CN107164315B - Construction method of recombinant epidermis model for in vitro skin irritation detection - Google Patents

Construction method of recombinant epidermis model for in vitro skin irritation detection Download PDF

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CN107164315B
CN107164315B CN201710460515.3A CN201710460515A CN107164315B CN 107164315 B CN107164315 B CN 107164315B CN 201710460515 A CN201710460515 A CN 201710460515A CN 107164315 B CN107164315 B CN 107164315B
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李润芝
卢永波
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Shaanxi Boxi General Testing Technology Co.,Ltd.
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Abstract

The embodiment of the invention provides a method for constructing a recombinant epidermis model for in-vitro skin irritation detection, which relates to the technical field of tissue engineering. The method comprises the following steps: step one, separating and culturing epidermal stem cells in a large scale; step two, constructing the tissue engineering epidermis.

Description

Construction method of recombinant epidermis model for in vitro skin irritation detection
Technical Field
The invention relates to the technical field of tissue engineering, in particular to a method for constructing a recombinant epidermis model for in-vitro skin irritation detection.
Background
Since cosmetics are generally in direct contact with human skin and the safety of cosmetics is a concern, it is essential to perform a skin irritation test. The traditional skin irritation test is mostly carried out by animals, the consumed animals have the defects of large batch, long time consumption, high cost, species difference and the like, and certain experiments can cause great pain to the animals and do not meet the requirements of animal protection and welfare. With the proposal of the principle of '3R', the research of animal experiment alternative methods is vigorously carried out in foreign countries on the basis of the principle. Through research on in vitro alternative methods of skin irritation tests in recent 20 years, methods for evaluating toxicity using tissue engineering skin constructed in vitro have been gradually approved, and the "technical standards of human skin models for in vitro skin irritation tests" guidelines issued by the European alternative verification Center (European Center for the Validation of alternative methods ECVAM) in 2007 month 5 and the "two in vitro skin irritation test verification methods" statements issued in 2008 month 11 both have shown that tissue engineering skin can be used as an alternative model for in vitro skin irritation tests.
To date, there have been over 30 tissue reconstruction skin models commercialized and to be commercialized, including an epidermal model formed of only epidermal cells, a full-thickness skin model having a dermal layer and an epidermal layer of fibroblasts, and a melanin skin model containing fibroblasts, epidermal cells, and melanocytes, and the like. But only four types of epidermal models available for in vitro skin irritation tests, including Episkin, episderm, SkinEthic and LabCyte, were certified by OECD. The number of validated epidermis models is so small, mainly because the stratum corneum of the constructed tissue engineering epidermis is not fully differentiated, resulting in a weak barrier function of the models.
The stratum corneum is the outermost layer of the epidermis and is composed of keratinocytes and a lipid matrix surrounding them, these intercellular lipids mainly containing ceramides, free fatty acids and cholesterol. Stratum corneum lipids play an important role in maintaining the skin barrier function, skin moisture, preventing water loss, and regulating the adhesion and desquamation of keratinocytes. Changes in the composition and content of stratum corneum lipids are both contributing factors to epidermal barrier function. Although the existing commercial epidermal model products have high similarity in tissue structure to human Skin, and typical basal, spinous, granular and stratum corneum structures, Ponec et al have other drawbacks, and found that the reconstructed epidermal model has a reduced ceramide content and a very low content of free fatty acids compared to natural human Skin (Ponec M, Boelsma E, Gibbs S, Mommaas M. Characterisation of reconstructed Skin models. Skin Pharmacol applied Skin physiol. 2002;15 (pl 1): 4-17.). The difference of lipid composition is the reason for causing the secretion of the lipid layer body in the model stratum corneum and the abnormality of the lipid layer body, and the barrier function is incomplete, and the defects cause the high permeability of the epidermis model to a tested object, thereby causing the influence on the accuracy of the in vitro safety test result, and the judgment of the irritation standard substance has certain misjudgment.
Disclosure of Invention
The embodiment of the invention provides a method for constructing a recombinant epidermis model for in vitro skin irritation detection, which has the advantages of simple and convenient operation, stable process and convenient large-scale production, the lipid composition of a product is similar to that of natural human skin, the barrier function is complete, and the method is convenient for detecting the irritation of cosmetics.
In order to achieve the purpose, the embodiment of the invention adopts the following technical scheme:
the embodiment of the invention provides a method for constructing a recombinant epidermal model for in-vitro skin irritation detection, which comprises the following steps:
step one, epidermal stem cell separation and large-scale culture
(1) Separating epidermal stem cells: placing human skin tissue in a culture dish, adding 10mL of 75% alcohol, rapidly washing, transferring into PBS solution, peeling off with scissors to remove subcutaneous loose connective tissue, washing with PBS, cutting tissue blocks into 0.3cm × 0.5cm, adding 15mL of 1% Dispase digestive juice, digesting overnight at 4 deg.C, separating dermis and epidermis, collecting epidermal skin pieces, cutting into pieces, adding 10mL of 0.025% EDTA-pancreatin digestive juice, digesting at 37 deg.C for 10 min to obtain epidermal cell solution separated into single cells, filtering epidermal fragments with 200 mesh stainless steel mesh, centrifuging at 1000rpm for 7 min, removing supernatant, washing lower layer cells with PBS, centrifuging at 1000rpm for 7 min, pouring out supernatant, adding epidermal cell culture solution, inoculating into IV type collagen-coated culture bottle at 2-4 × 106/bottle, 37 deg.C, 5% CO2Incubating for 10-15min in the incubator, sucking out the culture medium, replacing fresh epidermal cell culture solution, and continuing culturing, and replacing the culture solution once every 48 h;
(2) large-scale culture: when the cells reach 80% confluence, digesting the epidermal stem cells with pancreatin digestive juice, and re-suspending the cells with epidermal cell culture solution KC-growth at a ratio of 0.2-0.5 × 105 cells/cm2Inoculating into cell factory, adding epidermal cell culture solution, culturing at 37 deg.C for 48-72 hr, and changing the culture solution every other day;
step two, constructing the tissue engineering epidermis
(1) Inoculating epidermal stem cells: digesting the epidermal stem cells obtained in the step one with pancreatin digestive juice, preparing epidermal stem cell dispersion liquid with density of 1.25 × 106-2.5 × 106/ml with inoculation culture medium, inoculating 200 μ l into cell culture chamber containing polycarbonate filter membrane,shaking uniformly, 5% CO at 37 deg.C2Incubating for 20-30min in the incubator;
the inoculation culture solution comprises: adding 1.5-2.0mM/L of L-glutamine, 1-10 mu g/L of epidermal growth factor, 10-100 mu g/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin and 0.06-0.4mM/L of calcium chloride into KC base solution to promote epidermal stem cells to be rapidly and uniformly attached to a polycarbonate membrane;
(2) culturing under liquid: transferring the inoculated cell culture chamber into an epidermal model culture mold, adding a fresh epidermal cell TU culture solution into a culture dish outside the chamber, and culturing at 37 ℃ with 5% CO2Continuously culturing for 24-48h in the incubator;
the epidermal cell TU culture solution comprises: KC basic solution is added with 1.5-2.0mM/L of L-glutamine, 1-10 μ g/L of epidermal growth factor, 10-100 μ g/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin, 10-50mg/L of adenine, 5-12 μ g/L of transferrin and 0.06-0.4mM/L of calcium chloride;
(3) culturing on a gas-liquid surface: removing residual culture medium in the cell chamber, adding epidermal cell TA culture solution outside the chamber, and culturing at 37 deg.C with 5% CO2After the culture box is continuously cultured for 8-12 days, a tissue engineering epidermis model with the thickness of 100-150 mu m and with completely morphologically differentiated basal layer, spinous layer, granular layer and cuticle can be obtained;
the epidermal cell TA culture solution comprises: adding lipid mixture and/or peroxisome proliferator-activated receptor activator GW 5015160.1-1 mM/L, vitamin C30-50 μ g/L, and calcium chloride 2.0-3.0mM/L into epidermal TU culture solution.
Further, the epidermal cell culture solution comprises: KC base solution is added with 1% of L-glutamine and 0.2% of BPE, the concentration of the L-glutamine is 1.5-2.0mM/L, and the concentration of the BPE is 25-50 mg/L.
Further, the epidermis model culture mould comprises: a support for holding the chambers and a culture dish adapted to ensure that each chamber is in contact with the liquid surface at a uniform height.
Further, the lipid blend comprises: 0.5-1.0mM/L of L-serine, 8-10 mu M/L of palmitic acid, 0.02-0.1mM/L of linoleic acid, 0.05-0.1 mM/L of argininosuccinic acid and 0.02-0.1mM/L of bovine serum albumin.
The invention adopts the epidermal stem cells as seed cells, has strong cell proliferation and differentiation capacity, has a recombinant epidermal tissue structure formed by induced differentiation in a fine-adjustment culture solution which is highly similar to that of natural skin, has a basal layer, a spinous layer, a granular layer and a cuticle layer with complete morphological differentiation (see figure 1, and the figure shows that a recombinant epidermal model obtained by adopting the construction method used in the invention has a histological structure highly similar to that of natural human skin), and has long function and activity maintaining time; the polycarbonate membrane without toxicity and good biocompatibility is used as a support, so that the material standardization degree is high, the product uniformity and stability are high, the process is simple, the production period is short, and the production cost is effectively reduced; serum-free culture solution is adopted in the construction process, so that the influence of undefined components on toxicity test results is reduced; in the gas-liquid surface culture stage, according to the lipid composition characteristics of the model, the lipid components and the content added in the culture solution are adjusted, such as palmitic acid is reduced, the concentration of linoleic acid is increased, and oleic acid and peanut tetra-carboxylic acid are removed; meanwhile, the PPARs activator Peroxisome Proliferator-activated receptors (Peroxisome Proliferator PPARs) activator GW501516 is added to synergistically regulate lipid metabolism of epidermal cells and promote synthesis and secretion of ceramide, so that the lipid composition and lamellar structure of the stratum corneum are closer to the natural skin, the barrier function of the stratum corneum is effectively enhanced, the accuracy of chemical judgment is improved, the model sensitivity reaches 100%, the specificity reaches 80%, and the accuracy is improved to 90%.
Drawings
FIG. 1 is a comparison of histological staining results of in vitro recombinant epidermis models and natural human skin according to the present invention;
fig. 2 is a result of determining 20 skin irritation standards in the economic collaboration organization test file 439 by the in vitro recombinant epidermis model provided in the embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be described in detail below with reference to the accompanying drawings in the embodiments of the present invention.
In this embodiment of the present invention, the term "and/or" is only one kind of association relationship describing an associated object, and means that three relationships may exist, for example, a and/or B may represent: a exists alone, A and B exist simultaneously, and B exists alone.
Example 1
The embodiment of the invention provides a method for constructing a recombinant epidermis model for in-vitro skin irritation detection, which comprises the following steps:
1. isolation and large-scale culture of epidermal stem cells:
(1) separating epidermal stem cells: placing human skin tissue in a culture dish, adding 10mL of 75% alcohol, rapidly washing, transferring into PBS solution, peeling off with scissors to remove subcutaneous loose connective tissue, washing with PBS, cutting tissue blocks into 0.3cm × 0.5cm, adding 15mL of 1% Dispase digestive juice, digesting overnight at 4 deg.C, separating dermis and epidermis, collecting epidermal skin pieces, cutting into pieces, adding 10mL of 0.025% EDTA-pancreatin digestive juice, digesting at 37 deg.C for 10 min to obtain epidermal cell solution separated into single cells, filtering epidermal fragments with 200 mesh stainless steel mesh, centrifuging at 1000rpm for 7 min, removing supernatant, washing lower layer cells with PBS, centrifuging at 1000rpm for 7 min, pouring out supernatant, adding epidermal cell culture solution, inoculating into IV type collagen-coated culture bottle at 2-4 × 106/bottle, 37 deg.C, 5% CO2Incubating for 10-15min in the incubator, sucking out the culture medium, replacing fresh epidermal cell culture solution, and continuing culturing, and replacing the culture solution once every 48 h.
(2) Large-scale culture: when the cells reach 80% confluence, digesting the epidermal stem cells with pancreatin digestive juice, and re-suspending the cells with epidermal cell culture solution KC-growth at a ratio of 0.2-0.5 × 105 cells/cm2Inoculating into cell factory, adding epidermal cell culture solution, culturing at 37 deg.C for 48-72 hr, and changing the culture solution every other day.
The epidermal cell culture solution comprises: KC base solution is added with 1% L-glutamine and 0.2% BPE to make the concentration of L-glutamine at 1.5-2.0mM/L and the concentration of BPE at 25-50 mg/L.
2. Constructing a tissue engineering epidermis:
(1) inoculating epidermal stem cells: digesting the epidermal stem cells obtained in the step 1 with pancreatin digestive juice, preparing epidermal stem cell dispersion liquid with density of 1.25 × 106/ml with inoculation culture medium, inoculating 200 μ l into cell culture chamber containing polycarbonate filter membrane, shaking uniformly, and culturing at 37 deg.C with 5% CO2Incubating in incubator for 20-30 min.
The inoculation culture solution comprises: adding 1.5-2.0mM/L of L-glutamine, 1-10 mu g/L of epidermal growth factor, 10-100 mu g/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin and 0.06-0.4mM/L of calcium chloride into KC base solution to promote the epidermal stem cells to be rapidly and uniformly attached to a polycarbonate membrane.
(2) Culturing under liquid: transferring the inoculated cell culture chamber into an epidermal model culture mold, adding a fresh epidermal cell TU culture solution into a culture dish outside the chamber, and culturing at 37 ℃ with 5% CO2The incubator continues to culture for 24-48 h.
The epidermis model culture mould comprises: a support and a supporting culture dish for fixing the cell can guarantee that every cell is highly unanimous with the liquid level contact, conveniently trade liquid operation simultaneously, reduce the pollution risk.
The epidermal cell TU culture solution is as follows: KC basic solution is added with 1.5-2.0mM/L of L-glutamine, 1-10 μ g/L of epidermal growth factor, 10-100 μ g/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin, 10-50mg/L of adenine, 5-12 μ g/L of transferrin and 0.06-0.4mM/L of calcium chloride.
(3) Culturing on a gas-liquid surface: removing residual culture medium in the cell chamber, adding epidermal cell TA culture solution outside the chamber, and culturing at 37 deg.C with 5% CO2After the culture box is continuously cultured for 8-12 days, the tissue engineering epidermis model with the thickness of 100-150 mu m and complete morphological differentiation of the basal layer, the spinous layer, the granular layer and the cuticle can be obtained.
The epidermal cell TA culture solution is as follows: lipid mixture (L-serine 0.5-1.0mM/L, palmitic acid 8-10. mu.M/L, linoleic acid 0.02-0.1mM/L, argininosuccinic acid 0.05-0.1 mM/L, bovine serum albumin 0.02-0.1 mM/L) and vitamin C30-50. mu.g/L are added to the epidermal TU culture solution, and the concentration of calcium chloride is adjusted to 2.0-3.0 Mm/L.
Example 2
The embodiment of the invention provides a method for constructing a recombinant epidermis model for in-vitro skin irritation detection, which comprises the following steps:
1. isolation and large-scale culture of epidermal stem cells:
(1) separating epidermal stem cells: placing human skin tissue in a culture dish, adding 10mL of 75% alcohol, rapidly washing, transferring into PBS solution, peeling off with scissors to remove subcutaneous loose connective tissue, washing with PBS, cutting tissue blocks into 0.3cm × 0.5cm, adding 15mL of 1% Dispase digestive juice, digesting overnight at 4 deg.C, separating dermis and epidermis, collecting epidermal skin pieces, cutting into pieces, adding 10mL of 0.025% EDTA-pancreatin digestive juice, digesting at 37 deg.C for 10 min to obtain epidermal cell solution separated into single cells, filtering epidermal fragments with 200 mesh stainless steel mesh, centrifuging at 1000rpm for 7 min, removing supernatant, washing lower layer cells with PBS, centrifuging at 1000rpm for 7 min, pouring out supernatant, adding epidermal cell culture solution, inoculating into IV type collagen-coated culture bottle at 2-4 × 106/bottle, 37 deg.C, 5% CO2Incubating for 10-15min in the incubator, sucking out the culture medium, replacing fresh epidermal cell culture solution, and continuing culturing, and replacing the culture solution once every 48 h.
(2) Large-scale culture: when the cells reach 80% confluence, digesting the epidermal stem cells with pancreatin digestive juice, and re-suspending the cells with epidermal cell culture solution KC-growth at a ratio of 0.2-0.5 × 105 cells/cm2Inoculating into cell factory, adding epidermal cell culture solution, culturing at 37 deg.C for 48-72 hr, and changing the culture solution every other day.
The epidermal cell culture solution comprises: KC base solution is added with 1% of L-glutamine and 0.2% of BPE to ensure that the concentration of the L-glutamine is 1.5-2.0mmol/L and the concentration of the BPE is 25-50 mg/L.
2. Constructing a tissue engineering epidermis:
(1) inoculating epidermal stem cells: digesting the epidermal stem cells obtained in the step 1 with pancreatin digestive juice, preparing epidermal stem cell dispersion liquid with density of 1.25 × 106/ml with inoculation culture medium, and inoculating 200 μ lShaking the cells in a cell culture chamber containing a polycarbonate filter, and culturing the cells at 37 ℃ in 5% CO2Incubating in incubator for 20-30 min.
The inoculation culture solution comprises: adding 1.5-2.0mM/L of L-glutamine, 1-10 mu g/L of epidermal growth factor, 10-100 mu g/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin and 0.06-0.4mM/L of calcium chloride into KC base solution to promote the epidermal stem cells to be rapidly and uniformly attached to a polycarbonate membrane.
(2) Culturing under liquid: transferring the inoculated cell culture chamber into an epidermal model culture mold, adding a fresh epidermal cell TU culture solution into a culture dish outside the chamber, and culturing at 37 ℃ with 5% CO2The incubator continues to culture for 24-48 h.
The epidermis model culture mould comprises: a support and a supporting culture dish for fixing the cell can guarantee that every cell is highly unanimous with the liquid level contact, conveniently trade liquid operation simultaneously, reduce the pollution risk.
The epidermal cell TU culture solution is as follows: KC basic solution is added with 1.5-2.0mM/L of L-glutamine, 1-10 μ g/L of epidermal growth factor, 10-100 μ g/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin, 10-50mg/L of adenine, 5-12 μ g/L of transferrin and 0.06-0.4mM/L of calcium chloride.
(3) Culturing on a gas-liquid surface: removing residual culture medium in the cell chamber, adding epidermal cell TA culture solution outside the chamber, and culturing at 37 deg.C with 5% CO2After the culture box is continuously cultured for 8-12 days, the tissue engineering epidermis model with the thickness of 100-150 mu m and complete morphological differentiation of the basal layer, the spinous layer, the granular layer and the cuticle can be obtained.
The epidermal cell TA culture solution is as follows: GW 5015160.1-1 mM/L and vitamin C30-50 μ g/L are added into the epidermal TU culture solution, and the concentration of calcium chloride is adjusted to 2.0-3.0 Mm/L.
Example 3
The embodiment of the invention provides a method for constructing a recombinant epidermis model for in-vitro skin irritation detection, which comprises the following steps:
1. isolation and large-scale culture of epidermal stem cells:
(1) separating epidermal stem cells: placing human skin tissue in a culture dish, adding 10mL of 75% alcohol, rapidly washing, transferring into PBS solution, peeling off with scissors to remove subcutaneous loose connective tissue, washing with PBS, cutting tissue blocks into 0.3cm × 0.5cm, adding 15mL of 1% Dispase digestive juice, digesting overnight at 4 deg.C, separating dermis and epidermis, collecting epidermal skin pieces, cutting into pieces, adding 10mL of 0.025% EDTA-pancreatin digestive juice, digesting at 37 deg.C for 10 min to obtain epidermal cell solution separated into single cells, filtering epidermal fragments with 200 mesh stainless steel mesh, centrifuging at 1000rpm for 7 min, removing supernatant, washing lower layer cells with PBS, centrifuging at 1000rpm for 7 min, pouring out supernatant, adding epidermal cell culture solution, inoculating into IV type collagen-coated culture bottle at 2-4 × 106/bottle, 37 deg.C, 5% CO2Incubating for 10-15min in the incubator, sucking out the culture medium, replacing fresh epidermal cell culture solution, and continuing culturing, and replacing the culture solution once every 48 h.
(2) Large-scale culture: when the cells reach 80% confluence, digesting the epidermal stem cells with pancreatin digestive juice, and re-suspending the cells with epidermal cell culture solution KC-growth at a ratio of 0.2-0.5 × 105 cells/cm2Inoculating into cell factory, adding epidermal cell culture solution, culturing at 37 deg.C for 48-72 hr, and changing the culture solution every other day.
The epidermal cell culture solution comprises: KC base solution is added with 1% of L-glutamine and 0.2% of BPE to ensure that the concentration of the L-glutamine is 1.5-2.0mmol/L and the concentration of the BPE is 25-50 mg/L.
2. Constructing a tissue engineering epidermis:
(1) inoculating epidermal stem cells: digesting the epidermal stem cells obtained in the step 1 with pancreatin digestive juice, preparing epidermal stem cell dispersion liquid with density of 1.25 × 106/ml with inoculation culture medium, inoculating 200 μ l into cell culture chamber containing polycarbonate filter membrane, shaking uniformly, and culturing at 37 deg.C with 5% CO2Incubating in incubator for 20-30 min.
The inoculation culture solution comprises: adding 1.5-2.0mmol/L of L-glutamine, 1-10 mu g/L of epidermal growth factor, 10-100 mu g/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin and 0.06-0.4mM/L of calcium chloride into KC base solution to promote the epidermal stem cells to be rapidly and uniformly attached to a polycarbonate membrane.
(2) Culturing under liquid: transferring the inoculated cell culture chamber into an epidermal model culture mold, adding a fresh epidermal cell TU culture solution into a culture dish outside the chamber, and culturing at 37 ℃ with 5% CO2The incubator continues to culture for 24-48 h.
The epidermis model culture mould comprises: a support and a supporting culture dish for fixing the cell can guarantee that every cell is highly unanimous with the liquid level contact, conveniently trade liquid operation simultaneously, reduce the pollution risk.
The epidermal cell TU culture solution is as follows: KC basic solution is added with 1.5-2.0mmol/L of L-glutamine, 1-10 mug/L of epidermal growth factor, 10-100 mug/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin, 10-50mg/L of adenine, 5-12 mug/L of transferrin and 0.06-0.4mM/L of calcium chloride.
(3) Culturing on a gas-liquid surface: removing residual culture medium in the cell chamber, adding epidermal cell TA culture solution outside the chamber, and culturing at 37 deg.C with 5% CO2After the culture box is continuously cultured for 8-12 days, the tissue engineering epidermis model with the thickness of 100-150 mu m and complete morphological differentiation of the basal layer, the spinous layer, the granular layer and the cuticle can be obtained.
The epidermal cell TA culture solution is as follows: lipid mixture (L-serine 0.5-1.0mM/L, palmitic acid 8-10. mu.M/L, linoleic acid 0.02-0.1mM/L, argininosuccinic acid 0.05-0.1 mM/L, bovine serum albumin 0.02-0.1 mM/L), GW 5015160.1-1 mM/L, and vitamin C30-50. mu.g/L are added to the epidermal TU culture solution, and the concentration of calcium chloride is adjusted to 2.0-3.0 Mm/L.
Example 4
The application of the in-vitro recombinant epidermis model in the detection of evaluating the irritation of chemical substances to human skin comprises the following steps:
1. preparing the model
Chemicals to be tested are used as an experimental group, a doherty buffer solution is used as a negative control group (no irritation), 5% sodium dodecyl sulfate is used as a positive control group (irritation), 3 individual external recombinant epidermis models are prepared in each group, 16 pore plate is prepared for each 3 models, and 0.9ml of culture solution for tests is added into each pore.
2. Administration of drugs
For the liquid test object, 25 mul of the liquid test object is dripped on the surface of each epidermis model; for the semi-solid reagent, a volumetric pipette is used to suck 25 mul of test substance to be dripped on the surface of the model; for solid reagents, 25 μ l of a buffered solution of dornate is dropped onto the surface of the model to wet the surface of the epidermal model before administration, and about 25 mg of the weighed test substance is added using a spatula (or other suitable instrument) and the model is gently shaken to facilitate spreading of the solid test substance on the surface of the model. After dosing, all 6-well plates were transferred to 37 ℃ with 5% CO2After incubation in the incubator for 30 minutes, the washing procedure was started.
3. Cleaning of
The surface of the epidermis model was rinsed with sterile Du's phosphate buffer, and after the washing was completed, the in vitro reconstituted human epidermis model was immersed in 150 ml of Du's phosphate buffer and shaken to remove the residual test chemicals, and the rinsing was repeated 3 times. The remaining liquid was gently blotted off with sterile absorbent paper. The in vitro recombinant human epidermal model was then transferred to a new 6-well plate (with culture medium already added).
The negative control group and the positive control group were operated as above.
4. Post-dose incubation
After the completion of the washing, all 6-well plates were transferred to 37 ℃ with 5% CO2Culturing in an incubator for 24 h. All plates were then removed from the cell culture chamber, transferred to a sterile bench, and replaced with 0.9ml serum-free epidermal cell culture medium per well. Continuing at 37 + -1 deg.C and 5 + -1% CO2And after culturing for 18 hours under the culture condition of 95% relative humidity, starting to judge the irritation of the chemical.
5. Chemical irritation determination
The in vitro recombinant human epidermal model was removed from the cell culture chamber and transferred to a 24-well plate containing 1mg/mL of thiazole blue solution, 0.3mL per well, at 37 ℃ with 5% CO2After 3 hours of incubation in the cell incubator in the dark place, the thiazole blue solution was aspirated by a pipette, 2mL of isopropanol was added to each well to completely immerse the model, the model was sealed with a sealing film, and the cell was left to stand at 4 ℃ to extractAnd taking for 12-16 hours to obtain an isopropanol extract. The bottom of the culture chamber was punctured, the liquid in the chamber was allowed to flow into the culture well, 200. mu.l of the isopropyl alcohol extract was aspirated into a 96-well plate, and the OD value of absorbance at a wavelength of 550 to 570nm was measured with a 96-well plate spectrophotometer.
And (3) judging the irritation: and calculating the relative cell activity of the chemical by taking the absorbance value of the negative control group as a denominator, the absorbance OD value of the experimental group as a numerator and the percentage of the ratio as the relative cell activity of the experimental group, wherein the relative cell activity is higher than 50 percent, the detected chemical has no skin irritation, the relative cell activity is lower than 50 percent, and the detected chemical has skin irritation.
The results of the test for the tested chemicals and skin irritation are shown in Table 1.
Figure 81127DEST_PATH_IMAGE001
Referring to fig. 2, it can be seen that, the test result of the 20 skin irritation standard substance by using the recombinant epidermis model constructed by the invention shows that the accuracy of the chemical skin irritation test is obviously improved by using the recombinant epidermis model constructed by the invention, so that the test accuracy reaches 90%.
Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-described embodiments, which are merely illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.

Claims (2)

1. A method for constructing a recombinant epidermal model for in vitro skin irritation testing, comprising the steps of:
step one, epidermal stem cell separation and large-scale culture
Step two, constructing the tissue engineering epidermis
(1) Inoculating epidermal stem cells: digesting the epidermal stem cells obtained in the step one with pancreatin digestive juice, and preparing the cells into cells with the density of 1.25 multiplied by 10 by using an inoculation culture solution6-2.5×106The epidermal stem cell dispersion of each ml was inoculated in 200. mu.l into a cell culture chamber containing a polycarbonate filter, shaken well, and incubated at 37 ℃ with 5% CO2Incubating for 20-30min in the incubator;
the inoculation culture solution comprises: adding 1.5-2.0mM/L of L-glutamine, 1-10 mu g/L of epidermal growth factor, 10-100 mu g/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin and 0.06-0.4mM/L of calcium chloride into KC base solution;
(2) culturing under liquid: transferring the inoculated cell culture chamber into an epidermal model culture mold, adding a fresh epidermal cell TU culture solution into a culture dish outside the chamber, and culturing at 37 ℃ with 5% CO2Continuously culturing for 24-48h in the incubator;
the epidermal cell TU culture solution comprises: KC basic solution is added with 1.5-2.0mM/L of L-glutamine, 1-10 μ g/L of epidermal growth factor, 10-100 μ g/L of hydrocortisone, 2-20mg/L, BPE 25-50mg/L of insulin, 10-50mg/L of adenine, 5-12 μ g/L of transferrin and 0.06-0.4mM/L of calcium chloride;
(3) culturing on a gas-liquid surface: removing residual culture medium in the cell chamber, adding epidermal cell TA culture solution outside the chamber, and culturing at 37 deg.C with 5% CO2The thickness of the culture box is 100-150 μm after the culture is continued for 8-12 days;
the epidermal cell TA culture solution comprises: adding lipid mixture and peroxisome proliferator-activated receptor activator GW 5015160.1-1 mM/L, vitamin C30-50 μ g/L, calcium chloride 2.0-3.0mM/L into epidermal TU culture solution; the lipid mixture comprises: 0.5-1.0mM/L of L-serine, 8-10 mu M/L of palmitic acid, 0.02-0.1mM/L of linoleic acid, 0.05-0.1 mM/L of argininosuccinic acid and 0.02-0.1mM/L of bovine serum albumin.
2. The method for constructing a recombinant epidermal model for in vitro skin irritation detection according to claim 1, wherein the epidermal model culture mold comprises: a support for holding the chambers and a culture dish adapted to ensure that each chamber is in contact with the liquid surface at a uniform height.
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CN108048393A (en) * 2017-12-12 2018-05-18 谢举临 A kind of method of new rapid amplifying skin epidermis substrate stem cell
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103884835A (en) * 2014-03-12 2014-06-25 珀莱雅化妆品股份有限公司 Method for constructing tissue engineering epidermis for detecting irritation of cosmetics on polycarbonate film
CN104459145A (en) * 2013-09-13 2015-03-25 王阿慧 Cosmetic detection
CN105734009A (en) * 2016-04-01 2016-07-06 陕西博溪生物科技有限公司 In-vitro recombined human skin epidermis model and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104459145A (en) * 2013-09-13 2015-03-25 王阿慧 Cosmetic detection
CN103884835A (en) * 2014-03-12 2014-06-25 珀莱雅化妆品股份有限公司 Method for constructing tissue engineering epidermis for detecting irritation of cosmetics on polycarbonate film
CN105734009A (en) * 2016-04-01 2016-07-06 陕西博溪生物科技有限公司 In-vitro recombined human skin epidermis model and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Type IV Collagen and Fibronectin Enhance Human Keratinocyte Thymidine Incorporation and Spreading in the Absence of Soluble Growth Factors;David T. Woodley等;《THE JOURNAL OF INVESTIGATIVE DERMATOLOGY》;19901231;第139-143页 *
用于替代皮肤刺激试验的组织工程表皮模型的构建研究;李中良 等;《中国修复重建外科杂志》;20110228;第25卷(第2期);第129-132页 *

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