WO2024014752A1 - Method for forming hair-follicle-like structure through co-culturing of human-hair-follicle-derived immortalized dermal papilla cells and keratinocytes - Google Patents

Abstract

The present invention relates to a method for forming a hair-follicle-like structure through the co-culturing of human hair-follicle-derived immortalized dermal papilla cells and keratinocytes. Specifically, it has been identified that, when keratinocytes are three-dimensionally co-cultured with a dermal papilla cell line (SV40T-hTERT DPC) immortalized through the forced expression, in human-hair-follicle-derived dermal papilla cells, of SV40T, which inactivates cancer suppressor genes, and an hTERT gene, which is a telomerase promoting gene, a hair-follicle-like structure in which the stratum corneum grows to be formed similar to a hair-follicle-like structure of a combination of primary cultured hair-follicle-derived cells is formed. Therefore, the present invention can be applied in various fields such as those of non-animal alternative testing and efficacy evaluations of hair loss prevention/hair follicle growth promotion materials, can ensure result consistency higher than that of a conventional technology through immortalized dermal papilla cells, and enables a stable supply of hair-follicle-derived cells, and thus it is expected that high barriers to entry into cell-based efficacy evaluation of materials for hair loss can be removed.

Description

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

The present invention relates to a method of forming a hair follicle-like structure through co-culture of an immortalized human hair follicle-derived dermal papilla cell line and keratinocytes.

Hair loss is a disease that poses significant psychological and social negative factors to patients. Recently, in modern society, the number of patients is rapidly increasing due to various factors such as genetic predisposition, an increase in the elderly population, stress, and changes in eating habits. From this, in addition to the existing invasive treatments such as autologous hair transplantation and drug treatment, the demand for overcoming and preventing hair loss is steadily increasing, and related industries are also increasing. In addition to the pharmaceutical/quasi-drug industry, the importance of hair loss materials is also being highlighted in the functional cosmetics and health functional food industries.

Despite this demand and the growth of the industry, the current method for verifying the efficacy and safety of hair loss-related materials has several difficulties compared to other functional material verification methods. In the case of human application tests, recruiting subjects is more difficult than for other diseases, and as a result, the test period and cost are high. In the case of animal testing, the efficacy is indirectly estimated and proven due to differences in the physiological characteristics of the experimental model and the human body, and it is difficult to conduct animal testing due to the recent rise in animal ethics around the world. In the case of human-derived cell testing, it is difficult to secure consistent results due to differences between cell donors, and the gap between results from actual human application testing is the largest.

Various problems arising from these difficulties in verifying efficacy (discrepancies between human application test results and cell test results, high efficacy verification costs and time required, etc.) undermine the reliability of functional materials and products related to hair loss and hinder the growth of the functional product industry. It acts as a barrier to entry. Therefore, the need for a new cell-based efficacy evaluation system that complies with animal ethics, is similar to human application test results, and guarantees consistency and stability of results is emerging.

In a recent previous study, the possibility of using human hair follicle-derived cells to form a cell structure similar to the hair follicle structure in the body and to evaluate its efficacy was announced. However, the cells used in this study were primary culture cells, and there were differences in cells between donors. However, there is still a problem that it is difficult to obtain consistent results due to rapid changes in characteristics depending on the number of cell passages. In addition, there is a disadvantage in that it is difficult to stably secure hair follicle-derived primary cells due to limited hair follicle donations.

The purpose of the present invention is to provide a method of forming a hair follicle-like structure including the step of three-dimensional co-culturing immortalized dermal papilla cells and keratinocytes.

In order to achieve the above object, the present invention includes the steps of (1) separating dermal papilla cells from hair follicles; (2) immortalizing the isolated dermal papilla cells; and (3) providing a method of forming a hair follicle-like structure comprising the step of three-dimensional co-culturing the immortalized dermal papilla cells and keratinocytes.

The present invention relates to a method of forming a hair follicle-like structure through co-culture of an immortalized human hair follicle-derived dermal papilla cell line and keratinocytes, and specifically, SV40T, which inactivates a tumor suppressor gene in human hair follicle-derived dermal papilla cells, and a telomere elongation enzyme promoting gene. As a result of three-dimensional co-culture of keratinocytes with a dermal papilla cell line (SV40T-hTERT DPC) immortalized by forced expression of the hTERT gene, it was found that hair follicles with a keratinous layer growing in a form similar to the hair follicle analogue of the primary cultrue hair follicle-derived cell combination were found. It was confirmed that the structure was formed. Accordingly, the present invention can be applied to various fields such as animal replacement testing and efficacy evaluation of hair loss prevention/hair follicle growth promotion materials, and through immortalized dermal papilla cells, high consistency of results can be secured compared to existing technologies, and stable hair follicle-derived cells By enabling supply, it is expected that the high entry barrier to cell-based efficacy evaluation of hair loss materials can be resolved.

Figure 1 shows a schematic diagram of human hair follicle-derived dermal papilla cells and outer root sheath cell primary culture.

Figure 2 shows the process and results of dermal papilla cell immortalization.

Figure 3 shows a schematic diagram confirming the comparison of spheroid growth patterns between primary cultured dermal papilla cells and immortalized dermal papilla cells.

Figure 4 shows a schematic diagram confirming the formation of a hair follicle-like structure using primary cultured dermal papilla cells and immortalized dermal papilla cell spheroids.

Figure 5 shows a schematic diagram of confirming the formation of a hair follicle-like structure using only immortalized cells.

Figure 6 shows a schematic diagram confirming the cell arrangement shape of the formed hair follicle-like structure.

Figure 7 shows the results of comparing the growth patterns of primary cultured dermal papilla cell spheroids and SV40T-hTERT introduced dermal papilla cell line SV40T-hTERT DPC spheroids.

Figure 8 shows the results of spheroid size change according to culture time. In the case of the diameter according to the initial cell number and culture time of SV40T-hTERT DPC spheroids, spheroids with a diameter similar to the size of the previously known dermal papilla in hair follicles were formed under the conditions of initial cell number ≤ 1K and culture time ≤ 72 hr (A) , As a result of observation of spheroids cultured for 72 hours, compared to spheroids using primary culture cells, the spheroids using SV40T-hTERT DPC had a denser internal cell arrangement, and even though the initial cell number was small, the diameters were confirmed to be similar in size when cultured for 72 hours. (B to F).

Figure 9 shows a hair follicle-like structure using co-culture of primary cultured dermal papilla cells (Primary DPC) or immortalized dermal papilla cell line (SV40T-hTERT DPC) spheroids and human hair follicle outer root sheath cells ORSC. Results of co-culture with primary DPC or SV40T-hTERT DPC and human hair follicle-derived outer root sheath cells over time (A), hair follicle-like structures according to the start date of co-culture of immortalized dermal papilla cell line SV40T-hTERT DPC and outer root sheath cells (ORSC) Formation results (B), co-culture results of immortalized dermal papilla cell line SV40T-hTERT DPC and outer root sheath cells according to outer root sheath cell number (C).

Figure 10 shows the results of hair follicle-like structure formation by medium condition over time. Immortalized dermal papilla cell line SV40T-hTERT DPC Shows the results of co-culturing 1,000 (A) and 3,000 (B) keratinocyte lines on 1,000 cell number spheroids.

Figure 11 shows the results of confirming the cell arrangement status by quantity of keratin cell line in the hair follicle-like structure using only immortalized cells.

Figure 12 shows the results of confirming cell biomarkers within the hair follicle-like structure by frozen sectioning the hair follicle-like structure and performing immunohistochemical staining.

The present invention includes the steps of (1) separating dermal papilla cells from hair follicles; (2) immortalizing the isolated dermal papilla cells; and (3) providing a method of forming a hair follicle-like structure comprising the step of three-dimensional co-culturing the immortalized dermal papilla cells and keratinocytes.

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

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

Preferably, in step (3), the cell ratio of the immortalized dermal papilla cells and keratinocytes may be 1:1 to 1:4, but is not limited thereto.

Preferably, step (3) may be performed by adding the keratinocytes immediately after culturing the immortalized dermal papilla cells to within 72 hours of culturing them, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent 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. .

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Human hair follicle-derived cell culture and dermal papilla cell immortalization

Human hair follicle tissue was tested at the Kyungpook National University Hair Transplant Center with residual hair provided after autologous hair transplantation with the patient's consent.

Dermal papilla cells (DPC) were isolated by cutting hair bulbs from hair tissue and culturing them in DMEM (Hyclone) supplemented with penicillin (100U/ml), streptomycin (100ug/ml), and 20% heat-inactivated serum. When cells grew from the dermal papilla tissue settled in the culture medium, the cells were collected using 0.25% trypsin/10mM EDTA and maintained in DMEM supplemented with 10% heat-inactivated serum under conditions of 5% CO ₂ and 37°C.

After cutting the hair shaft from the hair tissue, penicillin (100U/ml), streptomycin (100ug/ml), and 20% heat-inactivated serum were added to a culture dish coated with collagen type 1, DMEM (Hyclone) with 5% CO ₂ and After culturing at 37℃, when the hair shaft settles on the bottom of the culture dish, keratinocyte-specific culture medium containing penicillin (100U/ml), streptomycin (100ug/ml), and EpiLife™ Defined Growth Supplement (Gibco) The cells were cultured in EpiLife (Gibco) medium under conditions of 5% CO ₂ and 37°C (Figure 1).

The method of immortalizing dermal papilla cells was described in our research team's previous research (BMB Reports. 44:8 512-516. 2011). Briefly, pSV3neo plasmid containing SV40T antigen and neomycin resistance gene was transfected, After culturing in neomycin-containing medium and selecting surviving cells, SV40T antigen expression was confirmed through immunoassay to obtain SV40T antigen-expressing dermal papilla cells. The pGRN145 plasmid containing the human telomere elongation enzyme reverse transcriptase hTERT gene and Hygrmycine was transfected into the obtained dermal papilla cells expressing the SV40T antigen, and then cultured in Hygrmycine-containing medium to select surviving cells, followed by immunoassay and reverse transcription polymerase. Immortalized dermal papilla cell line SV40T-hTERT DPC was obtained by confirming cells expressing SV40T antigen and hTERT through chain reaction (Figure 2).

Immortalized human skin keratinocyte cell line Ker-CT was purchased from ATCC and cultured in collagen type 1 coated culture medium with Lonza's KGM-Gold™ BulletKit™ under conditions of 5% CO ₂ and 37°C.

2. Comparative confirmation of spheroid growth pattern between dermal papilla cells and immortalized dermal papilla cells

In a previous study by the present inventors, the similarity between primary cultured dermal papilla cells and immortalized dermal papilla cells was confirmed by confirming gene expression and growth characteristics. As a notable result from this previous study, the growth rate between primary cultured dermal papilla cells and immortalized dermal papilla cells showed a significant difference, and the change in spheroid size according to the initial cell number was measured when forming hair follicle cell spheroids.

After plating the immortalized dermal papilla cell line SV40T-hTERT DPC and primary cultured dermal papilla cells (Primary DPC) in a culture dish with 0.25% trypsin/10mM EDTA, the immortalized dermal papilla cell line (SV40T-hTERT DPC) was plated on a U bottom low attachment 96 well plate. Inject 500, 1000, 3000 cells/well, primary cultured dermal papilla cells (Primary DPC) at 3,000 cells/well and culture for more than 16 hours in 5% CO ₂ and 37°C in DMEM 100μl/well with 10% heat-inactivated serum. This formed a spheroid. Afterwards, the diameter of the spheroid was measured and observed after taking pictures under a microscope every 24 hours (Figure 3).

3. Dermal papilla cells and immortalization papillae cell spheroid used Similar to hair follicles Confirm structure formation

To form spheroids, primary cultured dermal papilla cells plated on a culture dish and the immortalized dermal papilla cell line SV40T-hTERT DPC were removed with 0.25% trypsin/10mM EDTA and then placed in a U bottom low attachment 96 well plate at 1,000 cells/well. The cells were cultured at 5% CO ₂ and 37°C in 100 μl/well of DMEM supplemented with 10% heat-inactivated fetal bovine serum.

Afterwards, the outer root sheath cells plated on a culture dish were washed with 0.25% trypsin/10mM EDTA, and then incubated with 30,000 mL of William's E medium containing 100U/ml of penicillin/streptomycin, 10ng/ml of hydrocortisone, 10ug/ml of insulin, and 2mM of L-glutamin. Diluted to cells/ml, 100 μl/well was added to the low attachment 96 well plate described above, co-cultured under conditions of 5% CO ₂ and 37°C, and observed under a microscope over time.

To compare the degree of hair follicle-like structure formation according to cell combination, when forming spheroids, the number of immortalized dermal papilla cells, SV40T-hTERT DPC, was adjusted to adjust the number of immortalized dermal papilla cells per spheroid from a minimum of 300 to a maximum of 1000. To compare the degree of hair follicle-like structure formation according to the start time of cell-keratinocyte co-culture, the injection time of keratinocytes was adjusted from immediately after injection of dermal papilla cells to 72 hours after incubation after injection of dermal papilla cells. In addition, the experiment was conducted by adjusting the number of hair follicle-derived keratinocytes from the same number to three times that of the immortalized cells (Figure 4).

4. Confirmation of formation of hair follicle-like structures using only immortalized cells

After plate-cultured SV40T-hTERT DPC was removed with 0.25% trypsin/10mM EDTA, 1,000 cells/well were added to a U bottom low attachment 96 well plate at 5% CO ₂ and 37°C, and 100 μl/well of DMEM supplemented with 10% heat-inactivated serum. After culturing for 24 hours in well conditions, spheroids were formed.

Afterwards, the immortalized keratinocyte cell line Ker-CT plated on a culture dish was washed with 0.25% trypsin/10mM EDTA, and then cultured in DMEM containing 10% heat-inactivated fetal bovine serum, 100U/ml of penicillin/streptomycin, and hydrocortisone, which is a medium for dermal papilla cell growth. William's E, a tissue culture medium containing 10ng/ml, insulin 10ug/ml, and L-glutamin 2mM, and KGM Gold Medium Bulletkit, a medium for keratinocyte cell line growth, were diluted to 30,000 cells/ml under each condition and low attachment as described above. 100 μl/well was added to a 96 well plate and co-cultured under conditions of 5% CO ₂ and 37°C and observed every 24 hours (Figure 5).

5. Confirmation of cell arrangement form of formed hair follicle structure

The medium of the immortalized dermal papilla cell line SV40T-hTERT DPC plated on a culture dish was removed, and the cells were treated with phosphate-buffered saline (PBS) containing 1 μM CM-DiI, a cell tracker, for 30 minutes. After washing with 0.25% trypsin/10mM EDTA, 1,000 cells/well were added to a U bottom low attachment 96 well plate and incubated for 24 hours in 5% CO ₂ and 37°C in DMEM 100μl/well with 10% heat-inactivated serum. Spheroids were formed by culturing.

Afterwards, the keratinocytes plated on a culture dish were removed with 0.25% trypsin/10mM EDTA, and then 30,000 cells were placed in William's E medium containing 100U/ml of penicillin/streptomycin, 10ng/ml of hydrocortisone, 10ug/ml of insulin, and 2mM of L-glutamin. /ml and added to the low attachment 96 well plate described above at 100 μl/well, co-cultured under conditions of 5% CO ₂ and 37°C, and observed with an optical microscope and fluorescence microscope every 24 hours (FIG. 6).

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

It is known that the size of the dermal papilla within a human hair follicle is approximately 100 to 250 μm. From this, in order to form dermal papilla cell spheroids with a diameter of approximately 250um, 3,000 primary cultured dermal papilla cells (primary DPC) were injected per well of a 96-well U bottom plate. As a result, spheroids with a diameter of approximately 250um were confirmed. , it was confirmed that the size of the spheroids slightly decreased over time. Unlike Primary DPC, the spheroid growth pattern after injection of 3,000 SV40T-hTERT DPCs per well of a 96-well U bottom plate confirmed an increase in spheroid size after a certain period of time. It was confirmed that the ratio of cells in the spheroids was higher in density compared to primary cultured dermal papilla cells (Figures 7 and 8).

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

< Example 2> Dermal papilla cells and immortalization papillae cell spheroid used Similar to hair follicles Confirm structure formation

To form a hair follicle-like structure, outer root sheath cells, which are hair follicle-derived keratinocytes, were injected into the U bottom plate where spheroids were formed. As a result, a hair follicle-like structure with a structure similar to a hair follicle was obtained regardless of primary cultured dermal papilla cells and SV40T-hTERT DPC cells. The formation of was confirmed (Figure 9). As a result of testing by adjusting the administration time of outer root sheath cells, it was confirmed that the formation of hair follicle-like structures was smooth when the outer root sheath cells were injected within 24 hours after injection of dermal papilla cells to form spheroids (Figure 9B). As a result of testing by adjusting the injection amount of dermal papilla cells and outer root sheath cells, the growth of hair follicle-like structures was found under the conditions of an equal number of outer root sheath cells in the case of primary cultured dermal papilla cells and three times the outer root sheath cells compared to dermal papilla cells in the case of SV40T-hTERT DPC. It stood out. It was confirmed that the growth of hair follicle-like structures was noticeable when a certain ratio between spheroids formed from above and keratinocytes was maintained. It was confirmed that the optimal cell ratio between dermal papilla cells and outer root sheath cells was 1:3 to 1:4 to form the hair follicle structure (Figure 9C).

< Example 3> immortalization using only cells Similar to hair follicles Check structure formation and cell arrangement status

In order to form an entirely immortalized cell hair follicle-like structure, a hair follicle-like structure formation test was conducted using Ker-CT, a cell line that immortalized human new foreskin cells, and the results showed that the hair follicle-like structure cell body was similar to the hair follicle-like structure using outer root sheath cells. The formation of was confirmed. As a result of co-culturing 1,000 keratin cell lines and 3,000 keratin cell lines on SV40T-hTERT DPC spheroids with a cell count of 1,000, it was confirmed that both formed hair follicle-like cell structures (Figures 10 and 11 ). When comparing the three medium conditions, it was confirmed that the hair follicle-like structure extended the longest in the tissue culture medium, William's E medium, compared to other medium conditions (FIG. 10). In the case of the keratinocyte cell line Ker-CT, it was confirmed that hair follicle-like structures were formed even when 1,000 cells were injected, which were not formed when the same number of hair follicle outer root sheath cells were injected (Figure 9C, Figure 10). From the above results, the results of co-culturing under the conditions of William's E medium, a tissue culture medium, with a combination of 1,000 cells of immortalized dermal papilla cell spheroids and 3,000 cells of keratinocyte cell line in a tissue culture medium in which hair follicle-like structures were confirmed to be produced were the best results. The formation of a hair follicle structure similar to a hair follicle was confirmed (FIGS. 10B and 11).

By fluorescence observation of the hair follicle-like structure formed by attaching a cell marker to immortalized dermal papilla cells, it was confirmed that it had a cell arrangement structure similar to that of the dermal papilla located within the hair follicles in the body. As a result of the cell marker attachment test, it was confirmed that the spheroid and keratin cell line composed of the dermal papilla cell line formed a structure similar to a hair follicle (FIG. 11).

In addition, the hair follicle-like structures were frozen sectioned and immunohistochemical staining was performed to identify cellular biomarkers within the hair follicle-like structures. As a result of immunohistochemical staining, the expression of Vimentin, a dermal papilla cell line-specific biomarker, and keratin 14, a keratinocyte cell line-specific biomarker, was confirmed. From this, it was confirmed that cells were distributed inside the hair follicle-like structure in a form similar to human hair follicles (FIG. 12).

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

1. (1) separating dermal papilla cells from hair follicles;

   (2) immortalizing the isolated dermal papilla cells; and

   (3) A method of forming a hair follicle-like structure comprising the step of three-dimensional co-culturing the immortalized dermal papilla cells and keratinocytes.
2. The method of claim 1, wherein the immortalized dermal papilla cells are dermal papilla cells expressing simian virus 40T (SV40T) antigen and telomerase reverse transcriptase (hTERT).
3. The method of claim 1, wherein the keratinocytes are hair follicle-derived out root sheath cells (ORSC) or the skin keratinocyte cell line Ker-CT.
4. The method of claim 1, wherein in step (3), the cell ratio of the immortalized dermal papilla cells and keratinocytes is 1:1 to 1:4.
5. The method of claim 1, wherein step (3) is performed by adding the keratinocytes immediately after culturing the immortalized dermal papilla cells to within 72 hours of culturing the hair follicle-like structures.

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