CN112280732B - Hair follicle outer root sheath suspension and preparation method thereof - Google Patents
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- 239000000725 suspension Substances 0.000 title claims abstract description 33
- 210000003780 hair follicle Anatomy 0.000 title claims abstract description 21
- 210000004918 root sheath Anatomy 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 29
- 239000002244 precipitate Substances 0.000 claims abstract description 22
- 239000006285 cell suspension Substances 0.000 claims abstract description 18
- 230000003325 follicular Effects 0.000 claims abstract description 16
- 239000000706 filtrate Substances 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 14
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- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 13
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- 239000002609 medium Substances 0.000 claims abstract description 12
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 9
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- 238000011534 incubation Methods 0.000 claims description 5
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 4
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- 238000005406 washing Methods 0.000 claims description 4
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 claims 1
- 230000003115 biocidal effect Effects 0.000 claims 1
- 210000002752 melanocyte Anatomy 0.000 abstract description 11
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
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- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 2
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- 230000008859 change Effects 0.000 description 2
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- 238000000338 in vitro Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
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- 229960004393 lidocaine hydrochloride Drugs 0.000 description 2
- YECIFGHRMFEPJK-UHFFFAOYSA-N lidocaine hydrochloride monohydrate Chemical compound O.[Cl-].CC[NH+](CC)CC(=O)NC1=C(C)C=CC=C1C YECIFGHRMFEPJK-UHFFFAOYSA-N 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000037380 skin damage Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
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- 208000034826 Genetic Predisposition to Disease Diseases 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
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- 241000519995 Stachys sylvatica Species 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 238000001126 phototherapy Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 206010040872 skin infection Diseases 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
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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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0626—Melanocytes
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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
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a preparation method of a hair follicle outer root sheath suspension, which comprises the following steps: extracting follicular units and incubating; scraping the outer root sheath tissue of the hair follicle on the follicular unit, filtering to obtain a filtrate 1 and a cell suspension, centrifuging the cell suspension to obtain a precipitate, and blowing the precipitate to resuspension to obtain a suspension 1; adding the filter 1 into an EDTA solution containing trypsin for digestion, then adding a high-sugar DMEM medium containing antibiotics into the solution, filtering, obtaining a filter 2 and a cell suspension, centrifuging the cell suspension, collecting precipitate and blowing to resuspension to obtain a suspension 2; taking a filter material 2, and repeating the above operation to obtain a suspension 3; mixing suspensions 1,2 and 3, centrifuging to obtain cell precipitate, re-suspending, filtering to obtain filtrate, centrifuging, collecting precipitate, and re-suspending the precipitate. The method can effectively solve the problems of long culture time and safety of the solution used in the culture of the existing melanocytes.
Description
Technical Field
The invention relates to the technical field of hair follicle external root sheath suspension, in particular to hair follicle external root sheath suspension and a preparation method thereof.
Background
Vitiligo is a pigment skin disease caused by acquired melanocyte deficiency, and is clinically characterized by single or multiple white spots on skin. The incidence rate of vitiligo in China is about 0.09% -2.7%, and the incidence rate is in an increasing trend in recent years. The vitiligo does not endanger the life of the patient, but the lasting disease condition is not healed, and repeated attacks cause huge psychological burden to the patient, obstruct the interpersonal interaction of the patient, and even cause psychological diseases.
The etiology of vitiligo is not clear, and is thought to be related to several aspects: 1. genetic susceptibility, a certain family genetic aggregation phenomenon. 2. Autoimmune, often accompanied by other autoimmune diseases. 3. Melanocytes self-destruct, and the melanocytes are missing in the skin lesion area of vitiligo patients in different conditions. 4. Environmental factors, prolonged exposure to substances containing phenols can lead to increased incidence of vitiligo.
The treatment method of the vitiligo is imperfect, the vitiligo can be treated only by means of medicines, phototherapy and the like in the disease progression period, surgical means can be properly used in the stable period, the most commonly used surgical treatment mode at present is an autologous skin grafting operation, although skin grafting can make skin damage areas be multi-colored, the treatment area is completely limited by the size of the skin supply area, and the treatment can be realized only by multiple skin grafting on patients with large-area skin damage, thus causing great burden to the body and mind of the patients and economy.
At present, autologous melanocytes are used for transplantation treatment, but the melanocytes in the prior art can be used after being obtained and cultured, a large amount of substances such as growth factors, serum and the like are needed in the culture process, and the substances have great potential safety hazards when being used for a human body, and the melanocyte culture time is long, so that the treatment period is prolonged.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a preparation method of a hair follicle outer root sheath mixed solution, which can effectively solve the problems of long culture time and safety of a solution used for culturing existing melanocytes.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
A method for preparing a hair follicle outer root sheath suspension, comprising the following steps:
(1) Extracting follicular units of the head of the patient;
(2) Washing the follicular unit obtained in step (1) with a high sugar DMEM medium containing antibiotics;
(3) Placing the follicular unit cleaned in the step (2) into a high-sugar DMEM medium containing type II collagenase for incubation;
(4) Scraping the outer root sheath tissue of the hair follicle on the follicular unit after incubation in the step (3), filtering to obtain a filtrate 1 and a cell suspension, centrifuging the cell suspension, collecting a precipitate, adding a high-sugar DMEM culture medium containing antibiotics into the precipitate, and blowing and resuspension to obtain a suspension 1;
(5) Adding the filter 1 in the step (4) into an EDTA solution containing trypsin for digestion, then adding a high-sugar DMEM culture medium containing antibiotics into the EDTA solution to dilute trypsin, filtering, obtaining a filter 2 and a cell suspension, centrifuging the cell suspension, collecting a precipitate, adding the high-sugar DMEM culture medium containing antibiotics into the precipitate, and blowing and resuspension to obtain a suspension 2;
(6) Taking the filtrate 2 in the step (5), and repeating the operation in the step (5) to obtain a suspension 3;
(7) And (3) mixing the suspensions 1, 2 and 3 in the step (4), the step (5) and the step (6), centrifuging at a rotation speed of 2000r/min to obtain cell sediment, re-suspending the cell sediment, filtering by using a cell sieve to obtain filtrate, further centrifuging the filtrate at a rotation speed of 2000r/min for 5min, collecting the sediment, and re-suspending the sediment by using a high-sugar DMEM culture medium containing antibiotics.
Further, in step (3), the follicular unit was incubated at 37℃for 1h in a CO 2 incubator.
Further, the mass volume concentration of type II collagenase in the high sugar DMEM medium in step (3) is 1mg/ml.
Further, in step (4) and step (5), the cell suspension was centrifuged at 3000r/min for 5min, respectively.
Further, the filtrate 1 in the step (5) was digested in a CO 2 incubator at 37℃for 10min.
Further, the concentration of trypsin in the EDTA solution of step (5) is 0.25% by mass.
Further, the antibiotics in the high sugar DMEM of step (2), step (4), step (5) and step (7) are penicillin-streptomycin-amphotericin B suspension, wherein the concentration of penicillin is 100U/ml, the concentration of streptomycin is 0.1mg/ml, and the concentration of amphotericin is 0.25ug/ml.
Further, the pore size of the cell sieve in step (7) is 70um.
The hair follicle outer root sheath suspension is prepared by adopting the method.
The beneficial effects generated by adopting the scheme are as follows:
The culture medium does not contain any serum or growth factors, is harmless to human bodies, has high safety after transplantation, does not need to be subjected to in-vitro culture after the extraction of the melanocytes, and can be directly applied to the treatment of the human bodies.
In the preparation process of the external root sheath suspension, firstly, the culture medium containing penicillin-streptomycin-amphotericin B is used for immersing and washing the extracted melanocytes, so that polluted bacteria in the operation process can be effectively killed, then, the culture medium containing type II collagenase is used for digestion treatment, and then, EDTA solution containing trypsin is used for treatment, so that the cell tissues are digested through different action mechanisms, and further, the digestion effect is improved.
Detailed Description
Example 1
A method for preparing a hair follicle outer root sheath suspension, comprising the following steps:
(1) Selecting a patient with stable leucoderma in a disease period, preparing skin on the pillow part, selecting a dense hair follicle part, sterilizing by using iodophor, wiping by using normal saline, carrying out subcutaneous infiltration anesthesia by using lidocaine hydrochloride injection, obtaining complete hair follicle along the growth direction of the hair by using an extractor, and placing the removed hair follicle in the normal saline;
(2) Washing the follicular unit obtained in step (1) 3 times with a high sugar DMEM medium containing penicillin-streptomycin-amphotericin B;
(3) Placing the follicular unit cleaned in the step (2) into a high-sugar DMEM culture medium containing type II collagenase, and incubating the follicular unit in a CO 2 culture box at 37 ℃ for 1h, wherein the mass volume concentration of the type II collagenase in the high-sugar DMEM culture medium is 1mg/ml;
(4) Scraping the outer root sheath tissue of the hair follicle on the follicular unit after incubation in the step (3) by using a surgical knife, filtering to obtain a filtrate 1 and a cell suspension, centrifuging the cell suspension at a rotating speed of 3000r/min for 5min, collecting a precipitate, adding a high-sugar DMEM culture medium containing penicillin-streptomycin-amphotericin B into the precipitate, and blowing and resuspension to obtain a suspension 1;
(5) Adding the filter 1 in the step (4) into an EDTA solution containing trypsin, digesting the solution in a CO 2 incubator at 37 ℃ for 10min, then adding a high-sugar DMEM medium containing penicillin-streptomycin-amphotericin B into the solution to dilute trypsin, filtering, obtaining a filter 2 and a cell suspension, centrifuging the cell suspension at a rotating speed of 3000r/min for 5min, collecting a precipitate, adding the high-sugar DMEM medium containing penicillin-streptomycin-amphotericin B into the precipitate, and blowing and resuspension to obtain a suspension 2;
(6) Taking the filtrate 2 in the step (5), and repeating the operation in the step (5) to obtain a suspension 3;
(7) And (3) mixing the suspensions 1,2 and 3 in the step (4), the step (5) and the step (6), centrifuging at a rotating speed of 2000r/min to obtain cell sediment, re-suspending the cell sediment, filtering with a cell sieve with a pore diameter of 70um to obtain filtrate, further centrifuging the filtrate at a rotating speed of 2000r/min for 5min, collecting the sediment, and re-suspending the sediment with a high-sugar DMEM culture medium containing penicillin-streptomycin-amphotericin B.
The concentration of penicillin in penicillin-streptomycin-amphotericin B in the high sugar DMEM medium used in the above step was 100U/ml, the concentration of streptomycin was 0.1mg/ml, and the concentration of amphotericin was 0.25ug/ml.
Comparative example 1
Based on example 1, the type II collagenase in the high sugar DMEM medium in step (3) was replaced with a neutral protease.
Comparative example 2
Based on example 1, the mass-volume concentration of type II collagenase in the high-sugar DMEM medium in step (3) was changed to 3mg/ml.
Comparative example 3
Based on example 1, penicillin-streptomycin-amphotericin B in the high sugar DMEM medium in steps (2), (4) and (5) was replaced with penicillin.
Test examples
Sterilizing the affected area of the patient with iodophor, wiping with normal saline, performing subcutaneous infiltration anesthesia on the skin of the affected area with lidocaine hydrochloride injection, removing the epidermis of the affected area with a mechanical skin elimination mill, respectively sucking the hair follicle outer root sheath suspensions prepared in example 1 and comparative examples 1-3 with a syringe, mounting a blunt suction needle on the head of the syringe, uniformly smearing the hair follicle outer root sheath suspension on the affected area of the patient, covering the affected area with chitosan biomembrane, covering gauze thereon, and bandaging. After 2 weeks of injection, the affected part of the patient was observed, and the color change of the affected part was recorded, as shown in table 1.
Table 1: statistical table for skin color change of affected part of patient
The hair follicle external root sheath suspension prepared by the method can effectively treat the vitiligo of a patient, has good treatment effect, basically recovers the skin color of an affected part after treatment, has shorter preparation time, does not need an in-vitro culture process, and shortens the treatment period. And the melanocyte activity in the hair follicle outer root sheath suspension prepared by the method is higher, and the mixed bacteria in the suspension are less, so that skin infection is not easy to cause.
Claims (4)
1. A method for preparing a hair follicle outer root sheath suspension, which is characterized by comprising the following steps:
(1) Extracting follicular units of the head of the patient;
(2) Washing the follicular unit obtained in step (1) with a high sugar DMEM medium containing antibiotics;
(3) Placing the follicular unit washed in the step (2) into a high-sugar DMEM culture medium containing type II collagenase for incubation, wherein the mass volume concentration of the type II collagenase in the high-sugar DMEM culture medium is 1mg/ml;
(4) Scraping the outer root sheath tissue of the hair follicle on the follicular unit after incubation in the step (3), filtering to obtain a filtrate 1 and a cell suspension, centrifuging the cell suspension, collecting a precipitate, adding a high-sugar DMEM culture medium containing antibiotics into the precipitate, and blowing and resuspension to obtain a suspension 1;
(5) Adding the filter 1 in the step (4) into an EDTA solution containing trypsin for digestion, then adding a high-sugar DMEM culture medium containing antibiotics into the EDTA solution to dilute trypsin, filtering, obtaining a filter 2 and a cell suspension, centrifuging the cell suspension, collecting a precipitate, adding the high-sugar DMEM culture medium containing antibiotics into the precipitate, and blowing and resuspension to obtain a suspension 2; wherein, the filter 1 is digested in a CO 2 incubator for 10min at 37 ℃; the mass volume concentration of trypsin in the EDTA solution is 0.25%;
(6) Taking the filtrate 2 in the step (5), and repeating the operation in the step (5) to obtain a suspension 3;
(7) Mixing the suspensions 1, 2 and 3 in the step (4), the step (5) and the step (6), centrifuging to obtain cell precipitates, re-suspending the cell precipitates, filtering with a cell sieve to obtain filtrate, further centrifuging the filtrate at a rotating speed of 2000r/min for 5min, collecting the precipitates, and re-suspending the precipitates with a high-sugar DMEM culture medium containing antibiotics to obtain the antibiotic-containing high-sugar DMEM culture medium;
Wherein, the antibiotics in the high sugar DMEM in the step (2), the step (4), the step (5) and the step (7) are penicillin-streptomycin-amphotericin B suspension, the concentration of penicillin is 100U/ml, the concentration of streptomycin is 0.1mg/ml, and the concentration of amphotericin B is 0.25ug/ml.
2. The method of claim 1, wherein in step (3) the follicular unit is incubated at 37 ℃ in a CO 2 incubator for 1 hour.
3. The method of claim 1, wherein the cell suspension is centrifuged at 3000r/min for 5min in step (4) and step (5), respectively.
4. The method of claim 1, wherein the cell sieve in step (7) has a pore size of 70um.
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| CN106619505A (en) * | 2017-02-08 | 2017-05-10 | 北京爱富迪医药科技发展有限公司 | Injection used for hair growth, and preparation method thereof |
| CN107663512A (en) * | 2016-07-29 | 2018-02-06 | 长沙华山白癜风医院有限公司 | Leucoderma Autologous epidermis melanin transplanted cells moisture is into activating method |
| CN109576213A (en) * | 2018-12-28 | 2019-04-05 | 杭州恩格生物医疗科技有限公司 | A kind of preparation method and applications of melanocyte liquid |
| CN110564675A (en) * | 2019-09-30 | 2019-12-13 | 广东华夏健康生命科学有限公司 | Separation and extraction method of hair follicle stem cells |
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| EP2771454B1 (en) * | 2011-10-27 | 2019-07-24 | Universität Leipzig | Method for deriving melanocytes from the hair follicle outer root sheath and preparation for grafting |
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| CN107663512A (en) * | 2016-07-29 | 2018-02-06 | 长沙华山白癜风医院有限公司 | Leucoderma Autologous epidermis melanin transplanted cells moisture is into activating method |
| CN106619505A (en) * | 2017-02-08 | 2017-05-10 | 北京爱富迪医药科技发展有限公司 | Injection used for hair growth, and preparation method thereof |
| CN109576213A (en) * | 2018-12-28 | 2019-04-05 | 杭州恩格生物医疗科技有限公司 | A kind of preparation method and applications of melanocyte liquid |
| CN110564675A (en) * | 2019-09-30 | 2019-12-13 | 广东华夏健康生命科学有限公司 | Separation and extraction method of hair follicle stem cells |
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| Comparison between autologous noncultured extracted hair follicle outer root sheath cell suspension and autologous noncultured epidermal cell suspension in the treatment of stable vitiligo: a randomized study;C. Singh等;British Journal of Dermatology;第169卷;第289页左栏第2-3段 * |
| Noncultured extracted hair follicle outer root sheath cell suspension for transplantation in vitiligo;S. Mohanty等;British Journal of Dermatology;第164卷;第1242页左栏第1段至右栏第2段 * |
| 两种人毛囊外根鞘细胞培养方法的比较;唐建兵等;广东医学;第26卷(第1期);第44-46页 * |
| 白癜风的移植治疗进展;常心通等;中国医疗美容;第10卷(第2期);第130-134页 * |
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