KR101178032B1 - Hair grwoth material and product using fat stem cell disruped extract and manufacturing method of it - Google Patents
Hair grwoth material and product using fat stem cell disruped extract and manufacturing method of it Download PDFInfo
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- KR101178032B1 KR101178032B1 KR1020120042498A KR20120042498A KR101178032B1 KR 101178032 B1 KR101178032 B1 KR 101178032B1 KR 1020120042498 A KR1020120042498 A KR 1020120042498A KR 20120042498 A KR20120042498 A KR 20120042498A KR 101178032 B1 KR101178032 B1 KR 101178032B1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q7/00—Preparations for affecting hair growth
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
Abstract
Description
The present invention relates to a hair growth preparation and a method for producing the same, and more particularly, to a hair growth preparation using a crushed extract obtained by crushing fat stem cells and a preparation method thereof.
Hair is divided into anagen in which hair grows, catagen in which growth stops and hairball shrinks, and metabolism slows, and telogen in which hair follicles are pushed upward and hair falls out, As new hair is produced in the same place, the old hair falls out and has a hair cycle that converts into a growth phase.
There are about 100,000 hairs on the scalp of the human body, and the condition that occurs when the number of hairs that should be normally lacked or the number thereof decreases is called hair loss. Such hair loss is caused by various causes, and the condition is changed by factors such as stress, pollution, seasonal changes, diseases, and chemical procedures in addition to genetic factors.
Stem cells are cells that can multiply themselves into undifferentiated states and multiply into cells of other tissues. It has been shown to exist in many tissues of our body and can be divided into embryonic stem cells and adult stem cells. Adult stem cells are easier to obtain than embryonic stem cells, and many studies using them are underway. The first known adult stem cells are bone marrow-derived stem cells, and the most research is being conducted. Since then, it has been found that adult stem cells exist not only in umbilical cord blood but also in peripheral blood, placenta, skin, nerve tissue, fat, and muscle. Bone marrow-derived stem cells, which have been studied the most, have pain and morbidity during the procedure for collecting stem cells, and show a number of limitations in clinical applications due to low cell yield. Many studies have been conducted to extract and clinically apply. However, in order to use the stem cells themselves as a therapeutic agent, only autologous stem cells should be used due to the autoimmune rejection reaction, and it is difficult to use them for unspecified majority. To overcome this difficulty, stem cell culture is used as an alternative, but there is also much debate about the safety of other additives such as bovine serum and antibiotics added to the culture medium.
The present invention provides a hair growth agent using a crushed extract of fat stem cells and a method for producing the same.
Hair growth preparation according to the present invention has a configuration that is used by using adipose stem cell crushed extract. Adipose stem cell crushing extract consists of crushing stem cells using an ultrasonic crusher or a French press, bead millimg, a reagent that destroys stem cell membranes, and centrifugation to obtain the extract.
In another aspect, the present invention provides a hair growth agent and method for producing the same as an active ingredient stem cell crushing extract.
The hair growth preparation according to the present invention and a method for producing the same are composed of using adipose stem cell disruption extract, the adipose stem cell disruption extract is collected after culturing the stem cells, ultrasonic crusher and French press (French press), bead milling (bead miling), a method of crushing using a reagent that destroys the cell membrane.
The crushed fat stem cell crushed extract is separated only from the unique component concentrate of the stem cells, and has excellent regeneration effect of various tissues, and it can show the effects of hair loss prevention and hair growth by activating the hair of the resting or degenerative phase as the growth hair. .
In particular, the composition of the present invention is free from the legal or ethical problems that may occur when using stem cells by using the crushed extract of stem cells rather than stem cells, the specificity of the individual, for example, immune rejection in the human body It is possible to expect the effect that the restriction due to and the like does not occur. In addition, it does not contain additives such as antibiotics and bovine serum, which can prevent side effects.
1 is a photograph of stem cells crushed using an ultrasonic crusher.
Figure 2 is a scalp slope treated hair loss using adipose stem cell extract.
Hereinafter, the present invention will be described in more detail.
The hair growth preparation of the present invention is composed of adipose stem cell disruption extract, and the adipose stem cell disruption extract is obtained by separating the stem cells from the fat, and then obtained by separating the cells after culturing, the cells are obtained by crushing the cells Refers to the intrinsic component concentrate of fat stem cells.
Stem cells for obtaining the crushed extract refers to adult stem cells, and performing liposuction in plastic surgery, dermatology, etc. to separate the stem cells from the resulting fat. At this time, the fat is collected and separated in a sterile state, to remove impurities such as the two-methic solution and blood, and to separate only the pure fat tissue to separate the stem cells from the fat.
In order to achieve the object of the present invention, the present invention comprises the steps of (a) separating the stem cells from fat; (b) culturing the isolated adipose stem cells; (C) crushing the fat stem cells to separate the crushed extract; and comprises a method of manufacturing each step.
Step (a) is a step of separating fat stem cells from fat obtained through liposuction, such as plastic surgery or dermatology to centrifuged inhaled adipose tissue to obtain only pure adipose tissue. At this time, the obtained adipose tissue is washed with physiological saline and the like, and the collagenase solution prepared by mixing collagenase (SIGMA, SERVA) with physiological saline is mixed 1: 1 with adipose tissue. The mixed adipose tissue was enzymatically treated at 37 ° C. for 20-30 minutes, followed by centrifugation for 600G-1000G for 3-6 minutes to separate the layers into an oil layer, a fat layer and a fat stem cell layer. The fat stem cells are formed in the form of pellets. In this case, collagenase is commonly used SIGMA, but may use non-toxic SERVA. If SIGMA is used, the toxic neutralization process should be carried out separately. The separated fat stem cells are washed 2-3 times with 300-500G 3 minutes using physiological saline or P-phosphate buffered saline (PBS) to remove residual fat and oil. In order to remove cellular residues after washing 2-3 times, a filter having a thickness of 100 μm may be used, but the present invention is not limited thereto, and the process may be omitted.
Step (b) is a step of culturing the isolated adipose stem cells. Cultured fat stem cells isolated from the above is used to culture DMEM (dulbecco's modified eagle medium) medium is commonly used, added 10% bovine serum (FBS; fetal bovine serum) and penicillin and streptomycin as antibiotics Is added at 100 units / ml, 100 μg / ml. Human serum may be used instead of bovine serum, and other antibiotics or other nutrients may be added. The culture is carried out at 37 ° C. in a 5CO 2 environment and passaged when the cell density is 80-90%.
The step (c) is to separate the crushed extract used in the hair growth preparation of the present invention, the present invention is obtained and used in the step (b). The stem cell disruption extract can be separated at each subculture, and means that only the intrinsic components of the cells from which the cells have been removed after breaking the cell membranes by crushing the stem cells are removed. Obtained when the density of cells in step (a) is 80-90% or more, and obtained when passaged in 10cm plate (plate). Extracts can be obtained from 5 cm plates, 3 cm plates, 24 well plates and 12 well plates smaller than 10 cm plates, but the number of stem cells is small. The amount of extract is reduced. In step (b), when the density of the stem cells is 80-90%, the stem cells are removed from the plate using 0.25% trypsin-EDTA. Thus detached stem cells can be washed 2-3 times using physiological saline or phosphate buffered saline (PBS; phosphate buffer saline) to obtain only pure stem cells. The stem cells thus obtained are crushed to obtain crushed extracts. At this time, the crushed extract may be a conventional cell crushing method such as an ultrasonic crusher, bead milling, french press, a reagent, or the like. Other shredding methods that can break cell membranes are also available.
Separating the crushed extract of the (c) means the process of collecting the supernatant from the stem cell is broken cell membrane and the extract is removed in the above process to remove the stem cell residue. When the cell membranes of the stem cells are broken using conventional methods such as the ultrasonic crusher, bead milling, french press, and reagents, the cell membranes can be broken. The intrinsic component concentrates with are mixed together. In this state, centrifugation is performed to remove the stem cell residue, thereby collecting the supernatant in which only the components of the stem cell are concentrated. At this time, the stem cell residue may be removed using a filter.
Thus, only the supernatant obtained through the centrifugation process is called adipose stem cell crushed extract which is the active ingredient of the present invention. This stem cell lysate extract has excellent regenerative effect, thereby activating the resting and degenerative hair cells as the growing hair cells, thus preventing hair loss and hair growth.
In addition, the stem cell disruption extract thus extracted has no side effects such as legal or ethical problems and immune rejection reactions that may occur when stem cells are used directly. You can avoid the risk of side effects.
This configuration will be described in more detail with reference to the following examples. However, the following examples are merely illustrative for easy understanding of the present invention, and the contents of the present invention are not limited by the examples.
Example 1 Isolation of Adipose Stem Cells from Adipose Tissue
After obtaining consent from patients who underwent liposuction in plastic surgery or dermatology, sterile adipose tissue was collected. The collected fat is transferred to a 10cc syringe and centrifuged for 2-3 minutes at 3000-3500rpm. This centrifugation removes impurities such as tomecent solution, blood and oil, and then washes again with physiological saline to collect pure adipose tissue and place in a 10cc syringe. The collected adipose tissue was mixed 1: 1 with 0.66% to 0.1% collagenase mixed with physiological saline and enzymatically treated at 37 ° C. for 20-30 minutes. After centrifugation at 800G again for 5 minutes, the adipocyte layer and the adipocyte layer were separated. After removing the adipocyte layer in the upper layer, only the adipocyte layer in the lower layer was separated. The isolated fat stem cell layer was washed 2-3 times using saline solution, Hartman's solution, PBS, etc., and then filtered using a 100 μm filter to remove cellular residues to separate fat stem cells from fat. .
Example 2 Adipose Stem Cell Culture
Adipose stem cells isolated above were cultured. The cell culture process used a commonly used cell culture method.
The medium used for cell culture is a commonly used Dube (Dubecco's modified eagle medium), which contains 10% bovine serum (FBS; fetal bovin serum) and penicillin and streptomycin at 100 units / ml, 100 µg / ml. Was added as antibiotic. In addition to DMEM, any medium used for cell culture may be used, and additional nutrients such as vitamins may be added. The culture was incubated in 37 ℃, 5CO 2 environment. Adipose stem cells isolated from adipose tissue are separated with blood and cultured together with blood during primary culture. The lysis buffer (lysis buffer) is used to remove blood and culture. After 1 day of primary culture, phosphate buffered saline was used to remove fat stem cells and blood cells that did not adhere to the plate. Blood cells that were not completely removed were removed by trypsinization during subculture. Subcultured when the cell density was 80-90%, it was confirmed that the adipose stem cells are cultured by checking the morphology of the fibroblasts.
Example 3 Isolation of Adipose Stem Cell Fracture Extract
The method of separating the crushed extract by crushing fat stem cells can be divided into physical and chemical methods. Physical methods include ultrasonic crushers, bead milling, french presses, etc., and chemical methods are used for crushing using reagents.
(1) Crushing using ultrasonic crusher
After culturing stem cells through trypsinization, the cells were detached, and the cells were washed 2-3 times with physiological saline to prevent mixing of other impurities other than stem cells. The cells detached from one 10 cm plate were mixed with physiological saline each 10 ml and crushed using an ultrasonic crusher. At this time, the amount of physiological saline to be mixed is used in the same amount as the medium used for cell culture. Crushing is possible at a time of 10-40 seconds per 10ml, and the time increases as the amount of crushing increases, but the crushing time does not exceed a maximum of 5 minutes. At this time, it is crushed by an ultrasonic wave of 10-30kHz.
(2) Crushing using bead milling
In the method using bead milling, stem cells cultured in the same manner as above are removed through trypsinization, and then washed 2-3 times with physiological saline. Stem cells thus separated are also mixed with 10ml saline per 10cm plate, 0.20mm ~ 0.30mm glass bead (bead), the cell at a rotation speed of 6,500rpm at 5,500rpm for 2-4 minutes Will be broken.
(3) Crushing using french press
The method using the french press also removes the stem cells cultured in the same manner as above by trypsinization, and then washes 2-3 times with physiological saline. The isolated stem cells are used in a mixed state with saline, and the cells are crushed at a pressure of 14,000 ~ 16,000pci. At this time, the pressure may increase depending on the amount of stem cells.
(4) crushing using reagents
Disintegration using a reagent is used to obtain only stem cells through centrifugation at 1,100 rpm for 3 minutes after detaching the cells through trypsinization. In this case, the reagent used may include a lysis buffer, a lysozyme, an SDS solution (20% sodium dodesylsulfate), and the like. 50 mM Hepes, pH 7.4, 150 mM sodium chloride (NaCl), 1% deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonylfloon for lysis buffer Ride (PMSF) and 1 μg / ml aprotinin were mixed and treated for 30 minutes at 4 ° C. for crushing. Lysozyme is crushed in an amount of 5 mg per ml. In the case of SDS solution, the cells are crushed by mixing with stem cells and reacting in a 56 ° C. water bath for 2 hours. Other reagents may also be used.
Example 4 Separation Process of Shredded Extract
When the stem cells are crushed using other methods such as the ultrasonic crusher, bead milling, french press, reagents and the like, the crushed cell residues are mixed with the extract. do. Extracts obtained by physical methods such as ultrasonic crushers, bead milling, and french presses are mixed with physiological saline and extracts and cell residues. The extracts pulverized by physical methods were centrifuged at 1000G for 3 minutes to remove the crushed cell residues, and only the supernatant was collected. In addition to centrifugation, cell residues may be used by crushing only the extract using a filter. In the case of extracts chemically crushed using a reagent, it is difficult to completely separate the crushed reagents and the extracts in a form in which the extracts and the reagents are mixed together.
Example 5 Hair Proliferation Preparation Method
The hair growth agent of the present invention is not limited to one form but may be prepared in various forms. When the hair growth preparation is used as a pharmaceutical composition or a cosmetic composition, it is prepared in the form of liquids such as skins, toners, essences, sprays, creams such as creams, lotions, shampoos, and gels. These compositions can use 1-100% of the adipose stem cell extract, and additionally amino acids, growth factors such as cysteine for preventing hair loss, hair growth promoting iodine, and arginine for promoting hair growth, such as vitamin A, Chemical ingredients such as vitamin D, vitamin E, vitamin H, and vitamin C, and other additives that are effective in preventing hair loss and hair growth, can be added and can be used from 1 to 99%. Parabens such as methylparaben and ethylparaben may be used as preservatives for storage at room temperature, and do not exceed the allowable concentration of KFDA using those commonly used. In addition, additives such as emulsifiers and thickeners are used to control the concentration of the product.
For example, stearic acid to improve the oil feeling, betaine, glycerin, propylene glycol for moisturizing feeling and the like is added. Additives such as cyclopentasiloxane, cyclomethicone, and hydrogenated polyderate may be used as the softening agent, and as a thickener, pentaerythritol tetrahexanoate, sodupolyacrylate, V picopolymer, hydrogenated polyisobu A configuration in which an additive such as ten is used is also possible. When the form is a liquid, additives such as lotion, distilled water and mineral water may be used as the solvent.
The hair growth agent can be used as a method of applying to the scalp, it can also be used as a method of injection with injection.
Claims (1)
A second step of extracting the adipose stem cell debris extract by breaking the adipose stem cell membrane using an ultrasonic crusher, a french press, or a bead milling of the detached adipose stem cells;
A third step of centrifuging the fat stem cell crushed extract for 3 minutes at 1000 G to remove crushed residues;
A fourth step of formulating the fat stem cell crushed extract in the form of liquid, cream or gel: Method of producing a hair growth preparation using a fat stem cell crushed extract, characterized in that comprises a
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101838220B1 (en) * | 2017-05-22 | 2018-03-13 | (주) 굿모닝 바이오 | Container and method for extracting adipose stem cells |
| WO2020009147A1 (en) * | 2018-07-04 | 2020-01-09 | 正典 佐伯 | Stem cell filtrate formulation and method for preparing same |
| EP3756677A4 (en) * | 2018-02-23 | 2021-10-20 | Meis Technology Inc. | Erectile dysfunction therapeutic agent |
| KR20230040024A (en) | 2021-09-15 | 2023-03-22 | 주식회사 티스템 | Pharmaceutical composition for improving cognitive or memory ability comprising membrane free stem cell extract |
| KR20230040025A (en) | 2021-09-15 | 2023-03-22 | 주식회사 티스템 | Functional food composition for improving cognitive or memory ability comprising membrane free stem cell extract |
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| US20050048034A1 (en) | 2001-12-07 | 2005-03-03 | Fraser John K. | Methods of using regenerative cells to promote wound healing |
| KR100955212B1 (en) | 2006-01-27 | 2010-04-29 | (주)프로스테믹스 | Mass producing method of growth factor using adipose derived adult stem cells |
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2012
- 2012-04-24 KR KR1020120042498A patent/KR101178032B1/en active IP Right Grant
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20050048034A1 (en) | 2001-12-07 | 2005-03-03 | Fraser John K. | Methods of using regenerative cells to promote wound healing |
| KR100955212B1 (en) | 2006-01-27 | 2010-04-29 | (주)프로스테믹스 | Mass producing method of growth factor using adipose derived adult stem cells |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101838220B1 (en) * | 2017-05-22 | 2018-03-13 | (주) 굿모닝 바이오 | Container and method for extracting adipose stem cells |
| EP3756677A4 (en) * | 2018-02-23 | 2021-10-20 | Meis Technology Inc. | Erectile dysfunction therapeutic agent |
| WO2020009147A1 (en) * | 2018-07-04 | 2020-01-09 | 正典 佐伯 | Stem cell filtrate formulation and method for preparing same |
| CN112135606A (en) * | 2018-07-04 | 2020-12-25 | 佐伯正典 | Stem cell filtrate preparation and method for producing the same |
| JPWO2020009147A1 (en) * | 2018-07-04 | 2021-03-11 | 正典 佐伯 | Stem cell filtrate preparation and its preparation method |
| JP6999196B2 (en) | 2018-07-04 | 2022-01-18 | 正典 佐伯 | Stem cell filtrate preparation and its preparation method |
| EP3785721A4 (en) * | 2018-07-04 | 2022-03-02 | Saeki, Masanori | Stem cell filtrate formulation and method for preparing same |
| TWI823964B (en) * | 2018-07-04 | 2023-12-01 | 佐伯正典 | Stem cell filtrate preparation and preparation method thereof |
| KR20230040024A (en) | 2021-09-15 | 2023-03-22 | 주식회사 티스템 | Pharmaceutical composition for improving cognitive or memory ability comprising membrane free stem cell extract |
| KR20230040025A (en) | 2021-09-15 | 2023-03-22 | 주식회사 티스템 | Functional food composition for improving cognitive or memory ability comprising membrane free stem cell extract |
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