The two-layers hTECs were produced following the self-assembly approach. Briefly, dermal fibroblasts were cultured in the presence of ascorbic acid (50 μg/ml) for 35 days to promote the production of their own extracellular matrix (ECM). Two fibroblasts sheets were then superimposed to form a reconstructed stroma, on which keratinocyte were seeded. Reconstructed tissues were cultured for 7 days under submerged conditions in complete DME-HAM supplemented with ascorbate and then transferred to the air-liquid interface for 7 days in EGF-free DME-HAM to induce epithelial differentiation. At day 35, the previously polarized Th1 and Th17 cells were seeded to a total of 0.5 x 106 T cells per sheet on half of the dermal fibroblast sheets, with a ratio of 1:1. At the same time, keratinocytes were seeded on the other dermal fibroblast sheets. Keratinocytes and T cells were maintained in submerged culture in their respective medium for one additional week. Once T cells were added to the 3D skin model, the culture medium was supplemented with IL-2 (10 U/ml) and IL-23 (20 ng/ml) until the end of the culture period. At day 35, a fibroblast sheet with keratinocytes was superimposed on a T cell-containing fibroblast sheet, thus forming a skin substitute comprising a T-cell-enriched dermis and an epidermis. These newly formed skin substitutes were then raised at the air-liquid interface and kept in culture for a final 3 weeks in the keratinocytes culture medium depleted of EGF to allow complete differentiation of the epidermis.
Skin biopsies were washed in antibiotics-containing PBS, cut into 3 mm × 100 to 150 mm strips, and incubated overnight at 4°C in a 500 μg/mL thermolysin-HEPES solution. The epithelium was subsequently peeled from the dermis with sterile tweezers. Epidermal strips were then incubated for 30 minutes in a trypsin-ethylenediaminetretraacetic acid (EDTA) solution (0.05% w/v of porcine trypsin 1-250, 0.01% w/v of EDTA, 2.8 mM of D-glucose, 100 IU/mL of penicillin G, 25 μg/mL of gentamicin, and phenol red) in PBS at 37°C on a stirring plate. A DME-HAM was used to neutralize trypsin activity. DME-HAM was used to wash and resuspend the cell pellets. Keratinocytes were seeded at 1.4 × 104 cells/cm2 on iHFL (lethally irradiated (6000 rad) human fibroblasts feeder layer) in DME-Ham (Dulbecco–Vogt modification of Eagle's medium with Ham's F12 in a 3:1 ratio supplemented with 5% Fetal Clone II serum, 5 μg/ml of insulin, 0.4 μg/ml of hydrocortisone, 10 ng/ml of epidermal growth factor, 0.212 mg/ml of isoproterenol hydrochloride, 100 IU/ml of penicillin and 25 μg/ml of gentamycin.). Cells were kept in incubators at 37°C, 8% CO2, and 95 ± 5% humidity. Medium was changed three times per week. When cells attained 75-95% confluence, they were passaged. T cells were isolated directly from anticoagulated peripheral blood obtained from healthy donors by negative immunomagnetic selection. For this step, the EasySep™ Direct Human T Cell Isolation Kit was used according to the manufacturer’s instructions under sterile conditions and at room temperature. T cells were then cultured for 7 days at 37°C in an 8% CO2 atmosphere with various cytokines to induce their polarization in Th1 and Th17 cells. During the first 3 days, T cells were seeded in 12-wells plates at 2 x 106/ml with their respective cytokine mixture. Th1 polarization was achieved by adding IL-12 (20 ng/ml) and anti-IL-4 (5 µg/ml) while anti-IFNγ (10 µg/ml), anti-IL-4 (5 µg/ml), IL-1β (20 ng/ml) and IL-6 (40 ng/ml) were added in the medium for Th17 polarization. After these 3 days, Th1 and Th17 were separately exposed to 25 ng/ml of phorbol 12-myristate 13-acetate and to 1 µg/ml of ionomycin for 4 hours at 37°C. Following this stimulation, T cells were reseeded (2 x 106/ml) in 12-wells plates for an additional 4 days at 37°C under 8% CO2 with IL-2 (30U/ml) for Th1 cells and with IL-2 (10 U/ml) and IL-23 (20 ng/ml) for Th17 cells.
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Cat#74104)
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color LowInput QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions
600 ng of Cy3-labelled cRNA (specific activity >8.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole SurePrint G3 Human GE 8x60K (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
Slides were scanned immediately after washing on the Agilent SureScanner (G4900DA) using one color scan setting for 1x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
Gene expression of healthy keratinocytes in passage 3 co-cultured with T-lymphocyte from human tissue-engineered skin substitutes
The scanned images were analyzed with Feature Extraction Software 10,7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. gPixNormIQR : The normalized Inter-quartile range of all of the inlier pixels per feature. The range is computed independently in each channel.
Gene expression in human keratinocytes from psoriatic skin (directly into the lesion or in area without lesion) or healthy person (control) used for human tissue-engineered skin substitutes and co-cultured with T-lymphocyte