KR20230026871A - Composition for improving hair loss containing personalized dermal papilla cell culture medium - Google Patents

Abstract

The present invention relates to a composition for preventing hair loss containing a personalized dermal papilla cell culture medium, and more particularly, to a composition for preventing hair loss containing a personalized dermal papilla cell culture medium, in which a large number of growth factors effective for preventing and alleviating the hair loss are contained. Since the composition according to the present invention can play an important role in preventing the hair loss, the composition of the present invention can be used in various industrial fields such as the medical field and the beauty field.

Description

Composition for improving hair loss containing personalized dermal papilla cell culture medium}

The present invention relates to a composition for improving hair loss containing a dermal papilla cell culture solution, and more particularly, a composition for improving hair loss containing a dermal papilla cell culture solution containing a large number of growth factors effective for preventing and improving hair loss. It is an invention about

It is known that alopecia is caused by disease, malnutrition, aging, hormonal imbalance, and the like. Although many studies have been conducted, the mechanism for fundamental hair loss is not well known, but in general, hair goes through a certain hair cycle, and hair cells stimulated by dermal papilla (keratinocyte) It goes through the growth phase (anagen) in which hair grows through active division and proliferation, the catagen phase in which the blood supply to the hair bulb is cut off and the dermal papilla is separated from the hair follicle, and the telogen phase in which hair does not grow when cell proliferation stops. It goes into the growth phase again or goes to the exogenous phase in which the hair falls out from the scalp. Human hair has an independent growth cycle, so some enter the shedding phase and some enter the anagen phase, maintaining the same level of hair overall. is called alopecia.

Efforts have also been made to treat hair loss. Nevertheless, so far only two drugs (finasteride and minoxidil) have been approved by the Food and Drug Administration (U.S.A.) for the treatment of hair loss.

There are about 100,000 to 150,000 hairs in the human body, and they are formed in hair follicles. There is a papilla in the hair follicle, and small blood vessels are distributed in this area to supply nutrients necessary for hair growth, and there is a seborrheic gland that supplies oil to give luster to the hair on the side above the papilla. Hair follicles are composed of several different epithelial cells and dermal papilla cells. Dermal papilla cells are mesenchymally-derived fibroblasts located at the base of hair follicles and play an important role in hair growth. In particular, minoxidil has been reported to have proliferative and anti-apoptotic effects of dermal papilla cells. However, the invention of providing a composition for improving hair loss using such a culture medium of dermal papilla cells has not existed in the past.

Domestic Patent No. 10-2218727 Domestic Patent No. 10-2254911

The present invention is to provide a composition for improving hair loss containing a dermal papilla cell culture medium.

In order to solve the above problems, the present invention provides a composition for improving hair loss containing 1% by weight of dermal papilla cell culture medium.

In the present invention, the composition comprises perilla leaf extract 20% by weight, LES (Disodium Laureth Sulfosuccinate) 18% by weight, cocobetaine 14% by weight, olive surfactant 14% by weight, glucamate 3.2% by weight, dermal papilla cell culture medium 1% by weight %, euronapri 0.6% by weight, peppermint essential oil extract 0.4% by weight, rosemary essential oil extract 0.4% by weight, polyquater 0.4% by weight and purified water.

In addition, in the present invention, the composition may contain a growth factor (Growth Factor).

In the present invention, growth factors may be Human VEGF, Human KGF, Human HGF, Human Fibronectin, Human FGF, and Human Collagen.

In addition, in the present invention, the composition may be formulated into a shampoo, rinse, conditioner, tonic, essence, serum, treatment, lotion, wax, gel, spray, mousse, mist, or cream.

Since the composition according to the present invention exhibits a remarkable effect on hair loss improvement, the present invention can be used in various industrial fields such as the medical field and the beauty field.

Figure 1 shows a comparison of the difference in cell yield according to the method of separating dermal papilla cells from hair bulbs.
Figure 2 shows a comparison of the scratch assay (Scratch Assay) results according to the composition ratio of the dermal papilla cell culture medium.
Figure 3 shows a comparison of Cell Proliferation Assay results according to the composition ratio of dermal papilla cell culture medium.
Figure 4 shows the results of ELISA Assay for confirming the growth factors contained in the dermal papilla cell culture medium.

In one embodiment according to the present invention, it provides a composition for improving hair loss containing the dermal papilla cell culture medium.

In another embodiment according to the present invention, the composition may include 1% by weight of the dermal papilla cell culture medium.

In another embodiment according to the present invention, the composition comprises 20% by weight of perilla leaf extract, 18% by weight of LES (Disodium Laureth Sulfosuccinate), 14% by weight of cocobetaine, 14% by weight of olive surfactant, 3.2% by weight of glucamate, 1% by weight of dermal papilla cell culture medium, 0.6% by weight of euronapri, 0.4% by weight of peppermint essential oil extract, 0.4% by weight of rosemary essential oil extract, 0.4% by weight of polyquater, and purified water.

In another embodiment according to the present invention, the composition may contain growth factors.

In another embodiment according to the present invention, the growth factor may be Human VEGF, Human KGF, Human HGF, Human Fibronectin, Human FGF and Human Collagen.

In another embodiment according to the present invention, the composition may be formulated into a shampoo, rinse, conditioner, tonic, essence, serum, treatment, lotion, wax, gel, spray, mousse, mist, or cream.

Hereinafter, the present invention will be described in more detail through specific examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Isolation of hair bulbs from scalp tissue

After filling a sterilized 90 mm Petri dish with MEM alpha medium so that the scalp tissue (from the epidermis to the dermal layer) collected from the subject is submerged, MEM alpha medium was applied to the cover of the 90 mm Petri dish. A dog drop was formed, and hair bulbs in the dermal layer were separated one by one using microsurgical scissors and forceps, and transferred to the prepared drop. Thereafter, the hair bulb moved to the drop is observed under a stereo microscope, and the fatty tissue and hair shaft attached to the end of the hair bulb are removed using a 26G syringe and microsurgical forceps. It was removed to separate the hair bulb from the scalp tissue. In the present invention, “hair bulb” is a thick, club-shaped structure formed at the lower part of the hair root surrounded by hair follicles, and means a tissue in which capillaries, dermal papilla cells, and hair cells are located.

Example 2. Isolation of dermal papilla cells from hair follicles

In order to select the optimal method for separating dermal papilla cells from hair bulbs, the following comparative experiments were conducted.

First, in the separation method 1, 1 ml of dissection media was put in a 35 mm cell culture dish, about 50 hair bulbs were put in, and after chopping with fine scissors, the cells were placed in a tube. Collect and centrifuge at 2200 rpm for 5 minutes.

Next, in Separation Method 2, after adding 0.025% Collagenase type I reagent to a 35 mm cell culture dish containing about 50 hair bulbs, incubating for 2 hours and 30 minutes in a shaking incubator at 37 ° C. (tube) and centrifuged at 2200 rpm for 5 minutes.

In order to compare the results of the separation methods 1 and 2, each pellet was tapped and pipetting was sufficiently performed with 2 ml of Attachment media having the composition shown in Table 1 below, and then placed in a 35 mm cell culture dish. (seeding) in (cell culture dish), 37 ℃, 5% CO ₂ while exchanging the medium at intervals of 3 days in a large-scale proliferation until the culture dish is full of cells.

division Attachment Media
(attachment step) Component MEM alpha - 20% Fetal bovine serum Penicillin-Streptomycin Amphotericin B

As a result of the comparative experiment, compared to separation method 2 containing 0.025% Collagenase type I reagent, in separation method 1 in which only chopping was performed without any other treatment on the hair bulb itself, the size of the cells was smaller and more constant, and the cell yield was also higher. It was confirmed that it came out high (Fig. 1).

Example 3. Subculture step

The step of subculturing from Passage 0 to Passage 1 was performed as follows. First, after determining the culture dish to be subcultured according to the number of cells collected in the 35 mm cell culture dish, the medium contained in the 35 mm cell culture dish was discarded and washed once with PBS. did Next, it was treated with 0.25% Trypsin/EDTA and incubated for 5 minutes in a 37°C, 5% CO ₂ mass propagator. After that, for culture, MEM alpha medium containing 1% FBS was added, collected in a 50ml centrifuge tube, and centrifuged at 2200 rpm for 5 minutes. The supernatant was discarded and the pellet settled down sufficiently After tapping, an appropriate amount of Expansion Media 1 or 2 shown in Table 2 was added and the number of cells was confirmed.

division Expansion Media 1
(mass growth stage) Expansion Media 2
(mass growth stage) Component MEM alpha MEM alpha - Basic FGFs 10% Fetal bovine serum 10% Fetal bovine serum Penicillin-Streptomycin Penicillin-Streptomycin Amphotericin B Amphotericin B

After seeding in the culture dish in the next step to reach 1,500 cells/cm ² according to the number of counted cells, the medium is exchanged every 3 days in a 37°C, 5% CO ₂ mass propagator, while the culture dish grows into cells. Grow until full.

Thereafter, the step of subculturing from passage 1 to passage 4 was performed in the same order as above, and subculture was performed up to passage 4 according to the size of the culture dish according to the number of cells to be counted. In the case of 3,000 / cm ² It was seeded in a culture dish (seeding) to be.

Example 4. Preparation of dermal papilla cell culture solution

When the cells at the passage 4 stage proliferated by more than 90% on the bottom of the culture dish, the expansion media was completely removed and washed three times with a sufficient amount of PBS. Put an appropriate amount of MEM alpha phenol red free medium (serum-free, antibiotic-free medium) according to the capacity of the culture dish in the washed culture dish, collect and combine the cultured media on the 3rd and 6th days, and then dermal papilla cell culture medium was used as

Example 5. Preparation of hair loss shampoo containing dermal papilla cell culture medium

In preparing a personalized hair loss shampoo using an autologous culture solution, the contents of the raw materials contained in the shampoo are shown in Table 3 below.

division raw material name Weight (ml) water layer perilla leaf extract 100 Purified water 140 thickener polyquote 2 Glucamate 16 interface layer Disodium Laureth Sulfosuccinate (LES) 90 cocobetaine 70 olive surfactant 70 additive euro napri 3 Peppermint Essential Oil Extract 2 Rosemary Essential Oil Extract 2 Custom dermal papilla cell culture medium 5 total amount 500

Example 6. Confirmation of the effect according to the composition ratio of the dermal papilla cell culture medium

Example 6-1. Scratch Assay

First, 2000/cm ² cells per area were seeded in a 6-well plate, and 2 ml of a medium containing 10% FBS was dispensed, and cultured for 6 days at 37° C. and 5% CO ₂ conditions. After 6 days, place a ruler in the center of the well and apply a certain force using a tip to make a scratch, then suction all the medium and use basal media (2㎖) once. After washing, suction was performed once more. After that, after dispensing the medium (2 ml) prepared for each condition in each well, it was observed that the cells grew in the scratched area for 0 to 15 hours, and the results are shown in FIG. .

According to FIG. 2, as a result of DPC-CM (Dermal Papilla Cell Conditioned Media) observation, it was found that there was almost no difference from 0 hour even after 15 hours in the blank DMEM H / G only medium environment, and the control group In the DMEM H/G medium environment containing 10% FBS, it was observed that the cells grew slightly more than the blank. In the 0.1% DPC-CM treatment group, almost no cell growth was observed with similar results to Blank, and cell growth was observed in the 1, 10, and 30% DPC-CM treatment groups, especially in the 1% DPC-CM treatment group. I was able to confirm that it grew best. Cell growth was observed to be almost similar in the 10% DPC-CM treatment group and the higher treatment group, and it was confirmed that 1% was appropriate for the treatment concentration of DPC-CM to be used in the hair loss composition.

Example 6-2. Cell Proliferation Assay

First, 1000/cm ² cells per area were seeded in a 24 well plate, and 2 ml of medium containing 10% FBS was dispensed, followed by incubation at 37° C. and 5% CO ₂ for 24 hours. After 24 hours, all the medium was suctioned, and then the medium (2 ml) prepared for each condition was dispensed, and after 3 days, the medium prepared for each condition was suctioned and 10% FBS containing 10% CCK-8 solution was added. media (1 ml) was replaced. After 90 minutes, 100 ul was dispensed for each condition in a 96 well plate, and the absorbance was measured at 450 nm, as shown in FIG.

According to Figure 3, as a result of confirming cell proliferation using CCK-8 (= Cell Counting Kit - 8), cell proliferation was found to be the highest in the 1% DPC-CM treated group, similar to the scratch assay results. Confirmed. Specifically, almost no proliferation occurred in the Blank, Control group, 0.1, and 0.5% DPC-CM treatment groups, especially 1, 2, 4, 6, 8, 10, 30, 50, and 100% DPC-CM. As a result of confirming the treatment group, the cell proliferation was most active in the 1% DPC-CM treatment group, and as the concentration of DPC-CM increased, the proliferation did not increase proportionally, but rather decreased.

Example 6-3. ELISA Assays

First, 100ul of capture antibody (CA) was dispensed into a 96-well plate one day before the experiment, the CA-coated 96-well plate was sealed, incubated overnight at room temperature, and the 96-well plate was washed with a wash buffer. buffer). Thereafter, the 96-well plate was blocked with 300ul of reagent diluent (RD) for 1 hour at room temperature, and after 1 hour, the 96-well plate was washed with wash buffer. Next, 100ul/well duplication of the standard from high concentration to low concentration was performed through serial dilution, and reagent diluent (RD) was dispensed into the bottom well, and the remaining wells were duplicated by 100ul to sample ( sample) was dispensed, sealed, and incubated at room temperature for 2 hours. After 2 hours, the 96-well plate was washed with wash buffer, and then 100ul of detection antibody (DA) was dispensed into the 96-well plate. and sealed, incubated at room temperature for 2 hours, and then washed the 96 well plate with wash buffer after 2 hours. After that, Streptavidin-HRP was dispensed at 100ul/well and incubated for 20 minutes in a place without sunlight. After 20 minutes, the 96 well plate was washed with wash buffer, and after washing, Substrate Solution was dispensed at 100ul/well Color development was confirmed for about 15 minutes in a place without sunlight. When the color development of the standard and sample is confirmed, 50ul/well of Stop solution is dispensed, and the absorbance is measured at 450nm immediately after dispensing, and is shown in FIG.

According to FIG. 4, as a result of measuring the content of growth factors contained using DPC-CM, Human VEGF, which promotes the formation of blood vessels near hair follicle tissue, promotes cell proliferation of hair follicles by regulating the proliferation and differentiation of epithelial tissue Human KGF, a factor secreted by dermal papilla cells, which promotes the proliferation of epithelial follicle cells, Human FGF, which induces and increases the growth rate of early hair, and promotes the proliferation of dermal papilla cells and spheroid formation It was confirmed that Human Fibronectin, which promotes, and Human Collagen, which is a component of hair fiber, was secreted.

Claims (6)

A composition for improving hair loss containing a dermal papilla cell culture medium. According to claim 1, wherein the composition is a hair loss improvement composition containing a dermal papilla cell culture solution characterized in that it comprises 1% by weight of the dermal papilla cell culture solution. The method of claim 1, wherein the composition is perilla leaf extract 20% by weight, LES (Disodium Laureth Sulfosuccinate) 18% by weight, cocobetaine 14% by weight, olive surfactant 14% by weight, glucamate 3.2% by weight, dermal papilla cell culture medium A composition for improving hair loss containing a dermal papilla cell culture solution comprising 1% by weight, 0.6% by weight of euronapri, 0.4% by weight of peppermint essential oil extract, 0.4% by weight of rosemary essential oil extract, 0.4% by weight of polyquater and purified water. According to claim 1, wherein the composition is a composition for improving hair loss containing a dermal papilla cell culture medium characterized in that it contains a growth factor (Growth Factor). According to claim 4, wherein the growth factor is Human VEGF, Human KGF, Human HGF, Human Fibronectin, Human FGF and Human Collagen, characterized in that the hair loss improvement composition containing the papilla cell culture medium. The composition according to any one of claims 1 to 5, wherein the composition is formulated into a shampoo, rinse, conditioner, tonic, essence, serum, treatment, lotion, wax, gel, spray, mousse, mist, or cream. A composition for improving hair loss containing a papilla cell culture medium, characterized in that being.

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