CN103884835A - Method for constructing tissue engineering epidermis for detecting irritation of cosmetics on polycarbonate film - Google Patents
Method for constructing tissue engineering epidermis for detecting irritation of cosmetics on polycarbonate film Download PDFInfo
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Abstract
The invention relates to a method for constructing a tissue engineering epidermis for detecting the irritation of cosmetics on a polycarbonate film. The method is characterized by comprising the following steps: (1) carrying out separated culture on epidermization cells; (2) preparing a lipid mixture solution; and (3) constructing the tissue engineering epidermis on the polycarbonate film. The method has the advantages that the operation is easy and convenient and the cost is low; the method is good for synthesis and large-scale production of a product and the product has a basal layer, a spinous layer, a granular layer, a transparent layer and the cuticle tissue engineering epidermises which are in completely differentiated shapes, and the irritation of the cosmetics can be conveniently detected.
Description
Technical field
The present invention relates to a kind of method for detection of the irritating tissue engineering epidermis of cosmetics that builds on polycarbonate membrane.
Background technology
Tradition skin irritatin and corrosion test adopt animal to carry out more.In single body, skin irritant test generally carries out with rabbit, and 4 healthy adults are at least selected in each test.Have as expected irritant reaction, preliminary examination should consider to use 1 animal.As do not there is obvious positive reaction (erythema or oedema are scored and be greater than 2) time, should at least re-use 2 animals and test.Reactionless as expected, preliminary examination can be used 3 animals.Using after at least 3 animals, as being still doubtful reaction or indefinite, should consider to carry out retrial.Repeatedly the interior skin irritant test of body refers in 7-14 days animal used as test is carried out to repetition skin contamination, more effective than single contamination to detecting the irritating effect of cosmetics, is specially adapted to and personal care product skin long-term close contact or Reusability.More than three groups animals is selected in each test, and the each 5-10 of every treated animal male and female only, test by the each group of tested material of accepting respectively various dose.According to the character of product and purposes, repeatedly the adjustment of contamination program, mode and number of times is carried out in skin irritatin test.The zoopery of skin irritatin and corrosion test, the animal that consumes in batches large, cost is high.
Zooperal minimizing is proposed, substitute and optimizes (reduction from Russell in 1959 and Burch, replacement, and refinement, 3R) since theory, external 3R research and development is rapid, particularly nearly 20 years, no longer become biomedical important development direction take zootype as basic toxicology system, and accepted by government and research institution.Some zoopery alternative methods become the technology barriers that are related to international trade.Europe method of substitution authentication center (European Center for the Validation of Alternative Methods, ECVAM) " application on human skin model is for the technical standard of vitro skin irritant test " guide of issuing in May, 2007, and in " two the vitro skin irritant test verification methods " statement of issuing in November, 2008, all show that organization engineering skin can be used as the alternative model of vitro skin irritant test, as organization engineering skin Episkin, EpiDerm and SkinEthic etc., but these commercial products exist expensive, the defect such as originate limited, therefore under laboratory condition, build price comparatively cheap, the organization engineering skin model that can be used for skin irritatin test of being convenient to batch production can effectively address these problems.
Summary of the invention
The technical problem to be solved in the present invention is, a kind of method for detection of the irritating tissue engineering epidermis of cosmetics that builds on polycarbonate membrane is provided, the method is easy and simple to handle, be convenient to the synthetic and large-scale production of product, product has the intact cuticula of differentiation, convenient for detection of cosmetics pungency.
For solving the problems of the technologies described above, the present invention by the following technical solutions: a kind ofly on polycarbonate membrane, build for detection of the method for the irritating tissue engineering epidermis of cosmetics, it is characterized in that: adopt following steps:
(1) epidermis forms the separation cultivation of cell: under aseptic condition, get induced labor foetus skin of back 16cm2, be rolled into the skin bit of 0. 5cm × 0. 5cm size, wash with physiological saline, be that 0. 125% Dispase II enzyme was 20 ℃ of digestion 10 hours by 30ml mass percent, add again 10ml mass percent 0.1% trypsase 37 ℃ of digestion 30 minutes, acquisition is separated into the epidermal cell solution of individual cells, centrifugal 10 minutes of 5000rpm, remove supernatant liquor, with the lower confluent monolayer cells of physiological saline washing, then centrifugal 10 minutes of 5000rpm, after repeated washing and centrifugal 3 times, add 50ml to contain 30-60U/ml penicillin, the restricted epidermal cell serum free medium (DK-SFM) of 30-50ug/ml streptomysin, after shaking up, cultivate 24 hours at 37 ℃, obtain epidermis and form cell dispersion liquid, before inoculation, be 1-5 × 10 with normal saline dilution to cell density
5individual/ml,
(2) preparation of lipid mixture solution: take palmitic acid 150g, linoleic acid 100g, arachidonic acid 50g, be dissolved in 500ml 0.1 mol/L NaOH solution, be heated to 70 ℃, be incubated 30 minutes, then use 4-hydroxyethyl piperazine ethanesulfonic acid regulator solution pH value 6.5-7.0, the bovine serum albumin solution that is 10% by above-mentioned solution and mass percent by volume 1:1-2 mixes, and constant-temperature table mixes 20 minutes at 55 ℃;
(3) on polycarbonate membrane, build tissue engineering epidermis: get polycarbonate filtering membrane (thickness 0.2mm, aperture 0.4 μ m, area 0.64 cm
2) surperficial 10% the IV collagen aqueous solution that applies, after 37 ℃ of baking ovens placements are dried for 1 hour, the cell density of then preparing in surface seeding step 1 is 1-5 × 10
5the epidermis of individual/ml forms cell dispersion liquid 0.5ml, apply evenly, postvaccinal polycarbonate film is placed in to fresh Epilife nutrient culture media and carries out the cultivation of gas-liquid face, cultivate and after 48 hours, renew fresh Epilife nutrient culture media, by the 20-30ml lipid mixture solution that adds step 2 to prepare in every 100ml Epilife nutrient culture media, add CaCl
2and ascorbic acid, make Ca
2+concentration 1.5-2.0mmol/L, ascorbic acid concentrations is at 50-60 μ g/mL, continue cultivation and can be grown in polycarbonate membrane surface after 13 days, thickness is 0.3-0.6mm, has Morphological Differentiation basalis, spinous layer, stratum granulosum, hyaline layer and cuticular tissue's engineering epidermis completely.
The present invention adopts epidermis to form the method for cell and polycarbonate membrane structure tissue engineering epidermis model, sets up the tissue engineering epidermis model that can be used for skin irritatin test.Application Method of Tissue Engineering, take polycarbonate membrane as support, forms cell derived with application on human skin epidermis, adopts gas-liquid face culture technique, builds and can be used for detecting the irritating tissue engineering epidermis model of cosmetics.
Polycarbonate is due to its good biocompatibility and nontoxicity, building tumour cell metastasis model, make the medical domains such as medical dialysis membrane and be widely used.The tissue engineering epidermis model that this research builds on polycarbonate membrane, has the micromechanism similar to normal epidermis height, can be used as and detects the irritating tissue engineering epidermis of cosmetics.
The present invention has easy and simple to handle, and cost is low, is convenient to the synthetic and large-scale production of product, has Morphological Differentiation basalis, spinous layer, stratum granulosum, hyaline layer and cuticular tissue's engineering epidermis completely, convenient for detection of cosmetics pungency.
Embodiment
For further illustrating the present invention, specifically set forth with the following Examples:
embodiment 1:
On polycarbonate membrane, build the method for detection of the irritating tissue engineering epidermis of cosmetics, adopt following steps:
(1) epidermis forms the separation cultivation of cell: under aseptic condition, get induced labor foetus skin of back 16cm2, be rolled into the skin bit of 0. 5cm × 0. 5cm size, wash with physiological saline, be that 0. 125% Dispase II enzyme was 20 ℃ of digestion 10 hours by 30ml mass percent, add again 10ml mass percent 0.1% trypsase 37 ℃ of digestion 30 minutes, acquisition is separated into the epidermal cell solution of individual cells, centrifugal 10 minutes of 5000rpm, remove supernatant liquor, with the lower confluent monolayer cells of physiological saline washing, then centrifugal 10 minutes of 5000rpm, after repeated washing and centrifugal 3 times, add 50ml to contain 30U/ml penicillin, the restricted epidermal cell free serum culture (DK-SFM) of 30ug/ml streptomysin, after shaking up, cultivate 24 hours at 37 ℃, obtain epidermis and form cell dispersion liquid, before inoculation, be 1 × 10 with normal saline dilution to cell density
5individual/ml,
(2) preparation of lipid mixture solution: take palmitic acid 150g, linoleic acid 100g, arachidonic acid 50g, be dissolved in 500ml 0.1 mol/L NaOH solution, be heated to 70 ℃, be incubated 30 minutes, then use 4-hydroxyethyl piperazine ethanesulfonic acid regulator solution pH value 6.5, the bovine serum albumin solution that is 10% by above-mentioned solution and mass percent by volume 1:1 mixes, and constant-temperature table mixes 20 minutes at 55 ℃;
(3) on polycarbonate membrane, build tissue engineering epidermis: get polycarbonate filtering membrane (thickness 0.2mm, aperture 0.4 μ m, area 0.64 cm
2) surperficial 10% the IV collagen aqueous solution that applies, after 37 ℃ of baking ovens placements are dried for 1 hour, the cell density of then preparing in surface seeding step 1 is 1 × 10
5the epidermis of individual/ml forms cell dispersion liquid 0.5ml, apply evenly, postvaccinal polycarbonate film is placed in to fresh Epilife nutrient culture media and carries out the cultivation of gas-liquid face, cultivate and after 48 hours, renew fresh Epilife nutrient culture media, by the 20ml lipid mixture solution that adds step 2 to prepare in every 100ml Epilife nutrient culture media, add CaCl
2and ascorbic acid, make Ca
2+concentration is at 1.5mmol/L, and ascorbic acid concentrations, at 50 μ g/mL, continues cultivation and can be grown in polycarbonate membrane surface after 13 days, and thickness is 0.3mm, has Morphological Differentiation basalis, spinous layer, stratum granulosum, hyaline layer and cuticular tissue's engineering epidermis completely.
embodiment 2:
On polycarbonate membrane, build the method for detection of the irritating tissue engineering epidermis of cosmetics, adopt following steps:
(1) epidermis forms the separation cultivation of cell: under aseptic condition, get induced labor foetus skin of back 16cm2, be rolled into the skin bit of 0. 5cm × 0. 5cm size, wash with physiological saline, be that 0. 125% Dispase II enzyme was 20 ℃ of digestion 10 hours by 30ml mass percent, add again 10ml mass percent 0.1% trypsase 37 ℃ of digestion 30 minutes, acquisition is separated into the epidermal cell solution of individual cells, centrifugal 10 minutes of 5000rpm, remove supernatant liquor, with the lower confluent monolayer cells of physiological saline washing, then centrifugal 10 minutes of 5000rpm, after repeated washing and centrifugal 3 times, add 50ml to contain 35U/ml penicillin, the restricted epidermal cell free serum culture (DK-SFM) of 35ug/ml streptomysin, after shaking up, cultivate 24 hours at 37 ℃, obtain epidermis and form cell dispersion liquid, before inoculation, be 2 × 10 with normal saline dilution to cell density
5individual/ml,
(2) preparation of lipid mixture solution: take palmitic acid 150g, linoleic acid 100g, arachidonic acid 50g, be dissolved in 500ml 0.1 mol/L NaOH solution, be heated to 70 ℃, be incubated 30 minutes, then use 4-hydroxyethyl piperazine ethanesulfonic acid regulator solution pH value 6.5, the bovine serum albumin solution that is 10% by above-mentioned solution and mass percent by volume 1:1 mixes, and constant-temperature table mixes 20 minutes at 55 ℃;
(3) on polycarbonate membrane, build tissue engineering epidermis: get polycarbonate filtering membrane (thickness 0.2mm, aperture 0.4 μ m, area 0.64 cm
2) surperficial 10% the IV collagen aqueous solution that applies, after 37 ℃ of baking ovens placements are dried for 1 hour, the cell density of then preparing in surface seeding step 1 is 2 × 10
5the epidermis of individual/ml forms cell dispersion liquid 0.5ml, apply evenly, postvaccinal polycarbonate film is placed in to fresh Epilife nutrient culture media and carries out the cultivation of gas-liquid face, cultivate and after 48 hours, renew fresh Epilife nutrient culture media, by the 20-30ml lipid mixture solution that adds step 2 to prepare in every 100ml Epilife nutrient culture media, add CaCl
2and ascorbic acid, make Ca
2+concentration is at 1.5mmol/L, and ascorbic acid concentrations, at 50 μ g/mL, continues cultivation and can be grown in polycarbonate membrane surface after 13 days, and thickness is 0.4mm, has Morphological Differentiation basalis, spinous layer, stratum granulosum, hyaline layer and cuticular tissue's engineering epidermis completely.
embodiment 3:
On polycarbonate membrane, build the method for detection of the irritating tissue engineering epidermis of cosmetics, adopt following steps:
(1) epidermis forms the separation cultivation of cell: under aseptic condition, get induced labor foetus skin of back 16cm2, be rolled into the skin bit of 0. 5cm × 0. 5cm size, wash with physiological saline, be that 0. 125% Dispase II enzyme was 20 ℃ of digestion 10 hours by 30ml mass percent, add again 10ml mass percent 0.1% trypsase 37 ℃ of digestion 30 minutes, acquisition is separated into the epidermal cell solution of individual cells, centrifugal 10 minutes of 5000rpm, remove supernatant liquor, with the lower confluent monolayer cells of physiological saline washing, then centrifugal 10 minutes of 5000rpm, after repeated washing and centrifugal 3 times, add 50ml to contain 40U/ml penicillin, the restricted epidermal cell free serum culture (DK-SFM) of 40ug/ml streptomysin, after shaking up, cultivate 24 hours at 37 ℃, obtain epidermis and form cell dispersion liquid, before inoculation, be 3 × 10 with normal saline dilution to cell density
5individual/ml,
(2) preparation of lipid mixture solution: take palmitic acid 150g, linoleic acid 100g, arachidonic acid 50g, be dissolved in 500ml 0.1 mol/L NaOH solution, be heated to 70 ℃, be incubated 30 minutes, then use 4-hydroxyethyl piperazine ethanesulfonic acid regulator solution pH value 7.0, the bovine serum albumin solution that is 10% by above-mentioned solution and mass percent by volume 1:1.5 mixes, and constant-temperature table mixes 20 minutes at 55 ℃;
(3) on polycarbonate membrane, build tissue engineering epidermis: get polycarbonate filtering membrane (thickness 0.2mm, aperture 0.4 μ m, area 0.64 cm
2) surperficial 10% the IV collagen aqueous solution that applies, after 37 ℃ of baking ovens placements are dried for 1 hour, the cell density of then preparing in surface seeding step 1 is 3 × 10
5the epidermis of individual/ml forms cell dispersion liquid 0.5ml, apply evenly, postvaccinal polycarbonate film is placed in to fresh Epilife nutrient culture media and carries out the cultivation of gas-liquid face, cultivate and after 48 hours, renew fresh Epilife nutrient culture media, by the 30ml lipid mixture solution that adds step 2 to prepare in every 100ml Epilife nutrient culture media, add CaCl
2and ascorbic acid, make Ca
2+concentration is at 2.0mmol/L, and ascorbic acid concentrations, at 50 μ g/mL, continues cultivation and can be grown in polycarbonate membrane surface after 13 days, and thickness is 0.45mm, has Morphological Differentiation basalis, spinous layer, stratum granulosum, hyaline layer and cuticular tissue's engineering epidermis completely.
embodiment 4:
On polycarbonate membrane, build the method for detection of the irritating tissue engineering epidermis of cosmetics, adopt following steps:
(1) epidermis forms the separation cultivation of cell: under aseptic condition, get induced labor foetus skin of back 16cm2, be rolled into the skin bit of 0. 5cm × 0. 5cm size, wash with physiological saline, be that 0. 125% Dispase II enzyme was 20 ℃ of digestion 10 hours by 30ml mass percent, add again 10ml mass percent 0.1% trypsase 37 ℃ of digestion 30 minutes, acquisition is separated into the epidermal cell solution of individual cells, centrifugal 10 minutes of 5000rpm, remove supernatant liquor, with the lower confluent monolayer cells of physiological saline washing, then centrifugal 10 minutes of 5000rpm, after repeated washing and centrifugal 3 times, add 50ml to contain 55U/ml penicillin, the restricted epidermal cell free serum culture (DK-SFM) of 55ug/ml streptomysin, after shaking up, cultivate 24 hours at 37 ℃, obtain epidermis and form cell dispersion liquid, before inoculation, be 4 × 10 with normal saline dilution to cell density
5individual/ml,
(2) preparation of lipid mixture solution: take palmitic acid 150g, linoleic acid 100g, arachidonic acid 50g, be dissolved in 500ml 0.1 mol/L NaOH solution, be heated to 70 ℃, be incubated 30 minutes, then use 4-hydroxyethyl piperazine ethanesulfonic acid regulator solution pH value 7.0, the bovine serum albumin solution that is 10% by above-mentioned solution and mass percent by volume 1:2 mixes, and constant-temperature table mixes 20 minutes at 55 ℃;
(3) on polycarbonate membrane, build tissue engineering epidermis: get polycarbonate filtering membrane (thickness 0.2mm, aperture 0.4 μ m, area 0.64 cm
2) surperficial 10% the IV collagen aqueous solution that applies, after 37 ℃ of baking ovens placements are dried for 1 hour, the cell density of then preparing in surface seeding step 1 is 4 × 10
5the epidermis of individual/ml forms cell dispersion liquid 0.5ml, apply evenly, postvaccinal polycarbonate film is placed in to fresh Epilife nutrient culture media and carries out the cultivation of gas-liquid face, cultivate and after 48 hours, renew fresh Epilife nutrient culture media, by the 30ml lipid mixture solution that adds step 2 to prepare in every 100ml Epilife nutrient culture media, add CaCl
2and ascorbic acid, make Ca
2+concentration is at 2.0mmol/L, and ascorbic acid concentrations, at 60 μ g/mL, continues cultivation and can be grown in polycarbonate membrane surface after 13 days, and thickness is 0.53mm, has Morphological Differentiation basalis, spinous layer, stratum granulosum, hyaline layer and cuticular tissue's engineering epidermis completely.
embodiment 5:
On polycarbonate membrane, build the method for detection of the irritating tissue engineering epidermis of cosmetics, adopt following steps:
(1) epidermis forms the separation cultivation of cell: under aseptic condition, get induced labor foetus skin of back 16cm2, be rolled into the skin bit of 0. 5cm × 0. 5cm size, wash with physiological saline, be that 0. 125% Dispase II enzyme was 20 ℃ of digestion 10 hours by 30ml mass percent, add again 10ml mass percent 0.1% trypsase 37 ℃ of digestion 30 minutes, acquisition is separated into the epidermal cell solution of individual cells, centrifugal 10 minutes of 5000rpm, remove supernatant liquor, with the lower confluent monolayer cells of physiological saline washing, then centrifugal 10 minutes of 5000rpm, after repeated washing and centrifugal 3 times, add 50ml to contain 60U/ml penicillin, the restricted epidermal cell free serum culture (DK-SFM) of 50ug/ml streptomysin, after shaking up, cultivate 24 hours at 37 ℃, obtain epidermis and form cell dispersion liquid, before inoculation, be 5 × 10 with normal saline dilution to cell density
5individual/ml,
(2) preparation of lipid mixture solution: take palmitic acid 150g, linoleic acid 100g, arachidonic acid 50g, be dissolved in 500ml 0.1 mol/L NaOH solution, be heated to 70 ℃, be incubated 30 minutes, then use 4-hydroxyethyl piperazine ethanesulfonic acid regulator solution pH value 7.0, the bovine serum albumin solution that is 10% by above-mentioned solution and mass percent by volume 1:2 mixes, and constant-temperature table mixes 20 minutes at 55 ℃;
(3) on polycarbonate membrane, build tissue engineering epidermis: get polycarbonate filtering membrane (thickness 0.2mm, aperture 0.4 μ m, area 0.64 cm
2) surperficial 10% the IV collagen aqueous solution that applies, after 37 ℃ of baking ovens placements are dried for 1 hour, the cell density of then preparing in surface seeding step 1 is 5 × 10
5the epidermis of individual/ml forms cell dispersion liquid 0.5ml, apply evenly, postvaccinal polycarbonate film is placed in to fresh Epilife nutrient culture media and carries out the cultivation of gas-liquid face, cultivate and after 48 hours, renew fresh Epilife nutrient culture media, by the 30ml lipid mixture solution that adds step 2 to prepare in every 100ml Epilife nutrient culture media, add CaCl
2and ascorbic acid, make Ca
2+concentration is at 2.0mmol/L, and ascorbic acid concentrations, at 60 μ g/mL, continues cultivation and can be grown in polycarbonate membrane surface after 13 days, and thickness is 0.6mm, has Morphological Differentiation basalis, spinous layer, stratum granulosum, hyaline layer and cuticular tissue's engineering epidermis completely.
Claims (1)
1. on polycarbonate membrane, build the method for detection of the irritating tissue engineering epidermis of cosmetics, it is characterized in that adopting following steps:
(1) epidermis forms the separation cultivation of cell: under aseptic condition, get induced labor foetus skin of back 16cm2, be rolled into the skin bit of 0. 5cm × 0. 5cm size, wash with physiological saline, be that 0. 125% Dispase II enzyme was 20 ℃ of digestion 10 hours by 30ml mass percent, add again 10ml mass percent 0.1% trypsase 37 ℃ of digestion 30 minutes, acquisition is separated into the epidermal cell solution of individual cells, centrifugal 10 minutes of 5000rpm, remove supernatant liquor, with the lower confluent monolayer cells of physiological saline washing, then centrifugal 10 minutes of 5000rpm, after repeated washing and centrifugal 3 times, add 50ml to contain 30-60U/ml penicillin, the restricted epidermal cell serum free medium of 30-50ug/ml streptomysin, after shaking up, cultivate 24 hours at 37 ℃, obtain epidermis and form cell dispersion liquid, before inoculation, be 1-5 × 10 with normal saline dilution to cell density
5individual/ml,
(2) preparation of lipid mixture solution: take palmitic acid 150g, linoleic acid 100g, arachidonic acid 50g, be dissolved in 500ml 0.1 mol/L NaOH solution, be heated to 70 ℃, be incubated 30 minutes, then use 4-hydroxyethyl piperazine ethanesulfonic acid regulator solution pH value 6.5-7.0, the bovine serum albumin solution that is 10% by above-mentioned solution and mass percent by volume 1:1-2 mixes, and constant-temperature table mixes 20 minutes at 55 ℃;
(3) on polycarbonate membrane, build tissue engineering epidermis: get thickness 0.2mm, aperture 0.4 μ m, area 0.64 cm
2polycarbonate filtering membrane, surperficial coating quality is than the IV collagen aqueous solution that is 10%, 37 ℃ of baking ovens place 1 hour dry after, the cell density of then preparing in surface seeding step 1 is 1-5 × 10
5the epidermis of individual/ml forms cell dispersion liquid 0.5ml, apply evenly, postvaccinal polycarbonate film is placed in to fresh Epilife nutrient culture media and carries out the cultivation of gas-liquid face, cultivate and after 48 hours, renew fresh Epilife nutrient culture media, by the 20-30ml lipid mixture solution that adds step 2 to prepare in every 100ml Epilife nutrient culture media, add CaCl
2and ascorbic acid, make Ca
2+concentration 1.5-2.0mmol/L, ascorbic acid concentrations is at 50-60 μ g/mL, continue cultivation and can be grown in polycarbonate membrane surface after 13 days, thickness is 0.3-0.6mm, has Morphological Differentiation basalis, spinous layer, stratum granulosum, hyaline layer and cuticular tissue's engineering epidermis completely.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105158449A (en) * | 2015-10-08 | 2015-12-16 | 广州艾卓生物科技有限公司 | Method for detecting cosmetic and raw materials of cosmetic |
| CN107164314A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | A kind of construction method for the vitro recombination epidermis model that barrier is strengthened |
| CN107164315A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | A kind of construction method of the restructuring epidermis model detected for vitro skin excitant |
| WO2021243857A1 (en) * | 2020-06-03 | 2021-12-09 | 浙江大学 | In vitro digestion simulation box |
-
2014
- 2014-03-12 CN CN201410088817.9A patent/CN103884835A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105158449A (en) * | 2015-10-08 | 2015-12-16 | 广州艾卓生物科技有限公司 | Method for detecting cosmetic and raw materials of cosmetic |
| CN107164314A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | A kind of construction method for the vitro recombination epidermis model that barrier is strengthened |
| CN107164315A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | A kind of construction method of the restructuring epidermis model detected for vitro skin excitant |
| CN107164315B (en) * | 2017-06-18 | 2020-11-20 | 广东博溪生物科技有限公司 | Construction method of recombinant epidermis model for in vitro skin irritation detection |
| CN107164314B (en) * | 2017-06-18 | 2020-11-20 | 广东博溪生物科技有限公司 | Construction method of barrier-enhanced in-vitro recombinant epidermis model |
| WO2021243857A1 (en) * | 2020-06-03 | 2021-12-09 | 浙江大学 | In vitro digestion simulation box |
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