CN112280732A - Suspension of external root sheath of hair follicle and preparation method thereof - Google Patents
Suspension of external root sheath of hair follicle and preparation method thereof Download PDFInfo
- Publication number
- CN112280732A CN112280732A CN202011128816.4A CN202011128816A CN112280732A CN 112280732 A CN112280732 A CN 112280732A CN 202011128816 A CN202011128816 A CN 202011128816A CN 112280732 A CN112280732 A CN 112280732A
- Authority
- CN
- China
- Prior art keywords
- suspension
- filtrate
- cell
- precipitate
- centrifuging
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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 hair follicle outer root sheath tissue on the follicular unit, filtering to obtain a filtrate 1 and a cell suspension, centrifuging the cell suspension to obtain a precipitate, and blowing and resuspending the precipitate to obtain a suspension 1; adding the filtrate 1 into EDTA solution containing trypsin for digestion, adding high-sugar DMEM medium containing antibiotics, filtering to obtain filtrate 2 and cell suspension, centrifuging the cell suspension, collecting precipitate, and blowing and resuspending to obtain suspension 2; taking the filtrate 2, and repeating the operation to obtain a suspension 3; mixing the suspension 1, 2 and 3, centrifuging to obtain cell precipitate, resuspending, filtering to obtain filtrate, centrifuging, collecting precipitate, and resuspending the precipitate. The method can effectively solve the problems of long culture time and safety of culture solution of the existing melanocyte.
Description
Technical Field
The invention relates to the technical field of hair follicle outer root sheath suspension, in particular to hair follicle outer root sheath suspension and a preparation method thereof.
Background
Vitiligo is a pigmented skin disease caused by acquired melanocyte deficiency, and is characterized by single or multiple white spots on the skin in clinical. The incidence of the vitiligo in China is about 0.09% -2.7%, and the incidence of the vitiligo in recent years tends to rise. The vitiligo does not endanger the life of the patient, but the delayed and repeated attack of the disease causes huge psychological burden to the patient, hinders interpersonal communication of the patient, and even causes psychological diseases.
The etiology of vitiligo is not clear, and it is generally considered that the etiology is related to the following aspects: 1. the genetic susceptibility has certain familial genetic aggregation phenomenon. 2. Autoimmunity, often accompanied by other autoimmune diseases. 3. Melanocyte self-destruction, and melanocyte loss occurs in different conditions in skin damage area of vitiligo patients. 4. Environmental factors, prolonged exposure to phenolic-containing substances can lead to increased incidence of vitiligo.
The method for treating leucoderma is not perfect, only can the treatment be carried out by methods such as medicines, phototherapy and the like in the progressive stage of the disease, but the surgical means can be properly used in the stable stage, the most common surgical treatment mode at present is autologous skin transplantation, although the skin transplantation can enable a skin damage area to be discolored, the treatment area is completely limited by the size of a skin supply area, the operation can be realized only by repeated skin transplantation for patients with large-area skin damage, and the great burden is caused to the mind, the body and the economy of the patients.
At present, transplantation therapy is carried out through autologous melanocytes, however, melanocytes in the prior art need to be cultured after being obtained and then can be used, a large amount of substances such as growth factors and serum need to be used in the culturing process, and the substances are used for a human body, so that great potential safety hazards exist, and the treatment period is prolonged due to the fact that the melanocytes are cultured for a long time.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a preparation method of hair follicle outer root sheath mixed solution, which can effectively solve the problems of long culture time and safety of culture solution of the existing melanocyte.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a method for preparing an external root sheath suspension of hair follicle comprises the following steps:
(1) extracting follicular units of the head of the patient;
(2) washing the follicular unit obtained in the step (1) by using a high-glucose DMEM medium containing antibiotics;
(3) putting the follicular units cleaned in the step (2) into a high-glucose DMEM medium containing type II collagenase for incubation;
(4) scraping the hair follicle outer root sheath tissues on the follicular units incubated in the step (3), filtering to obtain a filtrate 1 and a cell suspension, centrifuging the cell suspension, collecting the precipitate, adding a high-sugar DMEM medium containing antibiotics into the precipitate, and blowing and resuspending to obtain a suspension 1;
(5) adding the filtrate 1 in the step (4) into EDTA solution containing trypsin for digestion, then adding high-sugar DMEM culture medium containing antibiotics to dilute the trypsin, filtering to obtain a filtrate 2 and cell suspension, centrifuging the cell suspension, collecting the precipitate, adding the high-sugar DMEM culture medium containing antibiotics into the precipitate, blowing and resuspending 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 steps (4), (5) and (6), centrifuging at the rotating speed of 2000r/min to obtain cell precipitates, re-suspending the cell precipitates, filtering by using a cell sieve to obtain filtrate, continuously centrifuging the filtrate at the rotating speed of 2000r/min for 5min, collecting the precipitates, and re-suspending the precipitates by using a high-sugar DMEM medium containing antibiotics to obtain the antibiotic.
Further, the hair follicle in the step (3) is positioned at 37 ℃ under CO2Incubate in incubator for 1 h.
Further, the mass volume concentration of collagenase type II in the high-glucose DMEM medium in the step (3) is 1 mg/ml.
Further, in the step (4) and the step (5), the cell suspension is centrifuged at 3000r/min for 5 min.
Further, the filtrate 1 in the step (5) is subjected to CO treatment at 37 DEG C2Digesting in incubator for 10 min.
Further, the mass volume concentration of trypsin in the EDTA solution in step (5) is 0.25%.
Further, in the step (2), the step (4), the step (5) and the step (7), the antibiotics in the high-sugar DMEM 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 0.25 ug/ml.
Further, the pore size of the cell sieve in the step (7) is 70 um.
The hair follicle external root sheath suspension is prepared by the method.
The beneficial effects produced by adopting the scheme are as follows:
the culture medium does not contain any serum and growth factors, is harmless to a human body, has higher safety after transplantation, does not need in-vitro culture after extraction of the melanocyte, and can be directly applied to the human body for treatment.
In the preparation process of the external root sheath suspension, the culture medium containing penicillin-streptomycin-amphotericin B is adopted to soak and wash extracted melanocytes, so that the polluted bacteria in the operation process can be effectively killed, then the high-sugar DMEM culture medium containing type II collagenase is used for digestion treatment, then EDTA solution containing trypsin is used for treatment, and cell tissues are digested through different action mechanisms, so that the digestion effect is improved.
Detailed Description
Example 1
A method for preparing an external root sheath suspension of hair follicle comprises the following steps:
(1) selecting a patient with stable leucoderma in a disease period, preparing the skin of the patient, selecting a dense part of a hair follicle, disinfecting the part with iodophor, wiping the part with normal saline, carrying out subcutaneous infiltration anesthesia on lidocaine hydrochloride injection, obtaining a complete hair follicle along the growth direction of the hair by adopting an extractor, and placing the removed hair follicle in the normal saline;
(2) washing the follicular unit obtained in the step (1) for 3 times by using a high-sugar DMEM culture medium containing penicillin-streptomycin-amphotericin B;
(3) placing the follicular unit cleaned in step (2) into high glucose DMEM medium containing collagenase type II at 37 deg.C under CO2Incubating for 1h in the incubator, wherein the mass volume concentration of the collagenase II in the high-glucose DMEM medium is 1 mg/ml;
(4) scraping the hair follicle outer root sheath tissues on the follicular units incubated in the step (3) by using a scalpel, filtering to obtain a filtrate 1 and a cell suspension, centrifuging the cell suspension for 5min at the rotating speed of 3000r/min, collecting precipitates, adding a high-sugar DMEM (DMEM) culture medium containing penicillin-streptomycin-amphotericin B into the precipitates, and blowing and resuspending to obtain a suspension 1;
(5) adding filtrate 1 of step (4) into EDTA solution containing trypsin at 37 deg.C under CO2Digesting in an incubator for 10min, adding high-sugar DMEM medium containing penicillin-streptomycin-amphotericin B to dilute trypsin, filtering to obtain filtrate 2 and cell suspension,centrifuging the cell suspension at 3000r/min for 5min, collecting precipitate, adding high-sugar DMEM culture medium containing penicillin-streptomycin-amphotericin B into the precipitate, blowing, and resuspending to obtain 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 steps (4), (5) and (6), centrifuging at the rotating speed of 2000r/min to obtain cell precipitates, re-suspending the cell precipitates, filtering by using a cell sieve with the pore diameter of 70um to obtain filtrate, continuously centrifuging the filtrate at the rotating speed of 2000r/min for 5min, collecting the precipitates, and re-suspending the precipitates by using a high-sugar DMEM medium containing penicillin-streptomycin-amphotericin B to obtain the penicillin-streptomycin-amphotericin B.
The concentration of penicillin in the penicillin-streptomycin-amphotericin B in the high-glucose DMEM culture medium used in the steps is 100U/ml, the concentration of streptomycin is 0.1mg/ml, and the concentration of amphotericin is 0.25 ug/ml.
Comparative example 1
Based on example 1, collagenase type ii in high glucose DMEM medium in step (3) was replaced with neutral protease.
Comparative example 2
Based on example 1, the mass volume concentration of collagenase type II in the high glucose DMEM medium in step (3) was changed to 3 mg/ml.
Comparative example 3
On the basis of example 1, penicillin-streptomycin-amphotericin B in high glucose DMEM medium in steps (2), (4) and (5) was replaced with penicillin.
Test examples
Disinfecting a skin lesion area of a patient with iodophor, wiping with normal saline, carrying out subcutaneous infiltration anesthesia on the skin of an affected part with lidocaine hydrochloride injection, then removing the epidermis of the skin lesion area by using a mechanical skin degritting machine, respectively sucking the hair follicle external root sheath suspension prepared in example 1 and comparative examples 1-3 by using a syringe, installing a blunt-end sucking needle at the head of the syringe, uniformly smearing the hair follicle external root sheath suspension on the skin lesion area of the patient, covering the affected part with a chitosan biological membrane, covering gauze on the affected part, and bandaging with a bandage. After 2 weeks of injection, the affected part of the patient was observed and the color change of the affected part was recorded, as detailed in table 1.
Table 1: statistical table for skin color change of affected part of patient
The above table shows that the suspension of the external root sheath of the hair follicle prepared by the method can effectively treat the vitiligo of a patient, has good treatment effect, basically restores the skin color of the affected part to be normal after treatment, and shortens the treatment period because the preparation time of the suspension of the external root sheath of the hair follicle prepared by the existing method is shorter, and the process of in vitro culture is not needed. The activity of melanocytes in the suspension of the external root sheath of the hair follicle prepared by the method is higher, and the content of foreign bacteria in the suspension is less, so that the skin infection is not easy to cause.
Claims (9)
1. A preparation method of the suspension of the external root sheath of the hair follicle is characterized by comprising the following steps:
(1) extracting follicular units of the head of the patient;
(2) washing the follicular unit obtained in the step (1) by using a high-glucose DMEM medium containing antibiotics;
(3) putting the follicular units cleaned in the step (2) into a high-glucose DMEM medium containing type II collagenase for incubation;
(4) scraping the hair follicle outer root sheath tissues on the follicular units incubated in the step (3), filtering to obtain a filtrate 1 and a cell suspension, centrifuging the cell suspension, collecting the precipitate, adding a high-sugar DMEM medium containing antibiotics into the precipitate, and blowing and resuspending to obtain a suspension 1;
(5) adding the filtrate 1 in the step (4) into EDTA solution containing trypsin for digestion, then adding high-sugar DMEM culture medium containing antibiotics to dilute the trypsin, filtering to obtain a filtrate 2 and cell suspension, centrifuging the cell suspension, collecting the precipitate, adding the high-sugar DMEM culture medium containing antibiotics into the precipitate, blowing and resuspending 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 steps (4), (5) and (6), centrifuging to obtain cell precipitates, re-suspending the cell precipitates, filtering by using a cell sieve to obtain filtrate, continuously centrifuging the filtrate at the rotating speed of 2000r/min for 5min, collecting the precipitates, and re-suspending the precipitates by using a high-sugar DMEM medium containing antibiotics to obtain the antibiotic.
2. The method of claim 1, wherein the follicular unit is maintained at 37 ℃ in step (3) under CO2Incubate in incubator for 1 h.
3. The method according to claim 1, wherein the mass volume concentration of collagenase type II in the high glucose DMEM medium in step (3) is 1 mg/ml.
4. The method according to claim 1, wherein the cell suspension is centrifuged at 3000r/min for 5min in each of the steps (4) and (5).
5. The method according to claim 1, wherein the filtrate 1 of the step (5) is subjected to CO treatment at 37 ℃2Digesting in incubator for 10 min.
6. The method of claim 1, wherein the mass volume concentration of trypsin in the EDTA solution in step (5) is 0.25%.
7. The method according to claim 1, wherein the antibiotics in the high-glucose DMEM in step (2), step (4), step (5) and step (7) are penicillin-streptomycin-amphotericin B suspension, wherein the concentration of penicillin is 80 to 120U/ml, the concentration of streptomycin is 0.05 to 0.15mg/ml, and the concentration of amphotericin is 0.2 to 0.3 ug/ml.
8. The method of claim 1, wherein the pore size of the cell sieve in step (7) is 70 um.
9. An epifollicular root sheath suspension prepared by the method of any one of claims 1 to 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011128816.4A CN112280732A (en) | 2020-10-21 | 2020-10-21 | Suspension of external root sheath of hair follicle and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011128816.4A CN112280732A (en) | 2020-10-21 | 2020-10-21 | Suspension of external root sheath of hair follicle and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112280732A true CN112280732A (en) | 2021-01-29 |
Family
ID=74424380
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011128816.4A Pending CN112280732A (en) | 2020-10-21 | 2020-10-21 | Suspension of external root sheath of hair follicle and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112280732A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150086513A1 (en) * | 2011-10-27 | 2015-03-26 | Universitat Leipzig | Method for deriving melanocytes from the hair follicle outer root sheath and preparation for grafting |
| 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 |
-
2020
- 2020-10-21 CN CN202011128816.4A patent/CN112280732A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150086513A1 (en) * | 2011-10-27 | 2015-03-26 | Universitat Leipzig | Method for deriving melanocytes from the hair follicle outer root sheath and preparation for grafting |
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| C. SINGH等: "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", BRITISH JOURNAL OF DERMATOLOGY, vol. 169, pages 2 - 3, XP071158067, DOI: 10.1111/bjd.12325 * |
| S. MOHANTY等: "Noncultured extracted hair follicle outer root sheath cell suspension for transplantation in vitiligo", BRITISH JOURNAL OF DERMATOLOGY, vol. 164, pages 1242 * |
| 唐建兵等: "两种人毛囊外根鞘细胞培养方法的比较", 广东医学, vol. 26, no. 1, pages 44 - 46 * |
| 常心通等: "白癜风的移植治疗进展", 中国医疗美容, vol. 10, no. 2, pages 130 - 134 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101757691B (en) | Preparation method of tissue engineering cornea | |
| CN107224617B (en) | Hydrogel taking spleen extracellular matrix as raw material and preparation method thereof | |
| CN104726406B (en) | It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell | |
| CN101856517B (en) | Tissue engineering material-based culture method and applications of melanophore | |
| CN108992466A (en) | A kind of injection and preparation method thereof using hair papilla cell excretion body treatment androgenetic alopecia | |
| CN108721606A (en) | A kind of striae of pregnancy reparation product and preparation method thereof based on stem cell factor | |
| CN108057116A (en) | Application of the stem cell composition in skin injury medicine | |
| CN114848911A (en) | Acellular dental pulp stem cell membrane and preparation method and application thereof | |
| CN110938587A (en) | Preparation method and application of epidermal stem cell suspension | |
| CN112353819A (en) | A preparation for promoting hair growth, and its preparation method | |
| CN111117965A (en) | Rapid purification method of high-purity primary tumor cells | |
| CN108066750A (en) | Stem cell and its secretion are used to treat the new application of skin burn | |
| CN113846064A (en) | FGF18 gene modified mesenchymal stem cell and preparation method and application thereof | |
| CN108619169A (en) | A kind of mesenchymal stem cell injection and preparation method for treating cerebral arterial thrombosis | |
| CN115537393B (en) | Preparation method and application of umbilical cord mesenchymal stem cell directional secretion group for promoting hair growth | |
| CN112280732A (en) | Suspension of external root sheath of hair follicle and preparation method thereof | |
| CN111110921A (en) | In-vitro construction method of tissue engineering posterior lamella cornea | |
| CN105963795A (en) | Method for preparing tissue engineering epidermis based on collagen | |
| CN112522180B (en) | Human scalp hair follicle single cell suspension and preparation method and application thereof | |
| CN111363716A (en) | Preparation of acellular amniotic membrane carrier composite autologous endometrial stem cells | |
| CN109771697B (en) | Dermal fibroblast skin sheet and construction method and application thereof | |
| CN114276986A (en) | Method for separating and purifying buffalo primary myoblasts and application thereof | |
| CN111117948A (en) | Fibroblast culture method | |
| KR100725133B1 (en) | Culture method of fibroblast using autologous serum mixed with placenta extract and composition for skin regeneration using the same | |
| CN114558119A (en) | Preparation and evaluation method of skin wound repair liquid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |