Follicle dermal papilla cell growth medium

Hair Follicle Dermal Papilla Cells (HFDPC) (C12071, Promocell, Heidelberg, Germany) were cultured at 10,000 cells/cm² from passage 2 to passage 6 in 75 cm² T-flasks in Follicle Dermal Papilla Cell Growth Medium (C26501, Promocell), which contains fetal calf serum, bovine pituitary extract, bFGF and insulin, supplemented with L-Glutamine (X0550, Biowest, Nuaillé, France) and 1% (w/v) Penicillin/Streptomycin (P/S) (L0022, Biowest). Human Foreskin Fibroblasts (HFF) and HeLa cells were cultured at 10,000 cells/cm² for not more than 10 passages in DMEM (DMEM-HXA, Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% FBS (S1810; Biowest), L-Glutamine and P/S. Adipose-derived stem cells (ADSC) were cultured from passage 2 to passage 6 at 10,000 cells/cm² in ADSC Basal Medium (PT-3273, Lonza, Basel, Switzerland) with SingleQuots^TM Growth Supplement Kit (PT-4503, Lonza). Cultures were maintained at 37 °C and 5% CO₂ in a humidified atmosphere and passaged at 80% confluency.

HFDP cells were cultured in follicle dermal papilla cell growth medium (PromoCell GmbH, Heidelberg, Germany) containing 10% FBS, 50 units/mL of penicillin and 50 mg/mL of streptomycin. HFDP cells were distributed at a concentration of 1 × 10⁴ cells/well to a 96-well plate and cultivated in a CO² incubator adjusted to 37 °C with 95% humidity and 5% CO². For cell viability, cells were treated with F1, F2, F3 (1, 10, 50 and 100 μg/mL) and hyaluronic acid (1 μg/mL) for 24 h. MTS solution (0.5 mg/mL, CellTiter 96^® Aqueous One Solution Cell Proliferation Assay kit) was added to each well for 4 h (5% CO² at 37 °C), and 100 μL of DMSO was added to solubilize purple formazan crystals. The mixed samples (F2 + F3 fraction, FFM) were dissolved in the new growth medium so that the final concentration was 1, 10, 50 and 100 μg/mL, followed by treatment with cytoplasm, and cultivation for five days. The proliferation rate of cells was measured using the Cell Titer 96^® Aqueous One Solution Cell Proliferation Assay purchased from Promega Co. (Madison, WI, USA). The absorbance was measured at 490 nm using the Spectramax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) [22 (link)].

hDPCs were cultured in Follicle Dermal Papilla Cell Growth Medium with Growth Medium Supplement Mix (PromoCell, Heidelberg, Germany) and 1% Gibco Antibiotic-Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). hORS cells were cultured in EpiLife medium with EpiLife Defined Growth Supplement (Gibco, New York, NY, USA) and 1% penicillin/streptomycin (Gibco). hDPCs and hORS cells were maintained at 37 °C in a humidified 5% CO₂ incubator. We used Nexinhib20 (Rab27a inhibitor; Tocris Bioscience, Bristol, United Kingdom). For chemical treatment, hDPCs and hORS cells were seeded, and the Nexinhib20 was treated after 24 h with normal mediums, including 1% penicillin/streptomycin or Gibco Antibiotic-Antimycotic. For cell proliferation assay and migration assay, Nexinhib20 was treated to the cells with different doses (2 or 20 μM) for 3 days, and the cells were then harvested.

Human hair follicle dermal papilla cells (HFDPCs) were purchased from Promocell (Heidelberg, Germany) and grown in follicle dermal papilla cell growth medium (Promocell, Sickingenstr, Heidelberg, Germany) supplemented with 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin (Gibco). The cells were cultured in a humidified atmosphere at 37 °C and 5% CO₂.