CN108203705A - The co-culture method of mouse melanin cell and keratinocyte - Google Patents
The co-culture method of mouse melanin cell and keratinocyte Download PDFInfo
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- CN108203705A CN108203705A CN201810142559.6A CN201810142559A CN108203705A CN 108203705 A CN108203705 A CN 108203705A CN 201810142559 A CN201810142559 A CN 201810142559A CN 108203705 A CN108203705 A CN 108203705A
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- 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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- 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
- C12N5/0629—Keratinocytes; Whole skin
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
- C12N2502/091—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells melanocytes
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
- C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
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Abstract
The invention discloses a kind of mouse melanin cells and the co-culture method of keratinocyte, it is to carry out the obtained mouse melanin cell of 1 secondary culture and keratinocyte as inoculating cell, first keratinocyte re-suspension liquid is seeded in the culture dish of paving cloth Collagen type-I in advance, inoculate melanocyte re-suspension liquid, and melanocyte special culture media and keratinocyte special culture media are added in, in 5% CO2It is co-cultured at 37 DEG C in cell incubator.Co-culture melanocyte and keratinocyte in the process of the present invention, not only realize keratinocyte mostly for secondary culture, and the melanin content in co-culture system dramatically increases.
Description
Technical field
The invention belongs to technical field of bioengineering, are related to a kind of extracorporeal culturing method of cell, more particularly to mouse
The co-culture method of melanocyte and keratinocyte.
Background technology
Keratinocyte is one of main ingredient of skin epidermis tissue.It can be generated there is many in mammalian epidermis
The melanocyte of melanin granule, and about 36~40 keratinocytes around each melanocyte, these are thin
Born of the same parents are closely coupled in form and function, collectively constitute " epidermis-melanocyte unit ".The melanin granule fortune that melanocyte generates
The defeated keratinocyte to surrounding completes the transport process of pigment deposition.
With flourishing for cosmetic industry, influence of the cosmetics to skin has been to be concerned by more and more people, and with
The Skin Cell that cosmetics are in direct contact is keratinocyte, therefore, to the assessment of chemical composition safety in cosmetics from not
Open keratinocyte.Meanwhile keratinocyte plays an important role in the pathology generating process of skin related disease.
Under normal circumstances, positioned at the melanocyte of basal layer of epidermis and keratinocyte it is a proliferation and its slowly steady
Determine population, but the proliferation of epidermis upper strata keratinocyte is relatively rapidly.It upwards pressure keratinocyte is thin together with angling
The melanin granule of born of the same parents' intake pushes skin surface to and forms a kind of critical barrier, so as to which organism be enable to resist from environment
Pressure.Research shows that in light skin, keratinocyte is intended to the melanin granule for assembling light color above core；And in depth
In color skin, dispersed distribution is presented in melanin granule in keratinocyte, so as to reach absorbance value to greatest extent.Melanin
The melanin granule that cell generates is transported to the intracellular quantity of neighbouring angling, and resisting extraneous ultraviolet irradiation to skin has one
Fixed decisive role.
Keratinocyte all plays highly important effect in the generation of melanin and transport process, melanin granule from
Melanocyte, which is migrated to the process study of keratinocyte, be unable to do without keratinocyte.Although the primary culture method of keratinocyte is
It is set up, but secondary culture is difficult to carry out.Therefore, it is necessary to establish a kind of method, keratinocyte is enable to pass more algebraically, simultaneously
Interaction research between keratinocyte and melanocyte provides material.
Invention content
The object of the present invention is to provide a kind of mouse melanin cells and the co-culture method of keratinocyte, can with offer
The method that keratinocyte is made to carry out passage existence.
The co-culture method of mouse melanin cell and keratinocyte of the present invention is obtained with carrying out 1 secondary culture
Keratinocyte re-suspension liquid is first seeded in paving cloth rat-tail in advance by the mouse melanin cell and keratinocyte arrived as inoculating cell
In the culture dish of collagen, melanocyte re-suspension liquid is inoculated, and adds in melanocyte special culture media and keratinocyte is special
With culture medium, in 5% CO2It is co-cultured at 37 DEG C in cell incubator.
The mouse melanin cell and keratinocyte of secondary culture of the present invention are both from C57BL/6J black
Mouse skin.
Specifically taking out C57BL/6J black mice skin of back 3~5 days raw, 0.25% Dispase enzymes II detach
Epidermis and corium, 0.25% trypsin digestion obtain cell suspension, respectively with melanocyte special culture media and keratinocyte
Special culture media is cultivated, and carries out 1 secondary culture.
It is that melanocyte is resuspended in the co-culture method of mouse melanin cell and keratinocyte of the present invention
After liquid is counted respectively with keratinocyte re-suspension liquid, according to melanocyte: keratinocyte=30~40: 1 cell quantity ratio
Example carries out fishplate bar.
The present invention provides mouse melanin cells and a kind of best model of keratinocyte co culture system in vitro, construct and grind
Study carefully the model of melanocyte and keratinocyte in the mechanism of action of cutaneous pigmentation.
Using construction method co culture system in vitro melanocyte of the present invention and keratinocyte, keratinocyte is not only realized
Mostly for secondary culture, and the melanin content in co-culture system dramatically increases, it was demonstrated that melanocyte there are diagonalization
The growth of cell has certain promotion, and keratinocyte has facilitation to the B16 cell in melanocyte again.
Description of the drawings
Fig. 1 is primary keratinocyte form.
Fig. 2 is primary melanocyte form.
Fig. 3 is the keratin expression in keratinocyte.
Fig. 4 is the gp100 expression in melanocyte.
Fig. 5 is that keratinocyte is expressed with the cell that melanocyte co-cultures.
Fig. 6 is independent melanin culture and melanocyte compared with the melanin content in keratinocyte co-cultivation.
Specific embodiment
Following embodiments are only the preferred technical solution of the present invention, are not used to carry out any restrictions to the present invention.For
For those skilled in the art, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made
Any modification, equivalent substitution, improvement and etc., should all be included in the protection scope of the present invention.
Embodiment 1.
Choose the raising of C57BL/6J black mices.
The skin of back of 3~5 days mouse after taking-up is raw is put into 1~2min of immersion in 75% alcohol, with precooling containing dual anti-
PBS embathes removal bloodstain, and sub-surface porosity connective tissue is rejected with eye scissors or scalpel.Skin is cut into small strip, then in advance
It is cold rinsed 3~5 times containing dual anti-PBS after, be put into advance prepared 0.25% Dispase enzymes II, the use of Dispase enzymes II
5 times for drawn materials volume are measured, 4 DEG C are digested overnight.
The strip skin digested is put into Tissue Culture Dish, is detached epidermis with corium with ophthalmic tweezers, with containing dual anti-
PBS rinses epidermis for several times, to remove the Dispase enzymes II contained in epidermis.The epidermis of separator well is put into a new cell
It in culture dish, is shredded with eye scissors, adds in prepared 0.25% trypsase (submerging skin chips), 37 DEG C of digestion
8min。
Trypsin digestion is terminated with the DMEM culture mediums containing 10% newborn bovine serum, skin is gently blown and beaten with elbow straw
Skin fragment, 200 mesh filter screens filtering, collects cell suspension.
Cell suspension is centrifuged into 10min with 1000rpm/min at 4 DEG C, discards supernatant, obtains epidermal cell.
Epidermal cell is divided into two parts, cell, part angle is resuspended with melanocyte special culture media in a part
Change cell special culture media and cell be resuspended, respectively obtain melanocyte suspension and keratinocyte suspension, and be inoculated in culture dish,
It observes afterwards for 24 hours, replaces culture medium.
Fig. 1 be primary keratinocyte form, isolated primary adherent cell be single epithelial cell, cell shape
State is in paving stone shape, is the representative configuration of keratinocyte；Fig. 2 is primary melanocyte, as shown in the figure, isolated is primary
Attached cell is single epithelial cell, and cellular morphology is in dendron shape, is the representative configuration of melanocyte.
Embodiment 2.
The upgrowth situation of primary melanocyte or primary keratinocyte in observation Tissue Culture Dish in time.When cell is close
When degree reaches about 80%, the culture medium in culture dish is sucked and is discarded.0.25% trypsin digestion prepared in advance is added in per hole
Liquid 1mL, covers culture dish and gently shakes, allow digestive juice that cellular layer is completely covered, and culture dish is put into CO2Cell culture
In case, 37 DEG C of 5~8min of digestion.
Culture dish is taken out from incubator, cellular morphology variation is observed under inverted microscope.When the cytoplasm form of cell
Retraction occurs to be rounded, gap becomes larger between cell, when gently shaking culture plate there are a large amount of cells to start shedding off, adds in equivalent immediately
1mL terminate liquids (90%DMEM/F12+10% newborn bovine serum) terminate digestion.Terminate liquid, which is drawn, with pipettor blows and beats culture repeatedly
The melanocyte or keratinocyte of ware bottom, are allowed to form suspension.Action is light during piping and druming, in case firmly excessive cause carefully
The unnecessary damage of born of the same parents.
Respectively by melanocyte suspension, keratinocyte suspension is collected into centrifuge tube, at 4 DEG C with 1000rpm/min from
Heart 10min.It discards supernatant, is separately added into suitable melanocyte special culture media and keratinocyte special culture media is resuspended carefully
Born of the same parents draw a small amount of cell suspension, are counted, according to cell concentration by cell suspension renewed vaccination to new Tissue Culture Dish
In, it is put into CO2In cell incubator, 37 DEG C of 1st generation secondary cultures for carrying out melanocyte and keratinocyte.
Embodiment 3.
When the melanocyte of 1st generation passage and the density of keratinocyte reach about 80%, melanocyte and angle are carried out
Change the co-cultivation of cell.
By the melanocyte of secondary culture and keratinocyte respectively with 0.25% trypsin digestion, terminate liquid, which terminates, to disappear
Change, centrifugation obtains melanocyte and keratinocyte, is separately added into melanocyte special culture media and the special training of keratinocyte
Base is supported, prepares melanoblast re-suspension liquid and keratinocyte re-suspension liquid.
After melanocyte re-suspension liquid and keratinocyte re-suspension liquid are counted respectively, according to melanocyte: angling
Cell=30~40: 1 ratio carries out fishplate bar.Required keratinocyte re-suspension liquid is seeded in paving in advance first and is furnished with Collagen type-I
Culture dish in, then required melanocyte re-suspension liquid is inoculated in this ware, finally adds the special training of melanocyte
Base and each 1mL of keratinocyte special culture media are supported, culture dish is gently shaken, makes melanocyte special culture media and keratinocyte
Special culture media is uniformly mixed.It is put into 5%CO2In cell incubator, 37 DEG C are continued to cultivate.
Embodiment 4.
When the keratinocyte density of 1st generation passage reaches about 80%, continue the secondary culture of keratinocyte.
0.25% trypsin digestion of keratinocyte will be passed on, terminate liquid terminates digestion, and centrifugation obtains keratinocyte, with
Keratinocyte special culture media is prepared into keratinocyte re-suspension liquid, is seeded in culture dish, adds in keratinocyte special culture media
1mL, in 5%CO2Continue to cultivate for 37 DEG C in cell incubator.
Cultivation results show that in the independent secondary culture of the present embodiment keratinocyte, keratinocyte tends to apoptotic state.And
In the co-cultivation of 3 keratinocyte of embodiment and melanocyte, keratinocyte growth state is preferable, illustrates depositing for melanocyte
There is certain facilitation in the growth of diagonalization cell.
Embodiment 5.
By keratinocyte and melanocyte according to the co-culture method of embodiment 3, it is added to and puts cell climbing sheet well in advance
Special glass piece and spread be furnished in 24 orifice plates of Collagen type-I, it is allowed to attach on sheet glass.
When cell density is suitable, original culture medium is sopped up, is rinsed 3 times with PBS, with 4% paraformaldehyde, 4 DEG C of fixations
20min, PBS are rinsed 3 times, each 3min；3% 37 DEG C of hydrogen peroxide shading handles 20min, and PBS is rinsed 3 times, each 3min；Envelope
Close 37 DEG C of closing 20min of liquid (5700 μ L PBS+0.3g BSA+18 μ L Triton-100).
Confining liquid is discarded, the mixing with the keratin antibody (1: 100) that PBS has diluted and gp100 antibody (1: 100) is added dropwise
Liquid, after 4 DEG C are incubated overnight, equilibrium at room temperature 30min；It is rinsed 3 times with PBS, then two diluted with PBS are added dropwise in each 5min
Anti- fluorescence secondary antibody (1: 200), 37 DEG C of incubation 30min；PBS is rinsed 3 times, each 3min.Gradient alcohol dehydration is simultaneously quenched with anti-fluorescence
Go out agent mounting, the relationship of the common keratinocyte of laser co-focusing FV1000 observations and melanocyte.
The keratinocyte and melanocyte of independent secondary culture are taken respectively, according to the method described above, respectively add in keratin
Antibody and gp100 antibody, the fluorescent immune method for carrying out keratinocyte and melanocyte are observed.
Fig. 3 gives the green fluorescent label qualification result of independent keratinocyte, and Fig. 4 then provides independent melanocyte
Red fluorescence Marker Identification result.From fig. 5, it can be seen that keratinocyte and two kinds of cells in melanocyte co-culture system
Equal great expression, works well.
Embodiment 6.
Keratinocyte with melanocyte according to the co-culture method of embodiment 3 is cultivated, while is cultivated individual
Melanocyte.After 3 generation of secondary culture, keratinocyte is measured respectively and deposits system and melanocyte with melanocyte symbiosis
In melanin content.
When cell length is to 80%, rinsed 3 times with PBS respectively, it, will after 0.25% trypsin digestion, terminate liquid terminate
Cell suspension centrifuges, and PBS purgings centrifuge again, adds in 0.2mol/L NaOH solutions, and 85 DEG C of effect 5min make melanin granule
Fully dissolving, melanin content is measured with ultraviolet specrophotometer under 475nm.
For measurement result as shown in fig. 6, MC is melanocyte culture in figure, MC+KC is melanocyte and keratinocyte
It co-cultures.Melanocyte is dramatically increased with the melanin content in keratinocyte co-culture system as seen from the figure, it was demonstrated that black
Chromatophore has certain facilitation with keratinocyte co-culture system to the B16 cell in melanocyte.
Claims (4)
1. the co-culture method of a kind of mouse melanin cell and keratinocyte is thin with the mouse melanin of 1 secondary culture
Keratinocyte re-suspension liquid is first seeded in the culture dish of paving cloth Collagen type-I in advance as inoculating cell by born of the same parents and keratinocyte,
Melanocyte re-suspension liquid is inoculated, and adds in melanocyte special culture media and keratinocyte special culture media, in 5%
CO2It is co-cultured at 37 DEG C in cell incubator.
2. the co-culture method of mouse melanin cell according to claim 1 and keratinocyte, it is characterized in that being used
Secondary culture mouse melanin cell and keratinocyte both from C57BL/6J black mice skins.
3. the co-culture method of mouse melanin cell according to claim 2 and keratinocyte, it is characterized in that taking out raw 3
The C57BL/6J black mice skin of back of~5 days, 0.25% Dispase enzymes II separation epidermis and corium, 0.25% trypsase
Digestion obtains cell suspension, is cultivated, gone forward side by side with melanocyte special culture media and keratinocyte special culture media respectively
1 secondary culture of row.
4. the co-culture method of mouse melanin cell according to claim 1 and keratinocyte, it is characterized in that by described in
Melanocyte re-suspension liquid and keratinocyte re-suspension liquid carry out fishplate bar according to 30~40: 1 cell quantity ratio.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951675A (en) * | 2019-12-23 | 2020-04-03 | 中国水产科学研究院珠江水产研究所 | Method for separating and culturing melanocytes of orange-colored double-crowned fish |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101969915A (en) * | 2008-03-11 | 2011-02-09 | 株式会社资生堂 | Skin whitening method and screening method for factors for skin wrinkle formation suppression and/or removal |
| CN105112353A (en) * | 2015-07-29 | 2015-12-02 | 赫柏慧康生物科技无锡有限公司 | Mixed cultivation method of keratinocyte and melanocyte and application |
| CN105132358A (en) * | 2015-07-29 | 2015-12-09 | 赫柏慧康生物科技无锡有限公司 | Culture method for acquisition of tissue-engineered epidermis and application of culture method |
| CN107151648A (en) * | 2017-06-18 | 2017-09-12 | 广东博溪生物科技有限公司 | A kind of melanocyte and keratinocyte co-cultivation culture medium |
-
2018
- 2018-02-11 CN CN201810142559.6A patent/CN108203705A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101969915A (en) * | 2008-03-11 | 2011-02-09 | 株式会社资生堂 | Skin whitening method and screening method for factors for skin wrinkle formation suppression and/or removal |
| CN105112353A (en) * | 2015-07-29 | 2015-12-02 | 赫柏慧康生物科技无锡有限公司 | Mixed cultivation method of keratinocyte and melanocyte and application |
| CN105132358A (en) * | 2015-07-29 | 2015-12-09 | 赫柏慧康生物科技无锡有限公司 | Culture method for acquisition of tissue-engineered epidermis and application of culture method |
| CN107151648A (en) * | 2017-06-18 | 2017-09-12 | 广东博溪生物科技有限公司 | A kind of melanocyte and keratinocyte co-cultivation culture medium |
Non-Patent Citations (5)
| Title |
|---|
| TAE-JIN YOON ET AL.,: "Co-Culture of Mouse Epidermal Cells for Studies of Pigmentation", 《PIGMENT CELL RES》 * |
| 张均田等: "《现代药理试验方法 上》", 31 July 2012, 中国协和医科大学出版社 * |
| 江蕾薇等: "人表皮黑素细胞与角质形成细胞体外共培养模型的构建", 《贵阳医学院学报》 * |
| 鄢良春等: "鼠尾胶原对Caco-2细胞模型建立的影响", 《中药药理与临床》 * |
| 马悦悦等: "小鼠皮肤组织原代黑色素细胞培养条件的优化", 《黑龙江畜牧兽医》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951675A (en) * | 2019-12-23 | 2020-04-03 | 中国水产科学研究院珠江水产研究所 | Method for separating and culturing melanocytes of orange-colored double-crowned fish |
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Application publication date: 20180626 |