CN111337494A - Preparation method and application of artificial epidermis cosmetic detection kit - Google Patents
Preparation method and application of artificial epidermis cosmetic detection kit Download PDFInfo
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- CN111337494A CN111337494A CN202010227486.8A CN202010227486A CN111337494A CN 111337494 A CN111337494 A CN 111337494A CN 202010227486 A CN202010227486 A CN 202010227486A CN 111337494 A CN111337494 A CN 111337494A
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- epidermal
- cell
- cosmetic
- artificial
- human skin
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
Abstract
The invention discloses a preparation method and application of an artificial epidermis cosmetic detection kit. The invention cultures human skin tissue cells on a polycarbonate membrane bracket material: human fibroblasts and epidermal cells, and preparing an artificial epidermis having a structure of a dermis layer and an epidermis layer. The artificial epidermis cosmetic detection kit prepared by the invention can detect the irritation, corrosivity, anti-aging property, anti-oxidation, whitening effect and the like of cosmetics on human skin.
Description
Technical Field
The invention relates to a preparation method and application of an artificial epidermis cosmetic detection kit, and belongs to the field of cell culture.
Background
With the development of society and the improvement of the living standard of modern people, more and more people pay more attention to the external image of the people, hope that the people can appear in various activity occasions with beautiful images to increase confidence, and meanwhile, the people more pursue the health of the skin, so more people hope to show the pursuit of the people for high-end life with healthy skin and beautiful images. Therefore, various efficacy cosmetics are always sought after, the market share of the cosmetics is larger and the brand types are increased. Therefore, in the face of the full-fleshed cosmetic product on the market, how consumers judge the anti-aging, anti-oxidation and whitening effects of the product and the irritation and corrosive damage to the skin become a concern.
According to the traditional cosmetic detection, an in-vivo eye stimulation method of a rabbit is utilized, and an experimental animal as a substitute of human plays an irreplaceable role in scientific development of human, and along with the development of scientific technology, particularly the use number of the experimental animal in the field of life science research is increased dramatically, the pain of the experimental animal and the right thereof attract great attention of the public. The optimized alternative detection scheme comprises computer simulation analysis, bovine eye turbidity permeability experiment, in-vitro rabbit eye experiment, in-vitro corn experiment, chick embryo chorioallantoic membrane experiment and the like. But facing the challenge of differences in animal ethics and ethnic attributes, Russell and Burch propose the 3R principle. Since Russell and Burch proposed the theory of reduction, replacement, and optimization of animal experiments in 1959, particularly in the last 20 years, toxicology systems that no longer are based on animal models have become important developments in biomedicine and are accepted by government and research agencies. Some animal experimental alternatives are technical barriers to international trade. The guidelines of the technical standards for in vitro skin irritation tests of the human skin model published by the european surrogate center at 5 months of 2007 and the statements of the two in vitro skin irritation test validation methods published by 11 months of 2008 have all shown that tissue engineering skin can be used as a surrogate model for in vitro skin irritation tests. Constructing tissue engineered skin models that can be used in skin irritation tests under laboratory conditions can therefore effectively address these issues.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a preparation method and application of an artificial epidermis cosmetic detection kit. The invention adds human skin tissue cells on the polycarbonate membrane bracket material: human fibroblasts and epidermal cells, and preparing an artificial epidermis having a structure of a dermis layer and an epidermis layer. The artificial epidermis cosmetic detection kit prepared by the invention can be applied to the detection of related effects of cosmetics.
A preparation method of an artificial epidermis cosmetic detection kit comprises the following steps:
(1) separating a human skin tissue original sample to obtain primary skin cells, wherein the primary skin cells can be selectively cultured by a dermal cell culture medium and an epidermal cell culture medium respectively to obtain human skin fibroblasts and human skin epidermal cells;
(2) subculturing the human skin fibroblasts obtained in the step (1) until the passage reaches P3, and obtaining a suspension of P3 generation human skin fibroblasts;
(3) subculturing the human skin epidermal cells obtained in the step (1) until the passage reaches P3 generation to obtain P3 generation human skin epidermal cell suspension;
(4) placing the chamber with the polycarbonate membrane in a six-hole cell culture plate, pretreating the chamber by using Coating Matrix, mixing 500 mu l of the P3 generation human skin fibroblast suspension prepared in the step (2) with type I collagen, adding the mixture into the pretreated chamber, keeping the mixed collagen with the final concentration of 0.8mg/ml for 20-60min in an incubator at 35-37 ℃, adding a dermis culture medium into the inner part and the outer part of the chamber after standing, culturing for 2-3 days in the incubator at 35-37 ℃, discarding the culture medium, and culturing dermis seepage for 2-3 times to obtain the chamber with the dermis of the artificial skin;
(5) adding 500 mul of the human skin epidermal cell suspension obtained in the step (3) into the small chamber with the artificial skin corium layer obtained in the step (4), standing for 20-60min in an incubator at 35-37 ℃, adding an epidermal culture medium inside and outside the small chamber after standing, culturing for 2-3 days in the incubator at 35-37 ℃ for epidermal layer culture, discarding the epidermal culture medium, replacing the artificial epidermal culture medium, culturing for 5-10 days in the incubator at 35-37 ℃, changing the culture medium every day, discarding the culture medium after finishing the culture, transferring to a 24-hole culture plate, adding a solid preservation culture medium at the bottom of a hole for preservation, sealing the plate, packaging and then placing at 2-4 ℃ for preservation.
Further, the cell concentration of the P3 generation human skin fibroblast suspension in the step (2) is 40-400 ten thousand/ml; the cell concentration of the P3 generation human skin epidermal cell suspension in the step (3) is 40-1000 ten thousand/ml.
Further, the dermal cell culture medium in the step (4) is a dermal cell culture medium containing 1-100 μ M rock inhibitor, and the dermal cell culture medium (patent No. 201610473348.1) comprises the following components: mixing 250ml of DMEM high-sugar medium and 250ml of F12 nutrient medium by 500ml, 5-20ml of B27 additive, 5-20ml of PC-1 additive, 10-100 mu l of gene recombinant human epidermal growth factor and 5ml of streptomycin;
the epidermal culture medium (patent number 201610473310.4) comprises 250ml of cutin-free serum-free culture medium, 125ml of DMEM culture medium, 125ml of Ham's F-12 culture medium, 200 mML-glutamine (final concentration is 1.5 mM), bovine pituitary extract (final concentration is 25 mug/ml), epidermal growth factor (final concentration is 0.2 ng/ml), dry cell growth factor (final concentration is 30 ng/ml), fibroblast growth factor (fibroblast growth factor) is 1.5ng/ml, transforming growth factor- β (final concentration is 0.8 ng/ml), vascular endothelial growth factor (final concentration is 10 ng/ml), 100X penicillic acid-streptomycin (final concentration is 1X), 0.3M calcium chloride solution (final concentration is 0.4 mM);
the artificial epidermal culture medium comprises the following components: 30-60% of DMEM basal medium, 30-60% of F12 basal medium, 0.1-4% of glutamine, 0.1-4% of adenine, 0.1-4% of calcium chloride and 0.05-2% of hydrocortisone.
Further, the solid preservation medium in the step (5) is the artificial epidermis medium added with 0.3% -0.6 agar.
The artificial epidermis cosmetic detection kit prepared according to the preparation method.
The application of the artificial epidermis cosmetic detection kit in the aspect of detecting the effect of cosmetics on human skin.
Further, the effect of the above cosmetics on human skin means irritation, corrosiveness, anti-aging, anti-oxidation, and whitening effects of the cosmetics on human skin.
Further, the method for detecting the irritation, corrosivity, anti-aging property, anti-oxidation and whitening effects of the cosmetics on human skin by using the artificial epidermis cosmetic detection kit comprises the following steps:
s1, transferring the chamber to a new six-hole cell culture plate, adding an artificial epidermal culture medium outside each hole, and incubating for 4-18h in a cell culture box at 35-37 ℃;
s2 incubating, adding the cosmetic to be tested into the incubated small chamber, reacting at room temperature for 3-30min, and stopping acting;
s3, transferring the chamber processed in the step S2 to a new six-hole cell culture plate containing artificial epidermal culture medium, and incubating for 46-50h in a cell culture box at 35-37 ℃;
s4, transferring the cell incubated in the step S3 to a new 24-well cell culture plate, and verifying the irritation, corrosivity, anti-aging, anti-oxidation and whitening effects of the cosmetics on the skin by using different dyeing methods.
Further, the cosmetic to be detected in the step S2 is a solid cosmetic or a liquid cosmetic, and the detection dosage of the solid cosmetic is 0.1-5g, and the detection dosage of the liquid cosmetic is 10-500 μ l.
Further, the different dyeing methods described in the above step S4 are: MTT direct staining method, comparison of viable cell number after MTT staining method and silver staining method.
Has the advantages that:
(1) after the related samples act on the artificial epidermis of the cosmetic detection kit, different effects of cosmetics or related chemicals on the skin can be verified by using different dyeing detection modes, and the effect detection of the irritation, corrosivity, anti-aging, anti-oxidation and whitening effects of the cosmetics on the skin can be realized.
(2) The small chamber used for preparing the artificial epidermis is a polycarbonate membrane small chamber, and when the artificial epidermis is immersed and cultured, the inner chamber and the outer chamber can exchange substances through the holes on the membranes; when gas-liquid culture is required, the volume of the culture solution can be reduced, and the culture solution is in contact with the bottom of the artificial epidermis to provide nutrition for the production of the artificial epidermis.
(3) Our artificial epidermis uses type I collagen as a scaffold material, which can form a three-dimensional structure after gelling, fibroblasts of the artificial epidermis can continuously proliferate in the three-dimensional space of collagen to form a dermis layer structure consisting of a collagen skeleton and dermal fibroblasts, epidermal cells are inoculated on the dermis layer, and the epidermis cells are continuously differentiated upwards through growth, proliferation and differentiation to form a plurality of layers of epidermal cells, and the epidermal cells are exposed in the air and continuously differentiated into a stratum corneum after an air-liquid surface culture process, and other cells maintain the characteristics of the epidermal cells, so that the artificial epidermis similar to human skin can be formed.
(4) The artificial epidermis provided by the patent is close to normal skin, can generate skin accessory organs such as hair follicle sweat glands and the like after transplantation to form complete functional skin, and has the advantages of simple preparation method and short period.
Drawings
FIG. 1 is a schematic view of a cosmetic detection kit culturing apparatus.
FIG. 2 is a schematic view showing the appearance of the cosmetic detection kit culturing process.
FIG. 3 is a comparison of the detection effect of the sample in the chamber of the cosmetic detection kit.
FIG. 4 is a structural composition of paraffin sections of artificial epidermis.
FIG. 5 is a comparative analysis chart of the structural composition of the paraffin sections of the artificial epidermis after the detection of two samples.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, which are only a part of examples of the present application, but not all examples, and the present invention is not limited by the following examples.
EXAMPLE 1 preparation of cosmetic detection kit
First, the manufacturing method
1. Separation of human skin cells: human primary skin cells are obtained by separating a human skin tissue primary sample.
(1) A raw sample of human skin tissue was taken into a laboratory, the tissue was placed in a 10cm cell culture dish in a clean bench and the subcutaneous fat of the tissue was carefully removed with sterile forceps and scissors (the thickness of the fat remaining was not more than 1 mm).
(2) Soaking the skin with epidermis facing downwards and dermis facing upwards in 75% alcohol for 3-10min in a new 10cm cell culture dish, soaking in PBS (phosphate buffer solution) containing 2 × gentamicin for 2 times and 3-10 min/time to achieve sterilization, weighing and recording.
(3) The tissues were cut into minced with 2 disposable sterile scalpels crossed 90 degrees, with no visible particles.
(4) Carefully transferring the minced skin tissue into a centrifuge tube containing mixed enzyme solution (1-3mg/ml Collagenase and 1-3mg/ml Dispase), mixing uniformly, performing digestion with 37 ℃ water bath and shaking at 80rpm, and shaking the centrifuge tube every 5-15min during digestion to shake out tissue lumps. When digestion is complete, no large tissue mass should be present in the centrifuge tube, and the tissue becomes a transparent, homogenous, and pulpous state.
(5) And (3) pancreatin digestion: adding 0.25% pancreatin into the centrifuge tube containing tissue, mixing, and digesting for 10-30min until the final concentration is 0.01-0.1%.
(6) DNA enzyme digestion: finally, 10mg/ml DNase, 100-.
(7) And (4) neutralizing enzyme liquid to stop digestion: adding the same volume of DMEM containing 2-15% hemotizan into the centrifuge tube to neutralize the enzyme digestion solution, and repeatedly beating for 20-50 times to stop digestion.
(8) Cell filtration: the 100 μm cell filter was placed in a 50ml centrifuge tube and the digested cell suspension was added to the filtration system.
(9) The filtered cell suspension was centrifuged at 1000rpm for 5min and the supernatant was discarded. The obtained cell sediment is the primary skin cell obtained by separation.
2. Primary and subculture of human skin fibroblasts:
(1) the primary skin cell pellet was resuspended in 10-40ml of DMEM medium containing 1 × gentamicin, the cell pellet was blown off and the cells counted.
(2) A corresponding amount of primary skin cell suspension was transferred to a new 50ml centrifuge tube, centrifuged at 1000rpm for 5min and the supernatant discarded.
(3) The primary fibroblasts were cultured in dermal medium at 7 ml/dish and 800 ten thousand/dish at 200-.
(4) When the density of primary fibroblasts reaches 80%, subculturing the cells, wherein 1 is added for each passage of 1 generation.
(5) Removal of old medium: old dermal media was removed from the dishes.
(6) Cleaning: the residual old medium was removed by washing once with PBS (5-10 ml).
(7) Enzymatic digestion TrypLE Express (1 ×) from gibco, 2 ml/dish, was added and digested for 3-8min at 37 ℃ in an incubator.
(8) Neutralizing: and after the digestion time is over, observing under a microscope, adding DMEM containing 5-15% hemotix to stop digestion after the cells completely fall off, blowing and uniformly mixing, and transferring the cells into a 50ml centrifuge tube.
(9) And (3) cell collection, namely centrifuging the cell suspension in the centrifugal tube at 1000rpm for 5min, discarding the supernatant, resuspending the cell precipitate by using 10-40ml of DMEM containing 1 × gentamicin, blowing and uniformly mixing, counting by using a counting plate or a cell counter, and carrying out cell passage or cryopreservation as required.
(10) Passage of human fibroblasts: taking a corresponding amount of fibroblast suspension to a new 50ml centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, suspending the fibroblast with a proper amount of dermal culture medium, 8ml/T75, 120-200 ten thousand/T75, and culturing in an incubator at 37 ℃.
3. Primary and subculture of human skin epidermal cells:
(1) a corresponding amount of primary skin cell suspension was transferred to a new 50ml centrifuge tube, centrifuged at 1000rpm for 5min and the supernatant discarded.
(2) The cell culture dish was pretreated with Coating Matrix, allowed to stand at room temperature for 10min, and the excess liquid was discarded.
(3) Adding a dermis culture medium containing 1-100 mu M rock inhibitor to suspend the cell sediment, placing the cell sediment in a 7 ml/dish and 800 ten thousand/dish at 200 ℃, placing the cell sediment in an incubator at 37 ℃ for culture, recording as EP0 generation, and changing the epidermal cell culture medium after 3 days of culture.
(4) When the density of the primary epidermal cells reaches 80%, subculturing the cells is needed, and 1 is added every 1 generation.
(5) Removal of old medium: the plates were cleaned of old epidermal medium.
(6) Cleaning: the residual old medium was removed by washing once with PBS (5-10 ml).
(7) Enzymatic digestion TrypLE Express (1 ×) from gibco, 2 ml/dish, was added and digested for 8-15min at 37 ℃ in an incubator.
(8) Neutralizing: and after the digestion time is over, observing under a microscope, adding DMEM containing 5-15% hemotix to stop digestion after the cells completely fall off, blowing and uniformly mixing, and transferring the cells into a 50ml centrifuge tube.
(9) And (3) cell collection, namely centrifuging the cell suspension in the centrifugal tube at 1000rpm for 5min, discarding the supernatant, resuspending the cell precipitate by using 10-40ml of DMEM containing 1 × gentamicin, blowing and uniformly mixing, counting by using a counting plate or a cell counter, and carrying out cell passage or cryopreservation as required.
(10) Passage of human epidermal cells: taking a corresponding amount of epidermal cell suspension to a new 50ml centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, suspending the epidermal cells with an appropriate amount of epidermal cell culture medium, 8ml/T75, 120-200 ten thousand/T75, and culturing in an incubator at 37 ℃.
4. Preparing and culturing an artificial epidermis model:
(1) the cell with the polycarbonate membrane and the diameter of 9mm is placed in a six-hole cell culture plate, the inside and the outside of the cell are pretreated by 200-. The chamber is a small circular device in the middle part shown in fig. 2, and the device is independent, movable and removable.
(2) Preparing an artificial epidermis dermis: the concentration range of the type I collagen is 0.05-1.50mg/ml, the concentration range of the human skin fibroblasts of the P3 generation is 40-400 ten thousand/ml, the collagen and the human skin fibroblasts are mixed evenly and then are carefully added into a chamber, 500 mu l/hole is formed after mixing, bubbles are avoided, and the gel is placed in an incubator at 37 ℃ for standing and gelling for 20-60 min.
(3) After the corium layer is gelled, adding a corium culture medium inside and outside the small chamber for culturing the corium layer, wherein the dosage of the culture medium in the small chamber is as follows: 100-: 3000-6000. mu.l of the culture broth were placed in an incubator at 37 ℃ for 2-3 days.
(4) Preparing an artificial epidermis layer: after the dermis layer is cultured, removing the dermis culture medium, infiltrating the dermis layer for 2-3 times for 10-20 min/time, uniformly adding the P3 generation human epidermal cells with the cell concentration of 400 plus 1000 ten thousand/ml above the dermis layer of the artificial epidermis to construct a uniform epidermis layer with 500 mul/hole of 100 plus, so as to avoid bubbles, and standing and attaching the epidermis layer in a 37 ℃ culture box for 20-60 min.
(5) After the epidermal layer is prepared, adding epidermal culture medium inside and outside the small chamber for culturing the epidermal layer, wherein the dosage of the culture medium in the small chamber is as follows: 100-: 3000-6000. mu.l of the culture broth were placed in an incubator at 37 ℃ for 2-3 days.
(6) Artificial epidermis keratinization culture: after the artificial epidermis layer is cultured, the epidermis culture medium is abandoned, the artificial epidermis culture medium is replaced, and the artificial epidermis culture medium is added outside the small chamber, so that the artificial epidermis is differentiated into a cuticle structure. The amount of the culture medium outside the chamber: 3000 μ l of 1000-.
(7) After the artificial epidermis culture is finished, the old culture medium is discarded, the culture medium is transferred to a 24-hole cell culture plate, the bottom of a hole is added with 200-.
Second, result in
As shown in fig. 1, a in fig. 1 is the stratum corneum (containing keratinocytes differentiated from epidermal cells); in the figure, b represents an epidermal layer (formed by epidermal cells); in the figure, c is the dermis layer (the cellular collagen layer, namely the dermis layer, is composed of collagen gel and a plurality of layers of dermal fibroblasts, and the whole batch of dermal fibroblasts present a three-dimensional growth pattern in collagen); d in the figure is a polycarbonate membrane; in the figure, e is a culture medium; in the figure f is the artificial epidermis cell. Fig. 4 is the structure composition of the paraffin section of the artificial epidermis, and the structure after the artificial epidermis is built can be seen. The structural composition of the paraffin section of the artificial epidermis is shown in figure 4.
As shown in FIG. 2, the appearance of the cosmetic detection kit during the culture process is shown, and the dry, smooth and liquid-tight skin surface can be observed in the artificial epidermis with a better structure in the final culture stage. The artificial epidermis small chamber with good structure is selected for storage, sealing and packaging, and can be used as a product for cosmetic or other chemical detection models.
The bottom of the small chamber is a layer of polycarbonate membrane, the artificial epidermis is cultured on the polycarbonate membrane, and the detection box is the small chamber filled with the artificial epidermis. The six-well plate in FIG. 2 is not a part of the cartridge of the present invention, but is used only for culturing, and the 12-well plate or 24-well plate described below is also a container for housing the chamber.
Example 2 experiment of cosmetic detection kit for detecting effects of test sample on skin
Firstly, detection step
The total of 5 selected test samples are as follows: non-irritating PBS buffer solution, non-irritating naphthylacetic acid solid, cosmetic original skin repairing solution, anhydrous ethanol and irritant substance 5% SDS.
Each sample was 3 replicates and required a total of 15 artificial epidermal cells.
(1) The cosmetic detection kit package is disassembled, the small chamber is transferred to a new six-hole cell culture plate, 500-.
(2) After the incubation is finished, adding a sample to be detected into the artificial epidermis small chamber, acting for 15min at room temperature, wherein the dosage of the solid sample is 0.2mg, and the dosage of the liquid cosmetic is 20 mul.
(3) After the action time of the sample to be detected is over, the cell is transferred to sterile absorbent paper by using sterile tweezers, the sample in the cell is carefully absorbed by using a sterile cotton swab, and then the inside and the outside of the cell are repeatedly washed by using sterile PBS for 20 times so as to stop the action of the sample.
(4) After the washing of the cell, excess liquid inside and outside the cell was blotted with a sterile cotton swab, and the cell was transferred to a new six-well cell culture plate containing 1000. mu.l of artificial epidermal medium, cultured in a cell culture chamber at 37 ℃ for 48 hours, and changed every day.
(5) After the end of the culture, the old medium was discarded, and the inside and outside of the chamber were washed 2 times with sterile PBS.
(6) The chamber was transferred to a new 24-well cell culture plate using sterile forceps, and 500. mu.l of MTT stain solution having a concentration of 1mg/ml was added to the 24-well cell culture plate, and a little was added dropwise to the chamber. The 24-well cell culture plate was placed in a 37 ℃ cell incubator for staining for 3 h.
(7) After MTT staining was completed, excess MTT staining solution was discarded, and the inside and outside of the chamber were washed 2 times with sterile PBS, and the staining results are shown in FIG. 4.
(8) Blue-violet crystalline formazan was dissolved in 1000. mu.l of dimethyl sulfoxide (DMSO) in a 24-well cell culture plate with a chamber, and a little was added dropwise to the chamber. The 24-well cell culture plate was placed on a shaker and shaken at low speed for 15 min.
(9) The crystals dissolved were transferred to a 96-well plate at 200. mu.l/well and the light absorption was measured at a wavelength of 570nm using an enzyme linked immunosorbent assay. The 5 samples had average OD570nm of 1.756, 1.755, 1.756, 0.801 and 0.052 respectively.
Second, result analysis
As shown in FIG. 3, it can be analyzed from the results of the tests that the negative control phosphate buffered saline PBS, the solid powder naphthylacetic acid and the cosmetic have no irritating damage to the artificial epidermis due to the skin-repairing solution, and the anhydrous ethanol and the irritant substance 5% SDS have different degrees of damage to the artificial epidermis, so that whether the samples have damage to the skin can be detected.
Different effects of the cosmetic on the skin can be verified using different dyeing methods in the above step (6). The direct MTT staining method is used for detecting the number of living cells, so that the results of the stimulation and the corrosivity of the cosmetics to the skin can be obtained, and compared with a control, after the cosmetics act on the artificial epidermis, the more deep and uniform MTT staining indicates that the stimulation and the corrosivity of the cosmetics to the skin are weaker or the stimulation and the corrosivity are lower; after the cosmetics act on the artificial epidermis, the artificial epidermis in the small chamber is cultured for different time points and then is subjected to an MTT staining method to detect the number of living cells, so that the anti-aging effect of the cosmetics on the skin can be obtained, and compared with a control, the lower the reduction degree of the number of the living cells after the cosmetics act on the artificial epidermis is, the better the anti-aging effect of the cosmetics on the skin is; the content of the melanin particles in the artificial epidermis is detected by a silver staining method, so that the whitening effect of the cosmetic on the skin can be obtained, and compared with a control, the lower the content of the melanin particles in the artificial epidermis after the effect of the cosmetic is realized, the better the whitening effect of the cosmetic on the skin is.
Example 3 cosmetic detection kit test efficacy validation
To further illustrate the detection efficacy of the cosmetic detection kit, paraffin sections are taken on the artificial epidermis after sample detection, and the structural composition is observed and analyzed by a microscope. The operation steps are as follows:
(1) sampling: two sample-tested epidermal cells (test samples: PBS and absolute ethanol) were selected, washed twice with PBS, and the epidermal tissue was carefully cut along the edges of the cells with a scalpel.
(2) Fixing: tissues were individually placed in small white boxes, pencil-labeled, 4% paraformaldehyde overnight for fixation, preferably in an amount that covered the tissue of the sample.
(3) Flushing with running water: flushing with running water for 1 h.
(4) And (3) dehydrating: pure water 5min → 50% ethanol 1h → 70% 1h → 80% 8h → 95% 1h → 95% 1h → 100% 1h → 100% 1h to complete dehydration.
(5) And (3) xylene transparency: the embedding machine should be opened up before this step is started, and the tissue is usually placed directly in pure xylene and operated in a fume hood. The typical residence time was 1h until the tissue changed color to an amber-like transparent color.
(6) Embedding: and melting the paraffin at the temperature of 62 ℃, putting the transparent sample into a melted paraffin box for 1h, taking out and putting into a second paraffin box for 1 h. If the user can not do the work in time, the user can always put the device in a paraffin box. Taking out a small amount of liquid paraffin, dripping the liquid paraffin into an embedding box, putting the tissue into the embedding box according to the requirement, and continuously adding the liquid paraffin until the tissue is full. And placing a cooling table for cooling.
(7) Slicing: the thickness of the paraffin section is generally 3-5 μm. Exhibition piece → roast piece (60 ℃ for 12 h).
(8) Paraffin sections were stored at room temperature and then HE stained.
(9) The xylene was dewaxed transparently for 5 min/time and 2 times.
(10) Removing dimethylbenzene: absolute ethanol → 95% ethanol → 80% ethanol → 75% ethanol for 3min respectively
(11) Fixing: fixing with 4% paraformaldehyde for 10min (Wet box, flat)
(12) Water washing 2s → hematoxylin staining (room temperature) 45s → water washing 10s → 1% ethanol hydrochloride 3s (differentiation) → water washing 5s → bluing liquid bluing 10s → water washing 15s → 0.5% red liquid staining 45s → water washing 2 s.
(13) And (3) dehydrating: 75% ethanol 2s → 80% ethanol 2s → 95% ethanol 2s → absolute ethanol 2 s.
(14) And (3) transparency: xylene 2s → xylene 2s or longer waiting for a seal.
(15) And sealing the neutral gum into a piece, drying in the air and observing under a mirror.
The observation results are shown in fig. 5: the absolute ethyl alcohol has certain irritant damage to the artificial epidermis and certain destructiveness to the skin structure, so that the skin tissue structure composition is destroyed. Therefore, it can be estimated that if the cosmetics or other samples contain components which are destructive to the skin, the skin structure composition can be damaged, and the artificial epidermis cosmetic detection kit can be used for well detecting whether the related cosmetics and the like are qualified or not.
In addition, the whitening effect of the cosmetics on the skin can be detected, the detection steps are the same as the irritation detection, the existence condition of the melanin particles in the artificial epidermis is dyed by a silver dyeing method during verification, and the reduction of the melanin particles indicates that the cosmetics have the corresponding whitening effect. Similarly, other related dyeing inspection modes can be utilized to find that the detection kit prepared by the invention can effectively detect various effects of the product on the skin.
Claims (10)
1. The preparation method of the artificial epidermis cosmetic detection kit is characterized by comprising the following steps:
(1) separating a human skin tissue original sample to obtain primary skin cells, wherein the primary skin cells can be selectively cultured by a dermal cell culture medium and an epidermal cell culture medium respectively to obtain human skin fibroblasts and human skin epidermal cells;
(2) subculturing the human skin fibroblasts obtained in the step (1) until the passage reaches P3, and obtaining a suspension of P3 generation human skin fibroblasts;
(3) subculturing the human skin epidermal cells obtained in the step (1) until the passage reaches P3 generation to obtain P3 generation human skin epidermal cell suspension;
(4) mixing the P3 generation human skin fibroblast suspension prepared in the step (2) and type I collagen, putting the mixture into a small chamber with a polycarbonate membrane, adding a dermis culture medium into the small chamber after standing, culturing for 2-3 days in an incubator at 35-37 ℃, and percolating the cultured dermis layer for 2-3 times to obtain the small chamber filled with the artificial skin dermis layer;
(5) adding the human skin epidermal cell suspension obtained in the step (3) into the small chamber with the artificial skin dermis obtained in the step (4), standing, adding an epidermal culture medium into the small chamber, culturing for 2-3 days in an incubator at 35-37 ℃, replacing the artificial epidermal culture medium, culturing for 5-10 days in the incubator at 35-37 ℃, adding a solid storage culture medium after the culture is finished, storing, sealing plates, packaging and storing.
2. The method according to claim 1, wherein the cell concentration of the suspension of P3 generation human skin fibroblasts in step (2) is 40 to 400 ten thousand/ml; the cell concentration of the P3 generation human skin epidermal cell suspension in the step (3) is 40-1000 ten thousand/ml.
3. The method according to claim 1, wherein the dermal medium in the step (4) is a dermal cell medium containing 1 to 100 μ M rock inhibitor; the dermal cell culture medium comprises the following components: mixing 250ml of DMEM high-sugar medium and 250ml of F12 nutrient medium by 500ml, 5-20ml of B27 additive, 5-20ml of PC-1 additive, 10-100 mu l of gene recombinant human epidermal growth factor and 5ml of streptomycin;
the epidermal culture medium comprises 250ml of cutin serum-free culture medium, 125ml of DMEM culture medium, 125ml of Ham's F-12 culture medium, 200 mML-glutamine, the final concentration of 1.5mM, the final concentration of 25 mug/ml of bovine pituitary extract, the final concentration of 0.2ng/ml of epidermal growth factor, the final concentration of 30ng/ml of stem cell growth factor, 1.5ng/ml of fibroblast growth factor, 0.8ng/ml of transforming growth factor- β, the final concentration of 10ng/ml of vascular endothelial growth factor, 100X penicillic acid-streptomycin, the final concentration of 1X, and the final concentration of 0.4mM of 0.3M calcium chloride solution, wherein the total concentration of the culture medium is calculated by 500 ml;
the artificial epidermal culture medium comprises the following components: 30-60% of DMEM basal medium, 30-60% of F12 basal medium, 0.1-4% of glutamine, 0.1-4% of adenine, 0.1-4% of calcium chloride and 0.05-2% of hydrocortisone.
4. The method according to claim 3, wherein the solid preservation medium in the step (5) is an artificial epidermal medium supplemented with 0.3% -0.6 agar.
5. The artificial epidermis cosmetic detection kit prepared by the preparation method according to any one of claims 1 to 4.
6. Use of the artificial epidermis cosmetic detection kit of claim 5 in detecting the effect of a cosmetic on human skin.
7. The use of claim 6, wherein the cosmetic effect on human skin means irritation, corrosivity, anti-aging, anti-oxidation, and whitening of the cosmetic on human skin.
8. The method for detecting the skin irritation, corrosion, anti-aging, anti-oxidation and whitening effects of cosmetics by using the artificial epidermis cosmetic detection kit according to claim 5, which comprises the following steps:
s1, transferring the chamber to a new six-hole cell culture plate, adding artificial epidermis culture solution outside each hole, and placing the cell culture plate in a cell culture box at 35-37 ℃ for incubation for 4-18 h;
s2 incubating, adding the cosmetic to be tested into the incubated small chamber, reacting at room temperature for 3-30min, and stopping acting;
s3, transferring the chamber processed in the step S2 to a new six-hole cell culture plate containing artificial epidermal culture solution, and incubating for 46-50h in a cell culture box at 35-37 ℃;
s4, transferring the cell incubated in the step S3 to a new 24-well cell culture plate, and verifying the irritation, corrosivity, anti-aging, anti-oxidation and whitening effects of the cosmetics on the skin by using different dyeing methods.
9. The method according to claim 8, wherein the cosmetic to be tested in the step S2 is a solid cosmetic or a liquid cosmetic, and the detected amount of the solid cosmetic is 0.1-5g and the detected amount of the liquid cosmetic is 10-500 μ l.
10. The method of claim 8, wherein the different dyeing methods in step S4 are: MTT direct staining method, comparison of viable cell number after MTT staining method and silver staining method.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686420A (en) * | 2022-04-08 | 2022-07-01 | 济南磐升生物技术有限公司 | Preparation method and application of 3D epidermis model |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1218837A (en) * | 1997-12-02 | 1999-06-09 | 中国科学院新疆化学研究所 | Detecting and screening method for biological active additive component of cosmetics |
| US20030096409A1 (en) * | 2001-11-22 | 2003-05-22 | Shigeru Yasumoto | Cultured skin and method of manufacturing the same |
| CN101297981A (en) * | 2008-07-03 | 2008-11-05 | 中国检验检疫科学研究院 | Tissue engineering skin construction method and tissue engineering skin produced by the same |
| US20090239254A1 (en) * | 2008-03-17 | 2009-09-24 | L'oreal | Functional pigmented skin equivalent |
| JP2011092179A (en) * | 2009-09-30 | 2011-05-12 | Toyobo Co Ltd | 3-dimensional cultured skin model containing melanocyte, and use of the same |
| JP2011092180A (en) * | 2009-02-26 | 2011-05-12 | Toyobo Co Ltd | Medium suitable for ultraviolet radiation experiment using 3-dimensional cultured skin model |
| WO2013191531A1 (en) * | 2012-06-20 | 2013-12-27 | Universiti Kebangsaan Malaysia | Autologous tissue-engineered human skin construct and a method for producing thereof |
| CN104459145A (en) * | 2013-09-13 | 2015-03-25 | 王阿慧 | Cosmetic detection |
| CN104667352A (en) * | 2015-02-03 | 2015-06-03 | 济南磐升生物技术有限公司 | Preparation method of tissue engineering epidermis having hypodermal cells |
| CN105861421A (en) * | 2016-06-24 | 2016-08-17 | 济南磐升生物技术有限公司 | Multifunctional serum-free cell culture medium and application thereof |
| CN105950542A (en) * | 2016-06-24 | 2016-09-21 | 济南磐升生物技术有限公司 | Human skin epidermal cell culture medium and application thereof |
| CN108693183A (en) * | 2018-03-27 | 2018-10-23 | 中南民族大学 | The method for detecting cosmetic composition sensitization by superweak bio-photon imaging system |
-
2020
- 2020-03-27 CN CN202010227486.8A patent/CN111337494A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1218837A (en) * | 1997-12-02 | 1999-06-09 | 中国科学院新疆化学研究所 | Detecting and screening method for biological active additive component of cosmetics |
| US20030096409A1 (en) * | 2001-11-22 | 2003-05-22 | Shigeru Yasumoto | Cultured skin and method of manufacturing the same |
| US20090239254A1 (en) * | 2008-03-17 | 2009-09-24 | L'oreal | Functional pigmented skin equivalent |
| CN101297981A (en) * | 2008-07-03 | 2008-11-05 | 中国检验检疫科学研究院 | Tissue engineering skin construction method and tissue engineering skin produced by the same |
| JP2011092180A (en) * | 2009-02-26 | 2011-05-12 | Toyobo Co Ltd | Medium suitable for ultraviolet radiation experiment using 3-dimensional cultured skin model |
| JP2011092179A (en) * | 2009-09-30 | 2011-05-12 | Toyobo Co Ltd | 3-dimensional cultured skin model containing melanocyte, and use of the same |
| WO2013191531A1 (en) * | 2012-06-20 | 2013-12-27 | Universiti Kebangsaan Malaysia | Autologous tissue-engineered human skin construct and a method for producing thereof |
| CN104459145A (en) * | 2013-09-13 | 2015-03-25 | 王阿慧 | Cosmetic detection |
| CN104667352A (en) * | 2015-02-03 | 2015-06-03 | 济南磐升生物技术有限公司 | Preparation method of tissue engineering epidermis having hypodermal cells |
| CN105861421A (en) * | 2016-06-24 | 2016-08-17 | 济南磐升生物技术有限公司 | Multifunctional serum-free cell culture medium and application thereof |
| CN105950542A (en) * | 2016-06-24 | 2016-09-21 | 济南磐升生物技术有限公司 | Human skin epidermal cell culture medium and application thereof |
| CN108693183A (en) * | 2018-03-27 | 2018-10-23 | 中南民族大学 | The method for detecting cosmetic composition sensitization by superweak bio-photon imaging system |
Non-Patent Citations (2)
| Title |
|---|
| 宋肖洁: "《3D皮肤模型的构建及其在化妆品原料活性物功效检测中的应用研究》", 《日用化学工业》 * |
| 李辉: "《细胞共培养人体皮肤类似物超微结构观察及其渗透性测试》", 《北京中医药大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686420A (en) * | 2022-04-08 | 2022-07-01 | 济南磐升生物技术有限公司 | Preparation method and application of 3D epidermis model |
| CN114686420B (en) * | 2022-04-08 | 2023-11-28 | 济南磐升生物技术有限公司 | Preparation method and application of 3D epidermis model |
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