KR102663430B1 - Composition for improving hair loss containing personalized dermal papilla cell culture medium - Google Patents

Abstract

The present invention relates to a composition for improving hair loss containing a dermal papilla cell culture solution, and more specifically, to a composition for improving hair loss containing a dermal papilla cell culture solution containing a large number of growth factors effective in preventing and improving hair loss. Since the composition according to the present invention can play an important role in improving hair loss, 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 specifically, to a composition for improving hair loss containing a dermal papilla cell culture solution containing a large number of growth factors effective in preventing and improving hair loss. It is an invention about.

Alopecia is known to be caused by disease, malnutrition, aging, hormonal imbalance, etc. Although much research has been conducted, the fundamental mechanism of hair loss is not well known. Generally, hair goes through a certain hair cycle, and keratinocytes stimulated by the dermal papilla It goes through the growth phase (anagen), when the hair actively divides and proliferates and the hair grows, the catagen phase, when the blood supply to the hair bulb is cut off and the dermal papilla separates from the hair follicle, and the resting phase (telogen), when cell proliferation stops and hair does not grow. It either goes into the anagen phase again or into the shedding phase (exogen), when the hair falls out from the scalp. Human hair has an independent growth cycle, with some entering the shedding phase and some entering the growing phase, maintaining the same level of hair overall. However, this balance tilts toward the shedding phase, causing hair to disappear from areas where hair should normally exist. It 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 (FDA) (U.S.A.) for the treatment of hair loss.

There are approximately 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. Next to the papilla, there is a seborrheic gland that supplies oil that gives hair shine. Hair follicles are made up 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 proliferation and anti-apoptotic effects on dermal papilla cells. However, there has been no existing invention providing a composition for improving hair loss using the culture medium of these dermal papilla cells.

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

The present invention seeks to provide a composition for improving hair loss containing 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 contains 20% by weight of perilla leaf extract, 18% by weight of LES (Disodium Laureth Sulfosuccinate), 14% by weight of coco betaine, 14% by weight of olive surfactant, 3.2% by weight of glucamate, and 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 polyquart, and purified water.

Additionally, in the present invention, the composition may contain growth factors.

Additionally, in the present invention, the growth factor may be Human VEGF, Human KGF, Human HGF, Human Fibronectin, Human FGF, and Human Collagen.

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

Since the composition according to the present invention has a significant effect on improving hair loss, 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 depending on the method of isolating dermal papilla cells from hair bulbs.
Figure 2 shows a comparison of 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 the dermal papilla cell culture medium.
Figure 4 shows the results of ELISA Assay to confirm growth factors contained in dermal papilla cell culture medium.

In one embodiment of the present invention, a composition for improving hair loss containing a dermal papilla cell culture medium is provided.

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

In another embodiment according to the present invention, the composition includes 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, It may include 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 polyquart, and purified water.

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

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

In another embodiment of the present invention, the composition may be formulated as 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 illustrating 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 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 to submerge the scalp tissue (from the epidermis to the dermal layer) collected from the subject, several layers were placed on the 90 mm Petri dish cover using MEM alpha medium. A drop was formed, and the hair bulbs in the dermal layer were separated one by one using microsurgical scissors and forceps and transferred to the prepared drop. Afterwards, the hair bulb moved to the drop is observed under a stereomicroscope, and the fatty tissue and hair shaft attached to the end of the hair bulb are removed using a 26G syringe and microsurgical forceps. The hair bulb was separated from the scalp tissue by removal. 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 refers to the tissue in which capillaries, dermal papilla cells, hair hair cells, etc. are located.

Example 2. Isolation of dermal papilla cells from hair bulbs

In order to select the optimal method for separating dermal papilla cells from hair bulbs, the following comparative experiment was performed.

First, for separation method 1, put 1 ml of dissection media in a 35 mm cell culture dish, add about 50 hair bulbs, chop them with precision scissors, and then place them in a tube. Collect and centrifuge at 2200 rpm for 5 minutes.

Next, in isolation method 2, 0.025% Collagenase type I reagent was added to a 35 mm cell culture dish containing about 50 hair bulbs, cultured at 37°C for 2 hours and 30 minutes in a shaking incubator, and cultured in a shaking incubator for 2 hours and 30 minutes. This method involves collecting them in a tube and centrifuging them at 2200 rpm for 5 minutes.

In order to compare the results of separation methods 1 and 2, each pellet was tapped and sufficiently pipetted with 2 ml of attachment media having the composition shown in Table 1 below, then placed in a 35 mm cell culture dish. They were seeded in a cell culture dish and grown in a mass propagator at 37°C and 5% CO ₂ while changing the medium every 3 days until the culture dish was filled with cells.

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

As a result of the above comparative experiment, compared to separation method 2 using 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 cell size was smaller and more consistent, and the cell yield was also higher. It was confirmed that it was high (Figure 1).

Example 3. Subculture step

The subculture step from Passage 0 to Passage 1 was performed as follows. First, determine the culture dish to be subcultured according to the number of cells collected in the 35 mm cell culture dish, then discard the medium in the 35 mm cell culture dish and wash once with PBS. did. Next, it was treated with 0.25% Trypsin/EDTA and incubated for 5 minutes in a mass propagator at 37°C and 5% CO ₂ . Afterwards, add MEM alpha medium containing 1% FBS for culture, collect in a 50ml centrifuge tube, and centrifuge at 2200 rpm for 5 minutes. Discard the supernatant and thoroughly shake off the pellet that settled at the bottom. After tapping, an appropriate amount of Expansion Media 1 or 2 shown in Table 2 was added and the number of cells was checked.

division Expansion Media 1
(Mass proliferation stage) Expansion Media 2
(Mass proliferation stage) Component MEM alpha MEM alpha - Basic FGF 10% fetal bovine serum 10% fetal bovine serum Penicillin-Streptomycin Penicillin-Streptomycin Amphotericin B Amphotericin B

Depending on the number of cells counted, seed the culture dish in the next step to reach 1,500 cells/cm ² and then change the medium every 3 days in a mass propagator at 37°C and 5% CO ₂ until the culture dish is transformed into cells. It was multiplied until full.

After this, the steps of subculturing from passage 1 to passage 4 are performed in the same order as above, and subculture was performed until passage 4 by adjusting the size of the culture dish according to the number of cells to be counted, and frozen and thawed at the intermediate passage stage. In this case, seeds were seeded in a culture dish to reach 3,000 cells/cm ² .

Example 4. Preparation of dermal papilla cell culture medium

When the cells at passage 4 had grown to 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. Add an appropriate amount of MEM alpha phenol red free medium (serum-free, antibiotic-free medium) to the washed culture dish according to the volume of the culture dish. Collect and combine each cultured medium on the 3rd and 6th days, and then add the dermal papilla cell culture fluid. It was used as.

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

In manufacturing personalized hair loss shampoo using autologous culture medium, the content of raw materials contained in the shampoo is shown in Table 3 below.

division Raw material name Weight (ml) water floor Perilla leaf extract 100 Purified water 140 thickener poly quarter 2 Glucarmate 16 interfacial layer LES(Disodium Laureth Sulfosuccinate) 90 Coco Betaine 70 olive surfactant 70 additive euro napri 3 Peppermint Essential Oil Extract 2 Rosemary Essential Oil Extract 2 Customized dermal papilla cell culture medium 5 total amount 500

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

Example 6-1. Scratch Assay

First, 2000/cm ² cells per area were seeded in a 6-well plate, 2 ml of medium containing 10% FBS was dispensed, and the cells were cultured at 37°C and 5% CO ₂ for 6 days. After 6 days, place a ruler in the center of the well and apply a certain amount of force using a tip to make a scratch. Then, suction all the medium and refill with basic media (2 ml) once. After washing, suction was performed once more. After that, the medium (2 ml) prepared according to the conditions was dispensed into each well, and the growth of cells in the scratched area was observed for 0 to 15 hours. The results are shown in Figure 2. .

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

Example 6-2. Cell Proliferation Assay

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

According to Figure 3, as a result of confirming cell proliferation using CCK-8 (=Cell Counting Kit - 8), cell proliferation was highest in the 1% DPC-CM treatment group, similar to the scratch assay results. Confirmed. Specifically, almost no proliferation occurred in the Blank, Control group, and 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 checking the treatment group, it was confirmed that cell proliferation was most active in the 1% DPC-CM treatment group, and that as the concentration of DPC-CM increased, proliferation did not increase proportionally, but rather decreased.

Example 6-3. ELISA Assay

First, a day before the experiment, 100 ul of capture antibody (CA) was dispensed into each 96 well plate, the CA-coated 96 well plate was sealed and cultured overnight at room temperature, and then the 96 well plate was washed with wash buffer. Washed with buffer). Afterwards, the 96 well plate was blocked with 300ul of Reagent Diluent (RD) at room temperature for 1 hour, and after 1 hour, the 96 well plate was washed with wash buffer. Next, 100ul/well of the standard was duplicated from high concentration to low concentration through serial dilution, and reagent diluent (RD) was dispensed into the bottom well, and 100ul of each well was duplicated into the remaining wells to obtain the 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 100 ul of detection antibody (DA) was dispensed into each 96 well plate. and sealed and incubated at room temperature for 2 hours. After 2 hours, the 96 well plate was washed with wash buffer. After that, 100ul/well of Streptavidin-HRP was dispensed and incubated for 20 minutes in a place without sunlight. After 20 minutes, the 96 well plate was washed with wash buffer. After washing, 100ul/well of Substrate Solution was dispensed. Color development was confirmed for about 15 minutes in a place out of sunlight. When the color development of the standard and sample was confirmed, 50ul/well of Stop solution was dispensed, and the absorbance was measured at 450nm immediately after dispensing, and is shown in Figure 4.

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

Claims (6)

A composition for improving hair loss containing dermal papilla cell culture medium,
The composition contains 20% by weight of perilla leaf extract, 18% by weight of LES (Disodium Laureth Sulfosuccinate), 14% by weight of coco betaine, 14% by weight of olive surfactant, 3.2% by weight of glucarate, 1% by weight of dermal papilla cell culture medium, and Eurona A composition for improving hair loss containing dermal papilla cell culture fluid, comprising 0.6% by weight of free, 0.4% by weight of peppermint essential oil extract, 0.4% by weight of rosemary essential oil extract, 0.4% by weight of polyquat, and purified water. delete delete The composition for improving hair loss containing dermal papilla cell culture medium according to claim 1, wherein the composition contains growth factors. The composition for improving hair loss containing papilla cell culture according to claim 4, wherein the growth factors are Human VEGF, Human KGF, Human HGF, Human Fibronectin, Human FGF, and Human Collagen. The composition according to claim 1, 4 or 5, wherein the composition is a shampoo, rinse, conditioner, tonic, essence, serum, treatment, lotion, wax, gel, spray, mousse, mist, or A composition for improving hair loss containing papilla cell culture medium, characterized in that it is formulated as a cream.