CN106568911A - In vitro simulated skin model - Google Patents
In vitro simulated skin model Download PDFInfo
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Abstract
The invention discloses an in vitro simulated skin model. According to an establishment method of the in vitro simulated skin model, a cell culture chamber and well plates are taken as the skeleton of the in vitro simulated skin model; the cell culture chamber is inoculated with immortalized epidermis cells, and a lower chamber well plate is inoculated with fibroblast for co-culture. The establishment method is capable of simulating the basic structure of human skin effectively; the in vitro simulated skin model can be applied in evaluation of transdermal absorption and functions in promoting fibroblast proliferation and migration of cosmetics and medicinal compounds effectively, can be used for replacing conventional animal experiments and in vitro skin tissue experiments, is capable of reducing experiment cost effectively, increasing experimental flux and efficiency, and preferably satisfying requirements on scientific research ethics with more and more attention.
Description
Technical field
The invention belongs to biological medicine and cosmetic field, are that the design and beauty of the simulated skin model of ex vivo situation should
With.
Background technology
Skin is located at the surface of human body, is the maximum organ of the human body for directly contacting with external environment.Be broadly divided into epidermis,
Corium, hypodermis, four parts of cutaneous appendages.Epidermis is divided into cuticula, hyaline layer, stratum granulosum etc. again；Cuticula is table
The outermost layer of skin, is formed by 4～8 layers of flat seedless angling dead cell close-packed arrays.Cuticular thickness is to affect skin pair
The important decisive factor that nutriment, cosmetic factor absorb.Corium is located under epidermis, is firmly connected in wavy with epidermis,
Its thickness is about 10 times of epidermis.By a large amount of fibrous connective tissues (collagenous fibres, elastic fibers, reticular fibre), cell (into fibre
Dimension cell, histocyte) and matrix (glutinous polysaccharide, protein and salinity) composition, the disappearance of this three part can cause skin
Flexibility decrease, presentation such as are dried, corrugate at the exsiccosis.Meanwhile, corium is also rich in blood vessel, lymphatic vessel, nerve, body of gland, piloerection
Flesh etc..Wherein, fibroblast is one of most important cell of skin corium.
For many cosmetic materials and external application medicinal compound, Transdermal absorption inefficiency is to restrict its effect
Most important reason.For example, EGF (epidermal growth factor, EGF) has and strongly promote cell increasing
The ability grown, can play skin repair, take good care of and go acne (to remove acne, sore of fullying recover from an illness and youth as cosmetic additive agent and medicine
Acne), Bearberry Extract.However, used as a kind of large biological molecule, Transdermal absorption difference always perplexs the great of its practical application and asks
Topic.Research shows that EGF smears accumulation skin permeation rate after 12h and also only has 0.993% with the dosage of 0.5 μ g/g, it was demonstrated that the overwhelming majority
EGF fails to pass through skin, as externally applied drug (such as beautifying drug) or when being added in cosmetics, its Transdermal absorption effect ten
Divide faint.Therefore, easy, efficient, sensitive, reliable Transdermal absorption merit rating model, for cosmetics and externally applied drug
The high flux screening of compound has great importance with evaluation.
However, enjoyed always using animal model evaluation cosmetic industry denouncing.Britain just passed through early in 1986《It is real
Test animal protection bill》(Animals (Scientific Procedures) Act, 1986 (ASPA)), forbid carrying out cosmetics
The zoopery of finished product and raw material.European Union also issued the ban of " cosmetic industry forbids zoopery " in 2003, clearly
Regulation " on March 11st, 2009, forbid sale composition and finished product to carry out zooperal cosmetics, but repeat toxicity, reproduction poison
Property and poison for it is toxicologic experiment exception；On March 11st, 2013, zoopery is completely forbidden, including repeat toxicity, reproduction poison
Property and poison are for toxicological experiment." at present, clearly forbidden the zooperal countries and regions of cosmetics to have Europe in the world
Alliance, Britain, Brazil, Norway, Turkey, Korea, India, New Zealand, Israel etc..Discussing and forbidding cosmetics zoopery
Countries and regions then include the U.S., Russia, Australia, Canada, Argentina, TaiWan, China etc..Therefore, set up non-dynamic
The cosmetic evaluation model of thing experiment has particularly significant and urgent realistic meaning.
The content of the invention
It is biological cosmetics and biological medicament it is an object of the invention to build a kind of simulated skin model of ex vivo situation
The efficient evaluations of thing Transdermal absorption ability and the high flux screening of potential compound provide a kind of new method.
The present invention realizes that the technical scheme of purpose is as follows：
The simulated skin model of a kind of ex vivo situation, by the use of cell culture cell and orifice plate as isolated skin model " bone
Frame ", epidermal cell is co-cultured with fibroblast, with simulated skin histological structure state.
And, the cell culture cell be can be placed in cell culture orifice plate, base be 0-4 μm of aperture microporous barrier
Cell.
And, the cell culture cell is Transwell or Thincert.
And, the orifice plate includes 96 orifice plates, 48 orifice plates, 24 orifice plates, 12 orifice plates, 6 orifice plates.
And, the epidermal cell includes people's immortalized cell line and detached primary epidermal cell.
And, the fibroblast includes immortalized cell line and detached primary fibroblast.
And, the epidermal cell be Hacat, it is described into fiber finer be Balb/c 3T3.
The simulated skin model of ex vivo situation is used for sample Transdermal absorption ability and its promotes fibroblast proliferation, moves
The application of the evaluation of shifting function.
A kind of Vitro Simulated skin model answering in cosmetics and bio-pharmaceutical raw material Transdermal absorption and efficacy assessments
With.
Advantages and advantages of the invention are：
1st, the present invention constructs the simulated skin mould under a kind of ex vivo situation for not relying on living animal or animal tissue
Type, can be used as cosmetics and the replacement evaluation model of the experiment of externally applied drug conventional animal or skin histology experiment.
2nd, the evaluation that can effectively realize to sample Transdermal absorption ability and biologically active of the invention, and experimental period is short, behaviour
Make easy, sensitivity high, with low cost.
3rd, the present invention constructs the simulated skin mould under a kind of ex vivo situation for not relying on living animal or animal tissue
Type, can effectively realize the evaluation to sample Transdermal absorption ability and biologically active, and experimental period is short, easy to operate, sensitivity
High, with low cost, the evaluation and compound high flux screening for cosmetics and the Transdermal absorption ability of externally applied drug provides one kind
The assessment model and method that the new experiment of alternative conventional animal or skin histology is tested.
Description of the drawings
Fig. 1 is that isolated skin simulation model builds schematic diagram, 1, Transwell insert；2nd, Tissue Culture Plate；3、
Hacat cells；4th, nutrient solution；5th, Balb/c 3T3 cells.
Fig. 2 is the BA detection that recombinant protein is carried out using isolated skin simulation model.
Fig. 3 is to carry out protein transmembrane transhipment and the detection of Transdermal absorption ability using isolated skin simulation model.
Specific embodiment
Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not limit
Qualitatively, it is impossible to which protection scope of the present invention is limited with this.
A kind of simulated skin model of ex vivo situation of present invention design, can be used to live sample Transdermal absorption ability and biology
The evaluation of property, particular content includes：
By the use of 0-4 μm of base aperture microporous barrier cell culture cell with corresponding orifice plate as isolated skin model " bone
Frame ", by immortalized cell lines such as the immortalized cell lines such as Hacat or detached primary epidermal cell and Balb/c 3T3 or separates
Primary fibroblast co-cultured, with simulated skin histological structure state.
Sample is added in upper room and is processed after certain hour, by sample concentration and fibroblast in the lower room of detection
Propagation and migration situation.
First, the structure of Vitro Simulated skin model
Fibroblast (by taking Balb/c 3T3 cells as an example) with containing 10% hyclone complete culture solution DMEM in
37 DEG C, 5%CO2Under the conditions of cultivate.Epidermal cell (by taking Hacat as an example) with containing 15% hyclone complete culture solution MEM in
37 DEG C, 5%CO2Under the conditions of cultivate；
(2) above-mentioned cell culture respectively takes 150 μ l, 500 μ l and is inoculated in cell culture cell (with 0.4 μm to the digestion of certain density
As a example by the Transwell of aperture) upper room and lower room in, culture 24h (cultivate 12h when, discard ventricular cell culture medium, make
Hungry culture is carried out with containing 0.4%DMEM culture mediums) make cell monolayer (Fig. 1).
2nd, evaluation of the Vitro Simulated skin model to sample Transdermal absorption and promotion fibroblast proliferation and transfer ability
(1) after Vitro Simulated skin model builds.Then by sample (with EGF standard items and fused cell cell-penetrating peptide
EGF, i.e. PEM, as a example by) serum free medium gradient dilution is used, make the final concentration of 20 μ g/ml of EGF mark product, pME final concentrations difference
For 10 μ g/ml, 20 μ g/ml, the culture medium of lower ventricular cell is replaced with, continue to cultivate 60h.
(2) the albumen positioning of upper and lower ventricular cell is analyzed using immunofluorescence technique, to determine the Transdermal absorption of sample
Ability (Fig. 2)；
(3) the proliferative conditions of lower ventricular cell are detected with MTT methods, to determine sample to fibroblast proliferation energy
The impact (Fig. 3) of power；If while analyzing impact of the sample to migration of fibroblast cells ability, can use in place of model construction
Sterile pipette hair washing draws a line to produce cut, different time after sample-adding process, under inverted microscope in lower confluent monolayer cells
Detection cut healing state.
Claims (9)
1. the simulated skin model of a kind of ex vivo situation, it is characterised in that：By the use of cell culture cell and orifice plate as in vitro skin
Skin model " skeleton ", epidermal cell is co-cultured with fibroblast, with simulated skin histological structure state.
2. Vitro Simulated skin model according to claim 1, it is characterised in that：The cell culture cell is to mount
In cell culture orifice plate, the cell that base is 0-4 μm of aperture microporous barrier.
3. Vitro Simulated skin model according to claim 1 and 2, it is characterised in that：The cell culture cell is
Transwell or Thincert.
4. Vitro Simulated skin model according to claim 1, it is characterised in that：The orifice plate includes 96 orifice plates, 48 holes
Plate, 24 orifice plates, 12 orifice plates, 6 orifice plates.
5. Vitro Simulated skin model according to claim 1, it is characterised in that：The epidermal cell includes people's immortalization
Clone and detached primary epidermal cell.
6. Vitro Simulated skin model according to claim 1, it is characterised in that：The fibroblast includes immortalization
Clone and detached primary fibroblast.
7. Vitro Simulated skin model according to claim 1, it is characterised in that：The epidermal cell is Hacat, described
It is Balb/c 3T3 into fiber finer.
8. the simulated skin model of the ex vivo situation described in claim 1 is used for sample Transdermal absorption ability and its promotes into fibre
Dimension cell propagation, the application of the evaluation of shift function.
9. a kind of Vitro Simulated skin model as claimed in claim 1 in cosmetics and bio-pharmaceutical raw material Transdermal absorption and
Application in efficacy assessments.
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| CN201610943395.8A CN106568911A (en) | 2016-10-26 | 2016-10-26 | In vitro simulated skin model |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287151A (en) * | 2017-06-18 | 2017-10-24 | 广东博溪生物科技有限公司 | A kind of construction method of the vitro skin test model containing melanocyte |
| CN108424852A (en) * | 2018-03-14 | 2018-08-21 | 张洪剑 | Transwell cell culture apparatus of the simulated blood vessel wall by force effect |
| CN108801871A (en) * | 2018-04-13 | 2018-11-13 | 北京航空航天大学 | A kind of device using membrane separation technique simulated skin sweating |
| CN109612898A (en) * | 2018-11-12 | 2019-04-12 | 禄根仪器(镇江)有限公司 | A kind of suspending agent flowing release test flowing lumen |
| CN114674734A (en) * | 2022-04-20 | 2022-06-28 | 无限极（中国）有限公司 | Method for evaluating antioxidant effect of cosmetic product and raw material |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287151A (en) * | 2017-06-18 | 2017-10-24 | 广东博溪生物科技有限公司 | A kind of construction method of the vitro skin test model containing melanocyte |
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| CN108424852A (en) * | 2018-03-14 | 2018-08-21 | 张洪剑 | Transwell cell culture apparatus of the simulated blood vessel wall by force effect |
| CN108424852B (en) * | 2018-03-14 | 2021-04-27 | 张洪剑 | Transwell cell culture device simulating stress effect of blood vessel wall |
| CN108801871A (en) * | 2018-04-13 | 2018-11-13 | 北京航空航天大学 | A kind of device using membrane separation technique simulated skin sweating |
| CN109612898A (en) * | 2018-11-12 | 2019-04-12 | 禄根仪器(镇江)有限公司 | A kind of suspending agent flowing release test flowing lumen |
| CN109612898B (en) * | 2018-11-12 | 2021-06-04 | 禄根仪器(镇江)有限公司 | Flowable release test circulation cavity for suspending agent |
| CN114674734A (en) * | 2022-04-20 | 2022-06-28 | 无限极（中国）有限公司 | Method for evaluating antioxidant effect of cosmetic product and raw material |
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