WO2022035023A1 - Method for transplanting dermal papilla cells through perforation - Google Patents

Method for transplanting dermal papilla cells through perforation Download PDF

Info

Publication number
WO2022035023A1
WO2022035023A1 PCT/KR2021/006413 KR2021006413W WO2022035023A1 WO 2022035023 A1 WO2022035023 A1 WO 2022035023A1 KR 2021006413 W KR2021006413 W KR 2021006413W WO 2022035023 A1 WO2022035023 A1 WO 2022035023A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
dermal papilla
papilla cells
transplanting
syringe
Prior art date
Application number
PCT/KR2021/006413
Other languages
French (fr)
Korean (ko)
Inventor
강다윗
윤정인
노정원
Original Assignee
한모바이오 주식회사
한모바이오메디테크 주식회사
강다윗
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 한모바이오 주식회사, 한모바이오메디테크 주식회사, 강다윗 filed Critical 한모바이오 주식회사
Publication of WO2022035023A1 publication Critical patent/WO2022035023A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B17/32—Surgical cutting instruments
    • A61B17/3205—Excision instruments
    • A61B17/32053—Punch like cutting instruments, e.g. using a cylindrical or oval knife
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B17/34—Trocars; Puncturing needles
    • A61B17/3468—Trocars; Puncturing needles for implanting or removing devices, e.g. prostheses, implants, seeds, wires
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B2017/00743—Type of operation; Specification of treatment sites
    • A61B2017/00747—Dermatology
    • A61B2017/00752—Hair removal or transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B2017/00969—Surgical instruments, devices or methods, e.g. tourniquets used for transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/18—Materials or treatment for tissue regeneration for hair reconstruction

Definitions

  • 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.
  • FUE Follcular Unit Extractor
  • 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), in which the hair grows due to 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) in which the hair growth stops due to cell proliferation. 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 anagen phase and maintain the same level of hair as a whole. is called alopecia.
  • hair follicles 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, it has been reported that minoxidil has an anti-apoptotic effect and proliferation of dermal papilla cells.
  • 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.
  • FUE Follcular Unit Extractor
  • 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; as a method of transplanting dermal papilla cells comprising; (C) the step of transplanting the dermal papilla cells to the scalp of the recipient is a Follcular Unit Extractor (FUE) ) Provides a method for transplanting dermal papilla cells, which is characterized by implanting the filled dermal papilla cells in a syringe after making a perforation using a punch.
  • FUE Follcular Unit Extractor
  • the dermal papilla cells may be spheroids or single cells.
  • 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 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, turning it over to make a hanging drop shape, and collecting the spheroid formed after
  • the drop of step (a5) may be characterized in that 16 to 25ul.
  • (C) the step of transplanting the dermal papilla cells to 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 1mm; may include; .
  • the perforation may be characterized in that the depth is 5 to 7 mm.
  • (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.
  • 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).
  • the syringe may be characterized in that the needle gauge is 24G.
  • the present invention can be utilized in various industrial fields such as the medical field and the cosmetic field.
  • 1 shows 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.
  • 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 hair bulb size according to the chopping stage.
  • Figure 9 shows the scalp photos by period after transplantation of dermal papilla cells by simple injection.
  • FIG. 10 shows scalp photos taken for a certain period after implantation of dermal papilla cells through perforation using a Follcular Unit Extractor (FUE) punch.
  • FUE Follcular Unit Extractor
  • FIG. 11 shows a comparison of the shape and size of a spheroid according to the syringe needle gauge.
  • FUE Follcular Unit Extractor
  • FUE Flullcular Unit Extractor
  • 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 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, etc., there are no aftereffects such as scars or pain, and it has the advantage of being able to return to life immediately.
  • the dermal papilla cells may be spheroids or single cells.
  • the spheroid is (a1) after seeding the cells in a flask, exchanging the medium in an incubator every 3 days to proliferate the cells; (a2) removing the medium 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, turning it over to make a hanging drop shape, and collecting the spheroid formed after cult
  • the drop of step (a5) may be characterized in that 16 to 25ul.
  • the step of transplanting the dermal papilla cells to 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;
  • FUE Frollcular Unit Extractor
  • the perforations may be characterized in that the depth is between 5 and 7 mm.
  • (c2) filling the syringe with 5.0 * 10 6 spheroid cells and injecting them into the perforation; may be characterized by further comprising.
  • 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).
  • the syringe may be characterized in that the needle gauge is 24G.
  • 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.
  • each pellet was tapped and pipetting was performed with 2 ml of attachment media having the composition shown in Table 1 below, then 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.
  • the step of subculture from Passage 0 to Passage 1 was performed as follows. First, a 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 °C, 5% CO 2 mass growth phase.
  • 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 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
  • case 1 is the result of large-scale proliferation in Expansion media 1 medium after isolating dermal papilla cells from hair follicles by chopping
  • case 2 is the result of large-scale proliferation in Expansion media 2 medium after separating dermal papilla cells from hair follicles by chopping.
  • 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
  • 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.
  • case 2 the cell size of case 2 was the smallest and kept evenly, and in the remaining groups except for case 1, the cell size 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.
  • FIG. 4 the secretion amount of growth factor related to dermal papilla cells according to subculture for each case is shown in FIG. 4 .
  • growth factors of dermal papilla cells such as HGF that stimulates human dermal papilla cells to promote 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
  • HGF that stimulates human dermal papilla cells to promote proliferation of epithelial follicle cells
  • VEGF that induces hair growth by participating in vasodilation
  • FGF FGF
  • 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.
  • hair bulbs with a size of 750 ⁇ m to 900 ⁇ m before chopping, 490 ⁇ m to 630 ⁇ m in the first chopping step, 300 ⁇ m to 410 ⁇ m in the second step, 180 ⁇ m to 260 ⁇ m in the third step, and 15 ⁇ m to 120 ⁇ m in the fourth step were obtained. was confirmed (FIG. 5).
  • the chopped hair bulbs were collected in a tube, centrifuged at 2200 rpm for 5 minutes, the supernatant was discarded, the pellet was collected, 1 ml of attachment media was put in a 35 mm cell culture dish, and 37 ° C, 5% CO 2 mass propagator Cell proliferation was confirmed, and the results are shown in FIG. 6 .
  • the cell proliferation and adhesion patterns were checked 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.
  • the chopping level was low, the cells were not evenly attached to the bottom of the cell culture dish and agglomerated growth occurred.
  • 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.
  • Example 5 Preparation step for transplantation of dermal papilla cells
  • spheroids and single cells were prepared as follows, respectively.
  • Example 5-1 Preparation of spheroid (Aggregation cell)
  • Expansion Media 2 After discarding the supernatant and tapping the pellet that settled at the bottom sufficiently, add an appropriate amount of Expansion Media 2, check the number of cells, and dilute the necessary amount of the cell suspension with Expansion Media 2 to contain 1000 to 3000 cells per drop. .
  • 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 an optimal hanging drop shape, and collect the spheroids formed after culturing for 1 to 7 days in an incubator at 37°C, 5% CO 2 .
  • Example 5-2 Determination of drop dose for spheroid formation
  • case 1 where the drop capacity is limited to 5 to 15ul, the cells are not aggregated and are widely dispersed or multiple 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.
  • case 2 where the drop capacity was limited to 16 to 25ul, and case 3 limited to 26 to 30ul, spheroids of a certain size were formed within 3 days.
  • case 3 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 dented shape when manufactured as a hanging drop type.
  • case 3 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 ⁇ m 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 5-4 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 an incubator at 37° C., 5% CO 2 for 1 to 7 days for spheroid formation, followed by T225 flask
  • the medium contained in the was removed and washed once with PBS. After that, treated with 0.25% Trypsin/EDTA, incubated at 37°C, 5% CO 2 in an incubator for 5 minutes, and then added MEM alpha medium containing 1% FBS for inactivation and collected in a 50ml centrifuge tube at 2200 rpm for 5 minutes. Centrifugation was performed for minutes. The supernatant was discarded and the pellet that had settled in the lower part 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 6-1 dermal papilla cell collection step
  • Example 6-2 dermal papilla cell transplantation stage
  • Example 6-3 syringe needle gauge determination
  • Example 6-2 In order to check the yield by the transplantation method of Example 6-2 using a needle having a needle gauge of 24G to 31G for cells collected with a 5mL syringe, spheroids of 208.5 ⁇ 7.71 size were collected according to the results 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. In addition, 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.
  • 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.
  • 24G it was confirmed that the cell came out of the syringe without deformation. In this case, there was a problem in that the needle became thick and caused severe pain in the recipient.
  • the collection using 24G is the best gauge to maintain the spheroid shape the most and to have a high yield, and to transplant cells with minimal scarring on the patient's scalp.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Surgery (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Transplantation (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Molecular Biology (AREA)
  • Dermatology (AREA)
  • Botany (AREA)
  • Epidemiology (AREA)
  • Medical Informatics (AREA)
  • Vascular Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Anesthesiology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Cardiology (AREA)
  • Pathology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Materials For Medical Uses (AREA)

Abstract

The present invention relates to a method for transplanting dermal papilla cells and, more specifically, to a method for transplanting mass-proliferated dermal papilla cells into the scalp of a transplant recipient through perforation using a follicular unit extractor (FUE) punch. Dermal papilla cells transplanted in large quantities according to the present invention can play an important role in hair growth, and thus, the present invention relating to an optimal method for transplanting dermal papilla cells can be used in various industrial fields such as the medical field and the beauty field.

Description

천공을 통한 모유두세포 이식방법Transplantation of dermal papilla cells through perforation
본 발명은 천공을 통한 모유두세포의 이식방법에 관한 발명으로, 더욱 상세하게는 대량증식된 모유두세포를 FUE(Follcular Unit Extractor) 펀치를 이용한 천공을 통하여 피이식자의 두피에 이식하는 방법에 관한 발명이다. 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. .
탈모증(alopecia)은 질병, 영양부족, 노화, 호르몬 불균형 등에 의해 야기되는 것으로 알려져 있다. 많은 연구가 진행되어 왔음에도 불구하고, 근본적인 탈모에 대한 메커니즘은 잘 알려져 있지 않으나, 일반적으로 모발은 일정한 모주기(hair cycle)를 거치게 되며, 모유두(dermal papilla)의 자극을 받은 모모세포(keratinocyte)가 활발히 분열·증식하여 모발이 자라는 성장기(anagen), 모구(hair bulb)에 혈액공급이 끊기고 모유두가 모낭으로부터 분리되는 퇴행기(catagen) 및 세포 증식이 멈춰 모발이 성장하지 않는 휴지기(telogen)를 거쳐 다시 성장기로 들어가거나 모발이 두피로부터 빠지는 탈락기(exogen)로 가게 된다. 사람의 모발은 각각 독립된 성장주기를 가져 일부는 탈락기로, 일부는 성장기로 들어가며 전체적으로 동일 수준의 모발수를 유지하게 되는데, 이러한 균형이 탈락기로 기울어 정상적으로 모발이 존재해야 할 부위에 모발이 없어지는 상태를 탈모증(alopecia)이라 한다. 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), in which the hair grows due to 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) in which the hair growth stops due to cell proliferation. 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 anagen phase and maintain the same level of hair as a whole. is called alopecia.
이러한 탈모를 치료하기 위한 연구도 오랜기간 동안 진행되어 왔다. 그럼에도 불구하고, 지금까지 오직 두 개의 약물들 (피나스테라이드(finasteride) 및 미녹시딜(minoxidil))만이 탈모 치료를 위해 FDA(Food and Drug Administration) (U.S.A)에서 인가되었다.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.
인체의 모발은 약 10만∼15만 개 정도이며 모낭(hair follicle)에서 형성된다. 모낭에는 유두가 있는데, 이 부위에는 작은 혈관이 분포되어 모발의 성장에 필요한 영양분을 공급하고 유두 위의 옆으로는 모발에 윤기를 주는 기름을 공급해주는 지루샘이 있다. 모낭은 여러 다른 상피세포들(epithelial cells) 및 모유두세포들로 이루어진다. 모유두세포는 모낭의 기저부에 위치한 중간엽-유래 섬유아세포(mesenchymally-derived fibroblasts)이며 육모에 중요한 역할을 한다. 특히, 미녹시딜(minoxidil)은 모유두세포의 증식 및 항-세포사멸 효과를 가지는 것으로 보고되었다. 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, it has been reported that minoxidil has an anti-apoptotic effect and proliferation of dermal papilla cells.
본 발명자는 육모에 중요한 역할을 하는 모유두세포를 대량증식하여 피이식자에게 이식하는 방법에 관하여 꾸준히 연구해 온 결과, 모유두세포 최적의 이식방법에 관한 본 발명을 완성하게 되었다. As a result of continuous research on a method for transplanting dermal papilla cells, which play an important role in hair growth, to transplant recipients, the present inventor has completed the present invention regarding an optimal transplantation method for dermal papilla cells.
본 발명은 대량증식된 모유두세포를 FUE(Follcular Unit Extractor) 펀치를 이용한 천공을 통하여 피이식자의 두피에 이식하는 최적화된 방법을 통하여 탈모를 치료하는 방법을 제공하고자 한다. 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.
상기와 같은 문제를 해결하기 위해 본 발명은 (A) 대량증식된 모유두세포를 준비하는 단계; (B) 모유두세포를 syringe에 수집하는 단계; (C) 모유두세포를 피이식자의 두피에 이식하는 단계;를 포함하는 모유두세포의 이식방법으로, (C) 모유두세포를 피이식자의 두피에 이식하는 단계는 피이식자의 두피에 FUE(Follcular Unit Extractor) 펀치를 이용하여 천공을 만든 후 syringe에 충진된 모유두세포를 이식하는 것을 특징으로 하는 모유두세포의 이식방법을 제공한다.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; as a method of transplanting dermal papilla cells comprising; (C) the step of transplanting the dermal papilla cells to the scalp of the recipient is a Follcular Unit Extractor (FUE) ) Provides a method for transplanting dermal papilla cells, which is characterized by implanting the filled dermal papilla cells in a syringe after making a perforation using a punch.
본 발명에 있어서, (A) 대량증식된 모유두세포를 준비하는 단계에서 모유두세포는 spheroid 또는 single cell일 수 있다.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.
또한 본 발명에 있어서, spheroid는 (a1) flask에 cell을 seeding한 후 배양기에서 3일 간격으로 배지를 교환해 주면서 세포를 증식시키는 단계; (a2) flask에 들어있던 배지를 제거하고, PBS로 세척하는 단계; (a3) 0.25% Trypsin/EDTA 처리 후 배양기에서 5분간 incubation한 다음, 1% FBS가 들어있는 MEM alpha 배지를 넣고 원심분리 하는 단계; (a4) 하부에 가라앉은 pellet을 tapping 한 후 Expansion Media 2를 넣고 세포수를 확인하여, 하나의 drop 당 1000 내지 3000 cell이 포함되도록 세포현탄액을 Expansion Media 2로 희석하는 단계; (a5) square dish 바닥에 PBS 40mL을 분주하고, square dish 덮개에 12채널 Multichannel Pipette으로 5 내지 30ul의 drop을 형성하는 단계; (a6) drop이 형성된 Square dish 모서리를 대각선으로 잡은 후 뒤집어서 hanging drop 형태를 만들고, 배양기에서 1 내지 7일 배양 후 형성된 spheroid를 수집하는 단계;를 포함할 수 있다.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 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, 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.
또한 본 발명에 있어서, (a5) 단계의 drop은 16 내지 25ul 인 것을 특징으로 할 수 있다. Also in the present invention, the drop of step (a5) may be characterized in that 16 to 25ul.
또한 본 발명에 있어서, (C) 모유두세포를 피이식자의 두피에 이식하는 단계는 (c1) FUE (Follcular Unit Extractor) 펀치를 이용하여 내경이 0.8 내지 1mm 인 천공을 만드는 단계;를 포함할 수 있다. In addition, in the present invention, (C) the step of transplanting the dermal papilla cells to 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 1mm; may include; .
또한 본 발명에 있어서, 천공은 깊이가 5 내지 7 mm인 것을 특징으로 할 수 있다. Also in the present invention, the perforation may be characterized in that the depth is 5 to 7 mm.
또한 본 발명에 있어서, (c2) 5.0 * 106개의 spheroid cell을 syringe에 충진하여 천공에 주사하는 단계;를 더 포함하는 것을 특징으로 할 수 있다. 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.
또한 본 발명에 있어서, 상기 syringe는 needle gauge의 내경이 (B)단계에서 수집된 모유두세포의 직경보다 큰 것을 특징으로 할 수 있다. 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).
또한 본 발명에 있어서, 상기 syringe는 needle gauge가 24G인 것을 특징으로 할 수 있다.In addition, in the present invention, the syringe may be characterized in that the needle gauge is 24G.
본 발명에 의하여 제공되는 방법에 의하여 대량으로 이식된 모유두세포는 육모에 중요한 역할을 할 수 있는 점에서 본 발명은 의료분야, 미용분야 등 다양한 산업분야에서 활용이 가능하다.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.
도 1은 모구로부터 모유두세포를 분리하는 방법에 따른 세포수득률의 차이를 비교하여 나타낸 것이다.1 shows a comparison of the difference in cell yield according to the method of separating dermal papilla cells from hair follicles.
도 2는 모유두세포 분리방법과 배지조성에 따른 대량증식결과의 차이를 비교하여 나타낸 것이다.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.
도 3은 case 별 계대배양에 따른 세포수득률 차이를 비교하여 나타낸 것이다. 3 shows a comparison of the cell yield difference according to the subculture for each case.
도 4는 case 별 계대배양에 따른 모유두세포와 관련된 growth factor 분비량의 차이를 비교하여 나타낸 것이다. 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.
도 5는 chopping의 단계에 따른 모구 크기 변화를 나타낸 것이다. Figure 5 shows the change in the hair bulb size according to the chopping stage.
도 6은 chopping의 단계에 따른 세포증식의 차이를 비교하여 나타낸 것이다. 6 shows a comparison of the difference in cell proliferation according to the chopping stage.
도 7은 drop 용량별 cell 수에 따른 Spheroid 형태를 비교하여 나타낸 것이다. 7 shows a comparison of the spheroid shape according to the number of cells for each drop capacity.
도 8은 면적당 seeding 된 세포수에 따른 증식 속도 및 수득률을 비교하여 나타낸 것이다.8 shows a comparison of the proliferation rate and yield according to the number of seeded cells per area.
도 9는 단순주사에 의한 모유두세포 이식 후 두피사진을 기간별로 나타낸 것이다. Figure 9 shows the scalp photos by period after transplantation of dermal papilla cells by simple injection.
도 10은 FUE (Follcular Unit Extractor) 펀치를 이용한 천공을 통하여 모유두세포를 이식한 후 일정 기간별로 촬영한 두피사진을 나타낸 것이다. 10 shows scalp photos taken for a certain period after implantation of dermal papilla cells through perforation using a Follcular Unit Extractor (FUE) punch.
도 11은 syringe needle gauge에 따른 spheroid 형태 및 크기를 비교하여 나타낸 것이다. 11 shows a comparison of the shape and size of a spheroid according to the syringe needle gauge.
본 발명에 의한 일 실시예에서는, (A) 대량증식된 모유두세포를 준비하는 단계; (B) 모유두세포를 syringe에 수집하는 단계; (C) 모유두세포를 피이식자의 두피에 이식하는 단계;를 포함하는 모유두세포의 이식방법으로, (C) 모유두세포를 피이식자의 두피에 이식하는 단계는 피이식자의 두피에 FUE(Follcular Unit Extractor) 펀치를 이용하여 천공을 만든 후 syringe에 충진된 모유두세포를 이식하는 것을 특징으로 하는 모유두세포의 이식방법을 제공한다.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; as a method of transplanting dermal papilla cells comprising; (C) the step of transplanting the dermal papilla cells to the scalp of the recipient is a Follcular Unit Extractor (FUE) ) Provides a method for transplanting dermal papilla cells, which is characterized by implanting the filled dermal papilla cells in a syringe after making a perforation using a punch.
FUE (Follcular Unit Extractor)는 비절개 모발이식시에 모낭을 채취할 때 사용하는 장비로 내경 0.8 내지 1mm 크기의 소형 펀치를 두피조직에 접촉시킨 후 선형으로 회전시켜 모낭이 포함된 두피조직을 포셉 등을 이용하여 천공된 모낭을 발췌할 수 있다. 발췌된 모낭은 두피의 탈모가 일어난 부위에 이식할 수 있으며, 메스 등을 이용하여 절개하지 않기 때문에 흉터나 통증과 같은 후유증이 없고 일생 생활로 바로 복귀 가능한 장점이 있다. 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 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, etc., there are no aftereffects such as scars or pain, and it has the advantage of being able to return to life immediately.
본 발명에 의한 다른 실시예에서, (A) 대량증식된 모유두세포를 준비하는 단계에서 모유두세포는 spheroid 또는 single cell일 수 있다.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.
본 발명에 의한 또 다른 실시예에서, spheroid는 (a1) flask에 cell을 seeding한 후 배양기에서 3일 간격으로 배지를 교환해 주면서 세포를 증식시키는 단계; (a2) flask에 들어있던 배지를 제거하고, PBS로 세척하는 단계; (a3) 0.25% Trypsin/EDTA 처리 후 배양기에서 5분간 incubation한 다음, 1% FBS가 들어있는 MEM alpha 배지를 넣고 원심분리 하는 단계; (a4) 하부에 가라앉은 pellet을 tapping 한 후 Expansion Media 2를 넣고 세포수를 확인하여, 하나의 drop 당 1000 내지 3000 cell이 포함되도록 세포현탄액을 Expansion Media 2로 희석하는 단계; (a5) square dish 바닥에 PBS 40mL을 분주하고, square dish 덮개에 12채널 Multichannel Pipette으로 5 내지 30ul의 drop을 형성하는 단계; (a6) drop이 형성된 Square dish 모서리를 대각선으로 잡은 후 뒤집어서 hanging drop 형태를 만들고, 배양기에서 1 내지 7일 배양 후 형성된 spheroid를 수집하는 단계;를 포함할 수 있다.In another embodiment according to the present invention, the spheroid is (a1) after seeding the cells in a flask, exchanging the medium in an incubator every 3 days to proliferate the cells; (a2) removing the medium 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, 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.
본 발명에 의한 또 다른 실시예에서, (a5) 단계의 drop은 16 내지 25ul 인 것을 특징으로 할 수 있다. In another embodiment according to the present invention, the drop of step (a5) may be characterized in that 16 to 25ul.
본 발명에 의한 또 다른 실시예에서, (C) 모유두세포를 피이식자의 두피에 이식하는 단계는 (c1) FUE (Follcular Unit Extractor) 펀치를 이용하여 내경이 0.8 내지 1mm 인 천공을 만드는 단계;를 포함할 수 있다. In another embodiment according to the present invention, (C) the step of transplanting the dermal papilla cells to 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
본 발명에 의한 또 다른 실시예에서, 천공은 깊이가 5 내지 7 mm인 것을 특징으로 할 수 있다. In another embodiment according to the invention, the perforations may be characterized in that the depth is between 5 and 7 mm.
본 발명에 의한 또 다른 실시예에서, (c2) 5.0 * 106개의 spheroid cell을 syringe에 충진하여 천공에 주사하는 단계;를 더 포함하는 것을 특징으로 할 수 있다. 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.
본 발명에 의한 또 다른 실시예에서, syringe는 needle gauge의 내경이 (B)단계에서 수집된 모유두세포의 직경보다 큰 것을 특징으로 할 수 있다. 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).
본 발명에 의한 또 다른 실시예에서, syringe는 needle gauge가 24G인 것을 특징으로 할 수 있다.In another embodiment according to the present invention, the syringe may be characterized in that the needle gauge is 24G.
이하, 구체적인 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.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.
실시예 1. 두피조직으로부터 모구의 분리Example 1. Isolation of hair bulbs from scalp tissue
멸균된 90 mm petri dish에 MEM alpha 배지를 피험자로부터 채취한 두피 조직(표피에서 진피층까지)이 잠기도록 채운 후, 90 mm petri dish 덮개에 MEM alpha 배지를 이용하여 여러 개의 drop을 형성시키고, 미세수술용 가위와 포셉(forcep)을 이용하여 진피층의 모구를 하나씩 분리하여 준비한 drop으로 옮겼다. 이 후, drop으로 이동된 모구를 실체현미경으로 관찰하며 26G syringe와 미세수술용 포셉을 이용하여 모구 말단에 부착되어 있는 fatty tissue 및 모간(hair shaft) 등을 제거하여 두피조직으로부터 모구를 분리하였다. 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, while observing the hair bulbs moved to the drop under a stereoscopic microscope, the hair bulbs were separated from the scalp tissue by removing the fatty tissue and hair shaft attached to the tip of the hair bulbs using a 26G syringe and microsurgery forceps.
실시예 2. 모구로부터 모유두세포의 분리Example 2. Isolation of dermal papilla cells from hair bulbs
모구로부터 모유두세포를 분리하는 최적의 방법을 선택하기위하여 하기와 같은 비교실험을 하였다. In order to select the optimal method for isolating dermal papilla cells from hair follicles, the following comparative experiment was conducted.
먼저, 분리 방법 1은 35 mm cell culture dish에 1 ml 의 Dissection media를 넣고 모구 약 50개를 넣은 후 정밀미세가위로 chopping한 후 tube에 모으고 2200 rpm에서 5 분간 원심분리하는 방법이다.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.
다음으로, 분리 방법 2는 모구 약 50개가 들어있는 35 mm cell culture dish에 0.025% Collagenase type I 시약을 넣은 후 37 ℃, shaking incubator에서 2시간 30분 간 incubation 하고 tube에 모아 2200 rpm에서 5 분간 원심분리하는 방법이다.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.
상기 분리 방법 1과 2의 결과를 비교하기 위하여, 각각의 Pellet을 tapping 하고 하기 표 1의 조성을 가지는 Attachment media 2 ml로 pipetting을 충분히 한 후 35 mm cell culture dish에 seeding 하고, 37℃, 5% CO2 대량증식기에서 3일 간격으로 배지를 교환해 주면서 culture dish가 세포로 가득 찰 때까지 증식시켰다. 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, then 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.
Figure PCTKR2021006413-appb-img-000001
Figure PCTKR2021006413-appb-img-000001
상기 비교실험 결과, 0.025% Collagenase type I 시약을 넣은 분리방법 2와 비교하여, 모구 자체에 다른 처리를 하지 않고 chopping만 진행한 분리방법 1에서 세포의 크기가 더 작고 일정하며 세포수득률도 높게 나오는 것을 확인할 수 있었다(도 1).As a result of the comparative experiment, compared to 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 cell size was smaller, constant, and the cell yield was high. was confirmed (FIG. 1).
실시예 3. 계대배양 단계Example 3. Passage step
Passage 0 에서 Passage 1 로 계대배양하는 단계는 다음과 같이 실시하였다. 먼저, 35 mm cell culture dish에서 수집된 세포수에 따라 계대배양 할 culture dish를 결정한 후, 35 mm cell culture dish에 들어있던 배지를 버리고, PBS로 1회 세척하였다. 다음으로, 0.25% Trypsin/EDTA을 처리하고 37℃, 5% CO2 대량증식기에서 5분간 incubation 하였다. 이 후, inactivation을 위해 1% FBS가 들어있는 MEM alpha 배지를 넣고 50ml centrifuge tube에 모은 후 2200 rpm에서 5 분간 원심분리 후, 상층액은 버리고 아래에 가라앉은 pellet을 충분히 tapping 한 후 표 2에 나타낸 적당량의 Expansion Media 1 또는 2를 넣고 세포수를 확인하였다.The step of subculture from Passage 0 to Passage 1 was performed as follows. First, a 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, for inactivation, add MEM alpha medium containing 1% FBS, 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.
Figure PCTKR2021006413-appb-img-000002
Figure PCTKR2021006413-appb-img-000002
계수된 세포수에 따라 1,500개/cm2가 되도록 다음 단계의 culture dish에 seeding 한 후, 37℃, 5% CO2 대량증식기에서 3일 간격으로 배지를 교환해 주면서 culture dish가 세포로 가득 찰 때까지 증식시켰다. After seeding the culture dish of the next step so that it becomes 1,500 cells/cm 2 according to the number of cells counted, exchange the medium every 3 days at 37℃, 5% CO 2 mass growth phase when the culture dish is full of cells was propagated until
이 후, passage 1 내지 passage 4 로 계대배양하는 단계는 상기와 동일한 순서로 이루어지며, 계수되는 세포수에 따라 culture dish의 크기를 맞추어 passage 4까지 계대배양하였고, 중간 passage 단계에서 동결하였다가 해동할 경우에는 3,000개/cm2가 되도록 culture dish에 seeding하였다.Thereafter, 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 thawed at the intermediate passage step. In this case, it was seeded in a culture dish to be 3,000 pieces/cm 2 .
실시예 4. 모유두세포 분리 및 대량증식 조건에 따른 결과 도출Example 4. Derivation of results according to dermal papilla cell isolation and mass proliferation conditions
실시예 4-1. 세포 분리방법과 배지조성에 따른 차이Example 4-1. Differences according to cell separation method and medium composition
모유두세포 분리방법과 배지 조성에 따른 대량증식결과를 도 2에 나타내었다. 도 2에서 case 1은 chopping에 의하여 모구로부터 모유두세포를 분리한 후 Expansion media 1 배지에서 대량증식한 결과이고, case 2는 chopping에 의하여 모구로부터 모유두세포를 분리한 후 Expansion media 2 배지에서 대량증식한 결과이며, case 3은 0.025% Collagenase type I 시약 처리에 의하여 모구로부터 모유두세포를 분리한 후 Expansion media 1 배지에서 대량증식한 결과이고, case 4 는 0.025% Collagenase type I 시약 처리에 의하여 모구로부터 모유두세포를 분리한 후 Expansion media 1 배지에서 대량증식한 결과를 나타낸 것이다. 각 case 별로 세포의 morphology를 확인해 본 결과 chopping에 의하여 모구로부터 모유두세포를 분리한 후 Expansion media 2를 사용한 경우인 case 2에서 세포의 형태가 잘 유지되며 모유두세포의 대량증식 증식률이 현저히 향상되는 것을 확인할 수 있었다.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 isolating 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, which is the case where Expansion media 2 was used after separating the dermal papilla cells from the hair follicles by chopping. could
또한, case 별로 계대 대량증식에 따른 세포 크기변화를 하기 표 3에 나타내었다. In addition, the cell size change according to the passage mass proliferation for each case is shown in Table 3 below.
Figure PCTKR2021006413-appb-img-000003
Figure PCTKR2021006413-appb-img-000003
상기 표 3에 따르면, case 2의 세포 크기가 가장 작고 고르게 유지되었고, case 1을 제외한 나머지 군에서는 계대배양수가 늘어남에 따라 세포크기도 커지는 경향을 나타내었다. 나아가, case 2의 경우에는 P1 내지 P4까지는 큰 변화 없이 세포크기가 유지되는 것으로 보아 세포의 수득률 또한 높을 것으로 예상을 해 볼 수 있었고, 이를 확인한 결과를 도 3에 나타내었다. 도 3에 따르면, 세포의 수득률 역시 전체 계대에 걸쳐 case 2가 가장 높은 것을 확인 할 수 있었고, 특히 P3에서 가장 많은 양의 세포가 수집된 것으로 확인되었다.According to Table 3, the cell size of case 2 was the smallest and kept evenly, and in the remaining groups except for case 1, the cell size 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.
다음으로, case 별로 계대배양에 따른 모유두세포와 관련된 Growth factor 분비량을 도 4에 나타내었다. 도 4에 따르면, 인체 모유두세포를 자극하여 상피 소낭 세포의 증식을 촉진시키는 HGF, 혈관확장에 관여하여 모발 성장을 유도하는 VEGF, 모낭조직의 성장을 촉진하는 FGF (KGF) 등 모유두세포의 성장인자로 알려져 있는 다양한 Growth factor의 분비량을 측정해 본 결과 전반적으로 case 2의 P3에서 가장 많은 양의 growth factor가 분비되는 것을 확인할 수 있었다.Next, the secretion amount 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 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
실시예 4-2. chopping 범위에 따른 차이Example 4-2. Differences by chopping range
상기 실험결과를 토대로, 최적의 chopping 범위를 결정하기 위한 실험을 진행하였다. 먼저, 두피 조직으로부터 펀칭되어져 나온 모구를 PBS 또는 basal media가 채워진 petri dish에 수집하고, 35 mm cell culture dish에 1 ml의 Dissection media와 모구를 넣고 정밀미세가위로 모구를 chopping한 후, chopping의 정도에 따른 모구 크기 변화를 현미경으로 관찰하였다. chopping 단계에 따른 모구의 크기를 측정한 결과 chopping 전은 750μm 내지 900μm, chopping 1단계에서는 490μm 내지 630μm, 2단계에서는 300μm 내지 410μm, 3단계에서는 180μm 내지 260μm, 4단계에서는 15μm 내지 120μm 크기의 모구를 확인할 수 있었다(도 5). 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).
이 후, chopping된 모구를 tube에 모으고 2200 rpm에서 5 분간 원심분리 한 후, 상층액은 버리고 pellet을 수집하여 35 mm cell culture dish에 1ml 의 Attachment media를 넣고 37℃, 5% CO2 대량증식기에서 세포의 증식을 확인하였으며, 그 결과는 도 6에 나타내었다.After that, the chopped hair bulbs were collected in a tube, centrifuged at 2200 rpm for 5 minutes, the supernatant was discarded, the pellet was collected, 1 ml of attachment media was put in a 35 mm cell culture dish, and 37 ° C, 5% CO 2 mass propagator Cell proliferation was confirmed, and the results are shown in FIG. 6 .
도 6에 따르면, chopping 범위에 따라 총 4단계로 나눠 세포의 증식 및 부착 형태를 확인해 본 결과 chopping의 정도에 따라 세포의 부착 형태가 상이한 것을 확인할 수 있었다. 도 6의 좌측은 chopping 후 부착한지 3일째 된 사진이고, 우측은 chopping 후 부착한지 5일째 된 세포 사진이다. 배양 3일째 관찰한 결과 chopping의 단계가 낮은 경우 cell culture dish의 바닥면에 세포들이 고르게 부착되지 못하고 뭉쳐 자라는 현상이 일어났다. 반면 chopping의 단계가 높아질수록 cell culture dish 바닥면에 세포들이 일정한 간격으로 고르게 부착되어 자랐으며 양끝이 뾰족한 방추 형태로 증식되었다. 나아가, 배양 5일째에 관찰한 결과 chopping의 단계가 낮은 상태의 경우 뭉쳐 자란 곳과 뭉치지 않은 곳에서의 세포 증식이 현저하게 차이가 났으며 세포가 군데군데 퍼져 자라는 경향을 보였으나, chopping의 단계가 높아질수록 cell culture dish 바닥면 전체에 고르게 세포가 가득차 있는 것을 확인할 수 있었다.According to FIG. 6 , the cell proliferation and adhesion patterns were checked 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 in the clumped and non-clumped regions was significantly different, 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.
또한, chopping의 단계에 따른 세포수득률에서도 차이를 나타내었는 바, 이를 하기 표 4에 나타내었다. In addition, there was also a difference in cell yield according to the chopping step, which is shown in Table 4 below.
Figure PCTKR2021006413-appb-img-000004
Figure PCTKR2021006413-appb-img-000004
상기 표 4에 따르면, chopping의 정도에 따라 세포의 부착 형태뿐만 아니라 수득률에서도 차이가 나는 것을 확인할 수 있었다. chopping 정도에 따라 세포가 cell culture dish에 부착되는 형태가 달라지며 세포 사이의 간격 및 증식되는 모양에 따라 수득률에 크게 영향을 미치는 것으로 보인다. 대략적으로 1~2단계 chopping 정도에서는 콜로니(colony)를 형성하여 증식되고, 그로인해 세포가 퍼져 자라는 경향이 있어 1.0E+05 ~ 1.3E+05 수준의 세포수득율을 보이며, 3단계 chopping 정도에서는 세포의 뭉침은 1~2단계 chopping 보다는 덜 하지만 약간의 뭉침으로 인해 초기 부착시 세포의 퍼짐이 관찰되고, 4단계 chopping 에서는 세포들이 초기부터 single 상태로 잘 부착하여 세포의 퍼짐이나 뭉침들은 전혀 관찰되지 않았으며 배양일수가 늘어남에 따라 cell culture dish 바닥 전체에 고르게 부착하여 증식되는 것을 관찰 할 수 있었고, 이로 인해 chopping 1단계와 비교 했을 때 수득율이 약 2배 정도 차이가 나는 것을 확인할 수 있었다. 그러나, 15μm 이하로 chopping을 실시한 경우에는 모구 내부의 모유두세포가 손상되어 세포수득률이 현저히 낮아지는 문제가 있었고, 120μm 이상으로 chopping을 실시한 경우에는 상기 실험에서 확인한 바와 같이 세포의 뭉침현상이 발생하여 세포가 culture dish 바닥 전체에 고르게 분포되지 아니하는 결과 세포증식이 현저히 저하되는 문제가 있었는데, 이는 고르게 분포되는 세포들에 비하여 뭉쳐진 세포들은 영양분 흡수율이 저하되고, 세포밀도 상승에 기인한 외부스트레스 증가의 결과로 보인다. According to Table 4, it was confirmed that there was a difference in the yield as well as the cell adhesion type according to the degree of chopping. Depending on the degree of chopping, the shape of the cells attached to the cell culture dish varies, and it seems that the yield is greatly affected by the spacing between the cells and the shape of proliferation. Roughly, in the first and second 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, and in the third stage of chopping, the 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 evenly attached to the bottom of the cell culture dish and proliferated. However, in the case of chopping to 15 μm or less, 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 in the aggregated cells compared to evenly distributed cells, and an increase in external stress caused by the increase in cell density. seems to be
실시예 5. 모유두세포 이식 준비단계Example 5. Preparation step for transplantation of dermal papilla cells
세포 형태에 따른 세포이식 효과를 평가하기 위하여 spheroid 및 single cell을 다음과 같이 각각 준비하였다. In order to evaluate the effect of cell transplantation according to the cell type, spheroids and single cells were prepared as follows, respectively.
실시예 5-1. spheroid (Aggregation cell) 준비 단계Example 5-1. Preparation of spheroid (Aggregation cell)
먼저, T225 flask 면적당(cm2) 대략 1300 내지 1500 개의 cell을 seeding 한 후, 37℃, 5% CO2 배양기에서 3일 간격으로 배지를 교환해 주면서 T225 flask가 세포로 가득 찰 때까지 증식시킨 다음, T225 flask에 들어있던 배지를 제거하고, PBS로 1회 세척하였다. 이 후, 0.25% Trypsin/EDTA을 처리하고 37℃, 5% CO2 배양기에서 5분간 incubation한 다음, Inactivation을 위해 1% FBS가 들어있는 MEM alpha 배지를 넣고 50ml centrifuge tube에 모은 후 2200 rpm에서 5분간 원심분리를 하였다. 상층액은 버리고 하부에 가라앉은 pellet을 충분히 tapping 한 후 적당량의 Expansion Media 2를 넣고 세포수를 확인한 후, 하나의 drop 당 1000 내지 3000cell이 포함되도록 필요한 만큼의 세포현탄액을 Expansion Media 2로 희석하였다. 다음, 온도와 습도를 유지하기 위해 Square dish 바닥에 PBS 40mL을 분주하고, Square dish 덮개에 12채널 Multichannel Pipette으로 5 내지 30ul의 drop을 형성하였다. drop이 형성된 Square dish 모서리를 대각선으로 잡은 후 뒤집어서 최적의 hanging drop 형태를 만들고, 37℃, 5% CO2 배양기에서 1~7일 배양 후 형성된 spheroid를 수집하였다. First, after seeding approximately 1300 to 1500 cells per area (cm 2 ) of the T225 flask, exchanging the medium in an incubator at 37° C., 5% CO 2 every 3 days until the T225 flask is full of cells. , The medium in the T225 flask was removed and washed once with PBS. After that, treated with 0.25% Trypsin/EDTA, incubated at 37°C, 5% CO 2 in an incubator for 5 minutes, and then added MEM alpha medium containing 1% FBS for inactivation and collected in a 50ml centrifuge tube at 2200 rpm for 5 minutes. Centrifugation was performed for minutes. After discarding the supernatant and tapping the pellet that settled at the bottom sufficiently, add an appropriate amount of Expansion Media 2, check the number of cells, and dilute the necessary amount of the cell suspension with Expansion Media 2 to contain 1000 to 3000 cells per drop. . 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 an optimal hanging drop shape, and collect the spheroids formed after culturing for 1 to 7 days in an incubator at 37°C, 5% CO 2 .
실시예 5-2. spheroid 형성을 위한 drop 용량 결정Example 5-2. Determination of drop dose for spheroid formation
바람직한 spheroid 형태를 위한 drop 용량 결정 실험결과를 하기 표 5 및 도 7에 나타내었다.The experimental results for determining the drop dose for the preferred spheroid shape are shown in Table 5 and FIG. 7 below.
Figure PCTKR2021006413-appb-img-000005
Figure PCTKR2021006413-appb-img-000005
도 7에 나타낸 바와 같이, drop 용량을 5 내지 15ul로 제한한 case 1의 경우 cell이 aggregation 되지 않고 넓게 분산되거나 여러 개의 spheroid가 형성되는 양상을 나타내었고, 일정하지 않은 크기의 spheroid 형성으로 표준편차가 매우 큰 것을 확인할 수 있었다. 반면, drop 용량을 16 내지 25ul로 제한한 case 2와 26 내지 30ul로 제한한 case 3는 3일 이내에 일정한 크기의 spheroid가 형성되었다. 다만, case 3의 drop 용량이 컨트롤하기 어려운 점에서, hanging drop 형태로 제조시 drop 형태가 일정하지 않고 흘러내리거나 찌그러진 모양으로 형성되는 문제를 확인할 수 있었다. 그 결과 case 3의 spheroid 크기는 case 2와 동일한 양의 cell임에도 불구하고 약 250um 정도로 case 2 보다 큰 형태로 형성된 것을 확인할 수 있었다. 이로써 case 2에서 형성된 Spheroid가 가장 잘 aggregation 되었으며 hanging drop 제조시 컨트롤도 용이하여 적절한 용량과 cell 수인 것을 확인하였다. 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 multiple 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 where the drop capacity was limited to 16 to 25ul, and case 3 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 dented shape when manufactured as a hanging drop type. 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.
실시예 5-3. seeding 세포밀도 결정Example 5-3. Determination of seeding cell density
효율적인 seeding 세포밀도를 결정하기 위하여 각 case 별로 면적당 세포 수를 다르게 seeding하여 세포증식 속도 및 세포수득률 비교실험을 진행하였고, 그 결과를 하기 표 6 및 도 8에 나타내었다. In order to determine efficient seeding cell density, a cell proliferation rate and cell yield comparison experiment were performed by seeding the number of cells per area differently for each case, and the results are shown in Tables 6 and 8 below.
Figure PCTKR2021006413-appb-img-000006
Figure PCTKR2021006413-appb-img-000006
실시예 5-4. single cell 준비 단계Example 5-4. Single cell preparation step
상기 spheroid 준비 단계에서 수집한 cell 중 일부를 T225 flask 면적당(cm2) 1000 내지 5000개가 되도록 seeding하고, spheroid가 형성되는 1 내지 7일 동안 37℃, 5% CO2 배양기에서 증식시킨 후, T225 flask에 들어있던 배지를 제거하고, PBS로 1회 세척하였다. 이 후, 0.25% Trypsin/EDTA을 처리하고 37℃, 5% CO2 배양기에서 5분간 incubation한 다음, Inactivation을 위해 1% FBS가 들어있는 MEM alpha 배지를 넣고 50ml centrifuge tube에 모은 후 2200 rpm에서 5분간 원심분리를 하였다. 상층액은 버리고 하부에 가라앉은 pellet을 충분히 tapping 한 후 적당량의 NS 배지를 넣고 세포수를 확인하고, 이식되는 모수에 따라 세포현탄액을 희석하였다. 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 an incubator at 37° C., 5% CO 2 for 1 to 7 days for spheroid formation, followed by T225 flask The medium contained in the was removed and washed once with PBS. After that, treated with 0.25% Trypsin/EDTA, incubated at 37°C, 5% CO 2 in an incubator for 5 minutes, and then added MEM alpha medium containing 1% FBS for inactivation and collected in a 50ml centrifuge tube at 2200 rpm for 5 minutes. Centrifugation was performed for minutes. The supernatant was discarded and the pellet that had settled in the lower part 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.
실시예 6. 모유두세포 이식Example 6. Transplantation of dermal papilla cells
실시예 6-1. 모유두세포 수집단계Example 6-1. dermal papilla cell collection step
먼저, 37℃, 5% CO2 배양기에서 3일 내지 7일 이내 형성된 spheroid의 크기와 형태를 확인하고, hanging drop이 형성되어있는 square dish를 뒤집은 후 scraper를 사용하여 아래 방향으로 긁어 한 곳으로 수집하여 petri-dish로 이동시켰다. 이 후, PBS를 분주하고 원형을 그리며 cell을 세척한 후 가운데로 모인 spheroid를 10mL pipette으로 수집하는 세척과정을 3회 반복한 다음 NS배지에 부유시킨 후 syringe에 수집하였다. 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 moved 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.
실시예 6-2. 모유두세포 이식단계Example 6-2. dermal papilla cell transplantation stage
먼저, 하기 표 7에 나타낸 바와 같이, 단순 주사방법에 의한 이식의 경우 피이식자의 두피에서 유분을 제거한 후, ① 이마의 M자 탈모가 형성된 두 부분을 선택하여 한 사이트 당 대략 4 * 4 cm = 16 cm2 의 면적에 5.0 * 106개의 세포를 single cell 상태로 syringe에 충진하고 ② 5.0 * 106개의 세포를 spheroid cell 로 만들어 aggregation된 상태로 syringe에 충진하여 두피에 바로 주사하였고, 주사 후 15일, 30일, 45일, 60일 경과 후 결과를 도 9에 나타내었다. 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 as single cells, 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 scalp, 15 after injection. After days, 30 days, 45 days, and 60 days, the results are shown in FIG. 9 .
[규칙 제91조에 의한 정정 03.06.2021]　
Figure WO-DOC-FIGURE-7
[Correction by Rule 91 03.06.2021]
Figure WO-DOC-FIGURE-7
도 9에 나타낸 바와 같이, 세포를 단순히 주사한 방법에서는 single cell과 spheroid cell 두 가지 세포 모두에서 뚜렷한 차이가 나타나지 않는 것을 확인할 수 있었다. As shown in FIG. 9 , it was confirmed that no clear difference was observed in both single cells and spheroid cells in the method of simply injecting cells.
[규칙 제91조에 의한 정정 03.06.2021]　
다음으로, 펀칭(punching) 후 주사방법에 의한 이식의 경우 피이식자의 두피에서 유분을 제거한 후, 정수리에 탈모가 형성된 부분을 선택하여 모낭이 없는 두피에 FUE (Follcular Unit Extractor) 펀치를 이용하여 내경은 약 0.8 내지 1mm , 깊이는 대략 5 ~ 7 mm 의 천공을 각 사이트 당 60개씩 만든 다음, 한 사이트 당 대략 ① 3 * 3 * 3.14 / 2 cm = 14 cm2 의 면적에 5.0 * 106개의 세포를 single cell 상태로 syringe에 충진하고 ② 5.0 * 106개의 세포를 spheroid cell 로 만들어 aggregation된 상태로 syringe에 충진하여 천공을 낸 부분에 바로 주사하였고(표 8), 주사 후 10일, 30일, 40일, 50일, 60일 경과 후 결과를 도 10에 나타내었다.(도13 참조).[Correction by Rule 91 03.06.2021]
Next, in the case of implantation by the injection method after punching, after removing the 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 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 into the syringe as a single cell, and ② 5.0 * 10 6 cells were made into spheroid cells, filled in the syringe in an aggregated state, and injected directly into the punctured area (Table 8). 10 days, 30 days after injection, The results after 40 days, 50 days, and 60 days are shown in FIG. 10 (see FIG. 13).
[규칙 제91조에 의한 정정 03.06.2021]　
Figure WO-DOC-FIGURE-8
[Correction by Rule 91 03.06.2021]
Figure WO-DOC-FIGURE-8
도 10에 나타낸 바와 같이, 펀칭 후 세포를 주사한 방법에서는 single cell과 spheroid cell 두 가지 세포에서 모발이 올라오는 것을 확인할 수 있었다. 특히 모낭이 없는 부분에 천공을 내고 세포를 주사했기 때문에 기존에 모발이 나고 빠진 부위가 아니며 새롭게 올라온 모발로 확인된다. 또한 초기 이미 있던 모발의 경우 가늘고 힘이 없는 상태이지만 세포를 주사하고 60일 정도 후에 관찰했을 때 모발에 힘이 있고 굵어진 것으로 보아 세포가 분비하는 영양분에 의해 세포를 주사한 위치뿐만 아니라 주변의 모발에도 영향을 미치는 것으로 확인되었다.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 is newly grown hair because a hole is made in the area 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 the injection of the cells, the hair appears to be strong and thick. was also found to have an effect.
실시예 6-3. syringe needle gauge 결정Example 6-3. syringe needle gauge determination
5mL syringe로 수집한 세포를 needle gauge가 24G 내지 31G인 needle을 사용하여 상기 실시예 6-2의 이식 방법으로 수득율을 확인하기 위하여, 실시예 5-2 결과에 따라 208.5±7.71 크기의 spheroid를 수집하였다. 도 11에 나타낸 바와 같이, 31G로 수집한 spheroid의 경우 퍼져있거나 찌그러진 형태로 표준편차가 매우 큰 것을 확인할 수 있었다. 또한 syringe needle을 spheroid가 통과하면서 여러개로 나눠지거나 needle 내부에 정체되는 현상이 발행하여 수득율이 현저하게 낮게 나타났다. 26G 또한 spheroid의 크기가 일정하지 않고 기존 spheroid 에서 떨어져 나온 spheroid를 여러 개 확인할 수 있었으며, 가장자리 형태 또한 불규칙적이고 찢어지거나 aggregation이 풀린 모습이 확인되었다. 반면 24G를 사용하여 수집한 spheroid는 수집하기 전의 모습을 유지하였으며 크기가 일정하고 수득율 또한 높은 것을 확인할 수 있었다. 이는 spheroid cell 직경보다 내경이 작은 gauge에서는 spheroid cell 이 syringe 외부로 나오는 과정에서 부스러지는 현상이 발생되며, 반면 24G에서는 cell의 변형 없이 syringe 외부로 나오는 것을 확인하였고, 24G보다 더 작은 수치의 Gauge를 사용할 경우 needle이 굵어져 피이식자의 심한 고통을 유발하게 되는 문제가 있었다. 이로써 24G를 사용하여 수집하는 것이 spheroid형태를 가장 우수하게 유지하고 수득율 또한 높으며 환자의 두피에 최소한의 흉터로 세포를 이식할 수 있는 최적의 gauge임을 확인하였다. In order to check the yield by the transplantation method of Example 6-2 using a needle having a needle gauge of 24G to 31G for cells collected with a 5mL syringe, spheroids of 208.5±7.71 size were collected according to the results 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. In addition, 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. On the other hand, in 24G, it was confirmed that the cell came out of the syringe without deformation. 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 best gauge to maintain the spheroid shape the most and to have a high yield, and to transplant cells with minimal scarring on the patient's scalp.

Claims (9)

  1. (A) 대량증식된 모유두세포를 준비하는 단계;(A) preparing the mass-proliferated dermal papilla cells;
    (B) 모유두세포를 syringe에 수집하는 단계;(B) collecting dermal papilla cells in a syringe;
    (C) 모유두세포를 피이식자의 두피에 이식하는 단계;를 포함하는 모유두세포의 이식방법으로, (C) transplanting the dermal papilla cells to the scalp of the recipient;
    상기 (C) 모유두세포를 피이식자의 두피에 이식하는 단계는 피이식자의 두피에 FUE(Follcular Unit Extractor) 펀치를 이용하여 천공을 만든 후 syringe에 충진된 모유두세포를 이식하는 것을 특징으로 하는 모유두세포의 이식방법.The step of (C) transplanting the dermal papilla cells to 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.
  2. 제1항에 있어서, 상기 (A) 대량증식된 모유두세포를 준비하는 단계에서 모유두세포는 spheroid 또는 single cell인 것을 특징으로 하는 모유두세포의 이식방법.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. 제2항에 있어서, 상기 spheroid는 3. The method of claim 2, wherein the spheroid is
    (a1) flask에 cell을 seeding한 후 배양기에서 3일 간격으로 배지를 교환해 주면서 세포를 증식시키는 단계;(a1) proliferating the cells while exchanging the medium every 3 days in an incubator after seeding the cells in the flask;
    (a2) flask에 들어있던 배지를 제거하고, PBS로 세척하는 단계;(a2) removing the medium in the flask and washing with PBS;
    (a3) 0.25% Trypsin/EDTA 처리 후 배양기에서 incubation한 다음, 1% FBS가 들어있는 MEM alpha 배지를 넣고 원심분리 하는 단계;(a3) after treatment with 0.25% Trypsin/EDTA, incubated in an incubator, and then centrifuged into MEM alpha medium containing 1% FBS;
    (a4) 하부에 가라앉은 pellet을 tapping 한 후 Expansion Media 2를 넣고 세포수를 확인하여, 하나의 drop 당 1000 내지 3000 cell이 포함되도록 세포현탄액을 Expansion Media 2로 희석하는 단계;(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) square dish 바닥에 PBS 40mL을 분주하고, square dish 덮개에 12채널 Multichannel Pipette으로 5 내지 30ul의 drop을 형성하는 단계;(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) drop이 형성된 Square dish 모서리를 대각선으로 잡은 후 뒤집어서 hanging drop 형태를 만들고, 배양기에서 1 내지 7일 배양 후 형성된 spheroid를 수집하는 단계;를 포함하는 것을 특징으로 하는 모유두세포의 이식방법.(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 collecting the spheroid formed after 1 to 7 days of incubation in an incubator; dermal papilla cell transplantation method comprising a.
  4. 제3항에 있어서, (a5) 단계의 drop은 16 내지 25ul인 것을 특징으로 하는 모유두세포의 이식방법.The method of claim 3, wherein the drop in step (a5) is 16 to 25ul.
  5. 제1항에 있어서, (C) 모유두세포를 피이식자의 두피에 이식하는 단계는 (c1) FUE (Follcular Unit Extractor) 펀치를 이용하여 내경이 0.8 내지 1mm 인 천공을 만드는 단계;를 포함하는 것을 특징으로 하는 모유두세포의 이식방법.The method according to claim 1, wherein (C) transplanting the dermal papilla cells into the scalp of the recipient comprises (c1) making a hole with an inner diameter of 0.8 to 1 mm using a Follcular Unit Extractor (FUE) punch; A method of transplantation of dermal papilla cells.
  6. 제5항에 있어서, 상기 천공은 깊이가 5 내지 7 mm인 것을 특징으로 하는 모유두세포의 이식방법.The method of claim 5, wherein the perforation has a depth of 5 to 7 mm.
  7. 제5항에 있어서, (c2) 5.0 * 106개의 spheroid cell을 syringe에 충진하여 천공에 주사하는 단계;를 더 포함하는 것을 특징으로 하는 모유두세포의 이식방법.[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 further comprises:
  8. 제1항에 있어서, 상기 syringe는 needle gauge의 내경이 (B)단계에서 수집된 모유두세포의 직경보다 큰 것을 특징으로 하는 모유두세포의 이식방법.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).
  9. 제8항에 있어서, 상기 syringe는 needle gauge가 24G인 것을 특징으로 하는 모유두세포의 이식방법.The method of claim 8, wherein the syringe has a needle gauge of 24G.
PCT/KR2021/006413 2020-08-12 2021-05-24 Method for transplanting dermal papilla cells through perforation WO2022035023A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020200100977A KR102254911B1 (en) 2020-08-12 2020-08-12 Method for transplanting dermal papilla cells through perforation
KR10-2020-0100977 2020-08-12

Publications (1)

Publication Number Publication Date
WO2022035023A1 true WO2022035023A1 (en) 2022-02-17

Family

ID=76152544

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2021/006413 WO2022035023A1 (en) 2020-08-12 2021-05-24 Method for transplanting dermal papilla cells through perforation

Country Status (2)

Country Link
KR (1) KR102254911B1 (en)
WO (1) WO2022035023A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102663430B1 (en) 2021-08-18 2024-05-17 강다윗 Composition for improving hair loss containing personalized dermal papilla cell culture medium

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07274950A (en) * 1994-04-11 1995-10-24 Res Dev Corp Of Japan Long-term subculture of hair papilla cell
JPH11180878A (en) * 1997-12-17 1999-07-06 Toyobo Co Ltd Trichogenous inducer and trichogenic method
KR20060059557A (en) * 2004-11-29 2006-06-02 박정극 Method for preparation of dermal papilla tissue inducing hair folicle formation
KR20120096185A (en) * 2011-02-22 2012-08-30 김영실 Alopecia(hair loss) treatment and hair growth using fat stem cell

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102454189B1 (en) 2018-05-04 2022-10-14 연세대학교 산학협력단 Method for preparing dermal papilla cell and cell spheroid under hypoxic culture condition
KR20200025210A (en) 2018-08-29 2020-03-10 주식회사 스템모어 Composition for culturing dermal papilla cells and the method for culturing dermal papilla cells using the same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07274950A (en) * 1994-04-11 1995-10-24 Res Dev Corp Of Japan Long-term subculture of hair papilla cell
JPH11180878A (en) * 1997-12-17 1999-07-06 Toyobo Co Ltd Trichogenous inducer and trichogenic method
KR20060059557A (en) * 2004-11-29 2006-06-02 박정극 Method for preparation of dermal papilla tissue inducing hair folicle formation
KR20120096185A (en) * 2011-02-22 2012-08-30 김영실 Alopecia(hair loss) treatment and hair growth using fat stem cell

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TOPOUZI HELENA, LOGAN NIALL J., WILLIAMS GREG, HIGGINS CLAIRE A.: "Methods for the isolation and 3D culture of dermal papilla cells from human hair follicles", EXPERIMENTAL DERMATOLOGY, BLACKWELL MUNSGAARD, COPENHAGEN; DK, vol. 26, no. 6, 1 June 2017 (2017-06-01), COPENHAGEN; DK , pages 491 - 496, XP055900115, ISSN: 0906-6705, DOI: 10.1111/exd.13368 *

Also Published As

Publication number Publication date
KR102254911B1 (en) 2021-05-24

Similar Documents

Publication Publication Date Title
CN104450611B (en) A kind of primary isolation and culture method of human amnion mesenchymal stem cell
JP5508715B2 (en) Method for isolating, proliferating and differentiating hair follicle stem cells and composition for treating buns
TW201827592A (en) A method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord and a cell culture medium for isolating mesenchymal stem cells
US20090305406A1 (en) Method of cultivation of human mesenchymal stem cells, particularly for the treatment of non-healing fractures, and bioreactor for carrying out this cultivation method
WO2021256663A1 (en) Method for isolating and mass proliferating dermal papilla cells derived from scalp tissue
WO2022035023A1 (en) Method for transplanting dermal papilla cells through perforation
WO2022245086A1 (en) Novel method for isolating and culturing dermal papilla cells
US20200149005A1 (en) The method of autologous primary hair follicles preparation in 3d culture
Chen et al. A cell line derived from tilapia ovary
JP2001302520A (en) Method for regenerating hair by cultured hair papilla cell
CN112626006B (en) Method for separating and culturing villous goat hair follicle hair papilla cells
CN116024159A (en) Method for constructing murine skin organoids
CN117157389A (en) Preparation method and application of mesenchymal stem cells of hair follicle
CN108359633A (en) A kind of beaver rabbit dermis of skin hair papilla cell isolated culture method
KR20220116805A (en) Method for transplanting dermal papilla cells through perforation
CN113564109A (en) Preparation method of menstrual blood mesenchymal stem cells
CN114480268B (en) Preparation method of human umbilical cord mesenchymal stem cells
RU2695364C1 (en) Method of treating non-scarring alopecia
WO2024010157A1 (en) Composition containing hair follicle cells
RU2681854C1 (en) Method for the treatment of nonscarring alopecia
CN114736862B (en) Hematopoietic stem cell culture medium and hematopoietic stem cell in-vitro amplification method
CN112300982B (en) Hair follicle microarray co-culture system and application thereof in medicine for treating pathological alopecia
CN115282166B (en) Application of pilose antler stem cells and exosomes thereof in preparing anti-scar products
US20240066066A1 (en) Method for treating alopecia with stem cell exosome
WO2022114727A1 (en) Culture medium composition for establishing or culturing keratinocytes comprising calcium, epideral growth factor, and albumin

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21856031

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21856031

Country of ref document: EP

Kind code of ref document: A1