Keratinocyte serum free medium

Total of 1×10⁵ adult pooled NHEK cells (Promo Cell, Heidelberg, Germany) were cultured in keratinocyte serum-free medium (Promo Cell) with epidermal growth factor plus bovine pituitary extract (Gibco, Gran Island, NY) and 1% antibiotics, and seeded at 1×10⁴ cells/ml in 25 cm² tissue culture flasks at 37°C in 5% CO₂. The medium was changed daily until the cells were confluent. NHEKs were detached using 0.25% trypsin and 0.04% EDTA. Total of 3×10⁵ NHEKs (2×10⁵ cells/ml) were seeded onto Transwell plates at passages 2-4 to co-culture with 1×10⁵ DMSCs from 12 psoriasis and 12 control (500μl cell suspension at a concentration of 2×10⁵ cells/ml), respectively. NHEKs were detached using 0.25% trypsin-0.04% EDTA after 72 h of co-culture. Afterward, NHEKs were harvested for further analyses. NHEKs cultured without any DMSCs for 72h alone served as “Untreated NHEKs”. The NHEKs co-cultured with psoriatic DMSCs and control DMSCs were labelled as “P-DMSC-Treated NHEKs” and “C-DMSC-Treated NHEKs”, respectively.

Human follicle dermal papilla cells (HFDPCs) (PromoCell GmbH, Heidelberg, Germany) were cultured in keratinocyte serum-free medium (PromoCell GmbH) with supplied supplement and 100 mg/mL penicillin and streptomycin (Gibco, Grand Island, NY, USA) in a 5% CO₂ incubator at 37°C. Cells were used within ten passages for the experiment. The HFDPCs (1.5 × 10⁵ cells/mL) were seeded in collagen type-I coated 96-well and 6-well plates and incubated overnight. The cells were treated with DHT (10 nM, Sigma-Aldrich, St. Louis, MO, USA) with or without finasteride (1 μM, Sigma, USA) or 50 μg/mL of S. hexaphylla extract for 24 and 72 h. Cellular morphology was observed at 200 magnification using a phase contrast microscope (Olympus, Japan).