KR102454189B1 - Method for preparing dermal papilla cell and cell spheroid under hypoxic culture condition - Google Patents

Abstract

The present invention relates to a method for producing dermal papilla cells, human dermal papilla cell-derived growth factors and cell spheroids with improved hair induction ability. According to the method for producing dermal papilla cells according to the present invention, it is possible to obtain dermal papilla cells excellent in cell proliferation ability, migration ability, aging inhibition ability, and hair growth induction ability despite continuous subculture, and by culturing dermal papilla cells in a hypoxic state A method for mass production of growth factors can be provided, and further, cell spheroids capable of inducing hair follicles can be obtained from dermal papilla cells of a passage in which hair follicles are not formed by the prior art, thus preventing or treating hair loss, hair growth Or it can be usefully used in the development of quasi-drugs and cosmetic compositions for promoting hair growth.

Description

Method for preparing dermal papilla cells and cell spheroids using hypoxic conditions {Method for preparing dermal papilla cells and cell spheroids under hypoxic culture condition}

The present invention relates to a method for producing dermal papilla cells, human dermal papilla cell-derived growth factors and cell spheroids with improved hair induction ability.

Dermal papilla cells (DPCs) are cells located in the dermal papilla at the bottom of the hair follicle, and capillaries are distributed in the dermal papilla cells. factor) to regulate the growth of hair follicle epithelial cells. In order for human hair to grow, the action of various factors around the hair follicle and the interaction of epidermal cells and dermal papilla cells in the hair follicle are very important, and hair growth and cycle are regulated by these interactions. During the growth phase, when hair is actively growing, active proliferation and differentiation of dermal papilla cells occur actively, hair growth is stopped, and these cells are killed during the regression, resting, and depilatory phases when hair loss occurs. Therefore, since the proliferation and death of dermal papilla cells are closely related to hair growth and hair loss, prolonging the growth phase or inhibiting apoptosis by inducing their proliferation and shortening the regressive, resting, and depilatory phases will be a treatment method for improving hair loss. is fed In addition, cell division and migration in the vicinity of the dermal papilla are closely related to hair growth. During the growth phase, new hair is generated from the dermal papilla. Cells are activated by various cytokines, hormones, etc., and cell movement to the dermal papilla appears. will affect the growth of

Currently, a technique for culturing and transplanting hair follicle cells in vitro is being attempted as a hair loss treatment method. However, if dermal papilla cells are isolated and cultured in vitro, there is a problem that the ability to induce hair follicles is gradually lost after the 3rd or 4th passage.

Under this background, the present inventors tried to discover a new dermal papilla cell activation method that can enhance the function of dermal papilla cells. As a result, it was confirmed that the proliferation ability, migration ability, aging inhibition ability, hair growth induction ability and hair growth promoting ability of human-derived dermal papilla cells cultured under hypoxic conditions were remarkably improved, and a conditioned medium prepared using human-derived dermal papilla cells. The effect on hair growth was confirmed. Furthermore, the hair growth-inducing effect of cell spheroids obtained by additionally hanging drop culture of dermal papilla cells cultured under normoxic or hypoxic conditions through mouse in vivo experiment was confirmed, and hair loss prevention and hair growth promotion ability using this The present invention was completed by developing a composition having

Accordingly, an object of the present invention is to pre-culture the dermal papilla cells; And to provide a method for producing dermal papilla cells comprising a; and sub-culturing the pre-cultured dermal papilla cells under hypoxic conditions.

Another object of the present invention comprises the steps of pre-culturing dermal papilla cells; And it is to provide a method for producing a large amount of human growth factors from the dermal papilla cells comprising a; and sub-culturing the pre-cultured dermal papilla cells in a hypoxic state having an oxygen partial pressure of 1 to 5%.

Another object of the present invention is to provide a conditioned medium composition of human dermal papilla cells obtained by the method for producing the human growth factor in large quantities.

Another object of the present invention is to provide a method for producing a dermal papilla cell spheroid, further comprising the step of culturing the subcultured dermal papilla cells (hanging drop culture).

Another object of the present invention is to provide a composition for preventing hair loss or promoting hair growth containing the dermal papilla cells, conditioned medium composition or dermal papilla cell spheroids prepared by the above method as an active ingredient.

In order to solve the above problems, the present invention comprises the steps of pre-culturing dermal papilla cells; and sub-culturing the pre-cultured dermal papilla cells under hypoxic conditions; provides a method for producing dermal papilla cells comprising.

The present invention also comprises the steps of pre-culturing dermal papilla cells; And it provides a method for producing a large amount of human growth factors from the dermal papilla cells, including; sub-culturing the pre-cultured dermal papilla cells in a hypoxic state having an oxygen partial pressure of 1 to 5%.

The present invention also provides a conditioned medium composition of human dermal papilla cells obtained by the above method.

The present invention also provides a composition for preventing hair loss or promoting hair growth containing the dermal papilla cells prepared by the above method or the conditioned medium composition as an active ingredient.

The present invention also provides a method for producing a dermal papilla cell spheroid, further comprising the step of culturing the subcultured dermal papilla cells under hypoxic conditions (hanging drop culture).

The present invention also provides a composition for preventing hair loss or promoting hair growth containing the dermal papilla cell spheroids as an active ingredient.

According to the method for producing dermal papilla cells according to the present invention, it is possible to obtain dermal papilla cells excellent in cell proliferation ability, migration ability, aging inhibition ability, and hair growth induction ability despite continuous subculture, Even in this case, there is an excellent effect in which the hair follicle inducing ability is significantly improved compared to the dermal papilla cells not cultured under hypoxia, so it can be usefully used as a hair loss prevention or treatment agent, quasi-drugs for hair growth or hair growth promotion, and a cosmetic composition.

In addition, according to the present invention, it is possible to provide a method for mass production of growth factors by culturing dermal papilla cells under hypoxic conditions, and furthermore, cells capable of inducing hair follicles from dermal papilla cells of a passage in which hair follicles are not formed in general culture. You can get spheroids.

1 is a result of confirming the degree of cell proliferation by measuring absorbance with an ELISA reader after culturing dermal papilla cells in hypoxia.
Figure 2 is the result of confirming the cell migration ability after culturing the dermal papilla cells in hypoxia.
3 is a result confirming the aging inhibitory effect after culturing the dermal papilla cells in hypoxia.
4 is a result of confirming the ALP1 level of the dermal papilla cells according to the hypoxic culture of the present invention.
5 is a result confirming the effect of increasing the growth factor of dermal papilla cells according to the hypoxic culture of the present invention.
Figure 6 is the result of confirming the hair growth effect by measuring the weight of the newly grown hair 14 days after subcutaneous injection of dermal papilla cells cultured under hypoxic conditions in an experimental animal.
7 is a result of subcutaneous injection of dermal papilla cells cultured under hypoxic conditions to an experimental animal and confirming the effect of inducing hair follicles and the rate of hair follicle growth.
8 is a result confirming the effect of improving the proliferation rate of dermal papilla cells according to hypoxic conditions by measuring absorbance with an ELISA reader after culturing dermal papilla cells in a hypoxic state with different oxygen concentrations.
Figure 9 is the result of confirming the hair follicle formation effect of dermal papilla cell spheroids (DPS) obtained by subculturing the dermal papilla cells up to passage 8 to 9 under normoxic conditions or hypoxic conditions, and then additionally culturing dermal papilla cells (DPS) by patch analysis. As a positive control, mouse-derived dermal papilla cells (DPC) were used.
10 is a result confirming the hair growth effect of the culture medium according to the hypoxic culture of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of pre-culturing dermal papilla cells; and sub-culturing the pre-cultured dermal papilla cells under hypoxic conditions; provides a method for producing dermal papilla cells comprising.

As used herein, the term "Dermal papilla cell (DPC)" is a cell present at the base of the hair follicle, and is responsible for hair growth and hair follicle cycle regulation by supplying oxygen and nutrients to the cells constituting the hair follicle. Therefore, when the proliferation of dermal papilla cells is promoted, hair becomes healthy, hair growth is promoted, and hair loss can be prevented. Because hair growth proceeds as the epithelial cells surrounding the dermal papilla divide to form a hair shaft, the dermal papilla cells play an important role in regulating the division of epithelial cells. Also, in androgenetic alopecia, the site where male hormones act on the hair follicle is also the dermal papilla, and the dermal papilla cells play a very important role in hair growth. It was confirmed that the growth, proliferation, and follicle-forming ability of human-derived dermal papilla cells were increased through additional suspension culture.

As used herein, the term "hypoxia" refers to the presence of an amount of oxygen less than that of general cell culture conditions, and conditions that can proliferate the dermal papilla cells of the present invention are included without limitation, Preferably in about 5% carbon dioxide, it is preferable to incubate for 6 hours to 72 hours under the conditions of 1% to 5% oxygen. According to an embodiment of the present invention, the hypoxic state can be induced by treating the culture medium with cobalt chloride (Cocl ₂ ). Cobalt chloride replaces iron in the heme moiety and maintains the reduced state, which can cause hypoxic state.

The dermal papilla cells of the present invention are characterized in that they are derived from humans.

In a specific example of the present invention, human dermal papilla cells (hDPC) were pre-cultured in FDPG (Folicle Dermal Papilla cell Growth media) medium, and then subcultured under hypoxic conditions to prepare dermal papilla cells (Example) One). As a result, when cultured in hypoxic conditions, it was confirmed that the proliferation of dermal papilla cells was significantly increased compared to when cultured in normoxic conditions (FIG. 1), cell migration ability was significantly increased (FIG. 2), and cellular senescence was inhibited and it was confirmed that the lifespan of the cells was increased (FIG. 3).

In addition, in a specific example of the present invention, it was confirmed that ALP1 mRNA expression increased in dermal papilla cells cultured in hypoxia, and the amount of ALP-positive cells increased ( FIG. 4 ). Alkaline phosphatase (ALP) is known as an indicator of angiogenesis in the hair growth cycle and is used as an indicator of hair growth because the enzyme activity increases when the hair is induced into the growth phase. Therefore, it can be confirmed that the hair follicle inducing ability of the dermal papilla cells is improved from the increase of ALP-positive cells.

The method for producing the dermal papilla cells of the present invention may be a method having one or more of the following characteristics compared to the case of culturing under normal conditions by culturing the dermal papilla cells in a hypoxic state.

i) increase in cell proliferative capacity;

ii) increased cell migratory capacity;

iii) inhibition of cellular senescence;

iv) induction of the hair growth phase;

v) increased expression of growth factors.

In another aspect, the present invention comprises the steps of pre-culturing dermal papilla cells; And it provides a method for producing a large amount of human growth factors from the dermal papilla cells, including; sub-culturing the pre-cultured dermal papilla cells in a hypoxic state having an oxygen partial pressure of 1 to 5%.

Specifically, the present invention relates to human growth factors such as BMP6, FGF17, FGF22, FIGF, GDNF, VEGFA, PSPN, JAG2, IL4, VEGF, etc. by culturing human-derived dermal papilla cells obtained from a bald patient in an appropriate medium and hypoxic conditions. It provides a way to manipulate it so that it can be synthesized. The growth factor may be preferably ALP1, VEGF and GDNF.

The conditioned medium of the present invention is preferably prepared by culturing for 6 to 72 hours in a hypoxic state containing 1 to 5% oxygen, and further culturing in a serum-free medium, more preferably 2% oxygen It may be prepared by culturing for 3 days in a hypoxic state comprising a, and culturing for 3 days in a serum-free medium.

According to an embodiment of the present invention, the dermal papilla cells obtained from humans are obtained by subculture in FDPG medium, then cultured in a hypoxic state of 2%, and the culture medium obtained by further culturing in a serum-free medium is centrifuged and filtered to condition de medium was prepared.

In another aspect, the present invention provides a conditioned medium composition obtained by culturing dermal papilla cells obtained from a human under hypoxic conditions and further culturing in a serum-free medium.

As used herein, the term "conditioned medium" means cell suspension cultured in liquid suspension and when reaching the logarithmic growth phase, the peak of cell division, the dividing cells are removed by centrifugation or filtration, and only the culture medium is collected and mixed with the culture substrate. It says one badge. This uses an unknown growth factor extracted from dividing cells in the medium, and is widely used for cell plating at low density or protoplast culture.

The conditioned medium composition in the present invention is a composition comprising a solution in which the dermal papilla cells are removed from a medium in which human-derived dermal papilla cells are cultured. Refers to a composition, preferably including BMP6, FGF17, FGF22, FIGF, GDNF, VEGFA, PSPN, JAG2, IL4, VEGF, and more preferably ALP1, VEGF and GDNF.

According to one embodiment of the present invention, in order to confirm the effect of the conditioned medium of the present invention, as a result of culturing human-derived dermal papilla cells in the conditioned medium prepared as above for 6 to 12 hours under hypoxia, 9 mRNA expression was increased, and it was confirmed that the expression of VEGF and GDNF increased (FIG. 5). In addition, as a result of photographing hair follicles after culturing normal mouse hair in a conditioned medium composition for 72 hours, an increase in hair length was confirmed ( FIG. 10 ). Therefore, it was found that the composition of the present invention is useful as a medium for hair growth.

In another aspect, the present invention comprises the steps of pre-culturing dermal papilla cells; And it provides a composition for preventing hair loss or promoting hair growth, comprising the dermal papilla cells produced through; and subculture of the pre-cultured dermal papilla cells in a hypoxic state.

The hypoxic conditions are the same as described above.

The composition of the present invention is effective in preventing hair loss, promoting hair growth, and hair growth.

In one embodiment of the present invention, as a result of subcutaneous injection of dermal papilla cells produced by hypoxic culture into mice, the weight of the grown hair increased more than 3 times compared to dermal papilla cells cultured under normoxic conditions, confirming that the hair growth effect is excellent. There was (Fig. 6).

In addition, as a result of transplanting dermal papilla cells produced by hypoxic culture into mice, it was confirmed that hypoxia-cultured dermal papilla cells had superior hair follicle-forming ability and hair follicle growth rate than dermal papilla cells cultured in normoxia (Fig. 7).

The composition for preventing hair loss or promoting hair growth of the present invention may further include the conditioned medium prepared above. That is, the composition may include only dermal papilla cells, only the conditioned medium composition, or both the dermal papilla cells and the conditioned medium composition.

The composition for preventing hair loss or promoting hair growth of the present invention may be a cosmetic composition.

Components included in the cosmetic composition of the present invention include components commonly used in cosmetic compositions other than dermal papilla cells, for example, conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. do.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing agent , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto.

According to another aspect of the present invention, the composition for preventing hair loss or promoting hair growth of the present invention may be a pharmaceutical composition for preventing hair loss or promoting hair growth comprising the dermal papilla cells and/or conditioned medium composition produced by hypoxic culture as an active ingredient have.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

In another aspect, the present invention comprises the steps of pre-culturing dermal papilla cells; Passaging the pre-cultured dermal papilla cells in normoxic or hypoxic state; And it provides a method for producing a dermal papilla cell spheroid, comprising the step of culturing the subcultured dermal papilla cells in a hypoxic state (hanging drop culture).

As used herein, the term "dermal papilla cell spheroid (DPS)" refers to the form of a three-dimensional cellular tissue, and refers to the existence of cells in an aggregated form without dissociation. In general, it refers to a three-dimensional structure in which cells are aggregated to such a degree that the cross section can be seen as circular or oval. .

The cell aggregate is a method capable of culturing the cell aggregate in a three-dimensional form such as hanging drop culture, spinner flask culture, and three-dimensional cell culture using a hemispherical microwell plate. can be manufactured.

In an embodiment of the present invention, dermal papilla cell spheroids were prepared through suspension culture. The dermal papilla cells were suspended in the culture medium to make them clump in the lower part, and the culture medium was added to keep the cells and suspension cells from drying out and cultured for 6 days.

After 6 days, the cell spheroid is preferably of a size that can maintain a three-dimensional shape while maintaining the activity of the cells in the cell aggregate, and specifically, the cell aggregate may have a diameter of 250 μm or less, 100 to 250 μm.

In an embodiment of the present invention, dermal papilla cell spheroids (DPS) obtained by additionally culturing dermal papilla cells subcultured up to passage 8 to 9 under hypoxic or normoxic conditions are mixed with epidermal cells to nude mice, etc. As a result of subcutaneous transplantation, it was confirmed that hair follicles or hairs were observed at a level similar to that of the positive control (mouse-derived dermal papilla cells, DPC) only in mice transplanted with dermal papilla cell spheroids (DPS-Hyp) obtained through suspension culture after hypoxic conditions. could be (Fig. 9). No hair follicles or hairs were observed when dermal papilla cell spheroids (DPS-Nor) obtained by suspension culture after culturing under normal oxygen conditions and dermal cells as a negative control were transplanted.

In the method for preparing dermal papilla cell spheroids of the present invention, the dermal papilla cells may be obtained from humans.

In an embodiment of the present invention, unlike mouse-derived dermal papilla cells (DPC), human-derived dermal papilla cells lost their ability to form hair follicles even after suspension culture after passage 8-9, but cultured under hypoxic conditions until passage 8-9. It was confirmed that hair follicles were formed when one dermal papilla cell was cultured in suspension, and from this, it can be seen that the hair follicle-forming ability of the dermal papilla cells was regenerated through the suspension culture under hypoxic conditions.

In another aspect, the present invention provides a composition for preventing hair loss or promoting hair growth containing the dermal papilla cell spheroids prepared by the above method as an active ingredient.

The composition may be a cosmetic composition or a pharmaceutical composition, and the components that may be additionally included depending on the type of composition are the same as described above.

The composition for preventing hair loss or promoting hair growth of the present invention can be used for cell transplantation.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these Examples and the like are intended to explain the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereby.

Example 1. Hypoxic culture of human dermal papilla cells (hDPC)

Follicle Dermal Papilla cell growth media (Promocell, Heidelberg, Germany) medium, and cultured in a 5% carbon dioxide incubator at 37 ℃ to prepare cells. hDPC prepared in 6-well was inoculated into Follicle Dermal Papilla cell growth media at a concentration of 1Х10 ⁴ /well, and further cultured for 3 days under hypoxic conditions with 1-5% oxygen partial pressure.

Example 2. Confirmation of the effect of hypoxic culture on dermal papilla cells

2-1. Confirmation of the proliferation effect of dermal papilla cells according to hypoxic culture (Fig. 1)

The dermal papilla cells obtained in Example 1 were observed under a microscope, and the cells were floated with trypsin and then centrifuged. The supernatant was discarded, leaving only the cells in the lower layer. Cells were well released by adding 1X PBS and then stained with trypan blue. Cell proliferation was confirmed by counting the number of cells using the formula.

As a result, when cultured under hypoxic conditions, the proliferation of dermal papilla cells was significantly increased compared to the control group (FIG. 1).

2-2. Confirmation of the migration effect of dermal papilla cells according to hypoxic culture (Fig. 2)

In a 60 mm culture dish, dermal papilla cells (hDPC) were inoculated at a concentration of 5Х10 ⁵ . After culturing overnight, the bottom of the dish was scratched using a blue (1000 μL) tip. Each was further cultured for 48 hours under normoxic and hypoxic conditions. Cell migration ability was confirmed by measuring the distance the cells on both sides moved toward the middle position of the scratch.

As a result, as shown in Figure 2, it was confirmed that the migration of dermal papilla cells cultured under hypoxic conditions was significantly increased compared to the control group.

2-3. Lifespan increase of dermal papilla cells according to hypoxic culture (Fig. 3)

In a 60 mm culture dish, dermal papilla cells (hDPC) were inoculated at a concentration of ^1Х104 . After overnight incubation, the syrup-free medium was replaced. After additional incubation for 5 days in normoxic and hypoxic conditions, cells were stained and analyzed using the X-Gal kit (Sigma-Aldrich, MO, USA).

Next, 12-well dermal papilla cells were inoculated at a concentration of 1x10 ⁴ and subcultured every 4 days with growth medium. At each passage, the cells were floated with trypsin and centrifuged. The supernatant was discarded, leaving only the cells in the lower layer. After the cells were well released by adding 1x PBS, they were stained with trypan blue (Sigma-Aldrich, MO, USA) and counted using a hemocytometer. This cycle was repeated up to 7 times (10 passages). The number of population doublings was calculated using the number of cells inoculated and harvested at the beginning of each passage.

As a result, it was confirmed that the aging of dermal papilla cells cultured under hypoxic conditions was suppressed and population doubling was increased (FIG. 3).

2-4. Confirmation of ALP1 level in dermal papilla cells following hypoxic culture (Fig. 4)

Cells were inoculated at a concentration of 2.5x10 ⁵ in a 60 mm culture dish. After overnight incubation, additional incubation was performed under normoxic and hypoxic conditions for 6-10 hours. Then, total RNA was extracted with an RNA extraction kit (RNeasy, Qiagen), and cDNA synthesis was performed from 1000 ng of total RNA with a cDNA synthesis kit (A3500, Promega) using 1,000 U reverse transcriptase and 50 ng/μl oligo (dT) primer. ) was used. qPCR was analyzed by Step One Plus real-time PCR system (Applied Biosystems, Invitrogen) using SYBR Green PCR Master Mix (Takara Bio, Japan).

Cells were inoculated at a concentration of 1Х10 ⁴ in a 60 mm culture dish. After overnight incubation, the syrup-free medium was replaced. After additional culture for 5 days in normoxic and hypoxic conditions, cells were stained and analyzed using ALP staining kit (Sigma-Aldrich, MO, USA).

As a result, it can be confirmed that the expression of ALP1 mRNA in dermal papilla cells is increased in hypoxic culture for 6-10 hours (FIG. 4A), and it can be confirmed that the amount of ALP-positive cells is increased in hypoxic conditions (FIG. 4) b).

2-5. Confirmation of increase in growth factor of dermal papilla cells according to hypoxic culture (FIG. 5)

The target gene under hypoxic culture conditions was analyzed using the human signal transduction pathway explorer RT ² profiler PCR array (signal transduction pathway: PAHS-041ZA). 2.5 x 10 ⁵ dermal papilla cells were inoculated into a 60 mm dish and cultured overnight. The next day, it was further cultured under normal and hypoxic conditions for 6 hours. Then, total RNA was extracted with an RNA extraction kit (RNeasy, Qiagen), and cDNA synthesis was performed from 500 ng of total RNA with a cDNA synthesis kit (A3500, Promega) using 1,000 U reverse transcriptase and 50 ng/μl oligo (dT) primer. ) was used. Gene expression was analyzed according to the method of the PCR array kit (Qiagen).

As shown in Figure 5, it can be confirmed that the expression of nine mRNAs increased in dermal papilla cells cultured for 6 hours under hypoxic conditions.

2-6. Confirmation of hair growth effect of dermal papilla cells according to hypoxic culture in experimental animals (Fig. 6)

In order to check the hair growth effect of dermal papilla cells cultured in hypoxic conditions, 1Х10 ⁵ was inoculated into a 100 mm culture dish and cultured for 5 days under normoxic and hypoxic conditions. Then, after floating the cells with trypsin, the supernatant was discarded through centrifugation, leaving only the cells in the lower layer. 1 ml of PBS was added and the cells were well lysed. Cells were counted by staining with trypan blue. After counting using the formula, 1x10 ⁶ cells were transferred to a new tube with a pipette and centrifuged again. The supernatant was removed and 100 μL of PBS was added to loosen the cells well. After hair removal on the back of 6-week-old female C3H mice, 100 μL of hDPC cultured in normoxic and hypoxic conditions was subcutaneously injected. After 14 days, the newly grown hair was pushed on the back and the degree of hair regeneration was examined by measuring the weight.

As a result, hair growth phase induction was different from the group administered with dermal papilla cells cultured under normoxic conditions, in the case of mice administered with dermal papilla cells cultured under hypoxic conditions, hair on the back grew and covered the back after 14 days. It can be confirmed (FIG. 6A). The weight of hair increased more than 3 times in dermal papilla cells cultured under normoxic conditions (FIG. 6B).

2-7. Confirmation of hair growth effect of dermal papilla cells according to hypoxic culture in experimental animals (Fig. 7)

After 3 weeks of transplantation, specimens were fixed in para-formaldehyde buffered solution (10% paraformaldehyde buffered solution) for histological examination, paraffin slides were made, and hematoxylin and eosin (H & E) staining was performed. The induced HF was observed and imaged using a microscope. The growth rate of hair follicles was observed using Ki67 immunostaining and fluorescence microscopy. The immunostaining control was used as DAPI.

As a result, it can be confirmed that the hair growth of mice transplanted with both breast milk cells cultured under hypoxic conditions is increased (FIG. 7).

Example 3. Confirmation of the effect of hypoxic culture conditions on dermal papilla cells (FIG. 8)

The hDPC prepared in 96-well was inoculated at 1x10 ⁴ /well concentration. They were cultured for 3 days under normoxic or hypoxic conditions of 1%, 2%, and 5%. Then, after removing the culture medium in each well, 10 μL of CCK8 reagent and 90 μL of PBS were added and incubated at 37°C for 2 hours. The absorbance of the supernatant was measured at 450 nm using an ELISA reader. The degree of cell proliferation was confirmed.

As a result, it can be confirmed that the proliferation capacity of hDPC is significantly increased in the hypoxic condition of 2% (FIG. 8).

Example 4. Confirmation of suspension culture effect of hypoxic cultured dermal papilla cells (FIG. 9)

4-1. Hanging drop culture of dermal papilla cells

In order to prepare dermal papilla cell spheroids, dermal papilla cells cultured up to passages 8 to 9 under normoxic or hypoxic conditions were additionally cultured in suspension. 1x10 ⁴ /20 μl of dermal papilla cells were cultured on the underside of the lid of a 100 mm culture dish. Human-derived dermal papilla cells (1x10 ⁴ cells) were suspended in 20 μL of culture medium, clumps on the lower part of a culture dish lid (100 mm), and 6 ml of culture medium was added to prevent drying of cells and drop spheres. The medium was replaced every 2 days and cultured for 6 days under normoxic or hypoxic conditions. The dermal papilla cells gather at the bottom of the droplet by gravity and become spherical.

4-2. patch assay

To determine the hair-inductivity of DPCs under hypoxia, an established patch assay was performed. Epidermal (EPC) cells were obtained from neonatal mouse (C3H/HeN) epidermis. Next, DPS (DP spheres) harvested from 3D hanging drop culture in normoxic or hypoxic culture (approximately 100 DP spheres, DPCs of passages 8 and 9 are used) were treated with 1 x 10 ⁶ epidermal cells. was mixed with and subcutaneously implanted into the back of 6-week-old female nude mice (BALB/cAJcl-nu). Neonatal mouse epidermal cells (1 x 10 ⁶ cells) or dermal cells mixed with epidermal cells (1 x 10 ⁶ cells) were used as controls. After 3 weeks of implantation, the dissected sample was first imaged through a microscope for visual observation. For further histological examination, specimens were fixed in para-formaldehyde buffered solution (10% paraformaldehyde buffered solution), paraffin slides were made, and hematoxylin and eosin (H & E) staining was performed. Induced hair follicles (HF) were observed and imaged under a microscope.

As a result, dark spots or hairs were observed in DPS-implanted mice obtained through the three-dimensional suspension culture method under hypoxic conditions (FIG. 9).

Example 5. Confirmation of effect of medium obtained by hypoxic culture of dermal papilla cells (FIG. 10)

Anagen vibrissal of normal mice was separated using a scalpel and tweezers. The mustaches of the isolated mice were mixed with quantitation medium (Williams E medium supplemented with 2 mM L-glutamine, 10 µg/mL insulin, 10 ng/mL hydrocortisone, 100 U/mL penicillin and 100 µg/mL streptomycin without serum) and DPC was administered together with a culture medium of normoxic (NorCM) or hypoxic (HypCM) culture in a 1:1 ratio. Individual hair follicles were photographed 72 hours after initiation of culture (Edmund Optics Ltd, UK). Changes in hair length were calculated from photographs and expressed as mean±SE of 8-10 hair follicles.

As a result, when the DPC culture solution under hypoxic conditions was administered compared to the group administered with the normal oxygen DPC culture solution, the length of the mustache of the separated mice was increased.

Claims (5)

Pre-culturing the dermal papilla cells in Follicle Dermal Papilla cell Growth media (FDPG) medium; and
BMP6, FGF17, FGF22 in dermal papilla cells in vitro including; culturing the pre-cultured dermal papilla cells in a hypoxic state with an oxygen partial pressure of 2% in FDPG (Folicle Dermal Papilla cell Growth media) medium , FIGF, GDNF, VEGFA, as a method of increasing the expression of human growth factors of PSPN, JAG2 and IL4,
In vitro (in vitro ( A method for increasing the expression of human growth factors of BMP6, FGF17, FGF22, FIGF, GDNF, VEGFA, PSPN, JAG2 and IL4 in dermal papilla cells in vitro .
delete delete delete delete

Images