Method for Separating and Culturing Novel Dermal Papilla Cells

Abstract

The present invention relates to a novel method for isolating and culturing hair papilla cells. More specifically, in the isolation and culturing method of the present invention, hair papilla tissue isolated from the scalp is cultured under hypoxic conditions, which has the effect of allowing hair papilla cells isolated from the hair papilla tissue to quickly attach to a culture plate, thereby shortening the period required for the establishment (harvesting) of hair papilla cells, and since the isolated and cultured hair papilla cells are immunocompatible, they can be effectively used for allogeneic transplantation of hair papilla cells.

Description

Technical Field

[0001] The present invention relates to a method for separating and culturing novel dermal papilla cells. More specifically, in the separation and culture method of the present invention, by culturing dermal papilla tissue separated from the scalp under hypoxic conditions, dermal papilla cells detached from the dermal papilla tissue rapidly adhere to the culture plate, and the establishment (acquisition) period of dermal papilla cells is shortened. The separated and cultured dermal papilla cells have immunocompatibility.

Background Art

[0002] Human hair is an aggregate of approximately 100,000 individual hairs, and hair is produced by hair follicles. Hair follicles function as a reservoir of stem cells that can generate all cell lines necessary for the hair follicle itself to reconstruct the epithelium and sebaceous glands. At the base of the hair follicle is the hair follicle bulb, and matrix cells that proliferate from the hair follicle bulb become hair. The hair follicle bulb contains dermal sheath cup (DSC) cells and dermal papilla cells (DRM). Dermal papilla cells are mesenchymally-derived fibroblasts located at the base of the hair follicle and are important cells responsible for hair generation and growth. Patent Document 1 discloses a method for separating dermal papilla cells from hair.

[0003] Hair grows, is maintained, and then shed through a three - stage cycle: anagen (growth phase), catagen (transitional phase), and telogen (resting phase). During the anagen phase, which is the period of hair growth, in adults, hair grows at an average rate of about 0.3 mm per day, about 1 cm per month, and is known to typically last from 3 to 7 years. Generally, after the anagen phase, over a period of 10 to 14 days, general apoptosis of hair follicle cells occurs, leading to the catagen phase where the hair follicle shrinks, and then through the telogen phase, which prepares for the next anagen phase lasting on average 3 months, the hair falls out. The reason why hair length varies by body part is that the duration of the anagen phase, which is a unique characteristic of each hair follicle for each body part, differs from one another.

[0004] Thus, since human hair has a certain hair growth cycle, a constant number of hairs is always maintained. However, as hair loss progresses, the dermal papilla cells present in the hair root become smaller. When the dermal papilla cells become smaller, the thickness of the hair becomes thinner, and at the same time, the hair growth cycle also becomes shorter. Therefore, as hair loss progresses, the hair becomes very thin, and the hair growth cycle becomes even shorter, so it grows a little and then falls out.

[0005] Causes of hair loss are known to be due to not only genetic causes and the action of male hormones, but also a complex interplay of factors such as endocrine disorders, nutritional deficiencies, drug use, childbirth, fevers, and severe physical and mental stress such as surgery. In recent years, not only male pattern baldness, but also due to changes in diet and increased stress due to social environment and other factors, the number of women suffering from hair loss is increasing, and the age is also getting lower.

[0006] Currently, as the hair removal therapeutic agents most frequently used in Korea, there are finasteride (trade name: Propecia (registered trademark)), dutasteride (trade name: Avodart (registered trademark)), minoxidil (trade name: Minoxyl or Rogaine), etc. However, these therapeutic agents have side effects such as decreased libido, erectile dysfunction, loss of driving and performance ability, itching, erythema, skin irritation, eye irritation, etc. in the application site, and unwanted hair growth may also be observed in other body parts other than the head. Also recently, hair transplantation has been attempted for severe alopecia patients, but limitations such as high cost and side effects after the operation have been pointed out.

[0007] On the other hand, in addition to the method of directly applying drugs to the skin, as a hair removal treatment method, a technique of culturing and transplanting hair follicle cells in vitro has been attempted, but there are several difficulties in using dermal papilla cells as a hair removal therapeutic agent. First, it is difficult to separate from the scalp, the culture conditions are strict, and it is not only difficult to culture a sufficient amount of cells, but also there are limitations such as a significant decrease in hair regeneration ability when subculturing multiple times. Summarizing the standard process of separating dermal papilla cells (see Figure 1, Non-Patent Document 2), the steps are: separating the hair bulb from the scalp tissue and separating the dermal papilla tissue where many dermal papilla cells are present; (a→b), attaching the dermal papilla cells from the dermal papilla tissue to a culture plate; (b→c), subculturing; (c→d), and from the obtained dermal papilla cells, a cell therapeutic agent or exosomes using cells can be separated. Among them, it takes an average of more than 12 days until the dermal papilla cells attach to the plate from the dermal papilla tissue, leading to a synergistic effect on the production period and production cost of the therapeutic agent by the dermal papilla cells. Therefore, in the development of cell therapeutic agents, an increase in the attachment rate of the dermal papilla cells separated and attached from the dermal papilla tissue and a shortening of the attachment time are eagerly desired.

[0008] Therefore, the present inventors have found a novel method for separating dermal papilla cells, which can increase the attachment of dermal papilla cells to the culture plate and shorten the establishment period of dermal papilla cells by culturing under hypoxic conditions (oxygen saturation of 0.5 to 5%) during the process of establishing dermal papilla cells from dermal papilla tissue in the separation and culture of dermal papilla cells, thus completing the present invention.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0011] An object of the present invention is to provide a novel method for separating and culturing dermal papilla cells. Further, an object of the present invention is to provide a composition for preventing or treating hair loss, which contains dermal papilla cells obtained by the separation and culture method of the present invention, or a cell culture solution or exosome obtained from dermal papilla cells.

Means for Solving the Problems

[0012] The present invention provides a method for separating and culturing dermal papilla cells, which includes culturing dermal papilla tissue separated from the scalp under hypoxic conditions and attaching dermal papilla cells to a culture plate.

[0013] The hypoxic conditions of the present invention may have an oxygen saturation of 0.5 to 5%, and preferably may have an oxygen saturation of 2%.

[0014] In the method for separating and culturing dermal papilla cells of the invention, the period until the dermal papilla cells adhere to the culture plate may be shortened by 5 days or more compared to culturing the dermal papilla tissue under normal conditions.

[0015] The dermal papilla cells separated and cultured according to the present invention may be immunocompatible dermal papilla cells, and the immunocompatible dermal papilla cells may inhibit the mRNA expression of at least one selected from the group consisting of HLA-A, HLA-B, CD55, IL-1RA, and CCL2 related to the immune response.

[0016] The present invention provides dermal papilla cells obtained by the method for separating and culturing dermal papilla cells, which includes culturing dermal papilla tissue separated from the scalp under hypoxic conditions and attaching dermal papilla cells to a culture plate, and the dermal papilla cells may be immunocompatible dermal papilla cells.

[0017] The present invention provides a pharmaceutical composition for preventing or treating hair loss, which includes dermal papilla cells obtained by the method for separating and culturing dermal papilla cells, which includes culturing dermal papilla tissue separated from the scalp under hypoxic conditions and attaching dermal papilla cells to a culture plate.

[0018] The present invention provides a pharmaceutical composition for preventing or treating hair loss, which includes a cell culture solution or exosomes obtained from dermal papilla cells obtained by the method for separating and culturing dermal papilla cells, which includes culturing dermal papilla tissue separated from the scalp under hypoxic conditions and attaching dermal papilla cells to a culture plate.

Advantages of the Invention

[0019] According to the novel method for separating and culturing dermal papilla cells of the present invention, in the process of separating dermal papilla cells from scalp tissue, when the dermal papilla tissue is cultured under hypoxic conditions (0.5 - 5%, preferably 2% oxygen saturation), the dermal papilla cells detached from the dermal papilla tissue can quickly adhere to the culture plate, and the primary cells of the dermal papilla cells can be rapidly established. After separating the primary cells of the dermal papilla cells, mass production of dermal papilla cells by subculture is possible, and the production period of cell therapy agents can be shortened. In addition, it is possible to ensure sufficient dermal papilla cells required in the hair removal treatment process, and it can be usefully utilized for hair removal treatment.

[0020] Furthermore, since the dermal papilla cells separated and cultured according to the present invention have immunocompatibility, it can reduce the immune response that may occur during allogeneic transplantation of dermal papilla cells, and can be used for hair removal treatment by transplantation of dermal papilla cells.

Brief Description of the Drawings

[0021]

Figure 1

Figure 2

Figure 3

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Mode for Carrying Out the Invention

[0022] Hereinafter, with reference to the accompanying drawings, embodiments and examples of the present application will be described in detail so that those having ordinary knowledge in the technical field to which the present invention pertains can easily implement it. However, since the present application can be implemented in various forms, it is not limited to the embodiments and examples described below.

[0023] Throughout the present specification, when a certain part "includes" a certain component, this means that, unless otherwise specified, it does not exclude other components, but may further include other components.

[0024] The present invention provides a novel method for separating and culturing dermal papilla cells.

[0025] In the present invention, "dermal papilla cell (DRM)" is a mesenchymally-derived fibroblast located at the base of the hair follicle and is an important cell responsible for hair generation and growth.

[0026] The term "hair loss" used in the present invention refers to a state where there is no hair in a site where hair should normally be present, regardless of the cause, and may be alopecia areata, hereditary male pattern alopecia, telogen effluvium, traumatic alopecia, trichotillomania, traction alopecia, anagen effluvium, pityriasis amiantacea, syphilitic alopecia, seborrheic alopecia, symptomatic alopecia, cicatricial alopecia, or congenital alopecia, but is not limited thereto.

[0027] As used herein, the term "treatment" means any act that advantageously changes alopecia symptoms such as improving alopecia symptoms by delaying or stopping the progression of hair loss by administration of a composition, or promoting hair growth, such as increased hair growth and number of hairs.

[0028] As used herein, "flow cytometry" is a laser-based technique widely used to analyze the characteristics of cells or particles, and can measure cell size, complexity, cell number, cell cycle, etc. By irradiating a laser beam onto an emulsion in which the cells to be measured flow, detecting and analyzing the light rays that pass through or are scattered by the cells, it is also possible to separate only specific cell populations from the parent population.

[0029] Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example

[0030] [Example 1] Separation of tissue The bottom surface of a sterilized 100 mm petri dish was filled with 20 mL of separation / medium containing 4% FBS so that the scalp tissue collected from the donor was immersed, and stored so that the scalp tissue did not dry out. After placing the tissue on the bottom surface of the petri dish, it was cut into strips using a disposable scalpel so that the hair follicles did not overlap, and the cut tissue strips were stored in a petri dish containing the separation medium in which the tissue was stored. After placing the cut tissue strips on the bottom surface of a new petri dish, the hair follicles were separated one by one using a disposable scalpel and stored in a petri dish containing fresh medium.

[0031] [Example 2] Separation of dermal papilla tissue from hair bulbs Place a 150-mm Petri dish on the stage of a stereomicroscope, place one hair follicle isolated in Example 1 on it, and then drop one drop of isolation medium using a 1-mL syringe to prevent drying. Using a disposable scalpel, cut out the boundary between the dermal sheath cup (DSC) and the upper dermal sheath (UDS) of the hair bulb of the hair follicle. To prevent drying of the excised dermal sheath cup of the hair bulb, add one drop of isolation medium, and then use two 1-mL syringes, one to fix the dermal sheath cup of the hair bulb and the other to pierce the bottom of the sheath, which is the sharpest part of the dermal sheath cup of the hair bulb, to turn the sheath inside out. When an elongated rhomboid or teardrop-shaped dermal papilla (DP) protrudes from the inside of the inverted dermal sheath cup of the hair bulb, cut out only the dermal papilla using a syringe.

[0032] [Example 3] Plate adhesion of dermal papilla cells in dermal papilla tissue Place the dermal papilla tissue (DP) isolated in Example 2 on the tip of a syringe needle and transfer it to a 6-well (CellBind surface) plate. Inoculate 12 pieces of dermal papilla tissue per well, place the plate in an incubator at 37 °C under normal conditions (5% CO2: 21% O2) and hypoxic conditions (5% CO2: 2% O2) without shaking, culture for 5 to 10 days, and then change the medium once every 2 to 3 days twice until the culture density (confluency) reaches 60% (p0). For the culture medium from isolation (p0) to the 5th passage (p5), use CellCor (XCELL therapeutics) or Follicle dermal papilla cell growth medium (Promaocell) / 4% Fetal bovine serum (Hyclone (registered trademark)) / 1% penicillin streptomycin (Thermo Fisher scientific).

[0033] [Example 4] Subculture of dermal papilla cells When the well was about 60% filled with dermal papilla cells detached from the dermal papilla tissue inoculated in Example 3, they were subcultured in a T75 flask. The medium in the 6-well plate was discarded, washed once with 1×DPBS, then 0.5 mL of Accutase was added per well and reacted in an incubator under each condition for 10 minutes. Then, 2 mL of separation medium was dispensed per well, and then all were collected in a 50 mL tube, followed by centrifugation at room temperature and 1300 rpm for 3 minutes to remove the supernatant. The cells remaining in the lower layer were suspended in 1 mL of medium, and then the cell count was performed by trypan blue staining. Based on the number of recovered cells, the number of T75 flasks for subculture was determined (2.5×10 5 cells / flask). 15 mL of fresh medium was added to the T75 flask, the suspended cell solution was calculated and dispensed, and then the flask was carefully rotated more than 3 times to uniformly mix the cells. Then, they were cultured in an incubator under normal conditions (5% CO2: 21% O2) and hypoxic conditions (5% CO2: 2% O2) for 4 days, and subcultured at a 1:5 magnification from the first passage to the fifth passage (p1 - p5) at 4-day intervals.

[0034] [Experimental Example 1] Comparison of the plate attachment level of dermal papilla cells Similar to Example 3, dermal papilla tissue (DP) isolated from the scalp was cultured under hypoxic conditions (5% CO2: 2% O2) and normal conditions (5% CO2: 21% O2), and then the culture plate attachment levels of dermal papilla cells (DRM) on the 5th to 10th days were compared. The results are shown in Figure 2.

[0035] As shown in Figure 2, when comparing the attachment rates on the 6th day after culture, it was confirmed that when cultured under hypoxic conditions rather than normal conditions, the number of dermal papilla cells attached to the culture plate increased significantly. This means that the establishment (acquisition) period of primary cells of dermal papilla cells, which takes an average of more than 12 days under normal conditions (oxygen saturation of 20%), is shortened by about 5 days on average, and further production processes during subculture can be shortened.

[0036] [Experimental Example 2] Analysis of the Attachment Mechanism of Dermal Papilla Cells As confirmed in Experimental Example 1, it can be seen that the dermal papilla cells isolated and cultured according to the present invention have an increased level of attachment to the culture plate. Therefore, in order to confirm the mechanism by which dermal papilla cells attach to the culture plate, the following was done.

[0037] 2-1. Analysis of Base Sequence (NGS) and Ontology The base sequence and ontology of the dermal papilla cell gene isolated and cultured under hypoxic conditions according to the present invention were analyzed. As a result, as shown in Figure 3, it was confirmed that the hypoxic condition affects the extracellular matrix (ECM) protein, and among them, it was confirmed that the collagen expression levels of Col18A1, Col13A1, and Col23A1 were increased. The collagen affects the activity of dermal papilla cells (see Non-Patent Document 1), and thus it can be seen that the increased expression of the collagen under hypoxic conditions affects the attachment of dermal papilla cells to the culture plate.

[0038] 2-2. Analysis of mRNA Expression of Extracellular Matrix and Adhesion Molecules The mRNA expression levels of the extracellular matrix and adhesion molecules of the dermal papilla cell gene isolated and cultured under hypoxic conditions according to the present invention were analyzed. As a result, as shown in Figure 4, among various genes, it was confirmed that the expressions of MMP1, MMP3, MMP11, MMP12, and LAMA1 were decreased. In particular, MMP (matrix metalloproteinase) is an enzyme that degrades collagen, and thus it can be seen that the decreased MMP expression under hypoxic conditions inhibits collagen degradation and affects the attachment of dermal papilla cells to the culture plate.

[0039] [Experimental Example 3] Evaluation of Dermal Papilla Cell Isolation and Process Shortening Period In order to evaluate the establishment (acquisition) period of the dermal papilla cells isolated and cultured according to the present invention, experiments were conducted as follows.

[0040] The dermal papilla tissue (DP) separated from the hair follicle was inoculated onto a culture plate and cultured under hypoxic conditions (5% CO2: 2% O2) and normal conditions (5% CO2: 21% O2), and stored as a cell stock at the third passage (filled). In a 6-well plate, among the total dermal papilla tissue (DP) inoculated per well, the period required until more than 80% adhered was defined as "separation → adhesion", the cell density reached 60% or more of one well, and the period required until the first passage culture was defined as "separation → first passage", after the first passage culture, the cell density reached 80% or more, and the period required until the second passage culture was defined as "separation → second passage", after the second passage culture, the cell density reached 80% or more, and the period required until the third passage culture was defined as "separation → third passage", after the third passage culture, the period until filling as a stock was defined as "separation → filling", and the required periods were evaluated. The results are shown in Table 1 below.

[0041]

Table 1

[0042] As shown in Table 1, when measuring the period required until the dermal papilla tissue was separated and filled, it was found that it took 41 days when cultured under normal conditions, but 26 days when cultured under hypoxic conditions, and it was confirmed that about 15 days were shortened. Also, the period required until dermal papilla cells adhered from the separated dermal papilla tissue was also shortened by 5 days when cultured under hypoxic conditions, and it was found to be significantly shorter.

[0043] Therefore, in the establishment (acquisition) of dermal papilla cells, when culturing the separated dermal papilla tissue under hypoxic conditions, it can be seen that the period is significantly shortened compared to culturing under normal conditions, and it is shortened by more than 15 days until the final filling.

[0044] [Experimental Example 4] Evaluation of marker expression of dermal papilla cells In order to confirm that the cells separated and cultured according to the present invention are dermal papilla cells, they were analyzed by flow cytometry using an antibody that binds to the surface antigen of dermal papilla cells.

[0045] Using DPBS, the dermal papilla cells of each passage dissociated in the culture solution were washed twice. 1×10 6 cells were resuspended with 500 μL of cell staining buffer (1% BSA in DPBS) per cell, and then the cell suspension was dispensed at 100 μL per test group tube. 1 μL of the antibody corresponding to each test group was dispensed, and then the light was blocked and reacted at 4°C for 15 minutes. Using DPBS, the test groups stained with the antibody were washed once, and then the cells were resuspended with 100 μL of DPBS and analyzed using flow cytometry. The results are shown in Table 2 below.

[0046] The antibodies corresponding to each test group are as follows: isotype antibody control group (APC Mouse IgG1_κ Isotype Ctrl Antibody), expression evaluation staining group (for dermal papilla cell confirmation: APC anti-human CD34 Antibody, APC anti-human CD45 Antibody), purity evaluation staining group (APC anti-human CD44 Antibody, APC anti-human CD90 Antibody).

[0047] When the distribution rate of the confirmation expression evaluation staining group was 90% or more and the distribution rate of the purity evaluation staining group was less than 10% in the gate where the distribution rate of the control group was less than 0.1%, it was evaluated as suitable.

[0048] [Table 2]

[0049] As shown in Table 2, when evaluating the marker expression in the dermal papilla cells of each passage (p), it was found that the purity antibody expression in the dermal papilla cells of all passages was less than 1%, and the confirmation antibody expression was 90% or more. Therefore, it was confirmed that the cells separated and cultured according to the present invention are dermal papilla cells.

[0050] [Experimental Example 5] Evaluation of the Expression of Immunoresponse Activity Genes in Dermal Papilla Cells In order to evaluate the degree of expression of genes related to the immunoresponse activity of cells isolated and cultured according to the present invention, experiments were conducted as follows.

[0051] Dermal papilla cells were isolated from hair follicles (dermal papilla tissues) of male donors in their 20s and 30s and cultured under hypoxic conditions (5% CO2: 2% O2) and normal conditions (5% CO2: 20% O2). After the third passage culture, RNA of the cultured dermal papilla cells was extracted and the mRNA expression levels of genes related to the immune reaction activity of T cells were analyzed. The results are shown in Fig. 5. As a control group, dermal fibroblasts (mRNA expression level: 1) were used.

[0052] As shown in Fig. 5, when analyzing the degree of expression of genes related to the immune reaction activity via the RNA of dermal papilla cells, it was confirmed that when dermal papilla cells were isolated and cultured under hypoxic conditions, the expression of HLA-A, HLA-B, CD55, IL-1RA, and CCL2 decreased significantly compared with the case under normal conditions. In particular, the expression of HLA-A, HLA-B, and CD55 decreased remarkably, from which it can be seen that dermal papilla cells isolated and cultured under hypoxic conditions can reduce the immunoresponse activity by T cells.

[0053] Therefore, it can be seen that when dermal papilla cells are isolated and cultured under hypoxic conditions according to the present invention, the immunoprivilege of dermal papilla cells, that is, the immune modulation function, can be improved, thereby reducing the possible immunoresponse during the allotransplantation of dermal papilla cells. Eventually, it can be seen that the dermal papilla cells isolated and cultured according to the present invention have immunocompatibility.

Claims

Claim 1 A method for separating and culturing dermal papilla cells, comprising the steps of culturing dermal papilla separated from the scalp under hypoxic conditions and attaching the dermal papilla cells to a culture plate, wherein the hypoxic conditions have an oxygen saturation of 2%. Claim 2 The method for separating and culturing dermal papilla cells according to claim 1, characterized in that the period until the dermal papilla cells adhere to the culture plate is shortened by 5 days or more compared to the case where the dermal papilla is cultured under normal conditions.

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