Method for forming hair follicle-like structures through co-culture of an immortalized dermal papilla cell line derived from human hair follicles and keratinocytes

Abstract

The present invention relates to an immortalized dermal papilla cell line derived from human hair follicles and a method for forming a hair follicle-like structure through co-culture with keratinocytes. Specifically, as a result of three-dimensional co-culture of an immortalized dermal papilla cell line (SV40T-hTERT DPC) obtained by forced expression of SV40T, which inactivates a tumor suppressor gene, and the hTERT gene, which is a telomerase-promoting gene, in dermal papilla cells derived from human hair follicles, and keratinocytes, it was confirmed that a hair follicle-like structure in which the stratum corneum part grows in a form similar to that of a hair follicle-like body of a primary cultured hair follicle cell combination is formed. Thus, the present invention is applicable to various fields such as animal alternative tests and efficacy evaluation of hair loss prevention / hair follicle growth promotion materials. Through the immortalized dermal papilla cells, it is expected to ensure higher result consistency compared to existing technologies, enable stable cell supply from hair follicles, and eliminate high entry barriers for efficacy evaluation of the cell-based hair loss materials.

Description

Technical Field

[0001] The present invention relates to an immortalized dermal papilla cell line derived from human hair follicles and a method for forming a hair follicle-like structure through co-culture with keratinocytes.

Background Art

[0002] Hair loss is a disease that gives patients very negative psychological and social factors. Recently, in modern society, the number of patients is increasing rapidly due to various factors such as not only genetic factors but also the increase in the elderly population, stress, and changes in eating habits. As a result, in addition to the conventional invasive treatment method of autologous hair transplantation and drug treatment methods, the demand for overcoming and preventing hair loss continues to increase, and related industries are also increasing. In addition, the importance of hair loss materials is attracting attention not only in the pharmaceutical / quasi-drug field but also in the functional cosmetics and health functional food industries.

[0003] Despite such demand and the growth of industries, currently, methods for verifying the efficacy and safety of hair loss-related materials have various difficulties compared to other methods for verifying functional materials. In the case of human application tests, it is more difficult to recruit subjects than for other diseases, which results in a long test period and high costs. In the case of animal tests, due to the difference in physiological characteristics between the experimental model and humans, it is at a level where the efficacy is indirectly estimated and proven. Recently, animal ethics has been attracting worldwide attention, making it difficult to conduct animal tests. In the case of cell tests derived from humans, it is difficult to ensure consistent results due to differences between cell donors, and in fact, the deviation from the results of human application tests is the largest.

[0004] Various problems caused by such difficulties in efficacy verification (such as the inconsistency between human application test results and cell test results, high efficacy verification costs and required time, etc.) inhibit the reliability of hair loss-related functional materials and products, and act as an entry barrier preventing the growth of the functional product industries. Therefore, there is a growing need for a new cell-based efficacy evaluation system that complies with animal ethics, is similar to human application test results, and ensures consistency and stability of results.

[0005] Recently, previous research has reported on a method for forming a cell structure similar to the in-vivo hair follicle structure and its applicability to efficacy evaluation using cells derived from human hair follicles. However, the cells used in this research are primary cultured cells, and there are still problems such as differences in cells among donors and rapid changes in characteristics due to the number of cell passages, making it difficult to obtain consistent results. In addition, there is a disadvantage that it is difficult to stably secure primary cells derived from hair follicles due to limited hair follicle donation.

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a method for forming a hair follicle-like structure including a step of three-dimensionally co-culturing immortalized dermal papilla cells and keratinocytes.

Means for Solving the Problems

[0007] To achieve the above object, the present invention provides a method for forming a hair follicle-like structure, including: (1) separating dermal papilla cells from hair follicles; (2) immortalizing the separated dermal papilla cells; and (3) three-dimensionally co-culturing the immortalized dermal papilla cells and keratinocytes.

Effects of the Invention

[0008] The present invention relates to an immortalized dermal papilla cell line derived from human hair follicles and a method for forming a hair follicle-like structure through co-culture with keratinocytes. Specifically, the present invention relates to a dermal papilla cell line (SV40T-hTERT DPC) immortalized by forcibly expressing SV40T, which inactivates tumor suppressor genes, and the hTERT gene, which is a telomerase promoting gene, in dermal papilla cells derived from human hair follicles, and keratinocytes through three-dimensional co-culture. As a result, it was confirmed that a hair follicle-like structure in which the stratum corneum part grows in a form similar to that of a hair follicle-like body of a cell combination derived from primary culture hair follicles was formed. Accordingly, the present invention is applicable to various fields such as animal alternative tests and efficacy evaluations of hair loss prevention / hair follicle growth promoting materials. Through immortalized dermal papilla cells, it is possible to ensure higher result consistency compared to existing technologies, enable stable cell supply from hair follicles, and is expected to eliminate high entry barriers to the efficacy evaluation of the cell-based hair loss materials.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0010] The present invention provides a method for forming a hair follicle-like structure, comprising: (1) separating dermal papilla cells from hair follicles; (2) immortalizing the separated dermal papilla cells; and (3) three-dimensionally co-culturing the immortalized dermal papilla cells and keratinocytes.

[0011] Desirably, the immortalized dermal papilla cells are dermal papilla cells expressing simian virus 40T (SV40T) antigen and telomerase reverse transcriptase (hTERT) (SV40T-hTERT DPC), but are not limited thereto.

[0012] Desirably, the keratinocytes are outer root sheath cells (ORSC) derived from hair follicles or the skin keratinocyte cell line Ker-CT, but are not limited thereto.

[0013] Desirably, in the step (3), the cell ratio of the immortalized dermal papilla cells and keratinocytes is 1:1 to 1:4, but is not limited thereto.

[0014] Desirably, in the step (3), the keratinocytes can be added for three-dimensional co-culture immediately after culturing the immortalized dermal papilla cells or within 72 hours of culturing, but is not limited thereto.

[0015] Hereinafter, the present invention will be described in more detail through examples. It is obvious to those skilled in the art that these examples are merely for more specifically explaining the present invention, and the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0016] <Experimental Example>

[0017] The following experimental examples are for providing experimental examples commonly applicable to each example according to the present invention.

[0018] 1. Cell culture derived from human hair follicles and immortalization of dermal papilla cells

[0019] Human hair follicle tissue was used for the experiment with the residual hair provided after autologous hair transplantation under the consent of the patient at the Hair Transplantation Center of Kyungpook National University in the Republic of Korea.

[0020] The dermal papilla separated by cutting the hair bulb from the hair tissue was cultured in DMEM (Hyclone) supplemented with penicillin (100 U / ml), streptomycin (100 μg / ml), and 20% heat-inactivated serum to isolate dermal papilla cells (DPC). When cells grew from the dermal papilla tissue placed in the culture medium, the cells were collected using 0.25% trypsin / 10 mM EDTA and maintained in DMEM supplemented with 10% heat-inactivated serum under the conditions of 5% CO2 and 37°C.

[0021] After cutting the hair shaft from the hair tissue, it was cultured in DMEM (Hyclone) supplemented with penicillin (100 U / ml), streptomycin (100 μg / ml), and 20% heat-inactivated serum in a culture dish coated with collagen type 1 under the conditions of 5% CO2 and 37°C. When the hair shaft was placed on the bottom of the culture dish, it was cultured in EpiLife (Gibco) medium, a keratinocyte-specific culture medium containing Defined Growth Supplement (Gibco), under the conditions of 5% CO2 and 37°C (Figure 1). TM (Figure 1)

[0022] The immortalization method of dermal papilla cells was described in previous studies by our research team (BMB Reports. 44:8 512-516. 2011). Briefly, after injecting the pSV3neo plasmid containing the SV40T antigen and the neomycin resistance gene, cells that survived culturing in a neomycin-containing medium were selected. Then, SV40T antigen expression was confirmed through immunoassay to secure SV40T antigen-expressing dermal papilla cells. After injecting the pGRN145 plasmid containing the human telomerase reverse transcriptase hTERT gene and hygromycin into the secured SV40T antigen-expressing dermal papilla cells, cells that survived culturing in a hygromycin-containing medium were selected. Then, cells expressing both SV40T antigen and hTERT were confirmed by immunoassay and reverse transcription polymerase chain reaction to secure the immortalized dermal papilla cell line SV40T-hTERT DPC (Figure 2).

[0023] The immortalized human keratinocyte cell line Ker-CT was purchased from ATCC and cultured in a collagen type 1-coated culture medium under conditions of 5% CO2 and 37°C using Lonza's KGM-Gold TM BulletKit TM and cultured.

[0024] 2. Comparative confirmation of spheroid growth patterns between dermal papilla cells and immortalized dermal papilla cells

[0025] In our previous research, we confirmed the gene expression and growth characteristics between primary cultured dermal papilla cells and immortalized dermal papilla cells to verify their similarity. As a notable result in this previous research, since there was a significant difference in the growth rate between primary cultured dermal papilla cells and immortalized dermal papilla cells, when hair follicle cell spheroids were formed, the change in spheroid size due to the initial cell number was measured.

[0026] The immortalized dermal papilla cell line SV40T-hTERT DPC and primary dermal papilla cells (Primary DPC) cultured on a petri dish were detached with 0.25% trypsin / 10 mM EDTA. Then, 500, 1000, and 3000 cells / well of the immortalized dermal papilla cell line (SV40T-hTERT DPC) and 3000 cells / well of primary dermal papilla cells (Primary DPC) were seeded into a U bottom low attachment 96 well plate and cultured for at least 16 hours under the conditions of 5% CO2, 37 °C, and 100 μl / well of DMEM supplemented with 10% heat-inactivated serum to form spheroids. Thereafter, photographs were taken with a microscope every 24 hours, and the diameter of the spheroids was measured and observed (Figure 3).

[0027] 3. Confirmation of the formation of hair follicle-like structures using dermal papilla cells and immortalized dermal papilla cell spheroids

[0028] To form spheroids, primary dermal papilla cells and the immortalized dermal papilla cell line SV40T-hTERT DPC cultured on a petri dish were detached with 0.25% trypsin / 10 mM EDTA. Then, 1000 cells / well were seeded into a U bottom low attachment 96 well plate and cultured under the conditions of 5% CO2, 37 °C, and 100 μl / well of DMEM supplemented with 10% heat-inactivated fetal bovine serum.

[0029] Thereafter, outer root sheath cells cultured on a petri dish were detached with 0.25% trypsin / 10 mM EDTA, diluted to 30,000 cells / ml in William’s E medium containing 100 U / ml of penicillin / streptomycin, 10 ng / ml of hydrocortisone, 10 μg / ml of insulin, and 2 mM of L-glutamin, and 100 μl / well were added to the above-described low attachment 96 well plate. Then, co-culture was performed under the conditions of 5% CO2 and 37 °C, and observations were made over time under a microscope.

[0030] To compare the degree of formation of hair follicle-like structures by cell combinations, the number of immortalized dermal papilla cells (SV40T-hTERT DPC) was adjusted during spheroid formation so that the number of immortalized dermal papilla cells per spheroid was adjusted from a minimum of 300 to a maximum of 1000. To compare the degree of formation of hair follicle-like structures according to the start time of dermal papilla cell-keratinocyte co-culture, the injection time of keratinocytes was adjusted from immediately after the injection of dermal papilla cells to 72 hours after the injection of dermal papilla cells. Also, the number of keratinocytes derived from hair follicles was adjusted to three times the same number compared to immortalized cells and the experiment was advanced (Figure 4).

[0031] 4. Confirmation of the presence or absence of formation of hair follicle-like structures using only immortalized cells

[0032] After the SV40T-hTERT DPC cultured on a flat plate was aspirated with 0.25% trypsin / 10 mM EDTA, 1,000 cells / well were placed in a U bottom low attachment 96 well plate and cultured for 24 hours under the conditions of 5% CO2, 37 °C, and 100 μl / well of DMEM supplemented with 10% heat-inactivated serum to form spheroids.

[0033] Thereafter, after the immortalized keratinocyte cell line Ker-CT cultured on a culture dish was aspirated with 0.25% trypsin / 10 mM EDTA, it was diluted to 30,000 cells / ml under the respective conditions of DMEM containing 10% heat-inactivated fetal bovine serum, which is a medium for dermal papilla cell growth, penicillin / streptomycin 100 U / ml, hydrocortisone 10 ng / ml, insulin 10 μg / ml, and L-glutamin 2 mM, William’s E, which is a tissue culture medium, and KGM Gold Medium Bulletkit, which is a medium for keratinocyte cell line growth. Then, 100 μl / well was added to the above-described low attachment 96 well plate, and co-cultured under the conditions of 5% CO2 and 37 °C, and observed every 24 hours (Figure 5).

[0034] 5. Confirmation of the cell arrangement morphology of the formed hair follicle structure

[0035] The medium of the immortalized dermal papilla cell line SV40T-hTERT DPC cultured on a petri dish was removed, and the cells were treated with phosphate-buffered saline (PBS) containing 1 μM of the Cell tracker CM-DiI for 30 minutes. Subsequently, after aspirating with 0.25% trypsin / 10 mM EDTA, 1,000 cells / well were seeded into a U-bottom low attachment 96-well plate and cultured for 24 hours under the conditions of 5% CO2, 37 °C, and 100 μl / well of DMEM supplemented with 10% heat-inactivated serum to form spheroids.

[0036] Subsequently, the keratinocytes cultured on a petri dish were aspirated with 0.25% trypsin / 10 mM EDTA, diluted to 30,000 cells / ml in William's E medium containing 100 U / ml of penicillin / streptomycin, 10 ng / ml of hydrocortisone, 10 μg / ml of insulin, and 2 mM of L-glutamine, and 100 μl / well was added to the above-described low attachment 96-well plate. Then, co-culture was performed under the conditions of 5% CO2 and 37 °C, and observation was carried out using an optical microscope and a fluorescence microscope every 24 hours (Figure 6).

[0037] <Example 1> Spheroid growth pattern between dermal papilla cells and immortalized dermal papilla cells

[0038] It is known that the size of the dermal papilla in the hair follicle has a size of about 100 to 250 μm. Accordingly, in order to form a dermal papilla cell spheroid with a diameter of around 250 μm, as a result of injecting 3,000 primary cultured dermal papilla cells (primary DPC) per well of a 96 well U bottom Plate, a spheroid with a diameter of around 250 μm could be confirmed, and it was confirmed that the size of the spheroid decreased slightly over time. After injecting 3,000 SV40T-hTERT DPC per well of a 96 well U bottom Plate, the spheroid growth pattern was different from that of Primary DPC, and an increase in spheroid size was confirmed after a certain period of time. It was confirmed that the ratio of cells in the spheroid was higher in density compared to primary cultured dermal papilla cells (Figs. 7 and 8).

[0039] Therefore, when forming a spheroid using immortalized dermal papilla cells for the production of a hair follicle-like structure, in order to produce a morphology similar to that of a primary cultured dermal papilla cell spheroid, the number of cells and the culture time must be adjusted. In the present invention, in the case of immortalized dermal papilla cells, it was confirmed that the growth pattern of immortalized dermal papilla cells was similar to that of dermal papilla cells based on a cell number that was 1 / 3 times that of existing primary cultured dermal papilla cells and within 72 hours of spheroid formation time (Fig. 8).

[0040] <Example 2> Confirmation of the formation of a hair follicle-like structure using dermal papilla cells and immortalized dermal papilla cell spheroids

[0041] To form a hair follicle-like structure, outer root sheath cells, which are keratinocytes derived from hair follicles, were injected into a U-bottom plate on which spheroids had been formed. As a result, it was possible to confirm the formation of a hair follicle-like structure with a structure similar to that of a hair follicle without differentiating between primary cultured dermal papilla cells and SV40T-hTERT DPC cells (Figure 9). As a result of testing by adjusting the administration timing of the outer root sheath cells, it was confirmed that when the outer root sheath cells were injected within 24 hours after injecting the dermal papilla cells for spheroid formation, the formation of the hair follicle-like structure was smooth (Figure 9B). As a result of testing by adjusting the injection amounts of the dermal papilla cells and the outer root sheath cells, in the case of primary cultured dermal papilla cells, the growth of the hair follicle-like structure was remarkable under the condition of three times the number of outer root sheath cells compared to the dermal papilla cells in the case of SV40T-hTERT DPC. It was confirmed that when a certain ratio between the spheroids formed from the above and the keratinocytes was maintained, the growth of the hair follicle-like structure was remarkable. It was possible to confirm that the cell ratio of dermal papilla cells to outer root sheath cells for the formation of the hair follicle structure is optimally about 1:3 to 1:4 (Figure 9C).

[0042] <Example 3> Confirmation of the presence or absence of formation of a hair follicle-like structure using only immortalized cells and the cell arrangement state

[0043] To form a whole immortalized cell hair follicle-like structure, a hair follicle-like structure formation test was conducted using the immortalized cell line Ker-CT of human neonatal epidermal cells. As a result, the formation of a hair follicle-like structure cell body with a morphology similar to that of an outer root sheath cell-utilizing hair follicle-like structure was confirmed. As a result of co-culturing 1,000 keratinocyte cell lines with 1,000 SV40T-hTERT DPC spheroids and co-culturing 3,000 keratinocyte cell lines, it was confirmed that both formed hair follicle-like cell structures (Figs. 10 and 11). When comparing three culture medium conditions, it was confirmed that the hair follicle-like structure grew the longest under the William’s E medium condition, which is a tissue culture medium, compared to other culture medium conditions (Fig. 10). In the case of the keratinocyte cell line Ker-CT, it was confirmed that the hair follicle-like structure was formed when the same number of outer root sheath cells of the hair follicle were injected and also when 1,000 cells that had not been formed were injected (Figs. 9C and 10). From the above results, as a result of co-culturing a combination of 1,000 immortalized dermal papilla cell spheroids with 3,000 keratinocyte cell lines in the William’s E medium condition, which is a tissue culture medium, in the tissue culture medium in which the generation of the hair follicle-like structure was confirmed, the formation of the hair follicle structure most similar to the hair follicle was confirmed (Figs. 10B and 11).

[0044] The hair follicle-like structure formed by attaching a cell label to the immortalized dermal papilla cells was observed by fluorescence, and it was confirmed that it had a cell array structure similar to that of the dermal papilla located in the hair bulb of the hair follicle in vivo. As a result of the test for attaching the cell label, it was confirmed that the spheroid composed of the dermal papilla cell line of the cell structure and the keratinocyte cell line formed a structure similar to the hair follicle (Fig. 11).

[0045] In addition, the hair follicle-like structure was cryosectioned and immunohistochemical staining was performed to confirm the cell biomarkers in the hair follicle-like structure. As a result of the immunohistochemical staining, the expression of vimentin, which is a dermal papilla cell line-specific biomarker, and keratin 14, which is a keratinocyte cell line-specific biomarker, was confirmed. Thereby, it was confirmed that the cells were distributed in a form similar to that of the human hair follicle inside the hair follicle-like structure (Fig. 12).

[0046] As described above in detail for specific parts of the present invention, it is clear to those skilled in the art that such specific descriptions are merely preferred embodiments and do not thereby limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the claims and their equivalents.

Claims

**Claim 1** (1) Separating dermal papilla cells from a hair follicle; (2) Immortalizing the separated dermal papilla cells; (3) Three-dimensionally co-culturing the immortalized dermal papilla cells and keratinocytes; A method for forming a hair follicle-like structure, comprising the above steps. **Claim 2** The method for forming a hair follicle-like structure according to claim 1, wherein the immortalized dermal papilla cells are dermal papilla cells expressing simian virus 40 T (SV40T) antigen and human telomerase reverse transcriptase (hTERT). **Claim 3** The method for forming a hair follicle-like structure according to claim 1, wherein the keratinocytes are outer root sheath cells (ORSC) derived from a hair follicle or a skin keratinocyte cell line Ker-CT. **Claim 4** The method for forming a hair follicle-like structure according to claim 1, wherein in step (3), the cell ratio of the immortalized dermal papilla cells to the keratinocytes is 1:1 to 1:

4. **Claim 5** The method for forming a hair follicle-like structure according to claim 1, wherein in step (3), the keratinocytes are added for three-dimensional co-culture immediately after culturing the immortalized dermal papilla cells or within 72 hours of culturing.

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