Method for forming hair follicle-like structures by co-culturing human hair follicle-derived immortalized hair papilla cell lines and keratinocytes

Abstract

The invention relates to a method for forming a hair follicle-like structure by co-culturing an immortalized hair papilla cell line derived from human hair follicles and keratinocytes. Specifically disclosed is a hair papilla cell line (SV40T-hTERT DPC) which is immortalized by forced expression of SV40T, which deactivates a cancer suppressor gene, in human hair follicle-derived hair papilla cells and hTERT gene, which is a telomerase elongase promoting gene, and keratinocytes, are co-cultured three-dimensionally. From the result, it has been confirmed that a hair follicle-like structure is formed in which a cuticle grows so that a hair follicle analogue in combination with a primary-cuticle hair follicle-derived cell has a shape similar to that of the primary-cuticle hair follicle-derived cell. Therefore, the method can be applied to multiple fields of animal replacement experiments, efficacy evaluation of materials for preventing alopecia / promoting hair follicle growth and the like. Through immortalized hair papilla cells, higher result consistency can be ensured compared with that in the prior art, and stable hair follicle-derived cell supply can be realized, so that the problem of high entry barriers of efficacy evaluation of alopecia materials based on cells is expected to be solved.

Description

Technical Field

[0001] The present application relates to a method for forming a hair follicle-like structure by co-culturing an immortalized hair papilla cell line derived from human hair follicles and keratinocytes. Background Art

[0002] Hair loss is a disease that brings very negative factors to the psychological and social life of patients. Recently in modern society, in addition to genetic factors, the number of patients has been increasing sharply due to various factors such as the increase in the elderly population, stress, and changes in eating habits. It can be seen that in addition to existing invasive treatments (such as autologous hair transplantation) and drug treatments, the demand for overcoming and preventing hair loss is continuing to increase, and related industries are also expanding. In addition, in addition to the medical and non-medical fields, the importance of hair loss-related materials in the functional cosmetics and health functional food industries is also becoming increasingly prominent.

[0003] Despite this demand and the growth of related industries, the current methods used to verify the efficacy and safety of hair loss-related materials still face more difficulties than the verification methods of other functional materials. For human-applicable experiments, the recruitment of subjects is more difficult than for other diseases, so the experiments are time-consuming and costly. For animal experiments, due to the difference between the experimental model and the physiological characteristics of the human body, its efficacy can only be inferred and proved indirectly, and with the emergence of animal ethics issues worldwide, it is difficult to conduct animal experiments. For human-derived cell experiments, due to the differences between cell donors, it is difficult to obtain consistent results, and the gap with the results of actual human-applicable experiments is the largest.

[0004] These various problems arising from efficacy verification (such as the inconsistency between human-applicable experimental results and cell-based experimental results, high efficacy verification costs and time consumption, etc.) not only weaken the credibility of hair loss-related functional materials and products, but also become an entry barrier that hinders the growth of the functional product industry. Therefore, there is an urgent need for a new cell-based efficacy evaluation system that complies with animal ethics, is similar to human-applicable experimental results, and can ensure the consistency and stability of results.

[0005] In recent previous studies, a method of using human hair follicle-derived cells to form a cell structure similar to the structure of hair follicles in vivo and the possibility of applying it to efficacy evaluation have been published. However, the cells used in this study were primary cultured cells, and there is still a problem of difficulty in obtaining consistent results due to differences in cells between donors and drastic changes in characteristics due to the increase in the number of cell passages. In addition, due to the limitation of hair follicle supply, there is a disadvantage that it is difficult to stably obtain primary cells derived from hair follicles. Summary of the invention

[0006] Problems to be solved by the invention

[0007] The purpose of the present application is to provide a method for forming a hair follicle-like structure, which comprises the step of three-dimensionally co-culturing immortalized hair papilla cells and keratinocytes.

[0008] Solutions for solving problems

[0009] In order to achieve the above-mentioned purpose, the present application provides a method for forming a hair follicle-like structure, comprising: a step of isolating hair papilla cells from hair follicles (1); a step of immortalizing the isolated hair papilla cells (2); and a step of three-dimensionally co-culturing the immortalized hair papilla cells and keratinocytes (3).

[0010] Effects of the Invention

[0011] The present application relates to a method for forming a hair follicle-like structure by co-culturing an immortalized hair papilla cell line and keratinocytes derived from human hair follicles. Specifically, the hair papilla cell line (SV40T-hTERT DPC) and keratinocytes immortalized by forcibly expressing the SV40T tumor suppressor gene in the hair papilla cells derived from human hair follicles and the hTERT gene as a telomerase elongation enzyme promoting gene are co-cultured in three dimensions, and the results confirm that a stratum corneum is formed to grow into a hair follicle analog with a similar morphology to the hair follicle analog combined with the primary culture (primary cultrue) hair follicle-derived cells. Therefore, the present application can be applied to multiple fields such as animal replacement experiments, efficacy evaluation of hair loss prevention / hair follicle growth promotion materials, etc. Through immortalized hair papilla cells, higher consistency of results can be ensured than the prior art, and a stable supply of hair follicle-derived cells can be achieved, thereby hopefully solving the high entry barrier problem of cell-based efficacy evaluation of hair loss materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A schematic diagram showing the primary culture of dermal papilla cells and outer root sheath cells derived from human hair follicles.

[0013] Figure 2 The immortalization process and results of dermal papilla cells are shown.

[0014] Figure 3 Schematic diagram showing the comparative confirmation of the spheroid growth pattern between primary cultured dermal papilla cells and immortalized dermal papilla cells.

[0015] Figure 4 A schematic diagram showing confirmation of the formation of hair follicle-like structures using primary cultured hair papilla cells and immortalized hair papilla cell spheroids.

[0016] Figure 5 A schematic diagram showing the confirmation of whether a hair follicle-like structure is formed using only immortalized cells.

[0017] Figure 6 A schematic diagram showing the confirmation of the cell arrangement morphology of the formed hair follicle-like structure.

[0018] Figure 7 The results of the comparison of the growth patterns of primary cultured spheroids of hair papilla cells and SV40T-hTERT DPC spheroids of the hair papilla cell line into which SV40T-hTERT was introduced are shown.

[0019] Figure 8 The results of the change in spheroid size according to the culture time are shown. Regarding the diameter of SV40T-hTERT DPC spheroids at different initial cell numbers and culture times, under the conditions of initial cell number ≤ 1K and culture time ≤ 72 hr, spheroids with a diameter similar to the known size of the hair follicle intradermal papilla were formed (A). By observing the spheroids cultured for 72 hours, it was confirmed that the cells inside the spheroids using SV40T-hTERT DPCs were more densely arranged than those using primary cultured cells, and even if the initial cell number was small, a diameter of similar size was formed after 72 hr of culture (B to F).

[0020] Fig. 9 The follicle-like structures obtained by co-culturing primary cultured hair papilla cells (Primary DPC) or immortalized hair papilla cell line SV40T-hTERT DPC spheroids with human hair follicle outer root sheath cells (ORSC) are shown. The co-culture results of primary cultured DPC or SV40T-hTERT DPC and human hair follicle-derived outer root sheath cells according to time (A), the formation results of hair follicle-like structures according to the start date of co-culture of immortalized hair papilla cell line (SV40T-hTERT DPC) and outer root sheath cells (ORSC) (B), and the co-culture results of immortalized hair papilla cell line SV40T-hTERT DPC and outer root sheath cells according to the cell number of outer root sheath cells (C).

[0021] Fig.10 The results of the formation of hair follicle-like structures under different culture medium conditions according to time are shown. The results of co-culturing 1000 immortalized hair papilla cell line SV40T-hTERT DPC spheroids with 1000 (A) and 3000 (B) keratinocytes are shown.

[0022] Fig.11 The results of confirming the cell arrangement state at different numbers of keratinocyte cell lines using only immortalized cells in a hair follicle-like structure are shown.

[0023] Fig.12 Shown are the results of cryosectioning of hair follicle-like structures and immunohistostaining to confirm cellular biomarkers within the hair follicle-like structures. DETAILED DESCRIPTION

[0024] The present application provides a method for forming a hair follicle-like structure, comprising: a step (1) of isolating hair papilla cells from hair follicles; a step (2) of immortalizing the isolated hair papilla cells; and a step (3) of performing three-dimensional co-culture on the immortalized hair papilla cells and keratinocytes.

[0025] Preferably, the immortalized dermal papilla cells may be dermal papilla cells expressing simian virus 40T (SV40T) antigen and telomerase reverse transcriptase (hTERT) (SV40T-hTERT DPC), but are not limited thereto.

[0026] Preferably, the keratinocytes may be hair follicle-derived outer root sheath cells (ORSC) or skin keratinocyte cell lines (Ker-CT), but are not limited thereto.

[0027] Preferably, in the above step (3), the cell ratio of the immortalized hair papilla cells to keratinocytes may be 1:1 to 1:4, but is not limited thereto.

[0028] Preferably, in the above step (3), the above keratinocytes may be added within 72 hours after the start of culturing the above immortalized hair papilla cells to perform three-dimensional co-culture, but the present invention is not limited thereto.

[0029] Best Mode for Carrying Out the Invention

[0030] The present application is further described below by way of examples. These examples are only for the purpose of more specifically describing the present application. It is obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0031] <Experimental example>

[0032] The following experimental examples are intended to provide experimental examples that are commonly applicable to various embodiments according to the present invention.

[0033] 1. Cultivation of human hair follicle-derived cells and immortalization of dermal papilla cells

[0034] Human hair follicle tissue was obtained from the Hair Transplant Center of Kyungpook National University with the patient's consent as residual hair after autologous hair transplantation.

[0035] The hair bulb was cut off from the hair tissue and the hair papilla was separated and cultured in DMEM (Hyclone) supplemented with penicillin (100U / ml), streptomycin (100ug / ml) and 20% heat-inactivated serum to isolate hair papilla cells (DPC). When cells grew out of the hair papilla tissue attached to the culture medium, the cells were collected using 0.25% trypsin / 10mM ethylenediaminetetraacetic acid (EDTA) and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated serum under the conditions of 5% CO2 and 37°C.

[0036] After cutting the hair roots from the hair tissue, they were placed in a culture dish coated with collagen type 1 and cultured in DMEM (Hyclone) supplemented with penicillin (100U / ml), streptomycin (100μg / ml) and 20% heat-inactivated serum at 5% CO2 and 37°C. After the hair roots attached to the bottom of the culture dish, they were placed in a culture dish containing penicillin (100U / ml), streptomycin (100μg / ml) and EpiLife TM Definition of Growth Supplement (EpiLife TM The cells were cultured in EpiLife (Gibco), a keratinocyte-specific culture medium containing Defined Growth Supplement (Gibco), at 5% CO2 and 37°C. Figure 1 ).

[0037] The immortalization method of dermal papilla cells has been described in a previous study by our research team (BMB Reports. 44: 8 512-516. 2011). In short, the pSV3neo plasmid containing the SV40T antigen and the neomycin resistance gene was first transfected, and then cultured in a medium containing neomycin, surviving cells were screened, and the expression of the SV40T antigen was confirmed by immunoassay, thereby obtaining dermal papilla cells expressing the SV40T antigen. The obtained SV40T antigen-expressing hair papilla cells were transfected with the pGRN145 plasmid containing the human telomerase reverse transcriptase (hTERT) gene and hygromycine, and then cultured in a medium containing hygromycine, surviving cells were screened, and cells expressing SV40T antigen and hTERT were confirmed by immunoassay and reverse transcription polymerase chain reaction, thereby obtaining the immortalized hair papilla cell line SV40T-hTERT DPC ( Figure 2 ).

[0038] Immortalized human keratinocyte cell line (Ker-CT) was purchased from ATCC and cultured in a medium coated with type 1 collagen at 5% CO2 and 37°C using KGM-Gold from Lonza. TM BulletKit TM Cultivated.

[0039] 2. Comparison and confirmation of the spherical growth pattern between dermal papilla cells and immortalized dermal papilla cells

[0040] In the previous study of the present inventors, the similarity between primary cultured hair papilla cells and immortalized hair papilla cells was verified by confirming their gene expression and growth characteristics. In this previous study, a noteworthy result was that the change in the size of the spheroid according to the initial cell number when the hair follicle cell spheroid was formed was measured, as the growth rate of the primary cultured hair papilla cells and the immortalized hair papilla cells showed a significant difference.

[0041] After collecting immortalized dermal papilla cells (SV40T-hTERT DPC) and primary dermal papilla cells (Primary DPC) cultured in a culture dish with 0.25% trypsin / 10mM EDTA, 500, 1000, and 3000 cells / well of immortalized dermal papilla cells (SV40T-hTERT DPC) and 3000 cells / well of primary dermal papilla cells (Primary DPC) were placed in a U bottom low attachment 96-well plate and cultured for more than 16 hours at 5% CO2 and 37°C in DMEM (100μl / well) supplemented with 10% heat-inactivated serum to form spheroids. Afterwards, the diameter of the spheroids was observed and photographed under a microscope every 24 hours ( Figure 3 ).

[0042] 3. Confirmation of the formation of hair follicle-like structures using dermal papilla cells and immortalized dermal papilla cell spheroids

[0043] In order to form spheroids, primary cultured dermal papilla cells and immortalized dermal papilla cell line (SV40T-hTERT DPC) cultured in culture dishes were collected with 0.25% trypsin / 10mM EDTA, and 1000 cells / well were placed into U bottom low attachment 96-well plates and cultured under 5% CO2 and 37°C in DMEM (100μl / well) supplemented with 10% heat-inactivated fetal bovine serum.

[0044] Afterwards, the outer root sheath cells cultured on the culture dish were collected using 0.25% trypsin / 10mM EDTA, diluted to a concentration of 30,000 cells / ml in William's E medium containing 100U / ml penicillin / streptomycin, 10ng / ml hydrocortisone, 10ug / ml insulin, and 2mM L-glutamin, and added to the above-mentioned low attachment 96-well plate at 100μl / well. Co-culture was performed under 5% CO2 and 37°C, and the cells were observed over time under a microscope.

[0045] In order to compare the degree of formation of hair follicle-like structures under different cell combinations, the number of immortalized hair papilla cell lines (SV40T-hTERT DPCs) was adjusted during spheroid formation so that the number of immortalized hair papilla cells in each spheroid was between a minimum of 300 and a maximum of 1000. In addition, in order to compare the degree of formation of hair follicle-like structures according to the starting time of hair papilla cell-keratinocyte co-culture, the injection timing of keratinocytes was adjusted from the injection of hair papilla cells to 72 hours of culture after the injection of hair papilla cells. In addition, when conducting the experiment, the number of hair follicle-derived keratinocytes was adjusted to the same as the number of immortalized cells to three times the number of immortalized cells ( Figure 4 ).

[0046] 4. Confirmation of the formation of hair follicle-like structures using only immortalized cells

[0047] SV40T-hTERT DPCs cultured on a plate were collected using 0.25% trypsin / 10mM EDTA, and then placed into a U-bottom low attachment 96-well plate at 1000 cells / well. The cells were cultured for 24 hours under the conditions of 5% CO2 and 37°C in DMEM supplemented with 10% heat-inactivated serum (100 μl / well) to form spheroids.

[0048] Afterwards, immortalized keratinocyte cell line (Ker-CT) cultured in a culture dish was collected with 0.25% trypsin / 10mM EDTA, and then diluted to a concentration of 30,000 cells / ml under the following three culture medium conditions: DMEM, which is used as a culture medium for hair papilla cells, contains 10% heat-inactivated fetal bovine serum; William's E, which is used as a tissue culture medium, contains 100U / ml penicillin / streptomycin, 10ng / ml hydrocortisone, 10ug / ml insulin, and 2mM L-glutamin; KGM Gold Medium Bulletkit, which is used as a culture medium for keratinocyte cell line growth. Then, it was added to the above-mentioned low attachment 96-well plate at 100μl / well, co-cultured under 5% CO2 and 37°C, and observed every 24 hours ( Figure 5 ).

[0049] 5. Confirm the cell arrangement morphology of the formed hair follicle structure

[0050] After removing the medium of immortalized dermal papilla cell line (SV40T-hTERT DPC) cultured on a plate in a culture dish, the cells were treated with phosphate buffered saline (PBS) containing 1 μM CM-DiI (used as a cell tracker) for 30 minutes. Afterwards, the cells were harvested with 0.25% trypsin / 10 mM EDTA, and 1000 cells / well were placed in a U bottom low attachment 96-well plate, and cultured for 24 hours under the conditions of 5% CO2 and 37°C, and DMEM (100 μl / well) supplemented with 10% heat-inactivated serum, to form spheroids.

[0051] After that, the keratinocytes cultured on the culture dish were collected with 0.25% trypsin / 10mM EDTA, diluted to a concentration of 30,000 cells / ml in William's E medium containing 100U / ml penicillin / streptomycin, 10ng / ml hydrocortisone, 10ug / ml insulin, and 2mM L-glutamin, and added to the above-mentioned low attachment 96-well plate at 100μl / well, and co-cultured under 5% CO2 and 37°C, and observed by optical microscopy and fluorescence microscopy every 24 hours ( Figure 6 ).

[0052] <Example 1> Spherical growth pattern between dermal papilla cells and immortalized dermal papilla cells

[0053] It is known that the size of the hair papilla in the human hair follicle is about 100 to 250 μm. In order to form a hair papilla cell spheroid with a diameter of about 250 μm, 3,000 primary cultured hair papilla cells (primary DPC) were injected into each well of a 96-well U bottom plate (96well U bottom Plate), and from the results, it was confirmed that there was a spheroid with a diameter of about 250 μm. As time went on, the size of the spheroid decreased slightly. Unlike the primary cultured DPC, after injecting 3,000 SV40T-hTERT DPC into each well of a 96-well U bottom plate (96well U bottom Plate), it was confirmed that the size of the spheroid increased after a certain period of time. It was confirmed that the proportion of cells in the spheroid is higher than that of the primary cultured hair papilla cells (density). Figure 7 and Figure 8 ).

[0054] Therefore, in order to produce hair follicle-like structures, when immortalized hair papilla cells are used to form spheroids, in order to make their morphology similar to that of the primary cultured hair papilla cell spheroids, it is necessary to adjust the number of cells and the culture time. In the present invention, it has been confirmed that when immortalized hair papilla cells are used, the number of cells is 1 / 3 of that of the primary cultured hair papilla cells, and under the standard of spheroid formation time of 72 hours, the growth pattern of immortalized hair papilla cells is similar to that of hair papilla cells ( Figure 8 ).

[0055] <Example 2> Confirmation of the formation of hair follicle-like structures using hair papilla cells and immortalized hair papilla cell spheroids

[0056] In order to form hair follicle-like structures, outer root sheath cells, which are keratinocytes derived from hair follicles, were injected into a U-shaped bottom plate formed with spheroids. From the results, it was confirmed that both primary cultured hair papilla cells and SV40T-hTERT DPC cells could form hair follicle-like structures with a similar structure to hair follicles ( Fig. 9 ). From the results of experiments conducted by adjusting the administration time of outer root sheath cells, it was confirmed that in order to form spheroids, the formation of hair follicle-like structures was more smooth when outer root sheath cells were injected within 24 hours of the injection of hair papilla cells ( Fig. 9 B). The results of the experiment by adjusting the injection amount of hair papilla cells and outer root sheath cells showed that the growth of hair follicle-like structures was particularly prominent under the following conditions: for primary cultured hair papilla cells, there were the same number of outer root sheath cells; for SV40T-hTERT DPCs, there were 3 times the number of outer root sheath cells compared to hair papilla cells. It was confirmed that when a certain ratio between the spheroids formed in the above manner and keratinocytes was maintained, the growth of hair follicle-like structures was particularly prominent. It was confirmed that when forming a hair follicle structure, preferably, the cell ratio of hair papilla cells to outer root sheath cells was about 1:3 to 1:4 ( Fig. 9 C).

[0057] <Example 3> Confirmation of the formation of hair follicle-like structures and the cell arrangement state using only immortalized cells

[0058] In order to form a whole immortalized cell hair follicle-like structure, a hair follicle-like structure formation experiment was conducted using the cell line Ker-CT immortalized from human newborn foreskin cells. From the results, it can be confirmed that a hair follicle-like structure cell body with a similar morphology to the hair follicle-like structure using outer root sheath cells was formed. By co-culturing 1000 SV40T-hTERT DPC spheroids with 1000 keratinocytes and 1000 SV40T-hTERT DPC spheroids with 3000 keratinocytes, it can be confirmed that both have formed a hair follicle-like cell structure ( Fig.10 and Fig.11 ). By comparing the three culture medium conditions, it was confirmed that the hair follicle-like structure was longest when William's E medium was used as a tissue culture medium compared to the other culture medium conditions ( Fig.10 ). For the keratinocyte cell line (Ker-CT), it was confirmed that when 1000 cells were injected, which failed to form a hair follicle-like structure when the same number of hair follicle outer root sheath cells were injected, they could still form a hair follicle-like structure ( Fig. 9 C, Fig.10). The above results confirmed that when a combination of 1000 immortalized hair papilla cell spheroids and 3000 keratinocytes was used in a tissue culture medium that has been confirmed to generate a hair follicle-like structure, and co-cultured under the conditions of William's E medium as a tissue culture medium, a hair follicle structure closest to a hair follicle was formed ( Fig.10 B and Fig.11 ).

[0059] By attaching cell markers to immortalized hair papilla cells and observing the hair follicle-like structure formed by fluorescence, it was confirmed that it has a cell arrangement structure with a similar morphology to the hair papilla located in the hair bulb of the hair follicle in vivo. The results of the cell marker attachment experiment confirmed that the spheroid composed of the hair papilla cell line in the cell structure and the keratinocyte line can form a structure similar to the hair follicle ( Fig.11 ).

[0060] In addition, the cell biomarkers in the hair follicle-like structure were confirmed by freezing sections and immunohistochemical staining of the hair follicle-like structure. The results of immunohistochemical staining confirmed the expression of vimentin, a biomarker specific to the hair papilla cell lineage, and keratin 14, a biomarker specific to the keratinocyte lineage. This confirmed that within the hair follicle-like structure, the cells were distributed in a morphology similar to that of human hair follicles ( Fig.12 ).

[0061] Above, the specific parts of the present application are described in detail. For those skilled in the art, it is obvious that such specific technology is only a preferred embodiment and the scope of the present invention is not limited thereto. Therefore, the actual scope of the present invention will be defined by the attached claims and their equivalents.

Claims

1. A method for forming a hair follicle-like structure, comprising: The step of isolating hair papilla cells from hair follicles (1); A step (2) of immortalizing the isolated dermal papilla cells; as well as The step (3) is to perform three-dimensional co-culture on the immortalized hair papilla cells and keratinocytes.

2. The method for forming a hair follicle-like structure according to claim 1, characterized in that: The immortalized dermal papilla cells are dermal papilla cells expressing simian vacuolating virus 40T antigen and telomerase reverse transcriptase.

3. The method for forming a hair follicle-like structure according to claim 1, characterized in that: The keratinocytes are outer root sheath cells derived from hair follicles or skin keratinocyte cell line Ker-CT.

4. The method for forming a hair follicle-like structure according to claim 1, characterized in that: In the step (3), the cell ratio of the immortalized hair papilla cells to keratinocytes is 1:1 to 1:

4.

5. The method for forming a hair follicle-like structure according to claim 1, characterized in that: In the step (3), the keratinocytes are added to perform three-dimensional co-culture within 72 hours after the immortalized dermal papilla cells are cultured.

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