CN106399225A - A clinical application based melanocyte culture method - Google Patents
A clinical application based melanocyte culture method Download PDFInfo
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- CN106399225A CN106399225A CN201610681698.7A CN201610681698A CN106399225A CN 106399225 A CN106399225 A CN 106399225A CN 201610681698 A CN201610681698 A CN 201610681698A CN 106399225 A CN106399225 A CN 106399225A
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- melanocyte
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- melanocytic
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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
Abstract
A clinical application based melanocyte culture method is disclosed. The method is characterized in that a medium used in the method comprises only a low concentration of fetal calf serum, a property that epidermal keratinocytes and melanocytes are different in protease-containing digestive juice tolerant time is utilized so as to achieve an objective of separating the two types of cells, and a culture process abandons an animal-derived cell dissociation buffer and adopts a plant-derived cell dissociation buffer. The method is a selective culture method the culture process of which is free of introduction of other animal-derived factors and/or harmful compounds, and therefore the method reduces risks of clinical application.
Description
Technical field
The present invention relates to cell injuring model technology, specifically a kind of melanocytic culture side based on clinical practice
Method.
Background technology
Leucoderma (vitiligo) is the depigmentation dermatoses of a kind of common local or general, and this sick incidence of disease is about
For 0.5-1%, with heredity, immunity, oxidative stress is unbalance etc., and factor has relation, at present for its pathogenesis research trend in
Think that the melanocyte that above comprehensive factor causes is destroyed, thus the depigmentation causing, and hickie is formed.This disease is controlled
Treatment method is a lot, such as medicine, laser, operation etc., but curative effect is all imprecise.
Traditional treatment means include endo-medicine, external application glucocorticoid, ultraviolet irradiation and surgical operation therapy
Deng.But the cure rate that drug therapy and ultraviolet irradiate is limited.
Surgical operation therapy method mainly includes the autologous black of AUTOEPIDERMIC GRAFTING, epidermal cell suspension transplanting and culture
Plain cell transplantation etc..
It is effective that these treatment methods confirm, but epidermic grafting needs large area to take skin, is not suitable for large area transplanting.Melanocyte
Cell suspension transplanting is the new treatment developing in recent years, but it is thin to expand highly purified melanocyte in needing the short time in vitro in a large number
Born of the same parents.
For this reason, forefathers devise culture medium and the purification process of multiple suitable melanocyte growths, including using containing height
The culture medium of concentration hyclone promotes cell growth, uses cell division accelerator phorbol -12- myristoyl -13- acetic acid
Ester (12-O-Tetradecanoylphorbol-13-acetate, TPA) promotes melanocyte proliferation and uses axoneme
(geneticin) non-melanocyte of selective removal etc..
There is potential medical-risk in these methods：Hyclone may contain crazy heifer disease virus, chemical substance TPA and base
Factor has certain carcinogenicity.
Content of the invention
Present invention aim to address in prior art, the not enough problem of melanocytic clinical practice security.
Employed technical scheme comprise that such for realizing the object of the invention, a kind of melanocytic based on clinical practice
Cultural method is it is characterised in that comprise the following steps：
1) take the skin abandoning in healthy population surgical site infections 1 hour as material；Material is placed in aseptic balance salt
In solution, prune away clot and subcutaneous fat；
2) by step 1) in obtain through pruning after the aseptic balanced salt solution of materials'use carry out rinsing 1～3 time；
3) by step 2) in obtain through rinsing after material be cut into the wide strip of 2mm；The material of strip will be trimmed to
Material is inserted in the EDTA that concentration is 0.25%, digests 8～12 hours under the conditions of 4 DEG C；
4) by step 3) in the postdigestive material that obtains pass through tweezers separation epidermis and corium, take epidermis, epidermis led to
Cross aseptic balanced salt solution to carry out rinsing 3 times；
5) by step 4) in the epidermis that obtains be cut into the laminated structure of several 2mm × 2mm；Laminated structure is placed in concentration
TrypLE for 0.25%TMIn (ThermoFisher Scientific 12563029), under the conditions of 37 DEG C digest 20min,
Obtain mixture A；
6) add aseptic balanced salt solution in mixture A, obtain cell suspension；The volume of described aseptic balanced salt solution
It is the twice of mixture A；
7) by step 6) in the cell suspension that obtains filtered by 200 eye mesh screens, centrifugation, collect precipitation；
8) by step 7) in the precipitation collected be resuspended in melanocyte culture medium；
9) by step 8) in melanocyte culture medium in cell density adjust to 1 × 105After individual/mL, cell is connect
Plant in blake bottle；
10) by step 9) in blake bottle be placed in 37 DEG C under the conditions of, concentration is 5% CO2Cultivated in incubator, 6h
Change culture medium afterwards and remove non-attached cell；
11) after step 10) in cell length after changing culture medium to 80% full when, cell is placed in concentration is
0.25% TrypLETMIn, digest 3～10 minutes, the melanocyte after being isolated and purified；
12) by step 11) in obtain isolate and purify after melanocyte, inoculated according to 1 2～1 3, passed on training
Support；
13) after step 12) in obtain isolate and purify after cell growth to enough clinical transplantations, retain standby.
Further, described step 5) and step 12) the middle TrypLE adoptingTMCell dissociation buffer for plant origin.
Further, described step 7) in centrifugal process centrifugation rate be 1000rpm, centrifugation time be 3min.
Further, described step 12) in carry out isolating and purifying melanocyte using the method for differential digestion.
Further, described step 8) in melanocyte culture medium formula include commercially available melanocyte basic culture solution
Medium 254, commercially available growth components HMGS-2, ATRA, 2 mercapto ethanol, vitamin C, penicillin and strepto-
Element；
Wherein, the volume of commercially available melanocyte basic culture solution Medium 254 is 1L；
Described commercially available growth components HMGS-2 volume proportion in the medium is 0.5～5%；
Described ATRA final concentration of 1～1000nM in the medium；
Described 2 mercapto ethanol final concentration of 1～1000nM in the medium；
Described vitamin C final concentration of 2～2000nM in the medium；
Described penicillin volume proportion in the medium is 0.5～5%；
Described streptomysin volume proportion in the medium is 0.5～5%；
Further, described step 1)～13) be obtained melanocyte preparation leucoderma medicament in application.
The solution have the advantages that mathematical, the present invention has the effect that：
1) the melanocytic purity that the present invention turns out is higher, cell viability strong, can preferably be applied to transplanting and control
Treat hypopigmentation or amelanotic dermatosis patient.
2) contain only the hyclone of low concentration in the culture medium of the present invention, and incubation will not bring other animals into
The factor in source and/or the selection cultural method of hazardous substances.Thus, method disclosed by the invention reduces clinical practice
Risk.
Brief description
Fig. 1 is the human melanocytes of in vitro culture；
Fig. 2 is the identified by immunofluorescence of the human melanocytes of in vitro culture.
In figure, A：After inoculation, 6h changes liquid, just adherent melanocyte；
B：Melanocyte after cultivating 3 days.
Specific embodiment
With reference to embodiment, the invention will be further described, but only should not be construed the above-mentioned subject area of the present invention
It is limited to following embodiments.Without departing from the idea case in the present invention described above, according to ordinary skill knowledge and used
With means, make various replacements and change, all should include within the scope of the present invention.
A kind of melanocytic cultural method based on clinical practice is it is characterised in that comprise the following steps：
1) skin abandoning after collecting healthy population surgery Wrapping annulus cuts operation is as material；Material is placed in aseptic balance
In salting liquid, prune away clot and subcutaneous fat；
2) by step 1) in obtain through pruning after the aseptic balanced salt solution of materials'use carry out rinsing 3 times；
3) by step 2) in obtain through rinsing after material be cut into the wide strip of 2mm；The material of strip will be trimmed to
Concentration inserted by material is in 0.25%EDTA (ethylenediamine tetra-acetic acid), digests 8 hours under the conditions of 4 DEG C；
4) by step 3) in the postdigestive material that obtains pass through tweezers separation epidermis and corium, take epidermis, epidermis led to
Cross aseptic balanced salt solution to carry out rinsing 3 times；
5) by step 4) in the epidermis that obtains be cut into the laminated structure of several 2mm × 2mm；Laminated structure is placed in concentration
TrypLE for 0.25%TM(ThermoFisher Scientific 12563029), digests 20min under the conditions of 37 DEG C, obtains
To mixture A；
6) add aseptic balanced salt solution in mixture A, obtain cell suspension；The volume of described aseptic balanced salt solution
It is the twice of mixture A；
7) by step 6) in the cell suspension that obtains filtered by 200 eye mesh screens, centrifugation, collect precipitation；Described from
The centrifugation rate of heart process is 1000rpm, and centrifugation time is 3min.
8) by step 7) in the precipitation collected be resuspended in melanocyte culture medium；
Described melanocyte culture medium formula includes commercially available melanocyte basic culture solution Medium 254, commercially available life
Long component HMGS-2, ATRA, 2 mercapto ethanol, vitamin C, penicillin and streptomysin；
Wherein, the volume of commercially available melanocyte basic culture solution Medium 254 is 1L；
Described commercially available growth components HMGS-2 volume proportion in the medium is 0.5；
Described ATRA final concentration of 1nM in the medium；
Described 2 mercapto ethanol final concentration of 1nM in the medium；
Described vitamin C final concentration of 2nM in the medium；
Described penicillin volume proportion in the medium is 0.5%；
9) by step 8) in melanocyte culture medium in cell density adjust to 1 × 105After individual/mL, cell is connect
Plant in blake bottle；
10) by step 9) in blake bottle be placed in 37 DEG C under the conditions of, concentration is 5% CO2Cultivated in incubator, 6h
Change culture medium afterwards and remove non-attached cell；Cell now is as shown in the A of Fig. 1.
11) after step 10) in cell length after changing culture medium to 80% full when, cell is placed in concentration is
0.25% TrypLETMIn, digest 10 minutes, the melanocyte after being isolated and purified；
12) by step 11) in obtain isolate and purify after melanocyte, inoculated according to 13, Secondary Culture；13)
By step 12) in obtain isolate and purify after cell carry out in vitro culture, after three days, cell quantity grows to enough clinical shifting
After plant, retain standby, cell now is as shown in the B in Fig. 1.
The cell turned out is identified, as shown in Fig. 2 all cells all express melanocyte Specific marker
Mitf_M, Trp-1, Trp-2, illustrate successfully to have turned out the human melanocytes purifying.
Claims (6)
1. a kind of melanocytic cultural method based on clinical practice is it is characterised in that comprise the following steps：
1) take the skin abandoning in healthy population surgical site infections 1 hour as material；Material is placed in aseptic balanced salt solution
In, prune away clot and subcutaneous fat；
2) by step 1) in obtain through pruning after the aseptic balanced salt solution of materials'use carry out rinsing 1～3 time；
3) by step 2) in obtain through rinsing after material be cut into the wide strip of 2mm；The material being trimmed to strip is put
Enter in the EDTA that concentration is 0.25%, digest 8～12 hours under the conditions of 4 DEG C；
4) by step 3) in the postdigestive material that obtains pass through tweezers separation epidermis and corium, take epidermis, epidermis passed through no
Bacterium balanced salt solution carries out rinsing 3 times；
5) by step 4) in the epidermis that obtains be cut into the laminated structure of several 2mm × 2mm；Laminated structure is placed in concentration is
0.25% TrypLETMIn, digest 20min under the conditions of 37 DEG C, obtain mixture A；
6) add aseptic balanced salt solution in mixture A, obtain cell suspension；The volume of described aseptic balanced salt solution is mixed
The twice of compound A；
7) by step 6) in the cell suspension that obtains filtered by 200 eye mesh screens, centrifugation, collect precipitation；
8) by step 7) in the precipitation collected be resuspended in melanocyte culture medium；
9) by step 8) in melanocyte culture medium in cell density adjust to 1 × 105After individual/mL, cell is inoculated in training
In foster bottle；
10) by step 9) in blake bottle be placed in 37 DEG C under the conditions of, concentration is 5% CO2Cultivated in incubator, after 6h more
Change culture medium and remove non-attached cell；
11) after step 10) in cell length after changing culture medium to 80% full when, it is 0.25% that cell is placed in concentration
TrypLETMIn, digest 3～10 minutes, the melanocyte after being isolated and purified；
12) by step 11) in obtain isolate and purify after melanocyte, inoculated according to 1 2～1 3, Secondary Culture；
13) after step 12) in obtain isolate and purify after cell growth to enough clinical transplantations, retain standby.
2. a kind of melanocytic cultural method based on clinical practice according to claim 1 it is characterised in that：Described
Step 11) the middle TrypLE adoptingTMCell dissociation buffer for plant origin.
3. a kind of melanocytic cultural method based on clinical practice according to claim 1 it is characterised in that：Described
Step 7) in centrifugal process centrifugation rate be 1000rpm, centrifugation time be 3min.
4. a kind of melanocytic cultural method based on clinical practice according to claim 1 it is characterised in that：Described
Step 11) in carry out isolating and purifying melanocyte using the method for differential digestion.
5. a kind of melanocytic cultural method based on clinical practice according to claim 1 it is characterised in that：Described
Step 8) in melanocyte culture medium formula include commercially available melanocyte basic culture solution Medium 254, commercially available growth
Composition HMGS-2, ATRA, 2 mercapto ethanol, vitamin C, penicillin and streptomysin；
Wherein, the volume of commercially available melanocyte basic culture solution Medium 254 is 1L；
Described commercially available growth components HMGS-2 volume proportion in the medium is 0.5～5%；
Described ATRA final concentration of 1～1000nM in the medium；
Described 2 mercapto ethanol final concentration of 1～1000nM in the medium；
Described vitamin C final concentration of 2～2000nM in the medium；
Described penicillin volume proportion in the medium is 0.5～5%；
Described streptomysin volume proportion in the medium is 0.5～5%.
6. a kind of melanocytic cultural method based on clinical practice according to claim 1 it is characterised in that：Described
Step 1)～13) be obtained melanocyte preparation leucoderma medicament in application.
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| CN201610681698.7A CN106399225A (en) | 2016-08-17 | 2016-08-17 | A clinical application based melanocyte culture method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108795842A (en) * | 2018-05-04 | 2018-11-13 | 江苏大学 | A kind of 3D suspensions induce method and the application of iPS Hemapoiesis autologous melanoma cells |
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