KR20220116805A - Method for transplanting dermal papilla cells through perforation - Google Patents

Abstract

The present invention relates to a method for transplanting dermal papilla cells, and more specifically, to a method for transplanting massively proliferated dermal papilla cells into the scalp of a recipient through perforation using a follicular unit extractor (FUE) punch. The dermal papilla cells massively transplanted according to the present invention can play an important role in hair growth, so that the present invention regarding an optimal method for transplanting dermal papilla cells can be utilized in various industrial fields such as a medical field and a skin care field.

Description

Method for transplanting dermal papilla cells through perforation

The present invention relates to a method for transplanting dermal papilla cells through perforation, and more particularly, to a method for transplanting massively proliferated dermal papilla cells into the scalp of a recipient through perforation using a Follcular Unit Extractor (FUE) punch. .

Alopecia is known to be caused by disease, malnutrition, aging, hormonal imbalance, and the like. Although many studies have been conducted, the underlying mechanism for hair loss is not well known, but generally hair goes through a certain hair cycle, and keratinocytes stimulated by dermal papilla It goes through the growth phase (anagen), where hair grows through active division and proliferation, the catagen phase (catagen), in which the blood supply is cut off to the hair bulb and the dermal papilla is separated from the hair follicle, and the telogen phase (telogen) when the cell proliferation stops and hair does not grow It goes back into the growth phase or goes into the exogen phase, where hair falls out of the scalp. Human hair has an independent growth cycle, some enter the decidual phase, and some enter the growth phase, maintaining the same level of hair overall. is called alopecia.

Research to treat such hair loss has also been conducted for a long time. Nevertheless, so far only two drugs (finasteride and minoxidil) have been approved by the Food and Drug Administration (U.S.A) for the treatment of hair loss.

There are about 100,000 to 150,000 hairs in the human body, and they are formed from hair follicles. There are nipples in the hair follicles, where small blood vessels are distributed to supply nutrients necessary for hair growth, and on the side above the nipples there are seborrheic glands that supply oil that gives shine to the hair. Hair follicles are made up of several different epithelial cells and dermal papilla cells. The dermal papilla cells are mesenchymally-derived fibroblasts located at the base of the hair follicle and play an important role in hair growth. In particular, minoxidil (minoxidil) has been reported to have an anti-apoptotic effect and proliferation of dermal papilla cells.

As a result of continuous research on a method for transplanting dermal papilla cells, which play an important role in hair growth, to a transplant recipient, the present inventor has completed the present invention regarding an optimal transplantation method for dermal papilla cells.

An object of the present invention is to provide a method for treating hair loss through an optimized method of transplanting massively proliferated dermal papilla cells into the scalp of a recipient through perforation using a Follcular Unit Extractor (FUE) punch.

In order to solve the above problems, the present invention comprises the steps of (A) preparing a mass-proliferated dermal papilla cell; (B) collecting dermal papilla cells in a syringe; (C) transplanting the dermal papilla cells to the scalp of the recipient; a method for transplanting dermal papilla cells, comprising the step of (C) transplanting the dermal papilla cells to the scalp of the recipient. ) Provides a method for transplanting dermal papilla cells, which is characterized by implanting dermal papilla cells filled in a syringe after making a perforation using a punch.

In the present invention, (A) in the step of preparing the mass-proliferated dermal papilla cells, the dermal papilla cells may be spheroids or single cells.

In addition, in the present invention, the spheroid comprises the steps of (a1) seeding the cells in a flask and then exchanging the medium in an incubator every 3 days to proliferate the cells; (a2) removing the medium contained in the flask and washing with PBS; (a3) after treatment with 0.25% Trypsin/EDTA, incubated for 5 minutes in an incubator, and then centrifuged into MEM alpha medium containing 1% FBS; (a4) diluting the cell suspension with Expansion Media 2 to contain 1000 to 3000 cells per drop by tapping the pellet that has subsided at the bottom, then inserting Expansion Media 2 and checking the number of cells; (a5) dispensing 40mL of PBS on the bottom of a square dish, and forming a drop of 5 to 30ul with a 12-channel multichannel pipette on the cover of the square dish; (a6) holding the corner of the square dish on which the drop is formed diagonally, turning it over to make a hanging drop shape, and collecting the spheroid formed after culturing for 1 to 7 days in an incubator; may include.

Also in the present invention, the drop of step (a5) may be characterized in that 16 to 25ul.

In addition, in the present invention, (C) the step of transplanting the dermal papilla cells into the scalp of the recipient (c1) using a FUE (Follcular Unit Extractor) punch to make a hole having an inner diameter of 0.8 to 1 mm; may include; .

Also in the present invention, the perforation may be characterized in that the depth is 5 to 7 mm.

In addition, in the present invention, (c2) 5.0 * 10 ⁶ filling the syringe with 6 spheroid cells and injecting into the perforation; may be characterized in that it further comprises.

In addition, in the present invention, the syringe may be characterized in that the inner diameter of the needle gauge is larger than the diameter of the dermal papilla cells collected in step (B).

In addition, in the present invention, the syringe may be characterized in that the needle gauge is 24G.

Since the dermal papilla cells transplanted in large quantities by the method provided by the present invention can play an important role in hair growth, the present invention can be utilized in various industrial fields such as the medical field and the cosmetic field.

1 is a comparison of the difference in cell yield according to the method of separating dermal papilla cells from hair follicles.
Figure 2 shows a comparison of the difference in mass proliferation results according to the method of separating dermal papilla cells and the medium composition.
3 shows a comparison of the cell yield difference according to the subculture for each case.
Figure 4 shows a comparison of the difference in the secretion amount of growth factor related to dermal papilla cells according to the subculture for each case.
Figure 5 shows the change in the size of the hair bulb according to the chopping stage.
6 shows a comparison of the difference in cell proliferation according to the chopping stage.
7 shows a comparison of the spheroid shape according to the number of cells for each drop capacity.
8 shows a comparison of the proliferation rate and yield according to the number of seeded cells per area.
Figure 9 shows the scalp photos by period after transplantation of dermal papilla cells by simple injection.
10 shows scalp photos taken for a certain period after implantation of dermal papilla cells through perforation using a Follcular Unit Extractor (FUE) punch.
11 shows a comparison of the shape and size of a spheroid according to the syringe needle gauge.

In one embodiment according to the present invention, (A) preparing a mass-proliferated dermal papilla cells; (B) collecting dermal papilla cells in a syringe; (C) transplanting the dermal papilla cells to the scalp of the recipient; a method for transplanting dermal papilla cells, comprising the step of (C) transplanting the dermal papilla cells to the scalp of the recipient. ) Provides a method for transplanting dermal papilla cells, which is characterized by implanting dermal papilla cells filled in a syringe after making a perforation using a punch.

FUE (Follcular Unit Extractor) is an equipment used to collect hair follicles during non-incisional hair transplantation. A small punch with an inner diameter of 0.8 to 1 mm is brought into contact with the scalp tissue and rotated linearly to remove the scalp tissue containing the hair follicles with forceps, etc. can be used to extract the perforated hair follicles. The extracted hair follicles can be transplanted to the area where hair loss has occurred, and since there is no incision using a scalpel or the like, there is no after-effects such as scars or pain, and it has the advantage of being able to return to life immediately.

In another embodiment according to the present invention, (A) in the step of preparing the mass-proliferated dermal papilla cells, the dermal papilla cells may be spheroids or single cells.

In another embodiment according to the present invention, the spheroid is (a1) after seeding the cells in a flask, exchanging the medium every 3 days in an incubator to proliferate the cells; (a2) removing the medium contained in the flask and washing with PBS; (a3) after treatment with 0.25% Trypsin/EDTA, incubated for 5 minutes in an incubator, and then centrifuged into MEM alpha medium containing 1% FBS; (a4) diluting the cell suspension with Expansion Media 2 to contain 1000 to 3000 cells per drop by tapping the pellet that has subsided at the bottom, then inserting Expansion Media 2 and checking the number of cells; (a5) dispensing 40mL of PBS on the bottom of a square dish, and forming a drop of 5 to 30ul with a 12-channel multichannel pipette on the cover of the square dish; (a6) holding the corner of the square dish on which the drop is formed diagonally, turning it over to make a hanging drop shape, and collecting the spheroid formed after culturing for 1 to 7 days in an incubator; may include.

In another embodiment according to the present invention, the drop of step (a5) may be characterized in that 16 to 25ul.

In another embodiment according to the present invention, (C) the step of transplanting the dermal papilla cells into the scalp of the recipient is (c1) making a hole with an inner diameter of 0.8 to 1 mm using a FUE (Follcular Unit Extractor) punch; may include

In another embodiment according to the invention, the perforations may be characterized in that the depth is 5 to 7 mm.

In another embodiment according to the present invention, (c2) filling the syringe with 5.0 * 10 ⁶ spheroid cells and injecting them into the perforation; may be characterized by further comprising.

In another embodiment according to the present invention, the syringe may be characterized in that the inner diameter of the needle gauge is larger than the diameter of the dermal papilla cells collected in step (B).

In another embodiment according to the present invention, the syringe may be characterized in that the needle gauge is 24G.

Hereinafter, the present invention will be described in more detail through specific examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Isolation of hair bulbs from scalp tissue

After filling a sterile 90 mm petri dish with MEM alpha medium to submerge the scalp tissue (from the epidermis to the dermis) collected from the subject, several drops are formed on the cover of the 90 mm petri dish using MEM alpha medium, and microsurgery is performed. Using scissors and forceps, the hair bulbs in the dermal layer were separated one by one and transferred to the prepared drop. Thereafter, the hair bulbs moved to the drop were observed under a stereoscopic microscope, and fatty tissue and hair shaft attached to the tip of the hair bulbs were removed using a 26G syringe and microsurgery forceps to separate the hair bulbs from the scalp tissue.

Example 2. Isolation of dermal papilla cells from hair bulbs

In order to select the optimal method for isolating dermal papilla cells from hair follicles, the following comparative experiment was conducted.

First, separation method 1 is a method of putting 1 ml of dissection media in a 35 mm cell culture dish, putting about 50 hair bulbs, chopping with precision fine scissors, collecting them in a tube, and centrifuging at 2200 rpm for 5 minutes.

Next, in separation method 2, 0.025% collagenase type I reagent is added to a 35 mm cell culture dish containing about 50 hair bulbs, incubated for 2 hours and 30 minutes in a shaking incubator at 37 ° C, collected in a tube, and centrifuged at 2200 rpm for 5 minutes. way to separate.

In order to compare the results of the separation methods 1 and 2, each pellet was tapped and pipetting was performed with 2 ml of attachment media having the composition shown in Table 1 below, seeded in a 35 mm cell culture dish, 37°C, 5% CO ₂ In the mass growth phase, the culture dish was grown until the culture dish was filled with cells while exchanging the medium every 3 days.

division Attachment Media
(Attachment step) component MEM alpha - 20% Fetal bovine serum Penicillin-Streptomycin Amphotericin B

As a result of the comparative experiment, compared with separation method 2 containing 0.025% collagenase type I reagent, in separation method 1 in which only chopping was performed without any other treatment on the hair bulb itself, the size of the cells was smaller, constant, and the cell yield was also high. was confirmed (FIG. 1).

Example 3. Passage step

The step of subculture from Passage 0 to Passage 1 was performed as follows. First, the culture dish to be subcultured was determined according to the number of cells collected in the 35 mm cell culture dish, and then the medium contained in the 35 mm cell culture dish was discarded and washed once with PBS. Next, 0.25% Trypsin / EDTA was treated and incubated for 5 minutes at 37 ℃, 5% CO ₂ mass growth phase. After that, add MEM alpha medium containing 1% FBS for inactivation, collect it in a 50ml centrifuge tube, centrifuge at 2200 rpm for 5 minutes, discard the supernatant, and sufficiently tap the pellet that has settled down as shown in Table 2 An appropriate amount of Expansion Media 1 or 2 was added and the number of cells was checked.

division Expansion Media 1
(mass propagation stage) Expansion Media 2
(mass propagation stage) component MEM alpha MEM alpha - Basic FGF 10% Fetal bovine serum 10% Fetal bovine serum Penicillin-Streptomycin Penicillin-Streptomycin Amphotericin B Amphotericin B

After seeding the culture dish of the next step so that 1,500 cells/cm ² according to the number of cells counted, the culture dish is filled with cells while exchanging the medium every 3 days at 37℃, 5% CO ₂ mass growth phase. was propagated until

After that, the step of subculture to passage 1 to passage 4 was performed in the same order as above, and the size of the culture dish was adjusted according to the number of cells to be counted and passaged to passage 4, and frozen and then thawed at the intermediate passage step. In this case, it was seeded in a culture dish to be 3,000 pieces/cm ² .

Example 4. Derivation of results according to dermal papilla cell isolation and mass proliferation conditions

Example 4-1. Differences according to cell separation method and medium composition

The mass proliferation results according to the dermal papilla cell separation method and medium composition are shown in FIG. 2 . In FIG. 2, case 1 is the result of large-scale proliferation in Expansion media 1 medium after separating dermal papilla cells from hair follicles by chopping, and case 2 is the result of large-scale proliferation in Expansion media 2 medium after separating dermal papilla cells from hair follicles by chopping. These are the results, case 3 is the result of large-scale proliferation in Expansion media 1 medium after separating dermal papilla cells from hair follicles by treatment with 0.025% collagenase type I reagent, and case 4 is the result of dermal papilla cells from hair follicles by treatment with 0.025% collagenase type I reagent. It shows the results of mass growth in Expansion media 1 medium after separation. As a result of checking the morphology of the cells for each case, it was confirmed that the cell shape was well maintained and the mass proliferation and proliferation rate of the dermal papilla cells was significantly improved in case 2, when Expansion media 2 was used after separating the dermal papilla cells from the hair follicles by chopping. could

In addition, the cell size change according to the passage mass proliferation for each case is shown in Table 3 below.

(um) P1 P2 P3 P4 P5 P6 case1 18.99 19 19.28 17.78 17.58 17.66 case2 13.2 13 13.7 13.88 15.14 15.2 case3 17.1 17 17.6 18.75 18.94 19.22 case4 15.3 15 15.5 16.6 17.03 18.06

According to Table 3, the cell size of case 2 was the smallest and uniformly maintained, and in the remaining groups except for case 1, the cell size also showed a tendency to increase as the number of subcultures increased. Furthermore, in case 2, the cell size was maintained without significant change from P1 to P4, so it could be expected that the cell yield would also be high, and the result of confirming this was shown in FIG. 3 . According to FIG. 3 , it was confirmed that the cell yield was also the highest in case 2 over the entire passage, and in particular, it was confirmed that the largest amount of cells was collected in P3.

Next, the amount of secretion of growth factor related to dermal papilla cells according to subculture for each case is shown in FIG. 4 . According to Figure 4, growth factors of dermal papilla cells such as HGF that stimulates human dermal papilla cells to promote the proliferation of epithelial follicle cells, VEGF that induces hair growth by participating in vasodilation, and FGF (KGF) that promotes the growth of hair follicle tissue As a result of measuring the secretion of various growth factors known as

Example 4-2. Differences by chopping range

Based on the experimental results, an experiment was performed to determine the optimal chopping range. First, the hair bulbs punched out from the scalp tissue are collected in a petri dish filled with PBS or basal media, 1 ml of dissection media and hair bulbs are put in a 35 mm cell culture dish, and the hair bulbs are chopped with precision fine scissors, and the degree of chopping Changes in the size of hair bulbs were observed under a microscope. As a result of measuring the size of hair bulbs according to the chopping step, hair bulbs with a size of 750 μm to 900 μm before chopping, 490 μm to 630 μm in the first chopping step, 300 μm to 410 μm in the second step, 180 μm to 260 μm in the third step, and 15 μm to 120 μm in the fourth step were obtained. was confirmed (FIG. 5).

After that, the chopped hair bulbs are collected in a tube, centrifuged at 2200 rpm for 5 minutes, the supernatant is discarded, the pellet is collected, 1 ml of Attachment media is placed in a 35 mm cell culture dish, and at 37 ° C, 5% CO ₂ mass propagator Cell proliferation was confirmed, and the results are shown in FIG. 6 .

According to FIG. 6 , it was confirmed that cell proliferation and adhesion patterns were confirmed by dividing the cells into four stages according to the chopping range. As a result, it was confirmed that the cell adhesion forms were different according to the degree of chopping. The left side of FIG. 6 is a picture of cells 3 days after chopping, and the right picture is a picture of cells 5 days after chopping. As a result of observation on the third day of culture, if the chopping level was low, the cells were not evenly attached to the bottom of the cell culture dish and agglomerated growth occurred. On the other hand, the higher the chopping stage, the more evenly the cells were attached to the bottom surface of the cell culture dish at regular intervals and grew in the form of spindles with pointed ends. Furthermore, as a result of observation on the 5th day of culture, when the chopping stage was low, the cell proliferation was significantly different in the clumped and non-clumped regions, and the cells tended to spread and grow, but the chopping stage was As the height increased, it was confirmed that the cells were evenly filled on the entire bottom surface of the cell culture dish.

In addition, there was also a difference in cell yield according to the chopping step, which is shown in Table 4 below.

T25 cell culture dish standard cell yield Chopping Step 1 1.0E+05 Chopping Step 2 1.3E+05 Chopping 3 steps 2.5E+05 Chopping 4 steps 2.8E+05

According to Table 4, it was confirmed that there was a difference in the yield as well as the cell attachment type according to the degree of chopping. The type of cell attachment to the cell culture dish varies depending on the degree of chopping, and it seems that the yield is greatly affected by the spacing between cells and the shape of proliferation. Roughly, in the 1st and 2nd stages of chopping, a colony is formed and proliferated, and as a result, the cells tend to spread and grow, showing a cell yield of 1.0E+05 ~ 1.3E+05 level, and at the 3rd stage of chopping, cells clumping is less than that of step 1 or 2 chopping, but due to slight clumping, cell spreading is observed at the time of initial attachment. As the number of incubation days increased, it could be observed that the cell culture dish was uniformly attached to the bottom of the cell culture dish and proliferated. However, when chopping to less than 15 μm, there was a problem that the cell yield was significantly lowered due to damage to the dermal papilla cells inside the hair bulb. As a result of not being evenly distributed over the entire bottom of the culture dish, there was a problem that cell proliferation was significantly lowered, which resulted in reduced nutrient absorption rate and increased external stress caused by increased cell density in aggregated cells compared to evenly distributed cells. seems to be

Example 5. Preparation step for transplantation of dermal papilla cells

In order to evaluate the effect of cell transplantation according to the cell type, spheroids and single cells were prepared as follows, respectively.

Example 5-1. Preparation of spheroid (Aggregation cell)

First, after seeding approximately 1300 to 1500 cells per area (cm ² ) of the T225 flask, the medium was exchanged in an incubator at 37° C., 5% CO ₂ every 3 days, and the T225 flask was grown until it was full of cells. , The medium in the T225 flask was removed and washed once with PBS. After that, treated with 0.25% Trypsin/EDTA and incubated for 5 minutes at 37°C, 5% CO ₂ in an incubator. For inactivation, MEM alpha medium containing 1% FBS was added, collected in a 50ml centrifuge tube, and 5 at 2200 rpm. Centrifugation was performed for minutes. The supernatant was discarded and the pellet submerged at the bottom was sufficiently tapped, and an appropriate amount of Expansion Media 2 was added and the number of cells was checked. . Next, in order to maintain temperature and humidity, 40 mL of PBS was dispensed on the bottom of the square dish, and a drop of 5 to 30 ul was formed on the cover of the square dish with a 12-channel multichannel pipette. After holding the corner of the square dish on which the drop was formed, turn it over to make the optimal hanging drop shape, and collect the spheroids formed after 1 to 7 days of incubation in an incubator at 37°C, 5% CO ₂ .

Example 5-2. Determination of drop dose for spheroid formation

The experimental results for determining the drop capacity for a preferred spheroid shape are shown in Table 5 and FIG. 7 below.

Case1
(drop capacity 5 to 15ul) Case2
(drop capacity 16 to 25ul) Case3
(drop capacity 26-30ul) Primary 172.76 206.91 260.05 Secondary 200.62 201.69 242.29 tertiary 201.42 216.88 254.4 spheroid mean diameter (um) 191.6±16.32 208.5±7.71 252.25±9.07

As shown in Figure 7, in case 1, where the drop capacity is limited to 5 to 15ul, the cells are not aggregated and are widely dispersed or several spheroids are formed, and the standard deviation is due to the formation of spheroids of non-uniform size. I could see that it was very large. On the other hand, in case 2 in which the drop capacity was limited to 16 to 25ul and case 3 in which the drop capacity was limited to 26 to 30ul, spheroids of a certain size were formed within 3 days. However, since the drop capacity of case 3 is difficult to control, it was confirmed that the drop shape was not constant and formed into a flowing or distorted shape when manufactured in a hanging drop shape. As a result, it was confirmed that the spheroid size of case 3 was formed in a larger shape than case 2 by about 250 μm despite the same amount of cells as case 2. As a result, the spheroid formed in case 2 was the best aggregated and it was easy to control when manufacturing a hanging drop, confirming that it had an appropriate capacity and number of cells.

Example 5-3. Determination of seeding cell density

In order to determine efficient seeding cell density, a cell proliferation rate and cell yield comparison experiment was performed by seeding the number of cells per area differently for each case, and the results are shown in Tables 6 and 8 below.

Case 1
(1000 cell/cm ² ) Case 2
(2000 cells/cm ² ) Case 3
(3000 cells/cm ² ) Case 4
(4000 cells/cm ² ) Case 5
(5000 cells/cm ² ) number of cells 2.75E+05 7.00E+05 8.25E+05 9.75E+05 1.38E+06

Example 5-4. Single cell preparation step

Some of the cells collected in the spheroid preparation step were seeded so that 1000 to 5000 cells per area (cm ² ) of the T225 flask were, and grown in a 37° C., 5% CO ₂ incubator for 1 to 7 days when spheroids were formed, followed by T225 flask The medium contained in the was removed and washed once with PBS. After that, treated with 0.25% Trypsin/EDTA and incubated for 5 minutes at 37°C, 5% CO ₂ in an incubator. For inactivation, MEM alpha medium containing 1% FBS was added, collected in a 50ml centrifuge tube, and 5 at 2200 rpm. Centrifugation was performed for minutes. The supernatant was discarded and the pellet submerged at the bottom was sufficiently tapped, an appropriate amount of NS medium was added, the number of cells was checked, and the cell suspension was diluted according to the number of cells to be transplanted.

Example 6. Transplantation of dermal papilla cells

Example 6-1. dermal papilla cell collection step

First, check the size and shape of the spheroid formed within 3 to 7 days in an incubator at 37°C, 5% CO ₂ , turn over the square dish on which the hanging drop is formed, and scrape it down using a scraper to collect it in one place and transferred to petri-dish. After that, PBS was dispensed, the cells were washed in a circular motion, and the washing process of collecting the spheroids collected in the center with a 10mL pipette was repeated 3 times, then suspended in NS medium and collected in a syringe.

Example 6-2. dermal papilla cell transplantation stage

First, as shown in Table 7 below, in the case of transplantation by the simple injection method, after removing the oil from the scalp of the recipient, ① select two parts with M-shaped hair loss on the forehead and approximately 4 * 4 cm per site = In an area of 16 cm ² , 5.0 * ^{10 6} cells were filled into the syringe in the form of single cells, After days, 30 days, 45 days, and 60 days, the results are shown in FIG. 9 .

As shown in FIG. 9 , it was confirmed that there was no significant difference in both single cells and spheroid cells in the method of simply injecting cells.

Next, in the case of implantation by injection method after punching, after removing oil from the scalp of the recipient, select the part where hair loss is formed on the crown of the head, and use a FUE (Follcular Unit Extractor) punch on the scalp without hair follicles. After making 60 perforations at each site with a depth of about 0.8 to 1 mm and a depth of about 5 to 7 mm, 5.0 * 10 ⁶ cells in an area of approximately ① 3 * 3 * 3.14 / 2 cm = 14 cm ² per site. was filled in the syringe as a single cell, and ② 5.0 * 10 ⁶ cells were made into spheroid cells and filled in the syringe in an aggregated state and injected directly into the perforated area (Table 8). 10 days, 30 days after injection, The results after 40 days, 50 days, and 60 days are shown in FIG. 10 .

As shown in FIG. 10 , in the method in which cells were injected after punching, it was confirmed that hairs came up from both single cells and spheroid cells. In particular, it is confirmed that it is not an area where hair has previously grown and has fallen out, but newly grown hair because a hole is made in a part without hair follicles and cells are injected. In addition, in the case of hair that was already present in the initial stage, it is thin and weak, but when observed about 60 days after injection of the cells, the hair appears to be strong and thick. was also found to have an effect.

Example 6-3. syringe needle gauge determination

In order to check the yield of the cells collected with a 5mL syringe by the transplantation method of Example 6-2 using a needle having a needle gauge of 24G to 31G, a spheroid having a size of 208.5±7.71 was collected according to the result of Example 5-2 did. As shown in FIG. 11 , in the case of the spheroids collected with 31G, it was confirmed that the standard deviation was very large in the form of spread or dents. Also, as the spheroid passes through the syringe needle, it is divided into several parts or stagnant inside the needle, resulting in a remarkably low yield. 26G also confirmed that the size of the spheroid was not constant and several spheroids separated from the existing spheroid were confirmed. On the other hand, it was confirmed that the spheroids collected using 24G maintained their shape before collection and had a constant size and high yield. This means that, in the gauge whose inner diameter is smaller than the diameter of the spheroid cell, the spheroid cell is broken while coming out of the syringe. In this case, there was a problem in that the needle became thick and caused severe pain in the recipient. Thus, it was confirmed that the collection using 24G is the optimal gauge for maintaining the spheroid shape the most, yielding a high yield, and transplanting cells with minimal scarring on the patient's scalp.

Claims (9)

(A) preparing the mass-proliferated dermal papilla cells;
(B) collecting dermal papilla cells in a syringe;
(C) transplanting the dermal papilla cells to the scalp of the recipient; a method for transplanting dermal papilla cells, including
The step of (C) transplanting the dermal papilla cells into the scalp of the recipient is to make a hole in the scalp of the recipient using a Follcular Unit Extractor (FUE) punch and then transplant the filled dermal papilla cells into a syringe. of transplantation method. The method according to claim 1, wherein the dermal papilla cells are spheroids or single cells in the step (A) of preparing the mass-proliferated dermal papilla cells. 3. The method of claim 2, wherein the spheroid is
(a1) proliferating the cells while exchanging the medium every 3 days in an incubator after seeding the cells in the flask;
(a2) removing the medium contained in the flask and washing with PBS;
(a3) after treatment with 0.25% Trypsin/EDTA, incubated in an incubator, and then centrifuged into MEM alpha medium containing 1% FBS;
(a4) diluting the cell suspension with Expansion Media 2 to contain 1000 to 3000 cells per drop by tapping the pellet submerged in the lower part, then inserting Expansion Media 2 and checking the number of cells;
(a5) dispensing 40mL of PBS on the bottom of a square dish, forming a drop of 5 to 30ul with a 12-channel multichannel pipette on the cover of the square dish;
(a6) After holding the corner of the square dish on which the drop is formed, turn it over to make a hanging drop shape, and collect the spheroid formed after 1 to 7 days of incubation in an incubator; dermal papilla cell transplantation method comprising a. The method of claim 3, wherein the drop in step (a5) is 16 to 25ul. According to claim 1, (C) the step of transplanting the dermal papilla cells into the scalp of the transplant recipient (c1) using a FUE (Follcular Unit Extractor) punch to make a hole having an inner diameter of 0.8 to 1 mm; characterized in that it comprises: A method of transplantation of dermal papilla cells. The method of claim 5, wherein the perforation has a depth of 5 to 7 mm. [Claim 6] The method of claim 5, wherein (c2) 5.0 * 10 ⁶ spheroid cells are filled in a syringe and injected into the puncture; The method of claim 1, wherein the syringe has an inner diameter of the needle gauge that is larger than the diameter of the dermal papilla cells collected in step (B). The method of claim 8, wherein the syringe has a needle gauge of 24G.

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