CN106047793A - Culture medium for skin keratinocyte and culture method thereof - Google Patents
Culture medium for skin keratinocyte and culture method thereof Download PDFInfo
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Abstract
The invention belongs to the field of cell culture, and in particular relates to a culture medium for skin keratinocyte and a culture method thereof. The culture medium for the skin keratinocyte provided by the invention comprises a DMEM (Dulbecco Modified Eagle Medium) basic culture medium, an epidermal growth factor, a transforming growth factor, vitamin H, recombinant human insulin, transferrin, hesperidin and resveratrol. By adopting the culture medium for the skin keratinocyte provided by the invention, the cell attachment time of the keratinocyte can be shortened, differentiation of the keratinocyte is prevented, and the pollution of keratinocyte caused by fibroblasts is lowered; meanwhile, by adopting the culture medium for the skin keratinocyte, the proliferation rate of the keratinocyte can be increased, and the vitality and cell characteristics of the keratinocyte in a passage process are maintained; the culture medium is a relatively ideal in-vitro culture medium for the skin keratinocyte.
Description
Technical field
The invention belongs to field of cell culture, be specifically related to a kind of skin keratin and form culture medium and the cultivation side thereof of cell
Method.
Background technology
The skin of human body is made up of epidermis, corium and subcutaneous tissue three part.Keratinocyte is the one of epidermis
Keratoprotein cell, is the important component part constituting epidermis.Under normal circumstances, human epidermal cell increases regularly
Grow, break up and come off, maintain skin sugar, protein, fat, water and the balance of electrolyte, there is protection self, regulation body
Temperature, the excretion function such as refuse and immunomodulating.
Keratinocyte is the key component of epidermis cell, is a kind of epithelial cell that can synthesize keratoprotein.
Keratinocyte to horn cell develop during, can be generally divided into four layers, i.e. basal layer, spinous layer, granular layer with
And horny layer.Someone calls stratum germinativum or Ma Er basic unit three first layers or the first two layer.Additionally, at some position, especially at the palm
Sole of the foot position, is also shown in clear layer below horny layer.
The In vitro culture of keratinocyte can be dermatoxicology, burn treating, cosmetology and dermatosis morbidity
The research of mechanism provides new approach.But, skin keratin formed cell in separation and Culture during there is easily differentiation, easily
Polluted by fibroblast, be difficult to the problems such as adherent, and the reproductive capacity of keratinocyte is relatively low, cell viability is more weak, carefully
The survival rate of born of the same parents is the highest.Therefore, set up a kind of stable system, be suitable for the cultivation that skin keratin formation cell growth is bred
Method becomes the task of top priority.
Zhang Xing etc. have delivered the paper of entitled " preparation of human keratinocytes and In vitro culture ", this opinion
Preparation and the In vitro culture of human keratinocytes mainly inquired in literary composition, and result shows: pancreatin+separation enzymic digestion can obtain
Purer keratinocyte, cell is well-grown in restricted KC-FSM culture medium, the D value recorded after cultivating 48h, 72h
It is significantly higher than DMEM culture medium and RPMI1640 culture medium.Therefore, pancreatin+separation enzymic digestion, restricted KC-FSM medium body
Outer cultivation is the good method preparing and cultivating human keratinocytes.But, this restricted KC-FSM culture medium is at fell
Still suffering from cell in skin keratinocyte Process of in vitro easily to break up, the cell attachment time is longer and cell proliferation in vitro rate
Relatively low defect, is unfavorable for the promotion and application of the method.
Chinese patent application 201510741542.9 discloses the training of a kind of culture medium and application thereof and keratinocyte
Breeding method, the culture medium of described keratinocyte include KC-FSM serum-free basal medium, KGF, EGF, glutamine and
Non essential amino acid.After the culture medium using above-mentioned offer is cultivated, the cutin that quantity is big, vigor is high can be obtained and formed carefully
Born of the same parents, cell characteristics keeps well in succeeding generations, and additionally this culture medium is to use culture medium based on serum-free medium,
On the premise of ensure that cell viability, it is to avoid the risk that allogeneic serum brings.But, the cultivation side of this keratinocyte
There is the defect of differentiation oxidizable, easy in method in cell cultivation process, greatly limits the cultivation side of this keratinocyte
The use of method.
Summary of the invention
Prior art exists in keratinocyte incubation in vitro cell easily break up, easily by fibroblast to overcome
The defect that dimension cell contamination, time length oxidizable, adherent and cell proliferation rate are low, it is an object of the invention to provide a kind of skin
The culture medium of keratinocyte and cultural method thereof, to solve the problems referred to above.
The invention provides a kind of skin keratin formed cell culture medium, including DMEM basal medium, also include as
Lower component and concentration thereof: epidermal growth factor 2-4ng/ml, transforming growth factor 3-5ng/ml, biotin 1-3ug/ml, weight
Group insulin human 6-9mg/ml, transferrins 5-8ug/ml, Hesperidin 10-20umol/L and resveratrol 6-12umol/L.
Further, described skin keratin forms the culture medium of cell and includes DMEM basal medium, also includes following group
Divide and concentration: epidermal growth factor 3ng/ml, transforming growth factor 4ng/ml, biotin 2ug/ml, recombinant human insulin
8mg/ml, transferrins 6ug/ml, Hesperidin 16umol/L and resveratrol 10umol/L.
Further, described DMEM basal medium is high glycoform DMEM culture medium.
Further, described transforming growth factor is transforming growth factor-β.
It addition, present invention also offers a kind of skin keratin to form the cultural method of cell, comprise the following steps:
S1 takes and wipes out hypodermic skin histology, and the fritter being cut into 0.5cm × 0.5cm is put in enzyme liquid I, places 4 DEG C
Under the conditions of digest overnight；
Skin histology after step S1 processes is separated into epidermis and corium by S2, is added by epidermis in enzyme liquid II and shreds,
15-20min is hatched under the conditions of placing 37 DEG C；
Epidermis after step S2 processes is blown and beaten 6-12 time by S3, filters with 200-240 mesh copper mesh, and filtrate is at 1200-
1500r/min is centrifuged, and abandons supernatant, obtains filtering residue；
The filtering residue that step S3 is obtained by S4 accesses the arbitrary described skin keratin of claim 1-4 and forms the culture medium of cell
In, in 37 DEG C, 5%CO2Under conditions of cultivate 5-8 days, to obtain final product.
Further, in described step S1 enzyme liquid I be mass concentration be the neutral protein enzymatic solution of 0.6-0.8%.
Further, in described step S1 enzyme liquid I be mass concentration be the neutral protein enzymatic solution of 0.7%.
Further, the enzyme liquid II in described step S2 is by the ethylenediaminetetraacetic acid that mass concentration is 0.3-0.5% and matter
Amount concentration is the pancreatin composition of 0.4-0.6%.
Further, the enzyme liquid II in described step S2 is dense by the ethylenediaminetetraacetic acid that mass concentration is 0.4% and quality
Degree is the pancreatin composition of 0.5%.
In the component of the culture medium that the skin keratin that the present invention provides forms cell, epidermal growth factor is a kind of multi-functional
Somatomedin, promote cell growth；Transforming growth factor-β can regulate cell growth and differentiation；Biotin is cell
The coenzyme of interior multiple enzyme, plays an important role, beneficially the life of keratinocyte in protein synthesis and substance metabolism
Long；Recombinant human insulin can improve anabolism ability, stimulates the growth of cell；Transferrins is that ferrum main in blood plasma passes
Pass albumen, the ferrum needed for providing for cell internalizing and cellular metabolism.
Hesperidin, molecular formula is C28H34O15, No. CAS is 520-26-3, resveratrol, and molecular formula is C14H12O3, No. CAS
For 501-36-0.Hesperidin and resveratrol have antioxidation, are conducive to maintaining the biological function of cell.
Further, finding through test, the skin keratin using the present invention to provide forms the cutin that cell culture medium is cultivated
Forming cell adherent in 12h, keratinocyte is rounded in 12h, then ovalize, and volume becomes big, about 2 days can
Seeing that several keratinocyte forms microcolony, within about 5 days, cell confluency is in blocks.And use the skin keratin that the present invention provides
The keratinocyte growth forming cell culture medium cultivation is vigorous, and profile understands, refractivity is good, becomes paving stone sample form, training
Very small amount fusiformis fibrocyte seen from the initial stage of supporting, along with the prolongation passed on incubation time, fibroblast-like cells fades away, can
To obtain the keratinocyte of higher degree.
Further, find through test, compared with high glycoform DMEM culture medium, use the skin keratin shape that the present invention provides
Become cell culture medium extremely significantly (P ＜ 0.01) rate of increase of keratinocyte can be improved in 48h and 72h, illustrate
The skin keratin that invention provides forms the mutually coordinated effect playing promotion keratinocyte growth of each component of culture medium of cell,
Can effectively improve the productivity of keratinocyte.
The skin keratin that the present invention provides forms mutually coordinated of each component of culture medium of cell and shortens keratinocyte
The adherent time, prevent Keratinocyte differentiation and reduce the effect polluted by fibroblast of keratinocyte；Simultaneously
This skin keratin forms cell culture medium can also improve the rate of increase of keratinocyte, maintains keratinocyte passing on
Vigor in journey and cell characteristics, be a kind of ideal keratinocyte in-vitro culture medium.
In a word, the skin keratin that the present invention provides forms the culture medium of cell, compared with prior art has the advantage that
1) skin keratin that the present invention provides forms cell culture medium and can effectively shorten the adherent of keratinocyte
Time, prevent the differentiation of keratinocyte, reduce keratinocyte by fibroblastic pollution；
2) skin keratin that the present invention provides forms cell culture medium and can be effectively improved the rate of increase of keratinocyte,
Maintain keratinocyte vigor in succeeding generations and cell characteristics, be that a kind of ideal keratinocyte is external
Culture medium.
Detailed description of the invention
Further describing the present invention below by way of specific embodiment, the present invention is not limited only to following example.At this
In bright scope or without departing from present disclosure, spirit and scope, change that the present invention is carried out, combine or replace
Change, will be apparent to the person skilled in the art, and be included within the scope of the present invention.Each in the present invention
Component is conventional commercial product, such as: epidermal growth factor is purchased from Heng Fei bio tech ltd, Shanghai, recombined human islets of langerhans
Element is purchased from Yi Sheng bio tech ltd, Shanghai, and neutral protease is molten and pancreatin is purchased from Sigma company, and DMEM culture medium is purchased from
Gibco company.
Embodiment 1, a kind of skin keratin form the culture medium of cell
Described skin keratin formed cell culture medium include high glycoform DMEM basal medium, also comprise the following components and
Concentration: epidermal growth factor 2ng/ml, Transforming growth factor-β3 ng/ml, biotin 1ug/ml, recombinant human insulin 6mg/
Ml, transferrins 5ug/ml, Hesperidin 10umol/L and resveratrol 6umol/L.
Preparation method:
In DMEM basal medium, add epidermal growth factor, transforming growth factor, biotin, weight by described concentration
Group insulin human, transferrins, Hesperidin and resveratrol, mixing, film is degerming excessively and get final product.
Embodiment 2, a kind of skin keratin form the culture medium of cell
Described skin keratin formed cell culture medium include high glycoform DMEM basal medium, also comprise the following components and
Concentration: epidermal growth factor 3ng/ml, transforming growth factor-β 4ng/ml, biotin 2ug/ml, recombinant human insulin 8mg/
Ml, transferrins 6ug/ml, Hesperidin 16umol/L and resveratrol 10umol/L.
Preparation method is similar to Example 1.
Embodiment 3, a kind of skin keratin form the culture medium of cell
Described skin keratin formed cell culture medium include high glycoform DMEM basal medium, also comprise the following components and
Concentration: epidermal growth factor 4ng/ml, transforming growth factor-β 5ng/ml, biotin 3ug/ml, recombinant human insulin 9mg/
Ml, transferrins 8ug/ml, Hesperidin 20umol/L and resveratrol 12umol/L.
Preparation method is similar to Example 1.
Embodiment 4, a kind of skin keratin form the cultural method of cell
S1 takes and wipes out hypodermic skin histology, and the fritter being cut into 0.5cm × 0.5cm is put in enzyme liquid I, described enzyme liquid
I be mass concentration be the neutral protein enzymatic solution of 0.7%, digest overnight under the conditions of placing 4 DEG C；
Skin histology after step S1 processes is separated into epidermis and corium by S2, is added by epidermis in enzyme liquid II and shreds,
Described enzyme liquid II is made up of the ethylenediaminetetraacetic acid that mass concentration is 0.4% and the pancreatin that mass concentration is 0.5%, places 37 DEG C
Under the conditions of hatch 18min；
Epidermis after step S2 processes is blown and beaten 10 times by S3, filters with 200 mesh copper mesh, and filtrate is centrifuged at 1200r/min,
Abandon supernatant, obtain filtering residue；
The filtering residue that step S3 is obtained by S4 accesses in the culture medium of the skin keratin formation cell that the present invention provides, in 37
DEG C, 5%CO2Under conditions of cultivate 5-8 days, to obtain final product.
Comparative example 1, a kind of skin keratin form the culture medium of cell
Described skin keratin formed cell culture medium include low-sugar type DMEM basal medium, also comprise the following components and
Concentration: epidermal growth factor 3ng/ml, transforming growth factor-β 4ng/ml, biotin 2ug/ml, recombinant human insulin 8mg/
Ml, transferrins 6ug/ml, Hesperidin 16umol/L and resveratrol 8umol/L.
Preparation method is similar to Example 1.
Difference with embodiment 2 is, basal medium is low-sugar type DMEM culture medium.
Comparative example 2, a kind of skin keratin form the culture medium of cell
Described skin keratin formed cell culture medium include high glycoform DMEM basal medium, also comprise the following components and
Concentration: epidermal growth factor 3ng/ml, transforming growth factor-β 4ng/ml, biotin 2ug/ml, recombinant human insulin 8mg/
Ml, transferrins 6ug/ml and Hesperidin 24umol/L.
Preparation method is similar to Example 1.
Difference with embodiment 2 is, does not add resveratrol, adds the concentration of Hesperidin.
Comparative example 3, a kind of skin keratin form the culture medium of cell
Described skin keratin formed cell culture medium include high glycoform DMEM basal medium, also comprise the following components and
Concentration: epidermal growth factor 3ng/ml, transforming growth factor-β 4ng/ml, biotin 2ug/ml, recombinant human insulin 8mg/
Ml, transferrins 6ug/ml and resveratrol 24umol/L.
Preparation method is similar to Example 1.
Difference with embodiment 2 is, does not add Hesperidin, adds the concentration of resveratrol.
Comparative example 4, a kind of skin keratin form the culture medium of cell
Described skin keratin formed cell culture medium include high glycoform DMEM basal medium, also comprise the following components and
Its concentration: epidermal growth factor 3ng/ml, transforming growth factor-β 4ng/ml, biotin 2ug/ml, recombinant human insulin
8mg/ml, transferrins 6ug/ml, Hesperidin 12umol/L and resveratrol 12umol/L.
Preparation method is similar to Example 1.
The difference of embodiment 2 is, the concentration of Hesperidin and resveratrol is 12umol/L.
Test example one, skin keratin form the culture experiment of cell
1, subjects: skin histology takes from the endometrium that healthy women comes off.
2, test material: embodiment 1, embodiment 2, embodiment 3, comparative example 1, comparative example 2, comparative example 3 and comparative example 4 are made
Standby skin keratin forms the culture medium of cell.
3, test method:
By the endometrium that comes off according to the step 1-3 process of embodiment 4, then the filtering residue after processing is put into reality
The skin keratin executing example 1, embodiment 2, embodiment 3, comparative example 1, comparative example 2, comparative example 3 and comparative example 4 preparation forms cell
Culture medium in, be masked as embodiment 1 group, embodiment 2 groups, embodiment 3 groups, comparative example 1 group, comparative example 2 groups, comparative example respectively
3 groups and comparative example 4 groups, in 37 DEG C, 5%CO2Under conditions of cultivate 5-8 days,
And under inverted microscope adherent time of observation of cell and growing state.
4, result of the test:
Result of the test is as shown in table 1.
Table 1 skin keratin forms the culture experiment of cell
As shown in Table 1, the skin keratin using embodiment of the present invention 1-3 to prepare forms the cutin of the culture medium culturing of cell
Forming cell adherent in 12h, rounded at 12h inner cell, ovalize then, volume becomes big, about 2 days visible several angles
Matter forms cell and forms microcolony, and within about 5 days, cell confluency is in blocks.And the skin keratin using comparative example 1-4 to prepare is formed carefully
The adherent time of the keratinocyte of born of the same parents' culture medium culturing and cell growth time all time than embodiments of the invention 1-3
Long, illustrate when the skin keratin formation cell culture medium that the present invention provides can effectively shorten keratinocyte adherent
Between, prevent Keratinocyte differentiation and minimizing keratinocyte from being polluted by fibroblast, be a kind of ideal angle
Matter forms cell injuring model base.
Test example two, skin keratin form Cell growth ability determination test
1, subjects: skin histology takes from the endometrium that healthy women comes off.
2, test material: embodiment 1, embodiment 2, embodiment 3, comparative example 1, comparative example 2, comparative example 3 and comparative example 4 are made
Standby skin keratin forms the culture medium of cell.
3, test method:
3.1, by the endometrium that comes off according to the step 1-3 process of embodiment 4, then the filtering residue after processing is put
The skin keratin entering embodiment 1, embodiment 2, embodiment 3, comparative example 1, comparative example 2, comparative example 3 and comparative example 4 preparation is formed
The culture medium of cell, is masked as matched group, embodiment 1 group, embodiment 2 groups, embodiment 3 groups, comparative example 1 group, comparative example 2 respectively
Group, comparative example 3 groups and comparative example 4 groups, in 37 DEG C, 5%CO2Under conditions of cultivate, be right with high glycoform DMEM basal medium
According to group, by primary cell with on identical dose inoculation to 96 orifice plates, the cell suspension in every hole is 200ul, cell number is 1 ×
104, every kind of culture medium sets 3 parallel holes, hatches 48h, 72h respectively；
3.2, MTT solution is dissolved in the PBS of 0.1mol/L so that it is concentration is 5g/L, by the original fluid of culture plate
Discarding, every hole adds 200ul culture fluid, containing MTT10ul in every 100ul culture fluid, is put into by culture plate and continues training in incubator
Supporting 4h, then every hole adds the DMSO stopped reaction of 20ul, is put by culture plate and the most fully mixes, measures by microplate reader
The optical density value (OD value) in every hole during 570nm wavelength, often group is repeated 3 times, and averages.
4, result of the test:
Result of the test is as shown in table 2.
Table 2 skin keratin formation Cell growth ability determination test (n=3,)
Note: compared with matched group*P ＜ 0.05,**P ＜ 0.01.
As shown in Table 2, compared with matched group, the skin keratin using embodiment of the present invention 1-3 to prepare forms the training of cell
Foster base extremely significantly (P ＜ 0.01) can improve the rate of increase of keratinocyte, and uses embodiment of the present invention 1-4 to prepare
Skin keratin forms the keratinocyte of cell culture medium cultivation cell proliferation rate in 48h and 72h all than the present invention's
The propagation rate variance of embodiment 1-3, illustrates that the skin keratin that the present invention provides forms mutually coordinated of each component of culture medium of cell
The effect promoting keratinocyte to increase, can effectively improve the productivity of keratinocyte.
Claims (9)
1. the culture medium of a skin keratin formation cell, it is characterised in that include DMEM basal medium, also include following group
Divide and concentration: epidermal growth factor 2-4ng/ml, transforming growth factor 3-5ng/ml, biotin 1-3ug/ml, recombined human
Insulin 6-9mg/ml, transferrins 5-8ug/ml, Hesperidin 10-20umol/L and resveratrol 6-12umol/L.
2. skin keratin as claimed in claim 1 forms the culture medium of cell, it is characterised in that include that DMEM basis is cultivated
Base, also comprises the following components and concentration: epidermal growth factor 3ng/ml, transforming growth factor 4ng/ml, biotin 2ug/
Ml, recombinant human insulin 8mg/ml, transferrins 6ug/ml, Hesperidin 16umol/L and resveratrol 10umol/L.
3. skin keratin as claimed in claim 1 or 2 forms the culture medium of cell, it is characterised in that described DMEM basis is trained
Foster base is high glycoform DMEM culture medium.
4. skin keratin as claimed in claim 1 or 2 forms the culture medium of cell, it is characterised in that described conversion growth because of
Son is transforming growth factor-β.
5. the cultural method of a skin keratin formation cell, it is characterised in that comprise the following steps:
S1 takes and wipes out hypodermic skin histology, and the fritter being cut into 0.5cm × 0.5cm is put in enzyme liquid I, places 4 DEG C of conditions
Lower digestion is overnight；
Skin histology after step S1 processes is separated into epidermis and corium by S2, is added by epidermis in enzyme liquid II and shreds, and places
15-20min is hatched under the conditions of 37 DEG C；
Epidermis after step S2 processes is blown and beaten 6-12 time by S3, filters with 200-240 mesh copper mesh, and filtrate is at 1200-1500r/
Min is centrifuged, and abandons supernatant, obtains filtering residue；
The filtering residue that step S3 is obtained by S4 accesses in the culture medium that the arbitrary described skin keratin of claim 1-4 forms cell,
In 37 DEG C, 5%CO2Under conditions of cultivate 5-8 days, to obtain final product.
6. skin keratin as claimed in claim 5 forms the cultural method of cell, it is characterised in that enzyme liquid in described step S1
I be mass concentration be the neutral protein enzymatic solution of 0.6-0.8%.
7. skin keratin as claimed in claim 6 forms the cultural method of cell, it is characterised in that enzyme liquid in described step S1
I be mass concentration be the neutral protein enzymatic solution of 0.7%.
8. skin keratin as claimed in claim 5 forms the cultural method of cell, it is characterised in that the enzyme in described step S2
Liquid II is made up of the ethylenediaminetetraacetic acid that mass concentration is 0.3-0.5% and the pancreatin that mass concentration is 0.4-0.6%.
9. skin keratin as claimed in claim 8 forms the cultural method of cell, it is characterised in that the enzyme in described step S2
Liquid II is made up of the ethylenediaminetetraacetic acid that mass concentration is 0.4% and the pancreatin that mass concentration is 0.5%.
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| CN110179903A (en) * | 2018-02-23 | 2019-08-30 | 大江生医股份有限公司 | The purposes that water perfume (or spice) rib extract leads to chafing reaction for reducing horn cell irradiating ultraviolet light, promotes skin keratin metabolism |
| CN110387348A (en) * | 2018-04-18 | 2019-10-29 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of application on human skin cutin cambial cell |
| CN111148829A (en) * | 2017-09-30 | 2020-05-12 | 上佳生物科技有限公司 | Cell culture medium |
| CN112980775A (en) * | 2021-03-19 | 2021-06-18 | 上海爱萨尔生物科技有限公司 | Culture solution for preparing keratinocytes based on differentiation of pluripotent stem cells |
| CN113046300A (en) * | 2021-03-19 | 2021-06-29 | 上海爱萨尔生物科技有限公司 | Culture method for preparing keratinocytes based on differentiation of pluripotent stem cells |
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| CN110179903B (en) * | 2018-02-23 | 2022-08-02 | 百岳特生物技术（上海）有限公司 | Application of aquilaria sinensis extract in reducing skin inflammation reaction caused by irradiation of ultraviolet light on keratinocytes and promoting skin cutin metabolism |
| CN110387348A (en) * | 2018-04-18 | 2019-10-29 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of application on human skin cutin cambial cell |
| CN112980775A (en) * | 2021-03-19 | 2021-06-18 | 上海爱萨尔生物科技有限公司 | Culture solution for preparing keratinocytes based on differentiation of pluripotent stem cells |
| CN113046300A (en) * | 2021-03-19 | 2021-06-29 | 上海爱萨尔生物科技有限公司 | Culture method for preparing keratinocytes based on differentiation of pluripotent stem cells |
| CN112980775B (en) * | 2021-03-19 | 2024-01-23 | 上海爱萨尔生物科技有限公司 | Culture solution for preparing keratinocyte based on differentiation of pluripotent stem cells |
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