitrarily combining any of the above lower limits and any of the above upper limits.

The mesenchymal cells are activated by culturing the mesenchymal cells adhering to the culture surface of the culture electrodes while applying an electric current through the culture electrodes. That is, by culturing while applying an electric current, activated mesenchymal cells can be obtained.

Here, activation of mesenchymal cells means improvement of hair growth-related properties of the mesenchymal cells.

Specifically, the improvement of the hair growth-related properties of the mesenchymal cells is, for example, an increase in the expression level of a hair growth-related gene in the mesenchymal cells and / or formed using the mesenchymal cells Improving the hair growth properties of the follicle primordium (eg, increasing the number and/or length of hairs growing from the follicle primordium implanted in the body).

That is, the activated mesenchymal cells obtained by the present method have, for example, an expression level of at least one hair growth-related gene that is that of mesenchymal cells cultured in the same manner except that no current is applied. It is a more increased mesenchymal cell.

The hair growth-related gene expressed in mesenchymal cells is not particularly limited as long as it is a gene related to the contribution of the mesenchymal cells to hair growth. For example, it consists of Versican, ALP, BMP4, Nexin and Notch1. It may be one or more selected from the group.

Specifically, the activated mesenchymal cells obtained by this method are, for example, mesenchymal cells cultured in the same manner except that the expression level of the ALP gene measured by RT-PCR is not applied. 1.5 times or more, or 2.0 times or more.

The activated mesenchymal cells produced by this method can be used for various purposes. That is, the activated mesenchymal cells are, for example, directly adhered to the culture surface of the culture electrode, or recovered from the culture surface and used for research purposes and medical purposes such as transplantation.

Specifically, for example, activated mesenchymal cells are preferably used for the formation of hair follicle primordia in vitro. That is, by co-culturing the activated mesenchymal cells produced by the present method and epithelial cells, it is possible to form hair follicle primordia having excellent hair growth-related properties.

The epithelial cells used for the formation of hair follicle primordium in this method have hair growth-related properties (e.g., expression of hair growth-related genes and/or formation of hair follicle primordium by co-culture with mesenchymal cells). ) is not particularly limited as long as it has The epithelial cells may be cells derived from hair follicle tissue (e.g., the outermost layer of the outer root sheath of the bulge region of the hair follicle tissue and/or the base of the hair matrix), or cells derived from skin tissue. They may also be cells derived ex vivo from stem cells (eg, iPS stem cells, ES cells, or EG cells). Epithelial cells may be primary cells collected from a living body, or pre-cultured cells (eg, subcultured cells and/or established cells). Epithelial cells are identified, for example, as cells that express cytokeratin. The epithelial cells are preferably epithelial stem cells. Epithelial stem cells are identified, for example, as cells expressing cytokeratin 15 and/or CD34.

Epithelial cells are preferably derived from humans, but may be derived from non-human animals (animals other than humans). Non-human animals are not particularly limited, but are preferably non-human vertebrates (vertebrates other than humans). Non-human vertebrates are not particularly limited, but preferably non-human mammals. Non-human mammals include, but are not limited to, primates (e.g., monkeys), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), carnivores (e.g., dogs, cats), or ungulates. (eg, porcine, bovine, equine, goat, ovine).

Co-culture of mesenchymal cells and epithelial cells is performed by mixing mesenchymal cells collected after being cultured while applying an electric current and epithelial cells prepared separately from the mesenchymal cells. It is carried out by culturing with

The method of co-culturing is not particularly limited as long as it is a method in which mesenchymal cells and epithelial cells aggregate to form hair follicle primordia. It is preferred to form the follicle primordium by co-cultivating on a non-adhesive surface.

Here, the cell non-adhesive surface is not particularly limited as long as it is a surface to which mesenchymal cells and epithelial cells do not substantially adhere. That is, the non-cell-adhesive surface is, for example, a surface to which mesenchymal cells and epithelial cells do not adhere and are maintained in a floating state, or a surface to which the mesenchymal cells and epithelial cells loosely adhere but is treated with trypsin. It is a surface from which the mesenchymal cells and epithelial cells can be easily detached by an operation such as pipetting to flow the culture solution without enzymatic treatment such as. As a culture vessel having a cell non-adhesive surface, for example, a commercially available multi-well plate having a cell non-adhesive coating applied to the bottom surface of each well can be used. Also, the micro-intaglio described in International Publication No. 2017/073625 can be preferably used.

By co-culturing mesenchymal cells and epithelial cells (for example, dispersing and mixing mesenchymal cells and epithelial cells in a culture solution and culturing them), the mesenchymal cells and epithelial cells are Spontaneously aggregate to form the follicle primordium. The formed hair follicle primordium is formed, for example, by a mesenchymal cell aggregation portion formed by spontaneous aggregation of mesenchymal cells and by spontaneous aggregation of epithelial cells, and and an epithelial cell aggregation portion, a portion of which is bound to a portion of the mesenchymal cell aggregation portion.

In the hair follicle primordium, for example, the mesenchymal cell aggregation part may constitute the inner core part, and the epithelial cell aggregation part may constitute the outer layer covering the outer circumference of the mesenchymal cell aggregate ( So-called core-shell type hair follicle primordium). In addition, in the hair follicle primordium, for example, mesenchymal cells and epithelial cells may be arranged separately (so-called random type hair follicle primordium).

On the cell non-adhesive surface, a non-adherent hair follicle primordium is produced. Here, the hair follicle primordium in a non-adhesive state refers to the hair follicle primordium that is in a floating state without adhering to the cell non-adhesive surface, or that is loosely adhered to the cell non-adhesive surface, but is treated with trypsin or the like. It is a hair follicle primordium that can be easily detached from the non-adhesive surface of the cell without enzymatic treatment by an operation such as pipetting to flow the culture solution.

The follicle primordium formed in this method may be, for example, a follicle primordium spheroid. Hair follicle primordium spheroids are roughly spherical cell aggregates. Follicle primordium spheroids are formed in a non-adherent state and are easily collected. For this reason, hair follicle primordium spheroids are preferably used for transplantation into a living body.

The hair follicle primordium formed by the co-culture in this method is formed in the same manner as the activated mesenchymal cells except that the mesenchymal cells are produced in the same manner as the activated mesenchymal cells except that no current is applied. It contains activated epithelial cells compared to the epithelial cells contained in the activated hair follicle primordium.

Specifically, the mesenchymal cells cultured while applying an electric current by this method are called activated mesenchymal cells, and are cultured in the same manner as the activated mesenchymal cells except that no electric current is applied. When the mesenchymal cells are called control mesenchymal cells and the hair follicle primordium formed by co-culturing the control mesenchymal cells and epithelial cells is called the control hair follicle primordium, the activated mesenchyme The hair follicle primordium formed by co-culturing the stromal cells and the epithelial cells is the control hair formed by the same method except that the control mesenchymal cells are used instead of the activated mesenchymal cells. It contains activated epithelial cells compared to the epithelial cells contained in the follicle.

That is, epithelial cells co-cultured with mesenchymal cells cultured while applying current by this method were co-cultured with mesenchymal cells cultured under the same conditions except that the current was not applied. Activated compared to epithelial cells. In other words, by using activated mesenchymal cells, hair follicle primordia containing activated epithelial cells can be produced as compared to the case of using control mesenchymal cells.

Here, activation of epithelial cells means improvement of hair growth-related properties of the epithelial cells. Specifically, the improvement of the hair growth-related properties of epithelial cells is, for example, an increase in the expression level of hair growth-related genes in the epithelial cells, and/or folliculogenesis formed using the epithelial cells. This includes increasing the hair growth properties of the root (eg, increasing the number and/or length of hairs growing from the hair follicle primordium implanted in the body).

That is, the activated epithelial cells contained in the hair follicle primordium formed by the present method, for example, are activated mesenchymal cells except that the expression level of at least one hair growth-related gene is not applied with an electric current. It is an epithelial cell that is more increased than that of the epithelial cells contained in the hair follicle primordium formed by the same method except for using mesenchymal cells produced by the same method.

The hair growth-related gene expressed in epithelial cells is not particularly limited as long as it is a gene related to the contribution of the epithelial cells to hair growth. Sonic hedgehog) and β-catenin may be one or more selected from the group consisting of.

Specifically, the activated epithelial cells contained in the hair follicle primordium produced by this method, for example, the expression level of the Wnt10b gene measured by RT-PCR is determined by the same method except that no current is applied. It may be 1.5 times or more, 2.0 times or more, or 2.5 times or more that of the epithelial cells co-cultured with the cultured mesenchymal cells. , or 3.0 times or more.

Thus, the effect of activating epithelial cells by co-culturing with mesenchymal cells activated by electrical stimulation is an unexpected effect that cannot be expected from the prior art.

In addition, the hair follicle primordium formed by the co-culture in this method is the same method except that the mesenchymal cells produced by the same method as the activated mesenchymal cells are used except that no current is applied. contains activated mesenchymal cells compared to the mesenchymal cells contained in the hair follicle primordium formed in . In other words, the activated mesenchymal cells produced by this method maintain their activated state even after forming hair follicle primordia by co-culturing with epithelial cells.

Specifically, the activated mesenchymal cells contained in the hair follicle primordium formed by this method are the mesenchymal cells produced by the same method as the activated mesenchymal cells except that no current is applied. Mesenchymal cells in which the expression level of at least one hair growth-related gene is increased compared to the mesenchymal cells contained in the hair follicle primordium formed by the same method except that the cells are used be.

Specifically, the activated mesenchymal cells contained in the hair follicle primordium produced by the present method, for example, the Versican gene expression level measured by RT-PCR is the same method except that no current is applied. It may be 1.5 times or more, or 2.0 times or more, that of the mesenchymal cells that were cultured in and formed the hair follicle primordium.

In addition, for example, the activated mesenchymal cells contained in the hair follicle primordium produced by this method are cultured in the same manner except that the expression level of the ALP gene measured by RT-PCR is not applied with an electric current. It may be 1.5 times or more, 2.0 times or more, or 2.5 times or more that of the mesenchymal cells that formed the hair follicle primordium. , 3.0 times or more.

Thus, the production of hair follicle primordium by the present method is the production of hair follicle primordium containing activated epithelial cells and/or activated mesenchymal cells , i.e., activated It can also be said to be the production of hair follicle primordia.

The hair follicle primordium produced by this method can be directly used for research purposes, but it is also preferably used for transplantation into a living body. That is, according to this method, a hair follicle primordium from which hair grows when transplanted into a living body is produced.

When the follicle primordium produced by this method is transplanted into a living body to grow hair from the follicle primordium, the living body is not particularly limited, and may be a human or a non-human animal. . Non-human animals are not particularly limited, but non-human mammals are preferred. Non-human mammals include, but are not limited to, primates (e.g., monkeys), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), carnivores (e.g., dogs, cats), or animals. Hoofed animals (eg, pigs, cows, horses, goats, sheep). The transplantation of hair follicle primordia into a living body is preferably transplantation into the skin of the living body. Implantation into the skin may be, for example, subcutaneous implantation or intradermal implantation.

The production of hair follicle primordium and its transplantation into animals may be for medical or research purposes. That is, for example, for the treatment or prevention of a disease associated with hair loss, a hair follicle primordium is produced for the purpose of transplantation into a human patient suffering from or likely to suffer from the disease, or the hair follicle primordium is It may be transplanted into the human patient.

Diseases associated with hair loss are not particularly limited, but for example, androgenetic alopecia (AGA), female androgenetic alopecia (FAGA), postpartum alopecia, diffuse alopecia, seborrhea Composed of alopecia areata, alopecia pityriasis, traction alopecia, metabolic alopecia, alopecia areata compressive, alopecia areata, alopecia areata, alopecia nervosa, trichotillomania, alopecia generalis, and symptomatic alopecia It may be one or more selected from the group.

In addition, for example, to search for substances that can be used for the treatment or prevention of diseases associated with hair loss and/or to search for substances involved in the mechanism of the disease, the hair follicle primordium is produced or the hair follicle The base may be implanted into a non-human animal.

Next, specific examples according to this embodiment will be described.

[Incubation device]
A culture device having the same structure as the culture device 1 shown in FIG. 1 was manufactured as follows. First, the substrate was washed. That is, an ammonia solution (25%) and a hydrogen peroxide solution (30%) were mixed at a volume ratio of 3:1 to prepare a cleaning solution. Next, the slide glass used as the substrate was immersed in a cleaning liquid boiled at 300° C. for 5 minutes to remove impurities adhering to the surface of the slide glass. After that, the cleaning solution was washed away by repeating the operation of immersing the slide glass in ultrapure water boiled at 300° C. for 5 minutes twice.

Next, an electrode for culturing mesenchymal cells on its surface was fabricated. That is, a chromium thin film was formed on the surface of the slide glass washed as described above using a sputtering apparatus, and a gold thin film was further formed on the chromium thin film. A slide glass on which a gold thin film is formed is immersed in a mixed solution of 0.1 M polypyrrole solution and 0.1 Mp-toluenesulfonic acid solution, and a current of 0.25 mA/cm ² is applied to the mixed solution for 4 minutes. Then, a polypyrrole film (conductive polymer film) was formed on the gold thin film. After that, the slide glass on which the polypyrrole film was formed was washed with ultrapure water and air-dried.

Then, culture wells were prepared. That is, by adhering a 4-well chamber (Lab Tek II Chamber Slide) to a part of the slide glass surface on which the polypyrrole film is formed, the 4-well chamber is partitioned by the frame (partition wall), and the polypyrrole film is formed. Four culture wells were formed with bottoms consisting of formed culture surfaces.

Thereafter, the inside of each culture well was sterilized by washing with ethanol. Next, a 0.03% collagen type I solution was added to each culture well and air-dried to form a collagen thin film on the polypyrrole film on the bottom surface of the culture well. Four culture surfaces were thus obtained, each with an area of about 2 cm ² .

Also, a resin base material having a size to block the upper opening of each culture well was prepared, and one surface of the resin base material was covered with a platinum mesh. In the culture of mesenchymal cells, which will be described later, this resin base material was used as the lid of the culture well, and was fitted into the upper opening of each culture well so that the platinum mesh surface faced the bottom surface of each culture well. .

A galvanostat device was used as a power source. That is, of the surface of the slide glass on which the polypyrrole film is formed, the portion where the culture wells are not formed (the portion where the frame of the 4-well chamber is not arranged) is electrically connected to the power supply via metal wiring. , the platinum mesh of each of the four resin substrates used as lids for the four culture wells was electrically connected to the power supply via metal wires. In this way, a culture device used for culturing mesenchymal cells was manufactured.

[Preparation of mesenchymal cells]
Dermal papilla cells were used as mesenchymal cells. That is, commercially available human dermal papilla cells (PromoCell) were subcultured, and the human dermal papilla cells of passage 4 to 6 (P4 to P6) were used in the experiment. As the culture medium, a commercially available dermal papilla cell culture medium (PromoCell) was used.

[Culture of mesenchymal cells]
As Example 1-1, dermal papilla cells were seeded in each culture well, and the dermal papilla cells were cultured on the bottom surface of each culture well (the culture surface of the culture electrode) for 3 days. During this culture period, a pulsed current was applied to each culture well for 16 minutes starting 24 hours after the dermal papilla cells were seeded.

Specifically, in this pulse current stimulation, (1) first, a current of “+200 μA (current value per unit area of culture surface: about +100 μA/cm ² )” is applied to each culture chamber for 0.05 seconds. (2) then apply a current of “−200 μA (current value per unit area of the culture surface: about −100 μA/cm ² )” for 0.05 seconds, and (3) then apply no current ( 0 μA) and held for 0.1 sec, a current was applied with the above (1), (2) and (3) as one pulse.

On the other hand, as Comparative Example 1-1, dermal papilla cells were cultured for 3 days in the same manner except that no current was applied. As Comparative Example 1-2, dermal papilla cells were cultured for 3 days in the same manner except that no current was applied, using a culture apparatus manufactured in the same manner except that the polypyrrole film was not formed.

[Life and death staining of mesenchymal cells]
On the third day of culture, viability staining of dermal papilla cells was performed using fluorescein diacetate (FDA) for staining viable cells and ethidium bromide (EB) for staining dead cells. That is, first remove the medium from each culture well on the third day of culture, then add a culture solution containing FDA (living cell staining solution) to each culture well and maintain for 30 minutes to stain living cells. gone. Next, the viable cell staining solution was removed from each culture well, and then a medium containing EBs (dead cell staining solution) was added to each culture well and maintained for several minutes to stain dead cells. Furthermore, after removing the dead cell staining solution from each culture well, the inside of each culture well was washed with a phosphate buffer solution (PBS). Then, the stained dermal papilla cells were observed under a phase-contrast microscope and a fluorescence microscope.

[result]
As a result of observing the stained dermal papilla cells with a phase contrast microscope and a fluorescence microscope, most of the hairs were observed in all of Example 1-1, Comparative Example 1-1 and Comparative Example 1-2, regardless of the presence or absence of electrical stimulation. Papilla cells were stained with FDA and confirmed to be viable. That is, it was confirmed that the pulse current stimulation used in this experiment did not affect the survival of dermal papilla cells.

[Incubation device]
The culture apparatus manufactured in Example 1 above was used.

[Preparation of mesenchymal cells]
Dermal papilla cells were used as mesenchymal cells. That is, commercially available human dermal papilla cells (PromoCell) were subcultured, and the human dermal papilla cells at P4 to P6 were used in the experiment. As the culture solution, a culture medium for dermal papilla cells (PromoCell) was used.

[Culture of mesenchymal cells]
As Example 2-1, each culture well was seeded with 1.2×10 ⁴ dermal papilla cells, and the dermal papilla cells were cultured on the bottom surface of each culture well (the culture surface of the culture electrode) for 3 days. bottom. In this Example 2-1, the timing of starting to apply the pulse current (5 hours after seeding of dermal papilla cells, 29 hours, 53 hours), the magnitude of the applied pulse current (0 μA (0 μA/cm ² minus culture surface), 45 μA (22.5 μA/cm ² ), 100 μA (50 μA/cm ² ), 200 μA (100 μA/cm ² ), and 300 μA (150 μA/cm ² )), pulse current continuously. Dermal papilla cells were cultured while applying a pulse current under a plurality of conditions with different application times (16 minutes, 3 hours, 24 hours). On the other hand, as Comparative Example 2-1, dermal papilla cells were cultured for 3 days in the same manner except that no current was applied.

[PCR analysis of hair growth-related genes]
Gene expression levels in dermal papilla cells cultured for 3 days were analyzed by RT-PCR. The base sequences of the primers used in RT-PCR were, for ALP (alkaline phosphatase), the forward primer was "5'-ATTGACCACGGGCACCAT-3'" and the reverse primer was "5'-CTCCACCGCCTCATGCA-3'", which were used as controls. For the GAPDH used, the Forward Primer was "5'-GCACCGTCAAGGCTgAGAAC-3'" and the Reverse Primer was "5'-TGGTGAAGACGCCAGTGGA-3'".

[result]
FIG. 2A shows the results of analyzing the ALP gene expression level of dermal papilla cells cultured with different timings for starting to apply a pulse current. Specifically, FIG. 2A shows the timing at which the pulse current was applied to the dermal papilla cells in Comparative Example 2-1 (“Control” in the figure) in which no current was applied, and Example 2-1. , an example that was 5 hours after seeding the dermal papilla cells ("Day 0" in the figure), an example that was 29 hours ("Day 1" in the figure), and 53 For each of the examples (“Day 2” in the figure) at which time had elapsed, the relative gene expression level (vertical axis “Relative gene expression”).

In each example shown in FIG. 2A, as the pulse current, (1) first apply a current of “+200 μA (approximately +100 μA/cm ² )” for 0.05 seconds, (2) then apply “−200 μA (approximately −100 μA /cm ² )” is applied for 0.05 seconds, and (3) thereafter, no current is applied (0 μA) and held for 0.1 seconds. One pulse of current was applied for 16 minutes.

As shown in FIG. 2A, when a pulse current was applied from 5 hours after seeding dermal papilla cells (“Day 0” in the figure), when no current was applied (“Control” in the figure) ), the ALP gene expression level of the dermal papilla cells decreased.

On the other hand, when the pulse current was applied from 29 hours or 53 hours after seeding the dermal papilla cells ("Day 1" and "Day 2" in the figure), the current was not applied. Compared to , the ALP gene expression level in dermal papilla cells was remarkably increased. Moreover, in particular, when the pulse current was applied from the 29th hour, the ALP gene expression level of the dermal papilla cells was greater than when the pulsed current was applied from the 53th hour.

FIG. 2B shows the results of analysis of ALP gene expression in dermal papilla cells cultured while varying the magnitude of the applied pulse current. Specifically, FIG. 2B shows the magnitude of the pulse current applied to the dermal papilla cells in Comparative Example 2-1 (“Control” in the figure) in which no current was applied, and Example 2-1. 45 μA per culture well (22.5 μA/cm ² per unit area of the culture surface (electrode surface) of each culture well) (“45 μA” in the figure), 100 μA (50 μA/cm ² ) (“100 μA” in the figure), an example of 200 μA (100 μA/cm ² ) (“200 μA” in the figure), and an example of 300 μA (150 μA/cm ² ) (“300 μA” in the figure). For each of them, the relative gene expression level (“Relative gene expression” on the vertical axis in the figure) is shown when the gene expression level in Comparative Example 2-1 is set to “1”.

In each example shown in FIG. 2B, the pulse current was applied for 16 minutes from 29 hours after seeding the dermal papilla cells. Further, in each example, as the pulse current, (1) first, a positive current of a predetermined value (for example, a current of “+45 μA (about +22.5 μA/cm ² )”) is applied for 0.05 seconds, and (2) a predetermined (e.g., a current of "-45 μA (approximately -22.5 μA/cm ² )") for 0.05 seconds, (3) followed by no current (0 μA) for 0.1 seconds. A current was applied with the (1), (2) and (3) of holding as one pulse.

As shown in FIG. 2B, in all cases where a pulse current of 45 μA (22.5 μA/cm ² ) or more was applied, the ALP gene expression level in dermal papilla cells increased compared to the case where no current was applied. In particular, when a pulse current of 100 μA (50 μA/cm ² ) or more was applied, the ALP gene expression level in dermal papilla cells was remarkably increased compared to when no current was applied.

FIG. 2C shows the results of analyzing the ALP gene expression level of dermal papilla cells cultured by changing the time of continuously applying the pulse current (also the total time of applying the pulse current during the culture period). Specifically, FIG. 2C shows Comparative Example 2-1 (“Control” in the figure) in which no current was applied, and Example 2-1, where the pulse current was applied to the dermal papilla cells for 16 minutes. For each of the example ("16min" in the figure), the example that was 3 hours ("3h" in the figure), and the example that was 24 hours ("24h" in the figure), the comparative example The relative gene expression level (“Relative gene expression” on the vertical axis in the figure) is shown when the gene expression level in 2-1 is set to “1”.

In each example shown in FIG. 2C, (1) first, a current of “+200 μA (approximately +100 μA/cm ² )” is applied for 0.05 seconds, and (2) then “−200 μA (approximately −100 μA/cm ² )” is applied. ” is applied for 0.05 seconds, and (3) then no current is applied (0 μA) and held for 0.1 seconds. A current was applied. In addition, the pulse current was applied 29 hours after seeding the dermal papilla cells.

As shown in FIG. 2C, in all cases where the pulse current was applied for 16 minutes or longer, the ALP gene expression level in dermal papilla cells was significantly increased compared to the case where no current was applied.

[Incubation device]
The culture apparatus manufactured in Example 1 above was used.

[Preparation of mesenchymal cells]
Dermal papilla cells were used as mesenchymal cells. That is, commercially available human dermal papilla cells (PromoCell) were subcultured, and the human dermal papilla cells at P4 to P6 were used in the experiment. As the culture solution, a culture medium for dermal papilla cells (PromoCell) was used.

[Culture of mesenchymal cells]
As Example 3-1, 1.2×10 ⁴ dermal papilla cells were seeded in each culture well, and the dermal papilla cells were cultured on the bottom surface of each culture well (culture surface of the culture electrode) for 3 days. bottom. During this culture period, a pulse current of 200 μA (100 μA/cm ² ) was applied to each culture well for 16 minutes starting 29 hours after seeding the dermal papilla cells.

[Collection of epithelial cells]
The back skin tissue was collected from C57BL / 6 mouse fetuses of 18 days of gestational age, and the method reported by Nakao et al. was performed at 4° C. for 1 hour under shaking conditions of 40 rpm to separate the epithelial layer and the mesenchymal layer of the skin tissue. Thereafter, the epithelial layer was treated with 100 U/mL collagenase for 1 hour and 20 minutes, and then treated with trypsin for 10 minutes to isolate epithelial cells.

[Production of hair follicle primordia]
A commercially available 96-multiwell, each well having a round bottom (concave curved, cell-non-adhesive bottom surface) with a cell-non-adhesive coating, as a culture vessel for the formation of follicle primordia. A plate was used.

Dermal papilla cells obtained by culturing while applying an electric current as described above and epithelial cells collected from skin tissue are each collected by trypsin treatment, fresh culture medium is added, and the A cell suspension containing dermal papilla cells and a cell suspension containing the epithelial cells were prepared.

Next, the dermal papilla cells and the epithelial cells are mixed so that the cell density of the dermal papilla cells is 5×10 ³ cells/well and the cell density of the epithelial cells is 1×10 ⁴ cells/well. to seed each well of a 96-multiwell plate. As the culture medium, a mixed medium prepared by mixing a mesenchymal cell culture medium (DMEM+10% FBS+1% penicillin/streptomycin) and HuMedia-KG2 at a volume ratio of 1:1 was used.

Then, in each well, hair follicle primordia were formed by culturing the dermal papilla cells and the epithelial cells in a mixed state for 3 days. That is, as the culture time passed, the cells spontaneously aggregated in each well to form a spherical, floating hair follicle primordium. The hair follicle primordium includes a dermal papilla cell aggregation portion mainly formed by aggregation of dermal papilla cells and an epithelial cell aggregation portion mainly formed by aggregation of epithelial cells. A part of the dermal papilla cell-aggregated part and a part of the epithelial cell-aggregated part were bound.

On the other hand, as Comparative Example 3-1, the same method was used except that instead of the dermal papilla cells cultured while applying the current, the dermal papilla cells were cultured in the same manner except that no current was applied. to form a hair follicle primordium.

[PCR analysis of hair growth-related genes]
Gene expression levels of hair follicle primordia formed by 3-day culture were analyzed by RT-PCR. The nucleotide sequences of the primers used in RT-PCR are, for Versican, Forward Primer is "5'-CCAGCAAGCACAAAATTTCA-3'", Reverse Primer is "5'-TGCACTGGATCTGTTTCTTCA-3'", LEF1 (Lymphoid Enhancer Factor-1 ), Forward Primer is “5′-CCCGATGACGGAAAGCAT-3′”, Reverse Primer is “5′-TCGAGTAGGAGGGTCCCTTGT-3′”, and for ALP, Forward Primer is “5′-ATTGACCACGGgCACCAT-3′”, Reverse Primer is '5'-CTCCACCGCCTCATGCA-3', for Wnt10b, Forward Primer is '5'-CCAAGAGCCGGGCCCGGTGA-3', Reverse Primer is '5'-AAGGGCGGAGGCCGAGACCG-3'', GAPDH used as a control, The Forward Primer was "5'-GCACCGTCAAGGCTGAGAAC-3'" and the Reverse Primer was "5'-TGGTGAAGACGCCAGTGGA-3'".

[Transplantation to mouse]
The hair follicle primordium formed by culturing for 3 days as described above was collected and implanted intradermally into nude mice. That is, a nude mouse was given isoflurane inhalation anesthesia, and its back was disinfected with isodine. Next, using a V-lance micro scalpel (Alcon Japan), a graft wound was formed from the epidermis layer to the lower dermis layer of the skin. Then, 20 hair follicle primordia were injected into this graft wound. The breeding and transplantation experiments of nude mice were conducted in compliance with the guidelines of the Yokohama National University Animal Care and Use Committee.

[result]
FIG. 3 shows the results of analysis of gene expression levels in hair follicle primordia. Specifically, in FIG. 3, for each of the genes (Versican, LEF1, ALP, and Wnt10b) indicated on the horizontal axis, dermal papilla cells cultured without applying current in Comparative Example 3-1 were used to form Dermal papilla cells cultured by applying current in Example 3-1, when the expression level (black bar graph) of the treated hair follicle primordium (“Control” in the figure) was set to “1”. The expression level of the hair follicle primordium (“200 μA” in the figure) formed by using this method (“Relative gene expression” on the vertical axis in the figure) (white bar graph) is shown.

As shown in FIG. 3, for both Versican and ALP, which are characteristic of dermal papilla cells, the gene expression levels of hair follicle primordia containing dermal papilla cells cultured with applied current were It was significantly larger than that of follicle primordia containing cultured dermal papilla cells.

That is, the dermal papilla cells cultured by applying an electric current are not only before forming the hair follicle primordium after the culture, but also after forming the hair follicle primordium by co-culturing with the epithelial cells. In the hair follicle primordium, it was confirmed that the expression levels of hair growth-related genes were increased.

In addition, LEF1 and Wnt10b, which are characteristic of epithelial cells, also showed that the gene expression levels of epithelial cells that formed hair follicle primordium together with dermal papilla cells cultured with applied current were cultured without applying current. In particular, the Wnt10b gene expression level was significantly higher than that of epithelial cells that formed hair follicle primordium together with dermal papilla cells.

That is, in epithelial cells that have formed hair follicle primordium by co-culturing with dermal papilla cells that have been cultured by applying an electric current, the expression level of the hair growth-related gene is remarkably increased in the hair follicle primordium. An unexpectedly remarkable effect was confirmed.

FIGS. 4A and 4B show hair follicle primordia formed using dermal papilla cells cultured with current applied and dermal papilla cells cultured without current applied. 30 shows a photograph of hair regenerated from the hair follicle primordium, taken 30 days after the hair follicle primordium was transplanted into the mouse.

FIG. 5 also shows hair follicle primordia formed using dermal papilla cells cultured with applied current (“Example 3-1” in the figure), and hair follicles cultured without applying current. 30 days after the transplantation of the hair follicle primordium formed using the dermal papilla cells (“Comparative Example 3-1” in the figure) into the mouse, the number of hairs grown from the hair follicle primordium The results of counting the number of hairs (vertical axis in the figure, "number of hair growth") are shown.

As shown in FIGS. 4A and 5, about 40 hairs were grown from hair follicle primordia which were formed using dermal papilla cells cultured by applying current and transplanted into mice. On the other hand, as shown in FIGS. 4B and 5, about 18 hairs were formed using dermal papilla cells cultured without applying current and grown from the hair follicle primordium transplanted into mice. was a book.

That is, the hair follicle primordium produced using dermal papilla cells cultured with applied current is higher than the hair follicle primordium formed using dermal papilla cells cultured without applying current. , it was confirmed that the hair growth ability is remarkably excellent when transplanted into a living body.

Claims (13)

20 μA/cm 2 or ^more of mesenchymal cells, which are dermal papilla cells and/or hair bulb root sheath cells adhered to the surface of the electrode, through the electrode after 10 hours or more have passed since seeding on the electrode surface. A method for culturing mesenchymal cells, comprising culturing while applying a pulse current of 400 μA/cm ² or less . applying the current via the electrode as a first electrode and a second electrode positioned opposite the first electrode;
The method for culturing mesenchymal cells according to claim 1 . 20 μA/cm 2 or ^more of mesenchymal cells, which are dermal papilla cells and/or hair bulb root sheath cells adhered to the surface of the electrode, through the electrode after 10 hours or more have passed since seeding on the electrode surface. By culturing while applying a pulse current of 400 μA/cm ² or less, the mesenchymal cells were activated compared to the mesenchymal cells cultured in the same manner except that the pulse current was not applied. A method for producing activated mesenchymal cells, comprising obtaining applying the current through the electrode as a first electrode and a second electrode positioned opposite the first electrode;
The method for producing the activated mesenchymal cells according to claim 3. In the activated mesenchymal cells, the expression level of at least one hair growth-related gene is increased compared to that in the mesenchymal cells cultured in the same manner except that the pulse current is not applied. are cells,
The method for producing the activated mesenchymal cells according to claim 3 or 4. A method for producing a hair follicle primordium, comprising: forming a hair follicle primordium by co-culturing mesenchymal cells produced by the method according to any one of claims 3 to 5 and epithelial cells. . forming the hair follicle primordium by co-cultivating the mesenchymal cells and the epithelial cells on a cell non-adhesive surface;
The method for producing a hair follicle primordium according to claim 6. The hair follicle primordium formed by the co-culture is formed by the same method except that the mesenchymal cells are produced by the same method as the mesenchymal cells except that the pulse current is not applied. comprising the activated epithelial cells compared to the epithelial cells contained in the activated hair follicle primordium,
The method for producing a hair follicle primordium according to claim 6 or 7. The activated epithelial cells used were mesenchymal cells produced by the same method as the mesenchymal cells except that the expression level of at least one hair growth-related gene was not applied with the pulse current. An epithelial cell that is more increased than that of the epithelial cell contained in the hair follicle primordium formed in the same manner except that
The method for producing a hair follicle primordium according to claim 8. The hair follicle primordium formed by the co-culture is formed by the same method except that the mesenchymal cells are produced by the same method as the mesenchymal cells except that the pulse current is not applied. comprising the activated mesenchymal cells compared to the mesenchymal cells contained in the activated hair follicle primordium,
The method for producing a hair follicle primordium according to any one of claims 6 to 9. The activated mesenchymal cells are hairs formed by the same method except that the mesenchymal cells are produced by the same method as the mesenchymal cells except that the pulse current is not applied. A mesenchymal cell in which the expression level of at least one hair growth-related gene is increased compared to the mesenchymal cell contained in the follicle primordium,
The method for producing a hair follicle primordium according to claim 10. 20 μA/cm 2 or ^more of mesenchymal cells, which are dermal papilla cells and/or hair bulb root sheath cells adhered to the surface of the electrode, through the electrode after 10 hours or more have passed since seeding on the electrode surface. , activating the mesenchymal cells by culturing while applying a pulse current of 400 μA/cm ² or less;
A method for activating mesenchymal cells. In the hair follicle primordium formed by co-culturing mesenchymal cells and epithelial cells,
activating the epithelial cells contained in the hair follicle primordium by using the mesenchymal cells produced by the method according to any one of claims 3 to 5 as the mesenchymal cells;
A method for activating epithelial cells.

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