JP2021083430A - Method for evaluating migration of dermal sheath cup cells - Google Patents

Abstract

To provide: a quality control method for dermal sheath cup cells provided for hair regenerative therapy; and a method or activator for activating the dermal sheath cup cells.SOLUTION: By finding out that the dermal sheath cup cells have migration capability and focusing on the migration capability, quality can be controlled. Also, by focusing on the migration capability, migration factors of the dermal sheath cup cells are identified.SELECTED DRAWING: Figure 6

Description

The present invention relates to the technical field of cell therapy for hair regeneration. More specifically, the present invention relates to a method for evaluating migration of hair bulb sheath cells used for cell therapy for hair regeneration.

Drugs have been mainly administered as a treatment for alopecia and thinning hair, but while continuous administration is required, sufficient effects may not be obtained depending on the subject. In addition, self-implantation surgery is also performed in which a graft is collected from the back of the head and transplanted to the hair loss area, but it is highly invasive, the total amount of hair does not increase even after the surgery, and the hair density that can be transplanted is limited. There is. Therefore, a technique is expected to regenerate hair more safely and effectively by proliferating cells that can become hair collected from the target scalp tissue and returning the cells to the target scalp by autologous transplantation. As the cells used for hair regeneration, hair papilla cells (DP cells), hair bulb sheath cells (Dermal Sheath Cup cells: DSC cells) and the like are used (Patent Document 1: Japanese Patent Application Laid-Open No. 2011). -101648).

The practical application of hair regrowth treatment by autologous transplantation of hair bulb sheath cells is approaching, and it is expected that the effectiveness will be further enhanced and stable hair regrowth will be realized. In order to enhance the effectiveness, it is important to control the quality of the cells to be transplanted, and it is important to obtain high-quality hair bulb sheath cells before transplantation. Quality control of the composition containing hair bulb sheath cells is performed by confirming that the composition is free of cells such as keratinocytes and melanocytes.

Japanese Unexamined Patent Publication No. 2011-101648

A. Tremel et al., Chemical Engineering Science (2009) Vol. 64, Issue 2, Pages 247-253

Conventional quality control is performed by preventing the contamination of keratinocytes and melanocytes into the hair bulb sheath cell composition. However, in such quality control, while it is possible to control the proportion of hair bulb sheath cells in the composition, the cell activity of the hair bulb sheath cells contained in the composition and the hair based on the cell activity The regenerative ability could not be evaluated.

As a result of diligent studies on the quality evaluation method of hair bulb sheath cells, the present inventors focused on the migration ability of hair bulb sheath cells, and it is possible to evaluate the quality of hair bulb sheath cells in vitro. The present invention was reached. Therefore, the present invention relates to the following:
[1] A method for evaluating the migration ability of hair bulb sheath cells in vitro by a migration assay using a hair papilla cell culture supernatant or a medium containing a migration factor of hair bulb sheath cells.
[2] The method of item 1, wherein the migration assay uses a Boyden chamber comprising a bottom chamber and a membrane chamber with a perforated membrane located within the bottom chamber.
[3] Below:
Step of seeding hairball root sheath cells in the membrane chamber;
A step of incubating with a membrane chamber placed in a bottom chamber into which a medium containing a dermal papilla cell culture supernatant or a migrating factor of hair bulb sheath cells is introduced;
The process of measuring hair bulb sheath cells that have moved to the opposite side of the membrane through the pores;
2. The method according to item 2.
[4] The method according to any one of items 1 to 3, wherein the migration factor of the hairball root sheath cell is at least 1 selected from the group consisting of SLP1, EDAA2, NID1 and IGFBP2.
[5] The method according to item 2, wherein the pore size of the perforated membrane is 5 μm to 10 μm.
[6] A method for determining the quality of a composition containing hair bulb root sheath cells based on the migration ability evaluated by the method according to any one of items 1 to 5.
[7] A method for determining the quality of a composition containing hairball root sheath cells based on the migration ability of hairball root sheath cells.
[8] A method for activating hair bulb sheath cells, which comprises culturing hair bulb sheath cells in a solution containing a hair papilla cell culture supernatant or a migration factor for hair root sheath cells.
[9] The method according to item 8, wherein the migration factor of the hairball root sheath cell is at least one selected from the group consisting of SLP1, EDAA2, NID1 and IGFBP2.
[10] The method according to item 8 or 9, which enhances the migration ability of hair bulb sheath cells.
[11] An activator of hairball root sheath cells, comprising at least one selected from the group consisting of SLP1, EDAA2, NID1, and IGFBP2.
[12] The activator according to item 11, which enhances the migration ability of hair bulb sheath cells.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to evaluate the migration ability of hair bulb sheath cells, and it is possible to control the quality of a cell composition containing hair bulb sheath cells based on the migration ability. In addition, by identifying the migration supernatant or migration factor of the hair papilla cell culture that increases the migration ability, it becomes possible to activate the migration of the hair bulb sheath cells or activate the hair root sheath cells using these. ..

FIG. 1 shows a cross-sectional view of one well of the Boyden chamber used in the migration assay. FIG. 1A shows a state in which the membrane chamber is arranged in the bottom chamber, and FIG. 1B shows a state in which the membrane chamber is separated from the bottom chamber. FIG. 2A shows a photograph showing the nuclei of migrating cells fluorescently stained on the lower surface of the membrane chamber in the migration assay. FIG. 2B is a graph showing the number of cells migrating to the underside of the membrane chamber in the migration assay. FIG. 3A is a photograph showing the polymerized actin of non-migrating cells existing on the upper surface of the membrane chamber by staining. FIG. 3B is a photograph showing the polymerized actin of the migrating cells existing on the lower surface of the membrane chamber by staining. FIG. 4A shows the nuclei of cells migrating to the lower surface of the membrane chamber by fluorescent staining by a migration assay using a DP-conditioned medium obtained by changing the DP cell density and an actin polymerization inhibitor and an actin polymerization activator. The photograph is shown. FIG. 4B is a graph showing the number of cells migrating to the underside of the membrane chamber in the migration assay. FIG. 5 shows the results of a membrane antibody assay for identifying secretory proteins such as cytokines or chemokines contained in the DP-conditioned medium and the fibroblast-conditioned medium, and shows the secretory proteins such as cytokines or chemokines that are increased in the DP-conditioned medium. .. FIG. 6 is a graph showing the number of cells that migrated to the lower surface of the membrane chamber after being subjected to a migration assay by changing the concentrations of SLP1, EDAA2, NID1, and IGFBP2, which are secretory proteins such as cytokines or chemokines contained in DP conditioned medium. Is.

One aspect of the present invention is an in vitro migration assay using a medium containing a migration factor of hair papilla cell culture supernatant or hair bulb root sheath cells (hereinafter, also referred to as DSC cells). Regarding the method of evaluating the migratory ability of.

The hair root sheath cell is a cell derived from the sheath cell surrounding the hair bulb among the sheath cells (Dermal Sheath cells: DS cells) surrounding the hair root. The hairball part is located in the deepest part of the hair follicle and refers to a swollen area of the hair root, and is mainly composed of a dermal papilla and a hair matrix cell. The hair follicle part of the scalp sample is incised, the hair bulb part hair root sheath is inverted, the hair papilla is excised, and the hair bulb part hair root sheath from which the hair papilla is removed is cultured to collect the hair bulb part hair root sheath cells. can do. By subculturing hair bulb sheath cells, the number of cells required for transplantation can be increased. Although not intended to be limited to theory, when hair bulb sheath cells are injected into the scalp, the hair bulb sheath cells migrate and localize around the hair follicle, and even the hair papilla. It is considered that it contributes to hair regrowth by differentiating into hair papillae after migrating to. On the other hand, it is considered that the hair bulb sheath cells that could not migrate to an appropriate place are naturally eliminated. Therefore, the migration ability of the hair bulb sheath cells contributes to the quality of the hair bulb sheath cells, that is, the hair regeneration ability of the hair bulb sheath cells when transplanted. It can be determined that the higher the migration ability of the hairball root sheath cells, the higher the quality of the hairball root sheath cells.

Migratory ability means the ability of cells to migrate in tissues or in a culture environment. Cell migration ability is roughly classified into chemotaxis (chemotaxis), haptotaxis (haptotaxis), wound healing, and cell infiltration. Of these, chemotaxis means that cells migrate according to the concentration gradient of chemotaxis factors such as chemokines, and it is considered that hair root sheath cells in the hair bulb migrate based on chemotaxis. Depending on the type of migratory ability to be measured, a method for measuring the migratory ability and an assay kit can be selected.

Cells exert migratory ability by controlling cell morphology and motility by polymerizing and depolymerizing actin. The cells migrating in this way form pseudopodia. Pseudopodia refers to the temporary protrusion of the cytoplasm that forms when cells move, and can be classified into stress fibers, lamellipodia, and filopodia based on their shape. When a cell migrates toward a chemotactic factor, a filamentous pseudopodia is formed in front of the cell (leading end) to search for the environment around the cell containing the chemotactic factor. In addition, the foliate pseudopodia acts to move the cell body, and the stress fiber acts to retract the posterior part (tail) of the cell, so that the cell migrates. Rho family proteins are involved in the polymerization and depolymerization of actin, and migration occurs due to the cooperation of multiple proteins such as RhoA, Rac1, and Cdc42. Therefore, it is known that the migration ability of cells is strongly related to the cell activity as it is, and that cells with high migration ability usually have high cell activity such as proliferative ability (Non-Patent Document 1: A. Tremel et al). ., Chemical Engineering Science (2009) Vol. 64, Issue 2, Pages 247-253).

The dermal papilla cell culture supernatant refers to the supernatant of a culture obtained by culturing dermal papilla cells in advance, and can also be referred to as a dermal papilla conditioned medium (DP conditioned medium). As the medium for culturing the dermal papilla cells, any medium used for culturing the dermal papilla cells can be used. As an example, serum-free Follicle Dermal Papilla Cell Growth Medium (PromoCell), AmnioMAX (Thermo Fisher Scientific) , Follicle Dermal Papilla Cell Basal Medium (Takara-bio), etc. are used. The dermal papilla cells can be seeded in medium at any density, eg 70% confluent, _{and the supernatant of the culture cultured in a 37 ° C. CO 2} atmosphere for at least 1 hour, eg 24-72 hours, can be used. In these hair papilla cell culture supernatants, various factors secreted by hair papilla cells, such as various proteins such as chemokines, cytokines, and enzymes, are secreted, and these factors alone or in collaboration with each other cause hair. Can attract bulbal chemo-sheath cells. In the present invention, the migration ability of hair bulb sheath cells can be evaluated by using the hair papilla cell culture supernatant in the migration assay. Further, instead of the hair papilla cell culture supernatant, a medium containing a migration factor of hair bulb sheath cells can also be used.

The migration factor of hairball root sheath cells refers to a substance capable of increasing the migration ability of hairball root sheath cells. As an example, it may be a component contained in the dermal papilla cell culture supernatant, or may be a component screened from a compound library or the like. Examples of growth factors for hair bulb sheath cells include stomatin-like protein-1 (SLP1), ectodysplasin-A2 (EDAA2), and Nidogen-1 (Nidogen-1:). NID1) and Insulin Like Growth Factor Binding Protein 2: IGFBP2. These components are thought to act via receptors expressed on hair bulb sheath cells.

The evaluation of migration ability can be evaluated by a migration assay. The specific measurement method can be selected according to the type of migratory ability to be measured. As an example, the migration ability of hair bulb sheath cells is evaluated in vitro using a Boyden chamber 1 including a bottom chamber 2 and a membrane chamber 3 having a perforated membrane arranged in the bottom chamber 2. be able to. The Boyden chamber 1 may further include a lid 4 that covers the membrane chamber 3. The configuration of the Boyden chamber 1 is shown in FIG. Although only one well is disclosed in FIG. 1, a plate with multiple wells, such as 24- or 96-well wells, can be used in the migration assay. The bottom chamber 2 is formed by the bottom chamber bottom 5 and the bottom chamber wall 6, and can hold the solution 10. The membrane chamber 3 has a perforated membrane as the bottom portion 7 of the membrane chamber, is formed by the bottom portion 7 of the membrane chamber and the wall portion 8 of the membrane chamber, and further includes a shoulder portion 9 at the upper end of the wall portion 8 of the membrane chamber. The cells 12 can be placed together with the cell culture medium 11 on the bottom 7 of the membrane chamber of the membrane chamber 3. The bottom 7 of the membrane chamber 3 is smaller than the bottom 5 of the bottom chamber of the bottom chamber 2, and the membrane chamber 3 can be arranged in the bottom chamber 2. Further, the height of the membrane chamber wall portion 8 of the membrane chamber 3 is formed lower than the height of the bottom chamber wall portion 6 of the bottom chamber 2. As a result, when the membrane chamber 3 is arranged in the bottom chamber 2, the shoulder portion 9 of the membrane chamber 3 contacts the bottom chamber wall portion 6, so that the membrane chamber bottom portion 7 of the membrane chamber 3 contacts the bottom chamber 2. It can be arranged in the bottom chamber 2 in a state where it is not. The size of the pores in the perforated membrane of the membrane chamber 3 is 3 μm to 14 μm, preferably 5 to 10 μm, for example 8 μm, and hairball root sheath cells can move through the pores. The surface on which the cells 12 are seeded in the membrane chamber 3 is the upper surface, and the opposite side is the lower surface. The amount of the solution 10 introduced into the bottom chamber 2 is adjusted so that the perforated membrane is immersed in the solution when the membrane chamber 3 is arranged in the bottom chamber 2.

In the migration assay using the Boyden chamber 1, the method for evaluating the migration ability of hairball root sheath cells is specifically described below.
Step of seeding hairball root sheath cells in membrane chamber 3;
A step of incubating a membrane chamber 3 in a bottom chamber 2 into which a medium containing a hair papilla cell culture supernatant or a migrating factor of hair bulb sheath cells is introduced;
The process of measuring hair bulb sheath cells that have moved to the opposite side of the membrane through the pores;
including.

The number of hairball root sheath cells seeded into the membrane chamber 3 can be appropriately determined according to the size of the membrane chamber 3. As an example, when a 24-well plate Boyden chamber is used, about 10,000 to 50,000 cells can be seeded per well, for example, about 40,000 cells. The cell medium arranged in the membrane chamber 3 may be any medium for culturing hair bulb sheath cells, but a serum-free medium is preferable, and for example, serum-free AmnioMax can be introduced together with the cells. When a serum-free medium is used, the medium of the culture containing the cultured hair bulb sheath cells or the freeze-thawed hair bulb sheath cells is replaced with a serum-free medium, and then seeded in the membrane chamber 3.

A solution containing a hair papilla cell culture supernatant or a migration factor of hair bulb sheath cells is introduced into the bottom chamber 2. The solution introduced into the bottom chamber 2 may be a medium, preferably a serum-free medium, such as serum-free AmnioMax. By arranging the membrane chamber 3 in the bottom chamber 2, the perforated membrane of the membrane chamber 3 is incubated in a state of being immersed in the solution of the bottom chamber 2. The incubation time can be appropriately adjusted according to the pore size of the perforated membrane of the membrane chamber 3 to be used and the number of cells to be used, and is 30 minutes to several hours, preferably 1 to 10 hours, more preferably 2 It can be incubated for ~ 8 hours, more preferably 3-6 hours. Incubate under normal culture conditions, eg 37 ° C. 5% CO _{2 atmosphere.}

The number of hair bulb sheath cells that have migrated to the opposite side of the membrane through the pores can be measured by any method. As an example, the membrane chamber 3 can be used as it is, or the perforated membrane can be taken out, nuclear staining is performed with Hoechst, DAPI, or the like, and the lower surface of the effective membrane can be photographed under a fluorescence microscope. The number of cells can be determined for the captured image. Such a determined number of cells can be evaluated as migration ability. Furthermore, an experiment was conducted on a composition containing a plurality of lots of hair bulb sheath cells under the same conditions, and the number of migrating hair root sheath cells existing on the lower surface of the perforated membrane and the composition used for the experiment. The relationship with the quality of the hair can be determined in advance. Thereby, for the newly tested composition, the quality of the composition containing the hairball root sheath cells can be determined based on the number of migrating hairball root sheath cells present on the lower surface of the perforated membrane. it can. The quality determination method can be performed on a composition containing hairball root sheath cells before transplantation, for example, a composition containing hairball root sheath cells after freeze-thaw. For a composition containing hair bulb sheath cells, a part thereof can be subjected to a quality determination method, and the use of the composition can be examined according to the determined quality.

A solution containing the hair papilla cell culture supernatant and the migration factor of the hair bulb sheath cell can enhance the migration ability of the hair bulb sheath cell. The migration ability of cells is strongly related to cell activity, and it is known that cells with high migration ability usually have high cell activity such as proliferative ability. Therefore, it can be said that at least one factor selected from the group consisting of hair papilla cell culture supernatant and hair bulb sheath cell migration factor, for example, SLP1, EDAA2, NID1, and IGFBP2, is a hair bulb sheath cell activator. it can. The hairball root sheath cell activator can be added at any time before, during, or after culturing the cultured hairball root small cells. In addition, the hairball root sheath cell activator may be directly administered to the scalp. Yet another aspect of the invention comprises culturing the hair bulb sheath cells in a solution containing the hair papilla cell culture supernatant or the migration factor of the hair bulb sheath cells. Regarding activation method. By this method, hairball root sheath cells can be activated in vitro, and the activated hairball root sheath cells have enhanced migration ability. The hairball root sheath cells may be activated when the hairball root sheath cells are cultured and proliferated, or the proliferated hair bulb sheath cells may be activated before transplantation. Cell compositions containing hair bulb sheath cells may be cryopreserved after proliferation, but cell activity usually temporarily decreases after freeze-thaw. In that case, after freezing and thawing, the hair bulb sheath cells frozen and thawed in a solution containing the hair papilla cell culture supernatant and the migration factor of the hair bulb sheath cells are cultured to cause the hair bulb sheath cells. Can be activated.

SLP1 refers to a stomatin-like protein 1 and refers to a membrane protein having a dichotomous structure containing a stomatin domain and a sterol carrier protein-2 domain (J Biol Chem. 2009 Oct 16; 284 (42): 29218-29). EDAA2 refers to ectodisplacin-A2 and is one of the members of the tumor necrosis factor family involved in ectoderm development (Structure. 2003 Dec; 11 (12): 1513-20.). NID1 refers to Nidgen-1, which is one of the cell adhesion factors (Genomics 1995 May; 27 (2) 245-250). IGFBP2 refers to insulin-like growth factor-binding protein 2, which binds to insulin-like growth factor (IGF) and regulates IGF affinity for receptors, distribution (local release), and metabolism (stabilization / degradation). Involved (Cancer Prev Res (Phila). 2010 Oct; 3 (10): 1222-34).

Yet another aspect of the invention relates to a method of screening for migration factors of hairball root sheath cells. In this screening method, the migration factor of the hair bulb sheath cell can be determined by subjecting the test substance to the migration assay and evaluating the migration ability of the hair bulb sheath cell. For the migration assay, it is preferable to use the Boyden chamber 1. The screening method using the migration assay using the Boyden chamber 1 is specifically described in the following steps:
Step of seeding hairball root sheath cells in membrane chamber 3;
A step of incubating a membrane chamber 3 in a bottom chamber 2 into which a solution containing a test substance is introduced;
It includes the step of measuring hair bulb sheath cells that have moved to the opposite side of the membrane through the pores. The solution containing the test substance is preferably a medium containing the test substance, and more preferably a serum-free medium. A solution containing no test substance can be used as a negative control, and a hair papilla cell culture supernatant can be used as a positive control, and the test substance depends on the number of hair bulb sheath cells that have moved to the opposite side of the membrane through the pores. It is possible to determine the activation effect on the migration ability of the hair bulb sheath cells of the hair bulb.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples, and various aspects can be taken as long as the problems of the present invention can be solved. All references referred to herein are incorporated herein by reference in their entirety.

Example 1: Detection of migration activity of hairball root sheath cells based on DP-conditioned medium
Acquisition of hair root sheath cells The hair follicle of the scalp sample was incised, the hair root sheath was inverted, and the dermal papilla was excised. Hairball root sheath cells from which the hair papilla was resected were cultured in AmnioMAX medium to obtain hairball root sheath cells.

The 1 × 10 ⁶ ~5 × 10 ⁶ cells of dermal papilla cells obtained from obtained from acquired human scalp conditioned medium hair follicles, were seeded in flasks (Corning Inc.) containing the AmnioMAX medium 10 ml, 37 The cells were cultured at ° C. in a 5% CO ₂ atmosphere for 2 days, and the culture supernatant was obtained and used as a DP-conditioned medium. Similarly, keratinocytes were cultured, and the culture supernatant was obtained to obtain a keratinocyte-conditioned medium. Similarly, fibroblasts were cultured, and the culture supernatant was obtained to obtain a fibroblast-conditioned medium.

Measurement of migration activity of hair bulb sheath cells The acquired 2 × 10 ⁵ to 1 × 10 ⁶ hair bulb sheath cells were seeded in a flask (manufactured by Corning) containing 10 ml of AmnioMAX medium. , Incubated for 2-5 days until subconfluent. The medium was replaced with a serum-free medium, and the cells were cultured overnight. Then, 40,000 hairball root sheath cells were seeded in the membrane chamber of the migration plate (manufactured by Corning). The test solution was placed in the bottom chamber and _{cultured at 37 ° C. in a 5% CO 2} atmosphere for 5 hours. As a control, serum-free AmnioMax (−) (manufactured by Thermo Fisher Scientific) was used, and DP-conditioned medium and keratinocyte-conditioned medium were used as test solutions. After culturing for 5 hours, the membrane chamber was removed and fixed with 4% paraformaldehyde. After fixation, it was stained with Hoechst (Thermo Fisher Scientific), and the lower surface of the membrane chamber was photographed with a fluorescence microscope (Olympus) (FIG. 2A). Hoechst-positive cells were counted (Fig. 2B). After fixation, the mixture was incubated with a fluorescently labeled phalloidin (Abcam) solution for 1 hour. The upper surface of the membrane chamber using serum-free AmnioMax (−) medium (FIG. 3A) and the lower surface of the membrane chamber using DP-conditioned medium (FIG. 3B) were photographed with a fluorescence microscope (Olympus), respectively. The intracellular polymerized actin staining was weak in the cells existing on the upper surface where the hairball root sheath cells were not migrating. On the other hand, the cells existing on the lower surface where the hair bulb sheath cells migrated were strongly stained with polymerized actin, and stress fibers, lamellipodia, and filopodia were observed.

In the measurement of the migration activity of hair bulb sheath cells, 70% confluent DP conditioned medium, 90% confluent DP conditioned medium, and actin polymerization inhibitor-containing AmnioMax (-) (Inhibitor: Rho GTPase Inhibitor, manufactured by R & D Systems) were used as test solutions. ), AmnioMax (−) containing an actin polymerization activator (Activator: Rho GTPase Actibator, manufactured by R & D Systems) was used. As the DP conditioned medium, a conditioned medium obtained by culturing 70% confluent DP cells and 90% confluent DP cells was used (FIG. 4).

Example 2: Identification of migration factors Chemokines and cytokines assay kits (manufactured by Ray-Bio) are applied with DP conditioned medium and fibroblast conditioned medium, and the cytokines and chemokines contained in each conditioned medium are applied according to the product instructions. Identification was performed (Fig. 5). Based on the literature, the effects of increased amounts of chemokines and cytokines as migration factors were estimated in DP-conditioned medium as compared to fibroblast-conditioned medium. As a result, EDA-A2, IGFBP-2, IGFBP-rp1, Latent TGF-βbp1, MMP1, Nidgen-1, MMP-20, SLP1, and Thrombospondin-1 were selected as candidates for hairball root sheath cell migration factor.

Determination of migration factors 2 × 10 ⁵ to 1 × 10 ^{6 obtained} hairball root sheath cells were seeded in a flask (manufactured by Corning) containing 10 ml of AmnioMAX medium until they became subconfluent. Incubated for days. The medium was replaced with a serum-free medium, and 40,000 hairball root sheath cells were seeded in the membrane chamber of a migration plate (manufactured by Corning). The test solution was placed in the bottom chamber and _{cultured at 37 ° C. in a 5% CO 2} atmosphere for 5 hours. As controls, serum-free AmnioMax (−) (manufactured by Thermo Fisher Scientific), PBS, and DP-conditioned medium were used. As a test solution, AmnioMAX medium was used in which SLP1, EDAA2, Nid1, and IGFBP2 were serially diluted to 1 nM, 10 nM, 100 nM, and 1 μM, respectively. After culturing for 5 hours, the membrane chamber was removed and fixed with 4% paraformaldehyde. After fixation, the cells were stained with Hoechst (Thermo Fisher Scientific), the lower surface of the membrane chamber was photographed with a fluorescence microscope (Thermo Fisher Scientific), and Hoechst-positive cells were counted (FIG. 6).

1 Boyden chamber 2 Bottom chamber 3 Membrane chamber 4 Lid 5 Bottom chamber bottom 6 Bottom chamber wall 7 Membrane chamber bottom 8 Membrane chamber wall 9 Shoulder 10 Solution 11 Cell culture 12 cells

Claims (12)

A method for evaluating the migration ability of hair bulb sheath cells in vitro by a migration assay using a hair papilla cell culture supernatant or a medium containing a migration factor of hair bulb sheath cells. The method of claim 1, wherein the migration assay uses a Boyden chamber comprising a bottom chamber and a membrane chamber with a perforated membrane located within the bottom chamber. below:
Step of seeding hairball root sheath cells in the membrane chamber;
A step of incubating a membrane chamber in a bottom chamber into which a medium containing a dermal papilla cell culture supernatant or a migrating factor of hair bulb sheath cells is introduced;
The process of measuring hair bulb sheath cells that have moved to the opposite side of the membrane through the pores;
2. The method of claim 2. The method according to any one of claims 1 to 3, wherein the migration factor of the hairball root sheath cell is at least one selected from the group consisting of SLP1, EDAA2, NID1, and IGFBP2. The method according to claim 2, wherein the pore size of the perforated membrane is 5 μm to 10 μm. A method for determining the quality of a composition containing hair bulb root sheath cells based on the migration ability evaluated by the method according to any one of claims 1 to 5. A method for determining the quality of a composition containing hairball root sheath cells based on the migration ability of hairball root sheath cells. A method for activating hair bulb sheath cells, which comprises culturing hair bulb sheath cells in a solution containing a hair papilla cell culture supernatant or a migration factor for hair bulb sheath cells. The method according to claim 8, wherein the migration factor of the hairball root sheath cell is at least one selected from the group consisting of SLP1, EDAA2, NID1, and IGFBP2. The method according to claim 8 or 9, which enhances the migration ability of hair bulb sheath cells. An activator of hairball root sheath cells, comprising at least one selected from the group consisting of SLP1, EDAA2, NID1, and IGFBP2. The activator according to claim 11, which enhances the migration ability of hair bulb sheath cells.

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