Methods for isolating and massively proliferating dermal papilla cells from scalp tissue

Abstract

This invention relates to a method for isolating and amplifying dermal dermal papilla cells, and more specifically, to a method for isolating dermal dermal papilla cells by cutting hair bulbs separated from scalp tissue, and then amplifying the isolated dermal dermal papilla cells by passage. The dermal dermal papilla cells amplified according to this invention can play an important role in hair growth; therefore, this invention can be used in various industrial fields, including the medical and cosmetic fields.

Description

Technical Field

[0001] This invention relates to a method for isolating and amplifying dermal papilla cells from scalp tissue, and more specifically, to a method for isolating dermal papilla cells from hair bulbs separated from scalp tissue by chopping, and then amplifying the isolated dermal papilla cells by passage. Background Technology

[0002] Hair loss is known to be caused by diseases, malnutrition, aging, hormonal imbalances, and more. Despite numerous studies, the fundamental mechanisms of hair loss remain unclear. Typically, hair undergoes a hair cycle consisting of three phases: the anagen phase, where hair grows by stimulating the dermal papilla to actively divide and proliferate keratinocytes; the catagen phase, where blood supply to the hair bulb is cut off and the dermal papilla separates from the hair follicle; and the telogen phase, where cell proliferation ceases and hair does not grow. After the telogen phase, the hair either re-enters the anagen phase or enters the exogenous phase, where it falls out of the scalp. In humans, hair has an independent growth cycle, with some hair entering the exogenous phase and others the anagen phase; therefore, the total number of hairs remains constant. Hair loss refers to this imbalance shifting towards the exogenous phase, and the loss of hair in areas where hair should normally be present.

[0003] Efforts have been made to treat hair loss. However, to date, only two drugs (finasteride and minoxidil) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of hair loss.

[0004] The average person has approximately 100,000 to 150,000 hairs, which form within hair follicles. Within these follicles are papillae, containing tiny blood vessels and providing nutrients necessary for hair growth. Above the papillae are sebaceous glands, which provide oil to give hair its shine. Hair follicles are composed of several different types of epithelial cells and dermal papilla cells. Dermal papilla cells are mesenchymal-derived fibroblasts located at the base of the hair follicle and play a crucial role in hair growth. In particular, minoxidil has been reported to have proliferative and anti-apoptotic effects on dermal papilla cells. However, dermal papilla cells, which play a vital role in hair growth, are difficult to isolate and culture from the scalp, and when large quantities of dermal papilla cells are cultured, reduced hair regeneration capacity occurs. Summary of the Invention

[0005] Technical issues

[0006] The present invention aims to provide a method for separating dermal dermal papilla cells from hair bulbs separated from scalp tissue by cutting, and for amplifying the separated dermal dermal papilla cells by passage.

[0007] Technical solution

[0008] To address the aforementioned problems, the present invention provides a method for isolating and amplifying dermal dermal papilla cells, the method comprising the following steps: (A) isolating hair bulbs from scalp tissue; (B) isolating dermal dermal papilla cells from said hair bulbs; and (C) passage.

[0009] In this invention, step (A) of separating hair bulbs from scalp tissue may include the following steps: (a1) filling a sterile Piper dish with MEMα medium such that scalp tissue (from the epidermis to the dermis) collected from the subject is submerged; (a2) forming a droplet on the lid of the Piper dish using MEMα medium, separating the hair bulbs from the dermis using microsurgical scissors and forceps, and transferring the separated hair bulbs onto the prepared droplet; and (a3) ​​while observing the hair bulbs transferred onto the prepared droplet under a stereomicroscope, removing adipose tissue and hair shafts from the ends of the hair bulbs using a syringe and microsurgical forceps.

[0010] In this invention, step (B) of isolating dermal papilla cells from hair bulbs may include the following steps: (b1) placing the dissection medium and the hair bulbs in a cell culture dish, and then cutting the hair bulbs using precision microscissors; and (b2) collecting the cut hair bulbs in a test tube and then centrifuging them.

[0011] In this invention, the pom-poms cut in step (b1) can have a size of 15 μm to 120 μm.

[0012] In this invention, the centrifugation can be performed at 2,200 rpm for 5 minutes.

[0013] In this invention, the passage in step (C) may include the following steps: (c1) determining the culture dish to be used for passage based on cell count, then discarding the culture medium from the culture dish and washing with PBS; (c2) treating the cells with 0.25% trypsin / EDTA and then culturing them in an incubator at 37°C and 5% CO2 for 5 minutes; (c3) adding MEMα medium containing 1% FBS, collecting the cells in 50 ml centrifuge tubes, and then centrifuging; (c4) discarding the supernatant, gently tapping off the particles, adding expansion medium 2 containing MEMα, basic FGF, 10% fetal bovine serum, penicillin-streptomycin, and amphotericin B, and performing cell counting; and (c5) based on the cell count, discharging the cells at a rate of 1,500 cells / cm³. 2 The cells were seeded at a density in the next step of the culture dish, and then expanded in an incubator at 37°C and 5% CO2 until the culture dish was full of cells, while the culture medium was changed every 3 days.

[0014] In this invention, the succession may include repeating steps (c1) to (c5) three times.

[0015] Beneficial effects

[0016] The dermal papilla cells amplified according to the present invention can play an important role in hair growth. Therefore, the present invention, which relates to a method for amplifying dermal papilla cells, can be used in various industrial fields, including the medical and cosmetic fields. Attached Figure Description

[0017] Figure 1 A comparison of cell yields between methods for isolating dermal papilla cells from hair bulbs is shown.

[0018] Figure 2 The results of amplification based on the dermal papilla cell isolation method and culture medium composition are shown.

[0019] Figure 3 A comparison of cell yields between cases based on various passages is shown.

[0020] Figure 4 The secretion levels of growth factors associated with dermal dermal papilla cells are shown in comparisons between cases from different passages.

[0021] Figure 5 The change in pom-pom size is shown according to the shearing stage.

[0022] Figure 6 A comparison of cell expansion between the cleavage phases is shown. Detailed Implementation

[0023] One embodiment of the present invention provides a method for isolating and expanding dermal dermal papilla cells, the method comprising the steps of: (A) isolating hair bulbs from scalp tissue; (B) isolating dermal dermal papilla cells from said hair bulbs; and (C) passage.

[0024] As used in this article, the term "hair bulb" refers to a thick, rod-shaped structure that forms the lower part of the hair root surrounded by a hair follicle, and contains capillaries, dermal papilla cells, keratinocytes, etc.

[0025] In another embodiment of the invention, step (A) of separating hair bulbs from scalp tissue may include the following steps: (a1) filling a sterile Piper dish with MEMα medium such that scalp tissue (from the epidermis to the dermis) collected from the subject is submerged; (a2) forming droplets on the lid of the Piper dish using MEMα medium, separating the hair bulbs from the dermis one by one using microsurgical scissors and forceps, and moving the separated hair bulbs onto the prepared droplets; and (a3) ​​removing the adipose tissue and hair shaft at the ends of the hair bulbs while observing the hair bulbs moved onto the droplets under a stereomicroscope.

[0026] In another embodiment of the invention, step (B) of isolating dermal papilla cells from the hair bulb may include the following steps: (b1) placing the dissecting culture medium and the hair bulb in a cell culture dish, and then cutting the hair bulb using precision microscissors; and (b2) collecting the cut hair bulb in a test tube and then centrifuging it.

[0027] In another embodiment of the invention, the pom-pom cut in step (b1) may have a size of 15 μm to 120 μm.

[0028] In another embodiment of the invention, the centrifugation can be performed at 2,200 rpm for 5 minutes.

[0029] In another embodiment of the invention, the passage of step (C) may include the following steps: (c1) determining the culture dish to be used for passage based on cell count, then discarding the culture medium from the culture dish and washing with PBS; (c2) treating the cells with 0.25% trypsin / EDTA, followed by culturing in an incubator at 37°C and 5% CO2 for 5 minutes; (c3) adding MEMα medium containing 1% FBS, collecting the cells in 50 ml centrifuge tubes, and then centrifuging; (c4) discarding the supernatant, gently tapping off the particles, adding expansion medium 2 containing MEMα, basic FGF, 10% fetal bovine serum, penicillin-streptomycin, and amphotericin B, and performing cell counting; and (c5) seeding the cells at a density of 1,500 cells / cm2 in the culture dish of the next step based on the cell count, and then expanding the cells in an incubator at 37°C and 5% CO2 until the culture dish is full of cells, while changing the culture medium every 3 days.

[0030] In another embodiment of the invention, the succession may include repeating steps (c1) to (c5) three times.

[0031] The present invention will now be described in more detail with reference to specific embodiments. These embodiments are for illustrative purposes only, and it will be apparent to those skilled in the art that the scope of the invention should not be construed as limited to these embodiments.

[0032] Example 1. Isolation of hair bulbs from scalp tissue

[0033] Sterilized 90 mm Piper dishes were filled with MEMα medium to immerse scalp tissue (from the epidermis to the dermis) collected from the subjects. Next, multiple droplets were formed on the lid of the 90 mm Piper dish using MEMα medium, and hair bulbs from the dermis were individually separated and transferred onto the prepared droplets using microsurgical scissors and forceps. Then, while observing the hair bulbs transferred onto the droplets under a stereomicroscope, adipose tissue and hair shafts were removed from the ends of the hair bulbs using a 26-G syringe and microsurgical forceps, thereby separating the hair bulbs from the scalp tissue.

[0034] Example 2. Isolation of dermal dermal papilla cells from hair bulbs

[0035] To select the optimal method for isolating dermal papilla cells from hair bulbs, the following comparative experiment was conducted.

[0036] First, separation method 1 is as follows: 1 ml of dissection culture medium is placed in a 35 mm cell culture dish, about 50 hair bulbs are placed in it, the hair bulbs are cut with precision microscissors, and the cut hair bulbs are collected in a test tube, and then centrifuged at 2,200 rpm for 5 minutes.

[0037] Next, separation method 2 is as follows: 0.025% type I collagenase reagent is added to a 35 mm cell culture dish containing about 50 hairballs, and then cultured in a shaking incubator at 37°C for 2 hours and 30 minutes. The hairballs are then collected in test tubes and centrifuged at 2,200 rpm for 5 minutes.

[0038] To compare the results of separation method 1 and separation method 2, each particle was gently tapped and thoroughly pipetted into a 35 mm cell culture dish with 2 ml of attachment medium having the composition shown in Table 1 below. The cells were then amplified in an incubator at 37°C and 5% CO2 until the dish was filled with cells, with the medium being changed every 3 days.

[0039] [Table 1]

[0040]

[0041] As a result of the comparative experiment, it can be confirmed that, compared with separation method 2 which uses 0.025% type I collagenase reagent, separation method 1, which only involves shearing without any other treatment of the hair bulb itself, produces smaller and more uniform cell sizes and higher cell yields.

[0042] Example 3. Subculture Steps

[0043] Passage 0 and Passage 1 were performed as follows. First, the culture dish used for passage was determined based on the number of cells collected in the 35 mm cell culture dish. The culture medium was then discarded from the 35 mm cell culture dish, and the cells were washed once with PBS. Next, the cells were treated with 0.25% trypsin / EDTA and amplified for 5 minutes in an incubator at 37°C and 5% CO2. Afterward, MEMα medium containing 1% FBS was added for cell inactivation, and the cells were collected in 50 ml centrifuge tubes and centrifuged at 2,200 rpm for 5 minutes. Next, the supernatant was discarded, particles were gently tapped, and the appropriate amount of amplification medium 1 or amplification medium 2 as shown in Table 2 was added, followed by cell counting.

[0044] [Table 2]

[0045]

[0046] After cell counting, the cells were stored at 1,500 cells / cm³. 2 The cells were seeded at a density in the next culture dish and then amplified in an incubator at 37°C and 5% CO2 until the culture dish was full of cells, with the culture medium being changed every 3 days.

[0047] Next, passages 1 through 4 were performed in the same manner as described above. Cells were passaged up to passage 4 in culture dishes determined based on cell counts. After each passage, cells were frozen and thawed, then cultured at 3,000 cells / cm². 2 The inoculation density was inoculated in the petri dish.

[0048] Example 4. Results based on the conditions for isolating and expanding dermal papilla cells.

[0049] Example 4-1. Based on differences in cell isolation methods and culture medium composition

[0050] Based on the amplification results of the dermal dermal papilla cell isolation method and culture medium composition, it is shown that... Figure 2 In. Figure 2In the examples, Case 1 shows the results of amplification of dermal papilla cells isolated from the hair bulb by shearing in amplification medium 1; Case 2 shows the results of amplification of dermal papilla cells isolated from the hair bulb by shearing in amplification medium 2; Case 3 shows the results of amplification of dermal papilla cells isolated from the hair bulb by treating them with 0.025% type I collagenase reagent in amplification medium 1; and Case 4 shows the results of amplification of dermal papilla cells isolated from the hair bulb by treating them with 0.025% type I collagenase reagent in amplification medium 2. As a result of the analysis of cell morphology in each case, it can be confirmed that in Case 2, where dermal papilla cells were isolated from the hair bulb by shearing and then amplified using amplification medium 2, the cell morphology was well maintained, and the amplification rate of dermal papilla cells was significantly increased.

[0051] In addition, the changes in cell size for each passage of amplification are shown in Table 3 below for each case.

[0052] [Table 3]

[0053] (urm) P1 P2 P3 P4 P5 P6 Case 1 18.99 19 19.28 17.78 17.58 17.66 Case 2 13.2 13 13.7 13.88 15.14 15.2 Case 3 17.1 17 17.6 18.75 18.94 19.22 Case 4 15.3 15 15.5 16.6 17.03 18.06

[0054] Referring to Table 3 above, the cell size in Case 2 was the smallest and remained at a uniform level, while in the other groups except Case 1, the cell size tended to increase with increasing passage number. Furthermore, in Case 2, the cell size from P1 to P4 remained stable without significant change, indicating that cell yield was also high. The cell yield measurement results are as follows... Figure 3 As shown in the image. (Refer to...) Figure 3 It can be confirmed that the cell yield was the highest in all passages in this case, and in particular, the largest number of cells were collected at P3.

[0055] Next, the secretion levels of growth factors involving dermal dermal papilla cells in each passage of each case are as follows: Figure 4 As shown. According to Figure 4 As a result of measuring the secretion levels of various growth factors known as dermal papilla growth factors, including HGF which promotes the proliferation of epithelial hair follicle cells by stimulating human dermal papilla cells, VEGF which induces hair growth by participating in vasodilation, and FGF (KGF) which promotes hair follicle tissue growth, it can be confirmed that, in these cases, P3 in Case 2 secreted the largest amount of growth factors.

[0056] Example 4-2. Based on the difference in shear range

[0057] Based on the above experimental results, experiments were conducted to determine the optimal shearing range. First, hairballs flushed from the scalp tissue were collected in Piper's dishes filled with PBS or basal culture medium. 1 ml of dissection culture medium and the hairballs were placed in a 35 mm cell culture dish, and the hairballs were cut using precision microscissors. The changes in hairball size according to the degree of shearing were then observed under a microscope. As a result of measuring hairball size according to the shearing stage, it was confirmed that the hairball size before shearing was 750 μm to 900 μm, shearing stage 1 was 490 μm to 630 μm, shearing stage 2 was 300 μm to 410 μm, shearing stage 3 was 180 μm to 260 μm, and shearing stage 4 was 15 μm to 120 μm. Figure 5 ).

[0058] The shredded hairballs were then collected in test tubes and centrifuged at 2,200 rpm for 5 minutes. The supernatant was discarded, and the particles were collected. 1 ml of attachment medium was placed in a 35 mm cell culture dish, and cell expansion was measured in an incubator at 37°C and 5% CO2. The results are as follows: Figure 6 As shown in the image.

[0059] Reference Figure 6 As a result of examining cell proliferation and attachment morphology across a total of four stages within the shear range, it can be confirmed that cell attachment morphology varies depending on the degree of shearing. Figure 6 The left side shows a photograph of cells on day 3 after shearing, and the right side shows a photograph of cells on day 5 after shearing. As observed on day 3, it can be confirmed that at a low shearing stage, cells unevenly adhered to the bottom surface of the cell culture dish and grew in an aggregated state. On the other hand, with increasing shearing stage, cells grew while attaching to the bottom surface of the cell culture dish at regular intervals, and in a spindle-shaped form with pointed tips. Furthermore, as observed on day 5, it can be confirmed that at a low shearing stage, there was a significant difference in cell proliferation between areas where cells grew in an aggregated state and areas where cells did not aggregate, and cells tended to spread and grow in certain places; however, with increasing shearing stage, cells were evenly distributed across the entire bottom surface of the cell culture dish.

[0060] Furthermore, cell yield varies depending on the cleavage stage, as shown in Table 4 below.

[0061] [Table 4]

[0062] T25 cell culture dish Cell yield Shearing stage 1 1.0E+05 Shearing stage 2 1.3E+05 Shearing stage 3 2.5E+05 Shearing stage 4 2.8E+05

[0063] Referring to Table 4 above, it can be confirmed that, depending on the degree of shearing, there are differences not only in cell attachment morphology but also in cell yield. It can be considered that the cell attachment morphology on the cell culture dish varies according to the degree of shearing, and the spacing between cells and the pattern of cell expansion have a significant impact on yield. In shearing phases 1 and 2, cells grow in a colony-like manner; therefore, cells tend to diffuse and grow, resulting in a cell yield of 1.0E+05 to 1.3E+05. In shearing phase 3, cell aggregation is less than in shearing phases 1 and 2, but cell diffusion is observed during the initial attachment process due to slight aggregation. In shearing phase 4, cells attach well in a single state from the beginning, without cell diffusion or aggregation, and as the number of culture days increases, cells attach uniformly to the entire bottom of the cell culture dish and grow. As a result, the cell yield is approximately twice that of shearing phase 1. However, when the hair bulb is sheared to a size less than 15 μm, the problem arises that the dermal papilla cells inside the hair bulb are damaged, leading to a significant decrease in cell yield. When the hairballs were sheared to a size greater than 120 μm, as confirmed by the experiments above, the problem was that cell aggregation occurred and the cells were not evenly distributed across the entire bottom of the culture dish, resulting in a significant decrease in cell growth. This is thought to be because nutrient uptake in aggregated cells is lower than in evenly distributed cells, and external pressure on aggregated cells increases due to increased cell density.

Claims

1. A method for isolating and expanding dermal dermal papilla cells, the method comprising the following steps: (A) Separating hair bulbs from scalp tissue; (B) Isolate dermal dermal papilla cells from the hair bulb; and (C) Transmission, Step (B), which involves separating dermal dermal papilla cells from the hair bulb, includes the following steps: (b1) Place the dissection medium and the hair bulb in a cell culture dish, and then cut the hair bulb using precision microscissors; and (b2) Collect the shredded hair balls in a test tube and then centrifuge them. In step (b1), the size of the fuzz ball being cut is between 15 μm and 120 μm. In this step (C), the passage was repeated three times using a medium containing MEM α, alkaline FGF, 10% fetal bovine serum, penicillin-streptomycin, and amphotericin B.

2. The method according to claim 1, wherein, Step (A) of separating hairballs from scalp tissue includes the following steps: (a1) Fill sterile Petri dishes with MEM α medium to immerse the scalp tissue collected from the subject; (a2) Using MEM α medium, droplets were formed on the lid of the Piper dish. Hair bulbs in the dermis were individually separated using microsurgical scissors and forceps, and the separated hair bulbs were transferred onto the formed droplets; and (a3) While observing the hair bulb under a stereomicroscope, use a syringe and microsurgical forceps to remove the adipose tissue and hair shaft from the end of the hair bulb that has moved onto the droplet.

3. The method according to claim 1, wherein, The propagation in step (C) includes the following steps: (c1) Determine the culture dish to be used for passage based on cell count, then discard the culture medium from the culture dish and wash with PBS; (c2) The cells were treated with 0.25% trypsin / EDTA and then incubated at 37°C and 5% CO2 for 5 minutes. (c3) Add MEM α medium containing 1% FBS, collect the cells in a 50 ml centrifuge tube, and then centrifuge to separate them; (c4) Discard the supernatant, gently tap off the particles, add amplification medium 2 containing MEM α, basic FGF, 10% fetal bovine serum, penicillin-streptomycin, and amphotericin B, and perform cell counting; and (c5) Based on cell count, the cells are divided into groups of 1,500 cells / cm³. 2 The cells were seeded at a density in the next step of the culture dish, and then expanded in an incubator at 37°C and 5% CO2 until the culture dish was full of cells, while the culture medium was changed every 3 days.

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