Method for constructing in-vitro recombinant epidermal model of analogous atopic dermatitis (AD) phenotype
A technology of atopic dermatitis and its construction method, which is applied in the field of in vitro recombinant epidermal model construction, which can solve the problems of insufficient accuracy and lack of atopic dermatitis-like phenotypes in animal AD models, shorten the modeling time, and avoid the genus differences, the effect of reducing individual differences
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Embodiment 1
[0032] Example 1: Construction of an in vitro recombinant epidermal model of atopic dermatitis-like phenotype.
[0033] Step (1): Preparation of Epidermal Cells
[0034] Take the primary keratinocytes, after recovery and expansion, use the P7 generation cells, digest with 2.5% trypsin digestion solution (100 mL PBS, 2.5 g trypsin), and make a single cell suspension with MCDB153 medium , according to 2.5×10 5 cells / mL, inoculated in culture flasks, placed at 37°C, 5% CO 2 cultivated under conditions;
[0035] Step (2): Submerged culture of epidermal cells
[0036] Preparation of cell culture medium I: Cell culture medium I was based on MCDB153, supplemented with insulin 2.5 μg / mL, isoproterenol 0.5 μM, hydrocortisone 0.1 μg / mL, epidermal growth factor 5 ng / mL, glandular Purine 30 μg / mL, transferrin 8 μg / mL, L-carnitine 0.05 mM and calcium chloride 0.2 mM.
[0037] Epidermal cells in the logarithmic growth phase were taken, and the cell density was 1.25×10 with cell culture...
Embodiment 2
[0042] Example 2: Construction of an in vitro recombinant epidermal model of atopic dermatitis-like phenotype
[0043] Step (1): Preparation of Epidermal Cells
[0044] Take the primary keratinocytes, after recovery and expansion, use the P3 generation cells, digest with trypsin digestion solution with a mass concentration of 2.5%, and make a single-cell suspension with MCDB153 medium, according to 1.0×10 5 cells / mL, inoculated in culture flasks, placed at 37°C, 5% CO 2 cultivated under conditions;
[0045] Step (2): Submerged culture of epidermal cells
[0046] Preparation of cell culture medium I: Cell culture medium I was based on MCDB153, supplemented with insulin 0.5 μg / mL, isoproterenol 0.1 μM, hydrocortisone 0.02 μg / mL, epidermal growth factor 1 ng / mL, glandular Purine 10 μg / mL, transferrin 5 μg / mL, L-carnitine 0.001 mM and calcium chloride 0.03 mM.
[0047] Epidermal cells in the logarithmic growth phase were taken, and the cell density was 1.25×10 with cell cultur...
Embodiment 3
[0052] Example 3: Construction of an in vitro recombinant epidermal model of atopic dermatitis-like phenotype
[0053] Step (1): Preparation of Epidermal Cells
[0054] Take the primary keratinocytes, after recovery and expansion, use the P7 generation cells, digest with trypsin digestion solution with a mass concentration of 2.5%, and make a single cell suspension with MCDB153 medium, according to 5×10 5 cells / mL, inoculated in culture flasks, placed at 37°C, 5% CO 2 cultivated under conditions;
[0055] Step (2): Submerged culture of epidermal cells
[0056] Preparation of cell culture medium I: Cell culture medium I was based on MCDB153, supplemented with insulin 5 μg / mL, isoproterenol 1 μM, hydrocortisone 0.2 μg / mL, epidermal growth factor 10 ng / mL, glandular Purine 50 μg / mL, transferrin 12 μg / mL, L-carnitine 0.1 mM and calcium chloride 0.2 mM.
[0057] Take the epidermal cells in the logarithmic growth phase, and make the cell density of 2.5×10 6 cells / mL of epiderma...
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