Hydrocortisone

Sulfonated-PRX triblock copolymers composed of sulfopropyl ether-modified α-CDs threaded onto a PEG chain as a middle PRX segment and poly(benzyl methacrylate) (PBzMA) at both terminals of the PEG as anchoring segments (SPE-PRXs) were prepared as described previously.^{33 (link)} SPE-PRXs with different numbers of threading CDs were obtained by altering the PEG/α-CD molar ratios. HBMSCs, an HBMSC Growth Medium BulletKit (HBMSC growth medium), HUVECs, endothelial growth medium-2 (HUVEC growth medium) supplemented with 0.1% VEGF, 0.1% human epidermal growth factor, 0.1% R3-insulin-like growth factor-1, 0.1% ascorbic acid, 0.04% hydrocortisone, 0.4% human fibroblast growth factor-2, 0.1% heparin, 2% fetal bovine serum, and 0.1% gentamicin were purchased from Lonza (Walkersville, MD, USA). Mesenchymal stem cell osteogenic differentiation medium (HBMSC differentiation medium) was purchased from Promo Cell (Heidelberg, Germany). Trypsin/ethylenediaminetetraacetic acid (EDTA) solution, phosphate buffered saline (PBS), 4% paraformaldehyde, alizarin red S, and dimethyl sulfoxide (DMSO) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Ammonia solution (28%) was purchased from Kanto Chemical Industry (Tokyo, Japan). A 24-well tissue culture polystyrene (TCPS) plate was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Neonatal normal human epidermal keratinocytes (NHEKs) were purchased from Lonza (Basel, Switzerland) and cultured in KGM-Gold (Lonza), supplemented with bovine pituitary extract, recombinant human epidermal growth factor, insulin, hydrocortisone, gentamycin–amphotericin, transferrin, and epinephrine (Lonza) at 37 °C in 5% CO₂. The medium was changed every 2 days. The cells reached 70–80% confluence and were passaged three times. The third passage of cells was used in all experiments. HaCaT cells (human keratinocyte cell line) were maintained in DMEM, supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were passaged at 70–80% confluence and used in the experiment of transfection of plasmids.

The murine dendritic cell line, JAWS II, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). JAWS II was cultured in the presence of alpha-minimum essential media (Lonza, Basel, Switzerland) complemented with 20% fetal bovine serum (FBS, Life Technologies; Grand Island, NY, USA), 1% penicillin-streptomycin (Sigma-Aldrich, St. Quentin Fallavier, France), 4 mM L-glutamine (Lonza), 1 mM sodium pyruvate (Lonza), and 5 ng/mL of Granulocyte-Macrophage Colony Stimulating Factor (Miltenyi Biotec; San Diego, CA, USA). The cells were maintained in culture in a humified incubator at 37 °C and 5% CO₂.
The MDA-MB-231 human breast cancer cells were purchased from ATCC and cultured at 5% CO₂ at 37 °C in Dulbecco’s modified Eagle media (Lonza), supplemented with 10% FBS, 2 mM glutamine, 10% penicillin-streptomycin.
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and grown at 37 °C and 5% CO₂ in endothelial basal cell growth medium, supplemented with 2% FBS, 1% streptomycin-penicillin, 0.1% ascorbic acid, 0.1% human epidermal growth factor, 0.1% heparin, 0.1% VEGF, 0.1% gentamicin-amphotericin B, 0.04% hydrocortisone, 0.4% human bFGF, and 0.1% R3-IGF-1 (all from Lonza).

Primary Caviae porcellus vascular endothelial cells were derived from segments of aortic tissue isolated from normal animals (guinea pigs) as previously described by Wang JM. et al. (2017) [71 (link)]. Human umbilical vein endothelial cells of the HUVEC line were obtained commercially (C2517A, Lonza, Walkersville, MD, USA). Cells were cultured in endothelial growth medium (EGM-2) enriched with fetal bovine serum (FBS), hydrocortisone, fibroblast growth factor (fFGF), vascular endothelial growth actor (VEGF), Epidermal Growth Factor (EGF), ascorbic acid, gentamicin sulphate, amphotericin (GA-1000) and heparin (Lonza, Poland), at 37 °C in a humidified atmosphere containing 5% CO₂. Every 2–3 days, the medium was changed, and the cells were passaged at 80–90% confluence. Cells were trypsinized, washed with phosphate buffered saline (PBS), and their viability was then assessed by trypan blue staining and cell suspensions were adjusted to working density.

Pooled HUVEC (from 3 to 6 individual donors, Lonza, Cologne, Germany) and HUAEC (from 250 individual donors, PeloBiotech GmbH, Planegg, Germany) were cultivated in EGM-2 Bullet Kit containing endothelial cell culture medium supplemented with 2% (v/v) fetal bovine serum, vascular endothelial growth factor, basic fibroblast growth factor, human epithelial growth factor, insulin-like growth factor-1, hydrocortisone, heparin, ascorbic acid, gentamycin, and amphotericin B (Lonza) in a standard humidified incubator at 37 °C with 5% (v/v) CO₂. Cells of passage 5 were seeded in 4-well PCA chamber slides (Sarstedt, Nürnbrecht, Germany) at a cell density of 15,000 cells/well with 1 mL medium per well, and cultivated for up to 7 days. Medium was exchanged at day 2 and day 4 of cultivation.

Normal human epidermal keratinocytes (NHEKs) obtained from Lonza (Basel, Switzerland) were grown in culture dishes at 37 °C in 5% CO₂^{35 (link)}. The NHEKs were cultured in serum‐free keratinocyte growth medium supplemented with bovine pituitary extract, recombinant epidermal growth factor, insulin, hydrocortisone, transferrin, and epinephrine (all from Lonza). Culture medium was replaced every 2 days. Near confluence (70%–90%), cells were disaggregated with 0.25 mg/mL trypsin/0.01% ethylenediaminetetraacetic acid (Lonza) and subcultured. Second‐ to fourth‐passage NHEKs were used in all experiments. The cells (1 × 10⁵) were seeded in 24‐well culture plates, allowed to attach for 24 h, and then subsequently treated with or without IL‐17 or TNF-α (PeproTech, Rocky Hill, NJ, USA) for 24 h.